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Endocrinology Vol. 147, No. 12 5708-5714
Copyright © 2006 by The Endocrine Society

Decreased Brown Adipocyte Recruitment and Thermogenic Capacity in Mice with Impaired Peroxisome Proliferator-Activated Receptor (P465L PPAR{gamma}) Function

Sarah L. Gray1, Edoardo Dalla Nora1, Emma C. Backlund, Monia Manieri, Sam Virtue, Robert C. Noland, Stephen O’Rahilly, Ronald N. Cortright, Saverio Cinti, Barbara Cannon and Antonio Vidal-Puig

Departments of Clinical Biochemistry (S.L.G., E.D.N., S.V., S.O., A.V.-P.) and Medicine (S.O., A.V.-P.), University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 2QR, United Kingdom; The Wenner-Gren Institute (E.C.B., B.C.), The Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden; Department of Normal Human Morphology (M.M., S.C.), Faculty of Medicine, Ancona University, 60020 Ancona, Italy; and Ward Sports Medicine Building and Brody School of Medicine (R.C.N., R.N.C.), Departments of Exercise and Sport Science and Physiology, East Carolina University, Greenville, North Carolina 27858

Address all correspondence and requests for reprints to: Antonio Vidal-Puig, Department of Clinical Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QR, United Kingdom. E-mail: ajv22{at}cam.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mice with a dominant-negative peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) mutation (P465L) unexpectedly had normal amounts of adipose tissue. Here, we investigate the adipose tissue of the PPAR{gamma} P465L mouse in detail. Microscopic analysis of interscapular adipose tissue of P465L PPAR{gamma} mice revealed brown adipocytes with larger unilocular lipid droplets, indicative of reduced thermogenic capacity. Under conditions of cold exposure, the brown adipose tissue of the PPAR{gamma} P465L mice was less active, a fact reflected in decreased uncoupling protein 1 levels. Analysis of the white adipocytes confirmed their normal cytoarchitecture and development, yet classical white adipose depots of the P465L PPAR{gamma} mice had a striking reduction in brown adipocyte recruitment, a finding supported by reduced expression of UCP1 in the perigonadal adipose depot. Taken together, these data suggest that whole animal impairment of PPAR{gamma} alters the cellular composition of the adipose organ to a more "white" adipose phenotype. Physiologically, this impairment in brown adipocyte recruitment is associated with decreased nonshivering thermogenic capacity after cold acclimation as revealed by norepinephrine responsiveness. Our results indicate that maintenance of oxidative brown-like adipose tissue is more dependent on PPAR{gamma} function for development than white adipose tissue, an observation that may be relevant when considering PPAR{gamma}-dependent strategies for the treatment of obesity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADIPOSE TISSUE IS primarily made up of adipocytes that store energy. However, two types of adipocytes, white and brown, are present in different proportions in most adipose tissue. In humans, the majority of adipocytes are white, energy-storing adipocytes, yet a small number of brown adipocytes can also be found in white fat depots, and these burn energy to produce heat. Rodents and other small mammals have adipose depots made up almost exclusively of these energy-burning adipocytes located in specific anatomical regions such as the interscapular depot (1, 2). Like in humans, brown adipocytes can also be found interspersed within white adipose tissue (WAT) (3, 4). It is unclear whether this phenomenon is the result of differentiation of specific preadipocyte precursors into brown adipocytes or alternatively from the transdifferentiation of white adipocytes to brown adipocytes (5, 6, 7, 8, 9, 10, 11).

Under certain environmental conditions, including cold and excess caloric intake, brown adipocytes become more active and increase in number (2, 10, 12). Given that the function of these brown adipocytes is to burn rather than store energy, increasing the number of brown adipocytes in WAT has been suggested as the basis for a novel strategy to prevent the development of obesity and related metabolic complications (13). Induction of brown adipocyte recruitment in WAT depots has been shown to decrease fat mass (14) and improve diet-induced insulin resistance in rodents (15) and humans (16).

A key protein to brown adipocyte function is uncoupling protein 1 (UCP1). UCP1 is expressed solely by brown adipocytes (17) and is required for uncoupling oxidative metabolism from the production of ATP, with the unused energy released in the form of heat. UCP1 expression and activity are increased in response to adrenergic stimulation, promoting increased energy expenditure. Several transcriptional regulators control UCP1 gene expression, including adrenergic activation of cAMP response binding protein (18), the CCAAT/enhancer binding proteins ß and {delta} (19), thyroid hormone receptors (20), and the peroxisome proliferator-activated receptors (PPARs). PPARs are members of the nuclear receptor superfamily and are known to regulate genes involved in lipid metabolism, carbohydrate metabolism, and energy balance (21). PPARs induce UCP1 expression through association with a PPAR response element in the proximal promoter of the UCP1 gene (22). The nuclear coactivator peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC1{alpha}) is induced in response to cold and associates with nuclear receptors to initiate pro-oxidative gene expression, including that of UCP1 (23). PPAR{gamma} agonism can induce the expression of UCP1 in brown adipocytes (24), and this induction of UCP1 improves the thermogenic capacity of the cell, demonstrated by the fact that norepinephrine-induced thermogenesis is enhanced in mice treated with PPAR{gamma} agonists (25, 26).

PPAR{gamma} is highly expressed in adipose tissue and has been shown to be important for the differentiation of both white and brown adipocytes (27, 28). PPAR{gamma} null mice die in utero at embryonic day 10 (28). As such models with tissue- and isoform- specific ablation of PPAR{gamma} have been used to examine PPAR{gamma} effects on adipogenesis and lipid and carbohydrate metabolism (29). Ablation of PPAR{gamma} specifically in mature adipocytes of mice results in progressive lipodystrophy. Specifically, the brown adipose tissue (BAT) in these mouse models is significantly reduced (30, 31) or absent (32). Isoform-specific ablation of PPAR{gamma}2 did not reduce the interscapular BAT depot (33, 34), yet brown adipocytes appeared to have reduced lipid content (33). Although these studies show that PPAR{gamma} is critical for BAT development and survival, the effects of PPAR{gamma} on brown adipocyte recruitment in WAT or the impact that these defects in brown adipocyte development have on adaptive thermogenesis were not examined in these mouse models.

Human patients with naturally occurring mutations in PPAR{gamma} have been identified (35, 36, 37). Patients with these dominant-negative mutations display a characteristic lipodystrophy, severe insulin resistance, and hypertension. Mouse models with equivalent heterozygous mutations in the ligand binding domain of PPAR{gamma} have been generated by homologous recombination (38, 39, 40) and provide an animal model with whole body impairment of PPAR{gamma} function. Due to the embryonically lethal phenotype associated with complete PPAR{gamma} knockout, a "clean" model of complete PPAR{gamma} ablation is not available. Here we use a dominant-negative PPAR{gamma} mouse model (P465L PPAR{gamma}) to demonstrate a role for PPAR{gamma} in the activation and recruitment of brown adipocytes in adipose tissue during cold exposure and its effects on thermogenic capacity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Animals heterozygous for the dominant-negative P465L PPAR{gamma} mutation (P465L PPAR{gamma} mice) were generated by homologous recombination as previously described (40). Briefly, in embryonic stem cells, one wild-type allele for PPAR{gamma} was homologously recombined with a targeting construct that contained a C to T point mutation in exon 6 of the PPAR{gamma} gene. Chimeric mice with germline transmission of the heterozygous P465L PPAR{gamma} mutation were bred to produce mice heterozygous for the P465L PPAR{gamma} mutation. All animals used in these studies were fed a standard chow diet ad libitum and housed at 24 C with 12-h light, 12-h dark cycles unless otherwise stated. Groups of experimental P465L PPAR{gamma} mice were paired with age- and sex-matched littermate wild-type controls. All experimental procedures were approved by the University of Cambridge and the UK Home Office.

Histology
Tissues were removed and fixed directly in 10% formalin, or animals were perfused with 4% paraformaldehyde. Fixed samples were embedded in paraffin and sectioned using a microtome. Sections were stained with hematoxylin/eosin, and immunohistochemical detection of UCP1 in adipose tissue was performed using a primary antibody (UCP1 1:4000) and a biotinylated secondary antibody (Amersham, Little Chalfont, UK) detected by the Vectastain ABC kit (Vector Laboratories, Peterborough, UK).

Perigonadal (four sections per mouse) and sc inguinal (two sections per mouse) adipose sections were stained for UCP1 and subjected to semiquantitative evaluations. All UCP1 immunoreactive adipocytes present in the section were counted with a Zeiss light microscope at x200 magnification. The percentage of multilocular adipocytes present in perigonadal fat sections was calculated using a morphological imaging system (LUCIA, version 4.5, Nikon Instruments, Florence, Italy). For each experimental group, the percentage of adipocytes was calculated as the mean of the percentages of each animal derived from 800 random cells taken from four different areas per animal.

Gene expression profiling
Isolation of RNA and generation of cDNA.
Individual tissues were dissected and snap-frozen immediately in liquid nitrogen. Total RNA was extracted using RNA STAT60 (Tel-Test "B" Inc., Friendswood, TX). RNA (500 ng) was reverse transcribed to cDNA using MMLV reverse transcriptase (Promega, Southampton, UK).

Taqman analysis of gene expression.
The expression of key thermogenic genes and PPAR{gamma} in perigonadal (g) WAT, and interscapular BAT was analyzed using real-time PCR (TaqMAN, Applied Biosystems, Warrington, UK) with primers and probes designed to specific mRNAs (Sigma, Pampisford, UK) using Primer Express 2.0 (Applied Biosystems). PCR conditions were as follows: 50 C for 2 min; 95 C for 10 min; 40 cycles of 95 C for 15 sec and 60 C for 1 min. Values were normalized against 18S RNA and expressed as fold change compared with wild-type controls.

Western blots
Protein was extracted from tissue and quantified (Dc protein assay, Bio-Rad, Hemel Hempstead, UK). Protein (10 µg BAT or 30 µg gWAT) was denatured and reduced, run on an SDS-PAGE gel (10%), and transferred to polyvinylidene difluoride membrane. Protein expression was determined using a primary antibody to UCP1 (1:2000) (17) labeled with a goat antirabbit IgG secondary antibody (Dako, Glostrup, Denmark) and detected by the enhanced chemiluminescence detection system (Amersham). Protein expression was quantified by densitometry using AlphaEaseFC image analysis software.

Thermoneutrality
To examine the expression of genes under conditions of thermoneutrality, animals were housed at 30 C for 3 wk. Tissues collected at dissection were snap-frozen or fixed in 10% formalin.

Energy expenditure under cold acclimation
Four-month-old female P465L PPAR{gamma} mice and wild-type littermates were exposed to thermoneutrality (30 C) for 3 wk, placed at room temperature for 1 wk, and then exposed to 4 C for 3 wk. For both 30 C and 4 C acclimated animals, resting metabolic rate was measured (awake animals) at 30 C. In addition, norepinephrine-stimulated (1 mg/kg body weight) energy expenditure was evaluated in anesthetized (pentobarbital) animals at 33 C. Animals recovered from the anesthesia at 33 C before being transferred back to the acclimation temperature (4 C). Here they recovered from norepinephrine treatment for at least 72 h before tissue collection to ensure no influence of norepinephrine on gene and protein expression analysis. Oxygen consumption measurements are expressed as ml O2/min/kg0.75 (41). Housing of animals at the various temperatures and metabolic experiments were approved by the North Stockholm Animal Ethics Committee.

Statistical analysis
Data are presented as mean ± SEM. Statistical analyses using two-tailed unpaired t tests were performed, and significance was declared for P values less than 0.05 (GraphPad Instat 3 or Microsoft Excel).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
P465L PPAR{gamma} mice have altered brown adipocyte morphology in the interscapular brown adipose depot
Histological analysis of the interscapular brown fat depot from animals raised at room temperature showed that P465L PPAR{gamma} mice had more lipid-replete BAT characterized by an increased number of unilocular adipocytes akin to white adipocytes (Fig. 1AGo). Gene expression of the adipocyte marker fatty acid binding protein, aP2, was reduced in interscapular BAT from P465L PPAR{gamma} mice compared with wild-type controls (Fig. 1BGo). Although there was no change in interscapular BAT UCP1 mRNA (Fig. 1BGo), P465L PPAR{gamma} brown adipocytes had a reduction in UCP1 immunostaining (Fig. 1AGo).


Figure 1
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  		FIG. 1. A, Hematoxylin and eosin (H+E) and UCP1 immunohistological staining of interscapular BAT from P465L PPAR{gamma} (P465L) and wild-type (WT) mice raised at room temperature. B, Gene expression levels of the fatty acid binding protein, aP2, and UCP1 in interscapular BAT and perigonadal adipose tissue (gWAT) of P465L PPAR{gamma} mice compared with wild-type controls. Values are expressed as mean fold change ± SEM compared with wild-type gene expression. RT, Room temperature. C, Gross morphology of adipose tissue depots in the P465L PPAR{gamma} (P465L) and wild-type (WT) mice. 1, Interscapular BAT; 2, sc WAT; 3, gWAT. D, UCP1 immunostaining of WAT. E, The percentage of multilocular "brown-like" adipocytes in gWAT of P465L PPAR{gamma} mice raised at room temperature compared with wild-type controls. Significance is represented by * (P < 0.05) and ** (P < 0.01).

 
Impaired PPAR{gamma} function reduces the number of brown adipocytes infiltrating WAT depots
PPAR{gamma} is a key molecular regulator of adipose tissue differentiation. Unexpectedly, P465L PPAR{gamma} mice are able to develop normal amounts of adipose tissue (Fig. 1CGo) (39, 40) and have normal levels of aP2 mRNA in their gonadal white adipose depot (Fig. 1BGo). However, histological analysis revealed a striking difference in the cellular composition of the classic WAT depots (inguinal sc and g) in the P465L PPAR{gamma} mice, which had a smaller number of brown adipocytes compared with wild-type controls. The more "white" adipose tissue phenotype of the P465L PPAR{gamma} mice is observed grossly by the color of the adipose tissue from animals housed at room temperature (Fig. 1CGo). The percentage of multilocular adipocytes among unilocular adipocytes in the perigonadal depot of P465L PPAR{gamma} mice was less than one third of that found in the same depot of wild-type mice (Fig. 1Go, D and E). Furthermore, in the gWAT depot of the P465L PPAR{gamma} mice UCP1 mRNA levels were reduced (Fig. 1BGo).

Impaired activation of thermogenic genes in perigonadal adipose tissue depots in cold-acclimated PPAR{gamma} mice
Thermogenic induction of UCP1 and PGC1{alpha} gene expression in response to cold acclimation was not impaired in interscapular BAT of P465L PPAR{gamma} mice compared with wild-type controls (Fig. 2AGo) yet protein levels of UCP1 were somewhat reduced in the BAT of cold-acclimated P465L PPAR{gamma} mice compared with wild-type controls (Fig. 2DGo). In the gWAT of cold-acclimated P465L PPAR{gamma} mice, UCP1 and PGC1{alpha} mRNA and UCP1 protein levels were significantly reduced compared with wild-type controls (Fig. 2Go, A and D). These differences were not observed in BAT or gWAT of P465L PPAR{gamma} mice housed at thermoneutrality (Fig. 2BGo). Expression of the ß3 adrenergic receptor was not altered in BAT or gWAT of cold-acclimated P465L PPAR{gamma} mice. Adenylate cyclase (subunit 3) was not significantly changed in P465L BAT or gWAT of cold-acclimated mice, although in gWAT an observed 1.7-fold decrease in expression was close to significance (P = 0.062) (Fig. 2AGo).


Figure 2
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  		FIG. 2. Altered expression of thermogenic genes in adipose tissue of cold-acclimated P465L PPAR{gamma} mice. A, UCP1, PGC1{alpha}, ß3 adrenergic receptor (B3), and subunit 3 of adenylate cyclase (AC) gene expression levels in interscapular BAT and perigonadal adipose tissue (gWAT) of mice acclimated to 4 C. B, Unaltered UCP1 gene expression levels in BAT and gWAT of P465L PPAR{gamma} mice housed at thermoneutrality (30 C) for 3 wk compared with wild-type controls. C, Quantification of WT PPAR{gamma} allele expression (mRNA) in cold-exposed wild-type and P465L PPAR{gamma} BAT and gWAT. D, UCP1 protein levels (assessed by quantified Western blots) in BAT (10 µg) and gWAT (30 µg) of animals housed at 4 C. Values are expressed as mean ± SEM. Significance is represented by ** (P < 0.01) or *** (P < 0.001).

 
Reduced thermogenic capacity of P465L PPAR{gamma} mice during cold exposure
Cold acclimation of P465L PPAR{gamma} mice to 4 C did not alter body weights or adipose tissue mass in P465L PPAR{gamma} mice compared with controls (Fig. 3Go, A and B). Despite decreased levels of UCP1 protein in cold-acclimated P465L PPAR{gamma} mice, both mutant and wild-type littermates had similar resting metabolic rates when acclimated to thermoneutrality or cold (4 C) (Fig. 3CGo), confirming that UCP1 activity does not contribute to resting metabolic rate (42). Norepinephrine treatment of thermoneutrality-acclimated animals, to determine the capacity for adrenergic-stimulated thermogenesis, did not reveal differences between wild-type and P465L PPAR{gamma} mice (Fig. 3CGo). However, administration of norepinephrine after cold acclimation showed that although the P465L PPAR{gamma} mice had significantly increased the magnitude of their response to norepinephrine compared with thermoneutrality, they showed a significantly reduced maximal thermogenic capacity compared with wild-type controls (Fig. 3CGo). The decrease in thermogenic capacity of about 25% was in good agreement with both the decrease in UCP1 protein levels seen in BAT of cold-acclimated animals (Fig. 2DGo) and the reduced expression of thermogenic genes in gWAT shown in the previous section of Results (Fig. 2AGo).


Figure 3
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  		FIG. 3. Decreased thermogenic capacity of P465L PPAR{gamma} mice cold acclimated to 4 C. Normal body mass (A) and adipose tissue mass (B) in P465L PPAR{gamma} mice housed at 4 C for 3 wk compared with wild-type controls. C, Basal and norepinephrine-stimulated oxygen consumption in wild-type and P465L PPAR{gamma} mice housed at thermoneutrality (30 C) or 4 C. Values are expressed as mean ± SEM, and significance is represented by ** (P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well established that PPAR{gamma} is important for the development of BAT, as evidenced by the fact that mouse models with adipose-specific PPAR{gamma} (both PPAR{gamma}1 and PPAR{gamma}2) ablation have reduced or absent interscapular BAT (30, 31, 32). Furthermore, UCP1 is a known transcriptional target of PPAR{gamma} (27), and induction of UCP1 by PPAR{gamma} agonists increases the thermogenic capacity of brown adipocytes (25, 26). Here we show that in a mouse model with whole-animal impairment of PPAR{gamma} function (P465L PPAR{gamma} dominant-negative mutation), brown adipocyte recruitment and induction of UCP1 protein in brown adipocytes is impaired in response to cold.

The P465L PPAR{gamma} mutation is expressed in the heterozygous state and acts in a dominant-negative manner to impair wild-type PPAR{gamma} function. In vitro, characterization of the biochemical activity of ligand binding domain PPAR{gamma} mutants has previously been described. Briefly, presence of the mutant PPAR{gamma} impairs interaction with coactivators, enhances corepressor recruitment, and impairs ligand-dependent corepressor release (35, 43, 44, 45)

In general, the P465L PPAR{gamma} adipose tissue has a more "white-like" appearance, suggesting that brown adipocyte development is more severely affected by reduced PPAR{gamma} function than is the development of white adipocytes. These changes were associated with reduced thermogenic capacity. These results are in accordance with studies showing the opposite effects by pharmacological activation of PPAR{gamma} (24, 26, 46).

We investigated the effects of the P465L PPAR{gamma} mutation on adipose tissue morphology and whole animal thermogenesis. Microscopic analysis of interscapular adipose tissue from P465L PPAR{gamma} mice revealed that the cellular composition of this depot was altered compared with controls. The P465L PPAR{gamma} animals have less well-recruited interscapular BAT with an increased number of unilocular white-like adipocytes. In addition to the "white-like" phenotype of the interscapular BAT depot, recruitment of brown adipocytes in classic WAT depots of P465L PPAR{gamma} mice is strikingly impaired compared with wild-type controls. This was shown by a specific reduction in the number of brown adipocytes observed within the P465L PPAR{gamma} WAT.

This observation was confirmed by a highly significant reduction in the expression of the brown adipocyte-specific gene, UCP1, and the thermogenic coactivator, PGC1{alpha}, in the perigonadal adipose depot of cold-acclimated P465L PPAR{gamma} mice compared with wild-type controls. These changes in UCP1 and PGC1{alpha} mRNA levels in mice with the P465L PPAR{gamma} mutation fit well with the fact that PPAR{gamma} agonism can promote brown adipocyte recruitment in classical white adipose depots (46) and can induce UCP1 gene expression in adipocytes (24). These observations clearly show that in vivo PPAR{gamma} is required for full activation and recruitment of brown adipocytes when increased thermogenic capacity is required. The origin of the brown adipocytes in WAT depots, whether from transdifferentiation of mature white adipocytes or from adipocyte precursors within the white adipose depots, is yet to be defined.

The morphological and biochemical observations that demonstrate a more "white" adipose tissue are associated with a reduced maximal thermogenic output in P465L PPAR{gamma} mice when compared with wild-type controls as measured by norepinephrine-stimulated oxygen consumption. Gene expression levels of the ß3 adrenergic receptor are not altered in cold-acclimated BAT or WAT. In cold-acclimated WAT, we did see a trend of reduced adenylate cyclase expression, suggesting that there may be some reduction in the activation of this pathway, a finding that would be consistent with our physiological measurements of reduced thermogenic capacity.

The ability to increase the number of brown adipocytes within WAT and increase thermogenic capacity may be of potential medical value. Genetic modification in mouse to induce UCP1 expression in WAT prevents genetic obesity (47). As such, the development of brown adipocytes at the expense of white adipocytes is the scientific basis for a potential therapeutic strategy to treat obesity (13). The "white-like" phenotype of adipose tissue in the P465L PPAR{gamma} mouse suggests that although partial PPAR{gamma} activity in vivo may be enough to sustain the differentiation of white adipocytes, the complete differentiation of a brown phenotype requires fully functional PPAR{gamma}. This agrees with the idea that the potency of PPAR{gamma} agonists may alter the physiological effects incurred by PPAR{gamma} agonism through altered cofactor recruitment (48). In addition to improving insulin sensitivity, currently used pharmacological PPAR agonists cause weight gain. Our results support the concept that altering the potency of PPAR{gamma} agonists to specifically increase recruitment of brown adipocytes into classic WAT depots may have a therapeutic advantage to current thiazolidinedione treatment.

In summary, we present morphological and biochemical evidence that whole-animal impairment of PPAR{gamma} function preferentially alters brown adipocyte recruitment over white adipocyte development. Thus, in the P465L PPAR{gamma} mice, brown adipocytes have a more "white-like" appearance, are present at lower numbers in WAT depots, and have reduced expression of thermogenic proteins. Physiologically, these morphological and biochemical observations are associated with a reduced maximal thermogenic output by P465L PPAR{gamma} mice when compared with wild-type controls. Use of the P465L PPAR{gamma} mouse model has provided an in vivo model system to explore the physiological relevance of PPAR{gamma} in the development of brown adipocytes and in UCP1 induction. Analysis of the P465L PPAR{gamma} BAT provides compelling in vivo evidence that PPAR{gamma} plays a role in the normal recruitment and function of mammalian BAT.


   Acknowledgments
 
We acknowledge Sylvia Shelton and Helen Westby for their exceptional technical assistance.


   Footnotes
 
Funding for this work was provided by Diabetes UK and an Medical Research Council Career Development Award. Work completed in the laboratory of B.C. was supported by the Swedish Research Council. Salary support was provided by the Natural Sciences and Engineering Research Council of Canada (to S.L.G.) and the O. Arlotti Trust, Ferrara (to E.D.N.).

Disclosure Statement: S.L.G., E.D.N., S.V., M.M., E.C.B., R.C.N., R.N.C., S.C., B.C., and A.V.-P. have nothing to declare. S.O. consults for Merck, has received lecture fees from NovoNordisk, and has equity interests in Prosidion.

First Published Online September 15, 2006

1 S.L.G. and E.D.N. contributed equally to this work. Back

Abbreviations: BAT, Brown adipose tissue; g, perigonadal; PGC1{alpha}, peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha}; PPAR, peroxisome proliferator-activated receptor; UCP1, uncoupling protein 1; WAT, white adipose tissue.

Received May 19, 2006.

Accepted for publication September 7, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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