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1 Tiraby et al. Acquirement of Brown Fat Cell Features by Human White Adipocytes Claire Tiraby, Geneviève Tavernier, Corinne Lefort, Dominique Larrouy, Frédéric Bouillaud , Daniel Ricquier , and Dominique Langin § From the Unité de Recherches sur les Obésités, Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 586, Institut Louis Bugnard, Centre Hospitalier Universitaire de Toulouse, Université Paul Sabatier, Toulouse, France and the Unité 9078, Centre National de la Recherche Scientifique (CNRS), Faculté de Médecine Necker-Enfants Malades, Paris, France § To whom correspondence should be addressed : Tel : (33)562172950. Fax : (33)561331721. E-mail: [email protected] Running title : Expression of UCP1 in white fat cells by guest on February 18, 2018 http://www.jbc.org/ Downloaded from

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Page 1: Induction of uncoupling protein 1 and fat oxidation in human

1 Tiraby et al.

Acquirement of Brown Fat Cell Features by Human White Adipocytes

Claire Tiraby, Geneviève Tavernier, Corinne Lefort, Dominique Larrouy, Frédéric

Bouillaud‡, Daniel Ricquier‡, and Dominique Langin§

From the Unité de Recherches sur les Obésités, Institut National de la Santé et de la

Recherche Médicale (INSERM) Unité 586, Institut Louis Bugnard, Centre Hospitalier

Universitaire de Toulouse, Université Paul Sabatier, Toulouse, France and the ‡Unité

9078, Centre National de la Recherche Scientifique (CNRS), Faculté de Médecine

Necker-Enfants Malades, Paris, France

§ To whom correspondence should be addressed : Tel : (33)562172950. Fax :

(33)561331721. E-mail: [email protected]

Running title : Expression of UCP1 in white fat cells

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Summary

Obesity, i.e. an excess of white adipose tissue (WAT), predisposes to the development

of type 2 diabetes and cardiovascular disease. Brown adipose tissue is present in

rodents but not in adult humans. It expresses uncoupling protein 1 (UCP1) that allows

dissipation of energy as heat. Peroxisome proliferator-activated receptor γ (PPARγ) and

PPARγ coactivator 1α (PGC-1α) activate mouse UCP1 gene transcription. We show

here that human PGC-1α induces the activation of the human UCP1 promoter by

PPARγ. Adenovirus-mediated expression of human PGC-1α increases the expression of

UCP1, respiratory chain proteins and fatty acid oxidation enzymes in human

subcutaneous white adipocytes. Changes in the expression of other genes were also

consistent with brown adipocyte mRNA expression profile. PGC-1α increased palmitate

oxidation rate by fat cells. Human white adipocytes can therefore acquire typical features

of brown fat cells. The PPARγ agonist rosiglitazone potentiated the effect of PGC-1α on

UCP1 expression and fatty acid oxidation. Hence, PGC-1α is able to direct human WAT

PPARγ towards a transcriptional program linked to energy dissipation. However, the

response of typical white adipocyte targets to rosiglitazone treatment was not altered by

PGC-1α. UCP1 mRNA induction was shown in vivo by injection of the PGC-1α

adenovirus in mouse white fat. Alteration of energy balance through an increased

utilization of fat in WAT may be a conceivable strategy for the treatment of obesity.

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INTRODUCTION

Two types of adipose tissues coexist in mammals. White adipose tissue (WAT)1 has

an essential role for storage of energy in the form of triacylglycerol. In situations of

energy deficit such as fasting, lipolysis in WAT controls the supply of energy to the body

through the release of fatty acids into the plasma. The uncontrolled expansion of WAT

seen in obesity predisposes to the development of an array of metabolic disturbances

leading to type 2 diabetes and cardiovascular disease. Though it shares many features

with WAT, brown adipose tissue (BAT) is specialized in adaptive thermogenesis (1).

Differences in gene expression between WAT and BAT, most notably at the

mitochondrial level, explain the thermogenic capacity of BAT. Fatty acid oxidation

enzymes and respiratory chain components are highly expressed in BAT contributing to

a high oxidative capacity. The activity of ATP synthase is low due to a defect in

expression of the P1 gene (2). However, the most distinguishing feature of BAT is the

expression of uncoupling protein 1 (UCP1) (3). UCP1 is a 32 kDa protein expressed in

the inner membrane of the mitochondria. UCP1 allows the dissipation of the proton

electrochemical gradient generated by the respiratory chain. Uncoupling between

oxygen consumption and ATP synthesis promotes energy dissipation as heat. The

mechanism of action of UCP1 is still controversial. One model depicts UCP1 as a true

proton transporter while another model states that UCP1 catalyzes a fatty acid

protonophoretic cycle (4). Fatty acids and retinoids have been shown to activate UCP1

(5,6). In neonatal mammals, hibernators and rodents, cold-induced thermogenesis in

BAT contributes to the maintenance of body temperature. Fuel is provided as fatty acids

deriving from BAT and WAT lipolysis. In rodents, BAT also participates in diet-induced

thermogenesis and may thereby control the energy efficiency of food (7).

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UCP1 biosynthesis is mainly controlled at the transcriptional level. During cold

exposure, sympathetic nervous system stimulation of BAT is the primary signal that

activates UCP1 gene expression. Retinoic acid (RA) and thyroid hormones are other

positive regulators. A critical enhancer has been characterized in rodent UCP1 genes

(8,9). This region is required for catecholamine and RA stimulation. The enhancer

contains a peroxisome proliferator-activated receptor γ (PPARγ) responsive element that

mediates the stimulation induced by thiazolidinediones (TZD) (10). In cooperation with

PPARγ, the PPARγ coactivator PGC-1α, has been shown to induce mouse UCP1 gene

transcription (11). It also stimulates the expression of electron transport chain genes and

mitochondrial biogenesis, through induction of nuclear respiratory factors (NRF) 1 and 2

(12). PGC-1α expression is increased in response to cold exposure and β-adrenergic

stimulation (11).

BAT is present throughout the life in rodents but disappears soon after birth in large

mammals. In human fetus and newborn child, it is found in the cervical, axillary,

perirenal and periadrenal depots (13). There are no BAT depots in adults and UCP1

mRNA is expressed at very low levels in WAT (14). BAT is not thought to contribute to a

significant part of thermogenesis (15). However, UCP1 is expressed in hibernomas and

in perirenal WAT of adult patients with phaeochromocytoma and primary aldosteronism

revealing that UCP1 expression can be induced in rare tumors and endocrinological

disorders (16,17). Pharmacotherapy targeted at molecular pathways that regulate

adaptive thermogenesis provides a plausible and safe means of increasing energy

expenditure (18). Reactivation of brown adipocytes is therefore an important goal.

Studies on human white adipocytes are mandatory to substantiate the proof of concept.

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In an attempt to promote a metabolic shift in white fat cells from lipid storage towards

fatty acid utilization, human subcutaneous white adipocytes were transduced with an

adenovirus expressing PGC-1α. The cells acquire features of brown adipocytes, i.e. an

induction of UCP1 and respiratory chain gene expression, and an increased capacity to

oxidize fatty acid. Conversion of white into brown adipocytes may therefore constitute a

strategy to regulate fat mass in humans.

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MATERIAL AND METHODS

Adenoviral expression system and adenofection experiment in CV-1 cells-

Recombinant adenovirus was generated as described (19). The full-length human PGC-

1α cDNA (20) was cloned into the pAdEasy parent plasmid. Recombination between the

pAdEasy and pAdTrack vectors and production of the PGC-1α adenovirus was

performed at the Laboratoire de Thérapie Génique de Nantes. The virus contains, in

tandem, the green fluorescent protein (GFP) gene and the PGC-1α cDNA downstream

of separate cytomegalovirus promoters. An adenovirus containing only the GFP gene

was used as control. Viral titers were, respectively, 1.7x1011 and 1.4x1011 infectious

particles per ml. Adenofection experiments were performed in CV-1 cells (ATCC,

Manassas, Virginia) cultured in DMEM containing 10% fetal calf serum (Invitrogen,

Cergy Pontoise, France). The 6.3 kb UCP1 promoter-chloramphenicol acetyl transferase

gene construct (21) was cotransfected with expression vectors for PPARγ2 (from Bruce

Spiegelman, Dana-Farber Cancer Institute, Boston, Massachussetts) and RXRα (from

Pierre Chambon, IGBMC, Strasbourg, France) and a cytomegalovirus promoter-β-

galactosidase gene vector to check for transfection efficiency. The PGC-1α or the GFP

adenoviruses were added to the Lipofectamine (Invitrogen) transfection mix at a

multiplicity of infection (moi) of 200. Cells were exposed for 6 h to the transfection mix.

Ligands dissolved in a vehicle containing 0.1% DMSO were added for 48 h.

Chloramphenicol acetyl transferase activity was assayed on cell extracts 72 h post-

adenofection.

Differentiation of human preadipocytes, adenovirus infection and flow cytometry-

Subcutaneous abdominal adipose tissue was obtained from female subjects undergoing

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plastic surgery in agreement with French laws on biomedical research. Human

adipocytes in primary culture were differentiated as described by Hauner et al. (22) with

modifications (23). Stromal cells prepared from WAT were cultured for 13 days in a

chemically defined medium. At day 13, 60-80% of cells were differentiated into lipid

droplet-containing adipocytes. UCP1 mRNA level in differentiated cells were similar to

the level found in native subcutaneous adipose tissue2. Hence, differentiation of

preadipocytes in primary culture did not result in dysregulation of UCP1 gene

expression. The cells were infected at a moi of 200 for 6 h. The day after infection, cells

were treated as indicated in text and figures with the following drugs at 1 µM unless

otherwise indicated: rosiglitazone (BRL49653, Smith Kline Pharmaceuticals, Harlow,

UK); Wy14643 (Cayman Chemical, Ann Arbor, Michigan); L165041 (Merck Research

Laboratories, Rahway, New Jersey); and 9-cis RA (Sigma). Cells were harvested after

48 h of treatment for mRNA assays and 72 h of treatment for protein and fatty acid

oxidation assays. To isolate adenovirus-transduced cells, GFP-positive cells were

sorted, after trypsinization, using an EPICS Altra Hypersort System (Beckman Coulter,

Roissy, France).

Quantitative RT-PCR analysis- Total RNA was isolated using RNeasy kit (Qiagen,

Courtaboeuf, France). Total RNA (1 µg) was treated with DNase I (DNase I amplification

grade, Invitrogen), then retrotranscribed using random hexamers and Thermoscript

reverse transcriptase (Invitrogen). Real time quantitative PCR was performed on

GeneAmp 7000 Sequence Detection System using SYBR green chemistry (Applied

Biosystems, Courtaboeuf, France) as described (24). Primers were designed using the

Primer Express 1.5 software (Table 1). Some mRNAs were quantified using Assay-on-

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Demand gene expression assays (Applied Biosystems). 18S ribosomal RNA was used

as control to normalize gene expression using the Ribosomal RNA Control Taqman

Assay kit (Applied Biosystems). Similar results were obtained using SYBR green- and

Assay-on-Demand-based detections for the quantification of PGC-1α and UCP1 mRNA

levels.

Western blot analysis- Mitochondria from mouse BAT and human adipocytes were

prepared by differential centrifugation in 10 mM Tris, pH 8, 1 mM EDTA, 250 mM

sucrose supplemented with a cocktail of protease inhibitors (Sigma). Mitochondrial

proteins (5 µg for human adipocytes and 0.2 µg for BAT) were subjected to 10% SDS-

PAGE, transferred onto nitrocellulose membrane (Hybond ECL, Amersham Biosciences,

Orsay, France) and probed with a polyclonal anti-rat UCP1 antibody (25) and an anti

cytochrome c antibody (Pharmingen, BD Biosciences, Le Pont de Claix, France).

Immunoreactive protein was determined by enhanced chemiluminescence reagent

(Amersham Biosciences).

In vivo adenovirus injection in mouse fat pad- Studies with mice followed the

INSERM and Louis Bugnard Institute Animal Care Facility guidelines. Male B6D2/JIco

mice (24-30 week-old, IFFA-CREDO, L'Arbresle, France) were anesthesized with

avertin (Sigma). Following dissection of the skin and body wall, one testis with attached

epididymal fat pad was pulled out. The adenoviral preparation (1.7x108 infectious

particles) was injected to 6 points in the fat pad. A fat pad was injected with PGC-1α

adenovirus and the contralateral fat pad with GFP adenovirus. After 5 days, total RNA

was prepared from the fat pads for quantitative RT-PCR analyses.

Palmitate oxidation experiment- Differentiated human adipocytes were incubated for

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3 h in a medium containing DMEM without glucose, 50 mM Hepes pH 7.8, 1% fatty acid-

free bovine serum albumin, 2 mM L-carnitine, 50 µM palmitate and 118 nM

[14C]palmitate (850 µCi/µmol, Amersham Biosciences). Medium was transferred in a

flask with a center well containing Carbosorb E (Perkin Elmer Life Sciences,

Courtaboeuf, France). 14CO2 was liberated by acidification with 5N HCl and collected

overnight on Carbosorb. 14CO2 was measured by scintillation counting. The acid soluble

fraction of the medium containing 14C-labelled β-oxidation metabolites were measured

by scintillation counting after 1-butanol extraction of palmitate. To inhibit fatty acid

oxidation, we used 50 µM of etomoxir (from Wolfgang Langhans, Swiss Federal Institute

of Technology, Zurich, Switzerland). To study the effect of mitochondrial uncoupling, m-

chlorocarbonylcyanide phenylhydrazone (Sigma), was added at 10 µM during the 3 h

incubation period.

Statistical analysis- Data are expressed as mean ± SEM. Statistical analyses were

performed using ANOVA with least-square difference post-hoc analysis or Student's t-

test.

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RESULTS

The nuclear receptor PPARγ2 and its coactivator PGC1α transactivate the human

UCP1 promoter- To test the transcriptional coactivation of the human UCP1 promoter by

PGC-1α, we utilized an adenovirus expressing human PGC-1α in combination with

expression vectors for PPARγ2 and its partner, retinoic acid X receptor α (RXRα). PGC-

1α functions as a transcriptional coactivator for the two nuclear receptors (11,26). The

6300 bp human UCP1 promoter region mediates the stimulation of transcription by TZD

in a murine brown adipocyte cell line (21). In simian CV-1 cells, the PPARγ2/RXRα

combination had no transactivation potency (Fig. 1). The expression of PGC-1α alone

induced a modest rise of UCP1 gene transcription. However, the addition of PGC-1α to

the PPARγ2/RXRα combination led to a marked increase in activity.

PGC-1α induces UCP1 expression in human white adipocyte- Human WAT express

low levels of PGC-1α (20). We used a human PGC-1α adenovirus to increase the

expression of the coactivator in primary culture of human subcutaneous adipocytes (Fig.

2A). Unlike retroviral vector systems that must be used on proliferating preadipocytes,

adenoviruses can transduce quiescent mature adipocytes (27). This method permits to

avoid the effect of continuous PGC-1α expression during adipogenesis. The PGC-1α

adenovirus efficiently transduced differentiated adipocytes as revealed by GFP labelling

of 40-50% of fat cells. Remarkably, adenoviral infection occurred exclusively in

differentiated cells (Fig. 2B). There was no GFP staining in undifferentiated fibroblasts.

To ascertain that preadipocytes were resistant to adenoviral infection, human fibroblasts

at day 3 of culture were infected with PGC-1α adenovirus at various mois. No increase

in PGC-1α mRNA was observed at an moi of 500. At moi of 1000 and 2000, PGC-1α

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mRNA were only increased by 3- and 5-fold, respectively whereas the induction was 100

to 150-fold in day 13 differentiated adipocytes at a moi of 200. The data reveal that

human adipocytes possess much more efficient plasma membrane binding and

internalization components for human serotype 5 adenovirus than preadipocytes. The

effects mediated by PGC-1α can thereby be ascribed to its selective overexpression in

adipocytes.

The robust overexpression of PGC-1α in human adipocytes was accompanied by an

induction of UCP1 mRNA expression (Fig. 3A). UCP1 mRNA was barely detectable in

cells infected with control GFP adenovirus. The marked increase associated with PGC-

1α expression was amplified in the presence of PPARγ and RXRα ligands (p < 0.01). As

shown on Fig. 3B, the mRNA level of UCP1 paralleled that of PGC-1α when cells were

transduced at different mois. The expression of GFP allowed us to select the transduced

cells using flow cytometry (Fig. 3C). Cells infected with PGC-1α adenovirus and treated

with TZD and RA co-expressed PGC-1α and UCP1 mRNA. These data show that the

induction of UCP1 is restricted to adipocytes expressing PGC1α. Western blot analysis

showed an increase of a 32 kDa immunoreactive band corresponding to UCP1 (Fig. 4).

Similar results were obtained using an antibody raised against the whole rat protein (25)

or an antibody directed against a 19 amino acid C-terminal UCP1 peptide2. As for mRNA

level, the addition of PPARγ/RXRα ligands further increased UCP1 protein expression.

The amount of UCP1 mRNA and protein in transduced and treated human adipocyte

cultures represented 1.0±0.4 and 1.6±0.6 % (n=4) of the corresponding levels in mouse

brown fat.

PPARγ cooperates with PGC-1α to induce UCP1 expression- To determine which

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PPAR was involved in PGC-1α coactivation of UCP1 expression, we tested ligands for

the three subtypes of nuclear receptors (Fig. 3D). Rosiglitazone, a PPARγ agonist, was

a potent stimulator of UCP1 expression. Agonists for PPARα and PPARβ and 9-cis RA

had poor inducing potency. The data show the pre-eminent role of the PPARγ/PGC-1α

association for the induction of UCP1 in human white adipocytes.

PGC-1α -expressing adipocytes show increased expression of mitochondrial

proteins and brown adipocyte markers- We wished to determine whether the

upregulation of UCP1 in TZD treated PGC-1α-overexpressing human adipocytes was

associated with other metabolically relevant adaptations in gene expression. PGC-1α

induces expression of components of the mitochondrial respiratory chain in several cell

types (11,12). The mRNAs for cytochrome c and cytochrome oxidase 4 were increased

(Fig. 5). An induction of cytochrome c was found at the protein level (Fig. 4). We also

observed an induction in the mRNA expression of mitofusin 2, a mitochondrial protein

essential for mitochondrial network architecture highly expressed in brown adipose

tissue (28). Ectopic expression of PGC-1α and PPARα in 3T3-L1 murine fibroblasts

leads to an increase in mitochondrial fatty acid oxidation enzyme gene expression (29).

We therefore determined the mRNA levels for muscle carnitine palmitoyltransferase I

(M-CPT1), the isoform of CPT1 expressed in human white adipocytes (30), and

medium-chain acyl coenzyme A dehydrogenase (MCAD). Treated PGC-1α-expressing

adipocytes showed higher M-CPT1 and MCAD mRNA levels than control cells. Glycerol

kinase activity is very low in WAT whereas relatively high levels are found in BAT (31).

PGC-1α in the presence of TZD and RA led to a 7-fold induction of glycerokinase mRNA

levels.

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PGC-1α does not alter TZD response of typical white adipocyte target genes- We

next investigated the effect of PGC-1α on the response of WAT genes regulated by TZD

(Fig. 6). Adipocyte lipid binding protein (ALBP) and cytosolic phosphoenolpyruvate

carboxykinase (PEPCK1) are direct targets of TZD in white adipocytes with identified

PPARγ responsive elements (32,33). Rosiglitazone increased ALBP and PEPCK1

mRNA levels in cells transduced with PGC-1α or GFP adenovirus. Hence, the

expression of PGC-1α was not accompanied by a loss of TZD response of target genes.

PGC-1α stimulates UCP1 mRNA expression in mouse white fat pads in vivo- We

then asked whether overexpression of PGC-1α could induce UCP1 in vivo. The PGC-1α

adenovirus was injected in the retroperitoneal fat pad of male mice. This fat pad was

selected because it contains very few brown fat cells (34). Compared to the contralateral

side injected with GFP adenovirus, the increase in PGC-1α mRNA level was

accompanied by a more than 10-fold induction of UCP1 mRNA expression (Fig. 3E).

The experiment reveals that PGC-1α is able to induce UCP1 gene expression in WAT in

vivo.

PGC-1α increases fat oxidation in human white adipocytes- We hypothesized that

an induction of UCP1, proteins of the respiratory chain and enzymes of fatty acid

oxidation may lead to an increased capacity of fat oxidation. Fig. 7 shows that PGC-1α-

expressing human adipocytes had higher total fatty acid oxidation than cells infected

with the GFP adenovirus. The effect was mimicked by the chemical mitochondrial

uncoupler, m-chlorocarbonylcyanide phenylhydrazone. This data reveals that, in human

adipocytes, uncoupling of the respiratory chain is associated with an increase in fatty

acid oxidation. There was both an increase in the production of CO2 and β-oxidation

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metabolites2. Addition of rosiglitazone potentiated the effect of PGC-1α (p < 0.01).

Compared to basal conditions, total oxidation was doubled. Addition of etomoxir, a CPT1

inhibitor, abolished palmitate oxidation.

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DISCUSSION

The transcriptional coactivator PGC-1α may play an important role in adaptive

thermogenesis in rodents through a positive regulation of mitochondrial proteins

(11,35,36). In this study, we show that expression of PGC-1α in human white adipocytes

specialized in storage of energy induces the expression of UCP1, respiratory chain

protein, fatty acid oxidation enzyme and other brown adipocyte markers. The most

salient observation is the marked induction of UCP1 expression. Expression of PGC-1α

is thus sufficient to trigger the expression of UCP1 in human white adipocytes. If only

transduced cells are considered, the level of UCP1 reaches 4-6 % of UCP1 amount in

mouse BAT. Ectopic expression of UCP1 in transgenic mice at 1 % of BAT UCP1

content impedes the development of obesity (37). Moreover, PGC1α induces

cytochrome c and cytochrome oxidase 4, two respiratory chain proteins. Interestingly, an

increase in mitofusin 2 mRNA expression was observed. The protein is highly expressed

in mitochondria from brown adipose tissue and skeletal muscle (28). Mitofusin 2

participates in the maintenance of the mitochondrial network architecture and controls

mitochondrial metabolism, most notably cellular respiration and mitochondrial proton

leak. We also show that TZD and PGC1α stimulate the expression of the mitochondrial

fatty acid enzymes CPT1 and MCAD. CPT1 catalyzes the initial reaction in the

mitochondrial import of long-chain fatty acids, a tightly regulated step in fatty acid

utilization. MCAD catalyzes a pivotal reaction of the β oxidation cycle. Other markers of

brown adipocytes such as glycerol kinase were induced (31).

The coordinated regulation of gene expression suggests a potential increase in the

capacity of fatty acid oxidation. Functional studies corroborate that view. Palmitate

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oxidation was indeed elevated in the modified adipocytes. These data are the first

demonstration of an increase in the fat oxidation capacity of human white fat cells. The

increase in CPT1 and MCAD expression is important as the two proteins control limiting

steps of β oxidation (38). However, adaptation at the level of the respiratory chain is

probably essential. Increase in respiratory chain protein and UCP1 content may lead to

an increase of cellular respiration with a stimulation of oxidative phosphorylation and

uncoupling. The marked upregulation of UCP1 and uncoupling capacity is important

since it enables the cells to oxidize fatty acids without the kinetic limitations imposed by

respiratory control (39). Accordingly, we show that addition of a chemical uncoupler

stimulates fatty acid oxidation. In TZD-treated adipocytes expressing PGC1α, free fatty

acids (e.g., derived from intracellular lipolysis) could furnish NADH and FADH2 to the

respiratory chain through β oxidation and also directly activate UCP1 as occurs during

cold exposure in BAT (40). Human white adipocytes can therefore acquire functional

features of brown adipocytes.

The molecular mechanisms involved in the regulation of the human UCP1 gene has

been partially elucidated. A 6300 bp 5’-flanking region mediates the positive effects of β-

adrenergic agonist, RA and TZD (21). The genomic fragment contains a 350 bp

enhancer organized as a multipartite response element partially homologous to the

mouse and rat enhancers (41). Our data demonstrate that PGC-1α can coactivate the

PPARγ2/RXRα heterodimer to stimulate the human UCP1 promoter. Stimulation of

transcription is associated with a PPARγ-dependent increase in UCP1 mRNA and

protein levels in human white fat cells. PPARα and PPARβ agonists have minor effects.

This is different from brown adipocytes where both PPARα and PPARγ cooperate with

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PGC-1α to activate UCP1 gene transcription (42,43). The regulation of classical white

adipocyte PPARγ-responsive genes is not altered by PGC-1α since ALBP and PEPCK

induction by TZD is preserved. Promoter-specific interactions between PPARγ and

coactivators may partially underlie the similarities and differences in gene expression

between brown and white adipocytes.

In response to cold, appearance of brown fat cells is observed in mouse visceral

WAT (44). An unanswered question is the origin of the novel brown fat cells. The cells

could originate from differentiation of a specific pool of precursor cells already present in

WAT. Accordingly, the presence of UCP1 was detected in about 10% of adipocytes

differentiated from a Siberian dwarf hamster white adipocyte precursor pool (45). It has

also been proposed that some unilocular white adipocytes are “masked” brown fat cells

that revert to the brown adipocyte phenotype (46). Finally, brown fat cells could derive

from direct conversion of white adipocytes (47,48). As PGC-1α was expressed

exclusively in fully differentiated white fat cells and not throughout the differentiation

process, our data reveal that transdifferentiation of mature white adipocytes into UCP1-

expressing cells can be activated in human WAT. The results could have implications on

drug discovery strategy as it brings the experimental proof for conversion of mature

white adipocytes into brown-like fat cells.

The notion of an activation of nonshivering thermogenesis in BAT to prevent

excessive fat storage in situations of high calorie intake was first introduced by Rothwell

and Stock (49). An apparent paradox comes from UCP1-deficient mice that show cold

intolerance but do not become obese (50,51). However, emergence of brown fat cells in

white fat depots is associated with a lean phenotype in several transgenic models (52-

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55). The mice have enhanced metabolic rate and insulin sensitivity and, are protected

against diet-induced obesity. Furthermore, transgenic mice expressing UCP1 in WAT

are protected against genetic and dietary obesity and show an increase in WAT oxygen

consumption (37,56). BAT of these mice is atrophied and the animals are cold sensitive

(57). UCP1 levels are low in retroperitoneal fat pads of genetically obese rats and mice

(34,46). It is therefore possible that the role of UCP1 and brown fat cells differ according

to the location in BAT or WAT, the latter being associated with obesity resistance.

The cooperation between PPARγ and PGC-1α may explain the effects of TZD on

thermogenesis. In vivo treatments with TZD have been reported to increase BAT mass

(58,59). The increased formation of BAT is accompanied by an increase in UCP1

expression (59-61). The effect is observed in lean animals but also in genetically obese

mice which are characterized by a defect in adaptive thermogenesis. A TZD, NC-2100,

has been shown to promote a robust antidiabetic effect on KKAy obese mice without the

increase in the weight of white fat depots reported with classical TZD (62). The lack of

weight gain may partially be explained by an induction of UCP1 in WAT that is stronger

for this compound than for classical TZD. Ectopic expression of PGC-1α in white

adipocytes is therefore sufficient to direct PPARγ towards a transcriptional program

linked to energy dissipation besides its classical role in adipogenesis and maintenance

of the white adipocyte phenotype (63,64). The in vivo data in rodents and our results

suggest that a combination of TZD and inducers of PGC-1α expression may enhance

the oxidative capacity of human WAT.

Obesity is commonly seen as a disorder of energy balance, where energy intake

exceeds energy expenditure. Mobilization of WAT without use of released fatty acids

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may be deleterious, both as the excess calories will be deposited in other organs as

seen in lipoatrophic models (65,66) and because excess fatty acids may play a role in

the development of insulin resistance and cardiovascular complications (67).

Therapeutic strategies to increase the expression and activity of PGC-1α in WAT could

contribute to the induction of UCP1 expression and fatty acid oxidation leading to a

decrease in fat mass.

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Acknowledgements- We deeply thank Dr. Anne-Marie Cassard-Doulcier, Dr. Bruno

Miroux (CNRS unit 9078, Paris), Dr. Max Lafontan, Dr. Nathalie Viguerie, Cedric

Jenkins, Stéphanie Bonhoure, Aline Mairal (INSERM U586, Toulouse) and Dr. Hubert

Vidal (INSERM U449, Lyon) for help and fruitful discussion. We also thank the Vector

Core of the University Hospital of Nantes supported by the Association Française contre

les Myopathies (AFM) for providing the Adenovirus vectors. We gratefully acknowledge

the Louis Bugnard Institute Animal Care Facility and Molecular Biology Platform (Dr. Y.

Bareira and J.J. Maoret, Toulouse) and, the Claude de Préval Institute Flow Cytometry

Core Facility (Dr. F. L'Faqihi, Toulouse). This work was supported by INSERM

PROGRES grant 4P007E.

FOOTNOTES AND ABBREVIATIONS

1 The abbreviations used are : ALBP, adipocyte lipid binding protein; BAT, brown

adipose tissue; CPT, carnitine palmitoyltransferase; GFP, green fluorescent protein; moi,

multiplicity of infection; MCAD, medium-chain acyl coenzyme A dehydrogenase; NRF,

nuclear respiratory factor; PEPCK, phosphoenolpyruvate carboxykinase; PGC, PPARγ

coactivator; PPAR, peroxisome proliferator-activated receptor; RA, retinoic acid; RXR,

retinoic acid X receptor; TZD, thiazolidinedione; UCP, uncoupling protein; WAT, white

adipose tissue

2 C.T. and D.L., unpublished information

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FIGURE LEGENDS

FIG. 1. PGC-1α-mediated transactivation of the human UCP1 promoter by PPARγ2

and RXRα. A chloramphenicol acetyl transferase reporter gene linked to the -6.3 kb

UCP1 promoter was transfected into CV-1 cells along with expression vectors for

PPARγ2 and RXRα and, PGC1α or control GFP adenovirus. The fold induction was

compared to the value observed in cells transfected with empty vectors and control

adenovirus. n = 3. **, P < 0.01.

Fig. 2 Transduction of human white adipocytes by an adenovirus expressing PGC-

1α. A, Schematic diagram of the experiments performed in human subcutaneous

adipocytes. B, Light microscopy and green fluorescence of human adipocytes

transduced with the PGC1α adenovirus. Only differentiated cells express GFP.

Fig. 3 Induction of UCP1 in white adipocytes expressing PGC-1α. A, Changes in

UCP1 mRNA levels induced by PGC-1α. Differentiated adipocytes were transduced with

PGC1α or control GFP adenovirus. Cells were treated with rosiglitazone (Rosi) and 9

cis-retinoic acid (RA). n = 4. **, P < 0.01. B, Effect of increasing expression of PGC1α

on UCP1 mRNA levels. Differentiated adipocytes were treated with Rosi and RA and

transduced with PGC1α adenovirus at increasing mois. n = 3. C, PGC-1α and UCP1

mRNA levels in cells expressing (gfp+) or not (gfp-) GFP. Differentiated adipocytes

transduced with GFP or PGC-1α adenovirus were trypsinized and sorted by flow

cytometry. n = 2. D, Effect of PPARγ and RXRα ligands on UCP1 mRNA expression.

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Differentiated human adipocytes were transduced with PGC1α adenovirus and treated

with Rosi, a PPARγ agonist; Wy14643 (Wy), a PPARα agonist; L165041 (L16), a

PPARβ agonist; or RA, an RXRα agonist. The fold induction was compared to the value

observed in untreated cells. Unless otherwise indicated, the drugs were used at 1 µM. n

= 6. *, P < 0.05. E, In vivo induction of UCP1 mRNA by human PGC1α in mouse white

fat pad. The PGC1α adenovirus was injected in epididymal fat pad. The control GFP

adenovirus was injected in the contralateral fat pad. Five days post-injection, mRNA

levels for human and murine PGC1α and, murine UCP1 were determined. n=6, **, P <

0.01.

FIG. 4. Western blot analysis of UCP1 and cytochrome c expression. Mitochondrial

proteins were prepared from mouse brown adipose tissue (BAT, 0.2 µg) and human

white adipocytes (5 µg). Adipocytes were treated with rosiglitazone (Rosi) and 9-cis

retinoic acid (RA).

FIG. 5. PGC-1α-mediated induction of mitochondrial protein and brown adipocyte

marker mRNA levels. Differentiated adipocytes were transduced with PGC1α or control

GFP adenovirus and treated with rosiglitazone (Rosi) and 9 cis-retinoic acid (RA). Cyt. c,

cytochrome c; COX4, cytochrome oxidase 4; M-CPT1, muscle carnitine

palmitoyltransferase I; GyK, glycerol kinase; MCAD, medium-chain acyl coenzyme A

dehydrogenase. n = 4. *, P < 0.05.

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FIG. 6. Effect of PGC-1α on TZD response of white adipocyte target genes.

Differentiated adipocytes were transduced with PGC1α or control GFP adenovirus. Cells

were treated with rosiglitazone (Rosi) and 9 cis-retinoic acid (RA). ALBP, adipocyte lipid

binding protein; PEPCK1, phosphoenolpyruvate carboxykinase 1. n = 4. *, P < 0.05; **,

P < 0.01.

FIG. 7. Increase of fatty acid oxidation in PGC-1α-expressing human adipocytes.

Differentiated adipocytes were transduced with PGC1α or control GFP adenovirus. Cells

were treated with rosiglitazone (Rosi). Hatched bars represent the inhibition of oxidation

obtained with the addition of the carnitylpalmitoyltransferase-1 inhibitor, etomoxir. To

study the effect of mitochondrial uncoupling on palmitate oxidation, we used m-

chlorocarbonylcyanide phenylhydrazone (CCCP). The fold induction was compared to

the value observed in cells transduced with control GFP adenovirus. n = 4. **, P < 0.01.

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TABLE 1 Primers used in real time quantitative PCR

ALBP, adipocyte lipid binding protein ; Cyt. c, cytochrome c ; M-CPT1, muscle carnitine palmitoyltransferase I ; GK, glycerokinase ; MCAD, medium-chain acyl coenzyme A dehydrogenase; hPGC-1, human peroxisome proliferator-activated receptor γ coactivator 1α ; hmPGC-1, primers for quantitation of human and murine PGC-1α ; hUCP1 and mUCP1, human and murine uncoupling protein 1. Other mRNA levels were quantified using Assay-on-Demand gene expression products (Applied Biosystems). Characteristics of the assays are available at www.allgenes.com. mRNA accession number sense primer antisense primer Amplicon size (bp) ALBP BC003672 5’-GCATGGCCAAACCTAACATGA-3’ 5’-CCTGGCCCAGTATGAAGGAAA-3’ 105 Cyt. c BC008477 5’-AGGCCCCTGGATACTCTTACACAG-3’ 5’-TCAGTGTATCCTCTCCCCAGATG-3’ 69 M-CPT1 D87812 5’-TACAACAGGTGGTTTGACA-3' 5'-CAGAGGTGCCCAATGATG-3' 105 GK NT_016354 5’-GCAGAAGGAGTCGGCGTATG-3’ 5’-CCCAACCCATTGACTTCATCA-3’ 145 MCAD AF251043 5’-AGCTCCTGCTAATAAAGCCTTTACTG-3' 5'-CATGTTTAATTCCTTTCTCCCAATC-3' 85 hPGC-1α AF159714 5’-CTGTGTCACCACCCAAATCCTTAT-3’ 5’-TGTGTCGAGAAAAGGACCTTGA-3’ 78 hmPGC-1α NM_008904 5’-AAAGGATGCGCTCTCGTTCA-3’ 5’-GGAATATGGTGATCGGGAACA-3’ 65 hUCP1 NM_021833 5’-TGCCCAACTGTGCAATGAA-3’ 5’-TCGCAAGAAGGAAGGTACCAA-3’ 80 mUCP1 NM_009463 5’-CCTGCCTCTCTCGGAAACAA-3’ 5’-TGTAGGCTGCCCAATGAACA-3’ 75

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Fig. 1

PPARγ + RXRα

PGC-1α030

60

90

120150

CA

T ac

tivity

GFP

**

**

+ +- -

- - + ++ + - -

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Fig. 2

Day 13Transduction with

PGC-α adenovirus orcontrol GFP adenovirus

6 h 48 h

Treatment with drugs

mRNAlevel

Protein expressionand fat oxidation

72 h

A B

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A

00.001

0.0020.003

0.004U

CP1

mR

NA

0

0.04

0.08

0.12

PGC

-1α

mR

NA

B

0100

300

500

700

PGC

-1α

mR

NA

0

20

40

60

80

UC

P1 m

RN

A

C

** **

**

**

0

0.01

0.02

0.03

PGC

-1α

mR

NA

0

0.002

0.004

0.006

UC

P1 m

RN

A+ -+ -

Rosi + RA

GFPPGC-1α

+-

- ++-

-+

Rosi + RA

GFPPGC-1α (moi)

+

- -+10-

-20 50 100 200

+ + + + +

- -

+ -+ -

Rosi + RA

GFPPGC-1α

+-

- ++-

-+

0

2

4

6

8

10

Ros

i +R

A

Ros

i

RA

Wy

1µM

L16-

Wy

10µM

**

UC

P1 m

RN

A

Fig. 3

D

GFPPGC-1α

-gfp+ -gfp-- gfp+

GFPPGC-1α

-gfp+ -gfp-- gfp+

PGC

-1α

( )

and

U

CP1

(

) mR

NA

0

5

10

15 ****

GFP

PGC-1α

-++-

E

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Fig. 4

BATGFP

GFP + Rosi + RA

PGC-1α+ Rosi + RA

PGC-1α

UCP1

Cytochrome c19.2 kDa

13.1 kDa

36.7 kDa

24.7 kDa

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0

0.5

1.01.5

2.0

2.5

Cyt

.C m

RN

A

CO

X4 m

RN

A0

0.5

1.01.5

2.0

2.5

0

0.5

1.01.5

2.0

2.5

Mito

fusi

n-2

mR

NA

0

2

4

6

M-C

PT1

mR

NA

Fig. 5

0

1

2

3

MC

AD

mR

NA

0

4

8

12

GyK

mR

NA

** *

***

Rosi + RA

GFPPGC-1α

+-

-++-

Rosi + RA

GFPPGC-1α

+-

-++-

Rosi + RA

GFPPGC-1α

+-

-++-

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Fig. 6

0

1

2

3

4

0

1

2

3

ALB

Pm

RN

A

PEPC

K1

mR

NA

****

**

Rosi + RA

GFPPGC-1α

+-

+ -++- -

-

+

-+ Rosi + RA

GFPPGC-1α

+-

+ -++- -

-

+

-+

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Page 38: Induction of uncoupling protein 1 and fat oxidation in human

Bouillaud, Daniel Ricquier and Dominique LanginClaire Tiraby, Geneviève Tavernier, Corinne Lefort, Dominique Larrouy, Frédéric

Acquirement of brown fat cell features by human white adipocytes

published online June 13, 2003J. Biol. Chem. 

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