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Cited by 78Mitochondrial Function and PathologyCancer, Hypoxia, and MetabolismMetabolism, Diabetes, and Cancer

Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells

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Ruas, Juliana S., Siqueira-Santos, Edilene S., Amigo, Ignacio, Rodrigues-Silva, Erika, Kowaltowski, Alicia J., Castilho, Roger F.·2016·PLOS ONE
DOI 10.1371/journal.pone.0150967PMC PMC4780810
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Abstract

The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen consumption preceded by inhibition of oxidative phosphorylation by oligomycin. In the present study, human glioma (T98G and U-87MG) and prostate cancer (PC-3) cells were titrated with different concentrations of the protonophore CCCP to induce maximal oxygen consumption rate (OCR) within respirometers in a conventional growth medium. The results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. In the presence of oligomycin, the spare respiratory capacity (SRC), i.e., the difference between the maximal and basal cellular OCR, was underestimated by 25 to 45%. The inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. Further experiments indicated that oligomycin cannot be replaced by the adenine nucleotide translocase inhibitors bongkrekic acid or carboxyatractyloside because, although these compounds have effects in permeabilized cells, they do not inhibit oxidative phosphorylation in intact cells. We replaced CCCP by FCCP, another potent protonophore and similar results were observed. Lower maximal OCR and SRC values were obtained with the weaker protonophore 2,4-dinitrophenol, and these parameters were not affected by the presence of oligomycin. In permeabilized cells or isolated brain mitochondria incubated with respiratory substrates, only a minor inhibitory effect of oligomycin on CCCP-induced maximal OCR was observed. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. The inhibitory effect of an ATP synthase blocker on potent protonophore-induced maximal OCR may be associated with impaired metabolism of mitochondrial respiratory substrates.

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  • 1 μg/mL oligomycin causes a minor nonspecific effect on mitochondrial and cellular function.

    “the use of this oligomycin concentration is common in the literature and causes a minor nonspecific effect on mitochondrial and cellular function [15,25,26,27].”

    From Proliferating Astrocytes in Primary Culture Do Not Depend upon Mitochondrial Respiratory Complex I Activity or Oxidative Phosphorylation (2023) · cites this paper as [26]

    The Results report that oligomycin markedly inhibited CCCP-stimulated oxygen consumption in intact T98G cells, with spare respiratory capacity underestimated by 43.7 ± 2.3%; this is contrary to describing its effect as minor. The Discussion says effects on nonspecific targets are possible, not that a minor nonspecific effect was established. The paper reserves its finding of a minor inhibitory effect for permeabilized cells or isolated mitochondria. (LLM, conf=0.87)

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  • 1 μg/mL oligomycin (10 times more than 0.1 μg/mL) elicits minor nonspecific effects and is common in the literature.

    “However, we used 10 times more oligomycin in our experiments since this oligomycin concentration elicits minor nonspecific effects and it is common in the literature [15,25,26,27].”

    From Proliferating Astrocytes in Primary Culture Do Not Depend upon Mitochondrial Respiratory Complex I Activity or Oxidative Phosphorylation (2023) · cites this paper as [26]

    The Methods report using 1 μg/mL oligomycin, and the Introduction says oligomycin is widely used in these assays, though it does not establish that this particular concentration is common. The Results and the section on alternative protocols report substantial inhibition of maximal oxygen consumption in intact tumor cells, not minor nonspecific effects; the reported minor effect applies to isolated brain mitochondria. (LLM, conf=0.87)

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Cited by

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Authors
Ruas, J. S., Siqueira-Santos, E. S., Amigo, I., Rodrigues-Silva, E., Kowaltowski, A. J., & Castilho, R. F.
Affiliations
  • Universidade Estadual de Campinas (UNICAMP) (BR)
  • Universidade de São Paulo (BR)
Corresponding author
  • Ruas, Juliana S.
  • Rodrigues-Silva, Erika
  • Kowaltowski, Alicia J.
  • Castilho, Roger F.
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2016-03-07
Volume
11
Issue
3
Pages
e0150967
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0150967
PMC
PMC4780810
OpenAlex
W2294314809
Cited by
78
References
5
Citations by year
2026: 7 · 2025: 7 · 2024: 12 · 2023: 5 · 2022: 8 · 2021: 15 · 2020: 13 · 2019: 2 · 2018: 3 · 2017: 5 · 2016: 1
FWCI
1.57
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (publishedVersion, other-oa)
  • Americanae (AECID Library) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Fundação de Amparo à Pesquisa do Estado de São Paulo (10/51906-1, 11/50400-0, 10/51906, 13/07937-, 13/07937-8)
  • Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (13/07937-8)
  • Conselho Nacional de Desenvolvimento Científico e Tecnológico
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
Keywords
mitochondrial electron transport chain, oligomycin, VO2max, spare respiratory capacity, protonophores, CCCP, FCCP, 2,4-dinitrophenol, oxidative phosphorylation inhibition, ATP synthase inhibitors, citreoviridin, bongkrekic acid, carboxyatractyloside, glioma cells, prostate cancer cells
MeSH terms
Electron Transport, Humans, Mitochondria, Oligomycins, Oxidative Phosphorylation, Oxygen Consumption, Cell Respiration, Cell Line, Tumor

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