Obesity
Original Article
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
ERRc Enhances UCP1 Expression and
Fatty Acid Oxidation in Brown Adipocytes
Karen Dixen1, Astrid L. Basse1, Maria Murholm1, Marie S. Isidor1,
Lillian H. L. Hansen1, M. Christine H. Petersen1, Lise Madsen2,3,
Natasa Petrovic4, Jan Nedergaard4, Bjørn Quistorff1 and Jacob B. Hansen1
Objective: Estrogen-related receptors (ERRs) are important regulators of energy metabolism. Here we
investigated the hypothesis that ERRc impacts on differentiation and function of brown adipocytes.
Design and Methods: We characterize the expression of ERRc in adipose tissues and cell models and
investigate the effects of modulating ERR? activity on UCP1 gene expression and metabolic features of
brown and white adipocytes.
Results: ERRc was preferentially expressed in brown compared to white fat depots, and ERRc was
induced during cold-induced browning of subcutaneous white adipose tissue and brown adipogenesis.
Overexpression of ERRc positively regulated uncoupling protein 1 (UCP1) expression levels during brown
adipogenesis. This ERRc-induced augmentation of UCP1 expression was independent of the presence of
peroxisome proliferator-activated receptor coactivator-1 (PGC-1a) but was associated with increased rates
of fatty acid oxidation in adrenergically stimulated cells. ERR? did not influence mitochondrial biogenesis,
and its reduced expression in white adipocytes could not explain their low expression level of UCP1.
Conclusions: Through its augmenting effect on expression of UCP1, ERRc may physiologically be
involved in increasing the potential for energy expenditure in brown adipocytes, a function that is
becoming of therapeutic interest.
Obesity (2013) 21, 516-524. doi:10.1002/oby.20067
Introduction
Whereas white adipose tissue (WAT) stores energy in the form of
triacylglycerol, brown adipose tissue (BAT) has a high capacity for
energy dissipation through adaptive thermogenesis. Characteristics
of BAT compared to WAT include the expression of uncoupling
protein 1 (UCP1) and high mitochondrial number and activity (1).
In response to, for example, cold or treatment with b-adrenergic
agonists, thermogenic brown-like adipocytes will appear in WAT, a
process termed adipose browning (2). Several studies in rodents
have shown that brown and brown-like adipocytes have a marked
anti-obesity effect and are involved in defending normal body temperature in response to cold (1).
Adipogenesis is controlled by numerous transcription factors of
which peroxisome proliferator-activated receptor c (PPARc) and
members of the CCAAT/enhancer-binding protein family are principal regulators (3). A number of transcription factors differentially
control the differentiation of brown and white preadipocytes, for
example, PPARc coactivator-1a (PGC-1a) (4), PGC-1b (5), and PR
domain containing 16 (PRDM16) (6) that stimulate brown adipocyte
differentiation, whereas, for example, receptor interacting protein
140 (RIP140) (7) and the retinoblastoma protein (8) inhibit brown
adipocyte formation.
The estrogen-related receptors (ERRs) are orphan nuclear receptors
with key functions in cellular energy metabolism. The ERR family
consists of ERRa, ERRb, and ERRc that are closely related to estrogen receptors (ERs). ERRc is structurally more closely related to
ERRb than to ERRa, but the expression pattern of ERRc resembles
that of ERRa, with abundant expression in mitochondria-rich tissues
with high energy demands, such as heart, brain, kidneys, BAT, and
1
Department of Biomedical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark. Correspondence: Jacob B. Hansen (jbhansen@sund.ku.dk)
Department of Biology, University of Copenhagen, Denmark, DK-2100 Copenhagen Ø, Denmark 3 National Institute of Nutrition and Seafood Research, N5817 Bergen, Norway 4 The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden
2
Disclosure: The authors declare no conflict of interest directly related to the data presented here.
Funding agencies: We appreciate the gift of valuable reagents from Bruce M. Spiegelman (Harvard Medical School, Dana Farber Cancer Institute, Boston), C. Ronald
Kahn (Joslin Diabetes Center, Harvard Medical School, Boston), Hueng-Sik Choi (Chonnam National University, Gwangju, Korea), Piia Aarnisalo (University of Helsinki,
Helsinki University Central Hospital, Finland), and Amgen (California). This work was supported by grants to J.B.H. from the EU FP7 project DIABAT (HEALTH-F2-2011278373), Danish Medical Research Council, the Novo Nordisk Foundation, the Carlsberg Foundation, the Aase and Ejnar Danielsen Foundation, the Augustinus
Foundation, the Hartmann Brothers’ Foundation and the Beckett Foundation, to B.Q. from the Danish Strategic Research Council (09-067124 and 09-059921) and the
European Union through the network of excellence, BioSim (contract no. LDHB-CT-2004-005137) and to J.N. from the Swedish Science Council.
Additional Supporting Information may be found in the online version of this article.
Received: 16 March 2012 Accepted: 14 August 2012 Published online 3 October 2012. doi:10.1002/oby.20067
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Original Article
Obesity
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
slow-twitch skeletal muscle (9). Both ERRa and ERRc are induced
during adipocyte differentiation (10-12).
ERRs act as constitutively active transcription factors that interact
with a number of coregulatory proteins modulating their transcriptional activity. Notably, the key regulators of energy metabolism
PGC-1a and -1b enhance the transcriptional activity of ERRs (13,14).
Moreover, several studies indicate that at least some of the metabolic
processes controlled by PGC-1a may be transduced by ERRs (15-17).
A natural ligand is apparently not required for ERR activity, suggesting that the relative concentration of ERRs and/or coregulators in a
tissue may determine their transactivation potential (9).
ERRs can regulate transcription of genes driven by ERR response
elements (ERREs) (9). An ERRE is present in the enhancer of the
UCP1 gene, and recruitment of ERRa to this ERRE can activate
transcription of the UCP1 gene (18). However, ERRa/ mice had
normal induction of UCP1 in BAT in response to cold, indicating
that ERRa is not essential for expression of UCP1 (19). It is not
known whether ERRb and ERRc regulate UCP1 expression and
adipose tissue function.
In the present study, we have therefore characterized the expression
of ERRc in adipose tissues and adipocytes as well as investigated the
impact of modulating ERRc activity on UCP1 gene expression and
metabolic features of brown and white adipocytes. We found that
ERRc markedly enhanced UCP1 expression and fatty acid oxidation
in brown adipocytes but that the low expression level of UCP1 in
white adipocytes was not explainable by their low ERRc levels.
blasts (MEFs), 3T3-L1 (22), and WT-1 preadipocytes (23) (kindly
provided by Dr. C. Ronald Kahn) were propagated and differentiated
as described (20,24). Briefly, 1-day postconfluent cells (designated
day 0) were induced to differentiate in DMEM containing 10% FBS
and supplemented with 1 lM dexamethasone, 0.5 mM methylisobutylxanthine, 5 lg/ml insulin, and 0.5 lM rosiglitazone for 2 days. From
day 2, medium consisted of DMEM containing 10% FBS and supplemented with 5 lg/ml insulin and 0.5 lM rosiglitazone, and the
medium was changed every other day. Immortalized PGC-1aþ/þ and
PGC-1a/ brown preadipocyte cell lines were kindly provided by
Dr. Bruce M. Spiegelman (5) and were cultured and differentiated like
WT-1 cells. For chronic treatment of Rb/ and WT-1 cells, 4-OHT
(10 lM) or vehicle was supplemented with the regular medium change
every other day during differentiation, starting at day 0. For treatment
of mature adipocytes, Rb/ and WT-1 adipocytes deprived of rosiglitazone and insulin from day 4, were exposed to 4-OHT (10 lM) or
vehicle at day 8, and harvested 48 h later.
Packaging and use of retrovirus were performed as described
(20,24). Transduced cells were selected with 8 lg/ml blasticidin S
HCl or 5 lg/ml puromycin, except for 3T3-L1 cells that were
selected with 5 lg/ml blasticidin S HCl or 3 lg/ml puromycin.
Lactate dehydrogenase release assay
The potential cytotoxicity of 4-OHT was assayed by lactate dehydrogenase (LDH) release into the medium using the in vitro toxicology assay kit according to the instructions of the manufacturer.
Medium from Rb/ and WT-1 adipocytes treated with vehicle,
4-OHT, or Triton X-100 (0.1%, 24 h) (positive toxicity control)
were diluted 10 times in water before measurement.
Materials and Procedures
Materials
Plasmids
Dexamethasone, methylisobutylxanthine, puromycin, 4-hydroxytamoxifen (4-OHT), isoproterenol, norepinephrine (NE), palmitoylcarnitine, and the in vitro toxicology assay kit were obtained from SigmaAldrich. Insulin and cloning enzymes were from Roche. Dulbecco’s
modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and
blasticidin S HCl were obtained from Life Technologies. Rosiglitazone and Adipolysis assay kit were from Cayman Chemical. [1-14C]palmitoylcarnitine was from Perkin Elmer and glucose from Merck.
The retroviral vectors pMSCVpuro link3, pMSCVbsd link3, and
pBabe-puro-TAg have been described (8,20). pcDNA3-mERRc and
pcDNA3-mERRc DAF2 were obtained from Dr. Hueng-Sik Choi
(25). The ERRc fragments were inserted into the HindIII/XhoI site
of pMSCVbsd link3, thereby creating pMSCVbsd-mERRc and
pMSCVbsd-mERRc DAF2. pCMX-mERRc WT, pCMX-mERRc
C125G, and pCMX-mERRc E429A were obtained from Dr. Piia
Aarnisalo (26). To create pMSCVbsd-mERRc WT, pMSCVbsdmERRc C125G, and pMSCVbsd-mERRc E429A, inserts were
inserted into the NotI/ApaI site of pMSCVbsd link3. pMSCVpuromPRDM16 has been described (6) and was purchased from Addgene
(Addgene plasmid 15504). The pcDNA3-hERRa, pcDNA3-hERRb,
and pcDNA-hERRc vectors were obtained from Amgen (27).
pMSCVbsd-hERRa was cloned by inserting the hERRa fragment
into the HindIII/NotI site of pMSCVbsd link3. pMSCVbsd-hERRb
and pMSCVbsd-hERRc were cloned by inserting the hERR fragments into the BamHI/XhoI site of pMSCVbsd link3.
Animals, cell culture, and packaging of virus
Interscapular and perirenal BAT (the latter only from rats) as well
as ovarian, inguinal, and omental WAT were obtained from five 3months old female C57BL/6 mice and Wistar rats (Taconic) kept at
ambient temperature and fed chow diet. The stromal-vascular and
adipocyte fractions (SVF and AF, respectively) were obtained as
described (20). The cold experiment was approved by the Norwegian Animal Health Authorities. Care and handling of mice were in
accordance with local institutional recommendations. Three-months
old male C57BL/6 mice were housed individually and kept at 22 C
(n ¼ 7) or exposed to 4 C (n ¼ 4) for 48 h.
Primary brown preadipocytes were isolated, cultured, and allowed to
undergo spontaneous differentiation essentially as described (21).
Briefly, primary preadipocytes were plated at day 0, reached confluence at day 3, and were considered mature adipocytes at day 7. Wildtype and retinoblastoma gene-deficient (Rb/) mouse embryo fibro-
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RT-qPCR
Real-time quantitative PCR (RT-qPCR) was performed as described
(24). Primers used are described in Supplementary Table S1.
Whole cell extracts and immunoblotting
Preparation of whole-cell extracts and immunoblotting were done as
described (24). Antibodies used have been described (24).
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ERRc and UCP1 Gene Expression Dixen et al.
FIGURE 1 ERRc is enriched in brown compared to white adipose tissues and is induced during cold-induced browning
of subcutaneous white fat. RNA from mouse and rat adipose tissues and mouse WAT and BAT fractions was analyzed
by RT-qPCR. Relative mRNA expression levels of ERRc and UCP1 were determined by normalization to expression
levels of TBP for mouse adipose tissues, whereas expression levels in rat adipose tissues were normalized to TFIIB. (A)
Mouse adipose tissues (n ¼ 5). (B) Rat adipose tissues (n ¼ 5). (C) Stromal-vascular fractions (SVF) and adipose fractions (AF) from mouse WAT and BAT. (D) Inguinal WAT and interscapular BAT from mice kept at 22 C (n ¼ 7) or at
4 C (n ¼ 4) for 48 h. In all panels, data represent mean þSEM. *, P < 0.05 versus interscapular (Int) BAT for adipose
tissues (panel A and B) or WAT at 22 C (or BAT at 22 C) versus WAT at 4 C (or BAT at 4 C). #, P < 0.05 versus perirenal (Re) BAT for rat adipose tissue. Ing, inguinal; Om, omental; Ov, ovarian.
Quantification of relative mtDNA copy numbers
and lipolysis
Determination of mtDNA copy numbers was carried out as
described (20). Adipolysis assay kit was applied for measuring glycerol content in undiluted medium according to the instructions of
the manufacturer.
Palmitoylcarnitine oxidation
Experiments were performed with cultured adipocytes gently transferred to conical flasks. Palmitoylcarnitine oxidation rate was determined after the addition of 1,000,000 dpm [1-14C]-palmitoylcarnitine together with cold palmitoylcarnitine to a final concentration of
50 lM and cold glucose to a final concentration of 25 mM. Radioactive CO2 was collected and measured. Flasks without cells were
run in parallel and used for background detection. For calculation of
palmitoylcarnitine consumption, the specific activity of [1-14C]-palmitoylcarnitine was determined and palmitoylcarnitine oxidation
was calculated as the sample count corrected for blank divided by
the specific activity of palmitoylcarnitine. Data were normalized to
protein content. Details of this procedure will be described elsewhere (Jørgensen et al., in preparation).
2A) where two dishes were harvested. For WT-1 cells transduced
with ERRc or empty control virus (Figure 4B), one dish was harvested in each of three independent experiments. Data shown for
primary cultures and WT-1 cells transduced with ERRc or empty
control virus are mean of three independent experiments. All other
data shown are from a representative experiment and presented as
mean of the harvested dishes (þSEM). All presented results were
confirmed in two to five independent experiments. Time-course studies (Figure 2B, 4D, and 6A) were analyzed for statistical significance
(P < 0.05) by multiple linear regression of means using PROC REG
(SAS 9.1.2, SAS Institute) with expression level as the dependent
variable and cell type and time as independent variables. It was
assumed that residual variance was identical for the two cell types (or
treatments), and a difference between means was considered statistically significant if there was no overlap between their 95% confidence intervals. All other relevant data were analyzed for statistical
significance (P < 0.05) using Student’s t-test on log-transformed
data. Bonferroni correction was used when multiple comparisons
were performed. Statistical analysis was not conducted on BAT fractions, as the measurements were performed on pools of RNA.
Results
Statistical analyses
For cell culture studies, three dishes were harvested at each time
point and/or treatment in each experiment, except for the PC consumption experiments (Figure 6D), for the treatment of mature adipocytes with 4-OHT (Figure 3B) in which four and six dishes,
respectively, were analyzed, and for the primary cultures (Figure
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ERRc is enriched in BAT compared to WAT and
is induced during cold-induced browning of WAT
and brown adipogenesis in vitro
Characterization of ERRc mRNA expression in different brown and
white adipose tissues and cell lines was performed by RT-qPCR.
Ovarian, inguinal, and omental WAT as well as interscapular and
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Original Article
Obesity
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
ERRc expression level in BAT of mice housed at ambient and cold
temperatures, NE did not influence ERRc expression in primary
brown adipocytes, indicating that the ERRc gene is not responsive to
adrenergic stimulation in brown adipocytes.
MEFs lacking the retinoblastoma gene (Rb/) were applied as a
model of brown adipogenesis (8). During differentiation of Rb/
MEFs, the ERRc mRNA level was robustly up-regulated (15-fold)
from the undifferentiated state (day 0) to the fully differentiated
state (day 8), in parallel with UCP1 (Figure 2B). In contrast, wildtype MEFs, which were used as a model of white adipocyte differentiation (8), have similar ERRc expression at days 0 and 8. Moreover, levels of ERRc mRNA were significantly higher in Rb/
compared to wild-type MEFs at days 4, 6, and 8 (Figure 2B).
FIGURE 2 ERRc is induced during brown adipogenesis in vitro. Total RNA was harvested at the indicated days and analyzed by RT-qPCR. Relative mRNA expression
levels of ERRc and UCP1 were determined by normalization to expression levels of
TBP. (A) Differentiation of primary brown preadipocytes stimulated or not with 1 lM
norepinephrine (NE) for 2 h. Primary cells spontaneously differentiated after reaching confluence at day 3 and became mature fat cells at day 7. (B) Differentiation of
wild-type and Rb/ MEFs as well as 3T3-L1 and WT-1 preadipocytes. Cells were
induced to differentiate at confluence (day 0) and considered mature adipocytes at
day 8. In all panels, data represent mean þSEM. #, P < 0.05 day 8 (day 7 for primary cells) versus undifferentiated state (day 0 for cell lines, day 3 for primary cells).
*, P < 0.05, day X in wild-type MEFs (or 3T3-L1) versus day X in Rb/ MEFs (or
WT-1) (panel B).
perirenal BAT (the latter only from rats) were isolated from threemonths old mice and rats. As expected, the key brown adipose
marker gene UCP1 was highly enriched in BAT depots of these animals (Figure 1A and B). ERRc mRNA was present at substantially
higher levels in BAT of both mouse (>16-fold) and rat (>4-fold)
compared to mouse and rat WAT depots, respectively.
To examine whether the enhanced ERRc expression was associated
with the preadipocyte or the differentiated brown adipocyte state,
we compared the expression of ERRc in the AF and the preadipocyte-containing SVF of mouse BAT and WAT. We found that
ERRc is expressed at 8-fold higher levels in the AF compared to
the SVF of BAT (Figure 1C). Moreover, the AF and SVF from
WAT have comparable expression levels of ERRc, which was markedly lower than in BAT SVF and AF.
Next, we measured adipose expression of ERRc in response to cold
exposure. In inguinal WAT, ERRc expression increased 3-fold after
48 h of cold exposure, concomitant with a robust induction of UCP1
mRNA (Figure 1D). Expression of ERRc in interscapular BAT
trended to increase in cold (P ¼ 0.06).
We further examined the expression of ERRc in primary and immortalized cells during adipogenesis. Primary brown preadipocytes were
isolated from mice and cultured to undergo spontaneous adipogenesis.
RNA was harvested at days 3 and 7 and analyzed by RT-qPCR. During conversion from the preadipocyte (day 3) to the mature adipocyte
state (day 7), expression of ERRc tended to increase (2-fold), both
with and without norepinephrine (NE) stimulation for 2 h prior to harvesting (Figure 2A). In mature primary brown adipocytes, UCP1 was
expressed at low basal levels, but NE stimulation causes a strong
induction of UCP1 mRNA (Figure 2A). Consistent with the similar
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In addition, ERRc expression was measured in the brown and white
preadipocyte cell lines WT-1 and 3T3-L1, respectively. ERRc was
expressed at comparable levels at day 0 in 3T3-L1 and WT-1 cells;
however, at day 8, ERRc expression was markedly increased in WT-1
cells compared to day 0, whereas it was decreased in 3T3-L1 cells (Figure 2B). UCP1 expression was strongly induced only in WT-1 cells at
day 8 (Figure 2B). Notice that despite the substantially increased
expression of ERRc in Rb/ and WT-1 brown adipocytes, the levels
are still low compared to BAT (compare Figure 1A, D, and 2B).
Collectively, these data demonstrate that ERRc expression is higher in
brown compared to white adipocytes and that it increases during browning of subcutaneous WAT and brown adipocyte differentiation in vitro.
An ERRc inverse agonist reduces UCP1
expression in brown adipocytes
Since ERRc was expressed at a higher levels in brown compared to
white adipocytes and was up-regulated during brown adipocyte differentiation, we investigated if lowering of ERRc activity would
affect UCP1 expression in the Rb/ and WT-1 models of brown
adipogenesis. We were not able to obtain significant knockdown of
ERRc by viral delivery of short hairpin RNA. Instead, we treated
Rb/ and WT-1 cells with 4-OHT, a compound displaying inverse
agonist activity toward ERRc (27,28). To rule out that 4-OHT
exerted toxic effects that might influence the interpretation of the
experiments, we measured cellular lactate dehydrogenase release in
the treatment regimens described below (Supplementary Figure S1).
From those measurements, we conclude that 4-OHT does not elicit
toxic effects in Rb/ and WT-1 cells.
Treatment with 4-OHT throughout the course of differentiation
(days 0-8) resulted in a 4- to 5-fold lower expression of UCP1 at
day 8 compared to cells treated with vehicle (Figure 3A). Treatment
with 4-OHT during differentiation had minor effects on expression
of adiponectin and FABP4 mRNAs (Figure 3A).
We also tested the effect of exposing mature Rb/ and WT-1
brown adipocytes to 4-OHT from days 8 to 10. Expression of UCP1
was reduced 2.5- to 3-fold in both Rb/ and WT-1 brown adipocytes by the 48 h of treatment with 4-OHT without a concomitant
effect on adiponectin and FABP4 mRNA levels (Figure 3B). Together, these data suggest that attenuating the activity of ERRc in
both differentiating and mature brown adipocytes results in
decreased UCP1 expression, with no effect on overall adipogenesis.
Obesity | VOLUME 21 | NUMBER 3 | MARCH 2013
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ERRc and UCP1 Gene Expression Dixen et al.
changes in expression at days 0 and 8 in response to forced ERRc
expression (data not shown). Comparison of UCP1 levels in Rb/
cells retrovirally transduced with ERRa, ERRb, or ERRc revealed
that only ERRc was able to increase expression of UCP1 mRNA, at
least under the conditions used here (Figure 4C).
To identify at what stage during differentiation the increased UCP1
expression became apparent in ERRc-transduced cells, we conducted
a time-course study of Rb/ cells transduced with ERRc or empty
control virus. Samples were harvested at days 0, 4, 6, and 8 and analyzed by RT-qPCR and immunoblotting. FABP4 protein levels
were similar in ERRc-transduced and control cells during differentiation (Figure 4E). Interestingly, UCP1 expression was not only
induced to higher levels in cells with increased ERRc expression,
but was also induced earlier during the course of differentiation
compared to vector cells. This was true both at the mRNA and protein level, the protein level of UCP1 being dramatically increased in
cells overexpressing ERRc (Figure 4D and 4E).
FIGURE 3 pi The ERRc inverse agonist 4-OHT reduces UCP1 expression in models of
brown adipogenesis. (A) Rb/ MEFs and WT-1 brown preadipocytes were treated
with 4-OHT (10 lM) or ethanol (EtOH) vehicle throughout the course of differentiation.
(B) Mature Rb/ and WT-1 brown adipocytes were deprived of rosiglitazone and insulin from day 4 and treated for 48 h with 4-OHT (10 lM) or ethanol vehicle from day 8.
Total RNA was harvested at day 8 (A) or 10 (B) and analyzed by RT-qPCR. Relative
expression levels of UCP1, adiponectin, and FABP4 were determined by normalization
to levels of TBP. Data represent mean þSEM. *, P < 0.05 versus vehicle-treated cells.
Forced expression of ERRc in brown adipocytes
increases UCP1 expression
The consequence of increased ERRc expression on brown adipogenesis was investigated using retroviral delivery of ERRc into the Rb/
and WT-1 cells. Overexpression of mouse ERRc in the two cell lines
was confirmed by RT-qPCR and resulted in an 500-fold increase in
ERRc mRNA, and the resulting average Ct value was 22.9 in Rb/
cells overexpressing ERRc compared to 25.9 in BAT. To verify that
the increased expression of ERRc in ERRc-transduced cells enhanced
ERRc activity, we measured at days 0 and 8 the mRNA levels of
ERRc target genes identified in other biological systems, including
PGC-1a (29), pyruvate dehydrogenase kinase 4 (PDK4) (30), small
heterodimer partner (SHP) (31), and ERRa (32). As expected, Rb/
cells transduced with ERRc have increased expression of PGC-1a,
PDK4, and SHP at confluence (day 0) compared to control cells (Figure 4A). ERRa expression was, however, not affected by overexpression of ERRc. On day 8, only SHP was expressed at elevated levels in
cells overexpressing ERRc, whereas the expression of PGC-1a,
PDK4, and ERRa was similar in vector- and ERRc-transduced cells
(Figure 4A). Interestingly, forced expression of ERRc resulted in a 4to 5-fold increase in the UCP1 mRNA expression in Rb/ and WT-1
adipocytes (Figure 4B). Differentiation per se appeared being similar
or slightly reduced in cells overexpressing ERRc, as determined by a
similar (WT-1) or moderately reduced (Rb/) expression of FABP4
and adiponectin mRNA in the adipose state (Figure 4B). Concerning
factors known to differentially regulate brown and white adipogenesis,
such as PGC-1b (5), PRDM16, and RIP140, we failed to detect
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Obesity | VOLUME 21 | NUMBER 3 | MARCH 2013
To examine whether the effect of ERRc on UCP1 expression was dependent on DNA-binding and/or the ligand-binding domain, we
expressed the DNA-binding mutant ERRc C125G or the activation
function-2 (AF2) mutant ERRc E429A (26) in parallel with wildtype ERRc in Rb/ cells. RNA was harvested at day 8 and analyzed by RT-qPCR. The levels of overexpressed wild-type and mutant ERRc were similar, and differentiation was comparable. As
expected, cells overexpressing wild-type ERRc displayed increased
UCP1 expression compared to control cells (Figure 4F). This ERRcinduced UCP1 expression was apparently dependent on functional
DNA-binding and AF2 domains, as cells expressing either one of the
two ERRc mutants exhibited an expression level of UCP1 comparable to control cells (Figure 4F). Similar results were obtained with a
truncated ERRc lacking the entire AF2 domain (data not shown).
ERRc promotes UCP1 expression in
the absence of PGC-1a
PGC-1a and PGC-1b are important for UCP1 gene expression and
proper brown fat cell function, and they associate with ERRs, stimulating their transcriptional activity (5,13,14). Thus, the increased
level of PGC-1a at day 0 caused by forced expression of ERRc in
Rb/ cells might explain the increase in UCP1 expression observed
on day 8. Therefore, we investigated the importance of PGC-1a
in the context of forced ERRc expression using immortalized
PGC-1aþ/þ and PGC-1a/ brown preadipocytes (5). Samples were
harvested at day 8 and analyzed by RT-qPCR and immunoblotting.
Overexpression of ERRc led to a 2-fold increase in UCP1 mRNA
and protein levels in PGC-1aþ/þ cells (Figure 5A and 5B). Of notice,
the level of PGC-1a mRNA was significantly higher in wild-type
cells overexpressing ERRc compared to vector-transduced cells at
day 0, but not at day 8 (data not shown), consistent with the situation
in Rb/ cells (Figure 4A). However, also in PGC-1a/ cells did
increased expression of ERRc cause increased expression of UCP1
mRNA and protein (Figure 5A and B). These data demonstrate that
ERRc promotes UCP1 expression independently of PGC-1a.
ERRc does not affect mitochondrial biogenesis
or lipolysis
Mitochondrial biogenesis is an important aspect of brown adipogenesis and involves replication of the mitochondrial DNA
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FIGURE 4 Forced expression of ERRc increases UCP1 expression in models of brown adipogenesis. Rb/ MEFs or
WT-1 preadipocytes were transduced with retroviruses and induced to differentiate. Total RNA and protein were harvested at the indicated days (or day 8 for Rb/ cells and day 6 for WT-1 cells) and analyzed by RT-qPCR or immunoblotting. Relative mRNA expression was determined by normalization to TBP. (A) Relative expression of ERRc target
genes PGC-1a, ERRa, PDK4, and SHP at days 0 and 8 in Rb/ cells transduced with mouse ERRc or empty control
virus (pMSCVbsd). (B) Relative expression of UCP1, adiponectin, and FABP4 in Rb/ and WT-1 cells transduced with
mouse ERRc or empty control virus (pMSCVbsd). Rb/ cells were harvested at day 8 and WT-1 cells at day 6. (C)
Relative expression of UCP1 in day 8 Rb/ cells transduced with control virus (pMSCVbsd) or retroviruses encoding
human ERRa, human ERRb or human ERRc. (D) Relative expression of UCP1 during differentiation of Rb/ MEFs
transduced with either mouse ERRc or empty control virus (pMSCVbsd). (E) Protein levels of UCP1 and FABP4 during
differentiation of Rb/ MEFs transduced with either mouse ERRc or empty control virus (pMSCVbsd). TFIIB was used
as a loading control. (F) Relative expression of UCP1 in day 8 Rb/ cells transduced with mouse ERRc wild-type
(WT), mouse ERRc C125G, mouse ERRc E429A or empty control virus (pMSCVbsd). Data represent mean þSEM. *,
P < 0.05 versus vector cells harvested at the same day; #, P < 0.05 day 8 versus day 0.
(mtDNA) (20,33). To determine whether this process is affected
by ERRc, we measured the ratio of mtDNA to nuclear DNA
(nDNA) by qPCR in Rb/ cells transduced with ERRc or empty
control virus. Total DNA was isolated at days 0, 4, and 8, and
as we have shown previously (20), Rb/ MEFs displayed a robust (13-fold) increase in relative mtDNA levels during adipose
conversion (Figure 6A). The mtDNA copy number was increased
with similar kinetics and to a similar extent in ERRc-transduced
and control cells (Figure 6A). Next, we determined citrate synthase (CS) activity as a surrogate measure of mitochondrial activity and biogenesis. CS activity was induced to similar levels dur-
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ing differentiation of Rb/ cells transduced with ERRc and
control retrovirus (Figure 6B). These data indicate that overexpression of ERRc has no effect on mitochondrial DNA replication, biogenesis, and activity in Rb/ cells.
We also analyzed if forced expression of ERRc would influence
b-adrenergic agonist-stimulated lipolysis. The amount of glycerol in
the medium was determined following a 2-h isoproterenol-stimulation of Rb/ adipocytes with or without forced expression of
ERRc. Cells overexpressing ERRc showed the same degree of
lipolysis as control cells (Figure 6C).
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ERRc and UCP1 Gene Expression Dixen et al.
compared to their respective control cells (data not shown). As
expected, TAg and PRDM16 significantly induced UCP1 expression
in wild-type MEFs at day 8 compared to their respective controls,
with TAg being the more potent of the two, increasing UCP1 levels
>100-fold (Figure 7A), although this level was still relatively low
(< 5% of the UCP1 expression in WT-1 adipocytes). However,
increased ERRc expression was not sufficient to induce UCP1
expression significantly in wild-type MEF-derived adipocytes (Figure 7A).
The experiment was repeated with 3T3-L1 cells with retroviral
delivery of ERRc, TAg, or the respective control viruses. Overexpression was confirmed, and adipose conversion was similar in all
cells as judged by the same criteria as for wild-type MEF-derived
fat cells. Again, TAg expression led to increased levels of UCP1
mRNA on day 8 (30-fold) (Figure 7B), but again the resulting
expression level was low (<0.5% of the UCP1 expression in WT-1
adipocytes). Overexpression of ERRc in 3T3-L1 cells significantly
FIGURE 5 ERRc promotes UCP1 expression in the absence of PGC-1a. PGC-1aþ/þ
(WT) and PGC-1a/ (KO) immortalized brown preadipocytes were transduced with
retroviruses encoding mouse ERRc or empty control virus (pMSCVbsd) and induced
to differentiate. Total RNA and protein were harvested at day 8 and analyzed by
RT-qPCR and immunoblotting, respectively. (A) Relative mRNA expression of UCP1
as determined by normalization to TBP. Data represent mean þSEM. *, P < 0.05
versus vector cells. (B) Protein levels of UCP1 and FABP4. TFIIB was used as a
loading control.
ERRc enhances adrenergically stimulated
palmitoylcarnitine oxidation
As Rb/ cells overexpressing ERRc had considerably increased
UCP1 levels (Figure 4), we investigated whether these cells can be
stimulated to display increased substrate oxidation by measuring oxidation of radiolabeled palmitoylcarnitine in mature adipocytes. Vector-transduced Rb/ adipocytes stimulated with isoproterenol for 2
h oxidized palmitoylcarnitine at roughly 5 pmol/min/mg protein,
whereas adipocytes overexpressing ERRc oxidized palmitoylcarnitine at 9 pmol/min/mg protein, an increase of 70% (Figure 6D).
This is in agreement with the higher UCP1 content in these cells
being activated by adrenergic stimulation.
Low levels of ERRc expression in white
adipocytes are not responsible for their
lack of UCP1 expression
The observations that ERRc is expressed at low levels in WAT and
models of white adipocytes (Figure 1 and 2) and that ERRc promotes UCP1 expression in models of brown adipocytes (Figure 4
and 5) raised the question whether the low levels of ERRc was causatively linked to the absence of UCP1 expression in the white adipocyte models. Hence, to test if overexpression of ERRc would suffice
to induce UCP1 expression in white adipocyte models, we transduced wild-type MEFs with retrovirus containing either ERRc or, as
positive controls, two factors that have been reported to increase
UCP1 expression in white adipocytes, namely simian virus 40 large
T antigen (TAg) (8) or PRDM16 (6). Cells were stimulated to
undergo adipogenesis, and RNA was harvested at day 8 and analyzed by RT-qPCR. Overexpression of the respective mRNAs was
confirmed, and a comparable degree of differentiation of all cell
types was verified by appearance of lipid droplets in >90% of the
cells as well as comparable expression levels of FABP4 and adiponectin mRNAs in ERRc, TAg and PRDM16 overexpressing cells
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Obesity | VOLUME 21 | NUMBER 3 | MARCH 2013
FIGURE 6 ERRc does not affect mitochondrial biogenesis and activity or lipolysis
but increases fatty acid oxidation. Rb/ MEFs were transduced with retroviruses
encoding mouse ERRc or empty control virus (pMSCVbsd) and induced to differentiate. (A) Total DNA was harvested at the indicated days, and mtDNA copy number
was determined by qPCR using primers specific for mtDNA (COX II) and nDNA
(RIP140). Relative mtDNA levels were calculated by normalizing COX II levels to
RIP140 levels. (B) CS enzyme activities (U) were determined at day 8 and normalized to protein contents. (C) Lipolysis was measured as glycerol levels in the
medium at day 8 after 2 h stimulation with 0.1 lM isoproterenol. (D) Fatty acid oxidation was measured at day 8 as described in the Materials and Procedures section using radiolabeled palmitoylcarnitine (PC) as substrate. Cells were stimulated
with 0.1 lM isoproterenol for 2 h before experiments were performed. The calculated PC oxidation per minute was normalized to protein content. Data represent
mean þSEM. *, P < 0.05 versus vector cells harvested at the same day; #, P <
0.05 day 8 versus day 0.
www.obesityjournal.org
Original Article
Obesity
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
(4,5). Studies have shown that PGC-1a can increase the transactivation potential of ERRc (13,14). However, although PGC-1a expression was increased at confluence by overexpression of ERRc (Figure
4A), the presence of PGC-1a was not required for the ERRc-mediated increase in UCP1 expression (Figure 5). A PGC-1a-independent
action of ERRc has previously been reported in ERRc-induced type
I muscle fiber specification (34). It has been shown that PGC-1a
and PGC-1b display functional redundancy in certain aspects of
brown adipocyte differentiation and function (5) and that ERRc
interacts with both PGC-1a and PGC-1b (13,14). However, the
expression of PGC-1b was unchanged in ERRc-transduced PGC1a/ cells compared with empty vector-transduced PGC-1a/
cells (data not shown), and therefore, it does not appear that PGC1b compensates for the lack of PGC-1a.
FIGURE 7 Low levels of ERRc expression in white adipocytes are not causative of
their lack of UCP1 expression. Wild-type MEFs and 3T3-L1 preadipocytes were
transduced with retroviruses encoding mouse ERRc, simian virus 40 TAg, mouse
PRDM16 (only wild-type MEFs), or the corresponding empty control virus
[pMSCVbsd for ERRc (white), pBabe-puro for TAg (black) and pMSCVpuro for
PRDM16 (grey)] and induced to differentiate. RNA was harvested at day 8 and analyzed by RT-qPCR. Relative mRNA expression of UCP1 was determined by normalization to TBP. (A) Wild-type MEF-derived adipocytes. (B) 3T3-L1 adipocytes.
Data represent mean þSEM. *, P < 0.05 versus the respective vector cells.
increased UCP1 expression (3.5-fold) in the adipose state (Figure
7B). This fold induction of UCP1 in 3T3-L1 cells in response to
forced expression of ERRc was thus principally similar to that
observed following forced expression in the three models of brown
adipogenesis (Rb/, WT-1, and PGC-1aþ/þ cells), albeit the
amount of UCP1 was very low in 3T3-L1 adipocytes compared to
levels in the brown adipogenesis models.
Discussion
Here, we describe the pattern of ERRc expression during white and
brown adipogenesis, in various brown and white adipose depots as
well as in fractionated WAT and BAT, and this clearly defines
ERRc as a BAT-enriched factor that is induced during brown adipogenesis (Figures 1 and 2). Moreover, ERRc expression is enhanced
in subcutaneous WAT during cold-induced browning, whereas its
expression remains unaltered in BAT in response to cold (Figure 1).
Treatment of Rb/ and WT-1 cells with the inverse ERRc agonist
4-OHT during differentiation or after differentiation to mature adipocytes caused a significant reduction in UCP1 expression (Figure
3). 4-OHT is known to inhibit the constitutive transcriptional activity of ERRc; however, 4-OHT is not specific for ERRc, as it also
inhibits the transcriptional activity of ERRb as well as ERa and
ERb, but not of ERRa (27,28). Of ERRb, ERa, and ERb, only ERa
seems to be expressed in significant amounts in BAT (www.nursa.org/10.1621/datasets.02001). ERa has, to our knowledge, not been
linked to expression of UCP1, but it cannot be ruled out that other
targets than ERRc have contributed to the effects observed with
4-OHT. Conversely, overexpression of ERRc consistently led to
increased UCP1 mRNA and protein expression in all brown adipogenesis models tested (Figures 4 and 5).
PGC-1a stimulates UCP1 expression in adipocytes and is required
for acquisition of the full thermogenic program in brown adipocytes
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ERRa has been shown to bind an ERRE in the UCP1 enhancer and
stimulate UCP1 expression, an effect dramatically potentiated by coexpression of PGC-1a or PGC-1b (18). Nevertheless, neither basal
UCP1 expression nor cold-induced induction of UCP1 expression in
BAT is compromised in ERRa-deficient mice (19,35). The promoter
of the ERRa gene contains a PGC-1a/ERRa response element that
can also be activated by ERRc (32). In our study, however, forced
expression of ERRc did not increase ERRa mRNA levels (Figure
4A), suggesting that the effects observed by ERRc in this study are
not mediated by increased expression of ERRa. Instead, we find it
likely that ERRc regulates UCP1 expression through direct binding
to the ERRE in the UCP1 enhancer.
Together with UCP1 expression, the high mitochondrial density of
brown fat cells is important for effective adaptive thermogenesis,
and mitochondrial density and mtDNA copy number are robustly
increased during brown adipogenesis (20,33). The ratio of mtDNA
to nDNA and activity of CS are increased and decreased, respectively, in hearts of ERRc/ mice (36). Mitochondrial biogenesis as
estimated by the mtDNA/nDNA ratio and activity of CS was not
influenced by forced expression of ERRc in Rb/ cells (Figures 6A
and B), suggesting that endogenous levels of ERRc are not limiting
for mitochondrial biogenesis during adipose conversion. Similarly,
we failed to detect an effect of ERRc overexpression on lipolysis in
response to b-adrenergic stimulation (Figure 6C). On the other hand,
we showed that ERRc overexpression in adrenergically stimulated
Rb/ adipocytes increased the rate of fatty acid oxidation by 70%
(Figure 6D). The enhanced rate of fatty acid oxidation in ERRctransduced brown adipocytes indicates that their increased level of
UCP1 protein is functionally active, as increased UCP1 levels
should increase the obtainable substrate oxidation rate in response to
adrenergic stimulation.
Even though overexpression of ERRc in differentiating 3T3-L1 cells
caused a modest increase in UCP1 expression, ERRc alone was not
sufficient to induce expression of UCP1 in differentiating wild-type
MEFs (Figure 7A and B). Thus, additional factors are required to
obtain UCP1 expression in wild-type MEFs, and the presence of
such additional factors in adipocyte-committed 3T3-L1 preadipocytes may explain why ERRc in these cells, but not in wild-type
MEFs, is able to induce UCP1 expression. The results in 3T3-L1
cells demonstrate that ERRc can stimulate UCP1 expression in differentiating white preadipocytes and thereby increase their potential
for energy expenditure. However, although ERRc can increase
UCP1 expression in white adipocytes, the level of UCP1 expression
obtained is low compared to the levels measured in brown
Obesity | VOLUME 21 | NUMBER 3 | MARCH 2013
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ERRc and UCP1 Gene Expression Dixen et al.
adipocytes. Thus, it can be concluded that whereas ERRc possess
the ability to promote UCP1 gene expression, it is not the low levels
of ERRc in white adipocytes that per se are responsible for their
inability to express high levels of UCP1.
The demonstration that BAT exists in a large fraction of adult
human subjects (37-39) together with the anti-obesity function of
BAT in rodents (1) has highlighted the importance of a better understanding of the development, activation, and recruitment of this tissue. Expression of ERRc increases during browning of subcutaneous
WAT, but it remains to be determined if ERRc plays an active role
in the browning process. Although ERRc levels do not change significantly in cold-activated BAT, it cannot be ruled out that ERRc
contributes to brown adipocyte activation by interacting with cofactors that themselves are regulated by adrenergic stimulation. Nevertheless, the findings that ERRc is able to increase UCP1 expression
and fatty acid oxidation in brown adipocytes are of substantial
interest.
In conclusion, this study demonstrates that ERRc is enriched in
brown adipocytes, that its expression increases during browning of
subcutaneous WAT, and that it is able to increase the potential for
energy expenditure in brown adipocytes by stimulating UCP1 gene
expression. Therefore, it is of interest and importance to clarify how
transcription of the ERRc gene is regulated and to identify interaction partners mediating the functions of ERRc in adipocytes.O
C 2012 The Obesity Society
V
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