Fig. 6

Effect of okadaic acid on the activation of key metabolic enzymes by Amaryl in rat adipocytes and diaphragms. Isolated rat adipocytes or diaphragms were treated with 100 nM okadaic acid for 5 min at 30°C prior to incubation with increasing concentrations of Amaryl or tolbutamide for 30 min. For determination of glycerol-3-phosphate acyltransferase (GPAT) activity, total microsomes isolated from the cell homogenates were incubated with [³H]glycerol-3-phosphate for 3 min at 25°C. For determination of glycogen synthase (GS) activity, a post-mitochondrial supernatant prepared from the cell homogenates was incubated with [¹⁴C]UDP-glucose for 10 min at 30°C. The activities were calculated from the radiolabeled acylglycerides in the butanol phase for GPAT and radiolabeled glycogen in the ethanol precipitate for GS as described previously (42,136). The specific activity (difference between presence and absence of enzyme source) in the absence of drug was set at 0% in each case. Each point represents the means ± standard deviation (SD) of (at least) three independent experiments.