{alpha}2- to {beta}3-Adrenoceptor switch in 3T3-L1 preadipocytes and adipocytes: modulation by testosterone, 17{beta}-estradiol, and progesterone

Marta Monjo, Esperanza Pujol, and Pilar Roca

Grup de Metabolisme Energètic i Nutrició, Departament de Biologia Fonamental i Ciències de la Salut, Institut Universitari d'Investigació en Ciències de la Salut, Universitat de les Illes Balears, Palma de Mallorca, Spain

Submitted 29 November 2004 ; accepted in final form 9 February 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sex steroid hormones are important factors in the determination of fat distribution and accumulation. The aim of this study was to investigate the effect of testosterone (T), 17{beta}-estradiol (17{beta}E), and progesterone (P) on adrenergic receptor (AR) gene expression in 3T3-L1 preadipocytes and adipocytes and their relation to the proliferation and differentiation processes. Our data clearly show that {alpha}2A-AR was the highest AR subtype expressed in preadipocytes, whereas in mature adipocytes was by far {beta}3-AR. In the differentiation process to adipocytes, {alpha}2A-AR expression was decreased to 0.3-fold (P < 0.01), whereas {beta}3-AR was upregulated 578-fold (P < 0.001) compared with preadipocytes. In addition, the expression of {alpha}2A-AR in preadipocytes was increased upon incubation with T, 17{beta}E, and P, and a stimulation of proliferation was also observed in 17{beta}E- and P-treated cells. In mature adipocytes, 17{beta}E and P enhanced both {alpha}2A- and {beta}3-AR gene expression (although the effects on {beta}3-AR mRNA levels could be more relevant, since {beta}3-AR was the most highly expressed), whereas T only increased {alpha}2A-AR mRNA levels. Leptin and adipocyte fatty acid-binding protein mRNA levels were higher after 17{beta}E and P treatment, possibly indicating a proadipogenic effect of these hormones. In conclusion, this study indicates that AR gene expression is affected by these hormones in both preadipocytes and adipocytes, which could have potential importance when considering the role of ARs in the mechanisms underlying the sex-related differences in adipose tissue regional distribution.

sex steroid hormones; {alpha}2A-; {beta}1-; {beta}2-; and {beta}3-adrenergic receptors; cell proliferation; 3T3-L1 adipocyte


FAT DISTRIBUTION AND ACCUMULATION are known to be under the control of sex steroid hormones (3). Android obesity, which is an important risk factor for cardiovascular diseases, is characterized by excessive adipose tissue deposition in abdominal and visceral regions, whereas in gynoid obesity fat accumulates in the femoral subcutaneous region (4).

The excessive accumulation of adipose tissue in different anatomical regions may be because of increased proliferation of preadipocytes (i.e., hyperplasia) and/or enlargement of existing adipocytes (i.e., hypertrophy). The latter is essentially related to altered balance between the triacylglycerol storage and lipolytic pathways (11).

The adrenergic system plays an important role in both the regulation of growth and lipolysis in white adipose tissue, through the action of catecholamines on adrenergic receptors (AR), which are coupled to several intracellular transduction pathways (27). With regard to the control of proliferation, cAMP modulates growth of many cell types, which could be mediated in part through interactions with the mitogen-activated protein (MAP) kinase pathway and the stimulation of {alpha}2A-AR (5). On the other hand, the regulation of lipolysis in mature adipocytes depends on the functional balance between inhibitory ({alpha}2A-AR) and stimulatory ({beta}1-, {beta}2-, and {beta}3-AR) adrenoceptors, which are coupled to cAMP production (27). Changes in the activity and/or amount of these receptors in mature adipocytes seem to be responsible, at least in part, for some of the site- and sex-related differences (25).

Sex steroid hormones have been proposed as key factors that could explain the sex differences observed in fat distribution and accumulation. The existence of sex steroid hormone receptors in adipocytes (8, 21, 23, 29) suggests that these hormones could have direct regulatory effects on the expression of some genes. In fact, testosterone, 17{beta}-estradiol, and progesterone have been shown to modulate the expression of the different adrenoceptor subtypes ({alpha}2A-, {beta}1-, {beta}2-, and {beta}3-AR) in primary cultures of brown adipocytes (22). Nevertheless, it is not known whether these hormones could have the same effects in the regulation of AR gene expression in white as in brown adipocytes.

The aim of the present study was to investigate in vitro the direct effects of testosterone, 17{beta}-estradiol, and progesterone on the expression of the different AR subtypes in 3T3-L1 cells. Because AR are known to play an important role both in proliferation and in mobilization of fat stores, we explored whether sex steroid hormones could directly affect AR expression both in preadipocytes and in mature adipocytes. In addition, we evaluated the influence of these hormones on the proliferation rate of preadipocytes, and on the expression of the differentiation marker genes, leptin and adipocyte fatty acid-binding protein (aP2/FABP), in mature adipocytes. We selected this 3T3-L1 cell line as a model, since steroid receptors are expressed and regulated by sex steroid hormones (29).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Testosterone, 17{beta}-estradiol, and progesterone were from Sigma (St. Louis, MO). Other cell culture reagents were supplied by Sigma and Invitrogen (Carlsbad, CA); routine chemicals were from Merck (Darmstadt, Germany) and Panreac (Barcelona, Spain).

Cell culture. Murine 3T3-L1 preadipocytes (American Type Culture Collection) were routinely cultured at 37°C in a humidified atmosphere of 8% CO2. The cells were maintained in growth medium with the following constituents: DMEM supplemented with 10% newborn calf serum, 4 mM glutamine, and antibiotics (50 IU penicillin/ml and 50 µg streptomycin/ml). For proliferation and mRNA studies in preadipocytes, 2% newborn calf serum was used.

Cell differentiation was initiated 2 days after confluence by incubation for 2 days in differentiating medium containing 10% FCS, 4 mM glutamine, and antibiotics (50 IU penicillin/ml and 50 µg streptomycin/ml), adding 0.5 mM IBMX, 0.25 µM dexamethasone (Dex), and 5 µM insulin (INS). This was followed by 2 days in differentiating medium containing 5 µM insulin and without insulin thereafter for 3 days, when cells presented a differentiated morphology (>90% of the cells) with important lipid accumulation. Once differentiated (day 10 postconfluence), the cells were placed in serum-free medium using DMEM-F-12 (1:1) and supplemented with 0.5% BSA, 4 mM glutamine, and antibiotics (50 IU penicillin/ml and 50 µg streptomycin/ml).

Treatments. Testosterone, 17{beta}-estradiol, and progesterone were dissolved in ethanol and added to the corresponding flasks/wells to a final concentration from 10–9 to 10–7 M. An equivalent volume of ethanol was added in untreated controls.

Cell proliferation. The proliferation rate of the cells was measured using [3H]thymidine. Subconfluent cells were incubated with DMEM supplemented with 2% newborn calf serum, 4 mM glutamine, and antibiotics (50 IU penicillin/ml and 50 µg streptomycin/ml), treated with testosterone, 17{beta}-estradiol, or progesterone (10–7 M), and pulsed with 37 kBq/ml (1 µCi/ml) [3H]thymidine. After 48 h, the medium was removed, and the cells were washed two times with ice-cold 1x PBS and two times with ice-cold 5% TCA to remove unincorporated [3H]thymidine. The cells were solubilized in 1 M NaOH (500 µl), and 400 µl of the solubilized cell solution were transferred to 4 ml of ACS Scintillation Cocktail (Amersham Pharmacia Biotech, Barcelona, Spain) and measured for 6 min in a liquid scintillation counter (Beckman LS 3801 scintillation counter).

RNA isolation. Total RNA was isolated using a monophasic solution of phenol and guanidine isothiocyanate (Roche Diagnostics, Manheim, Germany), following the instructions of the manufacturer. RNA was quantified using a spectrophotometer set at 260 nm.

Northern blot analysis. Total RNA (30 µg), denatured with formamide-formaldehyde, was fractionated by agarose gel electrophoresis, transferred to a Hybond nylon membrane (Roche Diagnostics) in 20x saline-sodium citrate buffer (1x buffer is 150 mM NaCl and 15 mM trisodium citrate, pH 7.0) by capillary blotting for 16 h, and fixed with ultraviolet light, as previously described (28).

aP2/FABP mRNA and 18S rRNA were analyzed sequentially on the same membrane, in the aforementioned order, by a chemiluminiscence procedure based on the use of antisense oligonucleotide probes end-labeled with digoxigenin and CDP-Star (Roche Diagnostics), essentially as in the protocols provided by Roche Diagnostics. We used the following probes obtained from Roche Diagnostics: for aP2/FABP mRNA, 5'-GCT ATG AGC CTC TGA AGT CCA GAT AGC TC-3'; and for 18S rRNA, 5'-CGC CTG CTG CCT TCC TTG GAT GTG GTA GCC G-3'.

Bands in films were analyzed by scanner photodensitometry and quantified using the KODAK 1D Image Analysis Software 3.5 (Eastman Kodak, Rochester, NY). The value of integrated optical density of each sample for aP2/FABP mRNA was divided by the integrated optical density of the corresponding 18S rRNA band to check for loading and transfer of RNA during blotting. Stripping between analyses was performed by exposing the membranes to boiling 0.1% SDS.

Real-time RT-PCR. Real-time PCR was done for two housekeeping genes (18S rRNA and {beta}2-microglobulin) and five target genes ({alpha}2A-AR, {beta}1-AR, {beta}2-AR, {beta}3-AR, and leptin).

Total RNA (1 µg) was reverse transcribed to cDNA at 42°C for 60 min with 25 units of murine leukemia virus RT in a 10-µl volume of RT reaction mixture containing 10 mM Tris·HCl (pH 9.0 at 25°C), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl2, 2.5 µM random hexamers, 10 units of RNase inhibitor, and 500 µM each dNTP. Each cDNA was diluted 1:10, and aliquots were frozen (–20°C) until the PCR reactions were carried out.

Real-time PCR was performed in the Lightcycler (Roche Diagnostics) using SYBR Green detection. Each reaction contained 1 µl of Lightcycler-FastStart DNA Master SYBR Green I (containing FastStart Taq DNA polymerase, dNTP mix, reaction buffer, MgCl2, and SYBR Green I dye; Roche Diagnostics), 0.5 µM of the sense and antisense specific primers (for all, except for {beta}2-AR, which was 0.25 µM), 2 mM MgCl2, and 3 µl of the cDNA dilution in a final volume of 10 µl. The amplification program consisted of a preincubation step for denaturation of the template cDNA (10 min, 95°C), followed by 40 cycles consisting of a denaturation step (5 s, 95°C), an annealing step (8 s, 60°C for all, except for {beta}1-AR and {beta}2-AR, which were 65 and 63°C, respectively), and an extension step (12 s 72°C). After each cycle, fluorescence was measured at 72°C (excitation wavelength 470 nm, emission wavelength 530 nm). A negative control without cDNA template was run in each assay.

Oligonucleotide primer sequences used for the real-time RT-PCR, amplification efficiencies, and the specific parameters [length and melting temperature (Tm)] of the resulting amplicons are shown in Table 1. Real-time efficiencies were calculated from the given slopes in the Lightcycler software (26) using serial dilutions, showing all the investigated transcripts high real-time PCR efficiency rates and high linearity (r > 0.99) when different concentrations were used. PCR products were subjected to a melting curve analysis on the Lightcycler and subsequently 2% agarose/Tris-borate-EDTA gel electrophoresis to confirm amplification specificity, Tm, and amplicon size.


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Table 1. Primers used and specific parameters of real-time PCR

 
To allow relative quantification after PCR, standard curves were constructed from the standard reactions for each target and housekeeping genes by plotting crossing point (Cp) values, i.e., the cycle number at which the fluorescence signal exceeds background vs. log cDNA dilution. The Cp readings for each of the unknown samples were then used to calculate the amount of either the target or housekeeping relative to the standard, using the Second Derivative Maximum Method with the Lightcycler analysis software 3.5 (Roche Diagnostics). For sex hormone experiments, both {beta}2-microglobulin and 18S rRNA housekeeping genes were used to normalize AR gene expression. To compare AR gene expression between preadipocytes and adipocytes, only the 18S rRNA housekeeping gene was used, since it was the only one that remained unchanged during adipocyte differentiation.

Statistics. All data are presented as mean values ± SE. Differences between groups were assessed by Student's t-test, or one-way ANOVA and least-significant difference for post hoc comparisons, using a statistical software package (SPSS, Chicago, IL).

In the ANOVA, possible effects are as follows: effect of testosterone, effect of 17{beta}-estradiol, and effect of progesterone treatment. Results were considered statistically significant at the P < 0.05 level.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of AR gene expression in 3T3-L1 preadipocytes and adipocytes. The regulation of AR subtypes during the differentiation to adipocytes has yet to be fully understood. Although the presence of the different AR subtypes in white adipocytes has been documented (14), no studies have been carried out to directly compare the expression of both {alpha}2A-AR and {beta}-ARs in preadipocytes and adipocytes.

As shown in Table 2, AR subtypes displayed different Cp, or specific cycle numbers, depending on the starting amount of mRNA in the preadipocyte or adipocyte samples. Taking into account the relationship where higher amounts of mRNA correlate with fewer cycles or Cp (26), and given their high PCR efficiencies, it is reasonable to suggest that {alpha}2A-AR was the highest AR subtype expressed in preadipocytes, whereas in adipocytes it would undoubtedly be {beta}3-AR.


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Table 2. Characterization of AR expression in 3T3-L1 preadipocytes and adipocytes

 
We analyzed the relative expression of the different AR subtypes in preadipocytes and adipocytes (Table 2). This relative expression was normalized by 18S ribosomal RNA, the expression of which was unchanged in the differentiation process to adipocytes, in contrast to {beta}2-microglobulin. {alpha}2A-AR was the only AR downregulated in adipocytes compared with preadipocytes, with a fold change of 0.31. In contrast, all {beta}-ARs were upregulated in adipocytes, with fold changes of 1.9, 3.7, and 578 for {beta}1-, {beta}2-, and {beta}3-AR, respectively.

Effect of testosterone, 17{beta}-estradiol, and progesterone on proliferation in 3T3-L1 preadipocytes. We next investigated the effect of testosterone, 17{beta}-estradiol, and progesterone (10–7 M) on the proliferation rate of 3T3-L1 preadipocytes, since sex hormones have been shown to affect proliferation in preadipocytes from different origins (1, 9, 15). For this purpose, 2% newborn calf serum was used in the medium because it contains essential mitogenic factors for proliferation, and this serum reduction was done to avoid hormonal interferences, together with the fact that serum-free medium was found to be unsuccessful for proliferation studies. However, extremely low levels of estradiol and progesterone in the calf serum (i.e., <10 pg/ml and <0.1 ng/ml, respectively) have been found (13). Incubation for 48 h with sex hormones was selected for the experiments, given that preliminary results indicated that maximum [3H]thymidine incorporation occurred after 48 h (data not shown). As seen in Fig. 1, 17{beta}-estradiol and progesterone had a significant stimulatory effect on the proliferation rate of preadipocytes, whereas no effect was seen after testosterone treatment.



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Fig. 1. Effects of testosterone, 17{beta}-estradiol, and progesterone on [3H]thymidine incorporation into 3T3-L1 preadipocyte DNA. Preconfluent cells were treated for 48 h with testosterone, 17{beta}-estradiol, and progesterone (10–7 M), or with the vehicle (ethanol), and pulsed with 37 kBq/ml. Data represent means ± SE of 3 separate experiments performed in duplicate. *Significant differences of hormone-treated vs. nontreated cells by t-test (P < 0.05).

 
Effect of testosterone, 17{beta}-estradiol, and progesterone on AR gene expression in 3T3-L1 preadipocytes. To find out whether these effects on proliferation were related to changes in AR gene expression, proliferating preadipocytes were treated with testosterone, 17{beta}-estradiol, and progesterone (10–7 M), and the expression of {alpha}2A-, {beta}1- and {beta}3-AR was determined by real-time RT-PCR, using the same cell culture conditions as in the proliferation studies. The {beta}2-AR gene was not analyzed because of its low gene expression in 3T3-L1 preadipocytes. An increase in {alpha}2A-AR mRNA levels was produced with all hormones tested (Fig. 2), with 17{beta}-estradiol (+31%) and progesterone (+31%) showing the greatest stimulatory effect compared with testosterone (+22%). Exposure to testosterone, 17{beta}-estradiol, and progesterone also resulted in increases in {beta}1-AR (i.e., +32 ± 19, +28 ± 13, and +46 ± 23%, respectively) and {beta}3-AR (i.e., +92 ± 73, +101 ± 38, and + 234 ± 124%) mRNA levels (data not shown), although most of these effects were not significant, probably because of the low expression of {beta}1-AR and {beta}3-AR in preadipocytes, which could be responsible for the higher variability found.



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Fig. 2. Effects of testosterone, 17{beta}-estradiol, and progesterone on {alpha}2A-adrenergic receptor (AR) mRNA levels in 3T3-L1 preadipocytes. Proliferating 3T3-L1 preadipocytes were treated for 48 h with testosterone, 17{beta}-estradiol, and progesterone (10–7 M). Total RNA was isolated, and mRNA expression was analyzed by real-time RT-PCR, as described in materials and methods. Ratios of the target gene relative to the housekeeping genes were expressed as a percentage of nontreated cell samples, which were set to 100%. Data represent means ± SE of 3 separate experiments performed in duplicate. Significant differences of hormone-treated vs. nontreated cells by t-test: *P < 0.05, **P < 0.01, ***P < 0.001.

 
Effect of testosterone, 17{beta}-estradiol, and progesterone on aP2/FABP and leptin gene expression in differentiated 3T3-L1 adipocytes. Seeing that an increased proliferation in preadipocytes was produced after 17{beta}-estradiol and progesterone treatment, we also looked into the effect of these hormones on differentiation. For this purpose, differentiated adipocytes were placed in serum-free medium and treated with testosterone, 17{beta}-estradiol, and progesterone at concentrations from 10–9 to 10–7 M. Because aP2/FABP is a critical marker for differentiation of preadipocytes into adipocytes (19), its expression was examined by Northern blotting (Table 3). An increased aP2/FABP gene expression was observed after 17{beta}-estradiol (10–9 and 10–7 M) and progesterone treatment, whereas testosterone did not seem to affect aP2/FABP mRNA levels.


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Table 3. Effects of testosterone, 17{beta}-estradiol, and progesterone on aP2/FABP and leptin mRNA levels in differentiated 3T3-L1 adipocytes

 
In parallel to the adipocyte marker aP2/FABP, leptin expression is induced as well during differentiation of 3T3-L1 preadipocytes to adipocytes, and with a time course comparable to other adipocyte markers (18). Real-time RT-PCR analyses were performed to detect leptin mRNA, since 3T3-L1 adipocytes have generally been reported to express low levels of leptin mRNA when differentiated by the traditional IBMX/Dex/INS protocol (32). The ability of testosterone, 17{beta}-estradiol, and progesterone to regulate leptin expression is shown in Table 3. Testosterone treatment was found to be ineffective in modulating leptin gene expression. On the other hand, progesterone had a significant stimulatory effect on leptin mRNA levels, and 17{beta}-estradiol showed a moderate tendency to increase leptin mRNA levels at the highest concentration tested, 10–7 M, although this did not reach significant levels (P = 0.07).

Effect of testosterone, 17{beta}-estradiol, and progesterone on AR gene expression in differentiated 3T3-L1 adipocytes. To address a potential role of ARs for the gender-related differences in white adipocytes, as pointed out by several in vivo studies (16, 17, 25), the influence of testosterone, 17{beta}-estradiol, and progesterone on {alpha}2A- and {beta}3-AR gene expression in differentiated 3T3-L1 adipocytes was also evaluated (Table 4).


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Table 4. Effects of testosterone, 17{beta}-estradiol, and progesterone on {alpha}2A-AR and {beta}3-ARmRNA levels in differentiated 3T3-L1 adipocytes

 
Testosterone treatment produced an upregulation in the mRNA levels for {alpha}2A-AR, as previously reported in white (6, 24) and in brown (22) adipocytes. Besides, 17{beta}-estradiol and progesterone brought about an important induction of mRNA levels for this receptor, contrary to what happens in brown adipocytes (22).

With regard to {beta}3-AR, the most abundant {beta}-AR in mature 3T3-L1 adipocytes, both progesterone and 17{beta}-estradiol showed stimulatory effects on its mRNA levels, whereas no effect was seen after testosterone treatment, in the same way as in brown adipocytes (22). Progesterone and 17{beta}-estradiol also produced an increase in {beta}1-AR and {beta}2-AR mRNA levels (data not shown); however, because of their low expression in mature adipocytes, as seen in Table 2, the physiological relevance of these receptors compared with {beta}3-AR was considered negligible.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present work, we have characterized AR gene expression in preadipocytes and adipocytes. Additionally, we have focused on the interplay between sex steroid hormones (testosterone, 17{beta}-estradiol, and progesterone) and AR gene expression in preadipocytes and adipocytes and their relation to the proliferation and differentiation processes.

In preadipocytes, {alpha}2A-AR was the highest AR subtype expressed, whereas in adipocytes {beta}3-AR has been found to be dramatically increased. This expression pattern agrees with the reported major role of {alpha}2A-AR during the proliferation process (5), whereas in mature 3T3F442A adipocytes {beta}3-AR has been shown to be the predominant receptor mediating lipolysis (10).

Several studies have provided evidence of a hormonal regulation of preadipocyte proliferation, which is important to understand the complex mechanisms by which growth of adipose tissue is regulated (12). We have reported that 17{beta}-estradiol and progesterone increased the proliferation rate of preadipocytes. The positive effect of 17{beta}-estradiol on proliferation is in accord with other studies in female rat preadipocytes (9) and in human preadipocytes (1). Additionally, stimulatory estradiol effects on proliferation have been shown to be both concentration and time dependent in 3T3-L1 preadipocytes (15). The stimulatory effect of 17{beta}-estradiol and progesterone observed on {alpha}2A-AR gene expression in 3T3-L1 preadipocytes (which was higher compared with testosterone), could contribute to the increase of proliferation by these hormones and would also agree with the important role of {alpha}2A-AR during proliferation. In this sense, it has been reported that catecholamines are involved in the control of white adipocyte proliferation through {alpha}2A-AR activation and linked to the MAP kinase pathway (5). Moreover, estradiol positive effects on proliferation have been shown to be related to MAP kinases as well (13). On the whole, our results provide evidence of a direct effect of testosterone, 17{beta}-estradiol, and progesterone inducing {alpha}2A-AR expression, suggesting an important role for this receptor in preadipocytes.

Sex hormones have also been reported to influence the differentiation process of adipocytes. The present increase observed in aP2/FABP gene expression in adipocytes treated with progesterone and 17{beta}-estradiol, together with the increase in leptin expression in progesterone-treated cells, could indicate a proadipogenic effect of these hormones in 3T3-L1 adipocytes. Supporting this hypothesis, there is a report showing that androgens elicit an antiadipogenic effect, whereas estrogens behave as proadipogenic hormones (9). These differences on leptin expression fit well with the sexual dimorphism that appears in plasma leptin levels (30) and also with the stimulatory effect of 17{beta}-estradiol on leptin expression in rat adipocytes (20). In addition, it has been pointed out that the increase in leptin levels during pregnancy could be explained by a stimulatory effect of gestational hormones (31).

Taken together, 17{beta}-estradiol and progesterone have been shown to enhance both preadipocyte proliferation and differentiation in the 3T3-L1 cell line, whereas these processes seem not to be influenced by testosterone. It is important to highlight that conversion between steroids in 3T3-L1 adipocytes cannot be discarded, since adipose tissue steroid conversion is a well-accepted phenomenon (2).

In mature 3T3-L1 adipocytes, 17{beta}-estradiol and progesterone enhanced both {alpha}2A- and {beta}3-AR gene expression, whereas testosterone only increased {alpha}2A-AR mRNA levels. The positive effect of the hormones tested in {alpha}2A-AR mRNA levels was reduced when administered at higher concentrations (10–7 M), indicating a different action depending on the levels of the hormone. Despite the fact that 17{beta}-estradiol and progesterone were able to affect the expression of inhibitory (i.e., {alpha}2A-AR) and stimulatory (i.e., {beta}3-AR) adrenoceptors, the effects on {beta}3-AR mRNA levels could be more important in the net balance on cAMP accumulation, because {beta}3-AR is by far the highest expressed. As a result, it could be argued that 17{beta}-estradiol and progesterone, mainly through {beta}3-AR, could increase lipolysis in mature 3T3-L1 adipocytes when stimulated with {beta}-AR agonists. This hypothesis would be consistent with the increase in {beta}3-AR mRNA levels and the stronger cAMP response to isoproterenol (a {beta}1-, {beta}2-, and {beta}3-AR agonist) induced by oleoyl-estrone in perovaric white adipocytes (7). On the other hand, without stimulation of {beta}3-AR by {beta}-AR agonists, progesterone and 17{beta}-estradiol have been found in this study to stimulate differentiation of the cells, together with an increase in {beta}3-AR gene expression, which is markedly induced in the differentiation process from preadipocytes to adipocytes, in parallel to lipid accumulation.

Sex hormones have displayed differential effects on the expression of AR subtypes in 3T3-L1 mature white adipocytes that differ from those effects previously observed in brown adipocytes (22). This is especially evident in the effect of progesterone and 17{beta}-estradiol on {alpha}2A-AR gene expression, downregulating or upregulating its levels in brown and white adipocytes, respectively. In contrast, testosterone behaved similarly in both systems, enhancing {alpha}2A-AR gene expression. As a result, the differential regulation of {alpha}2A-AR gene expression could have potential importance when the function of white and brown adipose tissue is considered, focusing on the storage of lipids or thermogenesis, respectively.

In summary, the present investigation shows that 17{beta}-estradiol and progesterone affect the in vitro proliferation of 3T3-L1 preadipocytes and suggests that the increased {alpha}2A-AR gene expression could contribute to promoting the proliferation by these hormones through mechanisms already described that might imply MAP kinase activation. In mature adipocytes, testosterone increased {alpha}2A-AR mRNA levels, whereas 17{beta}-estradiol and progesterone enhanced both {alpha}2A- and {beta}3-AR gene expression. The findings reported here could have potential importance when the role of ARs in the mechanisms underlying the sex-related differences in adipose tissue regional distribution is considered.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by Conselleria d'Innovació i Energia de la Comunitat Autònoma de les Illes Balears (PRDIB-2002GC4-24), Dirección General de Enseñanza Superior e Investigación Científica of the Spanish Government (BFI 2000-0988-C06-04), and Fondo de Investigaciones Sanitarias (PI021339) of the Spanish Government. M. Monjo was supported by a grant of the University of the Balearic Islands, and Esperanza Pujol was supported by a grant of the Spanish Government.


    ACKNOWLEDGMENTS
 
We thank Dr. F. García, Dr. M. Gianotti, Dr. I. Lladó, Dr. A.M. Proenza, and Dr. J. Oliver for critical reading of the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. Roca, Departament de Biologia Fonamental i Ciències de la Salut, Ed. Guillem Colom, Universitat de les Illes Balears, Cra. Valldemossa, Km 7.5, Palma de Mallorca, 07122, Spain (e-mail: pilar.roca{at}uib.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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