A dataset describing glycolytic inhibitors overcoming the underestimation of maximal mitochondrial oxygen consumption rate in oligomycin-treated cells

Abstract

Determination of oxygen consumption is one of the most valuable methodologies to evaluate mitochondrial (dys)function. Previous studies demonstrated that a widely used protocol, consisting of adding the ATP synthase inhibitor oligomycin before mitochondrial respiratory uncoupling by sequential addition of a protonophore (e.g., carbonyl cyanide 3-chlorophenyl hydrazone [CCCP]), may lead to underestimation of maximal oxygen consumption rate (OCR_max) and spare respiratory capacity (SRC) parameters in highly glycolytic tumor cell lines. In this dataset, we report the effects of the glycolytic inhibitors 2-deoxy-D-glucose, iodoacetic acid, and lonidamine on overcoming the underestimation of OCR_max and SRC in oligomycin-treated cells. We propose a protocol in which 2-deoxy-D-glucose is added after oligomycin and just before the sequential addition of CCCP to avoid underestimation of OCR_max and SRC parameters in A549, C2C12, and T98G cells. The oxygen consumption rates were determined in intact suspended cell lines using a high-resolution oxygraph device. The data can be used in several fields of research that require characterization of mitochondrial respiratory parameters in intact cells.

Keywords: 2-Deoxi-D-glucose; Mitochondria; Mitochondrial membrane potential; Oligomycin; Oxygen consumption; Spare respiratory capacity; Tumor cell.

Fig. 1

Effects of glycolytic inhibitors on the underestimation of maximal mitochondrial oxygen consumption rate (OCR_max) and spare respiratory capacity (SRC) in oligomycin-treated cells. T98G cells (1 × 10⁶ /mL) were resuspended in a standard incubation medium in the presence or absence of glycolytic inhibitors. A and B: Effects of the glycolytic inhibitors 40 mM 2-deoxy-D-glucose (2-DG), 200 µM iodoacetic acid (IAA), and 400 µM lonidamine (LON) on OCR_max (A) and SRC (B), determined in the presence and absence of 1 µg/mL oligomycin (Oligo). DMSO (the oligomycin solvent) was present at a final concentration of 0.025% (v/v). **P < 0.01, statistically significant difference versus respective DMSO group; ^##P < 0.01, statistically significant difference versus respective group without glycolytic inhibitors; two-way ANOVA/Bonferroni post-hoc test.

Fig. 2

Representative traces of OCR determinations in T98G cells in the presence or absence of 2-DG. T98G cells (1 × 10⁶ /mL) were resuspended in standard incubation medium. Oligomycin (1 µg/mL; Oligo; red traces), 0.025% (v/v) DMSO (blue traces), 40 mM 2-DG, and CCCP (400 nM each addition) were added where indicated by the arrows.

Fig. 3

2-DG effects on the determination of OCR_max and SRC in T98G cells. T98G cells (1 × 10⁶ /mL) were resuspended in standard incubation medium, and 0.025% (v/v) DMSO, 1 µg/mL oligomycin (Oligo), or 40 mM 2-DG were added to the experiments as indicated. A and B: 2-DG effects on OCR_max and SRC, respectively. C: OCR_max inhibition when cells were incubated in the presence of double-optimal CCCP concentrations. D: CCCP concentrations to reach OCR_max. **P < 0.01, statistically significant difference versus respective DMSO group; ^#P < 0.05 and ^##P < 0.01, statistically significant difference versus respective group without 2-DG; two-way ANOVA/Bonferroni post-hoc test (A-C) or Kruskal-Wallis test/Dunn's post-hoc test (D).

Fig. 4

2-DG effects on the determination of OCR_max and SRC in A549 cells. A549 cells (1.25 × 10⁶ /mL) were resuspended in standard incubation medium, and 0.025% DMSO (v/v), 1 µg/mL oligomycin or 40 mM 2-DG were added to the experiments as indicated. A and B: 2-DG effects on OCR_max and SRC. C: OCR_max inhibition when cells were incubated in the presence of double-optimal CCCP concentrations. D: CCCP concentrations to reach OCR_max. **P < 0.01, statistically significant difference versus respective DMSO group; ^#P < 0.05 and ^##P < 0.01, statistically significant difference versus respective group without 2-DG; two-way ANOVA/Bonferroni post-hoc test (A-C) or Kruskal-Wallis test/Dunn's post-hoc test (D).

Fig. 5

2-DG effects on the determination of OCR_max and SRC in C2C12 cells. C2C12 cells (1 × 10⁶ /mL) were resuspended in standard incubation medium, and 0.025% (v/v) DMSO, 1 µg/mL oligomycin or 40 mM 2-DG were added to the experiments as indicated. A and B: 2-DG effects on OCR_max and SRC. C: OCR_max inhibition when cells were incubated in the presence of double-optimal CCCP concentrations. D: CCCP concentrations to reach OCR_max. **P < 0.01, statistically significant difference versus respective DMSO group; ^#P < 0.05 and ^##P < 0.01, statistically significant difference versus respective group without 2-DG; two-way ANOVA/Bonferroni post-hoc test (A-C) or Kruskal-Wallis test/Dunn's post-hoc test (D).

Fig. 6

Simultaneous determination of OCR and mitochondrial membrane potential (∆Ψ) in T98G cells: 2-DG and oligomycin effects. T98G cells (1.5 × 10⁶/mL) were resuspended in standard incubation medium containing 500 nM TMRM and 1 µM TPB⁻. DMSO (0.025%, v/v), 1 µg/mL oligomycin (Oligo) or 40 mM 2-DG were added to the experiments as indicated. A and B: Graphic correlation of OCR and ∆Ψ values obtained in the absence and presence of 2-DG, respectively. The OCR data were normalized to the respective basal OCR with DMSO (OCR_basal = 1.0). ∆Ψ is expressed as ∆F/F, where F is the fluorescent intensity after the last CCCP-addition (i.e., completely dissipated ∆Ψ) and ∆F is F minus any given fluorescent intensity. C and D: The left ordinate axes represent the effects of DMSO and Oligo on normalized OCR_max (OCR_max/OCR_basal). The right ordinate axes represent ∆Ψ (∆F/F) values when OCR_max was reached in the presence of DMSO or Oligo. Cells were incubated in the absence (A and C) or presence (B and D) of 2-DG. E: Inhibition percentage of OCR_max when ∆Ψ was dissipated (≈0). **P < 0.01, statistically significant difference versus respective DMSO group, ^##P < 0.01, statistically significant difference versus respective group without 2-DG; unpaired Student's t-test (C and D) or two-way ANOVA/Bonferroni post-hoc test (E).