Mineralocorticoid and glucocorticoid receptors inhibit UCP expression and function in brown adipocytes

Say Viengchareun, Patrice Penfornis, Maria-Christina Zennaro, and Marc Lombès

INSERM U 478, Institut Fédératif de Recherche " Cellules épithéliales" IFR02, Faculté de Médecine Xavier Bichat, 75870 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Uncoupling proteins (UCP), specific mitochondrial proton transporters that function by uncoupling oxidative metabolism from ATP synthesis, are involved in thermoregulation and control of energy expenditure. The hibernoma-derived T37i cells, which possess functional endogenous mineralocorticoid receptors (MR), can undergo differentiation into brown adipocytes. In differentiated T37i cells, UCP1 mRNA levels increased 10- to 20-fold after retinoic acid or beta -adrenergic treatment. Interestingly, UCP2 and UCP3 mRNA was also detected. Aldosterone treatment induced a drastic decrease in isoproterenol- and retinoic acid-stimulated UCP1 mRNA levels in a time- and dose-dependent manner (IC50approx 1 nM aldosterone). This inhibition was unaffected by cycloheximide and did not modify UCP1 mRNA stability (half-life time = 5 h), indicating that it occurs at the transcriptional level. It involves both the MR and/or the glucocorticoid receptor (GR), depending on the retinoic or catecholamine induction pathway. Basal UCP3 expression was also significantly reduced by aldosterone, whereas UCP2 mRNA levels were not modified. Finally, as demonstrated by JC1 aggregate formation in living cells, aldosterone restored mitochondrial membrane potential abolished by isoproterenol or retinoic acid. Our results demonstrate that MR and GR inhibit expression of UCP1 and UCP3, thus participating in the control of energy expenditure.

aldosterone; mitochondria; steroids; thermogenesis; cell line; uncoupling proteins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

UNCOUPLING PROTEINS (UCP) are mitochondrial proteins that play a critical role in the regulation of thermogenesis and energy expenditure and thus participate in body weight homeostasis (33, 23). Recently, three distinct UCP [UCP1 (5), UCP2 (12), and UCP3 (4, 42)] have been identified and implicated as mediators of adaptive thermogenesis (33, 23). They function as specialized mitochondrial transporters that catalyze the reentry of protons into the mitochondrial matrix, thus bypassing complex V of the respiratory chain (ATP synthase), uncoupling oxidative phosphorylation, and producing heat. Brown adipose tissue (BAT) is now recognized as one of the most important sites for respiratory uncoupling and nonshivering thermogenesis (22). Its physiological importance was demonstrated by animal models in which in vivo BAT ablation resulted in marked obesity (24). BAT expresses all three UCP isoforms, but UCP1 seems to be the major factor responsible for heat generation in this tissue (10, 25).

UCP function in BAT seems to be predominantly regulated at the transcriptional level (36). Multiple hormones and factors have been shown to affect UCP transcription. Most of them, including catecholamines (6, 19), thyroid hormones (7, 14, 35), retinoids (1, 7, 30), fatty acids, insulin, thiazolidinedione (17, 32), and leptin (2, 8, 13), increase UCP1 expression both in vivo and in vitro. In contrast, glucocorticoids have been shown to decrease both thermogenesis and UCP1 gene expression in BAT in vivo (2, 27, 39). However, mechanisms responsible for the inhibition of UCP1 expression in BAT by corticosteroid hormones seem to be rather complex and involve multiple interactions, possibly through an effect on the central nervous system (hypothalamopituitary axis) (40, 46). Although direct action of glucocorticoids on BAT has been postulated (27, 40), analysis of the molecular mechanisms involved in this glucocorticoid-dependent inhibition of UCP1 gene expression has received little attention so far.

Recently, using a targeted oncogenesis strategy, we have been able to show that BAT is a novel target for aldosterone action (47). Hibernoma formation occurred in transgenic animals carrying a hybrid gene consisting of the proximal promoter of the human mineralocorticoid receptor (MR) gene fused to the SV40 large T antigen. A new cell line, T37i, was derived from one hibernoma. As expected from the transgenic approach used, the T37i cells maintain their expression of functional MR. These cells were also able to undergo terminal differentiation upon insulin and triiodothyronine treatment or thiazolidinedione stimulation. Differentiation was accompanied by morphological modifications, as evidenced by accumulation of lipid droplets and mitochondria and by biochemical changes with increased triglyceride content and sequential expression of adipogenic-specific genes (lipoprotein lipase or LPL, peroxisome proliferator-activated receptor or PPARgamma 2, and adipocyte-specific fatty acid binding protein or aP2). We also demonstrated that aldosterone induced T37i cell differentiation and that MR is involved in this process (29). More importantly, differentiated cells maintain their ability to express UCP1.

The aim of the present study was to investigate mineralocorticoid regulation of UCP gene expression in the T37i cellular model. By Northern blot analyses and ribonuclease protection assays, we showed that the three UCP isoforms are expressed in differentiated T37i cells. We also demonstrated that aldosterone inhibits UCP1 and UCP3 gene transcription without modifying UCP2 levels. Transcriptional repression seems to involve both the MR and the glucocorticoid receptor (GR), depending on whether the retinoic acid or adrenergic signaling pathway is implicated. Finally, using JC-1, a fluorescent probe of the mitochondrial membrane potential (37), we showed that aldosterone prevents beta -adrenergic or retinoic acid-induced decreases of the membrane potential.


    MATERIALS AND METHODS
TOP
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 (47). T37i cells were cultured in DMEM-Ham's F-12 (GIBCO-BRL) supplemented with 10% fetal calf serum (GIBCO-BRL; normal or dextran-coated charcoal-treated serum), 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 5-7 days. After this treatment, almost all cells were differentiated, with drastic morphological changes consisting of multiple small intracytoplasmic lipid droplets. All of the experiments were performed with T37i cells between passages 10 and 20.

For UCP expression, cells were incubated with medium supplemented with dextran-coated charcoal-treated serum and with various concentrations of hormones for diverse periods of time (as illustrated in Figs. 1-9). Generally, cells were incubated with 1 µM isoproterenol for 6 h, or with all trans-retinoic acid (RA) or 9-cis-RA overnight. Actinomycin D and cycloheximide were from Sigma.


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Fig. 1.   Aldosterone inhibits isoproterenol-stimulated uncoupling protein (UCP)1 expression at the transcriptional level. Differentiated T37i cells, grown in culture medium supplemented with 10% dextran-coated charcoal-treated serum, were incubated for 6 h with 1 µM isoproterenol (Iso), alone or in the presence of actinomycin D (Actino, 0.4 µM), cycloheximide (Cyclo, 5 µg/ml), aldosterone (Aldo, 100 nM), or a combination of these. Total RNA was extracted and processed for Northern blot analysis by use of specific probes for UCP1 and 28S rRNA. Signals were quantified by InstantImager, and the UCP1-to-28S ratio was calculated. Results are expressed as degree of induction of UCP1 expression and represent means ± SE of 3 independent determinations.



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Fig. 2.   Time-dependent expression of UCP1 and UCP2 in T37i cells in response to isoproterenol and aldosterone. Differentiated T37i cells, grown in culture medium supplemented with 10% charcoal-treated serum, were incubated for 1, 2, 4, or 6 h with 1 µM isoproterenol alone or in the presence of aldosterone (100 nM). Total RNA was extracted and processed for Northern blot analysis by use of specific probes for UCP1, UCP2, and glyceraldehyde-3-phosphate dehydrogenase (GADPH). Signals were quantified by InstantImager, and the UCP1-to-GADPH ratio (UCP1/GADPH) or UCP2/GAPDH was calculated. Quantitative results are presented only for UCP1 and are expressed as degree of induction of UCP1 expression. They represent means ± SE of 3 independent determinations.



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Fig. 3.   Retinoic acid stimulated UCP1 but not UCP2 expression. Differentiated T37i cells, grown in culture medium supplemented with 10% charcoal-treated serum, were incubated for 24 h with 10-7 or 10-6 M all-trans-retinoic acid (RA) or 9-cis-RA. Total RNA was extracted and processed for Northern blot analysis by use of specific probes for UCP1, UCP2, and GADPH. Signals were quantified by InstantImager, and UCP1/GADPH and UCP2/GAPDH were calculated. Quantitative results are presented only for UCP1 and are expressed as degree of induction of UCP1 expression. They represent means ± SE of >= 3 independent determinations.



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Fig. 4.   Aldosterone inhibits UCP1 transcription. Differentiated T37i cells were incubated overnight with 100 nM RA alone or in the presence of increasing concentrations of aldosterone, as indicated. Total RNA was extracted and processed for Northern blot analysis by use of specific probes for UCP1, UCP2, and 28S rRNA. Signals were quantified by InstantImager, and UCP1/28S or UCP2/28S was calculated. Quantitative results are presented only for UCP1 and are expressed as %maximum UCP1 expression. They represent means ± SE of >= 4 independent experiments.



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Fig. 5.   Effect of aldosterone on UCP1 mRNA stability. Differentiated T37i cells were incubated overnight with 100 nM 9-cis-RA to stimulate UCP1 expression. At time 0, actinomycin D (0.4 µM) was added to the culture medium alone () or in the presence of 100 nM aldosterone (). Total RNA was extracted at various periods of time, as indicated, and processed for Northern blot analysis with specific probes for UCP1 and 28S rRNA. Signals were quantified by InstantImager and expressed relative to UCP1 expression. The linear regression slopes between control and aldosterone-treated conditions were calculated using the GraphPad Prism program. Equations were, for control, y = -0.2507x + 2.57, r2 = 0.8925, and for aldosterone, y = -0.3149x + 3.022, r2 = 0.9183. The estimated half-life time (t1/2) values for UCP1 transcripts were not significantly different (control = 287 ± 45 min, aldosterone = 307 ± 69 min).



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Fig. 6.   Mineralocorticoid specificity of inhibitory effects of aldosterone on UCP1 expression. Top: Northern blot analysis of UCP1 expression. Differentiated T37i cells were incubated for 6 h with 1 µM isoproterenol (A) or for 24 h with 100 nM all-trans- RA (B) in the absence or presence of aldosterone (100 nM), RU-38486 (1 µM), or in combination. Total RNA was extracted and processed for Northern blot analysis with specific probes for UCP1 and GAPDH. Bottom: signals were quantified by InstantImager, and the UCP1/GAPDH ratio was calculated. Results, expressed as a percentage of UCP1 expression, are means ± SE of 3 different experiments. *P < 0.05 and **P < 0.01 vs. control conditions.



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Fig. 7.   Expression and hormonal regulation of UCP3 in T37i cells. Total RNA was extracted from T37i cells and analyzed by ribonuclease protection assays by use of antisense probes for UCP3 and GAPDH. A: lane 1, undifferentiated cells; lane 2, differentiated cells; lane 3, hibernoma. B: differentiated T37i cells (lane 1) were incubated overnight with 100 nM triiodothyronine (T3, lane 2), 100 nM RA (lane 3), 1 µM isoproterenol (lane 4), or 100 nM aldosterone alone (lane 5) or in combination with 1 µM RU-38486 (lane 6). C: signals corresponding to the long protected fragment (UCP3-L) and the short fragment (UCP3-S) were quantified by InstantImager and normalized to the GADPH signal. Results, expressed as a percentage of UCP3 expression under basal conditions, are means ± SE of 3 independent determinations. *P < 0.05 and **0.01 vs. control conditions.



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Fig. 8.   Uncoupling mitochondrial activity induced by isoproterenol is abolished by aldosterone treatment. Localization of JC-1 dye epifluorescence in differentiated T37i cells. A, C, and E: green fluorescence (527 nm) under blue excitation (485 nm) corresponds to JC-1 monomer fluorescence and is an index of fluorochrome loading into the cells. B, D, and F: red fluorescence (597 nm) under green excitation (546 nm) corresponds to fluorescence of intramitochondrial JC-1 J aggregates. Their presence indicates a high Delta Psi mitochondrial membrane potential, i.e., a functional coupled mitochondrial activity. A and B: untreated cells; C and D: 1 µM isoproterenol; E and F: 1 µM isoproterenol + 100 nM aldosterone. See details in MATERIALS AND METHODS. Note that the large differentiated T37i cells in D, which express UCP1 upon isoproterenol stimulation, do not exhibit any red-stained mitochondria. In contrast, aldosterone allows maintenance of highly coupled mitochondrial potential, as illustrated by multiple red JC-1 J aggregates within the isoproterenol-stimulated differentiated T37i cells (F).



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Fig. 9.   Schematic representation of signaling pathways for beta -adrenergic and RA activation of UCP1 expression in T37i cells. Catecholamines bind to beta 3-adrenergic receptors (beta 3-AR) and increase intracellular cAMP concentrations, followed by activation of protein kinase A (PKA) cascade. Phosphorylated CREB leads to stimulated UCP1 transcription. c-Jun binds to the proximal cAMP response element (-139/-122) and represses UCP1 expression. The glucocorticoid receptor (GR), by interacting with c-Jun, could inhibit catecholamine-induced UCP1 expression. RA via a retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimer stimulates UCP1 transcription. The mineralocorticoid receptor (MR) and/or GR inhibits the RXR/RAR activation on UCP1 expression.

RNA analysis. Total RNA was isolated from undifferentiated or differentiated T37i cells using Trizol (GIBCO-BRL/Life Technologies) reagent 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 (Megaprime, Amersham) of cDNAs for rat UCP1, rat UCP2, rat glyceraldehyde-3-phosphate dehydrogenase (GADPH), and mouse 28S RNA kindly provided by Dr. Ricquier (CEREMOD, Meudon, France), Dr. Escoubet (INSERM U426, France), and Dr. Thenet (INSERM U 505, France), respectively. Serial hybridizations with different probes were performed. Membranes were subjected to autoradiography, and specific hybridization signals were quantified by measuring radioactivity with a Packard InstantImager. All results are expressed in arbitrary units and correspond to the ratios of UCP specific counts normalized by gene control (28S or GAPDH) signal.

UCP1 mRNA stability was measured in differentiated T37i cells after exposure to RA for 16 h to induce UCP1 expression. Actinomycin D (400 nM) was added to the culture in the presence or absence of aldosterone, and cells were harvested at various periods of time. UCP1 expression was then assessed by Northern blotting.

Analysis of UCP3 mRNA transcripts by ribonuclease protection assay. Total RNA was isolated from cells with the Trizol reagent (GIBCO-BRL). For ribonuclease protection assay (RPA), a Bluescript (KS) plasmid containing the entire cDNA of the mouse UCP3 gene (kindly provided by Dr. D. Ricquier) was linearized by NdeI and alpha -32P-labeled antisense riboprobes synthesized with T7 RNA polymerase. The rat GAPDH probe, used as an internal control, was synthesized with T7 RNA polymerase after digestion of a Bluescript-SK-GAPDH plasmid by PvuII and StyI.

RPA were performed as described previously (16). Briefly, 50 µg of total RNA were hybridized overnight at 50°C with a 4 × 105 counts/min (cpm) UCP3 probe and a 5 × 104 cpm GADPH probe. Digestion with RNAse A and T1 was performed at 30°C for 1 h followed by a proteinase K-SDS treatment at 37°C for 30 min. After phenol-chloroform extraction and ethanol precipitation, protected fragments were electrophoresed on a 6% polyacrylamide-urea gel.

Unprotected GAPDH riboprobe migrated at 184 bases with a protected fragment migrating at 164 bases. Unprotected UCP3 riboprobe migrated at 462 bases. In our experimental conditions, we were able to detect two bands, which corresponded to a long UCP3 protected fragment (UCP3L) migrating at 322 bases and a short UCP3 protected fragment (UCP3S) migrating at 232 bases. These protected fragments might represent variants of the mouse UCP3 gene.

JC-1 analysis for determination of mitochondrial membrane potential. Differentiated T37i cells were cultured on circular glass coverslips overnight in fresh medium without insulin and T3. The next day, cells were pretreated with 100 nM aldosterone in medium containing insulin and T3 for 8 h followed by an overnight incubation with 1 µM isoproterenol or 100 nM all-trans-RA alone or in the presence of 100 nM aldosterone. At the end of the incubation, cells were rinsed twice with prewarmed PBS and incubated with 5 or 10 µg/ml JC-1 fluorochrome (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolocarbocyanine iodide, Molecular Probe, Interchim, Montlucon, France) in DMEM without serum for 20 min at 37°C. JC-1 stock solution, at a concentration of 1 mg/ml, was prepared in DMSO and kept in darkness at 4°C. Cells were then rinsed twice with prewarmed PBS and mounted and kept at 37°C. Epifluorescence was examined using a Zeiss Axioplan microscope equipped with a 100-W mercury lamp. For visualization of the green fluorescence of JC-1 monomers, a 450- to 490-nm exciter associated with an FT 510-nm dichroic mirror and an LP 520-nm barrier was used. The red fluorescence of JC-1 J aggregates was visualized using a BP 546-nm exciter, an FT 580-nm dichroic mirror, and an LP 590-nm barrier.

Statistical analysis. Student's t-tests were performed to analyze the data by use of the computer software InStat version 2.01 for Macintosh (GraphPad Software, San Diego, CA). Values were considered significantly different at P < 0.05. For measurement of UCP1 mRNA stability, linear regression slopes were calculated using a GraphPad Prism program.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Aldosterone inhibits UCP1 expression in T37i cells. As presented in Fig. 1, differentiated T37i cells under standard conditions do not express UCP1. Upon isoproterenol stimulation, a 15- to 20-fold increase in UCP1 expression was observed that was almost totally inhibited in the presence of actinomycin D, consistent with a transcriptional regulation. Simultaneous treatment of T37i cells with isoproterenol and aldosterone for 6 h induced a 50% inhibition of UCP1 expression. This effect was unaffected by cycloheximide treatment, indicating that inhibitory effects of aldosterone on beta -adrenergic stimulation of UCP1 expression do not require de novo protein synthesis.

We next examined UCP1 and UCP2 mRNA levels in T37i cells treated with isoproterenol alone or in the presence of aldosterone as a function of time. As shown in Fig. 2, a time-dependent increase of UCP1 expression was observed after isoproterenol stimulation. However, the inhibitory effects of aldosterone on UCP1 expression were observed as early as 1 h after stimulation, reaching a maximum inhibition after 6 h. It is worth noting that differentiated T37i cells express substantial amounts of UCP2 under basal conditions, which remain unchanged upon isoproterenol treatment (quantitative analysis not shown), as previously described in other animals or cellular models (10, 13, 43). Furthermore, UCP2 levels were not modified by aldosterone.

Retinoids are known to be important modulators of UCP1 expression and function in BAT (1, 7, 30). We therefore addressed the question of whether retinoids could modify UCP expression in our cellular model. As shown in Fig. 3, all trans-RA and 9-cis-RA stimulated UCP1 expression in T37i cells by 10- to 20-fold. The stimulatory effects of retinoids were more pronounced at 10-6 M than at 10-7 M but were not significantly enhanced with a combination of the two agents (data not shown). In contrast, UCP2 expression does not seem to be affected by retinoid treatment in T37i cells (quantification not shown). We next examined whether aldosterone could also affect this signal transduction pathway (Fig. 4). Aldosterone inhibited RA stimulation of UCP1. This inhibition was dose dependent, with an inhibitory concentration (IC50) estimated at 1 nM, a finding consistent with the affinity constant of MR for aldosterone. However, the shape of the dose-response curve was rather in favor of a biphasic effect, probably reflecting subsequent activation of MR and GR as a function of aldosterone concentrations. Under the same experimental conditions, UCP2 expression remained unchanged in the presence of increasing concentrations of aldosterone.

Finally, to test whether aldosterone modifies the stability of UCP1 transcripts, T37i cells were first stimulated by RA overnight, and actinomycin D was added at time 0 to the medium in the presence or absence of aldosterone. At various periods of time as indicated in Fig. 5, RNA was extracted and UCP1 mRNA levels were analyzed by Northern blot. In the absence of aldosterone, UCP1 mRNA levels rapidly decreased, with a half-life time (t1/2) calculated at ~5 h. Aldosterone treatment did not modify the t1/2 of UCP1 mRNA, suggesting that aldosterone affects transcriptional rate rather than accelerating degradation of UCP1 transcripts.

Collectively, our results demonstrate that aldosterone inhibits UCP1 transcription without affecting the transcription of UCP2.

Implication of the mineralocorticoid receptor on UCP1 transrepression. To determine whether MR was mediating aldosterone effects, we compared UCP1 mRNA levels after isoproterenol or RA treatment in the presence of 10 or 100 nM aldosterone alone or in combination with a 10- to 100-fold excess of RU-38486, a glucocorticoid antagonist, to prevent aldosterone binding to the glucocorticoid receptor. Figure 6 illustrates Northern blots (top) and quantitative analyses (bottom) expressed as percentages of UCP1 expression induced by isoproterenol (left) or RA exposure (right). After catecholaminergic or RA stimulation, a dose-dependent decrease in UCP1 expression was observed when T37i cells were simultaneously treated with increasing concentrations of aldosterone. However, in the presence of RU-38486, aldosterone, at both 10 and 100 nM concentrations, was no longer able to inhibit isoproterenol-stimulated UCP1 transcription, indicating that aldosterone action was mediated through its interaction with the GR. Consistent with these findings, dexamethasone, a GR agonist, totally blocked isoproterenol-stimulated UCP1 expression (data not shown). In contrast, after RA stimulation, even in the presence of the glucocorticoid antagonist RU-38486, a significant decrease of UCP1 was observed with 10 nM aldosterone. The higher aldosterone concentrations associated with a 10-fold excess of RU-38486 were still capable of reducing RA-stimulated UCP1 expression by 40%. These results demonstrate that inhibition of RA-activated UCP1 expression by aldosterone clearly involves the MR.

Expression of UCP3 in T37i cells. Another member of the UCP family, UCP3, has been identified recently and shown to be present in the skeletal muscle and BAT (4, 42). We investigated whether T37i cells also expressed UCP3. For this purpose, we used the sensitive RPA to examine the presence of UCP3 transcripts. As shown in Fig. 7A, by use of a 462-base long UCP3 riboprobe, two protected fragments of ~332 and 232 bases were observed in differentiated T37i cells (lane 2) as well as in a hibernoma, a malignant brown fat tumor (lane 3), whereas no specific signal could be detected in undifferentiated T37i cells (lane 1). These two protected fragments could correspond to different isoforms of mouse UCP3 transcripts. However, these fragments seem to be different from human short- and long-form UCP3 transcripts generated by utilization of distinct polyadenylation signal cleavage in intron 6 and exon 7 (38). Indeed, the mouse riboprobe used for the RPA corresponded to the 3' region of mouse UCP3 cDNA and contained part of the 3' untranslated sequence, which is highly homologous to the human exon 7, i.e., downstream of the putative by-passed signal. Nevertheless, our results demonstrate that UCP3 expression is associated with the differentiation state of brown adipocytes.

Hormonal regulation of UCP3 transcription was analyzed by RPA. UCP3 isoforms were quantified after overnight incubation of differentiated T37i cells with thyroid hormones, RA, beta -adrenergic agonists, and aldosterone, alone or in the presence of RU-38486. Figure 7, B and C, shows that T3 significantly increased both UCP3S and UCP3L isoform expression, whereas RA was ineffective. Interestingly, UCP3 expression was almost totally abolished by isoproterenol treatment and to a lesser extent by aldosterone. The aldosterone-induced inhibition of UCP3 expression was prevented by addition of RU-38486 and was therefore likely to be mediated by the GR. Along this line, dexamethasone was also able to inhibit UCP3 expression in these cells (data not shown). Altogether, both short and long UCP3 isoforms are expressed in differentiated T37i cells and submitted to regulation by a large variety of hormones.

Influence of aldosterone on mitochondrial membrane potential of T37i cells. Aldosterone was shown to inhibit transcriptional activation of both UCP1 and UCP3 expression. In a functional assay, we asked whether aldosterone treatment could affect the mitochondrial membrane potential Delta psi by modulating expression of UCP. For this purpose, we used the fluorochrome JC-1 as an index of the electrochemical gradient across the mitochondrial inner membrane of T37i cells submitted to various experimental conditions. As illustrated in Fig. 8, A, C, and E, the green fluorescence emitted by JC-1 monomers corresponds to the dye loading in cells. In nonstimulated differentiated T37i, which do not express UCP1, multiple red fluorescent JC-1 J aggregates were observed, consistent with a positive high Delta Psi mitochondrial membrane potential (Fig. 8B). In contrast, an overnight incubation with 1 µM isoproterenol, a condition known to drastically stimulate UCP1 expression at both mRNA and protein levels in differentiated T37i cells, totally abolished intramitochondrial red JC-1 J aggregates, thus reflecting the decrease of the mitochondrial membrane potential (Fig. 8D). Note that some undifferentiated T37i cells present at the bottom of Fig. 8D (characterized by their fibroblast-like appearance and the small size of the cytoplasmic compartment without lipid droplets) presented a few red-stained mitochondria coupled with high mitochondrial activity, due to the lack of expression of UCP1 in these undifferentiated cells. Interestingly, co-incubation with aldosterone and isoproterenol resulted in the persistence of red JC-1 J aggregates in differentiated cells (Fig. 8F). These results indicate that, under specific experimental conditions, aldosterone is able to prevent the mitochondrial depolarization (or deenergization) by impairing uncoupling functions of proton translocators in brown adipocytes.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Aldosterone, the principal mineralocorticoid hormone in humans, has been until recently associated prevalently with the control of vectorial ion transport in tight epithelia, such as the distal part of the nephron (3, 41). It is considered to be one of the major regulators of water and electrolyte balance, thus participating in the control of blood pressure. Its effects are mediated through the MR, which is a member of the nuclear receptor superfamily, and it acts as a ligand-dependent transcription factor. However, demonstration of the presence of MR in a large variety of tissues, such as the central nervous (9) and cardiovascular systems (21), and in various reproductive organs (uterus, epidydimus, testis, and placenta), strongly suggests that aldosterone might play a role in other important physiological functions. Indeed, aldosterone has been shown to influence behavioral adaptation, memorization, and the stress-response system (9) and to induce cardiac and vascular fibrosis independently of its pressure effect (34, 44). More importantly, we have recently demonstrated that MR is also expressed in brown fat (47) and that aldosterone is able to accelerate differentiation of preadipocytes into mature brown adipocytes (29).

In the present paper, we further analyze aldosterone effects on brown adipocyte physiology by use of the recently established T37i cell line (47). These cells represent an interesting model, because they exhibit all differentiated features of mature brown adipocytes, together with a functional MR signaling system. In addition to the inducible expression of UCP1, differentiated T37i cells also constitutively express the UCPs UCP2 and UCP3, although UCP3 levels are lower than those of the other homologues. Thus the relative abundance of UCPs detected in our cellular model is in accordance with that observed in normal BAT. We demonstrated that aldosterone inhibits both RA and isoproterenol-induced UCP1 expression, in a dose-dependent manner and as rapidly as 1 h after hormone treatment. This inhibitory effect seems to occur directly at the transcriptional level, because it is not affected by addition of the protein synthesis inhibitor cycloheximide and does not involve any modification of mRNA stability. Importantly, inhibition was confirmed by functional studies in living cells, showing that aldosterone relieves the uncoupling effects of catecholamines and RA on mitochondrial inner membrane potential. Moreover, aldosterone also represses basal UCP3 expression to a degree comparable with that obtained after catecholamine treatment, but it does not affect UCP2 levels. It is worth noting that, in T37i cells, beta -adrenergic agonists differentially regulate expression of UCP1 and UCP3. This is in accordance with recent data showing that UCP1 is the only physiological UCP for adrenergically induced thermogenesis (26).

Aldosterone effects on UCP1 expression in T37i cells appear to be mediated through both MR and GR, depending on the initial stimulatory pathway. In particular, inhibition of the RA-induced expression of UCP1 by aldosterone is mediated via its interaction with MR, because at low hormonal concentration it is not modified in the presence of RU-38486, a glucocorticoid antagonist. At high doses, aldosterone also binds to GR, and the inhibitory effect is only partially reversed by the addition of RU-38486. In contrast, this compound totally prevents aldosterone effects on UCP1 induction by catecholamines, suggesting the exclusive involvement of GR (Fig. 6). Indeed, in cellular models, both mineralo- and glucocorticoids can bind to both the MR and GR. In vivo, several mechanisms of hormonal selectivity allow aldosterone to bind specifically to the MR despite much higher circulating levels of glucocorticoids. The most important one is the enzyme 11beta -hydroxysteroid dehydrogenase type 2 (11HSD2), expressed in polarized epithelial cells, which converts glucocorticoid hormones into inactive 11-dehydro-compounds (11). In differentiated T37i cells, a NAD-dependent conversion of corticosterone has been detected [20% conversion of corticosterone (B) to 11-dehydrocorticosterone (A) over 60 min or 50 fmol (A)/2 × 105 cells]. This catalytic activity is lower than that found in classical epithelial target tissues. Further experiments are required to determine whether this enzymatic activity corresponds to 11HSD2.

Molecular mechanisms possibly involved in UCP1 transrepression by MR or GR are schematized in Fig. 9. Catecholamines are known to stimulate UCP1 expression through their interaction with beta 3-adrenergic receptors and activation of the protein kinase A (PKA) cascade. The final event in this pathway is the phosphorylation of CREB, which can bind to various potential cAMP response elements (CRE) located on the UCP1 gene promoter (6, 19, 32). Two major regions seem to be involved in cAMP stimulation of UCP1 expression, an upstream UCP enhancer located at -2.4 kb in the rat and mouse gene (6, 19) and a downstream sequence at approximately -100 b. Furthermore, it has been hypothesized that activated CREB directly induces expression of the transcriptional coactivator PGC-1 (23), which is essential for coactivation of transcription factors assembled on the UCP1 enhancer (31). Recently, c-Jun was shown to negatively regulate both basal and PKA-induced transcription of UCP1 in differentiated brown adipocytes (45). Given the known modulatory effects of GR on c-jun-mediated transcriptional activity, which depends on the cellular and/or promoter context (15), it is possible that GR represses UCP1 transcription by interacting with the c-jun-mediated pathway in T37i cells. Interestingly, MR is unable to inhibit AP-1-mediated transactivation (15, 28), consistent with the fact that MR has no effect on catecholamine induction of UCP1. Finally, the possibility that negative glucocorticoid-responsive elements exist on the UCP1 regulatory sequences cannot be excluded.

On the other hand, RA has been shown to stimulate UCP1 gene expression to levels as high as those elicited by norepinephrine in primary brown adipocytes (1, 7). RAR/RXR heterodimers are involved in RA-stimulated UCP1 expression through interaction with three potential RARE half-sites (located at -2357/2330 of the rat UCP1 gene) (20). It is possible that MR interferes with RAR/RXR transactivation by squelching of transcriptional coactivators or other factors necessary for full transcriptional activity. In particular, PGC-1, a coactivator of nuclear receptors highly expressed in BAT, binds to several members of the nuclear receptors including PPARgamma , RAR, and TR and greatly increases their transcriptional activities (31). In this context, it has recently been shown that PGC-1 also interacts with MR and GR (18).

One interesting finding is the recoupling effect of aldosterone on mitochondrial inner membrane potential in living cells. Not only does this observation confirm data on UCP1 repression, but it also underscores the importance of this UCP in the regulation of the mitochondrial electrochemical gradient, compared with the other UCP homologues. This is in accord with studies on bioenergetics of brown fat mitochondria from UCP1 knock-out mice, which suggest that although UCP1 clearly has a thermogenic function, UCP2 and UCP3 are more likely involved in fatty acid metabolism (24).

In conclusion, our data show that aldosterone inhibits expression and function of UCP1 and UCP3, but not of UCP2, in T37i cells via its interaction with MR and GR. In light of previous studies showing that aldosterone is able to induce T37i cell differentiation (29), we propose a model in which MR is involved in early differentiation events, allowing preadipocytes to enter into the brown adipocyte differentiation program. On the other hand, mineralocorticoids inhibit UCP1 expression, preventing the metabolic switch of thermogenic cells toward heat production. This ultimately leads to promotion of intracellular triglyceride accumulation rather than an increase in energy expenditure.

Although regulation of thermogenesis and lipid storage in rodents may not be completely superimposable over that in humans, it is tempting to speculate that in addition to the detrimental consequences of aldosterone excess on blood pressure, its effect on energy expenditure and lipid storage might represent an additional cardiovascular risk factor. Our findings open potential pharmacological perspectives, in that blockade of the mineralocorticoid and/or glucocorticoid signaling pathway could not only relieve the permanent inhibition on UCP1 expression but also facilitate action of other thermogenic compounds, such as beta -adrenergic agonists or thiazolidinediones.


    ACKNOWLEDGEMENTS

We thank Dr. D. Ricquier (Ceremod, Meudon, France) for providing us with UCP cDNAs and for very helpful discussions and support during this work. We also thank Dr. A. Lombès (INSERM U523) for the gift of the JC-1 compound and for useful advice. The assistance of J. Grellier, G. Delrue, and J. P. Laigneau for illustrations is also gratefully acknowledged.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Lombès, INSERM U 478, Institut Fédératif de Recherche "Cellules épithéliales" IFR02, 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. Section 1734 solely to indicate this fact.

Received 13 July 2000; accepted in final form 15 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 280(4):E640-E649
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