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
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ABSTRACT |
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sex steroid hormones; 2A-;
1-;
2-; and
3-adrenergic receptors; cell proliferation; 3T3-L1 adipocyte
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 2A-AR (5). On the other hand, the regulation of lipolysis in mature adipocytes depends on the functional balance between inhibitory (
2A-AR) and stimulatory (
1-,
2-, and
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-estradiol, and progesterone have been shown to modulate the expression of the different adrenoceptor subtypes (
2A-,
1-,
2-, and
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-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).
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MATERIALS AND METHODS |
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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-estradiol, and progesterone were dissolved in ethanol and added to the corresponding flasks/wells to a final concentration from 109 to 107 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-estradiol, or progesterone (107 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 2-microglobulin) and five target genes (
2A-AR,
1-AR,
2-AR,
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 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
1-AR and
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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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-estradiol, and effect of progesterone treatment. Results were considered statistically significant at the P < 0.05 level.
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RESULTS |
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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 2A-AR was the highest AR subtype expressed in preadipocytes, whereas in adipocytes it would undoubtedly be
3-AR.
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Effect of testosterone, 17-estradiol, and progesterone on proliferation in 3T3-L1 preadipocytes.
We next investigated the effect of testosterone, 17
-estradiol, and progesterone (107 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
-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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Effect of testosterone, 17-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
-estradiol, and progesterone on
2A- and
3-AR gene expression in differentiated 3T3-L1 adipocytes was also evaluated (Table 4).
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With regard to 3-AR, the most abundant
-AR in mature 3T3-L1 adipocytes, both progesterone and 17
-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
-estradiol also produced an increase in
1-AR and
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
3-AR was considered negligible.
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DISCUSSION |
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In preadipocytes, 2A-AR was the highest AR subtype expressed, whereas in adipocytes
3-AR has been found to be dramatically increased. This expression pattern agrees with the reported major role of
2A-AR during the proliferation process (5), whereas in mature 3T3F442A adipocytes
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-estradiol and progesterone increased the proliferation rate of preadipocytes. The positive effect of 17
-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
-estradiol and progesterone observed on
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
2A-AR during proliferation. In this sense, it has been reported that catecholamines are involved in the control of white adipocyte proliferation through
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
-estradiol, and progesterone inducing
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-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
-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-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-estradiol and progesterone enhanced both
2A- and
3-AR gene expression, whereas testosterone only increased
2A-AR mRNA levels. The positive effect of the hormones tested in
2A-AR mRNA levels was reduced when administered at higher concentrations (107 M), indicating a different action depending on the levels of the hormone. Despite the fact that 17
-estradiol and progesterone were able to affect the expression of inhibitory (i.e.,
2A-AR) and stimulatory (i.e.,
3-AR) adrenoceptors, the effects on
3-AR mRNA levels could be more important in the net balance on cAMP accumulation, because
3-AR is by far the highest expressed. As a result, it could be argued that 17
-estradiol and progesterone, mainly through
3-AR, could increase lipolysis in mature 3T3-L1 adipocytes when stimulated with
-AR agonists. This hypothesis would be consistent with the increase in
3-AR mRNA levels and the stronger cAMP response to isoproterenol (a
1-,
2-, and
3-AR agonist) induced by oleoyl-estrone in perovaric white adipocytes (7). On the other hand, without stimulation of
3-AR by
-AR agonists, progesterone and 17
-estradiol have been found in this study to stimulate differentiation of the cells, together with an increase in
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-estradiol on
2A-AR gene expression, downregulating or upregulating its levels in brown and white adipocytes, respectively. In contrast, testosterone behaved similarly in both systems, enhancing
2A-AR gene expression. As a result, the differential regulation of
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-estradiol and progesterone affect the in vitro proliferation of 3T3-L1 preadipocytes and suggests that the increased
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
2A-AR mRNA levels, whereas 17
-estradiol and progesterone enhanced both
2A- and
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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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