Cyclooxygenases and UCP1 Figure 3. Indomethacin prevents cold-induced UCP1 expression in iWAT. A. Stromal vascular fractions (SVFs) were isolated from mouse iWAT and iBAT, cultured and induced to differentiate as described in experimental procedures. Differentiated adipocytes were treated with vehicle or isoproterenol (100 nM) in the absence or presence of indomethacin (1 mM) for 24 h. Expression of UCP1 was measured by RT-qPCR in duplicates and normalized to PPARc. The bars represent mean 6 standard deviation. The experiment was performed in triplicates and repeated 3 times. B–D. C57Bl/ 6 mice were warm-acclimated at 28–30uC for 10 days and then transferred to 4–6uC for 48 h. Mice were injected with vehicle or indomethacin (2.5 mg/kg) 2 h prior transfer to 4uC and thereafter every 12 h. Rectal temperature was measured before the mice were transferred and after 24 and 48 h (B). Protein and RNA extractions were isolated after 48 h. UCP1 expression was measured by Western blotting (C) and expressions of genes were measured by RT-qPCR and normalized to TBP (D). The bars represent mean 6 error (n = 5–6). * indicates statistical significant difference (p,0.05). doi:10.1371/journal.pone.0011391.g003 affinity that is only 10–30 fold lower than that of PGF2a [51]. In order to probe the relative importance of these receptors in mediating the possible effect of PGE2 on induction of UCP1, expression of the EP and FP receptors was measured in adipose tissue and in Rb2/2 adipocytes. All receptors were expressed in both white and brown adipose tissue, whereas no expression of the EP3 receptor could be detected in Rb2/2 adipocytes (Figure 6A), implying a minor if any role of this receptor in mediating the PGE2 response in those cells. Thus, Rb2/2 adipocytes were treated with isoproterenol and 9-cis retinoic in the absence or presence of AL8810, AH6809, or AH23848, that are FP-, EP1/EP2 and EP4 receptor antagonists, respectively. Isoproterenol-stimulated UCP1 expression was not affected by the FP receptor antagonist, but slightly attenuated by the EP1/EP2 receptor antagonist and strongly attenuated by the EP4 antagonist (Figure 6B). Reduced expression of UCP1 was accompanied by reduced expression of PGC1a and Cidea (Figure 6B). To verify the importance of PGE2 signaling via the EP4 receptor with a possible minor contribution by the EP3 receptor, mice were injected with an EP3/EP4 receptor agonist [52], the stable PGE2 analogue 16,16-dimethyl-PGE2 As predicted, qPCR analysis revealed that UCP1 expression was induced in iWAT, but not in iBAT (Figure 6C). Together, the in vitro and in vivo results suggest that PGE2-induced UCP1 expression at least in part is mediated via the EP3/EP4 receptors with EP4 being the predominant receptor involved. Inhibition of COX activity increases adiposity and energy efficiency in obesity resistant Sv129 mice Diet-induced thermogenesis protects several mouse strains against obesity [53–55]. Since it appears that the protection against diet-induced obesity is related to increased occurrence of brown-like adipocytes in white depots [56;57], we aimed to investigate the hypothesis that indomethacin could also attenuate diet-induced UCP1 expression and thereby increase the propen- sity for diet-induced obesity in Sv129 mice. Since it was recently demonstrated that UCP1-deficient mice become obese when housed at thermoneutrality [58], we predicted that the most pronounced effect of COX inhibition would be observed for mice kept under thermoneutral conditions. Accordingly, we fed Sv129 mice a very high-fat diet with or without indomethacin PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11391