HSL Null Mice Figure 4. Morphological changes in WAT of HFD-fed HSL mice. (A) Representative morphology images of epididymal WAT from 10– 11 months old wildtype (I) and HSL (III) mice fed control diet and wildtype (II) and HSL (IV) mice fed HFD for 6 months. (B) The cell area was determined using morphometry and was based on a total of 268 cells in wildtype control, 477 cells in HSL control, 922 cells in wildtype HFD and 775 cells in HSL HFD. (C) Caspase 3/7 activity was investigated in the infranatant fraction of WAT from 10–12 months old female mice fed a control diet or a HFD for 6 months (n = 7–12). Values given are mean6SEM. doi:10.1371/journal.pone.0001793.g004 PCR. To confidently monitor the mRNA expression in adipocytes, without contribution from other cell types present in adipose tissue, these analyses were performed on isolated adipocytes obtained from animals that had been on HFD for 4 weeks. At this early time point in the study it is possible to obtain a representative population of adipocytes following collagenase treatment of adipose tissue, whereas this is difficult following a 6-month HFD regimen due to the presence of very large adipocytes that are more sensitive to collagenase treatment. As shown in Figure 9A, the UCP-1 mRNA levels were found to be increased 3.5-fold (p,0.01) in white adipocytes of HSL mice compared to wildtype mice. Also, carnitine palmitoyl transferase 1 (CPT1), the rate-limiting enzyme for long-chain acyl-CoA transport into mitochondria, known to be more highly expressed in brown than in white adipocytes [29], showed a higher expression in HSL than in wildtype white adipocytes (2.2-fold, p,0.05) (Figure 9B). A functional reflection of this could be the increased oxidation of palmitate observed in epididymal and periovarial isolated white adipocytes of 6 months old HSL mice fed a normal chow diet (28.21064.616 pmol CO2/ h/ml packed cell volume (PCV), n = 3) compared to wildtype littermates (9.44363.260 pmol CO2/h/ml PCV, n = 3, p,0.05), in the fed state. The mRNA levels of pRb, shown to act as a molecular switch determining white versus brown adipocyte differentiation [5], and RIP140, which also promotes differentiation into the white adipocyte lineage [9], were decreased by 36 and 40% (p,0.01), respectively, in white adipocytes of HSL mice compared to wildtype littermates (Figure 9C,D). The expression of PGC-1a, on the other hand, was not significantly different between the two genotypes (1.17, p = 0.36) (Figure 9E). In order to study the functional consequences of the increased UCP-1 expression, oxygen consumption was investigated on isolated WAT from HFD-fed mice. Basal oxygen consumption PLoS ONE | www.plosone.org 5 2008 | Volume 3 | Issue 3 | e1793