Figure 5.

DIMT1 and mitochondrial dysfunction in EndoC-βH1 cells. Representative immunoblots (A) and densitometric analyses (B) of mitochondrial complex protein I–V. Data are mean ± SD (n = 3). Glucose-induced (1 and 20 mM glucose) hyperpolarization of the inner mitochondrial membrane as shown by TMRM (C); average traces (thick lines) in cells treated with scramble siRNA compared to the difference in maximal hyperpolarization with FCCP in DIMT1-silenced cells (thin lines) are shown (scale bars: 20 μm) (D; data are mean ± SD; n = 3). E, cytosolic ATP/ADP ratios determined by Perceval HR (green fluorescence); average traces and the difference in the maximal rise in ATP/ADP ratio with oligomycin, (scale bars: 20 μm). (F; data are mean ± SD; n = 3). G, mitochondrial OCR is shown upon stimulation with 10 mM pyruvate as scramble cells (black lines) and as DIMT1-silenced cells (gray lines). The pyruvate-stimulated respiratory response, proton leak, ATP production and maximal mitochondrial respiration with FCCP and nonmitochondrial respiration (antimycin+rotenone) are each expressed as fold relative to basal (H–M; data are mean ± SD; n = 4). Statistical differences were compared by Student's t test and extreme studentized deviate test was used to determine outliers. ∗p < 0.03 or <0.05 (as indicated), ∗∗p < 0.01 and ∗∗∗p < 0.001. DIMT1, dimethyladenosine transferase 1; TMRM, tetramethyl-rhodamine methyl ester.