impairment in metabolic rate or gross substrate utilisation on a whole organism level as a result of a loss of L-PGDS in the PPARc2 KO background. Subtle Alterations in Eicosanoid Levels in Brown Adipose Tissue are not Consistent with a Prostaglandin Synthase Role for L-PGDS L-PGDS is capable of acting as both a secreted carrier of lipophylic molecules and as a prostaglandin synthase producing PGD2. To try to determine which role was important for the metabolic alterations seen in DKO mice we analysed eicosanoid levels by LC-MS/MS analysis. Several eicosanoid species were reduced in DKO mice compared to PPARc2 KO mice (Figure 2F), however these were principally lipoxygenase products. In brown adipose tissue, none of the prostaglandins detected showed significant differences, however their levels were, below the limit for accurate quantification (data not shown). Overall, the lack of changes in cycloxygenase products and their extremely low levels suggest that, L-PGDS may affect BAT metabolism by acting as a lipid-carrying lipocalin rather than a prostaglandin synthase. DKO Mice have Markers of Altered Lipid Metabolism in WAT The elevated circulating FFA levels present in the DKO mouse compared to the L-PGDS KO alone pointed to a potential alteration in adipose tissue function. To investigate this further, gene expression analysis was carried out on subcutaneous and epididymal white adipose tissue depots. Both DKO and PPARc2 KO mice had reductions in the adipogenic marker aP2 compared to WT. DKO mice also had significantly lower levels of aP2 to PPARc2 KO mice in epididymal WAT. Glut4 expression which can be related to both adipogenesis and insulin sensitivity was down regulated only in DKO mice when compared to WT (Figures 3 A and B). Analysis of the de novo lipogenic program in subcutaneous and epididymal WAT revealed a general reduction in the expression of lipogenic genes in mice lacking PPARc2, with significant reduc- tions in FAS, SCD1 and ELOVL6 in both depots of PPARc2 KO and DKO mice (Figure 3C and D). Notably, the magnitude of the reduction in the expression of the de novo lipogenic program was much more substantial in scWAT than epididymal. Mice lacking L-PGDS alone exhibited the opposite pattern, with increases in FAS and Insig1 expression exclusively in subcutaneous white adipose tissue (Figure 3D). Most strikingly, analysis of lipase expression demonstrated a reciprocal regulation of HSL and ATGL between epididymal and subcutaneous adipose tissue depots. DKO mice had increased expression of ATGL and HSL compared to L-PGDS KO mice alone in epididymal white adipose tissue (Figure 3E). Conversely, in subcutaneous white adipose tissue the expression of ATGL and HSL was decreased in DKO mice compared to WT mice (Figure 3F). Of note, the pattern of the expression of HSL and ATGL in epididymal white adipose tissue was similar to the pattern of levels of FFA observed in serum (Figure 1F). LPL expression was unchanged in epididymal white adipose tissue, whereas it was down regulated in subcutaneous white adipose tissue of DKO mice when compared to WT (Figure 3F). Overall these data were suggestive of a greater impairment in subcutane- ous adipose tissue function when compared to epididymal adipose tissue in terms of lipogenesis, lipid uptake and lipid release. To provide further evidence for alterations in adipose depot- specific lipid handling, we analysed protein levels of HSL and ATGL as well as the phosphorylation status of HSL as a surrogate L-PGDS Compensates for a Lack of PPARc2 of HSL activation [27]. In epididymal WAT, DKO mice had a significant increase in ATGL levels compared to PPARc2 mice, but both PPARc2 and DKO mice had lower ATGL levels than WT (Figure 4A). In subcutaneous WAT, DKO mice had significantly lower ATGL protein expression than PPARc2 KO or WT mice (Figure 4B). In terms of HSL, the absolute protein levels bore relatively little relation to the mRNA levels, with total HSL levels tending to be lower in the PPARc2 and DKO mice compared to wild-type, but no different to each other in both subcutaneous and epididymal white fat. However, analysis of the S660 (activatory) and S565 (inhibitory) phosphorylation sites on HSL revealed that in epididymal WAT, mice DKO mice had a significantly increased ratio of activatory to inhibitory phosphor- ylation levels compared to PPARc2 KO mice. In subcutaneous WAT the opposite was observed, with DKO mice having reduced levels of activatory HSL phosphorylation and a decreased activatory to inhibitory phosphorylation ratio compared to PPARc2 KO mice. Altered Lipid Metabolism in DKO Mice is Associated with Markers of Increased Insulin Resistance in Epididymal WAT Overall, epididymal WAT from DKO mice had a protein expression and phosphorylation pattern consistent with increased rates of lipolysis compared to PPARc2 KO mice, which contrasted with the situation in scWAT the reverse pattern was seen. In humans, intra-abdominal fat has been shown to have both increased lipolytic rate and to be more inflamed than subcutane- ous depots. We next investigated if the alterations in lipid metabolism observed in the DKO mouse were associated with inflammation in epididymal WAT. Analysis of inflammatory markers in epididymal WAT demonstrated a significant increase in the mRNA levels of T-cell markers CD3e, CD3g and CD4 in DKO mice over WT and PPARc2 KO mice (Figure 5A). Expression of macrophage markers and cytokines demonstrated a general elevation in mice lacking PPARc2; however there was a trend towards higher levels of pro-inflammatory markers (CD11c, TNFa) in DKO mice compared to PPARc2 KO alone and significant increases in IFNc and IL-12 (Figure 5B). In subcuta- neous adipose tissue the general trend was reversed, with increased expression of T-cell markers CD3e, CD3g and CD4 in PPARc2 KO mice, but not in DKO mice when compared to WT (Figure 5C). While F4/80 levels, a marker of macrophage infiltration, were similar between PPARc2 and DKO mice in both epididymal and subcutaneous white adipose tissue, levels of the M1 polarisation marker CD11c, which has been associated with a worsened adipose tissue metabolic profile and levels of IFNc were only induced in PPARc2 KO mice compared to WT mice (Figure 5D). Increases in the de novo Lipogenic Program in Liver from DKO Mice While the changes in FFA levels where most likely to have come from alterations in adipose tissue function, the alterations in TG levels could either have been down to reduced TG clearance or due to increased de novo synthesis in liver. Gene expression analysis of liver showed that when compared to other genotypes DKO mice had a significant increase in expression of FAS and ELOVL6 compared to wild-type mice (Figure 6A) they also tended (P = 0.059) to have increased expression of SREBP1c. Consistent with markers of increased rates of lipid synthesis, livers from DKO mice were significantly heavier at 4 months than wild-type mice (Figure 6B). Although relatively small, the changes in liver mass between the DKO and wild-type were significant in two PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e39512