Although it has been estimated that 50 g of brown adipocytes would be sufficient to burn 20% of the daily energy intake [14], BAT has traditionally been considered to be virtually absent and of no physiological relevance in adult humans. This view has recently changed dramatically with the demonstration of func- tional BAT in adult humans [15–19] adding to the observation of brown-like multilocular adipocytes expressing UCP1 interspersed within human WAT [20–22]. Actually, UCP1 mRNA has been detected in all adipose tissues in adult humans, and it has been estimated that 1 in 100–200 adipocytes in human intraperitoneal adipose tissue expresses UCP1 [23]. Classic interscapular brown adipocytes and brown-like adipo- cytes found in WAT depots appear to originate from distinct lineages. Brown pre-adipocytes derived from the interscapular region (iBAT) demonstrate myogenic gene expression [24] and classic brown adipocytes arise from Myf5 expressing progenitors [25]. In contrast, brown-like adipocytes appearing in white adipose tissue by b-adrenergic stimulation (‘‘brite adipocytes’’) appear to originate from another lineage, much closer to white adipocytes [26–29] and display different molecular markers [30]. Several lines of evidence suggest that the number of brown-like adipocytes in WAT depots might influence whole body energy balance. Increased occurrence of brown-like adipocytes within WAT depots is a feature of mouse strains resistant to dietary obesity, such as the A/J strain [31;32], and reduced adiposity associated with aP2-transgenic expression of UCP1 is linked to increased energy dissipation in white, but not interscapular brown, adipose tissue [33]. Human obesity is associated with a reduced expression of UCP1 and other thermogenesis related genes in WAT depots [34;35]. Thus, identification of factors controlling induction of UCP1 expression and an increase in the number of brown-like adipocytes in white depots obviously deserves further attention. It is intriguing that the cold-induced occurrence of brown-like adipocytes and UCP1 requires the presence of the b3-adrenocep- tor in previously white adipose tissue, but not in interscapular brown adipose tissue [36]. Furthermore, the presence of the b - 3 adrenoceptor is required for full stimulation of energy expenditure and oxygen consumption in white adipose tissue [37]. Adipocytes from lean rats have higher isoprenalin-stimulated prostaglandin E2 (PGE2) synthesis, than adipocytes from obese Zucker rats [38]. We therefore hypothesized that prostaglandins or related products synthesized by cyclooxygenases (COXs) might be involved in the recruitment of brown adipocytes in white depots. The COXs have previously been implicated in adipogenesis [39–41], but no specific role has been assigned. Here, we demonstrate that COX activity is crucially involved in the induction of UCP1 expression in WAT providing further evidence for a role of COXs in the control of energy balance and obesity development. In view of the worldwide epidemic of obesity and associated metabolic disorders it is obviously of importance to identify pathways that can be manipulated genetically or pharmacologically and regulate the induction of UCP1 expression and recruitment of brown-like adipocytes in white adipose tissues. Results COX1 and COX2 protein expression is upregulated in iWAT during cold treatment When mice are kept at 28uC, close to thermoneutrality, the majority of the adipocytes in the inguinal white adipose depot (iWAT) – the major subcutaneous depot in the mouse – appear as UCP1-negative, spherical unilocular adipocytes [42]. In iWAT from warm-acclimated mice, only endothelial cells and macro- Cyclooxygenases and UCP1 phages stained positive for COX1 and COX2, respectively (Figure 1A and B). However, when mice were transferred to a cold environment, the emerging multilocular adipocytes stained positive for COX1 and COX2. In particular, cells that appeared to be in a transition state between uni- and multilocular cells stained strongly (Figure 1A and B). Western blotting demonstrated increased expression of COX1 and COX2 in iWAT, and also in iBAT, after cold exposure (Figure 1C). Real time qPCR analysis verified that genes preferentially expressed in brown vs. white adipose tissue, such as UCP1, peroxisome proliferator activated receptor gamma coactivator 1a (PGC1a), type II thyroxine deiodinase (Dio2), cytochrome C oxidase subunit 8b (Cox8b), epithelial V like antigen 1 (Eva1) and Cidea were all highly induced in iWAT upon cold exposure, whereas expression of 4E- BP1 and of nuclear receptor interacting protein 140 (Nrip1/ RIP140) was reduced (Figure 1D). Immunohistochemical staining of iBAT from cold-treated mice demonstrated that adipocytes stained positive for COX2, whereas only endothelial cells stained positive for COX1 (Figure S1A). The lack of COX1 and COX2 expression in adipocytes from iBAT in warm-acclimated mice was verified by analysis of protein and RNA isolated from fractionated adipose tissue, in which COX1 and COX2 was detected solely in the stromal vascular fraction (Figure S1B). Inhibition of COX activity represses induction of UCP1 expression Differentiated mouse embryo fibroblasts (MEFs) lacking the retinoblastioma (Rb) gene, resemble brown or brown-like adipocytes in demonstrating b-adrenergic induction of UCP1 expression [43]. To achieve a robust induction of UCP1 expression, differentiated Rb2/2 MEFs were treated with a combination of isoproterenol and 9-cis retinoic acid [44]. Just as cold exposure increased COX1 and COX2 mRNA and protein levels in brown-like adipocytes (Figure 1), isoproterenol/9-cis retinoic acid treatment increased COX1 and COX2 mRNA and protein expression (Figure 2A and B) in this model system. Upregulation of COX1 and COX2 expression in Rb2/2 adipocytes was accompanied by increased production of PGE2, the primary prostaglandin produced by mature adipocytes [45;46], but not of PGF2a and 6-keto-PGF1a (Figure 2C). This indicates that Rb2/2 adipocytes resemble mature adipocytes in producing PGE2 as the major prostaglandin species. To investigate the importance of COX activity for induction of UCP1 expression, we treated differentiated Rb2/2 adipocytes with isoproterenol/9-cis retinoic acid in the absence or presence of the general COX inhibitor indomethacin. Indomethacin prevent- ed induction of UCP1 mRNA and protein expression (Figure 2D and E), thus suggesting the intriguing possibility that COX activity is required for induction of UCP1. To examine if COX activity was required also in primary adipocytes, we induced cells from the stromal vascular fraction of iBAT and iWAT to differentiate and then treated the mature adipocytes with the b-adrenergic agonist isoproterenol in the absence and presence of indomethacin. Interestingly, indometh- acin inhibited isoproterenol-induced UCP1 expression in cells derived from iWAT but not from iBAT (Figure 3A), indicating that COX activity is required for b-adrenergic induction of UCP1 expression in adipocytes from iWAT, but not in iBAT adipocytes. In keeping with this notion, indomethacin only marginally attenuated induction of UCP1 expression in the WT-1 cell model representing interscapular brown adipocytes (Text S1, Figure S2) [47]. To investigate the role of COX activity during induction of UCP1 expression in iWAT and iBAT in vivo, we treated warm- PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11391