Cyclooxygenases and UCP1 Figure 6. UCP1 expression is attenuated by an EP4 receptor antagonist in Rb2/2 adipocytes and induced by the EP4 receptor agonist 16,16dmPGE2 in vivo. A. Expressions of EP1, EP2, EP3, EP4 and FP receptors were measured by RT-qPCR in iBAT and iWAT isolated from warm- and cold-acclimated mice, and in Rb2/2 adipocytes treated with vehicle or isoproterenol (100 nM) and 9-cis-retinoic acid (1 mM). B. Rb2/2 adipocytes were treated with vehicle or isoproterenol (100 nM) and 9-cis-retinoic acid (1 mM) by RT-qPCR in absence and presence of AL8810, AH6809, or AH23848, which are FP, EP1/EP2 and EP4 receptor antagonists, respectively. Expressions of genes were measured by RT-qPCR. The bars represent mean 6 standard error. Different letters indicate statistically significant differences (p,0.05). C. C57BL/6J mice were subcutaneously injected with vehicle or 16,16dmPGE2 (50 mM/kg) every 12 h for 48 h. Expressions of genes were measured by RT-qPCR. The bars represent mean 6 standard error (n = 5). * indicates statistical significant difference between vehicle and 16,16 dmPGE2 treated mice (p,0.05). doi:10.1371/journal.pone.0011391.g006 mice. Also, injection of a stable analog of the COX2 downstream product PGE2, 16,16-dimethyl-PGE2, induced UCP1 expression in iWAT. Forced expression of COX2, alone or in combination with COX1, induced UCP1 expression in a cell model resembling inguinal adipocytes. Finally, the inhibition of COX activity not only attenuated diet-induced UCP1 expression in iWAT, but also increased weight gain in Sv129 mice kept at thermoneutrality. The association between diet-induced thermogenesis and the recruitment of brown adipose tissue was first noted more than 30 years ago and believed to involve the classical brown adipose tissue located in the interscapular region [59]. The anti-obesity role of UCP1 was challenged by the finding that UCP1 KO mice were not obese [60]. However, the recent demonstration that UCP1 ablation per se induced obesity when the mice were kept at thermoneutrality [61] clearly indicates that UCP1 is important in diet-induced energy dissipation at thermoneutrality. Our data indicate that inhibition of COX activity increased weight gain and concomitantly attenuated diet-induced UCP1 expression in iWAT, but not iBAT in Sv129 mice kept at thermoneutrality, pointing to a novel role of COX activity in the control of energy balance and the development of obesity. These results are in line with our earlier observation that enhanced cAMP signaling in response to an increased glucagon/insulin ratio led to an increased COX-mediated PGE2 production. This was accompanied by increased expression of UCP1 in iWAT, but not in iBAT, and decreased feed efficiency [62]. Although neither COX1 KO nor COX2 KO mice are obese, COX2+/2 KO mice have more adipose tissue than wild-type littermates when fed an obesogenic diet [48]. The reason why COX2+/2, but not COX2 KO mice were reported to be prone to obesity is not clear. However, these studies were not performed at thermoneutrality [48]. A similar phenomenon is actually seen in GLUT4 KO mice, where the majority of GLUT42/+, but not GLUT42/2 mice develops diabetes [63]. Moreover, release of PGE2 from adipose tissue in COX2+/2 mice was reported to be reduced compared to adipose tissue from wild-type mice [48] and adipose tissue cultures obtained from obese rats have lower PGE2 release rates than cultures from lean rats [38]. In addition, microsomal prostaglandin α Figure 7. Indomethacin prevents high-fat diet-induced UCP1 expression in iWAT but not iBAT in the obesity-resistant Sv129 mouse strain. Mice were fed a very high-fat diet (VHF) with or without indomethacin supplementation (16 ppm) for 4 weeks at a temperature of 28–30uC. One group of mice was killed before the experiment started. A. Body weight gain and energy intake relative to body weight gain. B. Different adipose tissue depots were dissected and weighed. C and D. Representative paraffin-embedded representative sections from iWAT and iBAT were stained with hematoxylin and eosin. The scale bars represent 50 mM. E. Expressions of genes in iWAT and iBAT were measured by RT-qPCR. The bars represent mean 6 standard error (n = 6). * indicates statistical significant difference (p,0.05) between different groups. doi:10.1371/journal.pone.0011391.g007 PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e11391