High glycolytic activity of tumor cells leads to underestimation of electron transport system capacity when mitochondrial ATP synthase is inhibited

Abstract

This study sought to elucidate how oligomycin, an ATP synthase blocker, leads to underestimation of maximal oxygen consumption rate (maxOCR) and spare respiratory capacity (SRC) in tumor cells. T98G and U-87MG glioma cells were titrated with the protonophore CCCP to induce maxOCR. The presence of oligomycin (0.3–3.0 µg/mL) led to underestimation of maxOCR and a consequent decrease in SRC values of between 25% and 40% in medium containing 5.5 or 11 mM glucose. The inhibitory effect of oligomycin on CCCP-induced maxOCR did not occur when glutamine was the metabolic substrate or when the glycolytic inhibitor 2-deoxyglucose was present. ATP levels were reduced and ADP/ATP ratios increased in cells treated with CCCP, but these changes were minimized when oligomycin was used to inhibit reverse activity of ATP synthase. Exposing digitonin-permeabilized cells to exogenous ATP, but not ADP, resulted in partial inhibition of CCCP-induced maxOCR. We conclude that underestimation of maxOCR and SRC in tumor cells when ATP synthase is inhibited is associated with high glycolytic activity and that the glycolytic ATP yield may have an inhibitory effect on the metabolism of respiratory substrates and cytochrome c oxidase activity. Under CCCP-induced maxOCR, oligomycin preserves intracellular ATP by inhibiting ATP synthase reverse activity.

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

“…2 A depicts traces of OCR obtained under standard conditions in the absence of 2-DG. As previously reported [1] , [2] , [3] and shown in Fig. 1 A, OCR max was underestimated in the presence of oligomycin ( Fig.…”

“… Data format Raw data analyzed and processed. Parameters for data collection Oxygen consumption rate and mitochondrial membrane potential were analyzed in suspended intact cells [1 , 2] . Measurements were performed under basal conditions, after ATP synthase inhibition by oligomycin, and under mitochondrial respiratory uncoupling by CCCP.…”

“… Data source location Laboratory of Bioenergetics and Cell Metabolism Faculty of Medical Sciences, University of Campinas Campinas - State of São Paulo Brazil Data accessibility The raw data are available in Mendeley Data repository. Data identification number: doi: 10.17632/33ybs25n7m.1 Direct URL to data: data.mendeley.com/datasets/33ybs25n7m/1 Related research articles [1 , 2] J.S. Ruas, E.S.…”

Lactate supports a metabolic-epigenetic link in macrophage polarization

“…As shown in fig. S3I, as compared to oligomycin A treatment, which blocks ATP production ( 40 ), ATP levels were not significantly affected by depletion of glucose or lactate. To determine whether inhibition of mitochondrial lactate metabolism with UK-5099 treatment and/or exogenous acetate supplementation affects cellular bioenergetics, we next conducted an ATP assay and found no discernable changes in ATP levels with UK-5099 in contrast to the depletion of ATP following oligomycin A pretreatment (fig.…”

“…S4B). Notably, to limit generalized metabolic dysfunction, minimal concentrations of oligomycin A were used to inhibit ATP synthase ( 40 ). Given that inhibition of cellular bioenergetics with oligomycin A induced significant generalized cellular dysfunction in BMDMs, in contrast to UK-5099 treatment, this suggests that while lactate oxidation is likely occurring, the predominant contribution of mitochondrial lactate metabolism in IL-4–induced M2 gene expression is for lactate-derived acetyl-CoA production and subsequent ACLY-dependent histone acetylation.…”