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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
steroid receptors; testosterone; 17-estradiol; progesterone
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 107 M dose of testosterone (T), 17-estradiol (17
-E2), or progesterone (P4).
Steroid Assays
Determination of T, 17-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
-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-E2, and P4) were dissolved in ethanol and added to the corresponding flasks to a final concentration of 107 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- (ER
), estrogen receptor
1 (ER
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).
|
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.
|
T, 17-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
Plasma T levels were significantly higher in male than in female rats (P = 0.031). The 17-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-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
-E2 levels than the rest of the adipose depots. No depot differences in P4 levels of tissue were observed (Table 4).
|
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.
|
The presence of ER 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
3 and ER
4 being detected and identified (Fig. 1A). In fact, ER
isoforms
1 and
2 were not detected in adipose tissue with classical PCR, denoting lower expression levels of these receptors, although in this present experiment ER
1, the most estradiol-sensitive isoform, but not ER
2, was detected by real-time PCR (Fig 1B).
|
For ER1 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
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-E2, and P4 on steroid receptor expression in cell culture. AR was upregulated under 17
-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
was upregulated under both 17
-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
expression. Finally, PR-A+B was not altered under sex hormone treatment at the dosage used.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-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
-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
-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-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
-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 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
knockout (
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 mRNA expression is also under the hormonal milieu control. We have observed in vitro that 17
-E2 increases ER
mRNA levels. However, ER
mRNA expression stimulation by 17
-E2 cannot itself explain why inguinal adipose tissue, with higher 17
-E2 levels, has the lowest ER
mRNA expression and subsequently points to multiple hormonal effects (6, 13, 22), although specific culture conditions affecting ER
mRNA expression cannot be discarded (40).
In vitro T treatment also triggered a trend to increase ER levels. These effects are in agreement with the ER upregulation reported in T-treated hamsters (20). Nevertheless, ER
mRNA expression was not stimulated in vitro by P4, which would be in agreement with the slightly higher ER
mRNA levels in male depots, at least in the retroperitoneal depot.
The exact function of ER in adipose tissue remains unclear. Our study reported some sex and location differences in ER
1 mRNA levels, although its expression was very low compared with that of ER
, suggesting that ER
has a scant role in adipose tissue metabolism, and this is in accord with
ERKO and
ERKO studies (33, 37). Recently, It has been hypothesized that ER
would act as modulator of ER
action (25), and therefore ER
signaling could modify the tissue- and sex-specific response to estrogens predominantely exerted by ER
.
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-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 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-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|