ERRγ enhances UCP1 expression and fatty acid oxidation in brown adipocytes

ERRc Enhances UCP1 Expression andFatty Acid Oxidation in Brown AdipocytesKaren 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

IntroductionWhereas 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-adrenergicagonists, 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 tem-

perature 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 princi-

pal 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 estro-

gen 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 ([email protected])2 Department of Biology, University of Copenhagen, Denmark, DK-2100 Copenhagen Ø, Denmark 3 National Institute of Nutrition and Seafood Research, N-5817 Bergen, Norway 4 The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden

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-F2-2011-

278373), 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

516 Obesity | VOLUME 21 | NUMBER 3 | MARCH 2013 www.obesityjournal.org

Original ArticleOBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

Obesity

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 transcrip-

tional 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, suggest-

ing 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.

Materials and ProceduresMaterialsDexamethasone, methylisobutylxanthine, puromycin, 4-hydroxyta-

moxifen (4-OHT), isoproterenol, norepinephrine (NE), palmitoylcarni-

tine, and the in vitro toxicology assay kit were obtained from Sigma-

Aldrich. 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. Rosiglita-

zone and Adipolysis assay kit were from Cayman Chemical. [1-14C]-

palmitoylcarnitine was from Perkin Elmer and glucose from Merck.

Animals, cell culture, and packaging of virusInterscapular and perirenal BAT (the latter only from rats) as well

as ovarian, inguinal, and omental WAT were obtained from five 3-

months 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 Norwe-

gian 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 conflu-

ence at day 3, and were considered mature adipocytes at day 7. Wild-

type and retinoblastoma gene-deficient (Rb�/�) mouse embryo fibro-

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 methylisobutyl-

xanthine, 5 lg/ml insulin, and 0.5 lM rosiglitazone for 2 days. From

day 2, medium consisted of DMEM containing 10% FBS and supple-

mented 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 rosigli-

tazone 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 assayThe potential cytotoxicity of 4-OHT was assayed by lactate dehy-

drogenase (LDH) release into the medium using the in vitro toxicol-

ogy 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.

PlasmidsThe 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-mERRcC125G, and pCMX-mERRc E429A were obtained from Dr. Piia

Aarnisalo (26). To create pMSCVbsd-mERRc WT, pMSCVbsd-

mERRc C125G, and pMSCVbsd-mERRc E429A, inserts were

inserted into the NotI/ApaI site of pMSCVbsd link3. pMSCVpuro-

mPRDM16 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-hERRband pMSCVbsd-hERRc were cloned by inserting the hERR frag-

ments into the BamHI/XhoI site of pMSCVbsd link3.

RT-qPCRReal-time quantitative PCR (RT-qPCR) was performed as described

(24). Primers used are described in Supplementary Table S1.

Whole cell extracts and immunoblottingPreparation of whole-cell extracts and immunoblotting were done as

described (24). Antibodies used have been described (24).

Original Article ObesityOBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

www.obesityjournal.org Obesity | VOLUME 21 | NUMBER 3 | MARCH 2013 517

Quantification of relative mtDNA copy numbersand lipolysisDetermination of mtDNA copy numbers was carried out as

described (20). Adipolysis assay kit was applied for measuring glyc-

erol content in undiluted medium according to the instructions of

the manufacturer.

Palmitoylcarnitine oxidationExperiments were performed with cultured adipocytes gently trans-

ferred to conical flasks. Palmitoylcarnitine oxidation rate was deter-

mined after the addition of 1,000,000 dpm [1-14C]-palmitoylcarni-

tine together with cold palmitoylcarnitine to a final concentration of

50 lM and cold glucose to a final concentration of 25 mM. Radio-

active 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]-pal-

mitoylcarnitine 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 else-

where (Jørgensen et al., in preparation).

Statistical analysesFor cell culture studies, three dishes were harvested at each time

point and/or treatment in each experiment, except for the PC con-

sumption experiments (Figure 6D), for the treatment of mature adi-

pocytes with 4-OHT (Figure 3B) in which four and six dishes,

respectively, were analyzed, and for the primary cultures (Figure

2A) where two dishes were harvested. For WT-1 cells transduced

with ERRc or empty control virus (Figure 4B), one dish was har-

vested 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 stud-

ies (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 statisti-

cally significant if there was no overlap between their 95% confi-

dence 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 frac-

tions, as the measurements were performed on pools of RNA.

ResultsERRc is enriched in BAT compared to WAT andis induced during cold-induced browning of WATand brown adipogenesis in vitroCharacterization 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

FIGURE 1 ERRc is enriched in brown compared to white adipose tissues and is induced during cold-induced browningof subcutaneous white fat. RNA from mouse and rat adipose tissues and mouse WAT and BAT fractions was analyzedby RT-qPCR. Relative mRNA expression levels of ERRc and UCP1 were determined by normalization to expressionlevels 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 frac-tions (AF) from mouse WAT and BAT. (D) Inguinal WAT and interscapular BAT from mice kept at 22�C (n ¼ 7) or at4�C (n ¼ 4) for 48 h. In all panels, data represent mean þSEM. *, P < 0.05 versus interscapular (Int) BAT for adiposetissues (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 peri-renal (Re) BAT for rat adipose tissue. Ing, inguinal; Om, omental; Ov, ovarian.

Obesity ERRc and UCP1 Gene Expression Dixen et al.


perirenal BAT (the latter only from rats) were isolated from three-

months old mice and rats. As expected, the key brown adipose

marker gene UCP1 was highly enriched in BAT depots of these ani-

mals (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 preadipo-

cyte-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 mark-

edly 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 immor-

talized 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. Dur-

ing 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 har-

vesting (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

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, wild-

type MEFs, which were used as a model of white adipocyte differ-

entiation (8), have similar ERRc expression at days 0 and 8. More-

over, levels of ERRc mRNA were significantly higher in Rb�/�

compared to wild-type MEFs at days 4, 6, and 8 (Figure 2B).

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 (Fig-

ure 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 brown-

ing of subcutaneous WAT and brown adipocyte differentiation in vitro.

An ERRc inverse agonist reduces UCP1expression in brown adipocytesSince ERRc was expressed at a higher levels in brown compared to

white adipocytes and was up-regulated during brown adipocyte dif-

ferentiation, 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 adipo-

cytes by the 48 h of treatment with 4-OHT without a concomitant

effect on adiponectin and FABP4 mRNA levels (Figure 3B). To-

gether, 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.

FIGURE 2 ERRc is induced during brown adipogenesis in vitro. Total RNA was har-vested at the indicated days and analyzed by RT-qPCR. Relative mRNA expressionlevels of ERRc and UCP1 were determined by normalization to expression levels ofTBP. (A) Differentiation of primary brown preadipocytes stimulated or not with 1 lMnorepinephrine (NE) for 2 h. Primary cells spontaneously differentiated after reach-ing confluence at day 3 and became mature fat cells at day 7. (B) Differentiation ofwild-type and Rb�/� MEFs as well as 3T3-L1 and WT-1 preadipocytes. Cells wereinduced to differentiate at confluence (day 0) and considered mature adipocytes atday 8. In all panels, data represent mean þSEM. #, P < 0.05 day 8 (day 7 for pri-mary 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 (orWT-1) (panel B).



Forced expression of ERRc in brown adipocytesincreases UCP1 expressionThe consequence of increased ERRc expression on brown adipogene-

sis 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 (Fig-

ure 4A). ERRa expression was, however, not affected by overexpres-

sion 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 4-

to 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

changes in expression at days 0 and 8 in response to forced ERRcexpression (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 an-

alyzed by RT-qPCR and immunoblotting. FABP4 protein levels

were similar in ERRc-transduced and control cells during differen-

tiation (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 pro-

tein level, the protein level of UCP1 being dramatically increased in

cells overexpressing ERRc (Figure 4D and 4E).

To examine whether the effect of ERRc on UCP1 expression was de-

pendent 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 wild-

type ERRc in Rb�/� cells. RNA was harvested at day 8 and ana-

lyzed by RT-qPCR. The levels of overexpressed wild-type and mu-

tant 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 ERRc-induced 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 compara-

ble 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 inthe absence of PGC-1aPGC-1a and PGC-1b are important for UCP1 gene expression and

proper brown fat cell function, and they associate with ERRs, stimu-

lating 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-1ain 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 biogenesisor lipolysisMitochondrial biogenesis is an important aspect of brown adipo-

genesis and involves replication of the mitochondrial DNA

FIGURE 3 pi The ERRc inverse agonist 4-OHT reduces UCP1 expression in models ofbrown adipogenesis. (A) Rb�/� MEFs and WT-1 brown preadipocytes were treatedwith 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 insu-lin 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. Relativeexpression levels of UCP1, adiponectin, and FABP4 were determined by normalizationto levels of TBP. Data represent meanþSEM. *, P < 0.05 versus vehicle-treated cells.



(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 ro-

bust (�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-transducedand control cells (Figure 6A). Next, we determined citrate syn-

thase (CS) activity as a surrogate measure of mitochondrial activ-

ity and biogenesis. CS activity was induced to similar levels dur-

ing differentiation of Rb�/� cells transduced with ERRc and

control retrovirus (Figure 6B). These data indicate that overex-

pression of ERRc has no effect on mitochondrial DNA replica-

tion, 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-stimula-

tion of Rb�/� adipocytes with or without forced expression of

ERRc. Cells overexpressing ERRc showed the same degree of

lipolysis as control cells (Figure 6C).

FIGURE 4 Forced expression of ERRc increases UCP1 expression in models of brown adipogenesis. Rb�/� MEFs orWT-1 preadipocytes were transduced with retroviruses and induced to differentiate. Total RNA and protein were har-vested at the indicated days (or day 8 for Rb�/� cells and day 6 for WT-1 cells) and analyzed by RT-qPCR or immuno-blotting. Relative mRNA expression was determined by normalization to TBP. (A) Relative expression of ERRc targetgenes PGC-1a, ERRa, PDK4, and SHP at days 0 and 8 in Rb�/� cells transduced with mouse ERRc or empty controlvirus (pMSCVbsd). (B) Relative expression of UCP1, adiponectin, and FABP4 in Rb�/� and WT-1 cells transduced withmouse 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 encodinghuman ERRa, human ERRb or human ERRc. (D) Relative expression of UCP1 during differentiation of Rb�/� MEFstransduced with either mouse ERRc or empty control virus (pMSCVbsd). (E) Protein levels of UCP1 and FABP4 duringdifferentiation of Rb�/� MEFs transduced with either mouse ERRc or empty control virus (pMSCVbsd). TFIIB was usedas 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.



ERRc enhances adrenergically stimulatedpalmitoylcarnitine oxidationAs 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 ox-

idation of radiolabeled palmitoylcarnitine in mature adipocytes. Vec-

tor-transduced Rb�/� adipocytes stimulated with isoproterenol for 2

h oxidized palmitoylcarnitine at roughly 5 pmol/min/mg protein,

whereas adipocytes overexpressing ERRc oxidized palmitoylcarni-

tine 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 whiteadipocytes are not responsible for theirlack of UCP1 expressionThe observations that ERRc is expressed at low levels in WAT and

models of white adipocytes (Figure 1 and 2) and that ERRc pro-

motes UCP1 expression in models of brown adipocytes (Figure 4

and 5) raised the question whether the low levels of ERRc was caus-

atively linked to the absence of UCP1 expression in the white adipo-

cyte models. Hence, to test if overexpression of ERRc would suffice

to induce UCP1 expression in white adipocyte models, we trans-

duced 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 ana-

lyzed 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 adipo-

nectin mRNAs in ERRc, TAg and PRDM16 overexpressing cells

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 (Fig-

ure 7A).

The experiment was repeated with 3T3-L1 cells with retroviral

delivery of ERRc, TAg, or the respective control viruses. Overex-

pression 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 significantlyFIGURE 5 ERRc promotes UCP1 expression in the absence of PGC-1a. PGC-1aþ/þ

(WT) and PGC-1a�/� (KO) immortalized brown preadipocytes were transduced withretroviruses encoding mouse ERRc or empty control virus (pMSCVbsd) and inducedto differentiate. Total RNA and protein were harvested at day 8 and analyzed byRT-qPCR and immunoblotting, respectively. (A) Relative mRNA expression of UCP1as determined by normalization to TBP. Data represent mean þSEM. *, P < 0.05versus vector cells. (B) Protein levels of UCP1 and FABP4. TFIIB was used as aloading control.

FIGURE 6 ERRc does not affect mitochondrial biogenesis and activity or lipolysisbut increases fatty acid oxidation. Rb�/� MEFs were transduced with retrovirusesencoding mouse ERRc or empty control virus (pMSCVbsd) and induced to differen-tiate. (A) Total DNA was harvested at the indicated days, and mtDNA copy numberwas determined by qPCR using primers specific for mtDNA (COX II) and nDNA(RIP140). Relative mtDNA levels were calculated by normalizing COX II levels toRIP140 levels. (B) CS enzyme activities (U) were determined at day 8 and normal-ized to protein contents. (C) Lipolysis was measured as glycerol levels in themedium at day 8 after 2 h stimulation with 0.1 lM isoproterenol. (D) Fatty acid oxi-dation was measured at day 8 as described in the Materials and Procedures sec-tion using radiolabeled palmitoylcarnitine (PC) as substrate. Cells were stimulatedwith 0.1 lM isoproterenol for 2 h before experiments were performed. The calcu-lated PC oxidation per minute was normalized to protein content. Data representmean þSEM. *, P < 0.05 versus vector cells harvested at the same day; #, P <0.05 day 8 versus day 0.



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.

DiscussionHere, 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 adipo-

genesis (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 adi-

pocytes caused a significant reduction in UCP1 expression (Figure

3). 4-OHT is known to inhibit the constitutive transcriptional activ-

ity 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 ERaseems to be expressed in significant amounts in BAT (www.nur-

sa.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 adipo-

genesis 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

(4,5). Studies have shown that PGC-1a can increase the transactiva-

tion potential of ERRc (13,14). However, although PGC-1a expres-

sion was increased at confluence by overexpression of ERRc (Figure

4A), the presence of PGC-1a was not required for the ERRc-medi-

ated increase in UCP1 expression (Figure 5). A PGC-1a-independentaction of ERRc has previously been reported in ERRc-induced type

I muscle fiber specification (34). It has been shown that PGC-1aand PGC-1b display functional redundancy in certain aspects of

brown adipocyte differentiation and function (5) and that ERRcinteracts with both PGC-1a and PGC-1b (13,14). However, the

expression of PGC-1b was unchanged in ERRc-transduced PGC-

1a�/� cells compared with empty vector-transduced PGC-1a�/�

cells (data not shown), and therefore, it does not appear that PGC-

1b compensates for the lack of PGC-1a.

ERRa has been shown to bind an ERRE in the UCP1 enhancer and

stimulate UCP1 expression, an effect dramatically potentiated by co-

expression 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, respec-

tively, 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 ERRc-transduced 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 preadipo-

cytes 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 dif-

ferentiating 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

FIGURE 7 Low levels of ERRc expression in white adipocytes are not causative oftheir lack of UCP1 expression. Wild-type MEFs and 3T3-L1 preadipocytes weretransduced with retroviruses encoding mouse ERRc, simian virus 40 TAg, mousePRDM16 (only wild-type MEFs), or the corresponding empty control virus[pMSCVbsd for ERRc (white), pBabe-puro for TAg (black) and pMSCVpuro forPRDM16 (grey)] and induced to differentiate. RNA was harvested at day 8 and ana-lyzed by RT-qPCR. Relative mRNA expression of UCP1 was determined by nor-malization to TBP. (A) Wild-type MEF-derived adipocytes. (B) 3T3-L1 adipocytes.Data represent mean þSEM. *, P < 0.05 versus the respective vector cells.



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 under-

standing of the development, activation, and recruitment of this tis-

sue. 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 sig-

nificantly in cold-activated BAT, it cannot be ruled out that ERRccontributes to brown adipocyte activation by interacting with cofac-

tors that themselves are regulated by adrenergic stimulation. Never-

theless, 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 interac-

tion partners mediating the functions of ERRc in adipocytes.O

VC 2012 The Obesity Society

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ERRγ enhances UCP1 expression and fatty acid oxidation in brown adipocytes