Talk:Moreno-Sanchez 2022 MitoFit

 Comments by Gnaiger Erich (2022-06-12)

This manuscript makes a highly significant contribution to the metabolic study of cancer cells, by emphasizing the proper conversion of glycolytic lactate production and oxidative phosphorylation into equivalents of ATP turnover.

It may be trivial for the specialist but too little appreciated by non-metabolically oriented scientists, that what is termed ‘OXPHOS-linked’ ATP production includes substrate-level phosphorylation of ATP-equivalent GTP production, which is integrated into P»/O₂ ratios. The discussion of P»/O₂ ratios shifts between fundamental considerations of H⁺/P» ratios of F₁F_O ATPase ― Watt IN, Montgomery MG, Runswick MJ, Leslie AG, Walker JE (2010) Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. https://doi.org/10.1073/pnas.1011099107 would be a more appropriate reference than refs 32 - 34) ― and cancer-specific considerations of P»/O₂ ratios. It would be helpful to draw a line of distinction between these fundamental aspects and point out, what is or is not specific about properly measured P»/O₂ ratios in cancer cells.

General

1. "Likewise, OxPhos, as determined by the rate of cellular oxygen consumption in studies with cancer cells, is also usually over-estimated since the activity of several non-mitochondrial oxygen-consuming enzymes (heme oxygenase and cytochromes P450) are significantly over-expressed in cancer cells [7, 8], and the inner membrane mitochondrial H+ passive diffusion." – This is certainly true with reference to several low-quality publications. But well documented state-of-the-art SOPs should be mentioned and referred to: These points are known as baseline corrections: (1) baseline correction for Rox (https://doi.org/10.26124/bec:2020-0001.v1; and (2) baseline correction for L (https://doi.org/10.26124/bec:2020-0002 ). There are numerous other references which can be listed here. For obvious reasons, I mention merely the two BEC publications, which provide the most explicit rationale for these baseline corrections.

2. What is the difference between net OxPhos (oligomycin-sensitive oxygen consumption) and OxPhos? If you make this difference explicit, then the statement in #1 is incorrect: OxPhos, as determined by the rate of cellular oxygen consumption in studies with cancer cells, is also usually over-estimated .. If net OxPhos = R-L, then OxPhos = R’_tot according to the argument in #1. Consistent terminology helps to avoid ambiguities.

3. What is the difference between OxPhos and OXPHOS. It is not always clear, if OxPhos is simply used for the R-L net ROUTINE respiratory rate, or for O₂ flux converted to the rate of ADP→ATP phosphorylation. P» has been introduced for ADP→ATP, as in P»/O₂ ratio. See https://doi.org/10.26124/bec:2020-0001.v1

4. Why do you switch from O₂ to O? This is confusing and not necessary.

5. What is the difference between P/O ratio and ATP/O ratio?

6. Since Ref 9 is paywall, I have no access to it. I would like to see the basis of the P»/O₂ ratio of 3 for FADH₂-linked pathways.

7. ngAt O/min/mg – neither ngAt nor min are SI units.

8. Oligomycin: The only apparent drawback of this molecule is that it is a slow inhibitor .. – See Ruas JS, Siqueira-Santos ES, Rodrigues-Silva E, Castilho RF (2018) High glycolytic activity of tumor cells leads to underestimation of electron transport system capacity when mitochondrial ATP synthase is inhibited. https://doi.org/10.1038/s41598-018-35679-8 . There is a well documented second drawback, namely inhibition of ET capacity. In the most careful evaluations, uncoupler titrations are performed in controls in the absence of Omy (https://doi.org/10.1007/978-1-4939-7831-1_3 ). See https://www.bioblast.at/index.php/Doerrier_2018_Methods_Mol_Biol#Correction : “the use of 2.5 µM oligomycin may show an inhibitory effect on ET capacity in some biological samples (e.g. platelets)". Therefore, we recommend to (1) test the inhibitory effect of oligomycin on ET capacity (by uncoupler titrations in the absence of inhibitor) and, (2) evaluate lower oligomycin concentration (5-10 nM) to replace the 'standard' 2.5 µM oligomycin concentration. See more details in Oligomycin.

9. Table 1: nmol/min/mg

10. Figure 2: The right axis does not show metabolic O₂ flux, but the (negative) time derivative of O₂ concentration (‘O2 slope uncorr.’). The large range of the Y₂ axis is due to inclusion of the peak caused by the Dith titration (see Dithionite). Use Omy as abbreviation (https://doi.org/10.26124/bec:2020-0001.v1). At the high volume-specific fluxes the instrumental background correction exerts only a small artefact in the O2k, but flux should always been shown after background correction. Note the difference between baseline correction and background correction.

11. Symbols recommended in https://doi.org/10.26124/bec:2020-0001.v1 help to make equations more readable:

a. Change J_ATP cellular production = J_ATPglyc + J_ATPOxPhos to J_P» = J_P»glyc + J_P»OxPhos

b. Change J_{ATP consumption} to J_P«.

12. Sections 4.2 and 4.4: “Data of the present work on glycolysis and OxPhos suggest that many cancer cells have an oxidative type of metabolism, which implies that mitochondria play a central role in supporting ATP-dependent processes such as cancer cell migration, invasiveness, or perhaps proliferation.” - The discussion would gain balance by including references to the more recent literature that demonstrated ‘high OxPhos rate in cancer cells’. By quickly drawing on our own research, I list the following references on OXPHOS in cancer cells (not necessarily even including glycolysis):

a. Smolková K, Bellance N, Scandurra F, Génot E, Gnaiger E, Plecitá-Hlavatá L, Ježek P, Rossignol R (2010) Mitochondrial bioenergetic adaptations of breast cancer cells to aglycemia and hypoxia. https://doi.org/10.1007/s10863-009-9267-x

b. Kristiansen G, Hu J, Wichmann D, Stiehl DP, Rose M, Gerhardt J, Bohnert A, ten Haaf A, Moch H, Raleigh J, Varia MA, Subarsky P, Scandurra FM, Gnaiger E, Gleixner E, Bicker A, Gassmann M, Hankeln T, Dahl E, Gorr TA (2011) Endogenous myoglobin in breast cancer is hypoxia-inducible by alternate transcription and functions to impair mitochondrial activity: a role in tumor suppression? https://doi.org/10.1074/jbc.M111.227553

c. Schöpf B, Schäfer G, Weber A, Talasz H, Eder IE, Klocker H, Gnaiger E (2016) Oxidative phosphorylation and mitochondrial function differ between human prostate tissue and cultured cells. https://doi.org/10.1111/febs.13733

d. Schöpf Bernd, Weissensteiner Hansi, Schäfer Georg, Fazzini Federica, Charoentong Pornpimol, Naschberger Andreas, Rupp Bernhard, Fendt Liane, Bukur Valesca, Giese Irina, Sorn Patrick, Sant’Anna-Silva Ana Carolina, Iglesias-Gonzalez Javier, Sahin Ugur, Kronenberg Florian, Gnaiger Erich, Klocker Helmut (2020) OXPHOS remodeling in high-grade prostate cancer involves mtDNA mutations and increased succinate oxidation. https://doi.org/10.1038/s41467-020-15237-5

e. For many more cancer-mito-related references see https://wiki.oroboros.at/index.php/O2k-Publications:_Cancer

13. Conclusions: “One of the prevailing dogmas in cancer biology is that glycolysis is the principal energy supplier for cancer cells.” – Would it be more up-to-date to say: One of the dogmas prevailing in the past in cancer biology is that glycolysis is the principal energy supplier for cancer cells.

Minor

1. 2% or 37°C - SI recommends a space between numerals and units, including %. It should be 2 %. Or 37 °C.

2. Figure 1: Lactate was enzymatically determined by using commercial lactate dehydrogenase (LDH) following the NADH formation at λexc= 340 nm [11] or following the changes in the NADH fluorescent signal at λem= 450-460 nm and λexc= 340 nm (Figure 1A). – This should be largely moved to the methods section, which then can avoid some duplication.

3. Ref 9: https://doi.org/http://10.1002/biof.31 DOI not found.