From the Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Avda Diagonal 645, 08028 Barcelona, Spain
Received for publication, July 14, 2000, and in revised form, October 3, 2000
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ABSTRACT |
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High expression of the peroxisome
proliferator-activated receptor The peroxisome proliferator-activated receptor BAT is a major site for nonshivering thermogenesis in mammals. Its
thermogenic capacity relies on the presence of an inner mitochondrial
protein uniquely expressed in brown adipocytes, the uncoupling protein
(UCP) (15), now referred to as UCP-1 since the discovery of the more
widely expressed UCP-2 and UCP-3 (for review, see Ref. 16). Brown fat
thermogenesis is mainly controlled by norepinephrine released from
sympathetic terminals innervating the tissue, although nuclear
receptor-mediated pathways have also been described. Thus, activation
of PPAR BAT highly coexpresses not only PPAR Materials--
Wy 14,643 (pirixinic acid) and
15-deoxy- Cell Culture--
Primary culture of differentiated brown
adipocytes was performed as described previously (19), and grown in 5 ml of Dulbecco's modified Eagle's medium-Ham's F-12 medium (1:1)
supplemented with 10% fetal calf serum, 20 nM insulin, 2 nM 3,5,3'-triiodothyronine, and 100 µM
ascorbate. Experiments were performed on day 9 of culture when 80-90%
of the cells were considered to be differentiated on the basis of lipid
accumulation and acquisition of brown adipocyte morphology. Brown
adipocytes were exposed to 10 µM Wy 14,643 for 24 h,
or at the concentrations and times indicated in the experiments. Cells
were also exposed to various PPAR agonists for 24 h, except for
15-deoxy-
HepG2 human hepatoma cells were grown in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum. The HIB-1B brown adipocyte
cell line, kindly provided by Dr. B. Spiegelman, was cultured in
Dulbecco's modified Eagle's medium/F-12 (1:1) supplemented with 10%
heat-inactivated fetal calf serum and 4 mg/liter biotin.
RNA Isolation and Northern Blot Analysis--
Total RNA was
extracted using the RNeasy Mini Kit (Quiagen). Northern blot
analysis and hybridization were carried out as described (24). Blots
were hybridized using as probes the full-length cDNA for rat UCP-1
(25) and 0.5 kb of the cDNA for mouse mitochondrial-genome-encoded cytochrome oxidase subunit II (COII) (26), which was used as a control.
Hybridization signals were quantified using Molecular Image System
GS-525 (Bio-Rad). Statistical analysis was performed by Student's
t test.
Oligonucleotides and Plasmids--
Oligonucleotides were
chemically synthesized by Roche Diagnostics. The UCP1-PPRE
double-stranded oligonucleotide corresponds to positions
The plasmid (
Mammalian expression vectors that contain the murine cDNAs of the
respective PPAR isoforms are driven by the simian virus-40 promoter (1,
4, 5). pRSV-RXR Transfection Assays--
Murine primary brown adipocytes
differentiated in culture were transiently transfected by the calcium
phosphate precipitation method on day 9 of culture (22). Each
transfection contained 12 µg of (
HepG2 and HIB-1B cells were transfected using the FuGENE 6 Transfection
Reagent (Roche Molecular Biochemicals) for 16 h and cells were
harvested 24 h later. Unless otherwise indicated, each transfection contained between 0.5 and 1 µg of UCP1-CAT vector, 0.1 µg of cytomegalovirus-
Analysis of CAT activity was determined by thin layer chromatography
(22) and quantified by radioactivity counting (AMBIS). The amount of
cell extract used was adjusted to maintain a percentage conversion of
chloramphenicol between 1 and 20%. The CAT activity was normalized for
variation in transfection efficiency using the DNA Binding Experiments--
Nuclear proteins were isolated from
rat BAT or differentiated primary brown adipocytes as described
elsewhere (21, 22). cDNAs for mPPAR
For the gel retardation assays, the UCP1-PPRE oligonucleotide
(10,000-20,000 cpm) was incubated for 30 min at 25 °C with 5 µg
of nuclear protein extract from BAT or differentiated brown adipocytes,
or 5 µl of in vitro transcribed/translated proteins. Reactions were carried out in a final volume of 20 µl containing 10 mM Tris-HCl (pH 8.0), 0.05% Nonidet P-40, 1 mM
dithiothreitol, 40 mM KCl, 6% glycerol, and 2 µg of
poly(dI)·(dC). Samples were analyzed by electrophoresis at 4 °C
for 60-80 min in nondenaturing 5% polyacrylamide gels in 0.5 × TBE (44.5 mM Tris, 44.5 mM borate, 1 mM EDTA). In the competition experiments, 100-fold molar
excess of unlabeled oligonucleotide was included in each respective
binding reaction. When indicated, 1 µl of antiserum against PPAR Tissue Samples--
BAT was extracted from two-month-old female,
15-day lactating, or newborn Swiss mice. Adult mice were treated with a
single intraperitoneal injection of Wy 14,643 (50 µg/g body weight)
or troglitazone (100 µg/g body weight) in 50% dimethyl
sulfoxide/saline. Controls were given equivalent volumes of the vehicle
and mice were studied 6 h after injections. Neonates were placed
in a humidified thermostated chamber at 28 °C, and injected
intraperitoneally 2 h after birth with Wy 14,643 (50 µg/g body
weight), BRL 49653 (50 µg/g body weight), or equivalent volumes of
the 20% dimethyl sulfoxide/saline vehicle solution. Pups were studied
15 h after treatment.
Activators of PPAR
Exposure to Wy 14,643 led to a dose-dependent increase in
UCP-1 mRNA expression (Fig.
2A) and maximum induction was
attained at 10 µM, a concentration at which it
selectively activates PPAR The Stimulation of ucp-1 Gene Expression by the PPAR
When the effects of the RXR-specific agonist methoprene were analyzed
(Fig. 3B), results showed that besides its reported direct
action upon UCP-1 mRNA expression (20), there was a synergistic effect when both the PPAR PPAR RXR CBP and PGC-1 Coactivate the PPAR PPAR The The PPAR
When newborn mice at thermoneutrality were analyzed, injection of pups
with Wy 14,643 caused a significant 3-fold rise in UCP-1 mRNA
levels whereas injection of the PPAR Here we have established that PPAR By deletion and mutation analysis we have identified the
PPAR Other PPAR target genes have been described to be induced by both
PPAR Here we also demonstrate that in vivo activation of PPAR Activation of BAT thermogenesis has been classically recognized to be
mediated by norepinephrine. Among other regulatory effects, there is a
cAMP-dependent activation of hormone sensitive-lipase, which rapidly hydrolyzes the stored triglycerides and releases high
concentrations of fatty acids. These fatty acids, in addition to be the
major substrate for thermogenesis and the inducers of UCP-1 uncoupling
activity, may also act as PPAR activators. Accordingly, cold exposure
and In conclusion, PPAR (PPAR
) differentiates brown fat
from white, and is related to its high capacity of lipid oxidation. We
analyzed the effects of PPAR
activation on expression of the brown
fat-specific uncoupling protein-1 (ucp-1) gene. Activators
of PPAR
increased UCP-1 mRNA levels severalfold both in primary
brown adipocytes and in brown fat in vivo. Transient
transfection assays indicated that the (
4551)UCP1-CAT construct,
containing the 5'-regulatory region of the rat ucp-1 gene,
was activated by PPAR
co-transfection in a
dose-dependent manner and this activation was potentiated by Wy 14,643 and retinoid X receptor
. The coactivators CBP
and PPAR
-coactivator-1 (PGC-1), which is highly expressed in brown fat, also enhanced the PPAR
-dependent regulation of the
ucp-1 gene. Deletion and point-mutation mapping analysis
indicated that the PPAR
-responsive element was located in the
upstream enhancer region of the ucp-1 gene. This
2485/
2458 element bound PPAR
and PPAR
from brown fat nuclei.
Moreover, this element behaved as a promiscuous responsive site to
either PPAR
or PPAR
activation, and we propose that it mediates
ucp-1 gene up-regulation associated with adipogenic
differentiation (via PPAR
) or in coordination with gene expression
for the fatty acid oxidation machinery required for active
thermogenesis (via PPAR
).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(PPAR
)1 is a fatty
acid-activated transcription factor that plays a key role in the
transcriptional regulation of genes involved in cellular lipid
metabolism (1). PPAR
together with PPAR
and PPAR
/
belong to
a subgroup of the nuclear hormone receptor superfamily that
heterodimerizes with the 9-cis-retinoic acid receptors
(RXRs) (2-5). The PPAR-RXR heterodimer binds to specific response
elements (PPREs), which consist of a direct repeat of the consensus
half-site motif spaced by one nucleotide (DR-1) (6). Fatty acids,
peroxisome proliferators, and fibrate hypolipidemic drugs can activate
PPAR
(1, 4), and natural (leukotrine B4) or synthetic
(fibrate Wy 14,643) specific ligands for PPAR
have been identified
(7). In contrast, 15-deoxy-
12,14-prostaglandin
J2 and thiazolidinedione antidiabetic agents are selective
ligands for PPAR
(8-10). In addition to ligand selectivity, PPAR
subtypes have been involved in different biological functions. PPAR
is mostly expressed in tissues with high rates of fatty acid oxidation
and peroxisomal metabolism, such as brown fat, liver, or heart (1, 11).
Recent studies of PPAR
-null mice have confirmed that PPAR
is
necessary in vivo for hepatic fatty acid oxidation and
ketone body synthesis during starvation (12). PPAR
, which is
ubiquitously expressed, seems to be involved in basic lipid metabolism
(11). High expression of PPAR
is mainly restricted to white (WAT)
and brown (BAT) adipose tissue (13). Hence, in contrast to the role of
PPAR
in cellular lipid catabolism, PPAR
regulates adipogenesis
(i.e. lipid deposition) (13, 14).
promotes HIB-1B brown adipocyte differentiation (17), and
up-regulates ucp-1 gene expression (18). Furthermore, we
demonstrated that retinoic acid is a powerful inducer of
ucp-1 gene transcription, acting through retinoic acid
receptors and RXRs (19, 20). The 5'-flanking region of the rat
ucp-1 gene contains the proximal regulatory promoter,
including C/EBP-regulated sites (21) and the main cAMP-regulatory
element (22), and an upstream enhancer involved in complex regulation
by retinoic acid receptors, RXR, and thyroid hormone nuclear receptors
(19, 20, 23). A site responsive to PPAR
activators has also been
located in the upstream enhancer of the murine ucp-1 gene
(18).
and PPAR
subtypes but also
PPAR
(24). BAT stores triglycerides but, in contrast to WAT, it uses
lipids as oxidative substrates to generate heat. Since PPAR
induces
the expression of fatty acid oxidation enzymes in tissues other than
BAT (6), it may do so in BAT in association with thermogenic
requirements. Here we report that PPAR
activators induce
ucp-1 gene expression in brown adipocytes and in BAT
in vivo, acting through a PPRE located in the upstream
enhancer of the ucp-1 gene that is also responsible for
PPAR
-dependent regulation. PPAR
is proposed to
coordinate the activation of lipid oxidation and thermogenic activity
in brown fat.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12,14-prostaglandin J2 were
obtained from Cayman Chemicals. Troglitazone and BRL 49653 were kind
gifts from Dr. T. Leff (Parke-Davis Research) and Dr. L. Casteilla
(Toulouse, France), respectively. Clofibrate, fenofibrate, bezafibrate,
Ly171883, 3,5,3'-triiodothyronine, insulin, norepinephrine, and
8-bromo-cAMP were obtained from Sigma. Methoprene was from Promochem.
12,14-prostaglandin J2 which was
added at a final concentration of 10 µM for 6 h. As
indicated, cycloheximide (Sigma) was used at a dose of 5 µg/ml as
reported (19).
2485 to
2458 of the rat ucp-1 gene and its sequence is
5'-GTGGGTCAGTCACCCTTGATCACACTGC-3', flanked by
HindIII/XbaI ends. The mutated version
mutUCP1-PPRE corresponds to the sequence 5'-AGTCACAATTGATCACACTGC-3' also flanked by
HindIII/XbaI ends.
4551)UCP1-CAT, in which the region
4551 to +110 of the
rat ucp-1 gene drives the promoterless chloramphenicol acetyltransferase (CAT) gene, was a kind gift from Dr. D. Ricquier (27). The derived plasmids containing 5'-deletion mutations (
3628)UCP1-CAT and (
141)UCP1-CAT, an internal deletion mutation (
-2469/
2283)UCP1-CAT, or the plasmid (
3628/
2283)UCP1-CAT
corresponding to the fragment
3628/
2283 linked to (
141)UCP1-CAT,
have been described previously (19). The plasmid
(
2767/
2283)UCP1-CAT was obtained by digesting the plasmid
(
3628/
2283)UCP1-CAT with SpeI. The plasmid
(
2534/
2283) was constructed by polymerase chain reaction using an
oligonucleotide corresponding to the
2534/
2522 fragment
(5'-ACATGGGCGGCGAG-3') and (
3628/
2283)UCP1-CAT as template. The plasmids (
172)UCP1-CAT and (
2494/
2318)UCP1-CAT, in which the
fragment
2494/
2318 was placed upstream in (
172)UCP1-CAT, are
described elsewhere (20). The mutated versions of
(
3628/
2283)UCP1-CAT and (
2494/
2318)UCP1-CAT, containing AA
instead of CC at sites
2473 and
2472, were generated using the
Quikchange site-directed mutagenesis kit (Stratagene).
was an expression vector for the
-subtype of
human RXR (28). Expression plasmids driving murine RXR isoforms were
kindly provided by Dr. P. Chambon. The expression vectors for human
CBP, pCMX-CBP (29), and murine PGC-1, pSV-PGC1 (30), are described elsewhere.
4551)UCP1-CAT and included or not
3 µg of the expression vector pSG5-PPAR
. When indicated 10 µM Wy 14,643, 10 µM BRL 49653, or 30 µM Ly171883 was added after transfection. 1 µg of
cytomegalovirus-
-galactosidase was also included to assess
the efficiency of separate transfections. The cells were incubated for
24 h and, for each condition, at least three plates were pooled.
-galactosidase, and included or not 0.3 µg of pSG5-PPAR
or pSG5-PPAR
expression vector, and/or 0.1 µg of pRSV-RXR
. When indicated, 0.1 µg of the expression vector pCMX-CBP or pSV-PGC1 was added.
-galactosidase
activity measured for each sample as a standard.
, mPPAR
, and hRXR
were
transcribed and translated in vitro by using the TNT Quick
Coupled Transcription/translation Systems (Promega) according to the
manufacturer's instructions.
(N-19), PPAR
(N-20), RXR
(D-20), or ETS (C-275) from
Santa Cruz was used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Induce the Expression of the ucp-1 Gene in
Differentiated Brown Adipocytes--
To analyze whether PPAR
agonists modulate the expression of the ucp-1 gene, primary
cultures of murine brown adipocytes were used since they express all
three PPAR subtypes (24). As shown in Fig.
1, exposure of brown adipocytes
differentiated in culture (day 9) to PPAR
activators resulted in a
2-fold (15-deoxy-
12,14-prostaglandin J2) to
8-fold (10 µM BRL 49653) increase in UCP-1 mRNA
levels. When PPAR
activators, such as several fibrates and the
PPAR
-specific ligand Wy 14,643, were tested an even higher (3-12-fold) increase in UCP-1 mRNA expression was detected. In contrast, COII mRNA expression did not respond to PPAR activators, thus indicating that the effect of PPAR activators is specific for
UCP-1 mRNA.
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Fig. 1.
Effects of PPAR and
PPAR
agonists on ucp-1 gene
expression in primary brown adipocytes. Brown adipocytes
differentiated in culture from stromal vascular cells (day 9) were
exposed for 24 h (except
15-deoxy-
12,14-prostaglandin J2 for 6 h) to the following concentrations of PPAR
-specific (10 µM 15-deoxy-
12,14-prostaglandin
J2, 10 µM BRL 49653) or PPAR
-specific (30 µM Ly171883, 500 µM clofibrate, 500 µM fenofibrate, 100 µM bezafibrate, 10 µM Wy 14,643) agonists. Northern blot analyses were
performed with 10 µg of total RNA extracted from three pooled plates.
Bars are means from two to four independent experiments on
different cultures and S.E. is indicated when n
3. An example of the Northern blot analysis is depicted in the
bottom of the figure. Arrows indicate the
position of the two UCP-1 mRNA species in mice (1.6 and 1.9 kb) and
the mitochondrial genome-encoded COII mRNA (0.8 kb), which was used
as a control.
(4), whereas at 100 µM it
activates all three PPAR subtypes (10). The effects of 10 µM Wy 14,643 were maximal after 12 h of exposure to
the PPAR
ligand, and maintained after 24 h (Fig. 2B). The maximal effect of 10 µM Wy 14,643 on
UCP-1 mRNA levels resulted in an induction that was around 40%
that of 0.5 µM norepinephrine (20-fold ± 2.7) and
180% that of 0.5 mM 8-bromo-cAMP, a nonmetabolizable cAMP
derivative (4.3-fold ± 0.6).
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Fig. 2.
Dose-response and time course curves for the
effects of the PPAR ligand Wy 14,643 on
ucp-1 gene expression. Brown adipocytes
differentiated in culture (day 9) were exposed to the indicated
concentrations of Wy 14,643 for 24 h (A) or exposed to
10 µM Wy 14,643 for the indicated times (B).
Total RNA (10 µg) was analyzed by Northern blot. Points are means
from three independent experiments with triplicate plates, in which the
variation within the experimental groups is less than 15%.
Ligand Wy
14,643 Is Independent of Protein Synthesis and Synergizes with the
Effects of an RXR-specific Agonist--
Brown adipocytes were exposed
for 12 h to 10 µM Wy 14,643 in the absence or
presence of 5 µg/ml cycloheximide, an inhibitor of protein synthesis
(Fig. 3A). Cycloheximide
treatment led to lower basal expression of UCP-1 mRNA, as already
described (19), but it did not affect the ability of Wy 14,643 to
increase UCP-1 mRNA.
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Fig. 3.
Effects of 10 µM Wy 14,643 in the presence of
cycloheximide or of an RXR-agonist on UCP-1 mRNA expression.
Brown adipocytes differentiated in culture (day 9) were exposed to 10 µM Wy 14,643 for 12 h in the presence or not of 5 µg/ml cycloheximide (CHX) (A) or exposed to 10 µM Wy 14,643 and/or 100 µM methoprene for
24 h (B). Total RNA (10 µg) was analyzed by Northern
blot. Treatments are indicated as + whereas untreated cells are shown
as . Bars are means from two to three independent
experiments on different cultures and are expressed relative to
untreated cells, which were set as 1.
and the RXR ligands were added, suggesting a PPAR
-RXR heterodimer-mediated effect on ucp-1 gene expression.
Induces the Rat ucp-1 Gene Promoter Activity--
Primary
brown adipocytes were transiently transfected with a plasmid containing
the upstream 4.5 kb of the rat ucp-1 gene fused to a CAT
reporter gene. As shown in Fig. 4,
PPAR
activators increased the (
4551)UCP1-CAT activity at least
2-fold, in the same range of the effect caused by BRL 49653. Responsiveness of (
4551)UCP1-CAT to PPAR activators was enhanced
6-fold by co-transfection of the expression vector for PPAR
. Thus,
expression of both endogenous ucp-1 gene and transfected
ucp-1 gene promoter are up-regulated by PPAR
activators
in primary brown adipocytes.
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Fig. 4.
Effects of PPAR and
PPAR
agonists on (
4551)UCP1-CAT expression
in transiently transfected brown adipocytes. Brown adipocytes
differentiated in culture (day 9) were transfected with 12 µg of
(
4551)UCP1-CAT. When indicated, 3 µg of the expression vector
pSG5-PPAR
was co-transfected. After transfection, cells were exposed
or not to 10 µM Wy 14,643, 10 µM BRL 49653, or 30 µM Ly171883. Results are expressed as CAT activity
relative to control, which is set to 1, and are means of two
independent experiments, each one performed in triplicate.
Enhances the PPAR
-dependent Induction of the
ucp-1 Gene Promoter--
To further investigate the transcriptional
regulation by PPAR
of the ucp-1 gene promoter, we used
the brown adipocyte-derived HIB-1B cell line. These cells express
PPAR
and PPAR
but not PPAR
(24). Thus, HIB-1B cells provide a
useful model of brown fat-derived cell in which
PPAR
-dependent regulation rely on transfected receptor.
In agreement, Wy 14,643 did not modify (
4551)UCP1-CAT activity (Fig.
5A). However, co-transfection
of pSG5-PPAR
induced (
4551)UCP1-CAT activity 3-fold in the absence
and nearly 7-fold in the presence of 10 µM Wy 14,643. Co-transfection of pRSV-RXR
caused a synergistic increase in the
PPAR
-dependent effect upon (
4551)UCP1-CAT activity. We
next performed co-transfection experiments using HepG2 cells to avoid
any interference of PPAR
in the observed effects. The HepG2 cell
line was chosen because, in contrast to HIB-1B cells, does not express
PPAR
nor PPAR
(31), and has been widely used to analyze PPAR
regulation of gene transcription (31-33). As shown in Fig.
5B, co-transfection of pSG5-PPAR
enhanced (
4551)UCP1-CAT activity and its responsiveness to Wy 14,643 in a
dose-dependent manner, and maximal effects were observed at 0.3 µg of pSG5-PPAR
. This amount of vector was the same at which the maximum synergistic enhancement by co-transfection of pRSV-RXR
was found (Fig. 5C), nearly 200-fold in the absence and
350-fold in the presence of 10 µM Wy 14,643. When
the other PPAR subtypes were tested, co-transfection of PPAR
in the
presence of RXR
caused a similar increase in (
4551)UCP1-CAT
activity than that of PPAR
, but no effect was observed due to
PPAR
co-transfection (data not shown).
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Fig. 5.
PPAR -dependent induction of
(
4551)UCP1-CAT expression in transiently transfected HIB-1B and HepG2
cells: influence of RXR co-transfection. A, HIB-1B
cells were transfected with 1 µg of (
4551)UCP1-CAT vector, and
included or not 0.3 µg of pSG5-PPAR
, and/or 0.1 µg of
pRSV-RXR
. After transfection, cells were exposed (dark
bars) or not exposed (open bars) to 10 µM
Wy 14,643 for 24 h. Results are shown as relative to the basal
expression of (
4551)UCP1-CAT, which is set to 1. Bars are
means of at least two independent experiments, each one done in
duplicate. B, HepG2 cells were transfected with 1 µg of
(
4551)UCP1-CAT vector together with increasing amounts of the
expression vector pSG5-PPAR
. After transfection, cells were exposed
(
) or not exposed (
) to 10 µM Wy 14,643 for 24 h. C, as in B, but 0.1 µg of pRSV-RXR
was
also co-transfected. Points are means of at least two
independent experiments, each one done in duplicate.
-dependent
Activation of the ucp-1 Gene Promoter--
We next analyze whether
co-regulators CBP and/or PGC-1 were involved in mediating PPAR
transcriptional regulation of (
4551)UCP1-CAT. Co-transfection of
pCMX-CBP or pSV-PGC1 alone enhanced basal (
4551)UCP1-CAT activity 5- and 7-fold, respectively (Fig. 6). When
co-transfected together with pSG5-PPAR
, an additive effect was
observed in the absence of PPAR
ligand, but when 10 µM
Wy 14,643 was added, a synergistic activation was detected (30-fold for
CBP and nearly 40-fold for PGC-1). When both coregulators were
co-transfected in the presence of PPAR
, a further increase in
(
4551)UCP1-CAT activity was observed. These results point to an
involvement of both CBP and PGC-1 in coactivating PPAR
and further
increasing responsiveness of the ucp-1 gene promoter to
PPAR
-ligand.
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Fig. 6.
Effects of CBP and/or PGC-1 co-transfection
on the PPAR -dependent induction of
(
4551)UCP1-CAT expression. HepG2 cells were co-transfected twith
1 µg of (
4551)UCP1-CAT vector, and included or not 0.3 µg of
pSG5-PPAR
. When indicated, 0.1 µg of pCMX-CBP and/or pSV-PGC1 were
also co-transfected. After transfection, cells were exposed (dark
bars) or not exposed (open bars) to 10 µM
Wy 14,643 for 24 h. Results are shown as relative to the basal
expression of (
4551)UCP1-CAT, which is set to 1. Bars are
means of at least two independent experiments, each one done in
duplicate.
- and PPAR
-dependent Regulation Require the
Same Element in the Upstream Region of the ucp-1 Gene Enhancer--
To
determine the site in the 5'-region of the rat ucp-1 gene
responsible for PPAR
action, the effects of PPAR
co-transfection on different deletion and double-point mutants of (
4551)UCP1-CAT were
studied in transfected HepG2 and HIB-1B cells (Fig.
7). For comparative purposes, parallel
co-transfection experiments were performed with PPAR
. Results in
both cell lines and for each PPAR subtype, indicated that both PPAR
subtypes share a responsive site located in the
2494/
2318 enhancer
region of the ucp-1 gene. When a double-point mutation, in
which the CC at positions
2472 and
2473 were changed to AA (see
Fig. 8A), was introduced in both (
3628/
2283)UCP1-CAT and (
2494/
2318)UCP1-CAT vectors, responsiveness to both PPAR
and PPAR
was abolished. Furthermore, when the
2494 to
2445 fragment was placed upstream (
172)UCP1-CAT or the HSV thymidine kinase promoter in pBLCAT2, it
conferred 6- and 3-fold responsiveness, respectively, to PPAR
and
PPAR
(data not shown).
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Fig. 7.
Analysis of the
PPAR - and
PPAR
-dependent regulation of
transiently transfected deletion or double-point mutants of the
(
4551)UCP1-CAT. HepG2 and HIB-1B cells were transiently
transfected with 1 µg of (
4551)UCP1-CAT or equivalent amounts of
the deletion or double-point mutants illustrated on the left.
Asterisks indicate the double-point mutated versions of
(
3628/-2283)UCP1-CAT and (
2494/-2318)UCP1-CAT, containing AA
instead of CC at sites
2473 and
2472. Transfections included 0.1 µg of the expression vector pRSV-RXR
, and included or not, 0.3 µg of pSG5-PPAR
(dark bars) or pSG5-PPAR
(open
bars). Results are expressed as the -fold induction caused by PPAR
co-transfection on each transfected construct. Bars are
means of at least two independent experiments, each one done in
duplicate.
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Fig. 8.
Electrophoretic mobility shift assays of the
2485/
2458 region of the rat ucp-1 gene.
A, sequence corresponding to the
2485/
2458 region of the
rat ucp-1 gene (UCP1-PPRE), was compared with a consensus
PPRE (34) and to the analogous regions in the murine (
2499/
2472)
and human (
3732/
3705) ucp-1 gene promoters (18, 35).
Asterisks indicated the double-point mutant derivative
version (mutUCP1-PPRE) in which the CC at positions
2472 and
2473
were changed to AA. The upper arrows show the putative
alignments of three motifs closely related to an idealized half-site.
B, gel mobility shift assay: the double-stranded
oligonucleotide
2485/
2458 was end-labeled and incubated with 5 µl
of in vitro transcribed/translated RXR
, alone or together
with PPAR
or PPAR
. Arrows indicate the corresponding
heterodimers bound to the probe. Lane 1 showed that the mock
lysate produced two nonspecific bands when incubated with the probe.
C, super-shift assay: the labeled UCP1-PPRE probe was
incubated with 5 µg of nuclear protein extract from differentiated
primary brown adipocytes. When indicated, 1 µl of antiserum against
RXR
, PPAR
, PPAR
, or ETS (as negative control) were added.
Arrows indicate the super-shifted bands. D,
protein extracts from rat brown adipose tissue nuclei (5 µg) were
incubated with the labeled UCP1-PPRE probe. Super-shift analysis was
performed by incubation with 1 µl of antiserum against PPAR
,
PPAR
, or ETS. Oligonucleotide competitors, UCP1-PPRE (WT) and
mutUCP1-PPRE (MUT), were added at a 100-fold molar excess relative to
probe concentration. A nonspecific-binding band was detected
(n.s.). Bracket indicates the specific-binding
bands and arrows the super-shifted bands.
2485/
2458 Site in the ucp-1 Gene Binds PPAR
and
PPAR
--
Analysis of the sequence required for PPAR responsiveness
in the rat ucp-1 gene promoter indicated the presence of a
direct repeat with 1-base pair spacing related to a consensus PPRE (34) (Fig. 8A, arrows indicate half-site-related
motifs). This
2485/
2458 sequence (UCP1-PPRE) in the rat
ucp-1 gene promoter is highly conserved when compared with
the previously reported PPAR
-responsive element in the murine
ucp-1 gene (18) and to the corresponding sequence in the
human ucp-1 gene promoter (35) (Fig. 8A).
Electrophoretic gel mobility shift assays were performed using the
UCP1-PPRE as labeled probe. As shown in Fig. 8B, in
vitro transcribed/translated RXR
alone (lane 2) did
not bind significantly to this sequence although two nonspecific bands
were detected as with the reticulocyte lysate (lane 1,
n.s.). However, incubation with a mixture of PPAR
or
PPAR
with RXR
resulted in the formation of the respective heterodimer complexes (lanes 3 and 4, respectively). To
further assess the interaction of UCP1-PPRE with PPAR
-RXR
or
PPAR
-RXR
heterodimers found in nuclear extracts from
differentiated brown adipocytes in primary culture (Fig. 8C)
or from BAT (Fig. 8D), supershift assays were performed
using specific antibodies against RXR
, PPAR
, or PPAR
.
Arrows indicate the supershifted complexes formed (that
contain RXR
and PPAR
or PPAR
). Incubation with an antibody
against ETS transcription factors, used as negative control, did not
result in any change in the pattern of bands. Competition experiments
performed in Fig. 8D with a 100-fold molar excess of
specific (UCP1-PPRE) or its mutated version (mutUCP1-PPRE, see legend
of Fig. 8A) confirmed the presence of a nonspecific band
(n.s.). Taken together, these findings demonstrate that both PPAR
and PPAR
are present in brown fat cell nuclei and bind to
UCP1-PPRE as heterodimers with RXR
.
Ligand Wy 14,643 Induces ucp-1 Gene Expression in Brown
Adipose Tissue in Vivo in Different Physiological Situations--
To
assess the in vivo significance of PPAR activators on the
expression of the ucp-1 gene, mice at different
physiological situations were injected with single doses of the
PPAR
-specific ligand Wy 14,643 or, for comparative purposes, of the
PPAR
activator troglitazone. We have previously reported that
sensitivity of gene expression to PPAR
activators in acute
treatments in vivo depends on the status of lipid metabolism
able to provide endogenous PPAR
ligands (36). In adult mice (Fig.
9), Wy 14,643 caused a moderate 1.5-fold
increase in UCP-1 mRNA abundance in BAT. When lactating mice were
analyzed, Wy 14,643 significantly increased (5-fold) UCP-1 mRNA
levels. During lactation, functional atrophy of BAT, including
diminished lipolytic and lipoprotein lipase activities, and reduced
expression of the ucp-1 gene contribute to energy sparing
(37, 38). In contrast, troglitazone only had a moderate effect on brown
fat UCP-1 mRNA abundance.
View larger version (30K):
[in a new window]
Fig. 9.
Effects of PPAR activators on UCP-1 mRNA
expression in brown fat of adult, lactating, or neonate mice.
A, representation of the relative abundance of UCP-1
mRNA in brown fat from adult female control and 15-day lactating
dams after 6 h of being injected intraperitoneally with Wy 14,643 (50 µg/g body weight), troglitazone (100 µg/g body weight), or
vehicle solution, and neonates that were injected intraperitoneally
with Wy 14,643 (50 µg/g body weight), BRL 49653 (50 µg/g body
weight), or vehicle solution (see "Experimental Procedures" for
details). Data are expressed as relative to the adult female control
which was set as 1. Statistical significance of comparisons between
groups of treated mice and their respective vehicle treated controls
are shown by: *, p 0.05; **, p
0.01. Comparison between Wy 14,643 and troglitazone treatment is shown
by
, p
0.05. B, representative
Northern blot analysis of equal amounts of brown fat RNA (20 µg/lane)
hybridized with the UCP-1 and COII probes, as described in the legend
to Fig. 1.
-ligand BRL 49653 did not
significantly change UCP-1 mRNA expression. The action of PPAR
agonists was specific for the ucp-1 gene since COII mRNA levels were essentially unaffected by PPAR activators in BAT (see Fig.
9, bottom). Present results demonstrate an acute regulation of the ucp-1 gene in vivo by the PPAR
-ligand
Wy 14,643 that is more potent than that observed for PPAR
ligands.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
activators regulate the
expression of the ucp-1 gene both in primary brown
adipocytes and in BAT in vivo. Brown adipocytes
differentiated in primary culture were used since they highly coexpress
all PPAR subtypes, equally to BAT (24). In contrast, the HIB-1B brown
adipocyte cell line lacks PPAR
expression (24), and therefore, the
results of previous studies using HIB-1B cells to determine the effects of PPAR activators on the expression of the ucp-1 gene must
be viewed with caution. Present results also demonstrate that PPAR
induces the rat ucp-1 gene promoter activity upon treatment
with its specific ligand Wy 14,643, but it can also activate
transcription in the absence of exogenously added ligand. This has been
widely described for other PPAR
-responsive gene promoters (32, 39), and could be explained by either the presence of endogenous activators, such as fatty acids or their metabolites, or by ligand-independent activity of these nuclear receptors (40). The responsiveness of the
ucp-1 gene promoter to PPAR
-ligand is increased by
co-transfection with expression vectors for either coactivator CBP or
PGC-1. Furthermore, the synergistic effect observed when adding both
coactivators points to the involvement at the same time of CBP and
PGC-1 in coactivating PPAR
. In this way, PPAR
can interact
directly with CBP (41) and also with PGC-1 (42). In addition, CBP can
form a complex with PGC-1 (43), thus providing multiple contact points to stabilize the complex assembly. Furthermore, CBP can also interact with other transcription factors, such as CREB and C/EBP, known to
regulate transcription of the rat ucp-1 gene through its
proximal regulatory region (22, 21).
-responsive element in the upstream enhancer region of the rat ucp-1 gene. This
2485/
2458 region contains a potential
PPRE consensus formed by two direct repeats separated by one nucleotide (DR-1). Highly comparable elements are also found in the human and
mouse ucp-1 genes (see Fig. 8A), indicating that
these sequences may have an important regulatory role in response to
PPAR
. In fact, the murine element has been described to mediate
PPAR
responsiveness (18). Our present results further demonstrate
that the
2485/
2458 element in the rat ucp-1 gene behaves
as a promiscuous responsive site to either PPAR
and PPAR
activation, but not PPAR
. From the analysis of various natural
PPREs, it has been reported that the binding strength and functional
transactivation for each PPAR subtype on the same PPRE was similar
(33). Only some significant PPAR
specificity was described, and it
was related to the 5'-flanking sequence with respect to the DR-1
element, which is essential for PPAR
binding (33). However, present
results indicate a similar capacity of PPAR
and PPAR
to bind and
activate ucp-1 transcription through the UCP1-PPRE. The
predominant role of any subtype at any one time may thus depend on: 1)
the relative amount of each subtype. For instance, PPAR
and PPAR
gene expression in brown adipocytes are under opposite regulation by
their ligands and retinoic acid: up-regulation of PPAR
but
down-regulation of PPAR
(24). 2) Cross-talk with other signaling
pathways, like regulation of PPAR transcriptional activity by MAP
kinase-dependent phosphorylation, which enhances PPAR
(44) but decreases PPAR
activity (45). 3) Ligand availability.
Several PPAR ligands have been described to be highly subtype-specific
(6), although identification of endogenous ligands and how their
synthesis is regulated, is far from being established. 4) Interaction
with coregulators. The interaction of PGC-1 with PPAR
is
ligand-dependent whereas that with PPAR
is not (42, 30).
These and other possible events may determine which PPAR subtype
activates transcription of ucp-1 in response to brown
adipocyte physiological condition, mainly PPAR
in association with
differentiation-dependent events or PPAR
in coordination
with increased lipid catabolism in active BAT.
and
activators through the same PPRE (39, 46). However,
since they have been studied in tissues such as liver, which highly
expresses PPAR
but not PPAR
, or WAT, which predominantly expresses PPAR
, tissue-specific regulation has been suggested. In
contrast, BAT provides a model to study whether PPAR subtypes specifically regulate a PPRE in a target gene or whether a unique element behaves as a common site, as shown by our present findings in
the ucp-1 gene promoter. For instance, the lipoprotein
lipase (LPL) gene is up-regulated by PPAR
(in liver) and PPAR
(in
WAT) through the same PPRE (46). During BAT differentiation, induction of LPL allows for increased fatty acids delivery to brown adipocytes, which results in triglyceride accumulation, thus promoting the adipocyte phenotype. However, thermogenic stimulus also up-regulates LPL to increase fatty acids uptake, which increases the supply of
substrate for oxidation. Expression of LPL mRNA is increased by
PPAR
and
activators in differentiated brown
adipocytes,2 suggesting that
LPL gene transcription in BAT could be activated by both PPAR
and
-
. Other genes, such as the fatty acid transport protein and the
acyl-CoA synthetase genes, which also regulate cell uptake of fatty
acids, might be similarly regulated in BAT since they are induced by
PPAR
and -
activators (39, 47).
by Wy 14,643 up-regulates UCP-1 mRNA expression in BAT. The effects of the acute administration of this synthetic ligand are higher in
those physiological situations (lactating dams and newborn pups at
thermoneutrality) in which endogenous PPAR
-ligands are expected to
be low, in agreement with previous findings that PPAR
sensitivity
in vivo depends on the status of lipid metabolism (36).
Furthermore, the higher ucp-1 gene responsiveness to acute treatments with PPAR
than PPAR
agonists underlines the in
vivo significance of PPAR
-dependent regulation of
ucp-1 gene expression. In contrast, it has been reported
that chronic exposure to PPAR
or PPAR
activators led to opposite
results: long-term oral treatment of rats with Wy 14,643 did not change
UCP-1 mRNA levels and thiazolidinedione administration resulted in
a slight up-regulation of UCP-1 mRNA (48). This behavior of
ucp-1 is similar to other bona fide PPAR-target genes in BAT, which remain unchanged by chronic exposure to PPAR
agonists (49). Positive effects of long-term treatment with thiazolidinediones on ucp-1 gene expression may be a
consequence of their reported action promoting overall BAT
differentiation (17, 48, 50).
-adrenergic stimulation of BAT result in activation of the PPAR
pathway (51). We have previously reported that norepinephrine directly
up-regulates transcription of the ucp-1 gene promoter, mainly through a cAMP responsive region in the proximal promoter region
(22). However, the upstream enhancer region of the rat ucp-1
gene is also responsive to norepinephrine, although it lacks a defined
cAMP responsive region. Several lines of evidence suggest a role for
PPAR
in mediating this regulation, although the involvement of
PPAR
cannot be ruled out. Mutation of the UCP1-PPRE affects the
response of the ucp-1 gene promoter to norepinephrine
(17).3 Furthermore, the
mitogen-activated protein kinase pathway is activated in BAT by
adrenergic stimulation (52). This may result in activation of PPAR
but inactivation of PPAR
, as discussed above. The coactivator PGC-1
is rapidly induced by cold-exposure through
-adrenergic pathways in
BAT (30). Present data demonstrate that PGC-1 coactivates PPAR
and
further increases ucp-1 gene responsiveness to
PPAR
-ligand dependent activation. Likewise, PGC-1 cooperates with
PPAR
in the transcriptional control of nuclear genes encoding
mitochondrial fatty acid oxidation enzymes (42), and it also induces
mitochondrial gene expression by regulating the nuclear respiratory
factor system (53). Taken together, these data point to PGC-1/PPAR
interaction as playing an important role in mediating changes in gene
expression in response to BAT thermogenic requirements. Although basal
expression of UCP-1 mRNA in PPAR
-null mice has been reported to
be unaltered (12), as also reported for other bona fide
PPAR-target genes in liver (6), further studies are in course to
determine whether ucp-1 gene expression is altered in these
mice in response to thermogenic stimulus.
directly regulates ucp-1 gene
transcription and we propose that this transcriptional regulatory
mechanism is a component of the coordinate control of thermogenic and
lipid oxidation pathways in active BAT. Recently, PPAR
has been
implicated in obesity (54) and selective PPAR
activators have been
described to improve insulin sensitivity and reduce WAT mass (55). Part of these effects could be due to an increase in energy expenditure in
BAT, and the positive action of PPAR
on ucp-1 gene
expression opens new perspectives on the molecular targets of PPAR
involved in mediating these effects.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Daniel Ricquier for
(4551)UCP1-CAT and Dr. B. Spiegelman for the HIB-1B cell line. We
also thank Drs. S. Green, P. Grimaldi, B. Spiegelman, P. Chambon, R. Evans, D. Ricquier, and N. Glaichenhaus for kindly supplying expression
vectors and probes.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants PM98-0188 from Ministerio de Educación y Ciencia, Spain, and SGR99-38 from Generalitat de Catalunya, Spain.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.
To whom correspondence should be addressed: Dept. de
Bioquímica i Biologia Molecular, Universitat de Barcelona, Avda
Diagonal 645, E-08028-Barcelona, Spain. Tel.: 34-93-4034613; Fax:
34-93-4021559; E-mail: giralt@porthos.bio.ub.es.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M006246200
2 A. Schlüter, M. Giralt, and F. Villarroya, unpublished observations.
3 M. J. Barberà, F. Villarroya, and M. Giralt, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PPAR, peroxisome
proliferator-activated receptor;
UCP-1, uncoupling protein-1;
PPRE, PPAR response element;
BAT, brown adipose tissue;
WAT, white adipose
tissue;
RXR, retinoid X receptor;
CBP, CREB-binding protein;
PGC-1, PPAR coactivator-1;
CAT, chloramphenicol acetyltransferase;
LPL, lipoprotein lipase;
kb, kilobase(s);
COII, cytochrome oxidase subunit
II.
![]() |
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