FIG. 4.

MitoSOX Red and MitoB responses in the presence of various inhibitors of carriers and metabolism, on silencing ME1, pyruvate carboxylase, or aspartate/glutamate carriers of the malate/aspartate shuttle. Mitochondrial matrix superoxide release rates J_m (A–C, H, J) and changes in 2 h-accumulation of H₂O₂/ROS (D–G, I, K, L) on transition between 3 and 25 mM glucose: (A, D) in the presence of RR (green) (7 μM), etomoxir (100 μM) (yellow bars), and CGP37157 (10 μM) (light green bar); (B, E) on blockage of components of the malate/aspartate shuttle (dark red bars), that is, silencing of aspartate/glutamate antiporters SLC25A12/AGC1/aralar and SLC25A13/AGC2. (C, F) PPP blockage using 1 mM 6AN or 40 μM oxythiamine (blue bars); (C, G) pyruvate carboxylase silencing (red/orange bars); for scrambled siRNA cf. (J, K); (H, I) assays with CitC (10 mM BTC; orange bars) and 2OGC inhibitor (5 mM n-butylmalonate; yellow-green bars); and (J–L) assays on silencing cytosolic malic enzyme (“siRNA ME1”) (dark blue or aquamarine bars) alone or together with CitC (“si ME1+ si CitC”) as compared with scrambled siRNA (gray bars). J_m rates obtained in the presence of 25 mM glucose (J²⁵_m) were normalized either to rates obtained in 11 mM glucose before GSIS (J¹¹_m0), rates obtained in 3 mM glucose before GSIS (J³_m0), or their respective initial glucose concentration before glucose addition (J_m^{before Glc addition}). ROS accumulation was normalized to 3 mM glucose. All preincubations in 3 mM glucose before the assay were performed for 2 h. ANOVA (n = 3–6): **p < 0.05; ***p < 0.001; Student's t-test: ^###p < 0.001. 6AN, 6-aminonicotinamide; PPP, pentose-phosphate pathway; RR, ruthenium red. Color images are available online.