A further layer of complexity regarding the regulation of lipid buffering comes from the fact that not all adipose tissue depots contribute equally to fatty acid uptake and release. Different depots have different rates of lipid release and uptake [9] and the size of different adipose tissue depots has been associated with insulin resistance. Subcutaneous adipose tissue mass has a neutral, or in the case of gluteofemoral adipose tissue, even a positive association with insulin sensitivity. Conversely, intra- abdominal fat mass is strongly associated with insulin resistance [10,11]. Interestingly both P465L DN and PPARc2 KO mice exhibited alterations in adipose tissue distribution with P465L DN mice having an increased subcutaneous to epididymal ratio [12] and PPARc2 KO mice having reduced intra-dermal lipid levels [5]. We and others have put forward the adipose tissue expand- ability hypothesis to suggest how obesity leads to metabolic complications. Essentially, a loss of adipose tissue capacity to store lipid leads to the accumulation of lipids in non-adipose tissues. As lipids accumulate ectopically, they cause toxic insults to these tissues resulting in pathologies such as insulin resistance [13,14]. Based on the adipose tissue expandability hypothesis we expected mice lacking PPARc2 to have severe impairments in insulin sensitivity. The impairments in in vitro adipogenesis in PPARc2 preadipocytes and the known roles for PPARc in lipid handling suggested that adipose tissue would be compromised in its ability to store lipid. Given the unexpectedly normal phenotype of the PPARc2 KO mouse we hypothesised that there must be mechanisms acting in vivo to protect PPARc2 animals from developing severe metabolic disturbances. To try to identify possible compensatory mechanisms we used microarray analysis to compare white and brown adipose tissue from WT and PPARc2 KO mice. From these microarray analyses we identified Lipocalin prostaglandin D synthase (L-PGDS). L-PGDS is a bi-functional molecule capable of synthesising D series prostaglandins and acting as a carrier of small lipophilic molecules [15]. In humans, serum L-PGDS levels have been positively associated with the severity of coronary artery disease [16], whereas in mice ablation of L-PGDS has been reported to aggravate the development of atherosclerosis [17]. However, its involvement in insulin sensitivity and glucose tolerance is less clear [17,18]. Furthermore, in humans L-PGDS expression is higher in scWAT than abdominal WAT [19], suggesting it may modulate depot-specific differences in adipose tissue function. To determine if L-PGDS was able to protect mice lacking PPARc2 from impairments in metabolism we generated a double knock-out mouse lacking both PPARc2 and L-PGDS (DKO). Removing L-PGDS from PPARc2 KO caused glucose intolerance and resulted in dyslipidemia. Materials and Methods Generation of Mice Homozygous for PPARc2 KO and L- PGDS KO Mice heterozygous for a disruption in exon B1 of PPARc2 on a C57Bl/6J background (PPARc2+/2) were crossed with mice heterozygous for a disruption in exons II–V in the L-PGDS gene [20] on a C57Bl/6J background to obtain mice heterozygous for both the PPARc2 and L-PGDS (PPARc2+/2 L-PGDS +/2). These mice were crossed to obtain the four experimental genotypes: 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). Mice were phenotyped on C57Bl/6J background (at least 8 generations). DKO mice were born at the expected Mendelian ratios (908 mice, Chi squared = 0.22). Genotyping for L-PGDS Compensates for a Lack of PPARc2 deletion of PPARc2 or L-PGDS was performed by PCR using previously described protocols [5,20]. Animal Care and Diets Male mice were housed at a density of four animals per cage in a temperature-controlled room (20–22uC) with 12-h light/dark cycles. Food and water were available ad libitum unless noted. All animal protocols used in this study were approved by the UK Home Office (License no. 80/2098) and the University of Cambridge. Animals were fed on a normal chow diet (10% of calories derived from fat; D12450B, Research Diets). Blood Biochemistry Enzymatic assay kits were used for determination of plasma FFAs (Roche) and triglycerides (Sigma-Aldrich, St. Louis, Mis- souri, USA). Elisa kits were used for measurements of Insulin (DRG Diagnostics International Limited) according to manufac- turer’s instructions. Serum biochemistry was analysed in male C57Bl/6 mice of 4 months of age, n = 8 per group. Hepatic Triglyceride Analysis Lipids were extracted from 100 mg of homogenised liver by heating to 85uC in 1 ml of 5% NP-40 in water. Triglyceride levels in extracts were assessed by enzymatic assay (Biovision) according to manufacturers instructions. Triglyceride content was analyzed in male C57Bl/6 mice of 5 months of age, n = 8 per group. Oxygen Consumption (VO2) and Respiratory Exchange Ratio (RER) from Indirect Calorimetry Animals were placed in a Comprehensive laboratory animal monitoring system (Columbus Instruments, Ohio, USA) attached to a custom-built oxygen and carbon dioxide monitoring system. The system was maintained at 22uC. Oxygen consumption (VO2) and respiratory exchange ratio (RER) (VCO2/VO2) were measured in mice at ages as described in figures. Oxygen consumption was measured over a 48–72 hour period using a custom built oxygen and carbon dioxide monitoring system. Airflow rates were 400 ml/min and measurements of oxygen concentration and carbon dioxide concentration in room air and air leaving each cage were measured every 18 minutes [21]. Indirect calorimetry was performed on male C57Bl/6 mice of 4 months of age, n = 8 per group. Insulin Tolerance Test (ITT) Mice were fasted for 6 hours from 8 am until 2 pm. Mice were injected intraperitoneally with 0.75 U/Kg of human insulin (Actrapid, Novo Nordisk). Blood glucose levels were measured using a one touch ultra glucose meter (Lifescan, CA, USA) [22]. Insulin tolerance tests performed on male C57Bl/6 mice of 4.5 months of age, n = 8 per group. Glucose Tolerance Tests (GTT) Mice were fasted overnight from 4 pm until 9 am the next day. Mice were injected intraperitoneally with glucose (1 g/kg) and blood glucose levels were measured using a one touch ultra glucose meter (Lifescan, CA, USA) [22]. Glucose tolerance tests performed on male C57Bl./6 mice of 4 months of age, n = 8 per group. Histology Samples for histology were placed in 10% buffered formalin overnight before transfer to 70% ethanol and later embedding in paraffin. Multiple sections were stained with haematoxylin and PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e39512