Glycerol tri 9 10 n 3h oleate

For the determination of TG hydrolase activity, lysates of Cos-7 cells overexpressing human and murine ATGL and CGI-58, respectively, or mouse tissue extracts in a total volume of 100 μl buffer A, were incubated with 100 μl substrate and different concentrations of Atglistatin in a water bath at 37 °C for 60 min. As a control, incubations under identical conditions were performed in buffer A alone. After incubation, the reaction was terminated by adding 3.25 ml of methanol/chloroform/heptane (10/9/7; vol/vol/vol) and 1 ml of 0.1 M potassium-carbonate/0.1 M boric acid (pH 10.5). The mixture was intensively vortexed and centrifuged at 800 g for 10 min, 0.2 ml of the upper aqueous phase was collected and the radioactivity was measured by liquid scintillation counting (Tri-Carb 2100TR). TG substrate was prepared by emulsifying 330 μM triolein (40,000 c.p.m. nmol⁻¹ glycerol tri[9,10(n)-3H]-oleate (PerkinElmer)) and 45 μM phosphatidylcholine/phosphatidylinositol (3:1) in 100 mM potassium phosphate buffer (pH 7.0) by sonication and adjusted to 5% essentially FA-free BSA (Sigma, St Louis, MO).

Fresh isolated tissues from re-fed mice were weighed, minced with scissors and transferred to ice-cold tubes containing 1 ml of DMEM, 2% (wt/vol) FA-free BSA and 2 IU heparin. Tissues were incubated at 37 °C for 1 h before the mixture was centrifuged and media was collected to determine heparin-releasable LPL activity. The substrate consisted of 1.2 × 10⁶ c.p.m. glycerol tri[9,10(n)-3H]-oleate (PerkinElmer) and 1.04 μmol triolein per sample. Lipids were evaporated under a stream of nitrogen and emulsified by sonication (Bandelin SONOPLUS) in 0.1 M Tris/HCl (pH 8.6) and 0.1% Triton X-100. Forty microlitres of heat-inactivated human serum and 40 μl of 10% BSA were added to the substrate. To determine LPL activities, 200 μl of substrate were mixed with 100 μl of media (heparin-releasable LPL) and incubated in a water bath at 37 °C for 1 h. The reaction was stopped and analysed as described for TG hydrolase activity.