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 |
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
-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 (IC50
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
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INTRODUCTION |
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 PPAR
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
-adrenergic or retinoic acid-induced decreases
of the membrane potential.
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MATERIALS AND METHODS |
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  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 -adrenergic
and RA activation of UCP1 expression in T37i cells. Catecholamines bind
to 3-adrenergic receptors ( 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.
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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.
-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
-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.
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RESULTS |
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
-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,
-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

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 
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.
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DISCUSSION |
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,
-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
11
-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
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 PPAR
,
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
-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.
 |
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