We thank J

We thank J. comparable between WT and Tg mice, activity of hormone-sensitive lipase (HSL) was 40C50% less in Tg than WT mice under both feeding and fasting conditions, suggesting interference of PLIN2 with HSL. Mice generated through crossing of PLIN2-Tg mice and HSL-Tg mice showed cardiac-specific HSL overexpression and complete lack of steatosis. The results suggest that cardiac PLIN2 plays an important pathophysiological role in the development of dynamic steatosis and that the latter was prevented by upregulation of intracellular lipases, including HSL. = 3), as described above. The extracted lipids were dissolved and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis performed with an Agilent 1200 system (Agilent Technologies) coupled to a Finnigan LTQ Orbitrap XL (Thermo Fisher Scientific), as described previously (18). The resultant data were analyzed using LipidSearch (Mitsui Knowledge Industry) and SIEVE (Thermo Fisher Scientific) software. References for each annotated compound were searched for the KNApSAcK and KEGG database (1). Microscopy. Electron microscopy was performed as described previously using a transmission electron microscope (Hitachi H-7500) (45). For confocal microscopy, tissues were fixed with 4% paraformaldehyde/PBS for 1 h and embedded. The sections were stained with PLIN2-specific antibody, heat shock cognate protein of 70 kDa (hsc70; NB120-2788; Novus) and fluorescent secondary antibodies (ALEXA), or LipidTOX reagent (Thermo Fisher Scientific) for lipid staining. The sections were analyzed with a confocal microscope system TGFβRI-IN-1 (Leica TCS Sp2). Echocardiography. Cardiac function was studied by echocardiography in awake mice using ultrasonography equipped with a 13-MHz linear transducer (ALOKA), as previously described (41). Gene expression analysis. Total RNA was extracted from cardiac ventricles using TRIzol reagent (Invitrogen) and reverse-transcribed Rabbit Polyclonal to Tubulin beta using a Quantitect reverse transcription kit (Qiagen). The target genes were amplified and analyzed in triplicate using TaqMan probes (Applied Biosystems), as described previously (45). Cardiac uptake of energy sources. Tissue glucose uptake was analyzed by injecting d-[14C]deoxyglucose (GE Healthcare) via the tail vein, as described previously (41). Briefly, the mouse was injected with 0.1 Ci/g body wt of d-[14C]deoxyglucose and euthanized 40 min later. Plasma glucose was measured, and cardiac ventricles and liver were excised. The tissues were then dissolved in Solvable (Perkin-Elmer), and radioactivity was measured using a liquid scintillation counter. Tissue glucose uptake was calculated from tissue radioactivity and plasma-specific activity of the tracer. Tissue FA uptake was analyzed by injecting [125I]-methyl iodophenyl pentadecanoic acid ([125I]BMIPP; Nihon Medi-Physics) (41). Briefly, 0.1 Ci/g body wt of [125I]BMIPP was injected via the TGFβRI-IN-1 tail vein, and mice were euthanzied 20 min later. Cardiac ventricles and liver were excised, tissue radioactivity and plasma FA concentration were measured, and tissue FA uptake was calculated using the plasma-specific activity of the tracer. For tissue VLDL uptake, VLDL was obtained by ultracentrifugation from rabbits fed a high-cholesterol diet (0.5% wt/wt). TGFβRI-IN-1 The VLDL was radiolabeled with [125I] (GE Healthcare) using IODOBEASE (Pierce) and purified as described previously (41). The mice were injected with 120,000 countsmin?1g body wt?1 [125I]VLDL via the tail vein and euthanized 20 min later. Cardiac ventricles and livers were collected, and VLDL uptake was calculated from tissue radioactivity and plasma-specific activity of the tracer. Lipase assays. Hearts were homogenized in 20 mM Tris and 1 mM EDTA, pH 7.4, containing 255 mM sucrose, 1 M leupeptin, and 0.1 M okadaic acid and centrifuged, and the supernatant was used for lipase assays, as described previously (41). ATGL activity was measured using a specific ATGL inhibitor atglistatin TGFβRI-IN-1 (Cayman Chemical) (29). Atglistatin was dissolved in DMSO at a concentration of 10 mM, and 5 l was added in the reaction mixture. Ninety-five microliters of the supernatant was incubated at 37C for 30 min in 200 l of a reaction mixture containing 105 M [3H]trioleoylglycerol (99.4 Ci/mol), 23.7 M lecithin, and 5 mM sodium taurocholate in 100 mM potassium phosphate buffer (pH 7.4) in the absence (DMSO only) or presence of 250 M atglistatin. ATGL activity was determined by subtracting the activity with atglistatin from that with DMSO only. Tissue homogenates of WT adipose tissue and hearts of homozygous ATGL-KO mice were included in the assay as positive and negative controls, respectively. HSL activity was determined as neutral cholesteryl ester hydrolase (NCEH) activity using 100 l of the heart samples and a micellar substrate, including cholesteryl-[14C]oleate, as described previously (23). Because hearts of HSL-KO mice have virtually no NCEH activity (41), cardiac NCEH activity represents the activity of HSL..