Figure 7.

Loss of the Rimonabant-induced lipolysis and HSL translocation in adipocytes by ongoing lipid synthesis. Primary rat adipocytes, which had been metabolically labelled with -((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoic acid (NBD-FA), were pretreated (20 min, 37 °C) in the absence (Control) or presence of 10 µM Rimonabant (RIM). Thereafter, 500-µL aliquots of the incubation mixtures were transferred into 2-mL Eppendorf vials and then subjected to centrifugation (500x g, 2 min). Subsequently, the infranatant was removed by suction and the adipocytes were suspended in 2 mL of fresh incubation medium lacking Rimonabant. After two additional flotation cycles, the washed adipocytes were incubated (37 °C) in medium harboring 10 mM glucose and 0.1 nM insulin in the absence or presence of cytochalasin B (10 µM; CytB) or triacsin C (30 µM; TriC) for the periods indicated. Subsequently, the adipocytes were washed by flotation, resuspended and then incubated (15 min, 37 °C). Upper panel: The amounts of NBD-FA and glycerol released into the incubation medium were measured by fluorometry and enzymatically, respectively. Mean ± SD of two different adipocyte preparations with incubations/measurements in triplicate. Middle and lower panels: The amounts of HSL associated with LD, which were prepared from the homogenates of the adipocytes by centrifugation through sucrose cushions, were determined. For this, proteins contained in the top fraction were extracted from the lipids, then separated by SDS-PAGE and analyzed for HSL by immunoblotting. The images of a typical experiment with LD preparations and electrophoretic runs in triplicate with quantitative evaluations (mean ± SD) are shown. * p ≤ 0.05 for Rimonabant-treated adipocytes between absence and presence of TriC or CytB; no significant differences for control adipocytes.