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,||,1
,
,,,2
* INSERM, U772, Paris, France;
Collège de France, Paris, France;
INSERM, U693, Faculté de Médecine Paris-Sud, Le Kremlin Bicêtre, France;
University Paris-Sud, Orsay, France; and
|| Department of Internal Medicine, Chair of Endocrinology, University Tor Vergata, Rome, Italy
2Correspondence: INSERM U772, Collège de France, 11, place M. Berthelot, 75005 Paris, France. E-mail: maria-christina.zennaro{at}college-de-france.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mRNA expression, which was blocked by the MR antagonist spironolactone. Under permissive culture conditions, specific MR down-regulation by siRNAs markedly inhibited 3T3-L1 differentiation by interfering with the transcriptional control of adipogenesis, an effect not mimicked by specific inactivation of the glucocorticoid receptor. These results demonstrate that MR represents an important proadipogenic transcription factor that may mediate both aldosterone and glucocorticoid effects on adipose tissue development. MR thus may be of pathophysiological relevance to the development of obesity and the metabolic syndrome.—Caprio, M., Fève, B., Claës, A., Viengchareun, S., Lombès, M., Zennaro, M-C. Pivotal role of the mineralocorticoid receptor in corticosteroid-induced adipogenesis.
Key Words: adipocyte differentiation • aldosterone • glucocorticoids • nuclear receptors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. The adipogenic stimulus of glucocorticoids is most evident in the central obesity of patients with Cushing’s syndrome, a disease of systemic glucocorticoid excess (3)
. In animal studies, adipose tissue-specific amplification of cortisol production in transgenic mice results in a full metabolic syndrome, including central obesity, glucose intolerance, insulin-resistant diabetes, and hypertension (2)
. In contrast, glucocorticoid inactivation is associated with resistance to metabolic dysfunction, including diet-induced obesity (4
, 5)
.
The mineralocorticoid receptor (MR, NR3C2) classically mediates aldosterone effects on electrolyte balance and blood pressure by regulating trans-epithelial sodium transport through tight epithelia (6)
. The MR belongs to the nuclear receptor superfamily and acts as a ligand-activated transcription factor regulating the expression of a coordinate set of genes ultimately eliciting physiological aldosterone responses. Classical MR-expressing tissues include distal parts of the nephron, colon, salivary, and sweat glands. However, MR possesses the same affinity for aldosterone and the physiological glucocorticoid hormones; in particular, its affinity for cortisol (in human) and corticosterone (in rodents) is >10-fold higher than that of the glucocorticoid receptor (GR) itself (7)
. Given that glucocorticoids circulate at 100- to 1000-fold higher concentrations than those of aldosterone, specificity of MR activation in epithelial target tissues requires the intracellular enzymatic actions of 11ß-hydroxysteroid dehydrogenase type 2 (11HSD2), which converts cortisol to MR-inactive cortisone (and corticosterone to 11-dehydrocorticosterone in rodents). It is now well documented that MR are also expressed in nonepithelial tissues, including the cardiovascular and central nervous systems and adipose tissue. In these tissues, glucocorticoids represent the predominant endogenous ligand given the absence of significant 11HSD2 activity (6)
.
In contrast to glucocorticoids, the role of aldosterone in controlling metabolic physiology is largely unexplored. Using a transgenic animal model, we recently showed that brown adipose tissue expresses MR, which can be activated by aldosterone (8)
. Subsequent studies of a brown adipose cell line derived from this original model have shown that aldosterone is able to both promote brown adipogenesis and inhibit thermogenesis (8
9
10)
. Little is known about the potential involvement of the mineralocorticoid system in white adipose tissue development. Previous pharmacological studies have suggested that aldosterone may promote adipogenesis (11
, 12)
, and recent work has reported that MR gene expression is induced during the course of 3T3-L1 adipose conversion (13)
. However, both the possible involvement of MR in adipogenesis and the physiological relevance of these findings await investigation. Further, the contribution of MR and GR in mediating glucocorticoid effects on adipocyte differentiation has not yet been distinguished.
In the present study we have explored the role of MR and aldosterone in the control of white fat cell differentiation. We show that aldosterone is able to induce white adipose differentiation in both 3T3-L1 and 3T3-F442A cells, which is accompanied by induction of relevant transcription factors and adipose genes. Our data demonstrate a crucial role for MR in mediating glucocorticoid-induced adipogenesis, suggesting a potential role for this nuclear receptor in the development of obesity and its metabolic complications.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM Glutamax, Invitrogen, Cergy Pontoise, France) supplemented with 10% newborn calf serum, 100 IU/ml penicillin and 100 µg/ml streptomycin in a 5% CO2 humidified atmosphere at 37°C and allowed to reach confluence. To achieve adipose differentiation under classical conditions, postconfluent 3T3-L1 preadipocytes were incubated with a cocktail of 170 nM insulin, 0.25 µM dexamethasone, and 100 µM 3-isobutyl-1-methylxanthine (IBMX) in DMEM supplemented with 10% fetal calf serum (FCS) for 48 h, with the culture medium replaced every 48 h with DMEM, supplemented with 10% FCS and 170 nM insulin. For 3T3-F442A cells, postconfluent cells were incubated directly with DMEM supplemented with 10% FCS and 170 nM insulin. To study corticosteroid hormone effects on adipose differentiation, a specific differentiation protocol in the absence of steroids was developed. From confluence, cells were transferred into DMEM containing 10% dextran-coated charcoal stripped fetal calf serum (DCC). 3T3-L1 cells were induced to differentiate for 2 days in the presence of 100 µM IBMX and 170 nM insulin, then incubated an additional 5 days in 10% DCC and 1 nM insulin. 3T3-F442A cells were cultured from confluence in 10% DCC and 1 nM insulin. Effects of corticosteroid hormones were investigated by incubating both 3T3-L1 and 3T3-F442A cells with the concentrations indicated of aldosterone or dexamethasone, in the presence or absence of a 100-fold excess of spironolactone.
For [3H]-thymidine incorporation experiments, day 2 postconfluent cells were cultured for 36 h in 6-well plates in 10% DCC in the presence or absence of 100 nM aldosterone. After overnight culture in a serum-free medium (DMEM/Glutamax, with or without aldosterone), cells were refed for 1 h with 10% DCC and 5 µCi/well of [3H]-thymidine (Amersham TRK 758, Amersham Biosciences, Orsay, France) at 37°C in 5% CO2. Cells were rinsed twice with ice-cold PBS and lysed in 1% SDS. DNA was precipitated in 10% trichloroacetic acid, collected on Whatman GF/C filters, and counted.
siRNA treatment of 3T3-L1 cells
Undifferentiated 3T3-L1 cells were seeded in a 6-well plate at a density of 7 x 104 cells/well and transiently transfected the next day at 75% confluence. For transfection, Lipofectamine 2000 (5 µl/well, Invitrogen, France) was mixed with Opti-MEM serum-free medium (Invitrogen) and siRNA (100 and 150 pmol/well, Stealth siRNA, Invitrogen, for MR and GR knockdown, respectively) in a total volume of 1 ml, according to the manufacturer’s recommendations. Six hours post-transfection, cells were washed twice with PBS, then induced to differentiate for 48 h in a permissive medium that included 10% FCS, 170 nM insulin, 0.25 µM dexamethasone, and 100 µM IBMX. Thereafter, cells were switched to 10% FCS and 170 nM insulin for an additional 4 days. The siRNA sense sequences for MR were 5'-CCUCUGUUUGCAGCCCGCUCAACAU-3' (si1_MR) and 5'-CCCGCUCAACAUGCCGUCUUCAGUA-3' (si2_MR). The control sense siRNA sequence for MR was 5'-CCCAGUACACGUUGCUUCACGCGUA-3' (scrambled, scr_MR). The siRNA sense sequences for GR were 5'-GCACCUUUGACAUCUUGCAGGAUUU-3' (si1_GR) and 5'-GCCAUUUCUGUUCAUGGCGUGAGUA-3' (si2_GR). The control sense siRNA sequence for GR was 5'-GCCUCUUCAUCGGUAGUGCAUAGUA-3' (scr_GR).
RNA isolation and real-time quantitative PCR
All reagents used were from Invitrogen unless otherwise specified. Total RNA was extracted at various time points after adipogenic induction in Trizol reagent according to the manufacturer’s recommendations. Total RNA (1 µg) was first digested for 15 min at 37°C with 1U of DNase I and quantified with the Ribogreen RNA quantitation kit as described previously (15)
. Total RNA (500 ng) was reverse transcribed with Superscript Reverse Transcriptase and random hexamers (Promega, Charbonnières, France) in a final volume of 20 µl. Real-time quantitative PCR analysis was carried out on a Chromo 4 PCR detection system (Bio-Rad, Marnes-la-Coquette, France), using the qPCR MasterMix Plus for SYBR® green I (Eurogentec, Seraing, Belgium), and analyzed with Opticon Monitor 3 analysis software. PCR assay was performed in a final volume of 25 µl, starting with 12.5 ng of reverse transcribed total RNA, in the presence of 300 nM of each sense and antisense oligonucleotide and 5 mM MgCl2. Sequences of PCR primers are given in Table S1. Reaction parameters were 95°C for 10 min, followed by 40 cycles at 95°C, 15 s and 60°C, 1 min. Controls without reverse transcriptase and without template were included to verify that fluorescence was not overestimated by residual genomic DNA amplification or from primer dimer formation. Moreover, RT-PCR products were analyzed in a postamplification fusion curve to ensure that a single amplicon was obtained. Ribosomal 18S RNA was used to normalize for RNA quality, quantity, and RT efficiency. Quantification was done by the standard curve method. Standard curves were generated by serial dilutions of a linearized plasmid containing the specific amplicon spanning six orders of magnitude, yielding a correlation coefficient of at least 0.98 in all experiments, as described previously (15)
. For all experiments, PCR efficiency was close to 1, indicating a doubling of DNA at each PCR cycle, as expected.
Cell extracts, biochemical determination, and enzyme assays
Cultured cells were washed twice with PBS 1x, harvested, and homogenized in 25 mM Tris, pH 7.5, 1 mM EDTA. A fraction of the homogenate was used to determine cell triglyceride content with the PAP 150 triglyceride kit (Biomerieux, Marcy l’Etoile, France). The remainder was centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was used to measure glycerol-3-phosphate dehydrogenase (G3PDH) activity by recording the initial rate of oxidation of NADH at 340 nm at 25°C (16)
.
Aliquots of homogenates and supernatants were used to determine protein content with the BCA Protein Assay kit (Pierce Biotechnology Inc., Rockford, IL, USA).
Protein studies
For binding assays, cells were grown 24 h before the assay in the presence of 10% DCC, then incubated for 1 h in a 5% CO2 humidified atmosphere at 37°C with 10 nM [3H]-aldosterone (Amersham Biosciences) in the absence or presence of a 100-fold excess unlabeled aldosterone. Bound and unbound steroids were separated by washing twice with ice-cold PBS. Cells were scraped in cold 100% ethanol and radioactivity was measured in a scintillation counter. Specific [3H]-aldosterone binding was calculated by the difference between radioactivity measured in the absence and presence of unlabeled competitor.
For Western blot analysis, cells were lysed in 2 x Laemmli buffer, then 30 µg of whole-cell extract was separated on 12% SDS-PAGE and electroblotted onto an Immobilon-P 0.2 µm transfer membrane (Millipore Corp., Billerica, MA, USA). Membranes were subsequently incubated overnight at 4°C with a rabbit polyclonal anti-mouse GR antibody [1:1000, GR(M-20):sc-1004 Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA]. After three washes, membranes were incubated for 1 h at room temperature with ECL peroxidase-labeled anti-rabbit IgG (1:5000, NA934-vs, Amersham Biosciences). Immunoreactivity was visualized by enhanced chemiluminescence, digitalized on a 2-dimensional gel scanner, and quantified with Quantity One software (Bio-Rad). Membranes were subsequently stripped in 0.2% NaOH and probed with monoclonal anti-
-tubulin antibody (1:2000, 124K4876 Sigma-Aldrich, Lyon, France) for protein content.
For immunocytochemistry, 3T3-L1 and 3T3-F442A cells were grown in 24-well plates, fixed in a 4% paraformaldehyde solution, and preincubated in 5% normal goat serum for 30 min. Two monoclonal anti-rat MR antibodies (1:100, 6G1 and 1D5, kindly provided by Dr. Celso E. Gomez-Sanchez) (17)
were used in 5% normal goat serum overnight at 4°C, followed by a secondary antibody (1:400, horse biotinylated anti-mouse IgG, BA-2000; Vector Laboratories Inc., Burlingame, CA, USA) for 30 min and by amplification with the avidin-biotin complex (Vectastain ABC kit, PK-6100, Vector Laboratories Inc.) for 30 min. The peroxidase reaction was then developed with diaminobenzidine tetrahydrochloride (peroxidase substrate kit, SK-4100, Vector Laboratories Inc.). Reactions were followed by optical microscopy and stopped by the addition of water.
Statistical analysis
Effects of aldosterone on adipogenesis were assessed by a between-subjects factorial analysis of variance (ANOVA) including the treatment effect, the time effect, and the interaction between both. When the F value of interaction was significant, the effect of treatment on MR measurements was tested at each time. Elsewhere, effects of treatment and time were established independently (comparison between marginal means). In the presence of a significant effect (main effect or interaction), pairwise comparisons were conducted by Tukey’s or Dunnet’s adjustment method of P values for more than three comparisons. Effects of other measurements were assessed with the same methodology. Binding was tested with nonparametric Kruskal-Wallis ANOVA test, followed by a nonparametric Mann-Whitney test. The estimable function used for between-subjects factorial analysis of variances was the type III sums of squares since data are unbalanced. In the case of non-respected hypotheses on residuals, an ANOVA on log-transformed data or a nonparametric approach was performed.
All analyses were performed using the SPSS version 13.0.1 Software (SPSS Inc., Chicago, IL, USA). P values of < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, we found that MR is induced during the course of 3T3-L1 adipose conversion at both the protein level, where MR specific immunostaining was visible within differentiation foci (Fig. 1
A), and the mRNA level (Fig. 1B
). The same pattern of MR mRNA expression was observed when cells were cultured in steroid-depleted medium (DCC) (Fig. 1B
), an essential condition for the appropriate study of corticosteroid-dependent effects on adipogenesis. GR mRNA expression increased slightly (but not significantly) during adipocyte differentiation under standard conditions while remaining essentially unchanged in steroid-depleted medium (Fig. 1C
). However, 3T3-L1 cells cultured in steroid-free medium retain their glucocorticoid responsiveness, as demonstrated by the induction of well-known target genes such as angiotensinogen and plasminogen activator inhibitor 1 (supplemental Fig. S1). GR mRNA expression was always higher (30- to 50-fold) than that of MR and comparable under permissive or steroid-depleted culture conditions (not shown). These initial experiments show that MR and GR expression patterns agree with those recently reported (13)
, and suggest that the differentiation-dependent expression of MR may play a significant role in the regulation of adipogenesis.
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Aldosterone induces adipose conversion of 3T3-L1 cells
Dexamethasone is classically used as a component of the initial differentiation cocktail of 3T3-L1 cells. To examine aldosterone effects on terminal adipocyte maturation, we set up an experimental protocol in which cells were cultured in the presence of DCC and dexamethasone was omitted from the differentiation cocktail to prevent interfering glucocorticoid effects. From day 2 following confluence, cells were exposed to either aldosterone or dexamethasone (Fig. 2
). Aldosterone was used at low doses known to selectively activate MR (18)
. These specific culture conditions, together with the hormone concentrations used, are essential to distinguish mineralocorticoid and glucocorticoid effects on adipose conversion. Morphological examination showed that when compared with untreated cells, aldosterone promotes intracellular accumulation of lipid droplets (Fig. 2A
), as well as a higher number of differentiation foci. In contrast, late exposure of 3T3-L1 cells to dexamethasone inhibited adipocyte differentiation. These effects of aldosterone and dexamethasone were confirmed by measuring glycerol-3-phosphate dehydrogenase (G3PDH) activity, an index of the magnitude of adipose conversion (19)
, which showed a significant increase in aldosterone-treated cells on day 7 (P=0.004) and was reduced in the presence of dexamethasone (day 4, P=0.007; day 7, P=0.006, Fig. 2B
). Glucocorticoid effects on 3T3-L1 cells were comparable with those reported for 3T3-F442A (20)
, indicating that whereas dexamethasone promotes adipogenesis when added at early stages of differentiation (11
, 21)
, it is inhibitory once cells are committed to this process. Aldosterone induced various molecular markers of adipose conversion (Fig. 2C, D
), including increases in adiponectin (P<0.001), leptin (P=0.001) and resistin (P<0.001) mRNA levels on day 7 following confluence. Dexamethasone inhibited adiponectin and leptin gene expression (P<0.001) but induced resistin to an extent lower than seen with aldosterone (Fig. 2C
, P=0.029). A significant reduction in aP2 transcript levels at 7 days postconfluence was seen with dexamethasone (P=0.024). Aldosterone induced an increase in PPAR mRNA levels (P=0.001), a key transcriptional regulator of adipogenesis (22)
(Fig. 2D
), whereas dexamethasone lowered PPAR gene expression (P<0.001). The aldosterone-dependent effect was specifically mediated by the MR, since it was completely reversed by the MR antagonist spironolactone, which had no effect alone. Given its intrinsic inducing effect on adipogenesis (data not shown and ref. 23
), the GR antagonist RU486 could not be used to explore GR involvement in aldosterone and dexamethasone regulatory actions on adipocyte terminal maturation. Interestingly, the inhibitory effect of dexamethasone on PPAR gene expression was also reversed by spironolactone, indicating that glucocorticoids may also act via MR to modulate adipose-specific gene expression. This would not be surprising, since aldosterone and glucocorticoids have been shown to recruit different coregulators to MR-responsive promoters, thus differentially affecting gene transcription (24)
.
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Differences in the degree of adipocyte differentiation induced by aldosterone may formally represent increased induction of the adipogenic transcriptional program or increased clonal expansion of preadipocytes. An effect on clonal expansion was excluded, however, as [3H]-thymidine incorporation showed similar proliferation rates in the presence of aldosterone (111±8 and 83±7% of untreated cells) in 3T3-L1 and 3T3-F442A, respectively (mean±SE of 9 independent cultures).
Aldosterone induces differentiation of 3T3-F442A cells
To better elucidate the mechanism of aldosterone action, we asked whether its proadipogenic effects were reproduced in 3T3-F442A cells, which are generally regarded as a model with a more advanced commitment in the adipose differentiation process (1)
; consistent with this, 3T3-F442A cell differentiation is independent of an early exposure to glucocorticoids. As in previous studies, chronic glucocorticoid exposure inhibited adipose terminal maturation (supplemental Fig. S2 and refs. 1
, 25
) and, as in 3T3-L1 cells, 3T3-F442A cells displayed a differentiation-dependent increase in MR mRNA levels (Fig. 3
A). Although this induction was a late event in complete FCS-containing medium, it occurred as early as day 2 postconfluence in DCC (P<0.001). MR protein was consistently induced during adipose conversion (Fig. 3B
).
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Next, 3T3-F442A cells were cultured from confluence in DCC in the absence or presence of aldosterone. Exposure to aldosterone for 7 days was accompanied by a dose-dependent increase in cell triglyceride accumulation and G3PDH activity (Fig. 3C
), with an EC50 value between 0.3 and 0.6 nM, consistent with the affinity of the MR for aldosterone (7)
. This effect is lessened by higher doses of aldosterone (10–7 M), known to activate GR, in line with the inhibitory effects of dexamethasone observed in our model. The aldosterone-induced increase of these biochemical markers was also time dependent, with an optimal effect on days 6 and 8 following confluence (Fig. 3D, E
). Aldosterone effects were antagonized by the addition of a 100-fold excess of spironolactone (Fig. 3F
), which completely reversed triglyceride accumulation and the increase in G3PDH activity. In 3T3-F442A cells, therefore, there is a clear distinction between positive aldosterone and negative glucocorticoid effects on terminal adipocyte maturation.
MR, but not GR, knockdown inhibits glucocorticoid-induced adipose conversion of 3T3-L1 cells
Given the overwhelming occupancy of MR by glucocorticoids in vivo and that we find only very low levels of 11HSD2 mRNA in 3T3-L1 cells compared with whole kidney (in which only 
5% of cells express 11HSD2), it would seem that under physiological conditions MR mediate glucocorticoid-dependent effects on adipose differentiation. In vivo, convincing evidence exists for a major role of glucocorticoids on adipose differentiation, as elegantly demonstrated in transgenic animal models (2)
. Therefore, we tested the involvement of MR activation in adipogenesis under permissive conditions in which 3T3-L1 cells were maintained in steroid-containing FCS and induced to differentiate with the classical effector cocktail, including dexamethasone at the very early steps of the differentiation program. MR or GR expression was down-regulated by specific siRNA. Compared with scrambled siRNA, an optimal and reproducible decrease in MR mRNA expression was obtained 60 h after transfection by each of two unrelated MR-specific siRNA (P=0.044) (Fig. 4
A), as well as both together (data not shown). Figure 4B
shows that the siRNA-induced decrease in MR mRNA expression was accompanied by a parallel 65–70% reduction in specific [3H]-aldosterone binding (P<0.001 for both si1_MR and si2_MR compared with cells exposed to lipofectamine alone), as well as by the disappearance of immunodetectable MR in 3T3-L1 cells (Fig. 4C
). This decrease in MR expression was specific since neither of these two siRNAs modified GR mRNA levels (Fig. 4D
). MR down-regulation induced a dramatic inhibitory effect on 3T3-L1 adipose conversion (Fig. 5
A), reflected by the sharp decrease in cell triglyceride content (Fig. 5B
) and G3PDH enzyme activity (Fig. 5C
), which correlated nicely with the concentration of the MR-specific siRNA (Supplemental Fig. S3). Lipoprotein lipase mRNA expression was also reduced (Fig. 5D
, an effect more pronounced with si2_MR and associated with a parallel decrease in PPAR and C/EBP transcript levels (Fig. 5E, F
), whereas C/EBPß gene expression was unaffected (Fig. 5F
). It is generally recognized that C/EBPß is involved early and transiently in the control of adipogenesis, while PPAR and C/EBP are involved more distally (26)
, and that expression of the relevant genes is regulated in a sequential and coordinated manner by environmental proadipogenic factors. Since our experimental time points were later than the transient expression peak in C/EBPß, as demonstrated by the time-dependent decrease in C/EBPß expression (see Fig. 5F
), we cannot exclude an effect of the MR knockdown on this early proadipogenic transcription factor. Regardless of the exact molecular events, however, the marked inhibition of PPAR and C/EBP expression after specific MR down-regulation provides further evidence that MR is intimately involved in controlling the adipogenic transcriptional program.
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We next examined whether specific reduction of GR expression also modulates adipocyte differentiation, and using two unrelated GR siRNAs we induced a marked decrease in GR mRNA (Fig. 6
A) and protein (Fig. 6B
) expression. The magnitude of GR protein down-regulation was similar to that obtained for MR with MR-specific siRNAs (74% and 58% for si1_GR and si2_GR, compared with scrambled siRNA). Most likely due to the higher endogenous GR gene expression, a higher dose of GR siRNAs was required to achieve this inhibition, and overall gene expression profiles were slightly delayed compared with experiments using MR siRNAs. The two GR siRNAs did not interfere with MR mRNA expression (Fig. 6C
). In contrast to the phenotype observed after MR down-regulation, GR repression did not modify the morphological appearance of differentiated cells (Fig. 6D
), and no significant reduction of cell triglyceride content was observed (Fig. 6E
). PPAR mRNA levels were unchanged 60 h after transfection (Fig. 6F
). These data are consistent with previous reports demonstrating that PPAR is necessary and sufficient to promote adipogenesis (27)
. Taken together, our experiments demonstrate that under permissive conditions (i.e., dexamethasone-containing differentiation medium), only the knockdown of MR inhibits glucocorticoid-induced adipose maturation in 3T3-L1 cells, and GR inactivation does not lead to major modification of the adipogenic process.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although it might seem unlikely (considering the low 11HSD2 expression in adipocytes and overwhelming circulating glucocorticoids levels) that, in vivo, aldosterone plays a physiological role early or late in adipogenesis, evidence is accumulating to support a pathophysiologic link between aldosterone and adipose tissue development and metabolism. In previous work we have shown that MR is expressed in brown adipose tissue and that aldosterone is able to induce differentiation of brown adipose cells (8
, 9)
and affect energy expenditure by regulating expression and function of uncoupling proteins (10)
. In vivo, a recent study by Fallo et al. describes a higher prevalence of the metabolic syndrome in patients with primary aldosteronism compared to patients with essential hypertension (28)
. Conversely, it was recently reported that human adipocytes secrete as yet unknown factors able to stimulate synthesis and secretion of aldosterone from adrenal cells (29)
, which might explain the relative hyperaldosteronism often observed in obese subjects.
Second, dexamethasone appears to elicit a dual effect, dependent on the stage of adipocyte commitment. When added at the first days of adipocyte differentiation, it is a potent proadipogenic factor for 3T3-L1 cells (1)
. Once 3T3-L1 cell differentiation is induced, or in 3T3-F442A preadipocytes, it significantly inhibits adipocyte terminal maturation. Given our results with MR antagonists, the latter effect may include negative regulation via MR of PPAR gene expression. Translated into a more general model, our results agree with a recent working hypothesis in which MR are chronically occupied by glucocorticoids (6)
. Depending on the environmental conditions, glucocorticoid binding to MR may elicit either positive or negative control of gene transcription (6)
. The mechanistic explanation of this phenomenon may involve dynamic coregulator exchange and recruitment by nuclear receptors on target gene promoters. Consistent with previous reports (30
, 31)
, one might speculate that, in 3T3-L1 cells, cyclic and coordinated expression of distinct nuclear receptor coactivators/corepressors during adipogenesis may switch glucocorticoids from repressors to activators of MR-driven gene expression.
Third, and most important, we have demonstrated the central role of MR in mediating glucocorticoid effects on adipose conversion. Indeed, down-regulation of GR could not reproduce the phenotype observed after specific MR inactivation, indicating that MR is the principal contributor to corticosteroid-induced adipose differentiation. Also supporting this hypothesis is recent evidence coming from anatomical profiling of nuclear receptor expression, showing that MR belongs to a cluster of receptors highly expressed in all tissues and is crucial for maintaining global basal metabolic functions (32)
. MR fall into a subcluster containing RXRß, which serve as obligate heterodimer partners to a number of endocrine and lipid-sensing receptors, and LXRß, which play central roles in the transcriptional control of lipid metabolism (33)
. In view of our results, it is tempting to speculate that in mice overexpressing 11HSD1, which develop the metabolic syndrome as a consequence of high adipose levels of corticosterone (2)
, MR activation may play a role in addition to the involvement of the GR. This is of particular interest given the cyclic circadian expression of GR in adipose tissue, as opposed to MR (34)
, which might modulate the intracellular receptor ratios and therefore their contribution to the regulation of metabolic processes. Adipose tissue-specific deletion of MR and/or GR will thus be of major importance in establishing the roles of the two receptors in developing subcutaneous and visceral fat mass.
Finally, it is worth noting that the two receptors, MR and GR, evolved earlier than the capacity to synthesize aldosterone, which appeared relatively recently (35
, 36)
. Aldosterone, indeed, is found only in terrestrial vertebrates and therefore cortisol is the original evolutionary driver for MR (36)
. Evolution of an MR that in epithelial cells can be selectively regulated by aldosterone thus allowed its specific role in fluid and electrolyte homeostasis, developing new functions in addition to those subserved by a high-affinity receptor essentially always occupied by glucocorticoids. By its complex involvement in adipose differentiation and energy storage, in addition to its more familiar roles on salt and water reabsorption, MR may thus represent a major contributor across metabolic homeostasis, including fluid, electrolytes, and energy balance. Finally, the development of specific modulators of the MR signaling pathway in adipocytes may open exciting new perspectives in the management of human obesity and the metabolic syndrome.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 4, 2007. Accepted for publication February 1, 2007.
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