Báo cáo khoa học: b3-Adrenoceptor knockout in C57BL/6J mice depresses the occurrence of brown adipocytes in white fat pptx

b 3 -Adrenoceptor knockout in C57BL/6J mice depresses the occurrence of brown adipocytes in white fat Maria Jimenez 1 , Giorgio Barbatelli 3 , Roberta Allevi 3 , Saverio Cinti 3 , Josiane Seydoux 2 , Jean-Paul Giacobino 1 , Patrick Muzzin 1 and Fre ´ de ´ ric Preitner 1 De ´ partements de Biochimie Me ´ dicale 1 et Physiologie 2 , Centre Me ´ dical Universitaire, Gene ` ve, Switzerland; 3 Istituto di Morfologia Umana Normale, Universita ` di Ancona, Italy White and brown adipocytes are usually located in distinct depots; however, in response to cold, brown adipocytes appear in white fat. This response is mediated by b-adrenoceptors but there is a controversy about the sub- type(s) involved. In the present study, we exposed to cold b 3 -adrenoceptor knockout mice (b 3 KO) on a C57BL/6J genetic background and measured in white adipose tissue the density of multilocular cells and the expression of the brown adipocyte marker uncoupling protein-1 (UCP1). In brown fat of b 3 KO mice, UCP1 expression levels were normal at 24 °C as well as after a 10-day cold exposure. Strikingly, under both conditions, in the white fat of b 3 KO mice the levels of UCP1 mRNA and protein as well as the density of multilocular cells were decreased. These results indicate that b 3 -adrenoceptors play a major role in the appearance of brown adipocytes in white fat and suggest that the brown adipocytes present in white fat differ from those in brown fat. Keywords: adipose tissue; cold exposure; differentiation; knockout; uncoupling protein 1. Brown adipose tissue (BAT) and white adipose tissue (WAT) both play an important role in the control of energy balance in mammals. Indeed, BAT is involved in the control of body temperature and body weight via nonshivering and diet-induced thermogenesis [1,2] whereas the primary func- tion of WAT is to store and release energy. BAT function is particularly important for thermoregulation in small mam- mals in which the surface to volume ratio is unfavorable [1]. Moreover, activation of BAT prevents obesity induced by overfeeding in rodents [2]. Cold exposure, via activation of the sympathetic nervous system, induces heat production in BAT [3,4] and lipolysis in WAT [5]. The effects of norepinephrine released at the nerve endings are mediated in brown and white adipocytes mainly by the b 3 -adrenoceptor subtype [6]. Brown and white adipocytes are usually located in distinct depots [4] and can be distinguished morphologically [7]: brown adipocytes contain multiple (multilocular) lipid droplets and numerous mitochondria whereas white adipo- cytes display a main (unilocular) lipid droplet and few mitochondria. Nevertheless, the morphological aspect alone is not sufficient to distinguish between these two cell types [8]. In fact, the only criterion is the specific expression in mitochondria of brown adipocytes, of the uncoupling protein-1 (UCP1), which uncouples the oxidative phosphorylations from electron transport, thus dissipating energy as heat [9]. An observation made initially by Young et al. [10] was the presence of multilocular mitochondria-rich brown adipocytes in depots previously defined as typical WAT. The emergence of these ectopic cells was found to be induced by cold acclimation in WAT of rats [11,12] and of mice [13,14]. The new cells were found to be sympathetically innervated [15] and to remain present as long as a sympathetic nervous system stimulation persisted [16]. Several reports also showed that the administration of selective b 3 -adrenoceptor agonists like CL 316, 243 induced the emergence of brown adipocytes in WAT depots [14,17–19]; and that this phenomenon was strongly depend- ent on the genetic background [14,18,20]. It was, however, recently shown that a transgenic over-expression of a human b 1 -adrenoceptor in WAT of mice also induced the appearance of abundant brown adipocytes in this tissue [21]. These results suggested that the b 3 -adrenoceptor might not be the only b-subtype controlling the emergence of brown adipocytes in WAT. Another open question is the origin and the real nature of the multilocular cells expressing UCP1 that appeared in WAT upon cold acclimation or b 3 -adrenoceptor stimula- tion. Himms–Hagen et al. [8], studying the effect of CL 316, 243 in rats, suggested that the multilocular cells expressing UCP1 that appeared in WAT were different from the classical brown adipocytes and postulated that these cells might derive, at least in part, from preexisting unilocular adipocytes. Mice with a targeted disruption of their b 3 -adrenoceptor (b 3 KO) have been generated [22,23]. Our b 3 KO model, first generated on a mixed background (C57BL/6J · 129Sv/ev) [23], was then backcrossed to obtain the mutation on the C57BL/6J genetic background. In the present work, we studied the effect of b 3 -adrenoceptor-deficiency on the Correspondence to M. Jimenez, De ´ pt. de Biochimie Me ´ dicale Centre Me ´ dical Universitaire 1, rue Michel-Servet CH-1211 Gene ` ve 4, Switzerland. Fax: + 41 22 702 55 02, Tel.: + 41 22 702 54 88, E-mail: Maria.Jimenez@medecine.unige.ch Abbreviations: b 3 KO, b 3 -adrenoceptor knockout; UCP1, uncoupling protein-1; BAT, brown adipose tissue; WAT, white adipose tisue. (Received 23 August 2002, revised 26 November 2002, accepted 10 December 2002) Eur. J. Biochem. 270, 699–705 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03422.x occurrence of brown adipocytes in WAT of C57BL/6J mice at 24 °C and after cold exposure. We therefore measured (a) the density of multilocular cells in WAT, and (b) the levels of UCP1 mRNA and protein in WAT and BAT, as compared to wild-type (WT) controls. Materials and methods Materials All organic and inorganic chemicals of analytical or molecular biology grade were purchased from Merck (Darmstadt, Germany), Boehringer Mannheim (Mann- heim, Germany), Sigma Chemical Co (St. Louis, MI) and Fluka (Bucks, Switzerland). Trizol and the RNA ladder were purchased form Gibco BRL (New York, NY), Electran Nylon membranes from BDH Laboratory Sup- plies (Poole, UK), Quikhyb from Stratagene Inc (La Jolla, CA), Hyperfilm ECL films and [a- 32 P]dCTP from Amer- sham International (Bucks, UK), cytochrome oxidase subunit IV from Molecular probes (Eugene, OR). Rabbit antisheep IgG, avidin and biotin complex (ABC) peroxidase conjugated were purchased from Vector Laboratories Burlingame (CA). Mice b 3 KO mice were initially generated on a mixed 129Sv/ ev · C57BL/6J background. Then, the b 3 KO mice were backcrossed six times with C57BL/6J (C57) mice (obtained from BRL Ltd, Fu ¨ llinsdorf, Switzerland), in order to get mutated mice with the purified C57 genetic background (98.4% homogeneity). Mice used in this study were offsprings of WT and b 3 KO founders of the C57 purified genetic background. The genotypes were determined by Southern blots as previously described [23]. Three- to four- month-old female C57 WT or b 3 KO mice with free access to water and standard laboratory chow diet (Nordos, Cergy, France) were used. The average weights of the mice were 22.5 ± 0.3 and 22.8 ± 0.3 for the C57 WT and b 3 KO, respectively. They were housed individually for 10 days at 24 ± 1 °C on a 12-h light : 12-h dark cycle (7:00–19:00 h) before the beginning of each experiment. They were then divided into three groups, one maintained at 24 °Candthe other two exposed to cold (6 °C) for either two or 10 days. The fat pads along the uterus and fallopian tube (param- etrial fat) and those around the ovaries (periovarian) were dissected and pooled. They are referred to in the text as genital WAT. Morphometry and immunochemistry Mice were killed by CO 2 inhalation and perfused intra- cardially with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Genital WAT was dissected, further fixed by immersion in the 4% paraformaldehyde solution overnight at 4 °C and then embedded in paraffin blocks. In each animal, two sections, at a distance of 200 lmfrom each other in the central part of the parametrial or periovarian depots were studied. The adipocytes were identified as unilocular (a unique prevalent vacuole) or multilocular (more than five small lipid droplets) and the respective number of these cells in each section counted. The average number of adipocytes counted per section and per animal were 8173 and 10774, respectively, in a total of 237036 cells. In some experiments, the mean area of 100 unilocular adipocytes randomly chosen on the total surface of a section was measured using a Nikon Eclipse E800 connected with a digital Camera DXM1200 at a magnification of 20·. The surface area of the cells was measured using the Nikon LUCIA IMAGE Version 4.61 program. Three-micrometer sections of tissue were deparaffinizated and rehydrated with distilled water and immunohistochem- ical detection of UCP1 was performed as follows: (a) 0.3% hydrogen peroxide in methanol for 30 min to inactivate endogenous peroxidase; (b) normal rabbit serum 1 : 75 in NaCl/P i for 20 min to reduce non-specific background staining; (c) polyclonal sheep antirat UCP1 diluted 1 : 5000 in NaCl/P i ,at4°C, overnight; (d) NaCl/P i ,twicefor 15 min; (e) biotinylated secondary antibody, rabbit anti- sheep IgG 1 : 200, 30 min at room temperature; (f) NaCl/P i , twice for 15 min; (g) ABC complex for 1 h; (h) NaCl/P i twice for 15 min; (i) enzymatic development of peroxidase with 0.05% diaminobenzidine hydrochloride and 0.02% hydrogen peroxide in 0.05 M Tris/HCl pH 7.6, for 4 min. Negative controls were performed by substituting the primary antibody by sheep IgG. No cross-reaction with UCP2 and UCP3 were observed in tissues expressing the highest levels of UCP2 (liver) and UCP3 (skeletal muscle). The specificity of the UCP1 antibody used in this study has been recently confirmed [24]. Northern blots The mice were killed by cervical dislocation followed by decapitation and the interscapular BAT as well as genital WAT were dissected and quickly frozen in liquid nitrogen. Total RNA was isolated using the Trizol technique and 15 lg of RNA were electrophoresed in a 1% agarose gel containing formaldehyde and transferred according to standard protocols. The probes used were a full-length mouse UCP1 cDNA (GenBank accession number U63418), a b 1 -adrenoceptor PCR probe (position 297–1053, GenBank accession number L10084) and a PGC-1 PCR probe (posi- tion 297–2308; GenBank accession number AF049330). They were labelled by random priming with [a- 32 P]dCTP to a specific activity of approximately 1 · 10 9 d.p.m.Ælg )1 DNA. Hybridizations were performed using Quikhyb TM solution as previously described [25]. Blots were exposed at )80 °C to Hyperfilm ECL films. RNA levels were quanti- fied by scanning photodensitometry of the autoradiograms using IMAGEQUANT Software version 3.3 of Molecular Dynamics (Sunnyvale, CA). Subsequent hybridization of the blots with a [a- 32 P]ATP labelled synthetic oligonucleo- tide specific for the 18S rRNA subunit was used to correct for the differences in the amounts of RNA loaded onto the gels. Isolation of mitochondria Other groups of mice were used for Western blot experi- ments. BAT and WAT mitochondria were prepared as previously described [26] and the pellet containing heavy 700 M. Jimenez et al.(Eur. J. Biochem. 270) Ó FEBS 2003 and light mitochondria resuspended in 400 lL distilled water. Mitochondrial protein concentrations were deter- mined according to Bradford et al. [27] using the Bio-Rad Protein Assay, with bovine serum albumin as a standard. Isolated mitochondria were stored at )20 °Cas15lg mitochondrial protein aliquots. Western blots Fifteen micrograms of purified BAT and WAT mitochon- dria were dried under vacuum and resuspended in 10 lLof a loading buffer containing 50% glycerol, 5% SDS, 2.5% bromophenol blue and 0.5 M Tris/HCl pH 6.8. The samples were electrophoresed on a 12% polyacrylamide, 0.1% SDS gel, and transferred to a poly(vinylidene difluoride) mem- brane using the standard dry transfer method with a buffer containing 10% methanol, 25 m M Tris/HCI pH 6.8 and 190 m M glycine. The membrane was blocked with a NaCl/ P i buffer containing 0.1% Tween and 2% nonfat dry milk. This same buffer was used for all subsequent hybridizations. UCP1 protein was detected using a sheep polyclonal primary antibody raised against rat UCP1 protein (gener- ously provided by D. Ricquier, Meudon, France) at a concentration of 0.5 lgÆmL )1 and a 1 : 4000 diluted sheep monoclonal anti-mouse peroxidase-labelled secondary antibody. The cytochrome oxidase protein was detected as above using a 1 : 1000 diluted monoclonal antibody specific for cytochrome oxidase subunit IV, and a 1 : 1000 diluted goat anti-mouse peroxidase-labelled secondary antibody. The signals were detected by chemiluminescence using a standard ECL kit, and developed on a Hyperfilm ECL. They were quantified by scanning photodensitometry. Cytochrome c oxidase activity BAT homogenate was prepared as previously described [26] and 10 lg of homogenate proteins were used to measure the cytochrome c oxidase activity by the method of Yonetani and Ray [28]. Statistics Data are presented as mean ± SE and were analyzed using a two-factor analysis of variance ( ANOVA <0.05)forthe main effects of group (b 3 KO vs. WT) and treatment condition (24 °Cvs.6°C 2 or 10 days) as well as for the group · treatment condition interaction effect, using the computer software STATISTIX , version 4.0 (Analytical Soft- ware,St.Paul,MN). The non parametric Kruskal–Wallis test was used to analyze morphometric studies. Results Morphometry and immunohistochemistry in the WAT The density of multilocular cells was measured in genital WAT sections. As shown in Fig. 1, in the WAT of C57 WT mice maintained at 24 °C, only 1.1% of the total adipocytes was multilocular. The effects of the lack of b 3 -adrenoceptor were striking. In mice kept at 24 °C, the density of multilocular cells was 36-fold (P < 0.01) lower in the WAT of b 3 KO mice as compared to WT mice. Ten days of cold-exposure increased the density of multilocular cells in both WT and b 3 KO mouse WAT (11-fold, P <0.03and ninefold, P < 0.01, respectively). However, the density of multilocular cells was 42-fold lower (P < 0.03) in the WAT of b 3 KO as compared to WT mice. UCP1 protein expression in multilocular cells was assessed by immuno- histochemistry. No UCP1 could be detected in the WAT multilocular cells of WT or b 3 KO mice either maintained at 24 °C or exposed to 6 °C for 2 days. A 10-day cold exposure, however, induced the emergence of UCP1 expression in WAT multilocular cells. Figure 2A and B show multilocular cells and UCP1 positive multilocular cells in WT and b 3 KO mouse parametrial depots. Note that the intensity of the UCP1 expression in multilocular cells varies from cell to cell. In b 3 KO mouse, UCP1-positive multilo- cular cells were heterogenously distributed in the WAT depot and it was therefore not possible to assess the number of these cells and to compare it to that in WT mouse WAT. Genital pads of b 3 KO mice were 1.5-fold heavier than those of WT (388 ± 33 vs. 251 ± 14, P < 0.005, n ¼ 12) despite a normal number of cells, as assessed by DNA content (1142 ± 93 vs. 1098 ± 69 lgpergenitalWAT, n ¼ 12), suggesting an increase in cell size. Consistent with this hypothesis, a morphometric study showed an increase of 2.4-fold of the unilocular adipocyte mean area in the genital WAT of b 3 KO as compared to WT mice (1594 ± 42 vs. 656 ± 49 mm 2 , n ¼ 4 and 3 sections, respectively). UCP1 mRNA expression in WAT and BAT Quantitative measurements were then performed on whole BAT and WAT. The expression of UCP1 mRNA was measured in genital WAT, and in interscapular BAT of C57 WT and b 3 KO mice (Fig. 3). In genital WAT of WT mice (Fig. 3A), the basal level of UCP1 mRNA at 24 °C, measured by Northern blot, was found to be increased Fig. 1. Morphometric analysis of genital WAT from C57BL/6J wild- type (WT) or b 3 KO female mice, maintained at 24 °Corexposedto 6 °C for 2 or 10 days (2 d and 10 d, respectively). The number of multilocular (ML) cells is expressed as a percentage of the total amount of adipocytes in the pooled sections of the two WAT depots. n ¼ 3–4. *P <0.05 and **P < 0.01 vs. respective WT; # P <0.05 and ## P < 0.01 vs. respective control at 24 °C. Ó FEBS 2003 b 3 -adrenoceptor and ectopic adipocytes (Eur. J. Biochem. 270) 701 2.6-fold (P < 0.05) and 4.8-fold (P < 0.001) after 2- and 10-day cold exposure, respectively. The effects of a lack of b 3 -adrenoceptor were striking. The UCP1 mRNA expres- sion levels in WAT of b 3 KO mice were strongly depressed by 20-fold (P < 0.01), 33-fold (P < 0.005) and 2.9-fold (P < 0.005) as compared to WT mice kept at 24 °Cor exposed to 6 °C for 2 or 10 days, respectively. An effect of cold exposure on UCP1 mRNA expression was also seen in WAT of b 3 KO mice but only after 10 days (P < 0.005). In BAT of C57 mice (Fig. 3B), 2- or 10-day cold exposure increased UCP1 mRNA expression 1.8-fold (P <0.05 and P < 0.005, respectively) in the WT, and 1.6-fold (P < 0.05) and 1.9-fold (P < 0.01) in the b 3 KO mice. It is noteworthy that the b 3 -adrenoceptor deficiency did not affect UCP1 mRNA expression under all conditions studied. In WT C57 mice, the expression level of UCP1 mRNA in WAT relative to that in BAT, assessed on a same Northern blot, was approximately 3% in mice kept at 24 °Cand11% after a 10-day cold exposure (data not shown). UCP1 protein expression in WAT and BAT The level of UCP1 protein was measured in isolated mitochondria from genital WAT and BAT of C57 WT and b 3 KO mice. The results represent the amount of UCP1 in a given amount of mitochondrial proteins (specific value). The mitochondrial marker cytochrome c oxidase was chosen as a standard to normalize UCP1 protein level. As illustrated in Fig. 4, whereas a 2-day cold-exposure did not increase UCP1 protein level in WAT mitochondria of WT mice, 10-day cold-exposure increased it 6.7-fold (P <0.02).InWAT,theeffectsofb 3 KO on UCP1 protein levels were comparable with those observed on UCP1 mRNA. Indeed, the UCP1 protein levels in b 3 KO mice kept Fig. 2. Genital WAT of C57BL/6J wild-type (WT) (A) and b 3 KO (B) mice exposed to 6 °C for 10 days. Note the presence of multilocular cells and of UCP1 positive multilocular cells. Bar, 31.74 lm. The mean area of the adipocytes in these sections was 2.4-fold higher in b 3 KO than in wild-type mice (P < 0.05). Fig. 3. Northern blot quantification of UCP1 mRNA expression in adipose tissues of C57BL/6J wild-type (WT) or b 3 KO female mice, maintained at 24 °Corexposedto6°C for 2 or 10 days (2 d and 10 d, respectively). (A), Genital WAT; (B), brown adipose tissue (BAT). The results are expressed as means ± SE of arbitrary values normalized using the corresponding 18S mRNA values. The ratio of the control values is considered as 1.0. n ¼ 6. **P <0.01 and ***P <0.005 vs. respective WT; # P <0.05and ### P < 0.005 vs. respective control at 24 °C. 702 M. Jimenez et al.(Eur. J. Biochem. 270) Ó FEBS 2003 at 24 °C or exposed to 6 °C for 10-days were by 7.7-fold (P < 0.005) and 11-fold (P < 0.01) lower than those in WT animals. It is interesting to note the parallel effects of the b 3 KO on the percentage of multilocular cells, and on the degree of UCP1 expression at the mRNA and protein levels. This suggests that UCP1 is mainly expressed in multilocular cells. In BAT of C57 mice, 10-day cold-exposure increased UCP1 protein level 1.9-fold (P < 0.05) in WT mice and 2.8-fold (P < 0.05) in b 3 KO mice. In agreement with the UCP1 mRNA results, b 3 KO did not affect the UCP1 protein level under all conditions studied (data not shown). Furthermore, cytochrome c oxidase total activity (as expressed per BAT) showed no difference between wild-type and b 3 KOC57micekeptat6°C (170 ± 1 vs. 140 ± 5 lUÆmg )1 ). This observation suggests that the total amount of mitochondria per BAT was not modified by the b 3 KO. The expression level of UCP1 protein in WAT relative to that in BAT, assessed on a same Western blot, was approximately 5% in mice kept at 24 °C and 11% after a 10-day cold exposure (data not shown). b 1 -adrenoceptor mRNA expression in WAT of C57 The possibility of a compensation by the b 1 -adrenoceptor in the b 3 KO mice was tested. This receptor is the second most abundant b subtype in WAT [29]. The b 1 -adreno- ceptor mRNA was not significantly affected by the b 3 KO in either the BAT or the WAT of C57 (100 ± 15 vs. 78 ± 8; 100 ± 9 vs. 88 ± 7). PGC-1 mRNA expression in WAT and BAT of C57 PGC-1 is a transcriptional coactivator induced by cold- exposure via the b-adrenergic signaling pathway in BAT and in skeletal muscle [30]. PGC-1 stimulates the transcrip- tional activity of PPARc, of the thyroid hormone receptors and of UCP1 in BAT. The expression of PGC-1 mRNA was measured in interscapular BAT and in genital WAT, using the same Northern blots as for UCP1 expression measurements (Fig. 5). In WT mice, PGC-1 was expressed in WAT but approximately 100-fold less than in BAT (data not shown). Moreover, a 2-day cold exposure increased PGC-1 mRNA expression in WAT by 1.8-fold (P < 0.001). Strikingly, the b 3 KO WAT exhibited normal PGC-1 mRNA levels in both conditions. Discussion Morphometric analyzes showed that the number of multi- locular cells present in WAT was strongly depressed in the b 3 KO mice maintained at 24 °C as compared to WT mice. The absence of UCP1 expression in multilocular cells under this condition might represent a cellular phenotype inter- mediary between white and brown adipocytes or might be due to the heterogeneity of the content of multilocular cells in different regions of a given WAT depot [11]. Ten days of cold-exposure increased the number of multilocular cells and induced the expression of UCP1 in some of them in both WT and b 3 KO mice. Mice maintained singly housed at 24 °C are under a mild cold stimulus. It is interesting to note in the WAT of C57 mice at 24 °C a significant level of expression of UCP1 mRNA reaching 3% of that observed in BAT. Our study shows for the first time that the lack of b 3 -adrenoceptor strongly depresses UCP1 mRNA and protein expression in WAT.Inmicekeptat24°C the UCP1 mRNA and protein levels in WAT are strongly depressed by the b 3 KO. Ten days of cold-exposure increased UCP1 mRNA and protein in WAT of both WT and b 3 KO mice but the differences between the two genotypes was conserved. InBATofC57mice,thelackofb 3 -adrenoceptor did not affect the basal or cold-induced expression of UCP1 mRNA and protein. The b 3 -adrenoceptor independency of UCP1 expression in BAT suggests that in brown Fig. 4. Western blot quantification of UCP1 protein expression in gen- ital WAT mitochondria of C57BL/6J wild-type (WT) or b 3 KO female mice, maintained at 24 °C or exposed to 6 °C for 2 or 10 days (2d and 10d, respectively). The results are expressed as means ± SE of arbi- trary values normalized using the corresponding cytochrome c oxidase values. The ratio of the control value is considered to be 1.0. n ¼ 6. **P <0.01 and ***P < 0.005 vs. respective WT; ## P <0.01 vs. respective control at 24 °C. Fig. 5. Northern blot quantification of PGC-1 mRNA expression in genital WAT of C57BL/6J wild-type (WT) or b 3 KO female mice, maintained at 24 °C or exposed to 6 °Cfor2(2d).(A), Genital WAT. The results are expressed as means ± SE of arbitrary values nor- malized using the corresponding 18S mRNA values. The ratio of the control values is considered as 1.0. n ¼ 6. # P <0.05 and ### P < 0.005 vs. respective control at 24 °C. Ó FEBS 2003 b 3 -adrenoceptor and ectopic adipocytes (Eur. J. Biochem. 270) 703 adipocytes, one or both of the other b-adrenoceptor subtypes are able to substitute and confirms previous findings in which we demonstrated that the lack of b 3 - adrenoceptor did not affect BAT metabolism [23,31]. Our study shows that there is no compensation at the level of the b 1 -adrenoceptor mRNA in BAT or WAT. Our in vivo studies are in contrast with in vitro experiments performed in cultivated adipocytes of male mice suggesting that UCP1 mRNA expression is under strict control of the b 3 - adrenoceptor [32,33]. A compensation by a-adrenoceptors cannot be excluded. Nevertheless, we and others, recently showed that, at least in BAT, a total lack of b-adrenergic signaling is not compensated by other adrenergic sub- types. Indeed, mice lacking the whole b-adrenoceptor family are dramatically cold intolerant with an abnormal BAT morphology [34,35]. The PPARc-1 coactivator, PGC-1, is induced by cold via b-adrenergic stimulation. It has been shown to stimulate brown adipocyte differentiation including UCP1 expression and was undetectable in WAT [30]. In our study, PGC-1 was expressed in genital WAT of WT mice, although at a much lower level than in BAT, and its expression was increased by cold exposure. In WT WAT, PGC-1 expres- sion varied in parallel with UCP1 expression, consistent with the reported control of UCP1 by PGC-1 in brown adipocytes. Strikingly, in b 3 KO WAT where UCP1 expres- sion was depressed, PGC-1 levels were normal. This suggests that b 3 -adrenoceptors are not essential for a normal expression of PGC-1. b 3 -adrenergic signaling might stimulate UCP1 expression in WAT by modulating a target molecule downstream PGC-1. This hypothesis deserves further studies. Together, our results suggest that the brown adipocyte- like cells present in WAT and the brown adipocytes constituting BAT are subjected to different control systems. The hypothesis of a different nature of BAT and WAT multilocular cells has already been proposed by Himms- Hagen et al. [8], who suggested that most of the multilocular cells appearing in WAT upon b 3 -adrenoceptor agonist stimulation derived from preexisting convertible unilocular white adipocytes. 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