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Laboratori de Bioquímica i Biologia Molecular, Departament de Biologia Fonamental i Ciències de la Salut, Universitat de les Illes Balears, Palma de Mallorca 07071, Spain
Address all correspondence and requests for reprints to: Pilar Roca, Departament de Biologia Fonamental i Ciències de la Salut, Edifici Guillem Colom, Universitat de les Illes Balears, Carretera Valldemossa, Km 7.5, Palma de Mallorca 07071, Spain. E-mail: pilar.roca{at}uib.es.
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Abstract |
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2A-, ß1-, ß2-, and ß3-AR) and lipolytic activity was investigated in differentiated brown adipocytes in culture. We report that the expression of each AR subtype gene was distinctively regulated by NE and sex hormones in brown adipocytes. Testosterone-treated cells had lower lipolytic activity and increased expression of antilipolytic receptors 2A-AR. Both 17ß-estradiol and progesterone decreased 2A-AR expression and 2A/ß3-AR protein ratio, but progesterone had higher potency than 17ß-estradiol, increasing ß-AR levels, mainly ß3-AR expression, and enhancing lipolysis stimulated by NE. In conclusion, our results support the idea that male and female sex hormones, as a part of the hormonal environment of BAT, have direct and opposite effects on the AR balance and lipolytic activity, and they might play a role in the gender dimorphism for the recruitment process in BAT. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2A-AR decreases cAMP levels (3). ß3-AR is known to be quantitatively the most abundant ß-AR in brown adipocytes (4), and 2A-AR is also highly expressed in these cells (5). The 2A/ß3-AR ratio is key in the regulation of thermogenesis and lipolysis (6, 7), through regulation of cAMP levels, which controls both UCP1 expression (2, 8) and the release of free fatty acids, which are positive UCP1 modulators (5). Thus, a lower 2A/ß3-AR ratio would lead to a greater thermogenic activity and lipolysis, and vice versa. The recruitment process in BAT is the basis for the enhancement of the thermogenic capacity in rodents adapted to cold and certain diets, and it includes proliferation from precursor cells within this tissue, differentiation of these cells into mature brown adipocytes, and mitochondriogenesis to provide thermogenic mitochondria, together with an increased UCP1 expression (9). Gender dimorphism has been established in the morphology, thermogenesis function and adrenergic response in BAT (7), and gender differences have also been found in the thermogenic response of this tissue to overfeeding, overweight, and cold (6, 10, 11, 12). As a consequence, sex hormones have been proposed as key factors that could explain, at least in part, the gender differences observed in the recruitment of BAT. However, it is not known whether sex hormones exert their effect through an indirect mechanism or directly at the adipocyte level. The existence of sex hormone receptors in adipocytes (13, 14, 15) would argue for a direct effect in the adipocyte, and in vitro methodology offers a powerful approach for the confirmation of such hypotheses.
We have recently reported, in primary cultures of brown adipocytes, distinct actions of testosterone, progesterone, and 17ß-estradiol on UCP1 expression and lipid accumulation (12). Nevertheless, it is not known whether these effects could be mediated via the modulation of the activity or the expression of 2A- and ß-adrenoceptors.
Moreover, ARs are thought to be affected by sex hormones in brown adipocytes, because 2A- and ß-ARs have been previously observed to show a gender dimorphism in BAT (6, 7). Therefore, whether the adrenergic regulation observed in vivo could be attributed to a direct effect of the sex hormones on the brown adipocytes is one of the questions that remain to be resolved.
The aim of this study was to investigate, under cell culture conditions, the direct effects of testosterone, 17ß-estradiol, and progesterone on the expression of ARs and lipolytic activity in male differentiated brown adipocytes. Moreover, norepinephrine (NE) regulation of gene expression for the different adrenoceptor subtypes was further characterized in this model by showing a specific regulation for each subtype. We find that in vitro sex hormones modulate distinctively AR expression for the different subtypes by a transcriptional mechanism and regulate lipolytic activity in differentiated brown adipocytes. Furthermore, the male and female sex hormones showed opposite effects regulating adrenoceptor expression and lipolysis. Thus, our findings provide support for the idea that sex hormones, as a part of the hormonal environment, might play a role in the recruitment process of BAT.
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Materials and Methods |
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Cell isolation, culture, and treatments
Brown fat precursor cells were isolated, as previously described (16), from 4-wk-old male NMRI mice (obtained from CRIFFA, Barcelona, Spain) and were cultured as described in Ref.12 . All animals were treated in accord with the principles and procedures outlined in the Guidelines for Care and Use of Experimental Animals. The cultured medium was DMEM, supplemented with 10% newborn calf serum, 4 nM insulin, 4 mM glutamine, antibiotics (50 IU penicillin/ml and 50 µg streptomycin/ml), 10 mM HEPES, and 25 µg sodium ascorbate/ml. This medium-containing serum was changed on d 1 (cells were previously washed with DMEM) and d 3. On d 6, the medium was discarded, and a serum-free medium was added, consisting of DMEM-F12 (1:1), free fatty acid BSA (0.5%), 4 nM insulin, 4 mM glutamine, antibiotics (50 IU penicillin/ml and 50 µg streptomycin/ml), 10 mM HEPES, and 25 µg sodium ascorbate/ml.
The different treatments were carried out on d 6, when cells presented a differentiated morphology and important lipid accumulation, and when they were placed in the serum-free medium to avoid hormonal interference due to the serum. Steroid hormones and inhibitors of steroid hormones were dissolved in ethanol (testosterone, 17ß-estradiol, progesterone, or flutamide) or dimethylsulfoxide (DMSO) (ICI 182.780 or RU486) and added to the corresponding flasks, never exceeding a final ethanol or DMSO concentration of 0.01%. An equivalent volume of ethanol or DMSO was added in untreated controls. On d 7, after 24 h treatment, without changing the medium, the cells were exposed to NE for 6 h. Then, an aliquot of the medium was taken for measurement of glycerol released, the medium was discarded, and the cells were harvested with a monophasic solution of phenol and guanidine thiocyanate (Roche Diagnostics, Mannheim, Germany) for isolation of RNA, DNA, and protein.
RNA, DNA, and protein isolation
Total RNA, DNA, and protein were isolated following the instructions of the manufacturer. RNA and DNA were determined using a spectrophotometer set at 260 nm. Proteins were determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL).
RT-PCR analysis
For semiquantitative RT-PCR analysis of ß1-, ß2-, ß3-, and 2A-AR, 0.5 µg of the total RNA was reverse transcribed to cDNA at 42 C for 60 min with 25 U MuLV reverse transcriptase in a 10-µl vol of reverse transcriptase 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 ribonuclease inhibitor, and 500 µM each deoxynucleotide triphosphate. cDNAs were denatured for 30 sec at 95 C and submitted to 30 amplification cycles. One PCR cycle consisted of 20 sec at 95 C, 20 sec at 60 C, and 30 sec at 72 C, followed by a final extension of 7 min at 72 C in a DNA thermal cycler 2400 (Applied Biosystems, Madrid, Spain). PCRs were performed in a final vol of 25 µl containing 10 µl cDNA, 10 mM Tris-HCl (pH 9.0 at 25 C), 50 mM KCl and 0.1% Triton X-100, 1.2 mM MgCl2, 1.25 U Taq polymerase, and 200 nM of both sense and antisense primers for adrenoceptors and 20 nM of both sense and antisense primers for ß-actin, which was used as the control of amplification.
The reliability of the RT-PCR assay as a semiquantitative tool was evaluated by testing the linearity of the reaction, relative to cycle number. The amplification reaction was essentially linear up to 30 cycles for ß1-AR, ß2-AR, ß3-AR, 2A-AR, and the coamplified control ß-actin.
Sequences of the sense and antisense oligonucleotides were as follows: 5'-TCGTGTGCACAGTGTGGGCC-3' and 5'-AAGCGGCGCTCGCAGCTGTCGATC-3' for ß1-AR; 5'-GTGTTGTGCGTCACAGCCAG-3' and 5'-CCTCCCATCCTG-CTCCACCT-3' for ß2-AR; 5'-TAGTCCTGGTGTGGATCGTGTCCGC-3' and 5'-GCGATGAAAACTCCGCTGG-GAACTA-3' for ß3-AR; 5'-GCGCCCCAGAACCTCTTCCTGGTG-3' and 5'-GAGTGGC-GGGAAGGAGATGACGGC-3' for 2A-AR; 5'-ACGGGCATTGTGATGGACTC-3' and 5'-GTGGTGGTGAAGCTGTAGCC-3' for ß-actin. These primers were derived from the sequences of the corresponding genes and cDNAs. Amplification products had the expected sizes of 265, 420, 514, 311, and 145 bp for ß1-, ß2-, ß3-, 2A-AR, and ß-actin, respectively.
The resulting PCR products were separated on a 2% agarose gel in 45 mM Tris-Borate-1 mM EDTA Buffer (pH 8.0) and visualized by ethidium bromide. PCR products were analyzed by video-densitometric scanning and quantified using the KODAK 1D Image Analysis Software 3.5 (Eastman Kodak Co., Rochester, NY). Levels of mRNA were expressed as the ratio of signal intensity for the target genes relative to that for ß-actin.
Western-blot analysis
Fifteen micrograms of total protein were fractionated by SDS-PAGE on 10% polyacrylamide gels according to Laemmli (17) and electrotransferred onto a nitrocellulose filter as described elsewhere (18). Ponceau S staining provided visual evidence for correct loading and electrophoretic transfer of proteins to the nitrocellulose filter. Blocking and development of the immunoblots were performed using an enhanced chemiluminescence Western blotting analysis system (Amersham, Barcelona, Spain). Polyclonal antibodies against 2A- and ß3-AR (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies. Autoradiograms of membrane revealed proteins with apparent molecular masses of 58 and 55 kDa, for ß3- and 2A-AR, respectively.
Bands in films were analyzed by scanner photodensitometry and quantified using the KODAK 1D Image Analysis Software 3.5 (Eastman Kodak Co.).
Measurement of lipolysis
On d 7, after 24 h treatment with sex hormones, brown adipocyte cultures were incubated for 6 h in the absence or presence of NE, without changing the medium, and lipolysis was monitored as the amount of glycerol released into the medium. Glycerol was determined enzymatically with a Sigma Chemical kit [Triglyceride (GPO-Trinder) no. 337]. The DNA content was determined as described above, and glycerol release was corrected per microgram of cell DNA. The results were expressed as a percentage of the values obtained in samples not incubated with hormones or NE, which were set at 100%.
Statistics
All data are presented as mean values ± SEM. Differences between groups were assessed by one-way ANOVA and Student’s t test for post hoc comparisons, using a statistical software package (SPSS, Inc., Chicago, IL).
In the ANOVA, possible effects are: effect of testosterone treatment, effect of 17ß-estradiol, effect of progesterone, and effect of NE. Results were considered statistically significant at the P < 0.05 level.
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Results |
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2A-AR were expressed in brown adipocytes. Other authors have described an apparent absence of ß2-AR expression, using Northern-blot analysis in a similar model (19). However, it is possible that the discrepancy with our results could be attributed to the lower sensitivity of Northern-blot compared with RT-PCR.
Differential effect of NE concentration on AR gene expression
Adrenergic receptor gene expression is known to be modulated by NE and to depend both on the receptor subtype and the cell type (19). The effects of NE concentration, i.e. 10-8, 10-7, 10-6, and 10-5 M, on ß1-, ß2-, ß3-, and 2A-AR mRNA levels and ß3- and 2A-AR protein levels are shown in Fig. 1
. ß1-AR mRNA levels were up-regulated by NE at all doses used, whereas ß3-AR showed an important dose-dependent down-regulation; both results are in accordance with earlier observations by other authors (19, 20, 21). The 2A-AR and ß2-AR mRNA levels showed a dose-dependent down-regulation, more moderate than ß3-AR, which was statistically significant at the highest doses of NE, i.e. 10-6 and 10-5 M.
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2A-AR at any concentration used. Therefore, the different AR subtypes were differentially modulated by NE in these cells, which is a fact physiologically important for the control of brown fat cell function.
Sex hormones have a direct and differential effect on AR gene expression
We have previously shown that sex hormones, mainly testosterone and progesterone, have opposite effects modulating UCP1 expression and lipid accumulation in brown adipocytes differentiated in culture (12).
To investigate whether these effects were mediated by changes in AR gene expression, differentiated brown adipocytes were treated with testosterone, 17ß-estradiol, and progesterone in serum-free medium for 24 h at concentrations from 10-9–10-7 M, because they are approximately physiological concentrations (22, 23). Results are shown in Table 1.
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2A-AR, as previously reported in white adipocytes (24, 25). On the other hand, both female sex hormones reduced mRNA levels for this receptor, and the effect was dose-dependent for progesterone. An increase in ß1-AR mRNA levels was produced with all the sex hormones tested, although in different magnitude, and progesterone had the greatest stimulatory effect.
Both testosterone and 17ß-estradiol decreased ß2-AR mRNA levels, whereas progesterone showed a tendency to increase mRNA levels for this receptor.
The female sex hormones showed stimulatory effects on ß3-AR mRNA levels, which were stronger for progesterone, whereas no effect was seen after testosterone treatment.
Next, we investigated whether sex hormone effects on AR expression were maintained after stimulating brown adipocytes with NE 10-7 M for 6 h (see Table 1
). For 2A-AR and ß3-AR, NE treatment gave a profile in testosterone, 17ß-estradiol, and progesterone-treated cells that was similar to that of the corresponding non-NE-treated cells. However, lower mRNA levels were observed (because of the NE regulation), particularly for ß3-AR. The down-regulating effects on ß2-AR mRNA levels by testosterone and 17ß-estradiol were reduced when NE was used. With regard to ß1-AR, only 17ß-estradiol in combination with NE appeared to have an effect on mRNA levels, reducing significantly its levels at 10-8 M.
On the whole, these results provide evidence that sex hormones have a direct and differential effect on AR expression. It is worth noticing that these effects were prevented when brown adipocytes where exposed at the same time to the transcriptional inhibitor, actinomycin D (1 µg/ml) (data not shown), suggesting a transcriptional mechanism for the sex hormone effects on adrenoceptor expression.
The differences found in some treatments between NE-treated and non-NE-treated cells, especially for ß1-AR and ß2-AR, could point toward a hormonal coordination in the brown adipocyte between NE and sex hormones, and the final effect may depend on the AR subtype.
Progesterone and 17ß-estradiol reduce 2A/ß3-AR protein ratio
Because gender differences have been observed in the 2A/ß3-AR protein ratio in BAT (6, 7), the influence of sex hormones on this ratio was also evaluated. Western blot analysis (Fig. 2) showed that 24 h treatments with progesterone and 17ß-estradiol were able to decrease the 2A/ß3-AR ratio in differentiated brown adipocytes. In contrast, testosterone did not alter this ratio at any concentration tested, although an increase in 2A-AR and ß3-AR protein levels at lower doses was also observed (data not shown). It is worth emphasizing that the reduced 2A/ß3-AR protein ratio for progesterone was due to an increase in ß3-AR protein together with a decrease in the 2A-AR (data not shown), whereas for 17ß-estradiol the effect was produced by an important decrease in the 2A-AR, results that were well correlated with above mRNA studies.
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), obtaining the typical dose-response curve in the glycerol release to the culture medium, reaching maximal stimulation at 10-6 M. The 10-7 M concentration of NE was selected for the experiments with sex hormones because it brought about important glycerol levels in the medium without reaching saturated values.
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), with a significant effect at its highest concentration, 10-7 M. In contrast, neither 17ß-estradiol nor progesterone influenced basal lipolysis in the range of concentrations assayed.
After stimulating lipolysis for 6 h with NE (Fig. 3C), glycerol release was significantly reduced in testosterone-treated cells at all concentrations tested. Lipolysis stimulated by NE was enhanced with progesterone but was not significantly affected by 17ß-estradiol, although a tendency to increase glycerol was also observed.
To find out the involvement of the sex hormone receptors in the regulation of the lipolytic activity, the effect of the sex hormones was antagonized by specific agents: flutamide for testosterone, ICI 182.780 for 17ß-estradiol, and Mifepristone (RU486) for progesterone (Fig. 4). The inhibitory effect of testosterone on lipolysis was prevented when brown adipocytes were exposed at the same time to the androgen receptor antagonist flutamide. No effect on glycerol release was observed in 17ß-estradiol-treated cells or with the addition to the cells of ICI 182.780. The progesterone antagonist RU486 decreased basal lipolysis and did not reverse progesterone enhancement in NE-stimulated lipolysis. These effects observed with RU486 could be due to the fact that this antagonist acquires agonist activity in response to stimulation of cAMP signaling pathways and seems to be through the progesterone receptor, although RU486 can bind with high affinity to either progesterone receptor or glucocorticoid receptor (26).
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Discussion |
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2A-, ß1-, ß2-, and ß3-AR).
Adrenergic receptor subtypes displayed a differential regulation by NE, i.e. up-regulation for ß1-AR and down-regulation for 2A-, ß2-, and ß3-AR. The marked down-regulation in ß3-AR mRNA levels found with NE treatment is consistent with the fact that ß3-AR expression regulation occurs only at a transcriptional level (21), because this adrenoceptor lacks consensus sequences for phosphorylation, unlike ß1-AR and ß2-AR (27), and is not regulated by desensitization. This dramatic down-regulation contrasted with the less marked regulation of 2A-AR and ß2-AR mRNA levels by NE. The differences in adrenoceptor regulation by NE could be due to the kind of mechanism involved. For instance, a suppression of transcription is the mechanism responsible for ß3-AR down-regulation; whereas, for ß2-AR, it is caused by an increase in mRNA degradation (28).
It is worth pointing out that NE exerted less pronounced effects on ß3-AR and 2A-AR protein levels than on their mRNA levels. These differences could indicate that longer stimulation times with NE would be necessary to appreciate greater changes, because adrenergic regulation of the gene expression for the different adrenoceptors has been shown to be both concentration and time dependent (19). In this sense, it has been reported that the half-life of the human ß3-AR in a heterologous expression system (murine L cells) is about 18 h and also that there is a lack of agonist-induced degradation of this protein (29), supporting again the hypothesis that the down-regulation of this receptor results mostly from the regulation of ß3-AR mRNA levels.
Under our experimental conditions, testosterone behaved as an antilipolytic hormone, and some of the effects could be mediated by an increase in 2A-AR mRNA and protein levels. The transcriptional regulation of 2A-AR by testosterone has been described to be androgen receptor dependent and limited to adipocytes (24, 25). Moreover, testosterone treatment brought about a lower lipolysis activation, both without NE and under stimulation with NE. These results would point toward a direct regulation of lipolysis by this hormone, and this would be mediated by the androgen receptor, because the testosterone effect on lipolysis was completely suppressed in the presence of flutamide. The inhibition of lipolytic activity by testosterone was found at almost all doses used, whereas the increase observed in the expression of 2A-AR was found only at some doses; thus, an effect of this hormone at other levels of the adrenergic pathway could not be discarded, as proposed in other studies on protein kinase A and hormone-sensitive lipase activity (30, 31, 32). Furthermore, in white adipocytes, androgens have been reported to have both inhibitory (i.e. up-regulation of 2A-AR) and stimulatory (i.e. enhancement of the adenylate cyclase activity, or up-regulation of ß-ARs) effects on lipolytic activity (32). As a consequence, it could be argued that the final effect for testosterone would be dependent both on the fat depot and on the experimental conditions used (in vivo vs. in vitro). It seems that in brown adipocytes and under our experimental conditions, the inhibitory pathway on lipolysis after testosterone treatment is the one to prevail.
In a previous report, we have observed in the same experimental model an antiadipogenic effect of testosterone, with cells showing fewer and smaller lipid droplets than control ones, and a decrease in UCP1 mRNA levels, which is a differentiation marker (12). The increase in 2A-AR expression found after testosterone treatment would lead to a lower activation of lipolysis and thermogenesis, which could also account for the testosterone-dependent inhibition mechanism of UCP1 mRNA expression.
In opposition to androgens, female sex hormones have been shown, by some studies (33), to behave as proadipogenic hormones. Moreover, a previous work with brown adipocyte cell cultures showed that cells treated with 17ß-estradiol and progesterone have more and larger lipid droplets, leading to an apparently more differentiated morphology (12). In this work, the female sex hormones had stimulatory effects on the expression of ß3-AR and inhibitory effects on the expression of 2A-AR, and resulted in a lower 2A/ß3-AR protein ratio. This would lead to a higher cAMP accumulation and, as a result, a greater activation of lipolysis and thermogenesis (the latter through an increase in UCP1 expression), as commented above. This behavior was more marked for progesterone, where higher glycerol levels were observed in the medium after NE treatment, and which correlates with greater UCP1 mRNA levels, reported in the same model (12). However, the changes obtained in AR expression induced by 17ß-estradiol were not great enough to stimulate this double axis (lipolysis-thermogenesis). Our findings are consistent with a report demonstrating that oleoyl-estrone increases ß3-AR mRNA expression and the cAMP response to isoprotenerol in BAT, indicating that the adrenergic pathway is modulated by oleoyl-estrone at the level of AR gene expression and cAMP accumulation as well (34).
Nevertheless, there seem to be conflicting results on the effects of female sex hormones on BAT in vivo studies. On the one hand, many studies support the notion that estrogen increases energy expenditure (35, 36, 37). Moreover, estrogen deficiency is followed by a reduced UCP1 expression in BAT (38); and additionally, it has been shown that estrogen receptor knockout mice have a decrease in thermogenesis (39). In contrast, other reports assert that 17ß-estradiol and progesterone inhibit BAT thermogenesis (40, 41), and change the affinity or density of ARs (42). From the present study, it seems to be clear that sex hormones can modulate the expression of AR in cultured brown adipocytes, and differences in vivo, compared with in vitro, could indicate a role for the sympathetic nervous system or changes in other hormones/metabolites.
As commented previously, gender differences have been reported in vivo in the thermogenic response of BAT (6, 10, 11, 12). Hence, female rats under usual housing temperature (22 C) have a more recruited BAT with a higher multilocular arrangement, greater mitochondrial machinery, and a higher thermogenic activity and capacity (7). This fact is accompanied by a lesser presence of 2A-AR in females and also a lower 2A/ß3-AR ratio (6, 7). In view of our findings, the gender differences found in vivo in this tissue could be dependent, at least in part, on the direct action of the sex hormones. Besides, the recruitment process, both in vivo and in vitro, is known to be mainly regulated by NE (9). In this sense, gender differences in NE release in BAT could not be ruled out either as determining factors for the recruitment differences found in vivo (43). However, in contrast with the recruitment process in vivo (where brown adipocytes are always under some degree of adrenergic stimulation), in vitro experiments avoid the influence of the sympathetic nervous system (44). Therefore, although NE is the trigger for the recruitment process, sex hormones could also influence it through a direct effect on the adrenergic control of brown adipocytes, by modulating both thermogenesis and lipolysis. Thus, our findings provide support for a role of the main sex hormones, as a part of the hormonal environment, in the recruitment process of BAT.
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Acknowledgments |
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Footnotes |
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Abbreviations: AR, Adrenergic receptor; BAT, brown adipose tissue; DMSO, dimethylsulfoxide; NE, norepinephrine; UCP1, uncoupling protein 1.
Received April 28, 2003.
Accepted for publication July 14, 2003.
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References |
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2-adrenoceptors: the regulation of fat cell function and lipolysis. Endocr Rev 16:716–738[CrossRef][Medline]
2-adrenoceptors by androgens is consequence of direct action on fat cells. Am J Physiol 267:C926–C931
2-adrenoreceptor protein and mRNA by a direct effect of androgens in white adipocytes. Biochem Biophys Res Commun 206:112–118[CrossRef][Medline]
2. Endocrinology 141:649–656 knockout mice. Proc Natl Acad Sci USA 97:12729–12734This article has been cited by other articles:
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  J. Harslund, O. L. Nielsen, N. Brunner, and H. Offenberg Gender-dependent physiological implications of combined PAI-1 and TIMP-1 gene deficiency characterized in a mouse model Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2007; 293(4): R1630 - R1639. [Abstract] [Full Text] [PDF]
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M. Monjo, E. Pujol, and P. Roca {alpha}2- to {beta}3-Adrenoceptor switch in 3T3-L1 preadipocytes and adipocytes: modulation by testosterone, 17{beta}-estradiol, and progesterone Am J Physiol Endocrinol Metab, July 1, 2005; 289(1): E145 - E150. [Abstract] [Full Text] [PDF]
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 S. Blanchette, K. Blouin, C. Richard, P. Dupont, V. Luu-The, and A. Tchernof Expression and Activity of 20{alpha}-Hydroxysteroid Dehydrogenase (AKR1C1) in Abdominal Subcutaneous and Omental Adipose Tissue in Women J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 264 - 270. [Abstract] [Full Text] [PDF]
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S. Rodriguez-Cuenca, M. Monjo, A. M. Proenza, and P. Roca Depot differences in steroid receptor expression in adipose tissue: possible role of the local steroid milieu Am J Physiol Endocrinol Metab, January 1, 2005; 288(1): E200 - E207. [Abstract] [Full Text] [PDF]
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