considering the small contribution of WAT to whole body energy expenditure compared to that of skeletal muscle, which accounts for the majority of energy consumption at the whole body level. The relative contribution of each organ to the increased energy expenditure in HSL mice will require the generation of mouse lines in which the HSL gene has been inactivated tissue-specifically. Altogether the phenotype of the HSL mice is very reminiscent of that of RIP140 mice, which also exhibit acquirement of BAT characteristics in WAT without increased PGC-1a expression [9]. Another similarity between HSL mice and RIP140 mice is that subcutaneous WAT depots are more reduced than visceral depots. RIP140 is expressed in both brown and white adipocytes [9] and seems to antagonize the function of PGC1 coactivators in these cells, thereby suppressing UCP-1 expression and ATP uncoupling [8]. It has been observed that in the absence of RIP140, altered expression of the PGC1 coactivators is not essential for UCP1 expression [39]. Thus, it can be speculated that downregulation of RIP140 in the HSL mouse model, allows the low levels of PGC1 coactivators in white adipocytes to promote the appearance of brown fat features. The mechanism whereby HSL is involved in the determination of cell fate during adipocyte differentiation remains to be explored. The fact that both pRb and RIP140 are transcriptionally controlled by retinoic acid [5,40,41], together with our recent observation that retinyl ester hydrolase activity is dramatically reduced in WAT of HSL mice [26], makes future studies into the role of the retinyl ester hydrolase activity of HSL highly warranted. Nevertheless, the key finding of the present paper, i.e. the attainment of BAT characteristics in WAT of HSL mice, emphasizes that the traditional view of HSL, as a fatty acid- mobilizing enzyme in WAT, has to be abandoned in favor of an emerging view of a multifunctional enzyme with several functions within and outside WAT. Materials and Methods Animal experiments The study was reviewed and approved by the Ethical Committees in Malmo¨/Lund, Lund, Sweden (license no. M162-05) and Gothenburg, Gothenburg, Sweden (license no. 294-2003) and is in accordance with the Council of Europe Convention (ETS 123). HSL mice were generated by targeted disruption of the HSL gene in SV129-derived embryonic stem cells as described elsewhere [42]. Animals used were all from the same embryonic stem cell colony and animals in the different groups were littermates and had a mixed genetic background from the inbred strains C57BL/6J and SV129 [17]. The animals were maintained in a temperature-controlled room (22uC) on a 12-h light-dark cycle. Mice, 16–20 weeks of age, were fed ad libitum a chow diet (control) (11% energy from fat) or a high fat diet (HFD) (58% energy from fat) (Research Diets; products no D12310 and D12309 respectively), for 6–10 months before the in vitro analyses were performed. Body weights were measured every third week during the study. A shorter HFD study was also performed, where four week old female mice were fed ad libitum a HFD (see above) for 4 weeks before the in vitro analyses were performed. From these mice, RNA was extracted from isolated periovarial white adipocytes and gene expression was analyzed by real-time quantitative PCR. For the comparison of food intake, female mice of the same genotype and feeding category were housed together and food consumption was measured as weight of the distributed food subtracted by weight of the remaining food over one week. Food consumption is presented as caloric intake per week. For measurements of stool lipids, female mice were put in metabolic HSL Null Mice cages and faeces were collected during a 24 h period. Total stool lipids were extracted and quantified according to Schwarz et al [43]. Registration of physical activity was measured during the dark-cycle by number of photo-beam disruptions using an open field (0.4 m60.4 m) 16616 photo-beam system (San Diego Instruments) and was performed using male mice housed individually in cages. The animals were allowed an adaptation period of 1 h before the recording started. Movements were then recorded for a period of 2 h with 4 individuals of each genotype. The parameters investigated were: horizontal motor activity in the periphery and in the central part of the cage, rear leg, fine (grooming), ambulatory and total movements. Collected tissues were rapidly dissected, snap frozen and stored in liquid nitrogen before analyses. Blood samples were drawn by retro- orbital puncture from female animals in the fed state and plasma leptin and adiponectin concentrations were measured using a leptin ELISA kit (Crystal Chem Inc.) and an adiponectin RIA kit (Linco). To analyze body fat distribution, computer tomography (CT) was performed in female mice, 4-mm proximal to the iliac crest, identified by a longitudinal prescan, with the Stratec peripheral quantitative CT (Norland Medical Systems) at 70-mm resolution. Images were analyzed using software from Bergstro¨m Instruments (Solna, Sweden). For measurement of energy expenditure, male wildtype and HSL mice, fed ad libitum the HFD, were put in metabolic cages, where oxygen consumption (VO2) (ml/kg/min) and carbon dioxide production (VCO2) (ml/kg/min) were measured for 2 h by an indirect calorimetry method (Oxymax, Columbus Instrument). To minimize influence of animal handling on the data, results from the last hour are presented. Morphology and morphometry of WAT Epididymal WAT was fixed in 4% buffered formaldehyde, pH 7.2, overnight, then dehydrated in graded ethanols and embedded in paraffin. Sections (10 mm) were cut and mounted on slides, after which they were stained with hematoxylin and eosin. Slides were analyzed using an Olympus BX60 microscope and images were captured with a DS-2Mv camera (Nikon). Calculation of the area of the adipocytes was performed using NIS-Elements Advanced Research software (Nikon) (106magnification). Transmission Electron Microscopy Periovarial WAT from HFD-fed wildtype and HSL mice were processed in parallel according to the following scheme. Following fixation in PBS containing 3% glutaraldehyde for 1 h, the tissues were cut into smaller pieces and then further fixed over night at 4uC. Specimens were post-fixed with osmium tetraoxide (1% in PBS-buffer) for 2 h at room temperature, followed by washing three times with MilliQ-water for 1 h. Dehydration was performed in acidified 2.2-dimethoxypropane (DMP) for 30 min at RT, followed by rinsing with acetone twice for 15 min. Spurr’s resin was infiltrated gradually and then cured at 70uC for 16 h. Sections (1 mm) were cut and studied in a light microscope (Leica ATC 2000) to locate well- fixed tissue in the blocks, for further trimming with a glass knife. Thin sections (70 nm) were cut with a diamond knife at an ultramicrotome (Leica UCT) and collected on pioloform-filmed copper grids. The sections were then observed in a Philips CM 120 Biotwin instrument at 120 kV. Morphometric analysis was performed semi-automati- cally in DigitalMicrograph (GATAN), by outlining profiles to obtain data about mitochondrial diameter. Tissue homogenization preparation Tissues were homogenized in 0.25 M sucrose, 1 mM EDTA pH 7.0, 1 mM dithiothreitol, 20 mg/ml leupeptin, 10 mg/ml antipain, 1 mg/ml pepstatin A using a glass/teflon homogenizer PLoS ONE | www.plosone.org 9 2008 | Volume 3 | Issue 3 | e1793