HSL Null Mice Figure 7. Increased mitochondrial size in adipocytes from WAT of HFD-fed HSL mice. (A) Representative electron microscope (TEM) images of periovarial WAT from 10–12 months old wildtype (I) and HSL mice (II) fed HFD for 6 months. (I)a shows a low-magnification overview image with details from the boxed-in areas shown in (I)b, c and d. For the HSL mice, (II)b is the overview image and (II)a, c, d and e are the details from the boxed-in areas. Black arrows indicate small lipid droplets in the cytoplasm, black short arrow indicates caveolae, black arrowhead indicates ER, white arrows indicate cytoplasm, white arrowheads indicate mitochondria and n indicates the nucleus. (B) Representative TEM image of periovarial WAT from 10–12 months old wildtype and HSL mice fed HFD for 6 months. Black arrows indicate location of mitochondria. Insets show enlargement of representative mitochondria. (C) The diameter of mitochondria in WAT was determined using morphometry and based on 140 wildtype and 105 HSL mitochondria. Values given are mean6SEM, *** p,0.001, analyzed with Mann-Whitney U tests and multiple regression analysis. doi:10.1371/journal.pone.0001793.g007 Figure 8. Induction of UCP-1 in WAT of HSL mice. Levels of UCP-1 protein in the mitochondrial fraction analyzed with western blot in periovarial WAT from 9–10 months old, wildtype and HSL mice fed HFD for 6 months (n = 5). Values given are mean6SEM, ** p,0.01, analyzed with Mann-Whitney U tests. doi:10.1371/journal.pone.0001793.g008 Several different mechanisms may underlie the decreased adiposity of HSL mice, including the attainment of BAT characteristics of WAT demonstrated in this study. Another mechanism suggested to contribute to decreased fat stores of HSL mice is the dramatic reduction in reesterification of fatty acids in WAT, which in turn is due to the coordinated alteration in the expression of enzymes involved in lipogenesis [27]. It was suggested that the downregulation in reesterification was compen- satory to the reduced lipolytic activity, thus serving to uphold the plasma concentrations of fatty acids even in the absence of HSL. Also the upregulation of lipoprotein lipase observed in the fasted state in WAT of HSL mice, may be compensatory to the lack of HSL activity. However, ATGL, which together with HSL has been shown to account for 95% of triglyceride lipase activity of murine WAT [34], appears to be unchanged in WAT of HSL mice [35]. Yet another mechanism that may contribute to decreased adiposity of HSL mice is the described adipocyte cell death in WAT of HSL mice [25], which appears to be necrosis rather than apoptosis [25] (Figure 4C). Emergence of brown adipocytes in WAT depots has been associated to a lean phenotype in several transgenic mouse models [4,36–38]. Even so, the increased oxygen consumption in WAT of HSL is likely to contribute little to the increase in total energy expenditure, PLoS ONE | www.plosone.org 7 2008 | Volume 3 | Issue 3 | e1793