The mineralocorticoid receptor mediates aldosterone-induced differentiation of T37i cells into brown adipocytes

Patrice Penfornis, Say Viengchareun, Damien Le Menuet, Françoise Cluzeaud, Maria-Christina Zennaro, and Marc Lombès

Institut National de la Santé et de la Recherche Médicale U 478, Faculté de Médecine Xavier Bichat, 75870 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

By use of targeted oncogenesis, a brown adipocyte cell line was derived from a hibernoma of a transgenic mouse carrying the proximal promoter of the human mineralocorticoid receptor (MR) linked to the SV40 large T antigen. T37i cells remain capable of differentiating into brown adipocytes upon insulin and triiodothyronine treatment as judged by their ability to express uncoupling protein 1 and maintain MR expression. Aldosterone treatment of undifferentiated cells induced accumulation of intracytoplasmic lipid droplets and mitochondria. This effect was accompanied by a significant and dose-dependent increase in intracellular triglyceride content (half-maximally effective dose 10-9 M) and involved MR, because it was unaffected by RU-38486 treatment but was totally abolished in the presence of aldosterone antagonists (spironolactone, RU-26752). The expression of early adipogenic gene markers, such as lipoprotein lipase, peroxisome proliferator-activated receptor-gamma , and adipocyte-specific fatty acid binding protein 2, was enhanced by aldosterone, confirming activation of the differentiation process. We demonstrate that, in the T37i cell line, aldosterone participates in the very early induction of brown adipocyte differentiation. Our findings may have a broader biological significance and suggest that MR is not only implicated in maintaining electrolyte homeostasis but could also play a role in metabolism and energy balance.

uncoupling proteins; peroxisome proliferator-activated receptor-gamma


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALDOSTERONE, THE MAJOR MINERALOCORTICOID hormone, is involved in the control of water and sodium homeostasis and participates in the regulation of blood pressure. It is known that aldosterone exerts its biological effects through its binding to the mineralocorticoid receptor (MR) present in sodium-transporting epithelia, such as the distal renal tubules, distal colon, and sweat and salivary glands (2). More recently, MR expression has been extended to a large variety of tissues, including nonepithelial cells of the central nervous system, the cardiovascular system, and also mononuclear leukocytes, skin, and lung (36), in which specific aldosterone effects have been described (5, 8, 30).

We have recently shown that expression of the human MR (hMR) is under the control of two alternative promoters, P1 and P2, located upstream of the two first untranslated exons 1alpha and 1beta of the hMR gene, respectively. In vitro studies have demonstrated that these two promoters differ strikingly by their basal transcriptional activity as well as their corticosteroid regulation (34). In an attempt to further characterize hMR promoter functions in vivo, we have established transgenic mice in which the proximal P1 and the distal P2 regions were used to direct expression of the SV40 large T antigen (35). This targeted oncogenesis strategy revealed important functional differences between these promoters, not only in terms of differential tissue-specific utilization but also with regard to their relative potency. Indeed, P1 is a strong promoter, active in all MR-expressing cells, whereas the P2 promoter is approximately ten times less potent, and its activity is restricted to a limited number of tissues. Interestingly, the phenotypic presentation of transgenic animals allowed identification of new sites of MR expression. The P1-TAg founder animals presented with malignant hibernomas originating from brown fat, indicating that the P1 promoter was transcriptionally active in brown adipose tissue (BAT). We subsequently demonstrated that endogenous MR was constitutively expressed in this tissue, suggesting that BAT was a previously unrecognized mineralocorticoid target (35).

Brown fat is a highly specialized tissue involved in thermoregulation in rodents (19) and has been shown to participate in energy balance and body weight control in humans (4). Brown adipocytes are the site of adaptive thermogenesis, because they increase energy expenditure and dissipate energy as heat. This results from the action of the BAT-specific protein uncoupling protein 1 (UCP1), which functions as a mitochondrial proton translocator, uncoupling oxidative phosphorylation and shutting down ATP synthase activity, the last enzymatic step of the respiratory chain (23). To determine whether mineralocorticoids could play a role in BAT function, we studied their effects on the T37i cell line, derived from one hibernoma of a P1-TAg transgenic mouse. T37i cells maintain the ability to undergo terminal differentiation and remain capable of expressing UCP1 upon retinoic acid and beta -adrenergic stimulation (35). More importantly, T37i cells express endogenous mouse MR at both mRNA and protein levels, thus constituting an ideal model to investigate aldosterone effects on brown adipocyte proliferation and differentiation. In this report, we show that aldosterone, through its interaction with MR, promotes T37i cell differentiation into mature brown adipocytes, as judged by morphological changes, increased triglyceride accumulation, and induction of early adipogenic gene expression. These results implicate another member of the nuclear receptor superfamily in the complex brown adipocyte differentiation process and further suggest that MR participates not only in hydroelectrolytic homeostasis but also in the regulation of energy balance.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. The T37i cell line was derived from a hibernoma (malignant brown fat tumor) of the transgenic mouse founder 37 carrying a hybrid gene composed of the human MR proximal promoter linked to the SV40 large T antigen (35). T37i cells were cultured in DMEM-Ham's F12 medium (GIBCO BRL) supplemented with 10% FCS (normal or treated with dextran-coated charcoal), 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 20 mM HEPES, and were grown at 37°C in a humidified atmosphere with 5% CO2. Differentiation into adipocytes was achieved under standard conditions by incubating subconfluent undifferentiated T37i cells with 2 nM triiodothyronine (T3; Sigma Chemical, St Louis, MO) and 20 nM insulin (GIBCO BRL) for >= 3 days. Thiazolidinedione (BRL-49653) was a generous gift of Dr. R. Negrel (Sophia Antipolis, France). The differentiation process was studied under different experimental conditions. Cells were generally incubated for various periods of time in medium supplemented with charcoal-treated serum together with different aldosterone or glucocorticoid concentrations, in the presence or absence of specific mineralocorticoid (spironolactone or RU-26752) or glucocorticoid (RU-38486) antagonists kindly provided by Roussel-Uclaf (Romainville, France). The mitochondrial UCP1 expression was induced by adding 1 µM isoproterenol (Sigma) for 6 h and then was analyzed by Northern blot. All of the experiments were performed with T37i cells between passages 10 and 20.

Cytological and electron-microscopic analyses. Cells were rinsed twice with PBS, incubated for 15 min at room temperature with a 0.3% solution of Oil Red 4beta (Merck) in isopropanol-distilled water (60:40, vol/vol). Cells were then rinsed with distilled water, counterstained with hematoxylin for 3 min, rinsed, and mounted. For transmission electron microscopy, cells were grown on transwell filters (Costar) for 7 days in the presence of 10 nM aldosterone. Cells were rinsed with PBS and then fixed for 30 min with 2.5% glutaraldehyde in PBS at room temperature. Cells were rinsed in PBS, postfixed with 1% osmic acid for 15 min, dehydrated in graded ethanol, and embedded in EPON 812 (Fluka, St Quentin, France). Ultrathin sections were realized on transversely oriented confluent cells and examined with a Philips EM 410 electron microscope.

Cell growth studies. Cell growth was estimated by measuring DNA synthesis in the T37i cells by incorporation of [3H]thymidine (925 GBq/mmol, Amersham). The cells were seeded in 12-well plates at a density of 104 cells per well in a medium containing 10% charcoal-treated serum supplemented or not with aldosterone. Two days later, cells were incubated with 0.5 µCi tritiated thymidine/well for 6 h. At the end of the incubation, cells were rinsed three times with cold PBS and lysed in 1 ml of 5% trichloroacetic acid for 30 min at 4°C followed by incubation with 1 ml 0.3 N NaOH overnight at 4°C. After neutralization by addition of 5 µl of 1 N acetic acid, the incorporated radioactivity was counted. Results are expressed by incorporated dpm corrected by the number of cells.

Triglyceride measurements. Undifferentiated T37i cells were plated in 12-well plates at a density of 5 × 104 cells/well and grown for 7 days in medium prepared with stripped FCS in the presence or absence of hormones. Cells were rinsed twice with cold PBS, harvested in a total volume of 250 µl of PBS with a rubber policeman, and counted. Cells were subsequently disrupted by sonication (2 × 10 s), and the homogenates were centrifuged for 10 min at 12,000 rpm at 4°C. Protein concentration of the supernatant was determined by the Bradford technique (3). Triglyceride content was measured using a colorimetric determination kit (Procedure 336, Sigma).

RNA analyses. Total RNA was isolated from undifferentiated or differentiated T37i cells by use of TRIzol (GIBCO BRL/Life Technologies) reagent extraction according to the manufacturer's recommendations. Fifteen micrograms of total RNA were analyzed by Northern blotting performed by standard techniques. alpha -32P-labeled probes were synthesized by random priming (Rediprime II random prime labeling system, Amersham Pharmacia Biotech, Buckinghamshire, UK) with cDNA for mouse lipoprotein lipase (LPL; 1.43 kb, from Dr. M. C. Schotz, UCLA), mouse adipocyte-specific fatty acid binding protein 2 (aP2; 0.6 kb, from Dr. B. Spiegelman, Harvard), mouse peroxisome proliferator-activated receptors-gamma 2 (PPARgamma 2; 0.85 kb, from Dr. E. Z. Amri, INSERM U470, Nice, France), rat UCP1 (from Dr. D. Ricquier, CEREMOD, Meudon, France), and rat glyceraldehyde-3-phosphate dehydrogenase (GADPH; 0.84 kb, from Dr. B. Escoubet, INSERM U426, Paris, France). Serial hybridizations with different probes were performed. Specific hybridization signals were quantified by measuring radioactivity with a Packard Instant Imager. Membranes were then subjected to autoradiography.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Aldosterone induces accumulation of triglycerides in T37i cells. Under standard culture conditions, T37i cells grow as undifferentiated cells with a fibroblast-like appearance and a unique nucleus (Fig. 1A). In contrast, after insulin and T3 treatment for 7 days, drastic morphological modifications were observed; cells became larger and spherical, accumulating multiple lipid droplets of various sizes as revealed by the Oil-Red-O staining (Fig. 1C). Similar results were obtained by incubation with thiazolidinedione (not shown). These morphological changes were consistent with cells entering into the adipocyte differentiation program. Note that the majority of differentiated cells presented with multiple nuclei. The degree of morphologically recognizable differentiation was almost 100% in the T37i cells. We tested the influence of aldosterone treatment on undifferentiated cells. As shown in Fig. 1B, 10 nM aldosterone induced the appearance of small intracytoplasmic lipidic vacuoles. This was a strong morphological indication that aldosterone may participate in the adipocyte differentiation process. Compared with undifferentiated cells (Fig. 2A), electron-microscopic examination of aldosterone-treated T37i cells (Fig. 2B) confirmed the presence of multiple lipid droplets, which never did coalesce, and a multilocular aspect, highly characteristic of brown adipocytes. Differentiated cells also presented numerous mitochondria, variable in size, bordering the fat vacuoles, a finding consistent with the high metabolic activity of brown adipocytes. Interestingly, we could also observe numerous mitochondrial cristae junctions in the differentiated T37i cells (Fig. 2C). Note that one cell clearly contained two distinctly visible nuclei, most probably due to the differentiated as well as neoplastic feature of T37i cells.


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Fig. 1.   Adipogenic differentiation of T37i cells. The T37i cells were cultured under standard conditions in the absence (A) or in the presence of either 10 nM aldosterone (B) or 20 nM insulin and 2 nM triiodothyronine (T3; C) for 7 days. After Oil-Red-O staining and hematoxylin counterstaining, light-microscopic examination revealed the presence of small intracytoplasmic lipid droplets in the aldosterone-treated cells, whereas fully differentiated cells accumulated numerous voluminous lipid droplets and presented with multiple nuclei.



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Fig. 2.   Transmission electron-microscopic examination of undifferentiated and aldosterone-induced differentiated T37i cells. A: undifferentiated T37i cells are long, fusiform cells with a unique flat nucleus. They are relatively poor in organelles, and no lipid droplet was observed. Magnification ×6,800. B: in contrast, T37i cells grown on filters and incubated with 10 nM aldosterone for 7 days exhibited numerous intracytoplasmic lipid vacuoles surrounded by multiple mitochondria. Note that one cell (left) presented two distinct nuclei. Magnification ×3,800. C: insert illustrates the presence of mitochondrial cristae junctions observed in differentiated T37i cells. Magnification ×24,000.

To quantify the morphological changes observed after aldosterone treatment, triglyceride content was measured in T37i cells cultured under basal conditions or incubated with various concentrations of aldosterone. Figure 3 shows that aldosterone induces a twofold increase in triglyceride accumulation in a dose-dependent manner. The maximum effect was achieved at a concentration of 10-8 M with a half-maximal effect at 10-9 M aldosterone, a value compatible with the dissociation constant (Kd) of aldosterone for endogenous mouse MR measured in T37i cells (35). Under the same experimental protocol, a fourfold increase in triglyceride content of T37i cells was observed after insulin-thyroid hormone treatment.


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Fig. 3.   Triglyceride accumulation in T37i cells. T37i cells were induced to differentiate with various concentrations of aldosterone (1-100 nM) in DMEM-Ham's F12 medium containing 10% charcoal-treated FCS for 7 days. Triglyceride content of the cells was measured with a triglyceride kit (Sigma). Results are means ± SE of 2-3 independent experiments performed in triplicate and are expressed as % above control levels (62.97 ± 5.5 mg triglycerides/g protein). *** Statistical significance (P < 0.001).

We next studied the effects of mineralocorticoid and glucocorticoid antagonists on the aldosterone-stimulated accumulation of triglycerides. Results are presented in Fig. 4. The significant increase in triglyceride content induced by 1 nM aldosterone (P < 0.01 compared with control) was unaffected by addition of 1 µM RU-38486, an anti-glucocorticoid compound, but was totally inhibited by the two mineralocorticoid antagonists, spironolactone and RU-26752. None of the anti-hormones alone modified the lipid content of the T37i cells. Under the same experimental conditions, when corticosterone was used at a concentration of 10 nM, a significant two- to threefold increase in triglyceride content of T37i cells was observed (118.79 ± 21.36 mg triglycerides/g protein, n = 3, vs. 44.37 ± 9.48, n = 6, P < 0.01). This increase was equivalent to that observed with aldosterone; however, cotreatment with RU-38486 inhibited by ~80% the glucocorticoid-mediated triglyceride accumulation. Collectively, our results demonstrate that activation of MR by aldosterone is able to stimulate adipocyte differentiation.


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Fig. 4.   Aldosterone-stimulated triglyceride accumulation in T37i cells is mediated by the mineralocorticoid receptor. T37i cells were grown for 7 days in the absence or presence of 1 nM aldosterone, alone or together with 1,000-fold excess of RU-38486 (RU 486), spironolactone (Spiro), or RU-26752 (RU 752). Triglyceride content was measured as described in MATERIALS AND METHODS. Results represent means ± SE for 6 determinations and are expressed as % above control levels (62.50 ± 4.0 mg triglycerides/g protein). Statistical significance: ** P < 0.01; * P < 0.05.

To determine whether aldosterone effects on cell differentiation occurred in part by stimulating cell growth, we measured the incorporation of tritiated thymidine during DNA synthesis. No significant difference was evidenced between untreated T37i cells (1.14 ± 0.11 × 104 dpm/106 cells, n = 6) compared with the aldosterone-treated cells (1.26 ± 0.11 × 104 dpm/106 cells, n = 6). These results indicate that aldosterone accelerates adipocyte differentiation of T37i cells without affecting cellular proliferation.

Aldosterone stimulates specific gene transcription in T37i cells. We first analyzed the temporal pattern of expression of known adipogenic regulators during adipocyte differentiation of T37i cells. Northern blot analysis was performed with total RNA samples from control or treated cells isolated daily at different times postconfluence. We examined the levels of mRNA for LPL, aP2, and PPARgamma , all genes known to be sequentially activated in a differentiation-specific manner, at least in most white adipocyte models (3T3-F24A, and the like) (18). GAPDH was used as an internal control. Figure 5 illustrates the sequential gene activation observed during differentiation induced by insulin and T3 treatment. LPL expression rapidly increased within the first 48 h and reached a maximum at day 6 to progressively decrease afterward. Similarly, exposure to insulin and T3 stimulated PPARgamma expression as early as day 2, reaching the maximum levels of PPARgamma 2 mRNA expression by day 6 and remaining unchanged thereafter. In constrast, aP2 was progressively expressed during this differentiation process, reaching a maximum expression at day 8. This coincided with the full expression of adipogenic genes. It has to be noted that T37i cells cultured for long periods of time (up to 15 days), even in the presence of charcoal-dextran-treated serum, could undergo a spontaneous differentiation process, as evidenced by the appearance of morphological changes and expression of substantial amounts of adipose-specific genes (not shown).


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Fig. 5.   Sequential activation of adipogenic gene expression during differentiation conversion of T37i cells into mature brown adipocytes. T37i cells were grown in the presence of 20 nM insulin and 2 nM T3. A: total RNA was extracted at various times as indicated during the differentiation process, and 15 µg of each RNA were analyzed by Northern blot hybridization with 32P-labeled cDNA probes for peroxisome proliferator-activated receptor-gamma 2 (PPARgamma 2), lipoprotein lipase (LPL), adipocyte-specific fatty acid binding protein 2 (aP2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: signal intensities were quantitated by InstantImager analysis, with GAPDH as internal control. Average values from 2 independent experiments are expressed as % maximum expression for each marker.

We next investigated whether aldosterone treatment was able to modify adipocyte-specific gene expression. Figure 6 shows that, compared with control, aldosterone induced an approximately twofold increase of LPL expression (1.95 ± 0.27, n = 8, P < 0.02) as early as day 2 postconfluence. In addition, PPARgamma 2 expression (2.56 ± 0.47, n = 6, P < 0.05) and aP2 expression (2.09 ± 0.27, n = P < 0.02) were also stimulated by aldosterone treatment, two- to threefold differences being detected between control and aldosterone-differentiated cells at days 4 and 5. Finally, we show that T37i cells exposed to aldosterone for 6 or 8 days were fully differentiated into brown adipocytes, because, after aldosterone had been washed out for 48 h, UCP1 mRNA levels rapidly increased after 6 h of isoproterenol stimulation (Fig. 7). Under the same experimental conditions, isoproterenol-induced UCP1 expression was undetectable in control cells but was clearly visible in insulin T3 differentiated cells. All together, these results indicate that aldosterone plays an important role in the differentiation process by inducing expression of specific genes determinant for brown adipocyte differentiation.


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Fig. 6.   Aldosterone induces expression of the early adipogenic gene markers LPL, aP2, and PPARgamma 2. T37i cells were grown in culture medium supplemented with 10% charcoal-treated serum in the absence (C) or presence of 10-7 M aldosterone (A). At various periods of time, as indicated by days 1-5 (D1...D5) during the differentiation process, total RNA was extracted, and Northern blot analysis proceeded using specific probes for LPL, PPARgamma 2, aP2, and GAPDH. Signals were quantified by Instant Imager and expressed as the ratio between specific gene expression and GAPDH in arbitrary units as a function of time. Open bars, control; filled bars, aldosterone.



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Fig. 7.   Induction of uncoupling protein 1 (UCP1) mRNA by isoproterenol in aldosterone-differentiated T37i cells. T37i cells were cultured for 6 days in medium supplemented with 10% charcoal-treated serum in the absence (control) or in the presence of aldosterone (Aldo) or insulin/T3 (IT) and maintained for 2 additional days in medium free of hormone. Before total RNA extraction at day 8, cells were incubated for 6 h in the absence (-) or presence (+) of 10-6 M isoproterenol, and Northern blot analysis was performed using a 32P-labeled UCP1 cDNA probe, a GADPH probe (for Aldo), and a 28S probe (for control and IT).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present article, we provide evidence of a role for aldosterone via the MR in the differentiation process of T37i cells into mature brown adipocytes. Up to now, aldosterone effects were considered to be restricted mainly to the control of sodium and water homeostasis through its interaction with the MR present in sodium-transporting epithelia of the distal nephron, colon, and the sweat and salivary glands. Strong lines of evidence indicate that aldosterone also exerts specific effects on nonepithelial cells of the central nervous and cardiovascular systems (5, 8), although the molecular basis of aldosterone action and the mineralocorticoid selectivity-conferring mechanisms remain elusive. More recently, using a targeted oncogenesis strategy in transgenic mice, we have identified brown adipose tissue as a novel mineralocorticoid target (35). The newly established T37i cell line was further examined in an attempt to characterize aldosterone's effects in this tissue. It is important to note that studies of the effects of aldosterone on cell lines have been hampered by the fact that most of them (primary cultures as well as immortalized cell lines) lose expression of MR. Our targeted oncogenesis strategy allowed us to circumvent this problem by placing the immortalizing oncogene, at least in part, under the same regulatory sequences as the endogenous MR.

We have now shown that aldosterone exposure at nanomolar concentrations was able to promote the conversion of T37i cells into fully differentiated brown adipocytes. Upon aldosterone treatment, major morphological changes were observed with the appearance of an adipocyte phenotype, associating multiple intracytoplasmic lipid vacuoles with an increase in the number and size of mitochondria. Interestingly, because it is known that brown adipocyte mitochondria architecture presents specific features such as crista junctions (21), the T37i cell could represent an ideal model for investigating inner mitochondrial membrane topology in relation to the metabolic and bioenergetic status of the cell. Differentiation-associated morphological changes correlated with biochemical modifications consisting of a twofold increase in triglyceride contents. Time-dependent variations in expression of adipogenic genes such as LPL, PPARgamma 2, and aP2 were clearly detected in aldosterone-stimulated cells compared with control. However, aldosterone was less potent with a delayed time course than insulin and T3 in inducing PPARgamma expression, which may in part explain the difference in the degree of adipose conversion of T37i cells between the two treatments. This might be due to the simultaneous activation of two different signaling pathways in cells treated by insulin and thyroid hormones, resulting in synergistic effects on PPARgamma expression at both transcriptional and posttranscriptional levels. More importantly, aldosterone-differentiated T37i cells maintained the ability to express UCP1 after beta -adrenergic stimulation, indicating terminal differentiation and full maturation of brown adipocytes. Preliminary results indicated that differentiated T37i cells are also able to synthesize and excrete leptin (M. Buyse, S. Viengchareun, A. Bado, and M. Lombès, unpublished results), confirming the major interest of this cellular model in the examination of regulatory mechanisms of leptin secretion and the effects of leptin on specific adipogenic gene expression and the differentiation process.

Several lines of evidence strongly support the involvement of MR in the differentiation of T37i cells. First of all, the low concentration of aldosterone (1 nM) used to induce triglyceride accumulation (see Fig. 3) was in accordance with the Kd of aldosterone for MR determined in this cellular model (35). In addition, aldosterone effects were still observed in the presence of RU-38486 but were abolished by addition of mineralocorticoid antagonists, which clearly indicates that aldosterone-stimulated differentiation was mediated by the MR.

Glucocorticoids are known to be potent activators of adipogenic differentiation. In our cellular model, glucocorticoids and mineralocorticoids have overlapping effects on adipocyte differentiation. Because glucocorticoid hormones bind to MR with affinities similar to aldosterone, mineralocorticoid selectivity of aldosterone action in target cells is ensured in part by the enzyme 11beta -hydroxysteroid dehydrogenase 2 (11HSD2), which catalyzes the rapid metabolism of glucocorticoids into inert 11-keto forms (6). Because we failed to detect in Northern blot analysis the presence of 11HSD2 transcripts in either normal or neoplastic BAT and in T37i cells (35), it is possible that, in this tissue, MR is predominantly occupied by glucocorticoids and functions as a glucocorticoid receptor. However, preliminary results performed in T37i cells indicate the presence of a metabolic conversion of corticosterone into 11-dehydrocorticosterone, suggesting that some MR-protecting enzymatic mechanisms might be operating in these cells. It remains to be established whether this activity corresponds to 11HSD type 2 or to another, as yet unidentified, tissue-specific isoform. In addition, the circadian fluctuations and the pulsatile variations of glucocorticoid levels may, in certain situations, allow aldosterone to access the MR even in the absence of specificity-conferring mechanisms (5). Finally, because heterodimerization between members of the nuclear receptor superfamily is well established, bringing about diverse complexes with specific transcriptional properties (7), MR could represent an important dimerization partner for the GR in mediating tissue-specific effects and even potentiate transcriptional activities, as already reported in different cellular and promoter contexts (17, 34).

The adipocyte differentiation program is an extremely complex process in which two major families of transcription factors are required, the CCAAT/enhancer-binding proteins (C/EBP) and PPAR. During adipogenesis, a cascade of gene activation for transcription factors and adipocyte-specific genes occurs in a coordinated and time-dependent manner (18). In the course of white adipocyte differentiation at least, it has been shown that C/EBPbeta and -delta are induced early and synergistically stimulate the expression of PPARgamma and C/EBPalpha (31). At the final stage of differentiation, expression of adipogenic genes such as LPL, aP2, or GLUT-4 is induced by PPARgamma alone or cooperatively with C/EBPalpha , which results in mature adipocyte phenotype (32). In contrast to white adipocytes, which function mainly as lipid storage, brown adipocytes have highly specialized thermogenic activity due to the presence of the mitochondrial UCP1, whose expression is regulated by multiple signaling pathways including PPARgamma , TR, RXR, and beta -adrenergic receptors (23). It has been reported that both beta 1- and beta 3-adrenergic receptors are expressed in BAT (13). We have not yet determined which subtypes are expressed in T37i cells. The temporal expression pattern of adipogenic genes in T37i cells reported in Fig. 5 is consistent with the sequential gene activation described above. The mitochondrial UCP1 is the last gene expressed during the differentiation process of T37i cells.

Knock-out animal models for C/EBPalpha (29), C/EBPbeta , and C/EBPdelta (26) demonstrate that these transcriptional factors are essential for brown adipocyte differentiation. However, C/EBPalpha and PPARgamma are not sufficient for full differentiation (26). Other factors, such as an estrogen-related receptor (ERRalpha ) (25, 28) and adipocyte determination and differentiation-dependent factor 1/sterol regulatory element-binding protein 1 (ADD1/SREBP1) (15), are also implicated in brown adipocyte differentiation. Recently, PPARgamma -coactivator 1 (PGC-1), a coactivator of PPARgamma that is exclusively expressed in BAT and skeletal muscle (22), has been clearly identified as a key factor in brown adipocyte differentiation. It will be of great interest to determine whether PGC-1 is also expressed in T37i cells. It was shown that PGC-1 binds to various members of the nuclear receptor superfamily, such as TR and ER, through an interaction outside the classical activation function 2 (AF2) domain located in the COOH-terminal part of the nuclear receptors. It may be of interest to determine whether PGC-1 interacts with and/or modulates the transcriptional activity of MR. Because it has been shown that another coactivator, steroid receptor coactivator 1 (SRC-1), is not expressed in BAT (12), it could be speculated that specific aldosterone effects on brown adipocytes and white fat (24) might depend in part on the relative levels of coactivators such as PGC-1 or SRC-1, respectively.

Our results suggest that aldosterone might play a role in a very early stage of the T37i cell differentiation program between adipoblast and preadipocyte, whereas terminal differentiation of immature to mature adipocytes does not seem to require the presence of MR. MR could regulate specific gene expression at the transcriptional level, but the nature of the primarily aldosterone-induced proteins remains to be determined. Of particular interest is the observation that brown adipocyte differentiation correlates with a decrease in c-jun content (33). Because activating protein 1 (AP-1) activity is modulated by interaction with nuclear receptors, including glucocorticoid receptor (GR) (11, 14, 20), such a mechanism could account for the MR-mediated responses in brown adipocytes. On the other hand, because a role of phosphatidylinositol (PI) 3-kinase has recently been demonstrated in aldosterone regulation of epithelial sodium channels in renal A6 cells (1), MR could also intervene by affecting the PI 3-kinase pathway, whose activation was shown to be an essential requirement for fetal brown adipocyte differentiation (27).

The physiological significance of aldosterone-induced differentiation of brown fat remains to be evaluated. However, an enlargement of the brown adipose tissue surrounding kidneys and adrenal glands has been observed in patients presenting hyperaldosteronism (9). Because emergence of brown adipocyte depots has been reported in white fat of both rodents (10) and humans (4, 16), our findings could be particularly relevant for human physiology, as an important factor determining the capacity to adapt thermogenesis and to mobilize triglyceride storage. Whether brown adipocytes arise from a preadipoblast capable of acquiring brown adipocyte phenotype under stimulation of specific factors, including aldosterone, remains to be determined.

In conclusion, the T37i cell line constitutes an interesting model for examining the regulatory mechanisms of differentiation and specific gene expression in brown adipocytes. Our results clearly demonstrate that MR participates in the differentiation of T37i cells, as evidenced by hormone-induced morphological, biochemical, and molecular modifications, and they indicate that MR is involved in this complex process. In addition to the well-known effect of aldosterone as a determinant factor implicated in sodium homeostasis, we provide first evidence of a role for aldosterone exerting specific functions on brown adipose tissue physiology, which thus constitutes a possible link between electrolyte and energy balance.


    ACKNOWLEDGEMENTS

We thank Drs. D. Ricquier and C. Forest (Ceremod, Meudon, France) for helpful discussions; Dr. R. Negrel (Nice, France) for the gift of thiazolidinedione; and Drs. A. Lombès and A. Baker for help in mitochondrial examination. The assistance of J. Grellier, G. Delrue, and P. Disdier in preparation of illustrations, and of Dr. A. Marette for critical reading of the manuscript, is also gratefully acknowledged.


    FOOTNOTES

Address for reprint requests and other correspondence: Marc Lombès, INSERM U 478, Faculté de Médecine Xavier Bichat 16, rue Henri Huchard, BP 416, 75870 Paris Cedex 18, France (E-mail: mlombes{at}bichat.inserm.fr).

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. §1734 solely to indicate this fact.

Received 19 November 1999; accepted in final form 16 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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