Fig. 3

Effect of methylene blue and NV118 on coupled respiration and lactate production in metformin-intoxicated human platelets. a Mitochondrial respiration was measured in human platelets with mitochondrial dysfunction induced by 60 min exposure to metformin (black circle; 50 mM). After subsequent addition of methylene blue (10 μM, black square) or the cell-permeable succinate prodrug NV118 (250 μM, black triangle), mitochondrial respiration due to coupled phosphorylation, here referred to as coupled respiration, was evaluated by addition of the ATP-synthase inhibitor oligomycin (1 μg/ml) to block the phosphorylation pathway, and calculated as the difference in respiration before and after the inhibition of the ATP-synthase. Subsequently, the complex III inhibitor antimycin A (1 μg/ml) followed by the complex IV inhibitor sodium azide (10 mM) were added. Control experiments were performed without the addition of oligomycin to account for background drift of oxygen consumption. A vehicle control to metformin was run with each experiment (white circle). b Lactate production of human platelets incubated with metformin (10 mM, black circle) was measured every 30 min over 4 h with or without co-treatment of methylene blue (10 μM, single dose, black square) or NV118 (250 μM, every 30 min, black triangle) starting at 60 min. A vehicle control was run with each experiment (white circle). Data are expressed as individual scatter plot and mean ± SD (a) or mean ± SD (b). All experiments were performed with n = 7, with exception of the control and metformin group of the lactate production assay run with methylene blue (n = 9) (b). One-way ANOVA with Dunnet post hoc test was used. ***p < 0.001, compared to metformin