WAT and brown adipose tissue (BAT), expressed also in steroidogenic tissues, skeletal muscle, macrophages and pancreatic b-cells. A unique property of HSL is its broad substrate specificity, with efficient hydrolysis of a large panel of substrates, including acylglycerols, cholesteryl esters [12] and retinyl esters (RE) [13]. In recent years several strains of HSL mice have been generated and characterized independently in several laboratories [14–17]. Although differences between the HSL models exist, several common features have been reported. First, and most unexpect- edly, HSL mice are not obese. In fact, these mice have unchanged or reduced adiposity when fed a normal chow diet (ND) [14,15,17] and exhibit a remarkable resistance to diet- induced obesity [18] when provoked with a long-term high-fat diet (HFD) regimen. Decreased expression levels of adipogenic transcription factors and lipogenic enzymes suggested impairment of adipogenesis in HSL mice, as also demonstrated in mice lacking both HSL and leptin [19]. A second striking feature that is shared between the existing HSL models is male infertility due to oligospermia, demonstrating that HSL expression is necessary for normal spermatogenesis. With regard to the control of glucose homeostasis in HSL animals conflicting data have been reported for both insulin sensitivity and insulin secretion. The strain of HSL mice investigated in the present study exhibits mild insulin resistance that is almost fully compensated by hypersecretion of insulin [17] and no signs of impairment of glucose-stimulated insulin secretion (GSIS) [20]. Insulin resistance in HSL mice could be a result of ectopic fat deposition, as increased amounts of intramyoceullular lipid droplets in soleus muscles [21] as well as increased amounts of triglycerides in liver [17,18], have been observed in HSL mice. However, in contrast to the observations in our strain of HSL mice, increased insulin sensitivity has been reported for two other strains of HSL mice [22,23] and Roduit et al. reported elevated basal insulin secretion and blunted GSIS [24]. The reason for these discrepancies remains to be resolved, but differences in nutritional state, gender, age, experimental setup, genetic background and intestinal microflora of the animals are likely to be contributing factors. Here we extend the phenotypic characterization of HSL mice and demonstrate that WAT of HSL mice attains BAT characteristics. The expression levels of markers that are characteristic of brown adipocytes are elevated, whereas markers of white adipocytes are decreased. Functionally, WAT of HSL mice exhibits increased oxygen consumption and increased uncoupling activity. Although the underlying mechanisms remain to be resolved, these data identify HSL as a determinant in the decision of cell fate during adipocyte differentiation. Results HSL mice are resistant to diet-induced obesity As already reported in an independently generated strain of HSL mice [18], our strain of HSL mice displayed a significant reduction in both body weight (Figure 1A) and WAT mass (Figure 1B,C) compared to wildtype mice following a 6- month HFD regimen. This was not due to decreased food intake (data not shown), impaired intestinal lipid absorption (Figure 1D) or increased physical activity (Figure 1E and [21]) as has been shown also for another HSL mouse model [18]. In fact, after 5 months of HFD feeding, the caloric intake per total body weight was increased by 40% in HSL mice compared to wildtype littermates (data not shown). Also, the lipid content in faeces from HFD-fed HSL mice was significantly lower than that of wildtype littermates (Figure 1D). Computer tomography (CT) scanning showed that the reduction in WAT mass in HSL HSL Null Mice mice was more dramatic for subcutaneous depots, which were virtually absent following HFD feeding, than for visceral depots (Figure 2). Plasma leptin levels in the mice on control diet were similar for the two genotypes (data not shown). After 24 weeks on HFD, wildtype mice showed an expected large increase in leptin levels, whereas this increase was absent in the HSL mice (Figure 3A). The plasma adiponectin level was dramatically lowered (0.41, p,0.01) already after 3 weeks of HFD feeding, decreasing even more (0.09, p,0.01) after 22 weeks of HFD, in the HSL mice compared to wildtype littermates (Figure 3B). This decrease was also observed in HSL mice fed a control diet (data not shown), although the effect was more pronounced following HFD feeding. Decreased expression of adipogenic markers in WAT of HFD-fed HSL mice Using light microscopy the size of periovarial WAT adipocytes was estimated (Figure 4A). In the control diet groups, the median adipocyte size, measured as cell area, was similar for the two genotypes (2774648 and 2568668 mm2 for wildtype and HSL null, respectively), and although an increased heterogeneity in cell size could be seen in adipocytes from HSL mice (Figure 4A, panel I and III), the size distribution pattern for the two genotypes was similar with a large peak in frequency seen at 1000– 3000 mm2 (Figure 4B). After HFD feeding, adipocytes from wildtype WAT showed the characteristic large and general increase in size (Figure 4A, panel II), reflected in a shift of the curve to the right, with the highest frequency at 7000–10000 mm2 (median area 6880686 mm2) (Figure 4B). This was not observed to the same extent in adipocytes from HSL mice, which instead exhibited an even greater size heterogeneity than that observed on the normal diet, with a markedly reduced median cell area compared to wildtype mice (31436127 mm2) (Figure 4A, panel IV) (Figure 4B). In WAT of HSL mice, HFD feeding also promoted the appearance of an increased number of eosinophilic-like cells, reflecting an inflammatory state of the tissue, as previously shown [25,26]. The inflammatory state was accompanied by macrophage infiltration and syncytia formation, supporting a necrotic-like cell death as also observed by immunohistochemistry [25]. This is further confirmed in the present study, showing no difference in caspase 3/7 activity between the two genotypes in either feeding category (Figure 4C). As already shown in independently generated HSL mouse strains [18,27], the mRNA expression levels of PPARc and C/ EBPa were several fold decreased in WAT from HFD-fed HSL male and female mice compared to wildtype littermates (data not shown). The downregulation of PPARc expression was confirmed at the protein level (0.21, p,0.05) using western blot analysis (Figure 5A). Stearoyl CoA Desaturase 1 (SCD-1), the expression of which is known to be positively correlated to the differentiation state of the adipocyte [28], was severely downreg- ulated at the protein level (0.027, p,0.01) in WAT from HSL mice (Figure 5B). HFD-fed HSL mice have increased energy expenditure and increased oxygen consumption in WAT Indirect calorimetry measurements revealed a significant increase in O2 consumption (Figure 6A) and CO2 production (Figure 6B) in HFD-fed HSL mice compared to wildtype littermates. No difference was seen between animals fed the control diet (data not shown). PLoS ONE | www.plosone.org 2 2008 | Volume 3 | Issue 3 | e1793