E synthase1 (mPGES1) expression is reported to be downregulated in iWAT and eWAT in obese mice [64], suggesting a dysregulation of prostaglandin synthesis in obesity. PGE2 mediates its action by interacting with four subtypes of PGE2 receptors, the EP1, EP2, EP3 and EP4 receptors [50]. Using the Rb2/2 brown-like adipocytes, we show that b-adrenergic stimulation of UCP1 expression is attenuated by an EP4 receptor antagonist. This combined with our finding that injection of the EP3/EP4 receptor agonist 16,16-dimethyl-PGE2 increased expres- sion of UCP1 in iWAT indicates that the action of PGE2 is predominantly mediated via the EP4 receptor with a possible minor contribution by the EP3 receptor. Most EP4 KO mice die shortly after birth, and no adipose tissue phenotype has been reported for the few surviving pups [65]. Similarly, to our knowledge, no adipose tissue phenotype has been reported for the EP3 KO mice. Collectively, our findings strongly indicate that both cold- and diet-induced expression of UCP1 in iWAT, but not in iBAT, requires COX activity and most likely PGE2 formation. Furthermore, our results point to differential roles of induction of UCP1 expression in iWAT and iBAT in the context of diet- and cold-induced thermogenesis. We suggest that whereas UCP1 in iWAT plays an important role in protection against obesity, UCP1 in iBAT is essential for temperature adaptation. Upon cold challenge, body temperatures were only slightly lower in wild-type mice treated with indomethacin and in COX2 KO mice compared to non-treated wild-type mice. This is in line with the earlier finding that UCP1 expression is blunted in iWAT, but not in iBAT, in cold-adapted b3 adrenoreceptor KO mice [66]. The importance of COX in diet-induced expression of UCP1 in iWAT and diet-induced thermogenesis is underscored by our demonstration that inclusion of the general COX inhibitor indomethacin in the diet augmented high-fat diet-induced obesity in Sv129 mice kept at thermoneutrality irrespectively of UCP1 expression in iBAT. Increased expression of UCP1 in WAT with accompanying increased thermogenic activity coupled with un- changed or even reduced BAT activity has been observed in several genetically modified lean mouse models such as RIP140 [67], Caveolin 1 [68], Fsp27 [69], hormone sensitive lipase [70] and vitamin D receptor [71] KO mice. Further, in RIIb mice [72], pRb- deficient mice [11;73] and in mice overexpressing FOXC2 [74], protection against diet-induced obesity is accompanied by an increased RIa/RIIb ratio, rendering PKA somewhat more sensitive to cAMP, which is accompanied by an increased occurrence of brown adipocytes in WAT. Last, it should be recalled that in aP2- UCP1 transgenic mice, both endogenous UCP1 expression and respiration are actually reduced in iBAT [75]. UCP1 expression, respiration and total oxidative capacity are, however, strongly induced in WAT and the oxidative capacity of WAT is sufficient for the changes of total energy balance induced by the transgene [76]. In keeping with the earlier notion that i) mouse strains that have more UCP1-expressing adipocytes in their WAT depots are protected against diet-induced obesity [77;78] and ii) brown-like multilocular adipocytes expressing UCP1 are detected interspersed within white adipose tissue in humans [20;21;79], we suggest that factors influencing UCP1 expression in white adipose tissue are of particular importance for the regulation of energy balance and the development of obesity also in humans. Materials and Methods Ethics Statement The animal experiments were approved by the Norwegian Animal Health Authorities, ID 819 and 888. Care and handling were in accordance with local institutional recommendations. Cyclooxygenases and UCP1 Cell culture, transduction and differentiation Mouse embryo fibroblasts (MEFs) were prepared from wild-type and Rb2/2 embryos [80]. The cells were grown and differentiated in AmnioMax Medium as described earlier [81]. Retrovira expressing pLXSN-hygro, pBabe-puro, pLXSN-COX1 or pBabe-COX2 were harvested from Phoenix–Eco cells, plated at 30–40% confluence in DMEM supplemented with 10% fetal bovine serum, and transductions performed as described [82]. Isolation of the stromal vascular fraction and adipocytes from mice The stromal vascular fraction and adipocytes were obtained from iWAT and iBAT dissected from 8-week old C57BL/6J mice as earlier described [83]. Contaminating erythrocytes were eliminated from the stromal-vascular fraction by a wash with sterile distilled water. Cells were plated and induced to differentiate as described [83]. Cold acclimation experiments Groups (n = 5–8) of 10-week old male mice were acclimated at a temperature of 28–30uC for at least 1 week and transferred to 4uC for 1, 2, 3 or 6 days. Where relevant, mice were injected with indomethacin (2.5 mg/kg) 2 h prior transfer to 4uC. The mice received a dose of indomethacin every 12 h. Injections were performed subcutaneously from a 0.75 mg/ml solution. Final dose was 5 mg/kg/day. Control mice received vehicle. Animals were housed individually with a 12 h light/dark cycle and free access to pellet food and water. Mice used for immunohistochemical analyses were immediately perfused intracardially with 4% paraformaldehyde. iBAT, iWAT, lung and skin were dissected and frozen for immunohistochemistry on cryosections. For morphology experiments, the mice were immediately perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 5 min. COX2 KO mice (B6;129P2 Ptgs2 tm1Unc) and correspond- ing wild-type littermates were obtained from Taconic. C57BL/6J used in indomethacin experiments were obtained from Møllega˚rd breeding laboratories. 16,16dmPGE2 injections Male, C57BL/6J approx 10-weeks old from Møllega˚rd breeding laboratories, Denmark were divided into two groups (n = 5). The mice received a dose of 50 mg/kg 16,16dmPGE2 every 12 h for 48 h. Injections were performed subcutaneously and the total dose was 0.1 mg/kg/day. Control mice received vehicle. Animals were housed individually with a 12 h light/dark cycle and free access to pellet food and water. High-fat feeding Male Sv129 mice, 11 weeks old, were obtained from Taconic. The mice were acclimated for 1 week at a temperature of 28–30uC and divided into three groups (n = 6 in each). One group of mice was sacrificed before dietary intervention while the remaining mice were fed a very high-fat diet (ssniff EF R/M acc D12492) with or without indomethacin supplementation (16 ppm) for 4 weeks at a temperature of 28–30uC. Body weight and feed intake were recorded twice a week. Mice were anesthetized using isoflurane, cardiac puncture was performed and mice were killed by cervical dissociation. Tissues were immediately frozen in liquid N2. Real time qPCR Total RNA was extracted from cultured cells or mouse tissue using TRIzol (Invitrogen). Reverse transcription and qPCR were PLoS ONE | www.plosone.org 10 June 2010 | Volume 5 | Issue 6 | e11391