osmiumtetroxide and epon embedded. Semi-thin sections (4 mi- crons) were cut to confirm longitudinal fiber orientation by light microscopy. Subsequently, ultrathin (80–90 nm) sections were cut and stained and contrasted with uranyl acetate and lead-citrate. Then, sections were examined in a Philips CM100 transmission electron microscope (Philips, Eindhoven, The Netherlands). Per muscle 15–20 images were grabbed and analyzed by an experienced electron microscopist blinded for the conditions. Using the NIH image analysis program all individual lipid droplets were identified, counted and encircled to compute lipid droplet area and derived morphometric parameters. Frequency distribu- tions were computed and plotted using Excel for Windows. DAG measurement Total lipids were extracted from frozen muscle using the method of Folch et al. [23]. The extracts were filtered and lipids recovered in the chloroform phase. DAG was isolated using thin- layer chromatography on Silica Gel 60 A plates developed in petroleumbenzin-diethyl ether-acetic acid (120:25:1.5 by volume) and visualized by rhodamine 6G. The DAG band was scraped from the plate and methylated using a mixture of toluene- methanol- (BF3/methanol 14%) (20%-55%-25% by volume), as described by Morrison and Smith [24]. The methylated fatty acids were extracted with hexane and analyzed by capillary gas liquid chromatography using a 50 m 60.25 mm CP-sil 88 silica column (varian) with helium as carrier gas at a flow of 130 kPa. The column oven was maintained at 165uC for 10 min and increased at a rate of 5uC/min to 190uC. This temperature was maintained for 15 min. Then temperature was increased to 230uC for 22 min with a flow rate of 2uC/min. Fatty acid methyl esters were identified by comparing retention times to those of known standards. Inclusion of an internal standard, 19:0 (dinonadecanoic acid), and an average molecular mass for each fatty acid methyl ester permits quantification of the amount of DAG in the sample, expressed as micromolar per gram of wet tissue weight. Ceramide measurement Ceramide content was determined as previously described (25) with slight modifications. Briefly, 50 ml of muscle homogenate was extracted with 600 ml of CHCl3/MeOH 1/2 (vol/vol). The extract was centrifuged for 10 min at 14,000 g and the pellet discarded. 500 ml of CHCl3/MQ-H2O 1/1.5 (vol/vol) was added, mixed, and centrifuged for 3 min at 14,000 g to separate the phases. The lower phase was collected and the upper phase re-extracted with 400 ml of CHCl3. The combined lower phases were dried under N2 flow, taken up in 500 ml of freshly prepared 0.1 M NaOH in MeOH, and deacetylated in a microwave (SAM-155, CEM corp.) for 60 min. 50 ml of this solution was derivatized with 25 ml o- phtaldehyde (OPA) reagent. The OPA-derivatized lipids were separated by HPLC and ceramides were quantified as previously described, using C17 sphinganine as an internal standard [25]. Western blotting Muscle samples were homogenized as described previously [26] and processed for standard SDS-PAGE and Western blotting. Protein concentration was assessed, and equal amounts of protein were loaded per lane. Actin was used as a loading control. Membranes were incubated with antibodies against OXPAT (GP31, Progen, Heidelberg, Germany), OXPHOS (MS601; MitoSciences, Eugene, OR, USA), PGC1a (516557; Calbiochem; VWR International BV, Amsterdam, The Netherlands) and mUCP3 [27], ADRP (GP40, Progen), ATGL (2138; Cell Signalling Technology; Bioke´; Leiden, The Netherlands) and CGI58 (NB110- 41576; Novus Biologicals, Littleton, CO, USA). Blots incubated DGAT1 Induced Lipid Turnover with OXPAT, OXPHOS, PGC1a and mUCP3 were probed with IRDye800-conjugated or IRDye700-conjugated secondary anti- bodies (Rockland, Gilbertsville, PA, USA and LICOR Biosciences, Westburg, Leusden, The Netherlands), and bands at a molecular weight corresponding to the control samples were quantified using the Odyssey infrared imaging system (LICOR Biosciences, Water- ingen, the Netherlands). Blots detecting ADRP, ATGL and CGI58 were probed with the appropriate horse radish-conjugated antibodies and after incubation specific protein bands were visualized by chemiluminescence and analyzed by Chemidoc XRS system (Bio-Rad, Veenendaal, The Netherlands). Statistics Results are presented as means 6 SEM. Differences between legs within one rat were analysed by paired samples t-test. Between groups, differences were analysed with an independent t-test. The accepted level of statistical significance was p,0.05 for all analyses. Al calculations were done using the Statistical Package for the Social Sciences (SPSS 16.0 software). Results Body mass Body mass gradually increased over the three weeks of dietary intervention in both the CHOW- and HFD-fed rats. However, this short-term dietary intervention did not result in differences in body mass between diets (300610 g and 290610 g in HFD- vs. CHOW-fed rats, p = 0.70). DGAT1 overexpression, intramyocellular lipid levels One week after electroporation, immunohistochemistry revealed a widely distributed DGAT1 overexpression in the muscle fibres of left TA muscle of rats; both in rats receiving the HFD as well as the CHOW. The mean green intensity of images from the DGAT1 electroporated leg was significantly higher than observed in the sham-electroporated contralateral control leg (25.7762.09 AU vs. 8.3261.68 AU in HFD-DGAT1 vs. HFD-control, p,0.001, and 25.7363.17 AU vs. 7.3761.79 AU in CHOW-DGAT1 vs. CHOW-control, p,0.001), indicating significant overexpression of DGAT1. The DGAT1 overexpression was associated with a ,52% increase in intramyocellular lipids in the HFD-DGAT1 overex- pressing leg and a ,66% increase in muscle triglyceride content in the CHOW-DGAT1 overexpressing leg, compared to control legs of the animals (p = 0.018, HFD-DGAT1 vs. HFD-control leg, and p = 0.044, CHOW-DGAT1 vs. CHOW-control leg, figure 1). Diacylglycerol content To determine whether the increased IMCL content in TA muscle overexpressing DGAT1 was accompanied by a reduction of DAG, we measured the latter in the legs overexpressing DGAT1 as well as in their empty vector control legs (figure 2). Three weeks of high-fat feeding was associated with a ,75% increase in DAG content (p = 0.039). Remarkably, overexpressing DGAT1 led to a ,30% increase in DAG content in the HFD- DGAT1 overexpressing leg and a ,35% increase in DAG content in CHOW-DGAT1 overexpressing leg, compared to control legs of the animals (p = 0.073, HFD-DGAT1 vs. HFD-control leg, and p = 0.045, CHOW-DGAT1 vs. CHOW-control leg). Ceramide content Ceramide content was measured in TA muscle overexpressing DGAT1 and in the contralateral empty vector electroporated legs to determine whether the increase in IMCL content is linked to changes in muscle ceramide levels (figure 3). Compared the PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e14503