Fig. 4.

Mitochondrial membrane potential, superoxide generation, and manganese superoxide dismutase (MnSOD) in WT and Akita^+/Ins2-derived β-cells. A: to detect mitochondrial membrane potential, WT and Akita^+/Ins2−-derived β-cells were treated with 100 nM tetramethylrhodamine methyl ester perchlorate (TMRM) for 30 min. FCCP and oligomycin controls were included to depolarize and hyperpolarize mitochondrial membrane potential, respectively. Results are expressed as mean arbitrary fluorescence units (AFU) ± SE; n = 3/group. B: WT and Akita^+/Ins2-derived β-cells were treated with 300 nM MitoSOX Red for 15 min and imaged using confocal microscopy (×100) to detect mitochondrial superoxide levels. The bar graph shows the arbitrary levels of mitochondrial superoxide detected in these cells per unit area. Results are means ± SE; n = 3/group. C: cell lysates from WT and Akita^+/Ins2-derived β-cells were resolved by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes, and probed for MnSOD. Western blot loading was controlled for by protein staining, and band intensities were quantified using AlphaView SA software. Results are expressed as mean fold change arbitrary units (AU) over WT β-cells ± SE; n = 3/group. D: MnSOD activity was determined in cell lysates using the cytochrome c reduction method. Results are expressed as mean fold change activity normalized to total protein over WT β-cells ± SE; n = 3/group. *P < 0.05 compared with WT β-cells; #P < 0.05 compared with respective controls.