Estrogen-related receptor alpha directs peroxisome proliferator-activated receptor alpha signaling in the transcriptional control of energy metabolism in cardiac and skeletal muscle.
ABSTRACT Estrogen-related receptors (ERRs) are orphan nuclear receptors activated by the transcriptional coactivator peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator 1alpha (PGC-1alpha), a critical regulator of cellular energy metabolism. However, metabolic target genes downstream of ERRalpha have not been well defined. To identify ERRalpha-regulated pathways in tissues with high energy demand such as the heart, gene expression profiling was performed with primary neonatal cardiac myocytes overexpressing ERRalpha. ERRalpha upregulated a subset of PGC-1alpha target genes involved in multiple energy production pathways, including cellular fatty acid transport, mitochondrial and peroxisomal fatty acid oxidation, and mitochondrial respiration. These results were validated by independent analyses in cardiac myocytes, C2C12 myotubes, and cardiac and skeletal muscle of ERRalpha-/- mice. Consistent with the gene expression results, ERRalpha increased myocyte lipid accumulation and fatty acid oxidation rates. Many of the genes regulated by ERRalpha are known targets for the nuclear receptor PPARalpha, and therefore, the interaction between these regulatory pathways was explored. ERRalpha activated PPARalpha gene expression via direct binding of ERRalpha to the PPARalpha gene promoter. Furthermore, in fibroblasts null for PPARalpha and ERRalpha, the ability of ERRalpha to activate several PPARalpha targets and to increase cellular fatty acid oxidation rates was abolished. PGC-1alpha was also shown to activate ERRalpha gene expression. We conclude that ERRalpha serves as a critical nodal point in the regulatory circuitry downstream of PGC-1alpha to direct the transcription of genes involved in mitochondrial energy-producing pathways in cardiac and skeletal muscle.
- SourceAvailable from: Chang Seok Lee[Show abstract] [Hide abstract]
ABSTRACT: Rapamycin at high doses (2-10 mg/kg body weight) inhibits mTORC1 and protein synthesis in mice. In contrast, low doses of rapamycin (10 micrograms/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with synthetic ligand for FKBP12 (SLF, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca2+ influx to enhance refilling of sarcoplasmic reticulum Ca2+ stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of Type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impacts multiple aspects of muscle function.Journal of Biological Chemistry 07/2014; · 4.60 Impact Factor
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ABSTRACT: Intramuscular creatine plays a crucial role in maintaining skeletal muscle energy homeostasis, and its entry into the cell is dependent upon the sodium chloride dependent Creatine Transporter (CrT; Slc6a8). CrT activity is regulated by a number of factors including extra- and intracellular creatine concentrations, hormones, changes in sodium concentration, and kinase activity, however very little is known about the regulation of CrT gene expression. The present study aimed to investigate how Creatine Transporter (CrT) gene expression is regulated in skeletal muscle. Within the first intron of the CrT gene, we identified a conserved sequence that includes the motif recognized by the Estrogen-related receptor α (ERRα), also known as an Estrogen-related receptor response element (ERRE). Additional ERREs confirming to the known consensus sequence were also identified in the region upstream of the promoter. When partnered with peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1α) or beta (PGC-1β), ERRα induces the expression of many genes important for cellular bioenergetics. We therefore hypothesized that PGC-1 and ERRα could also regulate CrT gene expression and creatine uptake in skeletal muscle. Here we show that adenoviral overexpression of PGC-1α or PGC-1β in L6 myotubes increased CrT mRNA (2.1 and 1.7-fold, P<0.0125) and creatine uptake (1.8 and 1.6-fold, P<0.0125), and this effect was inhibited with co-expression of shRNA for ERRα. Overexpression of a constitutively active ERRα (VP16-ERRα) increased CrT mRNA approximately 8-fold (P<0.05), resulting in a 2.2-fold (P<0.05) increase in creatine uptake. Lastly, chromatin immunoprecipitation assays revealed that PGC-1α and ERRα directly interact with the CrT gene and increase CrT gene expression.Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 08/2014; · 5.30 Impact Factor
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ABSTRACT: As a transcriptional coactivator, PGC-1α contributes to the regulation of a broad range of metabolic processes in skeletal muscle health and disease; however, there is limited information about the genes it transcriptionally regulates. To identify new potential gene targets of PGC-1α regulation, mouse C2C12 myotubes were screened by microarray analysis following PGC-1α overexpression. Genes with an mRNA expression of 2.5-fold or more (P < 0.001) were identified. From these, further genes were singled out if they had no previous connection to PGC-1α regulation or characterization in skeletal muscle, or were unannotated with no known function. Following confirmation of their regulation by PGC-1α using qPCR analysis, eight genes were focused on for further investigation (Akr1b10, Rmnd1, 1110008P14Rik, 1700021F05Rik, Mtfp1, Mrm1, Oxnad1 and Cluh). Bioinformatics indicated a number of the genes were linked to a range of metabolic-related functions including fatty acid oxidation, oxido-reductase activity, and mitochondrial remodeling and transport. Treating C2C12 myotubes for 6 h with AICAR, a known activator of AMP kinase and inducer of Pgc-1α gene expression, increased the mRNA levels of both Pgc-1α (P < 0.001) and of Mtfp1, Mrm1, Oxnad1 and Cluh (P < 0.05). Screening of the promoter and intron 1 regions also revealed all genes to contain either a consensus or near consensus response elements for the estrogen-related receptor α (ERRα), a key transcription factor-binding partner of PGC-1α in skeletal muscle. Furthermore, knockdown of endogenous ERRα levels partially or completely blocked the induction of gene expression of all genes by PGC-1α, while each gene was significantly upregulated in the presence of a constitutively active form of ERRα (P < 0.05) except for Akr1b10. These findings provide preliminary evidence for the novel regulation of these genes by PGC-1α and its signaling pathway in skeletal muscle.Molecular Biology Reports 09/2014; · 1.96 Impact Factor
MOLECULAR AND CELLULAR BIOLOGY, Oct. 2004, p. 9079–9091
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 20
Estrogen-Related Receptor ? Directs Peroxisome
Proliferator-Activated Receptor ? Signaling in the
Transcriptional Control of Energy Metabolism in
Cardiac and Skeletal Muscle
Janice M. Huss,1Ine ´s Pineda Torra,2† Bart Staels,2Vincent Gigue `re,3
and Daniel P. Kelly1*
Center for Cardiovascular Research, Washington University School of Medicine, St. Louis, Missouri1; De ´partment
d’ Athe ´roscle ´rose, Institut Pasteur de Lille, Universite ´ de Lille II, Lille, France2; and Molecular
Oncology, McGill University Health Centre, Montreal, Quebec, Canada3
Received 26 February 2004/Returned for modification 23 April 2004/Accepted 26 July 2004
Estrogen-related receptors (ERRs) are orphan nuclear receptors activated by the transcriptional coactivator
peroxisome proliferator-activated receptor ? (PPAR?) coactivator 1? (PGC-1?), a critical regulator of cellular
energy metabolism. However, metabolic target genes downstream of ERR? have not been well defined. To
identify ERR?-regulated pathways in tissues with high energy demand such as the heart, gene expression
profiling was performed with primary neonatal cardiac myocytes overexpressing ERR?. ERR? upregulated a
subset of PGC-1? target genes involved in multiple energy production pathways, including cellular fatty acid
transport, mitochondrial and peroxisomal fatty acid oxidation, and mitochondrial respiration. These results
were validated by independent analyses in cardiac myocytes, C2C12myotubes, and cardiac and skeletal muscle
of ERR??/?mice. Consistent with the gene expression results, ERR? increased myocyte lipid accumulation
and fatty acid oxidation rates. Many of the genes regulated by ERR? are known targets for the nuclear receptor
PPAR?, and therefore, the interaction between these regulatory pathways was explored. ERR? activated
PPAR? gene expression via direct binding of ERR? to the PPAR? gene promoter. Furthermore, in fibroblasts
null for PPAR? and ERR?, the ability of ERR? to activate several PPAR? targets and to increase cellular fatty
acid oxidation rates was abolished. PGC-1? was also shown to activate ERR? gene expression. We conclude
that ERR? serves as a critical nodal point in the regulatory circuitry downstream of PGC-1? to direct the
transcription of genes involved in mitochondrial energy-producing pathways in cardiac and skeletal muscle.
The essential role of nuclear receptors in regulating various
cellular metabolic pathways is becoming increasingly evident.
In recent years, various nuclear receptors that do not respond
to classical endocrine ligands, including peroxisome prolifera-
tor-activated receptors (PPARs), liver X receptors, farnesoid
X receptors, and retinoid X receptors, have been shown to be
activated by low-affinity diet-derived ligands (6, 11, 26, 44).
Activation of these receptors by metabolite ligands such as
fatty acids, oxysterols, and bile acids elicits downstream tran-
scriptional regulation of pathways involved in synthesis and
catabolism of these ligands. The remaining receptors, desig-
nated orphan receptors because endogenous ligands have not
been identified, comprise the largest subcategory of nuclear
receptors. It is likely that orphan receptors serve additional
roles in regulating intermediary metabolism. Linking orphan
receptors to target genes is an important goal in the field of
nuclear receptor biology. Target gene profiling will also pro-
vide insights for determining what metabolites serve as endog-
enous ligands for these receptors and, in turn, for developing
pharmacologic interventions designed to regulate cellular me-
One group of orphan receptors recently identified as candi-
date regulators of cellular metabolism are the estrogen-related
receptor (ERR) family. There are three members of the ERR
family, ERR?, ERR?, and ERR? (13, 16, 18). Early descrip-
tions of the tissue and developmental expression patterns of
ERR isoforms in mammalian organisms suggests that they are
involved in the regulation of cellular energy metabolism. For
example, in adults, ERR? and ERR? expression is enriched in
tissues that rely primarily on mitochondrial oxidative metabo-
lism for energy generation, such as heart, brown adipose, and
slow-twitch skeletal muscle (16, 43, 47). During embryonic
development, both isoforms are expressed in heart and skeletal
muscle, suggesting that they serve functional roles during de-
velopment as well as for maintaining function in differentiated
muscle. We demonstrated a dramatic increase in cardiac
ERR? expression after birth, coincident with the postnatal
switch to fatty acids as an energy substrate and the global
upregulation of enzymes involved in cellular fatty acid uptake
and mitochondrial oxidation (19). Moreover, ERR? binds the
5? regulatory region of the gene encoding medium-chain acyl
coenzyme A dehydrogenase (MCAD), which catalyzes the first
step of the mitochondrial ?-oxidation pathway and therefore
was implicated in the direct regulation of fatty acid oxidation
pathways (43, 47).
* Corresponding author. Mailing address: Center for Cardiovascular
Research, Washington University School of Medicine, St. Louis, MO
† Present address: New York University Medical Center, New York,
Early attempts failed to demonstrate ERR?-mediated acti-
vation of MCAD gene transcription in various cell culture mod-
els, suggesting that the cells lacked a key functional component
of ERR? signaling. Recently, we and others identified mem-
bers of the PPAR? coactivator 1 (PGC-1) family of transcrip-
tional coactivators as potent coactivators for ERR? and ERR?
(17, 19, 21, 42). Three PGC-1 isoforms have been character-
ized, PGC-1?, PGC-1?, and PRC. PGC-1? is a key regulator
of an array of cellular energy metabolic pathways, but its pri-
mary effect in target tissues is to enhance mitochondrial oxi-
dative metabolism (24, 37). PGC-1? increases cellular mito-
chondrial number, fatty acid oxidation, and respiration via
coactivation of a number of nuclear receptor and non-nuclear
receptor transcription factor partners (29, 38, 49). PGC-1? is
also thought to activate oxidative metabolism in tissues, though
it does so through a relatively restricted set of transcriptional
partners compared to PGC-1? (31, 45).
Shared PGC-1? and PGC-1? partners include ERR? and
ERR?, nuclear respiratory factor 1 (NRF-1), hepatocyte nu-
clear factor 4, estrogen receptor ?, and peroxisome prolifera-
tor-activated receptor ? (PPAR?) (17, 19, 21, 30, 42, 46, 49).
Hence, ERR isoforms likely confer PGC-1-mediated regula-
tion on ERR target genes in tissues where ERR?, ERR?, and
PGC-1 coactivators are coexpressed, such as heart and skeletal
muscle. Indeed, we demonstrated that the ERR?/PGC-1?
complex directly activated the MCAD gene promoter through
the ERR? binding site identified in earlier studies and that
ERR? overexpression activated endogenous MCAD gene ex-
pression in NIH 3T3 cells (19). Collectively, the published
results to date suggest that ERRs serve as a component of the
regulatory circuitry downstream of PGC-1 and have stimulated
interest in defining the metabolic roles of ERR? and related
isoforms. However, the specific target genes and related met-
abolic pathways regulated by ERR isoforms have not been
In order to identify potential target genes of ERR?, tran-
scriptional profiling studies were performed in rat neonatal
cardiac myocytes overexpressing ERR?. Validation studies
were performed in cell culture and in vivo in heart and skeletal
muscle of ERR? null mice. These studies unveiled several key
regulatory functions for ERR?. First, we found that ERR?
activates genes involved in multiple key energy production
pathways, including cellular fatty acid uptake, fatty acid oxida-
tion, and mitochondrial electron transport/oxidative phosphor-
ylation. Second, ERR?-mediated regulation of fatty acid uti-
lization genes occurs, at least in part, through direct activation
of PPAR? gene transcription, a mechanism that is coactivated
by PGC-1?. Collectively, these results identify ERR? as a
critical regulator of energy metabolism in heart and skeletal
MATERIALS AND METHODS
Plasmid constructs. The wild-type and mutated forms of the human PPAR?
promoter-reporter plasmids have been described (35, 36). The mammalian ex-
pression vectors expressing human ERR? and mouse PGC-1?, pcDNA3.1-
ERR? and pcDNA3.1-myc/his.PGC-1?, have also been described (19, 46). The
pSG5-HA-ERR? expression vector was a kind gift from M. Stallcup (University
of Southern California).
Mammalian cell culture and transient transfections. CV1 cells were main-
tained at 37°C and 5% CO2in Dulbecco’s modified Eagle’s medium–10% fetal
calf serum. Transient transfections were performed by the calcium phosphate
coprecipitation method (9). Reporter plasmids (4 ?g/ml) were cotransfected with
RSV-?Gal (0.5 ?g/ml), expressing the ?-galactosidase gene driven by the Rous
sarcoma virus promoter, to control for transfection efficiency. For cotransfection
experiments, mammalian expression vectors expressing ERR isoforms, PGC-1?,
or the corresponding empty vectors were used. Cells were collected and assayed
48 h posttransfection, and luciferase and ?-galactosidase activities were mea-
sured as described previously (5).
Ventricular cardiac myocytes were prepared from 1-day-old Harlan-Sprague-
Dawley rats as described previously (9). After 24 h, cells were infected with
adenovirus expressing green fluorescent protein (GFP), ERR?, PGC-1?, or
PPAR? driven by a cytomegalovirus promoter. The experimental constructs also
express GFP from an independent promoter. Infection rates of 90 to 95% were
achieved by 18 h as assessed by quantitation of GFP-expressing cells with fluo-
rescence microscopy. RNA and whole-cell protein extracts were prepared from
cells 48 and 72 h postinfection, respectively. The construction and propagation of
adenovirus expressing GFP, human ERR?, and mouse PGC-1? or mouse
PPAR? have been described (2, 15, 19, 20, 28).
Primary mouse fibroblasts were prepared from tail tissue of wild-type,
ERR??/?(ERR knockout), or ERR??/?PPAR??/?(double-knockout) mice.
Isolation and culturing of fibroblasts were performed in complete medium (Dul-
becco’s modified Eagle’s medium, 20% fetal calf serum, 2 mM L-glutamine, 1
mM nonessential amino acids, and 100 ?g of penicillin/streptomycin per ml). In
brief, tail clips (1 cm) were soaked 10 min in complete medium with 500 ?g/ml
penicillin/streptomycin per ml, rinsed, and minced in medium with no penicillin/
streptomycin. Tissue pieces were digested overnight at 37°C in complete medium
plus 500 U collagenase. Cells were liberated by triturating with a 5-ml pipette,
collected by centrifugation, and cultured in T25 flasks in complete medium. After
48 h cells were subcultured and expanded to determine appropriate viral titration
and used at passage two or three for overexpression experiments.
DNA microarray. Total RNA was isolated from rat neonatal cardiac myocytes
with RNAzol (Tel-Test Inc.) followed by an RNeasy kit (Qiagen Inc.) clean-up.
Double-stranded cDNA was synthesized from 12 ?g of total RNA that was first
reverse transcribed with Superscript II (Invitrogen Corp.) with a T7 promoter-
poly(A) primer (T7T24) followed by second-strand synthesis according to the
manufacturer’s protocol. Biotin-labeled cRNA was synthesized with T7-coupled
ENZO BioArray High-Yield RNA transcript labeling kit (ENZO Diagnostics
Inc.) following the manufacturer’s protocol.
The Alvin Siteman Cancer Center’s Multiplexed Gene Analysis Core at Wash-
ington University School of Medicine performed hybridization to the Affymetrix
rat U34A chip. Affymetrix MAS 5.0 software was used for the initial data analysis
and background normalization. Subsequent data manipulations were performed
in Excel. Probe sets that were called absent by Affymetrix software in both GFP
and ERR? overexpression conditions were excluded from subsequent data anal-
ysis. Signal intensities were normalized to the average intensity for all probe sets.
Signal intensity ratios were calculated as the ratio of ERR?-expressing to GFP-
expressing cells in order to detect changes due to ERR? overexpression. Three
independent trials were performed. Signal intensity ratios that increased ?2-fold
(induced) or decreased ?0.5-fold (repressed) in at least two trials were consid-
ered potentially regulated in ERR?-expressing cells.
Northern and immunoblot analyses. Total cellular RNA isolation and blotting
were performed as described previously (5). Blots were hybridized with radiola-
beled probes derived from cDNA mouse clones for MCAD, ERR?, PPAR?, and
PGC-1?. In addition, human ERR?, rat M-CPT I, and universal actin probes
were used. Protein extracts were resolved by sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (7.5%) and transferred to nitrocellulose membranes.
Immunodetection of MCAD and acyl-coenzyme A oxidase was performed with
the previously described anti-MCAD and anti-acyl-coenzyme A oxidase antibod-
ies (7, 22). The fatty acyl coenzyme A synthetase 1 antibody was generously
provided by J. E. Schaffer (Washington University School of Medicine).
Real-time quantitative reverse transcription-PCR. Real-time PCR was per-
formed as described previously (40). Total RNA isolated from the soleus muscle
of 8-week-old wild-type or ERR??/?mice was reverse transcribed with Taqman
reverse transcription reagents (Applied Biosystems) with oligo(dT) and random
hexamers (1:1 ratio). Reactions were performed in triplicate in the 96-well
format with Taqman core reagents and a Prism 7700 sequence detector (Applied
Biosystems). The PGC-1? primer-probe set has been described (3). The follow-
ing mouse-specific primer-probe sets were used to detect specific gene expres-
sion: MCAD forward, 5?-GGAAATGATCAACAAAAAAAGAAGTATTT-3?;
MCAD reverse, 5?-ATGGCCGCCACATCAGA-3?; MCAD probe, 5?-TGTCA
CACAGTAAGGACACATCATTGGCTG-3?; M-CPT I forward, 5?-TCTAGG
CAATGCCGTTCAC-3?; M-CPT I reverse, 5?-GAGCACATGGGCACCATA
C-3?; M-CPT I probe, 5?-TCAAGCCGGTCATGGCACTGG-3?; CD36 forward,
5?-CGGACATTGAGATTCTTTTCC-3?; CD36 reverse, 5?-TCCTTTAAGGTC
9080 HUSS ET AL.MOL. CELL. BIOL.
GATTTCAGATC-3?; CD36 probe, 5?-ACAGCGTAGATAGACCTGCAAAT
G-3?; ERR? forward, 5?-TGACTTGGCTGACCGAG-3?; ERR? reverse, 5?-CC
GAGGATCAGAATCTCC-3?; ERR? probe, 5?-CATATTCCAGGCTTCTCCA
PPAR? reverse, 5?-TTGTCGTACACCAGCTTCAGC-3?; PPAR? probe, 5?-A
GGCTGTAAGGGCTTCTTTCGGCG-3; PPAR? forward, 5?-TCACCGGCAA
GTCCAGCCA-3?; PPAR? reverse, 5?-ACACCAGGCCCTTCTCTGCCT-3?;
and PPAR? probe, 5?-AACGCACCCTTTGTCATCCACGA-3?. The rRNA
(VIC) probe set was included in all reactions as an internal correction control,
and corrected data were normalized to ?-actin expression (Applied Biosystems).
Electrophoretic mobility shift assays and chromatin immunoprecipitation.
Double-stranded complementary oligonucleotides corresponding to HNF-4-re-
sponsive element (5?GATCCTGGAGGGTGGGGCAAAGTTCACCATAGGT
A-3?) or HNF-4REmut (5?GATCCTGGAGGGTGCAGCAAAGTTCACCAT
AGGTA-3?) of the human PPAR? promoter were used to generate probes to
assay ERR isoform binding in vitro. Probes were32P labeled by a Klenow fill-in
reaction. Recombinant proteins for human ERR? and mouse ERR? were syn-
thesized with the TNT Quick T7-coupled translation kit (Promega). Synthesis of
appropriately sized proteins was verified in reactions incorporating [35S]methi-
onine followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
analysis and autoradiographic detection. Electrophoretic mobility shift assay
reactions were performed as described previously (9).
Chromatin immunoprecipitation assays were performed following previously
published methods in which specific buffer recipes used in the protocol can be
found (4, 48). In brief, L6 rat myoblasts (1 ? 107to 2 ? 107) were infected with
the adenovirus construct Ad-ERR?. Cross-linking was performed 48 h postin-
fection by incubating cells with 1% formaldehyde (10 min) followed by addition
of 0.125 M glycine to halt cross-linking. Cells were washed and collected in cold
phosphate-buffered saline and pelleted by centrifugation, followed by lysis and
disruption via Dounce homogenization. Nuclei were pelleted and resuspended in
a 1% sodium dodecyl sulfate nuclear lysis buffer. Chromatin was sheared by four
30-s sonication cycles with a Branson 250 sonifier. Aliquots were analyzed by
agarose gel electrophoresis to verify enrichment of the 400- to 1,000-bp sub-
population. Sheared chromatin was precleared with bovine serum albumin-
blocked Pansorbin (Calbiochem), followed by dilution of an aliquot in immuno-
precipitationdilution buffer and
immunoglobulin G or polyclonal antibody against ERR? (43).
Antibody-chromatin complexes were bound with Pansorbin and pelleted by
centrifugation. An aliquot from the immunoglobulin G sample was reserved as
“input” control. The pellets were washed twice in dialysis buffer followed by four
washes in a 500 mM LiCl immunoprecipitation buffer. Immunoprecipitated
complexes were eluted by two 15-min incubations with elution buffer. Cross-links
were reversed by incubating samples at 65?C for 5 h, during which time samples
were also RNase A treated. Samples were phenol and CHCl3extracted, precip-
itated overnight, and then proteinase K treated. A final phenol-CHCl3purifica-
tion and precipitation was performed to yield template for PCR (30 cycles with
standard temperature conditions). Primers were designed to amplify a 310-bp
amplicon corresponding to the region of the rat PPAR? promoter flanking the
HNF-4-responsive element (nucleotides ?1304 to ?1613). Primers amplifying
250 bp of the TFIID gene were used to control for nonspecific enrichment of
genomic DNA in the immunoprecipitation. PCR-amplified bands were analyzed
on a 1.3% agarose gel, and relative band intensities were quantified by densi-
Animal studies. All animal protocols were approved by the Animal Studies
Committee at Washington University School of Medicine. The ERR??/?mice
have recently been described (32). The original background strain of the
ERR??/?mice was a hybrid strain (C57BL/6/SvJ129). For baseline comparisons,
littermate wild-type and ERR??/?mice were generated from heterozygous
breeders to control for strain background. Heart and skeletal muscle (gastroc-
nemius and soleus) were isolated from fed wild-type and ERR??/?mice during
the daytime (1000 to 1200 h). ERR??/?backcrossed to C57BL/6 were bred with
a C57BL/6 strain of PPAR??/?mice (3, 27) to generate doubly heterozygous
mice that were then intercrossed to generate the ERR??/?PPAR??/?(double-
knockout) mouse lines that were used to isolate primary fibroblasts.
Palmitate oxidation assays. Measurement of palmitate oxidation rates was
performed with [9,10-3H]palmitic acid as described previously (10). Cells (?5 ?
103) were cultured in 24-well plates and infected with adenovirus expressing GFP
(Ad-GFP), Ad-ERR?, Ad-PGC-1?, or Ad-PPAR? 24 h later. At 84 h postin-
fection, palmitate oxidation assays were performed. To demonstrate the speci-
ficity of the assay for measuring fatty acid oxidation, 50 ?M sodium etomoxir, a
CPT I inhibitor, was added to half of the labeling reactions. Incubations were
performed for 2 h at 37°C. Data were normalized for background and for total
cellular protein quantitated by the bicinchoninic acid method. Rates were cal-
culated as nanomoles of [3H]palmitate oxidized per hour per milligram of pro-
Neutral lipid detection. Rat neonatal cardiac myocytes were infected with
Ad-GFP or Ad-ERR? for 24 h before addition of 35 ?M oleic acid conjugated
to bovine serum albumin. Oil red O staining was performed by the Digestive
Disease Histology Core at Washington University School of Medicine as de-
scribed previously (1).
ERR? induces expression of genes involved in cellular fatty
acid uptake, oxidation, and mitochondrial respiration. To
characterize the regulatory effects of ERR? on cellular metab-
olism, ERR? was overexpressed in primary rat neonatal car-
diac myocytes and gene expression changes were profiled with
the Affymetrix rat U34A array in three independent trials. The
criteria for designating a gene regulated by ERR? was a
present call in ERR?-expressing myocytes and a signal inten-
sity twofold or greater above that of GFP-expressing myocytes
in at least two of three independent trials. Overall, ERR?
induced 90 distinct genes, a significant number of which en-
code enzymes involved in cellular energy metabolic pathways
(Table 1). A complete list of ERR?-upregulated genes is avail-
able from the authors (unpublished data).
Notably, we found a number of genes involved in the cellular
uptake and mitochondrial oxidation of fatty acids. In addition,
several genes involved in mitochondrial respiratory function
were also activated. These results were of interest because
mitochondrial fatty acid oxidation serves as the chief source of
energy in adult heart. Specifically, genes encoding lipoprotein
lipase, the fatty acid transporter, CD36/fatty acid transporter,
and heart-specific fatty acid binding protein FABP3 were up-
regulated in rat cardiac myocytes. In addition, fatty acyl coen-
zyme A synthetase, which facilitates fatty acid uptake by cou-
pling transport to esterification at the plasma membrane, was
induced in response to ERR? overexpression.
As shown previously, the gene encoding MCAD, a key en-
zyme in the fatty acid oxidation cycle, was increased by ERR?.
Additional genes encoding enzymes of mitochondrial (very
long chain acyl-coenzyme A dehydrogenase, long-chain hy-
droxyacyl coenzyme A dehydrogenase) and peroxisomal (acyl
coenzyme A oxidase) ?-oxidation were also activated in par-
allel with MCAD. A subset of the genes encoding enzymes and
proteins involved in mitochondrial electron transport and
oxidative phosphorylation were also induced, including cyto-
chrome c, the muscle-specific cytochrome oxidase VIIIh
subunit, NADH (ubiquinone) dehydrogenase, and flavopro-
tein-ubiquinone oxidoreductase. Additional mitochondrion-as-
sociated enzymes, such as aminolevulinic acid synthase, in-
volved in heme synthesis, hydroxymethylglutaryl coenzyme A
synthase, creatine kinase, and aldehyde dehydrogenase, were
also induced in response to ERR? overexpression. Notably,
components of other metabolic pathways, such as glycolysis
and cholesterol metabolism, were also activated by ERR?.
However, no metabolic pathways appeared to be more uni-
formly regulated by ERR? in cardiac myocytes than those
involved in fatty acid uptake and oxidation. Finally, a number
of muscle-specific genes associated with the contractile appa-
ratus were upregulated in ERR?-expressing cardiac myocytes,
including Ca2?-transporting ATPase, phospholamban, and
myosin heavy-chain isoforms. Collectively, the results suggest
VOL. 24, 2004ERR? ACTIVATES FATTY ACID METABOLISM9081
that ERR? upregulates genes involved in cellular fatty acid
utilization and mitochondrial oxidation.
The putative ERR? targets were validated by independent
analytical methods (Fig. 1). Upregulation of lipoprotein lipase,
CD36/fatty acid transporter, and FABP3 in ERR?-expressing
myocytes was demonstrated in RNA prepared from indepen-
dent overexpression trials performed in cardiac myocytes (Fig.
1A). ERR?-mediated regulation of genes involved in mito-
chondrial energy production was compared with that of PGC-
1?, which has been shown to increase the expression of a
number of nucleus- and mitochondrion-encoded genes in-
volved in mitochondrial fatty acid oxidation and electron trans-
port/oxphos (Fig. 1B) (28). Expression of genes encoding mi-
tochondrial fatty acid oxidation enzymes (muscle carnitine
palmitoyltransferase I [M-CPT I, CPT I?] and MCAD) and
the peroxisomal enzyme acyl coenzyme A oxidase was in-
creased in response to ERR? or PGC-1? expression. In addi-
tion, ERR? modestly induced the expression of cytochrome
oxidase IV, cytochrome c, and ATP synthase ?. Although less
robust, these results paralleled that of PGC-1?-mediated reg-
TABLE 1. Genes upregulated by ERR? in rat neonatal cardiac myocytes
CategoryAccession no. Identification or gene symbola
Cellular fatty acid import/oxidationAA799326/AB005743
CD36 antigen (Cd36)
Fatty acyl-CoA ligase, long chain 2 (Facl2)
Acyl-CoA dehydrogenase, medium chain (Acadm)
Acyl-CoA dehydrogenase, very long chain (Acadvl)
Lipoprotein lipase (Lpl)
Fatty acid binding protein, heart (Fabp3)
Acyl-CoA oxidase (Acox1)
Peroxisomal multifunctional enzyme II (Hsd17b4)
Stearoyl-CoA desaturase 2 (Scd2)
Cytochrome b5, soluble (Cyb5)
Aldehyde dehydrogenase, phenobarbital inducible
Cytochrome c (Cycs)
Cytochrome c oxidase VIIIh (Cox8h)
NADH dehydrogenase (ubiquinone)
Acyl carrier chain (ACP/CI-SDAP)
Flavoprotein-ubiquinone oxidoreductase (Etfdh)
Mitochondrial carrier homolog 2 (Mtch2)
Protein phophatase 2A, ? regulatory subunit
Protein phosphatase 2C (Pp2c2)
Hydroxymethylglutaryl-CoA synthase I (Hmgcs1)
Hydroxymethylglutaryl-CoA reductase (Hmgcr)
Isopentenyl diphosphate-dimethylallyl diphosphate
Cholesterol esterase, lysosomal acid lipase (Lip1)
Pyruvate dehydrogenase phosphatase I (Pdp1)
Creatine kinase B (Ckb)
Aminolevulinic acid synthase (Alas1)
Choline kinase (Chk)
Cytochrome P450 subfamily 51 (Cyp51)
N-Acylsphingosine amidohydrolase (acid
Aspartate aminotransferase, soluble (Got1)
Smooth muscle cell LIM protein, cysteine-rich
High-mobility-group box 2 (Hmgb2)
Brahma homolog, SWI/SNF-related actin-dependent
regulator of chromatin, subfamily a, member 4
Transformer-2-related, splicing factor arginine/
serine-rich 10 (Sfrs10)
Ca2?transporting ATPase, cardiac, slow twitch 2
Myosin heavy-chain ? (Myh6)
Myosin heavy-chain ? (Myh7)
Mitochondrial respiratory chainK00750
Other metabolic pathways
aCoA, coenzyme A.
9082HUSS ET AL.MOL. CELL. BIOL.
ulation. Analysis of whole-cell protein extracts from ERR?-
overexpressing cells verified induction of MCAD, acyl coen-
zyme A oxidase, and fatty acyl coenzyme A synthetase 1
protein by ERR? (Fig. 1C). Collectively, the results indicate
that ERR? regulates a subset of PGC-1? targets, mainly genes
involved in mitochondrial oxidative fatty acid catabolism or
ERR? increases cardiac myocyte fatty acid uptake and ox-
idation. We next investigated the physiologic relevance of the
observed induction of lipid transport and uptake and mito-
chondrial oxidative enzyme genes by ERR?. Cardiac myocytes
overexpressing GFP (control) or ERR? were incubated with
oleic acid complexed to bovine serum albumin and stained with
Oil Red O to detect intracellular neutral lipid (Fig. 2). ERR?-
overexpressing myocytes showed increased accumulation of
small lipid droplets. Increased lipid accumulation was also
observed at the same concentration of bovine serum albumin-
oleate in cardiac myocytes overexpressing either PGC-1? or
PPAR?, which were included as positive controls (data not
To determine if fatty acid utilization was also increased,
oxidation of [3H-9,10]palmitic acid was measured (Table 2). A
significant increase in mean palmitate oxidation rates (49.3 ?
7.8%) was observed in the ERR?-overexpressing cells com-
pared to GFP. Oxidation rates were inhibited by 85% (?
0.4%) by the CPT I inhibitor etomoxir, verifying that the assay
was specifically measuring mitochondrial fatty acid oxidation
(data not shown). With the same assay, overexpression of
PPAR? or PGC-1? increased palmitate oxidation 41% and
92%, respectively, as expected (Table 2). These data are con-
sistent with the observed effects of PPAR?/PGC-1? on fatty
acid oxidation in NIH 3T3 cells (46). These physiologic effects
reflect the observed gene expression changes and demonstrate
that ERR? increases cardiac myocyte fatty acid uptake and
Effects of ERR? gene deletion on in vivo expression of fatty
acid utilization genes in cardiac and skeletal muscle. The
ERR? knockout mouse was recently described (32). The phe-
notype of ERR? knockout mice includes a derangement in
white adipocyte lipid metabolism that manifests as resistance
to diet-induced obesity. The results shown above suggest that
ERR? plays a role in the regulation of genes involved in
skeletal muscle and cardiac energy metabolism in vivo. To
explore this possibility, the expression of several putative
ERR? target genes involved in cellular fatty acid uptake and
oxidation was characterized in cardiac and skeletal muscle of
wild-type and littermate ERR? knockout animals (Fig. 3). The
levels of transcripts encoding MCAD or PPAR? were not
different in the hearts of ERR? knockout mice compared to
wild-type controls (Fig. 3A). Interestingly, the levels of
PGC-1? and ERR? mRNA were significantly increased in
FIG. 1. Validation of putative ERR? target genes involved in cel-
lular fatty acid utilization and mitochondrial respiratory pathways. (A)
Expression analysis of fatty acid uptake enzyme genes. Northern blot-
ting was performed with 15 ?g of total RNA isolated from Ad-GFP
(GFP) or Ad-ERR? (ERR?)-infected cardiac myocytes. Blots were
sequentially hybridized with probes specific for ERR?, fatty acid trans-
porter (FAT)/CD36, FABP3, and lipoprotein lipase (LPL). (B) In-
duced expression of mitochondrial enzymes by ERR? or PGC-1?.
Northern analysis was performed as above with probes against PGC-
1?, MCAD, acyl coenzyme A oxidase (ACO), M-CPT I, cytochrome
oxidase IV (COXIV), cytochrome c (Cyt. c), and ATP synthase ?, with
RNA from cardiac myocytes overexpressing either GFP, ERR?, or
PGC-1?, as indicated. (C) Western analysis of 30 ?g of whole-cell
extract (WCE) prepared from rat neonatal cardiac myocytes infected
with adenovirus vectors expressing GFP or ERR?. FACS-1, fatty acyl
coenzyme A synthetase 1.
VOL. 24, 2004 ERR? ACTIVATES FATTY ACID METABOLISM 9083
ERR? knockout hearts compared to wild-type hearts, suggest-
ing a compensatory response mediated by the ERR?/PGC-1?
The effect of ERR? gene deletion on the expression of the
putative ERR? target genes was different in skeletal muscle
compared to heart. As a preliminary step, the relative expres-
sion levels of ERR isoforms were assessed in the vastus late-
ralis, which contains mostly glycolytic fast-twitch fibers, and
soleus, which is predominantly a slow-twitch oxidative muscle,
in wild-type mice (Fig. 3B). Expression of ERR isoforms,
PPAR?, PGC-1?, and MCAD (used as a marker of mitochon-
drial fatty acid oxidative capacity) was significantly higher in
the soleus compared to the vastus in the wild-type mice. These
results suggested that any effects of ERR? deletion on expres-
sion of putative ERR? targets would most likely be observed in
In contrast to the heart, transcript levels for MCAD, a
known PPAR? gene target, were lower in ERR??/?soleus
compared to wild-type control soleus. However, no change in
several other known PPAR? targets (CD36/fatty acid trans-
porter or M-CPT I) was observed in the soleus of ERR? null
compared to wild-type mice (Fig. 3C). Regarding possible
compensatory changes, PGC-1? expression was significantly
increased, similar to the effect of ERR? deletion in the heart.
However, ERR? and PPAR? were unchanged in the
ERR??/?mouse soleus. This distinct pattern of compensatory
gene regulation, particularly the lack of induction of the ERR?
gene, may contribute to the differential effects of ERR? dele-
tion on PPAR? targets in the soleus compared to the heart.
Taken together, the results of the ERR? deletion and overex-
pression studies support a role for ERR? in the regulation of
muscle mitochondrial oxidative metabolism.
ERR? activates the PPAR? gene regulatory pathway. The
ERR? overexpression studies described above revealed up-
regulation of a number of genes involved in fatty acid utiliza-
tion pathways, most of which are known target genes for the
fatty acid-responsive nuclear receptor PPAR? (8). These re-
sults suggested that ERR? drives a metabolic regulatory pro-
gram that overlaps PPAR? or that ERR? might regulate the
PPAR? signaling pathway. To explore this potential mecha-
nism, the effect of ERR? on the expression of PPAR? and
related transcriptional activators was investigated.
The PPAR? transcript was called absent in the array anal-
yses, suggesting that this assay was not sensitive enough to
detect endogenous levels of PPAR? in cardiac myocytes.
Therefore, we used more sensitive Northern and real-time
PCR analyses to look at changes in PPAR? expression (Fig. 4).
Forced expression of ERR? increased endogenous PPAR?
transcript levels 8.3-fold in cardiac myocytes. The ERR?-me-
diated regulation was most robust for the PPAR? isoform,
although we also observed an approximately twofold increase
in PPAR? expression in the ERR?-overexpressing cardiocytes
(Fig. 4B). PPAR? expression was also upregulated by ERR?
in C2C12myotubes. The expression of endogenous PGC-1?
was unaffected by ERR?, indicating that the metabolic effects
of ERR? were not mediated through the known effects of
PPAR? coactivation by PGC-1? (Fig. 4A) (46). In contrast,
forced expression of PGC-1? led to upregulated of expression
of both ERR? and PPAR? in cardiac myocytes, suggesting a
potential feedforward regulation involving PGC-1?, ERR?,
To determine whether ERR? directly regulates the tran-
scriptional activity of the PPAR? gene, a human PPAR? pro-
moter-reporter construct, p?(H-H)pGL3, containing 1,664 bp
of the 5?-flanking region, was cotransfected with ERR? in the
absence or presence of PGC-1? into CV1 cells. Cotransfection
of ERR? alone activated the PPAR? promoter almost fivefold
(Fig. 5A). As expected, addition of the coactivator, PGC-1?,
significantly enhanced ERR?-mediated activation of the
PPAR? promoter. We also tested the related isoform, ERR?,
which shares many target genes with ERR? (Fig. 5A). ERR?
activated the PPAR? promoter to a similar magnitude as
ERR?. ?lthough we have shown that PGC-1? and ERR? form
a functional complex (19), the activity of ERR? was only mod-
estly enhanced by addition of PGC-1? on the PPAR? pro-
FIG. 2. ERR? expression causes lipid accumulation in primary cardiac myocytes. Oil Red O staining of primary rat neonatal cardiac myocytes
expressing either GFP (Ad-GFP) or ERR? (Ad-ERR?) and cultured in the presence of 35 ?M bovine serum albumin-complexed oleic acid. The
red droplets (inset) represent accumulated neutral lipid. Magnification, ?400.
TABLE 2. Palmitate oxidation in rat neonatal cardiac myocytes
Mean ? SEM [3H]palmitate
oxidation rate (nmol [mg of
Mean % changea
(relative to GFP) ? SEM
11.25 (? 0.83)
16.80 (? 0.88)
21.58 (? 1.66)
15.90 (? 0.70)
149.3 (? 7.8)*
191.7 (? 14.8)*
141.3 (? 6.2)*
a*, P ? 0.05 compared to the control (Ad-GFP).
9084HUSS ET AL.MOL. CELL. BIOL.
FIG. 3. Deletion of the ERR? gene has differential effects on expression of fatty acid utilization enzyme genes mouse heart and skeletal muscle.
(A) Northern blot studies performed with 15 ?g of total RNA isolated from the hearts of wild-type (WT) or ERR??/?mice. Blots were hybridized
sequentially with probes corresponding to MCAD, PGC-1?, PPAR?, and ERR?. Phosphorimage quantification of Northern signal intensities is
shown on the right. Data represent mean intensity values (? standard error) normalized to wild-type values (? 1.0). (B) Northern analysis of total
RNA comparing expression of ERR isoforms, PGC-1?, PPAR?, and MCAD in the vastus lateralis muscle, comprised predominantly of fast-twitch
glycolytic fibers, versus the soleus muscle, comprised of slow-twitch oxidative fibers. (C) (Left) Northern analysis of total RNA isolated from the
soleus of wild-type and ERR??/?mice. Representative pairs of samples from each genotype are shown. (Right) Real-time PCR (Taqman) analysis
of soleus gene expression in wild-type (n ? 6) and ERR??/?(n ? 6) mice. In addition to the transcripts detected in the Northern panel,
quantitative analysis of mRNA encoding the cellular fatty acid utilization enzyme M-CPT I and the PPAR? isoform is shown. Data represent mean
arbitrary units (? standard error) corrected to the ?-actin transcript and normalized to values in the wild type (? 1.0). Asterisks indicate significant
differences (P ? 0.05) compared to the control.
VOL. 24, 2004ERR? ACTIVATES FATTY ACID METABOLISM9085
The human PPAR? gene promoter contains two indepen-
dent nuclear receptor response elements within the 1,664-bp
region shown to be responsive to ERR?. The distal site com-
prises direct repeat 1 (DR1), through which several receptors,
including hepatocyte nuclear factor 4 (HNF-4) and chicken
ovalbumin upstream promoter-transcription factor I, as well as
PPAR? itself, activate or repress the activity of the promoter
(36). The proximal site binds the farnesoid X receptor and
confers bile acid responsiveness on the PPAR? promoter (35).
To determine whether either of the previously characterized
nuclear receptor binding sites was involved in ERR?/PGC-1?-
mediated activation of the PPAR? gene, cotransfection exper-
iments were repeated with full-length mutant PPAR? pro-
moter constructs in which either the HNF-4 or farnesoid X
receptor had been disrupted (Fig. 5B). FXREmut was acti-
vated by ERR?/PGC-1? to the same degree as the wild-type
promoter. However, disruption of the HNF-4-responsive ele-
ment (HNF4-REmut) abolished induction of the human
PPAR? promoter by ERR?/PGC-1?. Interestingly, the basal
activity of the HNF4-REmut construct was 20-fold higher than
that of the wild type, suggesting that the HNF-4-responsive
element is occupied by a repressor in CV1 cells that is dis-
placed by activators, like ERR? or HNF-4. Despite the en-
hanced baseline activity of HNF4-REmut, induction by the
farnesoid X receptor ligands chenodeoxycholic acid and tau-
rocholic acid, which occurs through the downstream farnesoid
X receptor, was still observed, demonstrating that the altered
baseline activity does not prevent further activation through
other elements (data not shown).
We then sought to determine whether ERR isoforms bound
directly to the PPAR? promoter. Electrophoretic mobility shift
assays were performed with radiolabeled probes corresponding
to the wild-type and mutated HNF-4-responsive element and
recombinant ERR? and ERR? proteins synthesized in a re-
ticulocyte lysate. ERR? and ERR? bound to the HNF-4-re-
sponsive element in a concentration-dependent manner (Fig.
5C). In contrast, the mutated HNF-4-responsive element,
which contains the same mutation that abolished ERR? re-
sponsiveness, did not form a complex with either ERR iso-
form. Parallel binding assays were performed with wild-type
and mutated farnesoid X-responsive element probes, but no
binding to ERR? or ERR? was observed (data not shown).
To determine whether ERR? bound the PPAR? promoter
in cells, chromatin immunoprecipitation assays were per-
formed on cross-linked chromatin isolated from L6 myoblasts
expressing ERR? (Fig. 5D). Precipitation with ERR? anti-
body enriched for chromatin containing the HNF-4-responsive
element (approximately threefold) compared to precipitations
performed in parallel with nonspecific antibody (immunoglob-
ulin G). Collectively, these studies show that ERR? activates
PPAR? gene expression via transcriptional regulation through
a nuclear receptor response element that is conserved in the
human and rodent PPAR? genes.
ERR?-mediated activation of fatty acid oxidation enzyme
genes is dependent on the presence of PPAR?. Our data indi-
cated that ERR? overexpression induces PPAR? targets in-
volved in cellular fatty acid utilization and is capable of directly
activating PPAR? gene transcription. Therefore, the observed
metabolic regulation in response to ERR? may occur through
direct regulation of metabolic target genes or through modu-
lation of the PPAR? pathway. To determine whether certain
ERR?-mediated regulatory events are dependent on PPAR?,
we evaluated the effects of ERR? overexpression on PPAR?
target gene expression in the presence and absence of PPAR?.
To this end, ERR? was overexpressed with adenovirus in
primary fibroblasts isolated from ERR??/?(ERR? knockout)
mice or PPAR??/?ERR??/?(double-knockout) mice (Fig.
6). ERR? knockout cells were used as the PPAR?-expressing
control in order to maximize detection of ERR?-mediated
activation of downstream targets. As expected, the expression
of known PPAR? targets involved in fatty acid oxidation,
MCAD and acyl coenzyme A oxidase, was induced by ERR?
in the ERR? knockout cells (Fig. 6A). In striking contrast,
expression of these fatty acid oxidation target genes was not
induced by ERR? in double-knockout cells. Similar results
were observed with an additional PPAR? target, M-CPT I,
although induction by ERR? in ERR? knockout cells required
coexpression of the ERR? coactivator PGC-1? (Fig. 6B). As
expected, endogenous PPAR? was induced in ERR?-express-
ing ERR? knockout fibroblasts relative to the GFP control
(data not shown). In addition, PPAR? target genes (MCAD
and acyl coenzyme A oxidase) were induced in response to
PPAR? overexpression in double-knockout cells (data not
shown). Thus, ERR?-mediated induction of at least a subset of
FIG. 4. ERR? induces endogenous PPAR? expression. (A) North-
ern analyses to characterize the expression of regulators of mitochon-
drial fatty acid oxidation, PPAR?, PGC-1?, and ERR? in cardiac
myocytes expressing GFP, ERR?, or PGC-1?. (B) Quantification of
PPAR? and PPAR? mRNA levels in response to ERR? overexpres-
sion in cardiac myocytes and C2C12myotubes by real-time PCR. Data
represent mean arbitrary units (? standard error) corrected to the
glyceraldehhyde-3-phosphate dehydrogenase (GAPDH) transcript
and normalized to the values in GFP-expressing myocytes (? 1.0).
9086HUSS ET AL.MOL. CELL. BIOL.
FIG. 5. ERR isoforms activate the PPAR? gene promoter through a conserved nuclear receptor binding site. (A) Transient cotransfection
studies in CV1 cells to analyze ERR regulation of the PPAR? promoter. (Left) The reporter construct p?(H-H)-pGL3, containing the ?1664 to
?83 region of the human PPAR? gene promoter, was cotransfected with empty expression vector (?ERR?) or pcDNA3.1-hERR? (?ERR?) in
the presence or absence of the coactivator PGC-1?. (Right) Activation by ERR? was analyzed with the same conditions as for ERR?. A
?-galactosidase (?-gal) expression construct was cotransfected to control for transfection efficiency. (B) Transient transfections were performed
with either the wild-type PPAR? promoter-reporter construct or with constructs mutated at the HNF-4-responsive element (HNF4-REmut) or
farnesoid X-responsive element (FXREmut) sites. Experiments were performed as described for panel A. All bars represent mean (? standard
error) corrected relative light units (RLU) for ?-galactosidase normalized to the activity of the reporter cotransfected with pcDNA3.1(?) (? 1.0).
Data represent at least three independent trials performed in triplicate. (C) Electrophoretic mobility shift assays were performed with32P-labeled
probes corresponding to the wild-type HNF-4-responsive element contained in the human PPAR? promoter or a mutated HNF-4-responsive
element (Mut). The Mut probe contains the same nucleotide substitutions as the HNF4-REmut promoter-reporter analyzed in B. Probes were
incubated with 1 or 3 ?l of recombinant human ERR? (left) or mouse ERR? (right) synthesized in a rabbit reticulocyte lysate. Control reactions
included probe alone (?) and probe incubated with unprogrammed reticulocyte lysate (rl). (D) (Top) L6 myoblasts were infected with Ad-ERR?
for 48 h. Cross-linked chromatin was immunoprecipitated with nonspecific antibody (immunoglobulin G) or anti-human ERR? (ERR?) antibody.
Amplicons corresponding to the 310-bp region of the PPAR? promoter containing the HNF-4-responsive element (PPAR?) or a 210-bp
nonspecific region of the TFIID promoter (control, Cont) were amplified by PCR. Input represents 0.2% of the total chromatin used in the
immunoprecipitation reactions. A representative trial from multiple experiments is shown. (Bottom) Band intensities from chromatin immuno-
precipitation experiments were quantified by densitometry. Data represent mean band intensity in arbitrary units (? standard error) from three
independent trials with PCR performed in triplicate normalized to immunoglobulin G (? 1.0). The asterisk indicates a significant difference
compared to the control (immunoglobulin G).
VOL. 24, 2004 ERR? ACTIVATES FATTY ACID METABOLISM 9087
genes involved in cellular fatty acid utilization requires
The effects of ERR? overexpression on palmitate oxidation
rates were also measured in the ERR? knockout and double-
knockout fibroblasts (Fig. 6C). In the ERR? knockout cells,
ERR? overexpression increased oxidation rates by 63%. In
contrast, ERR? expression elicited only a 15% increase in the
palmitate oxidation rate in double-knockout fibroblasts. Inter-
estingly, PGC-1? overexpression, used as a positive control,
was competent to induce palmitate oxidation in both ERR?
knockout and double-knockout cells (data not shown), suggest-
ing that another PGC-1? partner besides ERR? and PPAR? is
able to mediate activation of fatty acid oxidation. These results,
which are consistent with the gene expression studies, demon-
strate that ?RR?-mediated induction of fatty acid oxidation in
fibroblasts requires PPAR?.
Despite the fact that the ERRs were the first family of
orphan nuclear receptors cloned, their biological function has
remained uncertain (12, 13). Recent evidence has implicated
ERR? and ERR? in the transcriptional regulation of cellular
energy metabolism. ERR? is enriched in adult mammalian
tissues with high oxidative metabolic capacity, such as the
heart, slow-twitch skeletal muscle, and brown adipose. ERR?
and ERR? have also recently been shown to serve as func-
tional partners for the PGC-1 family of coactivators (17, 19, 21,
42), which have emerged as key regulators of mitochondrial
metabolism and biogenesis (23, 37). We hypothesized that
ERR isoforms serve as key regulators of heart and skeletal
muscle energy metabolism downstream of PGC-1?. To this
end, gene expression profiling experiments were conducted in
cardiac myocytes. ERR? overexpression was shown to increase
the expression of genes involved in multiple pathways involved
in cellular fatty acid utilization, including fatty acid uptake and
intracellular binding and mitochondrial oxidation. In addition,
a subset of genes involved in mitochondrial electron transport
and oxidative phosphorylation were upregulated by ERR?.
Heart and slow-twitch skeletal muscle meet high ATP de-
mands predominantly through oxidation of fatty acids in mi-
tochondria. Our results demonstrating ERR? as an activator
of oxidative metabolism in myocytes are consistent with the
enriched expression of this nuclear receptor in heart and slow-
twitch skeletal muscle. Indeed, ERR? null mice exhibit a com-
pensatory increase in PGC-1? and ERR? expression in heart
and a reduction in expression of MCAD, a key fatty acid
metabolic target gene, in the soleus, where no change in ERR?
expression was observed. These findings strongly suggest that
ERR isoforms contribute to the high-level basal expression of
fatty acid utilization genes in oxidative tissues.
Consistent with this conclusion, ERR? was recently shown,
in a combined proteomic and gene expression profiling study,
to be coregulated with proteins and enzymes physically asso-
FIG. 6. ERR? induction of ?-oxidation enzymes genes is depen-
dent on the presence of PPAR?. Primary fibroblasts isolated from
ERR??/?PPAR??/?(double-knockout, DKO) or ERR??/?(ERR
knockout, ERRKO) mice were infected with an adenoviral construct
expressing GFP (?) or ERR? (?) as indicated. PGC-1? was included
in some conditions to enhance ERR? activity. Real-time PCR was
used to quantify the expression of endogenous MCAD and acyl coen-
zyme A oxidase (ACO) (A) or M-CPT I (B) in total RNA isolated
from these cells. Data are reported as mean (? standard error) arbi-
trary units normalized to the GFP condition (? 1.0) for three inde-
pendent trials performed in triplicate. (C) Palmitate oxidation rates
were measured in the same primary fibroblasts as above expressing
GFP or ERR?. Data were normalized to GFP (? 1.0), and values
represent means (? standard error) for three overexpression trials
performed with cells from two independent isolations. Asterisks indi-
cate a significant difference compared to the controls (minus ERR?).
9088HUSS ET AL.MOL. CELL. BIOL.
ciated with the mitochondria, further supporting its role as a
regulator of energy metabolism (33). Interestingly, metabolic
function in white adipose, a predominantly glycolytic tissue, is
impaired in ERR? null animals and is associated with in-
creased MCAD expression (32). These apparently disparate
results support a complex tissue-selective metabolic regulatory
function for ERR?, with the activity of ERR? being depen-
dent on the complement of cofactors coexpressed in a given
tissue. However, our in vivo gene expression data do not ex-
clude indirect effects, such as metabolic derangements related
to the ERR?-deficient state, influencing the expression of
some putative gene targets.
We found that the metabolic regulatory effects of ERR?
overexpression in cardiac myocytes displayed considerable
overlap with those of the nuclear receptor PPAR?. The fol-
lowing lines of evidence indicate that this overlap is due, at
least in part, to direct activation of PPAR? gene expression by
ERR?: (i) overexpression of ERR? induced PPAR? gene
expression in cardiac myocytes, C2C12skeletal myotubes, and
primary mouse fibroblasts; (ii) ERR? directly activated the
PPAR? gene promoter in transient cotransfection assays
through a conserved nuclear receptor response element to
which it bound in vitro and in cells; and (iii) ERR?-mediated
regulation of a subset of its fatty acid oxidation targets in
primary fibroblasts absolutely required the presence of
PPAR?. Specifically, ERR? overexpression in PPAR? null
fibroblasts had no effect on the expression of PPAR? targets,
including M-CPT I, MCAD, or acyl-coenzyme A oxidase, yet
ERR? activated these targets in PPAR?-expressing cells.
These results strongly suggest that in tissues where ERR? and
PPAR? are coexpressed, like skeletal muscle and heart, acti-
vation of PPAR? by ERR? is an important mechanism to
control the expression of certain genes involved in cellular fatty
Collectively, our data and the results of recently published
studies suggest that ERR? regulates mitochondrial metabo-
lism through the activation of several downstream transcrip-
tional regulatory cascades. While this manuscript was in re-
view, two studies presented additional evidence for ERR? as a
key component of the PGC-1?-mediated regulation of mito-
chondrial metabolism. Schreiber et al. demonstrated that in-
duction of mitochondrial proliferation and respiratory chain
enzyme gene expression by PGC-1? is impaired by RNA in-
terference inhibition of ERR? expression (41), indicating that
ERR? is downstream of PGC-1? in regulating certain mito-
chondrial biogenic programs. Studies by Mootha et al. found a
similar role for ERR? downstream of PGC-1? in C2C12myo-
tubes (34). The latter study suggested that ERR? activates the
NRF cascade through direct activation of the Gabpa gene
promoter, which encodes a component of the NRF-2 complex.
These data are consistent with our findings, which demonstrate
that ERR? activates the mitochondrial fatty acid oxidation in
cardiac myocytes by converging on the PPAR? regulatory
Our data do not exclude the possibility that, in addition to
activating PPAR?, ERR? plays a direct role in the regulation
of certain target genes. The results of our gene expression
profiling studies demonstrated that, in addition to the fatty
acid oxidation enzyme genes, a number of genes involved in
cellular fatty acid uptake and mitochondrial respiration were
also activated by ERR?. It is likely that a number of these
genes are directly regulated by ERR?. Indeed, our previous
work demonstrated that ERR? with PGC-1? directly activates
the MCAD gene promoter in transient transfection assays
through NRRE-1, a pleiotropic nuclear receptor response el-
ement that has been shown to bind both PPAR? and ERR?
(14, 19, 43, 47). We also observed modest activation of the
lipoprotein lipase promoter by ERR? (J. Huss, unpublished
observation). Furthermore, recent studies have demonstrated
that ERR? with PGC-1? directly activates the ATP synthase ?
and cytochrome c gene promoters through consensus ERR?
response elements (41). It is therefore likely that ERR? reg-
ulates cellular metabolism through multiple pathways, includ-
ing indirect regulation via other transcription factors and direct
regulation of metabolic target genes.
Our results and studies by others have shown that ERR?
expression is upregulated by PGC-1? in cultured cells (Fig.
4A) (42) and in vivo in the hearts of mice overexpressing
PGC-1? (39; L. Russell and J. Huss, unpublished observation).
These results place ERR? in a central position within the
PGC-1? regulatory network (Fig. 7). We propose that ERR?
transduces PGC-1?-derived signals to transcription factors
such as PPAR? and NRFs as well as directly to target genes
involved in energy metabolism (34, 41). The extent of the role
of ERR isoforms in mediating the actions of PGC-1? on cel-
lular metabolism and physiology is unknown. However, the
inducibility of PGC-1? by fasting, exercise, and cold exposure
suggests that this regulatory circuit serves a critical role in the
physiologic regulation of cardiac and skeletal muscle energy
metabolism. Given that derangements in mitochondrial oxida-
tive metabolism occur in pathophysiologic states such as skel-
etal muscle insulin resistance and cardiac hypertrophy, ERRs
may prove to be an attractive therapeutic target for common
human diseases such as diabetes and heart failure.
This work is supported by NIH grants DK45416 and HL58493,
Digestive Diseases Core Center grant P30DK52574 (D.P.K), and an
operating grant from the Canadian Institutes for Health Research
FIG. 7. Role of ERR? in regulating cellular oxidative capacity.
PGC-1? coactivates and regulates the expression of a number of tran-
scription factors, including ERR?, PPAR?, and NRFs, involved in
mediating PGC-1? effects on cellular metabolism. PGC-1? regulation
of ERR? and NRF-2 expression involves both cross- and auto-regu-
latory mechanisms (25, 34). Data presented in the current study dem-
onstrated that ERR? likely directs PGC-1? upregulation of PPAR?.
In response to PGC-1?, activation of the NRF cascade regulates genes
involved in mitochondrial respiration and biogenesis, whereas activa-
tion of the PPAR? pathway regulates fatty acid uptake and mitochon-
drial oxidation enzymes. ERR? may also directly regulate metabolic
target genes in both pathways. FAO, fatty acid oxidation; ACO, acyl
coenzyme A oxidase; mtTFA, mitochondrial transcription factor A;
oxid. phos., oxidative phosphorylation.
VOL. 24, 2004ERR? ACTIVATES FATTY ACID METABOLISM 9089
(V.G.). J.M.H. is supported by NIH 1K01 DK063051-01 and the Wash-
ington University School of Medicine Diabetes Research Training
Center P60 DK20579.
We thank the Alvin Siteman Cancer Center Multiplexed Gene
Analysis Core at Washington University School of Medicine for per-
forming DNA microarray analyses and the Digestive Disease Histol-
ogy Core for histologic studies. We thank Mary Wingate for expert
assistance in preparing the manuscript.
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