HSL Null Mice Figure 5. Impairment of adipogenesis in WAT of HFD-fed HSL mice. Protein levels of PPARc (A) and SCD-1 (B), analyzed with western blot in WAT from 10–12 months old female, wildtype and HSL mice fed HFD for 6 months (n = 4–6). Values given are mean6SEM, * p,0.05, ** p,0.01, analyzed with Mann-Whitney U tests. doi:10.1371/journal.pone.0001793.g005 was almost 3-fold increased (p,0.001) in WAT from HSL mice compared to wildtype littermates (Figure 10A). The level of electron transport chain uncoupling in WAT from the two genotypes was investigated by adding the chemical uncoupler FCCP. Addition of FCCP increased the oxygen consumption by almost 50% in WAT from HFD-fed wildtype mice, whereas the same uncoupler had almost no effect in WAT from HFD-fed HSL mice (Figure 10B). Taken together, these data suggest that WAT of HSL mice fed a HFD attains BAT characteristics that are functionally manifested as increased oxygen consumption and increased uncoupling activity. Discussion Resistance to diet-induced obesity, accompanied by impaired adipogenesis, has been reported previously in two independently generated lines of HSL mice [18,19,30] and confirmed here in our line of HSL mice. Furthermore, we have used indirect calorimetry to demonstrate that HFD-fed HSL mice indeed exhibit increased energy expenditure, as previously suggested by the observed fasting-induced weight loss and increased body temperature [18]. A novel and key finding of our paper is the demonstration of attainment of BAT characteristics of WAT of HSL mice. There are several manifestations of acquisition of BAT character- istics in WAT of HSL mice. First, already macroscopically WAT of HSL mice has a more brownish appearance than WAT from wildtype littermates. Second, the expression of pRb and RIP140, which directly and indirectly, respectively, promote differentiation into the white versus the brown adipocyte lineage (for a review see [8,31–33]), is downregulated in white adipocytes from HSL mice. Third, the expression of UCP-1 and CPT1 is increased in adipocytes from classical WAT depots of HSL mice compared to wildtype littermates. Fourth, and most importantly, oxygen consumption and uncoupling activity are increased in WAT of HSL mice. In BAT, on the other hand, there were no signs of increased oxygen consumption in HFD-fed HSL mice (data not shown). The increased size of mitochondria of white adipocytes may be an additional manifes- tation of attainment of brown adipocyte features in white adipocytes, although cristae appeared less dense in cells. Figure 6. HFD-fed HSL mice have increased energy expenditure. Indirect calorimetry measurements of 10 months old wildtype and HSL male mice fed HFD for 6 months. Whole body oxygen consumption (A) and carbon dioxide production (B) measured for 2 h. Data from the last hour is presented (n = 7). Values given are mean6SEM, ** p,0.01, analyzed with Mann-Whitney U tests. doi:10.1371/journal.pone.0001793.g006 PLoS ONE | www.plosone.org 6 2008 | Volume 3 | Issue 3 | e1793