Fig. 3.

Effect of glucotoxicity and mTOR inhibition on the oxidized status, and mitochondrial superoxide accumulation of MIN6 cells. After a 72 h-exposure of the cells to medium containing 5 or 33 mM glucose and 0.1, 1 or 10 nM rapamycin, DCF fluorescence was measured. Rapamycin dose-dependently reduces the basal oxidized status of MIN6 cells under glucotoxic conditions (33 mM glucose) (a). The relative-to-basal ROS production was assessed by an acute glucose stimulation (15 mM glucose, added immediately after determining basal fluorescence, at t = 0 min) and monitored every 5 min over a total period of 60 min (b). The area under the curve (AUC) of the accumulated ROS during the first 5 min of the glucose stimulation was calculated, and shows that the decreased acute reactivity of cells exposed to glucotoxicity is not prevented by rapamycin (c). Mitochondrial superoxide levels after 72 h-culture in glucotoxic conditions are decreased compared to control regardless of the presence of rapamycin (d). MFI: mean fluorescence intensity. The circles indicate the number of independent experiments performed for each condition. *p < 0.05; **p < 0.01