eosin for morphological analysis. Percentage lipid content was determined by morphometric analysis by measuring the propor- tion of white area (lipid) per slide. The average lipid area in BAT per mouse was calculated from at least 3 separate sections. Analysis was carried out using CEL LˆP analysis software (Olympus, Southend-on-Sea, UK). BAT histology performed at 4 months, liver histology at 5 months. All; male C57Bl/6 mice, n = 8 per group. Q-RT-PCR RNA was isolated from ground tissues using STAT-60 reagent (TEL-TEST) followed by chloroform extraction and isopropanol precipitation. Complimentary DNA was generated from 500 ng of RNA using M-MLV reverse transcriptase and master mix (Promega) in a 20 ml reaction with 2.5 mM MgCl2, 1.25 mM dNTPs and 5 mg/ml random hexamers at 37uC for 1 hour. cDNA was diluted 75 fold and 5 ml of diluted cDNA was used in a 12 ml real time PCR (RT-PCR) reaction using TaqMan primers and probes or SYBR green reagent (Applied Biosystems) according to manufacturers instructions. Reactions were run in duplicate for each sample and quantified in the ABI Prism 7900 sequence detection system (Applied Biosystems). Data was normalised to 18s rRNA. Primer sequences available on request. eWAT, scWAT and BAT analyzed at 4 months, liver at 5 months. All; male C57Bl/6 mice, n = 8 per group. Western Blotting All western blots were carried out using 10% polyacrylamide gels (Biorad). 20 ug of protein was used for all samples. All primary antibodies from Cell Signalling (catalogue number in brackets) pHSL S600 (#4216), pHSL S565 (#4137), tHSL(#4107), ATGL (#2138), pAMPKa T172 (#2535) and tAMPKa (#2532). All antibodies used at a 1:1000 dilution in 5% BSA. Quantification was carried out using ImageJ (NIH). All; male C57Bl/6 mice, 4 months of age n = 6 per group except scWAT ATGL, n = 3. Eicosanoid Extraction and Quantification Lipidomic analysis of lipid mediators in brown adipose tissue was performed according to our published protocol [23]. In summary: Brown adipose tissue from WT (PPARc2+/+ L-PGDS +/+), PPARc2 KO (PPARc22/2 L-PGDS +/+), L-PGDS KO (PPARc2+/+ L- PGDS 2/2) and DKO (PPARc22/2 L-PGDS 2/2) were ground on liquid nitrogen and homogenised in 15% (v/v) methanol in water, followed by the addition of internal standards 12-HETE-d8 and PGB2-d4 (40 ng) (Cayman Chemicals,Ann Arbor, MI, USA). The resulting solutions were then acidified with 0.1 M hydrochloric acid to pH 3.0 and immediately applied to pre-conditioned SPE cartridges (C18-E, 500 mg, 6 mL) from Phenomenex (Macclesfield, UK).The cartridges were washed with 15% (v/v) methanol in water (20 ml) followed by 20 ml of water (20 ml) and hexane (10 ml); the lipid mediators were eluted in methyl formate (15 ml). The organic solvent was evaporated using a fine stream of nitrogen and the remaining residue was re-dissolved in ethanol (100 ml) stored at – 20uC awaiting analysis [24]. LC-MS/MS Analysis of Eicosanoids Chromatographic analyses were performed using Accela UHPLC (Thermo Scientific, Hemel Hempstead, UK) and Acquity UPLC systems (Waters, Hertsfordshire, UK). The bioactive lipids were separated on a C18 reversed-phase (RP) LC column (Phenomenex Luna, 3 mm particles, 15062 mm) using a linear mobile phase gradient (A, 0.02% glacial acetic acid in water; B, 0.02% glacial acetic acid in acetonitrile). Mass spectrometry L-PGDS Compensates for a Lack of PPARc2 analyses were carried out on LTQ Velos (Thermo, Hemel Hempstead, UK) linear ion trap (LIT)-orbitrap and QTRAP 4000 (AB Sciex, Concord, ON) quadrupole-linear ion trap (QqLIT) mass spectrometers [24]. Eicosanoids analyzed in male C57Bl/6 mice of 5 months of age, n = 8 per group. Shotgun Lipidomic Analysis of Liver Approximately 5 mg of liver, which was frozen directly in liquid N2, were weighed and liver tissue was diluted with 50 ml of PBS buffer and WAT was diluted with 150 ml of methanol and vertexed for 2 min. All samples were spiked with an internal standard (10 ml for liver) [25]. Lipids were extracted from the liver samples with 200 ml of chloroform:Methanol (2:1) solvent. After vortexing for 2 min and incubating for 1 h for liver samples at room temperature, the lower layer (approximately 100 ml for liver samples) was separated by centrifugation at 10,000 rpm for 3 min at room temperature. Ten microliters of labelled standard mixture (three stable isotope-labeled reference compounds) for liver samples was added to the lipid extracts and 0.5 - 1.0 ml injection was used for LC/MS analysis. Sample order for analysis was established by randomization. Lipid extracts were analyzed on a Q-ToF Premier mass spectrometer (Waters) combined with an Acquity Ultra Performance Liquid chromatography (UPLC/MS). Mass spectrometry was carried out on Q-Tof Premier (Waters, Inc.) run in ESI+ mode. The data was collected over the mass range of m/z 300–1200 with scan duration of 0.2 sec. The source temperature was set at 120uC and nitrogen was used as desolvation gas (800 L/h) at 250uC. The voltages of the sampling cone and capillary were 39 V and 3.2 kV, respectively. Reserpine (50 mg/L) was used as the lock spray reference compound (5 ml/ min; 10 sec scan frequency). The obtained data was converted into netCDF file format using Dbridge software from MassLynx (Waters, Inc.). The converted data was processed using MZmine software version 0.60 [26]. Lipids were identified based on their retention time (RT) and mass to charge ratio (MZ) using our in- house built lipid database as previously described [25]. All the identified lipids were quantified by normalizing with correspond- ing internal standards. Liver lipids analyzed in male C57Bl/6 mice of 5 months of age, n = 8 per group. Statistics Results were expressed as mean 6 SEM. Statistical analysis was performed using SPSS 17.0 (IBM, New York). Pair wise comparisons were carried out by students t-test. For data with more than two groups ANOVA was carried out followed by Tukeys Post-Hoc Test. Results Lack of PPARc2 Results in Increased L-PGDS mRNA Expression Selectively in BAT and Subcutaneous WAT The sv129 PPARc2 KO mouse represents a model of impaired lipid handling but with apparently normal glucose tolerance. To try to identify mechanisms that may protect mice lacking PPARc2 from metabolic impairments we used microarrays to identify genes up regulated in BAT and WAT. L-PGDS was found to be highly up regulated in brown adipose tissue (data not shown). We confirmed these results by RT-PCR analysis on major metabolic tissues from PPARc2 KO mice. L-PGDS expression was increased in BAT and scWAT from PPARc2 KO mice but down regulated in epididymal WAT and liver (Figure 1A). PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e39512