Depot differences in steroid receptor expression in adipose tissue: possible role of the local steroid milieu

S. Rodriguez-Cuenca, M. Monjo, A. M. Proenza, and P. 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 23 June 2004 ; accepted in final form 7 September 2004


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Sex hormones play an important role in adipose tissue metabolism by activating specific receptors that alter several steps of the lipolytic and lipogenic signal cascade in depot- and sex-dependent manners. However, studies focusing on steroid receptor status in adipose tissue are scarce. In the present study, we analyzed steroid content [testosterone (T), 17{beta}-estradiol (17{beta}-E2), and progesterone (P4)] and steroid receptor mRNA levels in different rat adipose tissue depots. As expected, T levels were higher in males than in females (P = 0.031), whereas the reverse trend was observed for P4 (P < 0.001). It is noteworthy that 17{beta}-E2 adipose tissue levels were higher in inguinal than in the rest of adipose tissues for both sexes, where no sex differences in 17{beta}-E2 tissue levels were noted (P = 0.010 for retroperitoneal, P = 0.005 for gonadal, P = 0.018 for mesenteric). Regarding steroid receptor levels, androgen (AR) and estrogen receptor (ER){alpha} and ER{beta} densities were more clearly dependent on adipose depot location than on sex, with visceral depots showing overall higher mRNA densities than their subcutaneous counterparts. Besides, expression of ER{alpha} predominated over ER{beta} expression, and progesterone receptor (PR-B form and PR-A+B form) mRNAs were identically expressed regardless of anatomic depot and sex. In vitro studies in 3T3-L1 cells showed that 17{beta}-E2 increased ER{alpha} (P = 0.001) and AR expression (P = 0.001), indicating that estrogen can alter estrogenic and androgenic signaling in adipose tissue. The results highlighted in this study demonstrate important depot-dependent differences in the sensitivity of adipose tissues to sex hormones between visceral and subcutaneous depots that could be related to metabolic situations observed in response to sex hormones.

steroid receptors; testosterone; 17{beta}-estradiol; progesterone


VISCERAL AND SUBCUTANEOUS adipose tissues display different metabolic properties, manifested by differences in the expression level of genes involved in fat cell metabolism, and in the secretion of adipose factors that could be involved in some pathologies in both rodents and humans (2, 3, 51, 59). Moreover, the existence of sex differences in adipose tissue metabolism affecting distribution pattern is well known. In this respect, we have previously investigated in rats the overall effect of sex and regional locations of the adipose tissue on some of the mechanisms affecting the lipolytic capacity, such as adrenergic receptor balance (45, 48) and several cascade steps at the postreceptor level, such as adenylyl cyclase, protein kinase A and hormone-sensitive lipase (45). It has been suggested that these sex-dependent differences are due to variations in the hormonal environment, as sex hormones play an important role in the adipose tissue metabolism (32, 48).

Previous studies in rodents and humans have demonstrated that sex hormones are involved in the direct modulation of adipose tissue metabolism at multiple levels, acting at different steps of the lipolytic and lipogenic pathways (18, 23); coactivating or modulating the expression of adipogenic transcription factors (23); and altering the adipocyte proliferation (1), the glucose metabolism (15), and the expression of several adipocyte hormones (26, 35, 50, 56).

In addition to serving as a steroidal reservoir, adipose tissue is one of the most important extragonadal sources of steroids, due to the specific expression of steroidogenic enzymes, such as aromatase (4), suggesting a potential impact on the local adipose tissue metabolism that would be independent of the sex hormone plasma milieu (53). Steroid hormones exert their effects through specific receptors that belong to the superfamily of nuclear hormone receptors, which are widely expressed in several tissues including adipose tissue (7, 10, 20, 21, 31, 36). The steroid receptor action is tripartite, involving a receptor with its corresponding ligands and corregulator proteins that forms a complex that interacts with basal transcription factors that ultimately regulate transcription of target genes (5).

In this study, sex hormone content and steroid receptor mRNA were determined in adipose tissues from different regions in male and female rats to elucidate their different sensitivities to hormonal action. In addition, an in vitro model was used to further address the role of steroid hormones modulating steroid hormone receptor expression.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

One hundred ten-day-old male and female Wistar rats were used. Males and females were housed separately three per cage in the same room at 22°C, with a 12:12-h light-dark cycle, with free access to drinking water and standard chow pellets (Panlab, Barcelona, Spain). All animals were killed by decapitation at the start of the light cycle. Animal studies were in agreement with the Universitat de les Illes Balears Bioethics Committee guidelines for animal care.

Experimental Setting 1

Sex hormone content and receptor mRNA were analyzed in male and female white adipose tissue from different anatomic locations to assess possible depot and sex differences. Inguinal (subcutaneous depot), gonadal, and mesenteric (visceral depot) and retroperitoneal tissues (nonsubcutaneous nonvisceral) were dissected and stored at –80°C until use.

Experimental Setting 2

To investigate whether the sex hormone content could be involved in the regulation of the steroid receptor mRNA expression in adipocytes, we designed an in vitro model using the murine 3T3-L1 preadipocyte cell line. Differentiated adipocytes were treated with a 10–7 M dose of testosterone (T), 17{beta}-estradiol (17{beta}-E2), or progesterone (P4).

Steroid Assays

Determination of T, 17{beta}-E2, and P4 levels from adipose tissue samples was adapted from methods previously described (58). Briefly, 600 mg of frozen tissue were homogenized in 1 ml of phosphate buffer, pH 7.4. The homogenate was extracted twice with 5 ml of ethanol-acetone (1:1, vol/vol). The extract was concentrated under N2 at 37°C and delipidated with 8 ml of 70% methanol at –20°C. After evaporation of the methanol under N2 at 37°C, the residue was dissolved in phosphate buffer containing 2% BSA. Levels of T, 17{beta}-E2, and P4 in tissue extracts and plasma were analyzed using a immunoenzymatic determination kit.

Cell Culture and Treatments

Murine 3T3-L1 preadipocytes (American Type Culture Collection) were routinely cultured at 37°C in a humidified atmosphere with 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).

Cell differentiation was initiated 2 days after confluence by incubation for 2 days in a differentiation medium containing 10% fetal calf serum, 4 mM glutamine, and antibiotics (50 IU penicillin/ml and 50 µg streptomycin/ml) and additionally 0.5 mM 3-isobutyl-1-methylxanthine, 0.25 µM dexamethasone, and 5 µM insulin. This was followed by a 2-day incubation in a differentiation medium containing 5 µM insulin and in the absence of insulin thereafter for 3 days, when cells presented a differentiated morphology with important lipid accumulation. Once differentiated, the cells were placed in serum-free medium consisting of 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) for hormone treatments.

The treatments were carried out on day 10 postconfluence. Sex hormones (T, 17{beta}-E2, and P4) were dissolved in ethanol and added to the corresponding flasks to a final concentration of 10–7 M for 24 h.

Total RNA Extraction

Total RNA was isolated from rat adipose tissues and murine 3T3-L1 adipocytes using Tripure reagent as described earlier (47). The integrity of RNA was verified by optical density (OD) absorption ratio OD260/OD280 between 1.7 and 1.8.

Reverse Transcription

For reverse transcription (RT) of estrogen receptor-{alpha} (ER{alpha}), estrogen receptor {beta}1 (ER{beta}1), androgen receptor (AR), and progesterone receptor (PR-B for the specific isoform B and PR-A+B for both isoforms), 1 µg of total RNA was reverse transcribed to cDNA at 42°C for 15 min with 25 U of MuLV reverse transcriptase 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 U of RNase inhibitor, and 500 µM of each dNTP in a DNA Gene Amp 9700 thermal cycler (Applied Biosystems, Barcelona, Spain).

Real-Time RT-PCR

The primers used were designed with specific primer analysis software Primer3 (Whitehead Institute for Biomedical Research) and Oligo Analyzer 3.0 (Integrated DNA Technologies), and the specificity of the sequences was analyzed by Fasta in the BLAST database (http://www.ncbi.nlm.nih.gov/BLAST/) (Table 1).


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Table 1. Oligonucleotide primer sequences and amplification conditions

 
Real-time PCR was performed using a LightCycler rapid thermal cycler system (Roche Diagnostics, Barcelona, Spain). Reactions were performed in a 10-µl volume containing 3 µl of cDNA sample (diluted 1:10) with 0.5 µM primers and 3 mM MgCl2+ as well as dNTPs, Taq DNA polymerase, and reaction buffer provided in the LightCycler FastStart DNA Master SYBR Green I mix. All real-time conditions are summarized in Table 1. Product specificity was confirmed in initial experiments by agarose gel electrophoresis and routinely by melting curve analysis. PCR efficiencies were calculated from the given slopes in LightCycler Software 3.3.

For the mathematical analysis, crossing point (CP) values were used for each transcript. CP is defined as the point at which fluorescence rises appreciably above the background fluorescence. "Fit Point Method" was performed in the LightCycler software 3.3 (Roche Diagnostics), at which CP was measured at constant fluorescence level. It should be noted that lower CP means higher expression rate and that a difference of 1 cycle number means theoretically a twofold difference in mRNA levels, although this is dependent on the amplification rate (between 1.7 and 2.2 in our study). Statistical data analysis was performed using the Relative Expression Software Tool (REST) for groupwise comparison and statistical analysis of relative expression results in real-time PCR, as previously described (43). Coefficient of variation based on the variation of CP from CP mean value expressed as percentage was given as test variability (Table 2). Test reproducibility for all investigated transcripts was low in intertest experiments and even lower in intratest experiments.


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Table 2. Precision and variability test of PCR amplifications

 
Drugs and Chemicals

T, 17{beta}-E2, and P4 were supplied by Sigma (Madrid, Spain). Tripure reagent and LightCycler FastStart DNA Master SYBR Green I mix were provided by Roche Diagnostics (Madrid, Spain). The reaction reagents for the retrotranscriptase were supplied by Applied Biosystems (Barcelona, Spain), and the sex hormone immunoenzymatic determination kit was supplied by DIAMETRA (Milan, Italy). Cell culture reagents were supplied by Sigma and Invitrogen (Barcelona, Spain), and routine chemicals were from Merck (Barcelona, Spain) and Panreac (Barcelona, Spain).

Statistical Analysis

Adipose tissue weight and sex steroid concentration data are presented as group mean values ± SE. Differences between groups were assessed by Student's t-test in adipose tissue weight data and sex hormone plasma levels, and, in the case of sex hormone tissue levels, two-way analysis of variance (ANOVA) was used, followed by a post hoc least significant difference comparison when a depot effect (D) was shown. The analysis was performed with SPSS 10.0 for Windows. The level of probability was set at P < 0.05 as statistically significant for both ANOVA and Student's t-test.

The statistical PCR data analysis was performed using REST. The statistical model used was the Pair Wise Fixed Reallocation Randomisation Test (43). Differences in expression between groups were assessed using the means for statistical significance by randomization tests, a proper model to avoid the normal distribution assumption of the data. The level of probability was set at P < 0.05 as statistically significant.


    RESULTS
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 ABSTRACT
 METHODS
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Adipose Tissue Weight

As seen in Table 3, adult male rats showed larger adipose depots than females in all different locations (P < 0.001 for inguinal, P = 0.001 for retroperitoneal and gonadal, P = 0.003 for mesenteric). When relative values with respect to body weight are considered, male rats showed higher values only for the inguinal depot (P = 0.014), and no sex differences were observed in gonadal, mesenteric, and retroperitoneal depots.


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Table 3. Adipose tissue weight of different locations

 
Steroid Concentrations in Plasma and Adipose Tissues

Plasma T levels were significantly higher in male than in female rats (P = 0.031). The 17{beta}-E2 and P4 plasma concentrations were higher in female than in male rats, although these differences were of statistical significance only for P4 (P < 0.001).

The same sex profile was depicted when sex hormone contents of the adipose tissue were analyzed, as T levels were higher in males (sex effect P < 0.001), no differences between sexes were observed in 17{beta}-E2 levels, and P4 levels were higher in females (sex effect P < 0.001; Table 4). Moreover, some depot-specific differences were shown, and T adipose tissue levels were lower in the inguinal depot than in the retroperitoneal (P = 0.030) and gonadal depots, although the latter did not reach statistical significance (P = 0.097). Moreover, the inguinal depot showed higher 17{beta}-E2 levels than the rest of the adipose depots. No depot differences in P4 levels of tissue were observed (Table 4).


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Table 4. Effect of sex and tissue location on T, 17{beta}-E2, and P4 concentrations in plasma and white adipose tissues

 
Regional and Sex Differences in Steroid Receptor mRNA Expression

AR. As shown in Table 5, male inguinal and retroperitoneal depots showed a lower AR expression (shown as CP, an inverse of expression data) than male gonadal and mesenteric depots (inguinal vs. gonadal, P = 0.035; retroperitoneal vs. gonadal, P = 0.003; retroperitoneal vs. mesenteric, P = 0.004). In contrast, in female rats, no tissue differences were found. Although AR mRNA levels were higher in almost all cases in males than in females, sex differences were not statistically significant for any of the adipose depots, probably due to the low mRNA levels and the high variability of AR showed by females.


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Table 5. Effect of sex and tissue location on steroid receptor mRNA expression in white adipose tissues

 
ER. Two main estrogen receptors are involved in the estrogenic action, ER{alpha} and ER{beta} (34), and it is well established that both receptors have several splice variants whose presence can modify tissue-estrogenic responsiveness. Thus we decided first of all to find out which was the major form of both receptors in adipose tissues by use of the primers and protocols previously described by classical RT-PCR (38, 44) before going further in the study of mRNA steroid receptors in the adipose depots.

The presence of ER{alpha} spliced variants, previously described for other tissues (38), was almost negligible compared with the full-length form present in all of the adipose tissue analyzed, only ER{delta}3 and ER{delta}4 being detected and identified (Fig. 1A). In fact, ER{beta} isoforms {beta}1 and {beta}2 were not detected in adipose tissue with classical PCR, denoting lower expression levels of these receptors, although in this present experiment ER{beta}1, the most estradiol-sensitive isoform, but not ER{beta}2, was detected by real-time PCR (Fig 1B).



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Fig. 1. A: presence or absence of mRNA of estrogen receptor (ER){alpha} full-length and {delta}3- and {delta}4-forms in several tissues. B: ER{beta}1 and ER{beta}2 in ovary and adipose tissue by classical RT-PCR.

 
Both ER{alpha} full-length and ER{beta}1 mRNA analyses were undertaken using real-time PCR. Thus the male inguinal depot showed lower ER{alpha} mRNA levels compared with retroperitoneal (P = 0.005), gonadal (P = 0.001), and mesenteric depots (P = 0.002); the same tendency was observed in females but was statistically significant only for the gonadal depot (P = 0.001; Table 5). Moreover, in both sexes, the gonadal depot showed higher ER{alpha} mRNA levels than the retroperitoneal one (P = 0.040 for males, P = 0.037 for females). In addition, retroperitoneal, gonadal, and mesenteric adipose tissue differed in both sexes in ER{alpha} mRNA levels, showing only in the retroperitoneal adipose tissue of males a statistically significant threefold higher mRNA levels than females (P = 0.040).

For ER{beta}1 mRNA levels, only females showed differences between depots (Table 5), as inguinal adipose tissue had lower mRNA levels than mesenteric and retroperitoneal adipose tissues (P = 0.009 and P = 0.007, respectively). Moreover, male depots showed higher ER{beta}1 levels than females for the retroperitoneal depot (P = 0.040), whereas the opposite profile was shown in the mesenteric depot (P = 0.048).

PR. Because PR-A is a truncated form of PR-B, it is not possible to amplify the PR-A isoform independently from PR-B, which has physiologically different functions (19). Thus two independent PCR protocols were used to amplify the PR-B isoform and total receptor (PR-A+B). As seen in Table 5, no differences between sexes or adipose tissue depots in the expression of both PR-B and PR-A+B were observed.

Effect of Sex Hormones on Steroid Receptor Expression in 3T3-L1 Adipocytes

Table 6 shows the effect of T, 17{beta}-E2, and P4 on steroid receptor expression in cell culture. AR was upregulated under 17{beta}-E2 treatment (+30%, P = 0.001) and slightly downregulated by P4 (–20%, P = 0.040), whereas T did not modify the AR mRNA levels. ER{alpha} was upregulated under both 17{beta}-E2 and T treatment (+230%, P = 0.001, and +80%, P = not significant, respectively), although the latter did not reach statistical significance. On the other hand, P4 treatment did not exert any effect on ER{alpha} expression. Finally, PR-A+B was not altered under sex hormone treatment at the dosage used.


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Table 6. Effect of sex hormone treatment on ER{alpha}, AR, and PR-A+B mRNA levels in 3T3-L1 adipocyte cell line

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, we provide evidence for a variation in the accumulation patterns of the main sex hormones in adipose tissues as well as in the distribution of their respective receptor mRNAs, which have been found to be both location and sex dependent. Moreover, we have tested in vitro the effect of sex hormones on their receptor mRNA levels in 3T3-L1 adipocytes. The data reported here could help to establish differences in the adipose tissue sensitivity to sex hormone action as well as further elucidate the sex-related differences in the adipose tissue metabolism.

To date, several studies demonstrate a role of the adipose tissue as reservoir and sex hormone extragonadal biosynthesis site (4, 9, 55). However, the differential role of the different depots in steroid metabolism remains unclear.

Adipose tissue sex hormone levels were markedly high, which is in agreement with the function of adipose tissue acting as a sex hormone reservoir. Nevertheless, it is noteworthy that no differences in 17{beta}-E2 tissue levels were noted between females and males, which could suggest a local estrogen synthesis system independent of gonadal action (4, 54). In both sexes, inguinal adipose tissue, which is characterized by scarce vascularization (12), had a 17{beta}-E2-to-T ratio two- to threefold higher than in other adipose tissues (P < 0.001, data not shown). To this extent, McTernan and coworkers (28, 29) have recently demonstrated that human subcutaneous preadipocytes showed a higher aromatase activity compared with visceral ones. Thus the higher 17{beta}-E2-to-T ratio shown by the inguinal depot could be related to the higher proliferative capacity of this tissue, especially when it is taken into account that estrogens can increase preadipocyte proliferation rate (1), whereas androgens can inhibit both proliferation and differentiation (11).

We have also observed variations in the mRNAs of steroid receptors that are adipose tissue location and sex dependent, which could be key in the understanding of adipose tissue steroid responsiveness.

The higher AR mRNA content observed in the gonadal and mesenteric adipose tissue (visceral depots), in contrast to inguinal (subcutaneous) and retroperitoneal tissues (nonvisceral nonsubcutaneous), is in agreement with the higher AR contents found in other studies (10, 21). These differences in the AR expression along with the differences found in T content suggest a higher responsiveness of visceral depots to androgens than the subcutaneous ones, which has also been previously reported (8) and could explain the depot-related differences in the androgen-modulated adipokines (26, 35, 50). This androgenic state in visceral depots could also be responsible for the higher lipolytic activity of this depot compared with the subcutaneous ones previously described (45, 57), as some studies have demonstrated that androgens increase the lipolytic capacity and specifically decrease lipoprotein lipase (LPL) in visceral depots (8, 46). These data are in agreement with the higher lipid accumulation shown by an AR knockout (ARKO) model compared with wild type (49).

T did not stimulate mRNA expression of its own receptor at the concentration tested. However, AR mRNA was slightly upregulated by 17{beta}-E2 in this study, as reported as well in mouse uterus (42), and downregulated by P4. This could explain why the tendency to lower AR mRNA levels observed in female depots showed higher P4 levels and no differences vs. males in the 17{beta}-E2 levels.

Studies carried out in rodent models show that the effect of estrogens is location and hormonal status dependent (39). The differences in the ER{alpha} expression reported here could suggest a higher estrogen sensitivity in visceral than in subcutaneous depots, which could explain the higher estrogen-stimulated lipolysis in visceral adipose cells that is absent in subcutaneous ones (14) and also the higher adipose deposition present only in the visceral location in the absence of ER as occurred in the ER{alpha} knockout ({alpha}ERKO) mice (17). These data, in conjunction with the findings of higher LPL by the aromatase knockout model (30), support the idea that both the local estrogen synthesis and ER mRNA expression reported in this study could be key factors in the adipose tissue metabolism and highlight an important depot-dependent estrogen-ER signaling.

ER{alpha} mRNA expression is also under the hormonal milieu control. We have observed in vitro that 17{beta}-E2 increases ER{alpha} mRNA levels. However, ER{alpha} mRNA expression stimulation by 17{beta}-E2 cannot itself explain why inguinal adipose tissue, with higher 17{beta}-E2 levels, has the lowest ER{alpha} mRNA expression and subsequently points to multiple hormonal effects (6, 13, 22), although specific culture conditions affecting ER{alpha} mRNA expression cannot be discarded (40).

In vitro T treatment also triggered a trend to increase ER{alpha} levels. These effects are in agreement with the ER upregulation reported in T-treated hamsters (20). Nevertheless, ER{alpha} mRNA expression was not stimulated in vitro by P4, which would be in agreement with the slightly higher ER{alpha} mRNA levels in male depots, at least in the retroperitoneal depot.

The exact function of ER{beta} in adipose tissue remains unclear. Our study reported some sex and location differences in ER{beta}1 mRNA levels, although its expression was very low compared with that of ER{alpha}, suggesting that ER{beta} has a scant role in adipose tissue metabolism, and this is in accord with {alpha}ERKO and {beta}ERKO studies (33, 37). Recently, It has been hypothesized that ER{beta} would act as modulator of ER{alpha} action (25), and therefore ER{beta} signaling could modify the tissue- and sex-specific response to estrogens predominantely exerted by ER{alpha}.

The presence of PR in adipose tissue is a matter of controversy. Several studies have suggested an absence of PR in male rat adipose depots, whereas in female adipocytes it has been proposed that PR is present due to estrogen stimulation (16, 41). However, in our study, the presence of PR mRNA, even in male adipose depots, has been clearly demonstrated. Nevertheless, no depot and sex differences were observed either for the specific B isoform or for the A+B combination. To this extent, our in vitro model failed to show any modulation of A+B mRNA expression under treatment with sex hormones, including 17{beta}-E2, which has been previously shown to upregulate PR in sheep adipose tissue (27).

P4 seems to have a marked effect on adipose tissue metabolism (24, 52). Studies carried out by our group (Monjo M, unpublished results) have demonstrated that P4 stimulates both the proliferation rate in preadipocytes and the {beta}3-adrenergic receptor mRNA expression in mature adipocytes. Thus the higher levels of P4 reached in female adipose tissues could be responsible, at least in part, for the higher lipolytic capacity of females (45). From our data, it could be hypothesized that P4 adipose tissue responsiveness is principally controlled by the differences in the P4 content (higher in females than in males) instead of their nuclear receptor isoforms (A or B form), of which no differences in mRNA expression were observed between males and females. All in all, posttranscriptional regulation of steroid receptors by sex hormones that could alter the steroid receptor pool in the cell cannot been discarded.

In summary, our study demonstrates important depot- and sex-dependent differences in the sensitivity of adipose tissues to the sex hormones, with visceral depots having an overall higher androgen and estrogen receptor expression than subcutaneous ones. Furthermore, important depot-dependent differences in the sex hormone contents, especially 17{beta}-estradiol, between subcutaneous and visceral depots have also been presented. These results could be key to understanding the sometimes contradictory effects of sex steroids on adipose tissue metabolism, which are dependent on factors such as hormone status, sex, and depot location.


    GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by Fondo de Investigaciones Sanitarias of the Spanish Government (PI 021339) and Comunitat Autònoma de les Illes Balears (PRDIB-2002GC4-24). S. Rodriguez-Cuenca and M. Monjo were funded by grants from the Universitat de les Illes Balears, Spain.


    ACKNOWLEDGMENTS
 
We thank Drs. F. J. Garcia, M. Gianotti, I. Lladó, and 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, CP 07122, Palma de Mallorca, Balearic Islands, 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.


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