Biochem. J. (2012) 443, 799–810 (Printed in Great Britain)
799
doi:10.1042/BJ20111714
Pref-1 in brown adipose tissue: specific involvement in brown adipocyte
differentiation and regulatory role of C/EBPδ
Jordi ARMENGOL*, Josep A. VILLENA†, Elayne HONDARES*, Marı́a C. CARMONA*, Hei Sook SUL‡, Roser IGLESIAS*,
Marta GIRALT* and Francesc VILLARROYA*1
*Departament de Bioquı́mica i Biologia Molecular, Institut de Biomedicina de la Universitat de Barcelona (IBUB) and CIBER Fisiopatologia de la Obesidad y Nutrición (CIBEROBN),
08028 Barcelona, Spain, †Laboratory of Metabolism and Obesity, Unit of Diabetes and Metabolism, Institut de Recerca Vall d’Hebron, 08035 Barcelona, Spain, and ‡Department of
Nutritional Science and Toxicology, University of California, Berkeley, Berkeley, CA 94720, U.S.A.
Pref-1 (pre-adipocyte factor-1) is known to play a central role
in regulating white adipocyte differentiation, but the role of
Pref-1 in BAT (brown adipose tissue) has not been analysed.
In the present study we found that Pref-1 expression is high
in fetal BAT and declines progressively after birth. However,
Pref-1-null mice showed unaltered fetal development of BAT,
but exhibited signs of over-activation of BAT thermogenesis
in the post-natal period. In C/EBP (CCAAT/enhancer-binding
protein) α-null mice, a rodent model of impaired fetal
BAT differentiation, Pref-1 was dramatically overexpressed, in
association with reduced expression of the Ucp1 (uncoupling
protein 1) gene, a BAT-specific marker of thermogenic
differentiation. In brown adipocyte cell culture models, Pref1 was mostly expressed in pre-adipocytes and declined with
brown adipocyte differentiation. The transcription factor C/EBPδ
activated the Pref-1 gene transcription in brown adipocytes,
through binding to the proximal promoter region. Accordingly,
siRNA (small interfering RNA)-induced C/EBPδ knockdown
led to reduced Pref-1 gene expression. This effect is consistent
with the observed overexpression of C/EBPδ in C/EBPαnull BAT and high expression of C/EBPδ in brown preadipocytes. Dexamethasone treatment of brown pre-adipocytes
suppressed Pref-1 down-regulation occurring throughout the
brown adipocyte differentiation process, increased the expression
of C/EBPδ and strongly impaired expression of the thermogenic
markers UCP1 and PGC-1α [PPARγ (peroxisome-proliferatoractivated receptor γ ) co-activator-α]. However, it did not alter
normal fat accumulation or expression of non-BAT-specific
genes. Collectively, these results specifically implicate Pref-1 in
controlling the thermogenic gene expression program in BAT,
and identify C/EBPδ as a novel transcriptional regulator of Pref1 gene expression that may be related to the specific role of
glucocorticoids in BAT differentiation.
INTRODUCTION
lipid content of both brown and white adipocytes has led to
the traditional concept that these two cell types differentiate
from closely related precursor cells. However, recent research
indicates that the brown adipocyte differentiation lineage is
substantially different from the white adipocyte lineage and, in
fact, brown adipocytes share mesenchymal precursor cells that
closely correspond to the myogenic lineage of differentiation
[4]. Several transcription factors, including PPARγ (peroxisome
proliferator-activated receptor γ ), play similar roles in BAT and
WAT, essentially promoting lipid accumulation, whereas other
transcription factors and co-regulators, such as PGC-1α (PPARγ
co-activator-α) or PRDM16 (PR-domain-containing 16), act at
distinct stages of the commitment of mesenchymal cells, driving
specific differentiation towards a brown adipocyte phenotype [5].
Moreover, transcription factors such as C/EBP (CCAAT/enhancer
binding protein) β are specifically involved in driving common
precursors of brown and myogenic lineages toward the brown
adipocyte differentiation pathway [6].
Pref-1 (pre-adipocyte factor-1), also known as Dlk-1 (deltalike-1 homologue) and FA-1 (fetal antigen-1), is an EGFrepeat-containing transmembrane protein. The biologically
active soluble form of Pref-1 is produced by cleavage
of the extracellular domain of Pref-1 by TACE [TNFα
BAT (brown adipose tissue) is the main site of adaptive nonshivering thermogenesis and allows small mammals as well
as neonates to adapt to cold environments. Moreover, BAT
thermogenesis contributes to energy expenditure in response
to overfeeding, and BAT thermogenic activity protects against
obesity [1,2]. In humans, BAT has traditionally been considered
physiologically relevant only in the neonatal period [1]. However,
a previous study demonstrated the presence of significant amounts
of active BAT in adult humans and showed an association of BAT
with lowered body mass index [3].
The cell type specialized in non-shivering thermogenesis is the
brown adipocyte, which contains a large number of mitochondria
with a high oxidative capacity. Brown adipocyte mitochondria are
naturally uncoupled due to the presence of UCP (uncoupling
protein) 1, a protein exclusively expressed in brown adipocytes
that confers on BAT mitochondria their capacity to produce heat.
Brown adipocytes also contain a large number of lipid droplets
capable of providing the fuel for oxidation and heat production
[1]. Despite the widely recognized opposite physiological roles
of BAT and WAT (white adipose tissue) (energy dissipation
and energy accumulation respectively), the high intracellular
Key words: brown adipose tissue, CCAAT/enhancer-binding
protein δ (C/EBPδ), glucocorticoid, Pref-1, thermogenesis.
Abbreviations used: aP2, adipose protein 2; BAT, brown adipose tissue; C/EBP, CCAAT/enhancer-binding protein; ChIP, chromatin immunoprecipitation;
COII, cytochrome c oxidase subunit 2; DMEM:F12, 1:1 mixture of Dulbecco’s minimal essential medium/Ham’s F12; FBS, fetal bovine serum; PPARγ,
peroxisome-proliferator-activated receptor γ; PGC-1α, PPARγ co-activator-α; Pref-1; pre-adipocyte factor-1; RT, reverse transcription; siRNA, small
interfering RNA; T3, 3,3 ,5-tri-iodothyronine; TE buffer, Tris/EDTA buffer (10 mM Tris/HCl, pH 8.0, and 1 mM EDTA); TEM, transmission electron microscopy;
UCP, uncoupling protein; WAT, white adipose tissue.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
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J. Armengol and others
(tumour necrosis factor α)-converting enzyme] [7]. Pref-1
is highly expressed in embryonic tissues and is associated with the undifferentiated status of multiple cell
types of the mesenchymal lineage. Pref-1 is highly
expressed in white pre-adipocytes, and its expression is
strongly down-regulated in association with the initiation of the
white adipogenic differentiation process [8]. In fact, Pref-1 is
known to play a pivotal role in inhibiting the differentiation of
pre-adipocytes into white adipocytes both in vitro and in vivo
[8–11] by mechanisms involving MAPK (mitogen-activated
protein kinase)/ERK (extracellular-signal-regulated kinase)
signalling, induction of the Sox (SRY-box-containing) gene and
subsequent inhibition of C/EBPβ and C/EBPδ [12]. Some studies
also suggest that Pref-1 could exert its action on adipocyte
differentiation through modulation of Notch pathways [13].
The effects of Pref-1 on white adipocyte differentiation may
be distinctly different depending on the extent of commitment
of precursor cells to the adipogenic pathway. For instance,
in C3H10T1/2c mesenchymal cells, Pref-1 does not exert the
powerful anti-adipogenic action that it displays in 3T3-L1 preadipocytes or mouse embryonic fibroblasts [14].
The biological role and regulation of Pref-1 in BAT has not
yet been directly addressed. In the present study, we analysed
the regulation of Pref-1 expression during brown fat development
and differentiation, and assessed the effects of manipulating Pref-1
levels on BAT and brown adipocyte differentiation. We established
that Pref-1 is preferentially involved in controlling the specific
thermogenic program of BAT, and also identified the transcription
factor C/EBPδ as a novel transcriptional regulator of Pref-1 gene
expression.
MATERIALS AND METHODS
Animals
The care and use of mice were in accordance with the European
Community Council Directive 86/609/EEC, and were approved
by the Comitè Ètic d’Experimentació Animal of the University of
Barcelona. Mice were housed in same-sex groups of three animals
per cage, in a room with constant temperature (21 ◦ C) and relative
humidity (30–40 %), and a 12h/12h light/dark cycle. Food pellets
(A03, Panlab) and water were available ad libitum. Swiss Webster
mice were used for studies of development changes. The day of
pregnancy was determined by the presence of a vaginal plug (day
0). For studies in fetuses, Caesarean sections of pregnant mice
were performed at the indicated days of gestation. Neonates (0 h
to 21 days after birth) were killed by decapitation. Interscapular
BAT was harvested, immediately frozen in liquid nitrogen and
stored at − 80 ◦ C. For studies using Pref-1-null mice [9]
and C/EBPα-null mice [15], heterozygous female mice were
mated with heterozygous males. Studies in Pref-1-null neonates
and C/EBPα-null neonates were performed in pups 24 h after
birth and 4 h after birth respectively. Mice were killed by
decapitation. Interscapular BAT, liver and heart were harvested,
and, where indicated, an aliquot was immediately frozen in liquid
nitrogen and stored at − 80 ◦ C until RNA or proteins were
isolated, and a separate aliquot was fixed for TEM (transmission
electron microscopy) analysis. Wild-type, heterozygous and
homozygous mice were obtained from the same litter in each
experiment. Where possible, samples from two or three pups were
pooled for each experimental condition. At least three different
litters were analysed independently for each developmental age.
Transgenic mice that express C/EBPα under the control of
the albumin enhancer/promoter were generated as described
previously [16]. This line was bred with the C/EBPα-null strain to
c The Authors Journal compilation c 2012 Biochemical Society
generate mice that express C/EBPα exclusively in the liver. Pups
were studied on days 2 or 7 after birth and genotyped as described
previously [16]. Transgenic wild-type (TG + , C/EBPα + / + ) and
homozygous (TG + , C/EBPα − / − ) mice were obtained from the
same litter in each experiment, and at least three different litters
were analysed independently for each postnatal age.
TEM and stereological analysis
BAT samples were fixed in 2.5 % glutaraldehyde and 2 %
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and
postfixed in 1 % OsO4 and 0.8 % FeCNK in phosphate buffer.
After dehydration in a graded acetone series, tissue samples
were embedded in Spurr resin. Ultrathin sections were stained
with uranyl acetate and lead citrate and examined with a Hitachi
H600AB transmission electron microscope at 75 kV. A total of
two to three mice were analysed for each developmental age
and genotype. For stereological quantification, the proportion
of lipid with respect to cell volume was estimated using the
volume density method [17], and mitochondrial inner membrane
surface density was calculated employing a vertical sections
method [18,19]. At least six distinct images from each mouse
at each developmental age and genotype were analysed.
Cell culture
Differentiation of brown adipocytes in primary culture was
performed as described previously [20]. Precursor cells were
isolated from the interscapular, cervical and axillary depots of
BAT from 21-day-old mice. Experiments were performed on day
8 or 9 of culture (a point at which 90 % of cells were considered
to be differentiated on the basis of lipid accumulation and brown
adipocyte morphology under standard conditions of culture), or
at earlier stages of differentiation, as indicated. For studies on
the effects of corticosteroids, pre-adipocytes were exposed to
30 nM dexamethasone (Sigma) from culture day 4 onwards.
Mouse HIB-1B cells [21] were grown in DMEM:F12 (1:1 mixture
of Dulbecco’s minimal essential medium/Ham’s F12, Gibco)
containing 10 % (v/v) FBS (fetal bovine serum), 100 units/ml
penicillin G and 100 μg/ml streptomycin. Differentiation was
induced by growing HIB-1B cells in DMEM:F12 supplemented
with 20 nM insulin and 1 nM T3 (3,3 ,5-tri-iodothyronine).
Once cells had reached 100 % confluence, they were placed in
DMEM:F12 supplemented with 20 nM insulin, 1 nM T3, 0.5 mM
IBMX (3-isobutyl-1-methylxanthine), 0.5 μM hydrocortisone
and 0.125 mM indomethacin for 24 h. HIB-1B cells were then
maintained for 5–7 days in DMEM:F12 containing 5 % (v/v)
FBS, 20 nM insulin and 1 nM T3 to allow acquisition of a
differentiated morphology. The cell diferentiation state was
determined by assessing cytoplasmic fat accumulation using
intracellular triglyceride staining with Oil Red O (Sigma).
RNA isolation, Northern blotting and quantitative real-time PCR
analyses
Total RNA was extracted using the RNeasy Mini Kit (Qiagen).
For Northern blot analysis, 10–15 μg of total RNA was
denatured, electrophoresed on 1.5 % formaldehyde/agarose gels
and transferred on to positively charged nylon membranes
(N + Boehringer Mannheim). Equal loading of gels was
confirmed by ethidium bromide staining and hybridization with
an 18S rRNA probe. Prehybridization and hybridization were
carried out as described previously [22]. Autoradiographs
were quantified by densitometric analysis (Phoretics, Millipore).
Pref-1 in brown adipose tissue
mRNA expression was quantitatively analysed using TaqMan
real-time RT (reverse transcription)–PCR. The RT reaction
was performed using 0.5 μg of RNA in a 25 μl volume
containing 1.25 units/μl MultiScribeTM Reverse Transcriptase,
0.4 units/μl RNase inhibitor, 500 μM dNTP mixture, 2.5 μM
random hexamers, 1× RT buffer and 5.5mM MgCl2 solution
(all from Applied Biosystems). TaqMan Real-Time PCR
reactions were performed using TaqMan Universal PCR Master
Mix and the following standardized (‘Assay-on-Demand’)
gene expression primers/probes: C/EBPα (Mm00514283_s1),
C/EBPβ (Mm00843434_s1), C/EBPδ (Mm00786711_s1),
UCP1 (Mm00494069_m1), aP2 (adipose protein 2)/FABP4
(fatty acid binding protein 4) (Mm00445880_m1), Pref-1
(Mm00494477_m1) and PGC-1α (Mm00447183_m1). COII
(cytochrome c oxidase subunit 2) was detected using the customdesigned primers 5 -CAAACCACTTTCACCGCTACAC-3 (forward) and 5 -GGACGATGGGCATGAAACTGT-3 (reverse)
and the FAM (6-carboxyfluorescein)-labelled probe 5 AAATCTGTGGAGCAAACC-3 . The amount of mRNA for the
gene of interest in each sample was normalized to that of
the housekeeping reference 18S rRNA (Hs99999901, Applied
Biosystems). Samples were run in duplicate on the ABI/Prism
7700HT Sequence Detection System (Applied Biosystems).
801
vectors for the active (LAP) C/EBPβ isoform (pCMV-LAP) and
truncated inhibitory (LIP) C/EBPβ isoform (pCMV-LIP) were
a gift from Professor Ueli Schibler (Department of Molecular
Biology, University of Geneva, Geneva, Switzerland). Cells
were incubated for 48 h after transfection. Firefly luciferase and
Renilla luciferase activities were measured in a Turner Designs
Luminometer (Model TD20/20) using the Dual Luciferase
Reporter Assay System kit (Promega) as described by the
manufacturer. Luciferase activity elicited by Pref-1 promoter
constructs was expressed relative to the Renilla luciferase activity
to normalize for variations in transfection efficiency.
C/EBPδ-knockdown assays
For knockdown of C/EBPδ expression, 40 pmol/plate siRNA
(small interfering RNA) duplex specific for mouse C/EBPδ (sc37723, Santa Cruz Biotechnology) and scrambled control siRNA
were transfected into proliferating HIB-1B cells using transfection
reagent (sc-29528, Santa Cruz Biotechnology) and following the
manufacturer’s instructions. After 48 h, cells were harvested and
C/EBPδ protein (immunoblot) and Pref-1 mRNA and aP2 mRNA
(quantitative RT–PCR) were measured.
Immunoblot analysis
ChIP (chromatin immunoprecipitation) assay
Samples of cell homogenates were separated by SDS/PAGE on
12.8 % gels and transferred on to PVDF membranes (ImmobilonP, Millipore). Membranes were then incubated with primary
antibodies against C/EBPα (sc-61X, 1:1000 dilution; Santa
Cruz Biotechnology), C/EBPβ (sc-150, 1:300; Santa Cruz
Biotechnology), C/EBPδ (sc-151X, 1:1000 dilution; Santa
Cruz Biotechnology) or β-actin (AC-15, 1:10 000 dilution;
Sigma). β-Actin detection was used to ensure equal protein
loading. Bound antibodies were detected using horseradish
peroxidase-coupled anti-mouse (170-6516, 1:3000 dilution; BioRad) or anti-rabbit (sc-2004, 1:3000 dilution; Santa Cruz
Biotechnology) secondary antibodies and an ECL (enhanced
chemiluminescence) detection kit (GE Healthcare). When
required, quantitative analysis was performed by densitometry
(Phoretics ID software).
ChIPs were performed as described previously [24] with a few
modifications. For ChIPs using tissue, brown fat from fetuses at
term was dissected and minced to a homogeneous consistency.
Samples of BAT or HIB-1B cells were fixed for 10 min with 1 %
formaldehyde. Formaldehyde was then quenched by incubating
with 125 mM glycine for 5 min. Cells were washed with PBS, and
immediately scraped off the dishes. In both cases (tissue and cells)
samples were harvest by centrifugation at 11 000 g for 2 min.
Nuclei were isolated by means of incubation with WB1 buffer
(10 mM Tris/HCl, pH 8.0, 0.25 % Triton X-100, 1 mM EDTA and
0.5 mM EGTA) for 10 min, centrifugation at 11 000 g for 2 min
and incubation with WB2 (10 mM Tris/HCl pH 8.0, 200 mM
NaCl, 1 mM EDTA and 0.5 mM EGTA) for 10 min. After a final
centrifugation at 11 000 g for 2 min, the pellet was resuspended
in SDS lysis buffer (15 mM Tris/HCl, pH 8.0, 10 mM EDTA
and 1 % SDS) and incubated for 10 min. Lysates were then
sonicated on ice using a VC50 50 W sonicator (Sonics &
Materials) to obtain 400–2000 bp chromatin fragments, and
immunoprecipitated using an antibody against C/EBPδ (sc-151X,
Santa Cruz Biotechnology) by incubating overnight at 4 ◦ C
using Dynabeads M-280 Sheep anti-Rabbit IgG (Invitrogen).
Pre-immune serum was used as a negative control to confirm
the specificity of the assay. Thereafter, magnetic beads were
washed once with low-salt buffer (20 mM Tris/HCl, pH 8.0,
0.1 % SDS, 1 % Triton X-100, 2 mM EDTA and 150 mM NaCl)
and then twice with TE (Tris/EDTA) buffer (10 mM Tris/HCl,
pH 8.0, and 1 mM EDTA). After elution with 1 % SDS in 0.1 M
NaHCO3 for 15 min, the DNA–protein cross-links were reversed
by addition of NaCl to a final concentration of 200 mM and
incubation at 65 ◦ C for 5 h. Finally, after adjusting the volume to
achieve final EDTA and Tris/HCl concentrations of 10 mM and
40 mM respectively, proteins were digested by adding 40 μg/ml
Proteinase K and incubating at 45 ◦ C for 1 h. The DNA was
extracted with phenol/chloroform and resuspended in 20 μl of
TE buffer. PCR was performed using 2 μl of DNA, 2.5 units of
Taq DNA Polymerase, 1× PCR buffer, 1× Q-Solution, 200 μM
dNTP mixture, 1.5 mM MgCl2 and 0.5 μM of each primer in a
final volume of 25 μl. Amplification products were resolved on
2 % agarose gels and visualized by ethidium bromide staining.
Transient transfection assays
Transfection assays were carried out in HIB-1B cells at
50 % confluence using FuGene6 Transfection Reagent (Roche)
according to the manufacturer’s instructions. Each experimental
point was assayed in triplicate in a six-well plate. Cells
were transfected with plasmids (1.5 μg) containing promoter–
reporter constructs in which expression of the firefly (Photinus
pyralis) luciferase gene was driven by variable-length promoter
regions of the rat Pref-1 gene [ − 2.5KPref-1–Luc, − 412Pref1–Luc and − 42Pref-1–Luc; gifts from Dr Hiroshi Takemori
(Osaka University Medical School, Osaka, Japan)], as described
previously [23]. Empty pGL3 vector (1.5 μg, Promega) was
used as a negative control. In each transfection, cells were
co-transfected with phRL-TK sea pansy (Renilla reniformis)
luciferase expression vector (0.5 ng, Promega) as a control
for transfection efficiency. Where indicated, cells were cotransfected with 0.5 μg of plasmid vectors driving the expression
of C/EBPα (pMSV-C/EBPα) and C/EBPδ (pMEX-C/EBPδ),
kindly provided by Professor Steven McKnight (University of
Texas Southwestern Medical Center, Dallas, TX, U.S.A.) and Dr
Peter Johnson (Laboratory of Protein Dynamics and Signaling,
NCI-Frederick, Frederick, MD, U.S.A.) respectively. Expression
c The Authors Journal compilation c 2012 Biochemical Society
802
Figure 1
J. Armengol and others
Expression of the Pref-1 gene in developing BAT
(A) Pref-1 mRNA expression during BAT development was measured in parallel with mRNA expression of UCP1, a differentiation marker. Each point corresponds to the mean of mRNA expression of
at least three independent experiments for each time point of development, expressed as percentages of the mean value at the time of maximal expression, which was set to 100 (arbitrary units). (B)
Representative Northern blot of Pref-1 mRNA changes during BAT development. Liver and lung samples from adult mice were included as negative controls.
The − 381/ − 37 fragment of mouse Pref-1 was amplified with
the primer pair 5 -GCGCGGGACTCCAGCCCTAAGT-3 and
5 -GCGGTGCAGGGGCTGCTCCGGG-3 . A primer set that
amplified a + 6438/ + 6825 fragment of Ucp3 (5 -CATAGGCAGCAAAGGAAC-3 and 5 -CTATATGGTTTACACAGC-3 )
was used as a control for Pref-1 enrichment. When required,
quantitative analysis was performed by densitometry (Phoretics
ID software).
Statistical analysis
Results are expressed as means +
− S.E.M. Differences between
means were analysed using Student’s t test; P < 0.05 was
considered to be statistically significant.
Table 1 Stereological analysis of lipid content and mitochondrial inner
membrane surface ratio in BAT from wild-type and Pref-1-null mice
Values are means +
− S.E.M. for at least six samples. **P < 0.01 and ***P < 0.001 indicate
statistically significantly differences between Pref-1-null and wild-type mice; # P < 0.05 and
##
P < 0.01 indicate statistical significance for comparisons with respect to fetuses; and
+++
P < 0.001 indicates statistical significance for comparisons with respect to newborns.
SIMM /SEMM , ratio of surface area of inner mitochondrial membrane to surface area of external
mitochondrial membrane.
Developmental stage
Genotype
Lipid content (%)
Mitochondrial inner membrane
surface ratio SIMM /SEMM
Fetus
Wild-type
Pref-1-null
Wild-type
Pref-1-null
Wild-type
Pref-1-null
15.3 +
− 2.7
15.1 +
− 5.0
18.2 +
− 3.8 ,#
4.9 +
− 0.7**
+++
84.9 +
− 2.8 ,##, + + +
46.0 +
7.4***
−
5.32 +
− 1.15
5.20 +
− 0.36
5.22 +
− 1.38
5.18 +
− 0.56
6.14 +
− 0.72
6.68 +
− 0.51
Newborn
Day 21
RESULTS
Developmental regulation of Pref-1 gene expression in BAT
The analysis of Pref-1 mRNA levels by quantitative RT–PCR
(Figure 1A) or Northern blotting (Figure 1B) revealed that Pref-1
was highly expressed in fetal BAT. The highest levels occurred
on fetal day 16 and decreased somewhat in the late fetal period,
prior to birth. The level of Pref-1 mRNA progressively decreased
after birth, becoming almost undetectable in brown fat from 21day-old mice (Figure 1A) and adult BAT, even using highly
sensitive RT–PCR methods. In contrast, Pref-1 mRNA was readily
detected by conventional Northern blotting in BAT from fetuses
and young neonatal mice (Figure 1B). The pattern of Pref1 mRNA expression during BAT differentiation/development
was compared with that of UCP1, a specific marker of brown
adipocytes. Consistent with previous reports [25,26], we found
that Ucp1 mRNA expression was initiated in late fetal life and
continued to increase in early neonatal life, peaking on day 2 after
birth, before gradually declining in adulthood.
Pref-1 is required for an appropriate development of the
thermogenic gene expression program in neonatal BAT
In order to determine the role of Pref-1 in BAT development,
we analysed the impact of targeted deletion of the Pref1 gene in brown fat at distinct stages of development. In
the late fetal period, brown adipocytes from Pref-1-null mice
appeared normal in terms of lipid droplet accumulation and
mitochondrial morphology (Figure 2A and Table 1) However,
c The Authors Journal compilation c 2012 Biochemical Society
at 2 days after birth, brown adipocytes from Pref-1-null mice
showed significantly less accumulation of lipid droplets, although
mitochondria appeared well developed, as in wild-type mice
(Table 1). In young (21-day-old) mice, lipid droplets in Pref-1-null
BAT were smaller than those in wild-type brown fat (Figure 2A)
and occupied less cellular space (Table 1). The mean surface
of inner mitochondrial membrane with respect to the surface of
external mitochondria, an index of mitochondrial maturation in
BAT [19,27], was not significantly altered in Pref-1-null mice
(Table 1). A gene expression analysis revealed that marker genes
of specific brown fat thermogenesis were abnormally overinduced
in brown fat from Pref-1-null mice after birth: in newborns PGC1α was overexpressed, whereas both PGC-1α and UCP1 were
overexpressed in 21-day-old mice (Figure 2B). A similar trend
was observed for 5 -deiodinase, another marker gene of BAT
thermogenic activation (results not shown). In contrast, markers
of overall adipogenesis, which do not distinguish between brown
and white adipocyte phenotypes, such as aP2, were unaltered at
the distinct stages of brown fat development analysed.
Pref-1 mRNA expression in BAT from C/EBPα-null fetuses and
neonates
In order to further analyse the role of Pref-1 in BAT development,
we studied C/EBPα-null mice, a model of specific impairment in
Pref-1 in brown adipose tissue
Figure 2
803
Effects of Pref-1 gene ablation in developing BAT
Analysis of BAT from homozygous Pref-1-null mice (Pref-1 KO) and wild-type (WT) littermates at fetal day 18 (fetus), and 2 days (Newborn) and 21 days after birth. (A) TEM analysis. Two or three
mice were analysed for each genotype and developmental day. Scale bars: 2 μm (upper panels) and 0.4 μm (lower panels). N, nucleus; m, mitochondrion; L, lipid droplet. (B) Each bar shows the
mean +
− S.E.M. of the relative mRNA level for three independent experiments from each time point of development and genotype, expressed as a percentage of the mean value at the time of maximal
expression, which was set to 100 (arbitrary units). Statistical significance of the comparisons between WT and Pref-1-null samples is shown as *P < 0.05.
BAT differentiation during fetal development [15,27]. As shown
in Figure 3(A) and Table 2, brown adipocytes from C/EBPαnull mice showed defective differentiation, characterized by
reduced accumulation of lipids and impaired mitochondrial
maturation. Consistent with this, expression of the Ucp1 gene
was significantly impaired in brown fat from C/EBPα-null fetuses
and neonates (Figure 3B, left-hand panel). Brown fat from mice
lacking C/EBPα showed a dramatic induction of Pref-1 mRNA
levels, both in early fetal life (day 17) or in the immediate postnatal
period (Figure 3B, middle and right-hand panels).
(TG + , C/EBPα + / + ) mice (Figure 4). In fact, Pref-1 mRNA levels
in BAT declined progressively after birth in TG + , C/EBPα − / −
mice and, in 7-day-old transgenic pups, Pref-1 mRNA levels were
less than 5 % of the overexpressed levels in C/EBPα-null fetuses.
A parallel assessment of UCP1 indicated that the level of UCP1
was normalized by postnatal day 7, but not yet by day 2 (results
not shown).
Changes in the expression of C/EBP transcription factors during
BAT development: effects of Cebpa gene invalidation
Pref-1 mRNA expression in BAT during the post-natal period in
mice lacking expression of C/EBPα in BAT
C/EBPα-null mice cannot be studied after birth because the
impairment in liver gluconeogenesis caused by the lack of
C/EBPα leads to mortality in the postnatal period [15].
To overcome this limitation, we used C/EBPα-null mice
incorporating liver-specific transgenic expression of C/EBPα
(TG + , C/EBPα − / − mice) which therefore have normalized
glucose homoeostasis [16]. An analysis of the expression of
Pref-1 in BAT from neonatal TG + , C/EBPα − / − mice showed
that on both days 2 and 7 after birth, Pref-1 mRNA levels were
still overexpressed in these mice relative to wild-type transgenic
The pattern of expression of members of the C/EBP family of
transcription factors during BAT development was determined
(Figure 5). C/EBPα and C/EBPβ expression followed a similar
pattern: low in fetuses and beginning to rise after birth, reaching
high levels at neonatal days 15 and 21. C/EBPδ expression showed
a quite different pattern of developmental regulation. Although
Cebpd mRNA levels were also low in early fetuses, they rose
dramatically a few days before birth and attained the highest
levels at term and in 1-day-old neonates. This was followed by a
rapid decay, such that within a few days after birth and through
day 21 Cebpd mRNA levels were again low. In accordance
with a previous report [27], we confirmed that the expression of
c The Authors Journal compilation c 2012 Biochemical Society
804
Figure 3
J. Armengol and others
Pref-1 mRNA expression in BAT from C/EBPα-null mice
(A) TEM analysis. Scale bars: 8 μm (fetus), and 2 μm, (newborn and day 21). N, nucleus; m, mitochondrion; L, lipid droplet. (B) Left-hand and middle panels: Ucp1 mRNA and Pref-1 mRNA levels
in interscapular BAT from homozygous C/EBPα-null mice (KO) and wild-type (WT) littermates in fetal day 17 and newborn mice. Each bar shows the mean +
− S.E.M. of the relative mRNA level for
three independent experiments from each time point of development and genotype, expressed as a percentage of the mean value at the time of maximal expression, which was set to 100 (arbitrary
units). Right-hand panel: representative Northern blot of Pref-1 mRNA expression. Statistical significance of the comparisons between WT and C/EBPα-null samples are shown as *P < 0.05, and
those between newborns and fetuses is shown as #P < 0.05.
Table 2 Stereological analysis of lipid content and mitochondrial inner
membrane surface ratio in BAT from wild-type and C/EBPα-null fetuses
Values are means +
− S.E.M. for at least six samples. ***P < 0.001 indicates a statistically
significant difference between C/EBPα-null and wild-type mice; # P < 0.05 and ## P < 0.01
indicate statistical significance for the comparison between newborns and fetuses at day 17.
SIMM /SEMM , ratio of surface area of inner mitochondrial membrane with respect to surface area
of external mitochondrial membrane.
Developmental stage
Genotype
Fetus day 17
Wild-type
C/EBPα-null
Wild-type
C/EBPα-null
Newborn
Mitochondrial inner membrane
Lipid content (%) surface ratio SIMM /SEMM
16.4 +
− 4.4
0.4 +
− 0.2***
14.8 +
− 1.4 ,##
1.4 +
− 0.4***
4.30 +
− 0.27
0.41 +
− 0.02***
#
5.22 +
− 0.31 ,##
1.40 +
0.18***
−
C/EBPβ and C/EBPδ proteins were overinduced in brown fat from
C/EBPα-null neonates compared with wild-type neonates. The
active LAP form of C/EBPβ was induced 3-fold, the inhibitory
LIP form of C/EBPβ protein was induced 1.7-fold and C/EBPδ
was induced 3.7-fold. Moreover, the expression of C/EBPδ, but
not C/EBPβ, remained slightly increased in BAT from 7-day-old
TG + , C/EBPα − / − mice (2.2-fold induction), also in agreement
with previous reports [16].
c The Authors Journal compilation c 2012 Biochemical Society
Pref-1 mRNA expression in primary brown adipocytes
differentiating in culture
The mouse model studies above suggested that Pref-1 regulation
is modulated by C/EBPα and/or by effects secondary to Cebpa
gene ablation (e.g. overexpression of other C/EBPs). To gain
insight into the relationship between Pref-1 expression and
C/EBPs in BAT differentiation, we undertook further studies
using a cell culture model of brown adipocyte differentiation.
For primary cultures, non-differentiated precursor cells were
obtained from mouse BAT and cultured under conditions that
lead to brown adipocyte differentiation. Day 3 cultured cells
had not yet differentiated, showing a fibroblast-like appearance
and no lipid accumulation (Figure 6A). At this stage of culture,
expression of brown adipocyte differentiation markers (Ucp1,
Co2 or aP2) was low, whereas Pref-1 mRNA levels were high.
As differentiation progressed, cells acquired a brown adipocyte
morphology, characterized by lipid accumulation and increased
expression of differentiation markers, and Pref-1 mRNA was
dramatically down-regulated (Figure 6A).
In this experimental setting, Cebpa and Cebpb mRNA
expression increased as cells differentiated, more abruptly for
Cebpa and progressively for Cebpb. In contrast, Cebpd expression
was highest in non-differentiated cells (days 3 and 5 of culture)
and decreased markedly with brown adipocyte differentiation
(Figure 6B). These changes at the mRNA level were confirmed at
the protein level (Figure 6C). Thus, Cebpa gene-encoded proteins
Pref-1 in brown adipose tissue
805
Figure 4 Pref-1 mRNA expression in BAT from trangenic C/EBPα-null mice
expressing C/EBPα in liver
Pref-1 mRNA in interscapular BAT from transgenic homozygous C/EBPα-null mice (Tg + ,
C/EBPα-KO) and wild-type (Tg + , WT) at neonatal days 2 and 7. Each bar shows the
mean +
− S.E.M. of the relative mRNA level from three independent experiments for each time
point of development and genotype, expressed as a percentage of the mean value at the time
of maximal expression, which was set to 100 (arbitrary units). Statistical significance of the
comparisons between wild-type (Tg + , C/EBPα + / + ) and knockout (Tg + , C/EBPα − / − )
samples are shown as *P < 0.05.
(specifically the 42 kDa form) were higher in differentiated brown
adipocytes (day 7) than in non-differentiated brown adipocytes
(day 4). The same was observed for the C/EBPβ proteins
LAP and LIP, although in fact the increase in LIP expression
(measured as band density in immunoblots) was more marked
in differentiated brown adipocytes. However, the opposite was
observed for C/EBPδ, which was dramatically down-regulated
in differentiated brown adipocytes (Figure 6C). These results
indicated that, during differentiation of brown adipocytes, the
expression profile of Pref-1 is inversely related to C/EBPα and
C/EBPβ, but positively correlated with that of C/EBPδ.
In primary cultures of brown adipocytes, proliferation arrest
and differentiation occur within a short period of time (between
day 3/4 and day 7 of culture) making it impossible to distinguish
events that occur in association with each process. To circumvent
this limitation, we used HIB-1B cells as a second model of
brown adipocyte differentiation. These cells can be studied during
active proliferation and upon reaching confluence and ceasing
proliferation, but in the absence of differentiation. They can also
be studied several days after the induction of differentiation
of confluent cells when they have acquired morphological
(lipid accumulation) and molecular (induction of Ucp1 mRNA
expression by noradrenaline) features of differentiated brown
adipocyte [21,22]. Using this model, we found that C/EBPα
proteins were significantly expressed only in differentiated HIB1B brown adipocytes, and C/EBPβ LAP and LIP proteins
were also mostly expressed in differentiated cells. In contrast,
C/EBPδ expression was high in non-differentiated HIB-1B
cells, both proliferating and confluent, and was reduced in
association with differentiation (Figure 7A). Pref-1 mRNA
levels were lower in differentiated HIB-1B brown adipocytes
than in non-differentiated cells, either proliferating or confluent
(Figure 7B), consistent with the positive association between Pref1 and C/EBPδ expression observed in primary brown adipocyte
cultures. These results indicate that the expression of C/EBPδ, in
contrast with C/EBPα and C/EBPβ, is negatively associated with
differentiation and positively associated with Pref-1 expression in
the HIB-1B brown adipocyte cell model.
Figure 5
Expression of C/EBP genes in developing BAT
Changes in Cebpa (C/EBPα), Cebpb (C/EBPβ) and Cebpd (C/EBPδ) mRNA expression. Each
point corresponds to the mean for at least three independent experiments at each time point of
development, expressed as a percentage of the mean value at the time of maximal expression,
which was set to 100 (arbitrary units).
Transcriptional regulation of the Pref-1 gene by C/EBPs:
transcriptional activation by C/EBPδ
The previous results prompted us to investigate the direct effects
of C/EBPs on Pref-1 gene expression. To this end, we transiently
transfected HIB-1B with a Pref-1 gene promoter–luciferase
reporter construct and studied the effects of co-transfection of
expression vectors for individual C/EBP proteins (Figure 8). As
shown in Figure 8(A), whereas C/EBPα did not significantly affect
Pref-1 promoter activity, the inhibitory LIP form of C/EBPβ
reduced Pref-1 promoter activity and the activating LAP form
significantly induced it. However, the strongest induction of the
Pref-1 gene promoter was observed with co-transfection of an
expression vector for C/EBPδ. An in silico analysis of the Pref-1
gene promoter sequence revealed the presence of several potential
C/EBP-binding sites in the proximal promoter region, between
− 412 and − 47. Deletion analysis showed that most of the
responsiveness to C/EBPδ was lost in a short construct containing
c The Authors Journal compilation c 2012 Biochemical Society
806
Figure 6
J. Armengol and others
Expression of Pref-1, marker genes of brown adipocyte differentiation markers and C/EBPs in brown adipocytes differentiating in primary culture
(A) Pref-1, Co2 (COII), aP2 and Ucp1 mRNA levels (left-hand panel), and representative micrographs of cultures of non-differentiated brown pre-adipocytes (day 3) and differentiated brown
adipocytes (day 7) (right-hand panel). (B) Cebpa (C/EBPα), Cebpb (C/EBPβ) and Cebpd (C/EBPδ) mRNA levels. Each point corresponds to the mean for three experiments from independent cell
cultures expressed as a percentage of the mean value at the time of maximal expression of each gene, which was set to 100 (arbitrary units). (C) Immunoblot analysis of C/EBPα, C/EBPβ and
C/EBPδ proteins at 4 and 7 days of culture. Molecular mass in kDa is indicated on the right-hand side of each blot.
only the − 47 region, thus indicating that C/EBPδ responsiveness
lies in this − 412/ − 47 region (Figure 8B). Moreover, a longer
construct, containing − 2.5 kb of the Pref-1 promoter did not
exhibit enhanced responsiveness to C/EBPδ relative to that of the
− 412 construct (results not shown). Thus it is unlikely that other
sites in the 5 region of the promoter upsteam of − 412 were
significantly involved in mediating the C/EBP responsiveness of
the Pref-1 gene.
In order to ascertain whether the activating role of C/EBPδ
on the Pref-1 gene transcription occurs in vivo and affects
endogenous Pref-1 gene expression, a loss-of-function approach
was followed through siRNA-mediated knockdown of C/EBPδ.
By these means, a siRNA reduction in C/EBPδ protein expression
was achieved in HIB-1B cells, and this resulted in a significant
and specific reduction in Pref-1 mRNA, but not in aP2 mRNA
levels (Figure 9), thus confirming the involvement of C/EBPδ in
Pref-1 gene regulation.
ChIP assays reveal C/EBPδ binding to the endogenous Pref-1 gene
promoter
Next, we studied whether C/EBPδ effectively binds to the
proximal promoter region of the Pref-1 gene using ChIP assays.
Because Pref-1 mRNA is highly expressed in BAT from fetuses at
c The Authors Journal compilation c 2012 Biochemical Society
term, a time that corresponds to the point in brown fat development
when C/EBPδ expression is highest (see Figure 5), we used BAT
from fetal day 19 mice for these assays, and we compared the
results with those obtained using BAT from 21-day-old mice,
a condition of almost negligible expression of the Pref-1 gene.
As shown in Figure 10(A), the fragment of DNA corresponding
to the 5 region of the Pref-1 gene was specifically enriched
when chromatin from fetal BAT was immunoprecipitated with
a C/EBPδ-specific antibody, but not when 21-day-old BAT was
analysed. Identical enrichment due to anti-C/EBPδ antibody was
observed in HIB-1B cells (Figure 10B).
Effects of dexamethasone on Pref-1, C/EBPδ expression and
differentiation of brown adipocytes
The finding that C/EBPδ was a direct activator of Pref-1 gene
transcription in brown adipocytes led us to investigate the role of
glucocorticoids, considering that C/EBPδ is considered a direct
target of dexamethasone, at least in the context of white adipocyte
differentiation [28]. Exposure of brown adipocyte precursors
to dexamethasone during the differentiation process completely
prevented the decay in Pref-1 mRNA expression (Figure 11A).
However, the appearance of brown adipocyte morphology was
completely preserved (Figure 11B). Dexamethasone caused
Pref-1 in brown adipose tissue
Figure 8
807
Effects of C/EBPs on Pref-1 promoter activity
(A) HIB-1B cells were co-transfected with − 412-Pref-1–Luc (luciferase) and C/EBPα,
LAP, LIP or C/EBPδ expression vectors, or empty vector (control). (B) HIB-1B cells were
transfected with − 412-Pref-1–Luc or the deletion-mutant form − 47-Pref-1–Luc, and
co-transfected with a C/EBPδ expression vector. Results are means +
− S.E.M. for at least three
independent experiments performed in triplicate. Statistical significance of differences with
respect to controls (C) is shown as *P < 0.05, and that for the comparison of the activity of the
two promoter constructs at a similar co-transfection condition is shown as #P < 0.05.
Figure 7 Changes in C/EBP proteins across the three stages of H1B-1B cell
differentiation
(A) Representative immunoblot of three independent assays of protein extracts from proliferating,
confluent and differentiated HIB-1B cells (see the Materials and methods section). Estimated
sizes of C/EBP proteins in kDa are indicated on the right-hand side. CRM, cross-reactive material.
(B) Bars show mean Pref-1 mRNA levels +
− S.E.M. for at least three independent experiments
performed in triplicate. Statistically significant differences compared with proliferating cells are
shown as **P < 0.01.
a significant increase in Cebpd expression, an effect that
was associated with dramatically impaired induction of the
specific brown adipocyte markers Ucp1 and Ppargc1a (encoding
PGC-1α) (Figure 11C). In accordance with the morphological
observations, the marker gene of overall adipocyte differentiation,
aP2, was unaltered.
DISCUSSION
During development of rodents, BAT develops much earlier than
WAT. In fact, brown adipocytes are considerably differentiated at
birth, a time when WAT is practically absent. BAT activation
and differentiation progress after birth, achieving maximal
activity and differentiation in neonates and young pups [1,25,26].
During this period time, we observed that Pref-1 expression
in BAT was progressively down-regulated, from very high
levels in fetuses to practically undetectable levels several weeks
after birth. This would be consistent with a repressive role
of Pref-1 on brown adipocyte differentiation, similar to what
is known to occur in white adipocytes. However, the fact
that mitochondriogenesis, lipid accumulation and expression of
marker genes of thermogenesis in BAT from Pref-1-null fetuses
were normal suggests that, during fetal life, brown adipocyte
differentiation in the absence of environmental thermal stress is
not responsive to changes in Pref-1 levels. In contrast, Pref-1null mice showed clear signs of thermogenic over-activation in
BAT from neonates and young pups, including overexpression of
Figure 9
Effects of C/EBPδ knockdown on Pref-1 gene expression
C/EBPδ protein (A), and Pref-1 and aP2 mRNA levels (B) in HIB-1B cells previously treated with
siRNA for C/EBPδ or scrambled siRNA [control (Ctrl)]. An example of a C/EBPδ immunoblot is
shown in the right-hand panel in (A). Results are means +
− S.E.M. for at least three independent
experiments performed in triplicate. Statistical significance of differences with respect to controls
is shown as *P < 0.05 and **P < 0.01.
thermogenic marker genes and evidence of enhanced mobilization
of lipids previously accumulated during fetal development. These
findings indicated that Pref-1 could have a specific role in the
acquisition of the thermogenic pattern of gene expression in
BAT, without significantly impacting other features of the brown
adipocyte phenotype. Although the specific role of Pref-1 in
BAT had not previously been directly addressed, the present
findings are consistent with observations in some rodent models
c The Authors Journal compilation c 2012 Biochemical Society
808
J. Armengol and others
Figure 10 ChIP analysis of the binding of C/EBPδ to the endogenous Pref-1
gene promoter in BAT and HIB-1B cells
Anti-C/EBPδ antibody or pre-immune serum was used to immunoprecipitate protein–DNA
complexes in late fetal BAT and 21-day-old BAT (A), and in HIB-1B cells (B). Representative
images of PCR amplification are shown; arrowheads indicate the 530 bp Pref-1 gene PCR
product and the 400 bp Ucp3 gene PCR product (Control). Bars show means +
− S.E.M. for four
independent experiments as arbitrary units of fold enrichment with respect to pre-immune serum
after densitometric scanning of PCR products. Statistical significance of differences between
anti-C/EBPδ antibody and pre-immune incubation is shown as *P < 0.05.
of lipodystrophy in which BAT depots show enhanced levels of
Pref-1 in association with a specific loss of thermogenic properties
but enhanced fat accumulation [29]. Supporting this notion,
expression of the thermogenic gene Ucp1 is strongly impaired
in BAT from transgenic mice overexpressing Pref-1 in both WAT
and BAT, whereas other genes related to fat accumulation, such
as aP2, are only mildly reduced in BAT [10].
These observations prompted us to further analyse the role
and regulation of Pref-1 in the process of BAT development
and differentiation using C/EBPα-null mice, a well established
model of inhibition of BAT differentiation in the perinatal period
[27]. These analyses revealed a dramatic induction of Pref-1
gene expression in association with impaired expression of Ucp1
and delayed acquisition of the specific features of thermogenic
activation of BAT. The high levels of Pref-1 mRNA expression in
the BAT from C/EBPα-null mice could potentially reflect relief
from a repressive effect of C/EBPα on the Pref-1 gene by the
targeted disruption of the Cebpa gene. This possibility would
be compatible with the hypothesis that sustained high levels of
C/EBPα are required for maintaining low Pref-1 gene expression
in the differentiated brown adipocyte. However, this hypothesis
is inconsistent with the time-course of Pref-1 and Cebpa gene
expression throughout brown adipocyte differentiation, observed
in the cell culture models reported in the present study. In
these models, C/EBPα induction is a late phenomenon in the
differentiation of brown adipocytes, whereas down-regulation
c The Authors Journal compilation c 2012 Biochemical Society
of the Pref-1 mRNA occurs earlier. Notably, the analysis of
the transcriptional regulation of the Pref-1 gene revealed that
C/EBPα did not have a significant impact on Pref-1 gene
promoter transcription, whereas another subtype of the C/EBP
family, C/EBPδ, had a powerful inductive effect. Moreover,
experimentally induced reduction of C/EBPδ led to a specific
reduction in Pref-1 gene expression. The identification of C/EBPδ
as a novel and powerful factor in the control of Pref-1 gene
transcription strongly suggests that the overexpression of C/EBPδ
expression that takes place in the BAT of C/EBPα-null mice ([27]
and the present study) could elicit the persistently high levels of
Pref-1 mRNA observed in BAT from these mice.
The positive action of C/EBPδ on the Pref-1 gene suggests
a specific role for this transcription factor in brown adipocyte
differentiation. In white adipocytes, C/EBPδ is induced in the
first stages of the differentiation process, a period when Pref1 expression is undergoing down-regulation [30]. The pattern
of expression of C/EBP subtypes during brown adipocyte
differentiation reported in the present study, both ‘in vivo’ (late
fetal development) and in the two cell culture models analysed
(primary cultures of murine brown pre-adipocytes and the HIB1B brown adipocyte cell line) indicated that C/EBPδ is highly
expressed in the brown pre-adipocyte stage, in association with
high levels of Pref-1. Given the repressive role of Pref-1 on
thermogenic gene expression which could be inferred from results
obtained with Pref-1-null mice, we hypothesized that C/EBPδ
could play an inhibitory role in thermogenic gene expression in
brown adipocytes. This potential role of C/EBPδ could be related
to the differential effects of glucocorticoids on brown adipocyte
differentiation, especially compared with white adipocytes.
Dexamethasone is a powerful inducer of Cebpd gene expression
[28] as well as a repressor of Pref-1 gene expression [31] in
white pre-adipocytes, events that are considered to favour white
adipocyte differentiation. In contrast, glucocorticoids are known
to repress the expression of BAT-specific thermogenic genes in
brown adipocytes [32], without altering other features common
to white adipocyte differentiation such as lipid accumulation.
In fact, it has been proposed that, in some cell models,
glucocorticoids specifically block brown adipocyte differentiation
of precursor cells, driving their differentiation pattern towards
a white-adipocyte-like phenotype [33]. Our investigation of the
action of dexamethasone in brown adipocyte differentiation
and the C/EBPδ–Pref-1 axis established that dexamethasone
blunted the Pref-1 down-regulation that occurred with normal
brown fat differentiation. These actions were associated with
an induction of C/EBPδ expression, maintained acquisition
of adipocyte morphology and induction of non-BAT-specific
genes on the one hand, and dramatic repression of thermogenic
genes such as Ucp1 or Ppargc1a on the other hand. These findings
indicate that maintenance of high Pref-1 levels is not compatible
with the acquisition of the brown-adipocyte-specific pattern of
thermogenic gene expression. Again, these observations in cell
culture models are fully consistent with the over-induction of
thermogenic genes observed in BAT from Pref-1-null mice, and
with previous reports of the association of high Pref-1 with
impaired expression of UCP1 but near-normal fat accumulation
in BAT in distinct rodent models [10,29].
In summary, we report in the present study that Pref-1 is an
important negative regulator of the specific thermogenic pattern
of gene expression in BAT. C/EBPδ is a powerful inducer of Pref1 gene transcription, and its induction by glucocorticoids is likely
to provide an indirect mechanism for glucocorticoid induction
of the Pref-1 gene in brown pre-adipocytes. The C/EBPδmediated induction of Pref-1 by glucocorticoids is thus expected
to contribute to the specific inhibitory action of glucorticoids
Pref-1 in brown adipose tissue
Figure 11
809
Effects of dexamethasone on the differentiation of brown adipocytes, and the expression of Pref-1 and C/EBPδ
(A) Pref-1 mRNA expression during the differentiation of brown adipocytes in primary culture exposed to 30 nM dexamethasone (Dexa) from day 4 onwards. Each point corresponds to the
mean +
− S.E.M. for four experiments from independent cell cultures expressed as a percentage of the mean value at the time of maximal expression, which was set to 100 (arbitrary units). (B)
Representative micrographs of brown adipocytes on day 8 of culture, grown in the absence (control) or presence of 30 nM dexamethasone. (C) mRNA expression of C/EBPδ and marker genes
of brown adipocyte differentiation in pre-adipocytes (day 5) and differentiated (day 8) brown adipocytes. Each bar is the mean +
− S.E.M. of the relative mRNA level for at least four independent
experiments for each condition, expressed as a percentage of the mean value at the time of maximal expression, which was set to 100 (arbitrary units). Statistically significant differences due to
dexamethasone at each time of culture are shown as *P < 0.05, **P < 0.01, ***P < 0.001 and those between day 5 and day 8 are shown as #P < 0.05.
on BAT thermogenic gene expression. The specific role and
regulation of Pref-1 in BAT shown here further highlight the
distinct processes controlling differentiation of the white and
brown adipocyte cell lineages, findings that could inform efforts to
modulate BAT activity and differentiation as a means to promote
energy expenditure, decrease white fat accumulation and alleviate
adiposity in humans.
AUTHOR CONTRIBUTION
Jordi Armengol, Josep Villena, Elayne Hondares and Marı́a Carmona performed the
experiments and analysed the data. Hei Sook Sul, Roser Iglesias, Marta Giralt and Francesc
Villarroya designed the research and analysed the data. Jordi Armengol, Josep Villena
and Francesc Villarroya co-wrote the paper. Francesc Villarroya supervised the research.
ACKNOWLEDGEMENTS
We thank Hiroshi Takemori (Osaka University Medical School, Osaka, Japan) for Pref-1
promoter plasmid constructs, and Steven McKnight (University of Texas Southwestern
Medical Center, Dallas, TX, U.S.A.), Peter Johnson (Laboratory of Protein Dynamics
and Signaling, NCI-Frederick, Frederick, MD, U.S.A.) and Ueli Schibler (Department of
Molecular Biology, University of Geneva, Geneva, Switzerland) for plasmid expression
vectors. We also acknowledge Gretchen Darlington (Baylor College of Medicine, Houston,
TX, U.S.A.) for providing C/EBPα-null mice.
FUNDING
This study was supported by the Ministerio de Ciencia e Innovación, Spain [grant numbers
SAF2008-01896 and SAF2011-23636].
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Received 26 September 2011/25 January 2012; accepted 13 February 2012
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