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Difference between revisions of "Talk:Chicco 2022 MitoFit"

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:::: Abbreviations: 'N-pathway = NADH-producing pathway'
:::: Abbreviations: 'N-pathway = NADH-producing pathway'
::::::* It is important to distinguish a single step from the pathway. The dehydrogenase step produces NADH, the CI-catalyzed step consumes NADH. The pathway produces (in case of complete oxidation) CO<sub>2</sub> and H<sub>2</sub>O. Respiratory N-pathway capacities are measured at (pseudo-) steady states, when O<sub>2</sub> flux is constant over time. Under these conditions, the N-pathway keeps the redox sate of the NADH + NAD<sup>+</sup> pool constant. As such, the N-pathway is not a NADH-producing pathway.
::::::* It is important to distinguish a single step from the pathway. The dehydrogenase step produces NADH, the CI-catalyzed step consumes NADH. The pathway produces (in case of complete oxidation) CO<sub>2</sub> and H<sub>2</sub>O. Respiratory N-pathway capacities are measured at (pseudo-) steady states, when O<sub>2</sub> flux is constant over time. Under these conditions, the N-pathway keeps the redox sate of the NADH + NAD<sup>+</sup> pool constant. As such, the N-pathway is not a NADH-producing pathway.
== Reply by Adam Chicco ==
:::: As for the abstract, it has been difficult for me to weave together the ‘Rotenone paradox’ with the malate and malonate studies given the strict word limit – but if we are allowed some flexibility here, I have presented a revised version below my responses for your review. 
:::: 1. It is trivial that the S-pathway – particularly with GM and PGM – is not fully substrate-deprived, and malonate inhibits the contribution of the S-pathway which is supported by incomplete TCA-intermediate depletion. Does malonate inhibition of P(G)M-supported respiration address the rotenone paradox?
:::: It is important to realize that in our manuscript we only begin with presentation of the “rotenone paradox” in liver mitochondria (which we can all agree involves oxaloacetate inhibition of CII) as a means of introducing the broader question of how succinate production/oxidation interacts with N-pathway substrate oxidation to influence JO2 in common SUIT protocols.  We extend the “paradox” observation in liver mt by showing that rotenone elicits a less-than-expected inhibition of NS-supported JO2 in muscle and cardiac mitochondria (based on the N-supported JO2 prior addition of S).  We hypothesize that this involves an interaction of malate concentration (and downstream oxaloacetate) on succinate oxidation even in the absence of exogenous succinate (i.e., during N-supported JO2).  This leads to our malate and malonate experiments, which further extend the observations of the tissue-specific interactions of malate concentration (via fumarate and oxaloacetate) with succinate oxidation in the N-pathway state.  These interactions are at the heart of the “rotenone paradox”, and extend its relevance beyond a curious observation in liver mitochondria.
:::: While these concepts may be obvious to us, I don’t think it they trivial for the majority of HRR users in the field.  I know many scientists who assume that either:  1) JO2 supported by N-substrates exclusively involves electron flow to Q through CI (due to fully inhibitory effects of malate-fumarate on CII), or 2) JO2 supported by N-substrates involves full TCA cycle flux, just like the text books show.  Even if we acknowledge that the S-pathway is “not fully substrate deprived with P(G)M”, I think the variation of this effect across tissues and its dependence on small mM changes in malate concentration merit broad publication and discussion in our field. 
:::: 1. Increasing external malate inhibits the S-pathway by product inhibition (fumarate) of the S-pathway. With Zuzana Sumbalova we presented in several MiPevents the data (unfortunately still not fully published) showing that increasing malate concentrations increase the PM- and GM-pathway capacity while inhibiting the S-pathway contribution (in the absence of malonate). This was published by John Lemasters long ago. This S-pathway inhibition is competitive and is prevented by high succinate concentrations. But then we switch from the N-pathway to the NS-pathway.
:::: Yes, I have seen some of these abstracts (I have cited Komlodi 2017 in the MS, but please others so I can include them all in the revised MS and Bioblast talk), and I am aware of these observations extending at least as far back as Harris and Manger in 1968 (also cited in the MS).  I would never argue that we are uncovering a new biochemical phenomenon here - only attempting to bring it to light for further consideration and discussion among current and future HRR users.- and what better platform for this than BEC and Bioblast 2022!  In addition, we have many new experiments that I have not presented that extend these studies by targeting oxaloacetate handling more directly  (e.g., via mPEPCK inhibition), which are alluded to in the manuscript as potential areas of future experimentation.
:::: I hope these responses make sense and provide a better foundation for understanding my revised abstract.
:::: Best wishes, Adam

Latest revision as of 11:58, 26 May 2022

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Comments by the Editor on Introduction and Figure 1

Gnaiger E 2022-05-23
Title and Abstract: 'Multi-substrate respirometry protocols'
  • The term 'multi-substrate respirometry protocols' does not include inhibitors (rotenone) and uncouplers, both of which are important in the present respirometric protocols. For these, the term substrate-uncoupler-inhibitor-titration protocols has been introduced, which is more appropriate than 'multi-substrate respirometry protocols'.
Abstract: 'rotenone (a selective CI inhibitor) is utilized in the presence of N+S substrates to deduce the contribution of N-pathway flux to the total (N+S-pathway) JO2. .. elucidating the biological bases for variations in NS-pathway flux in multi-substrate respirometry protocols.'
  • The nomenclature in the abstract is confusing. What is the difference between "N+S-pathway JO2" and "NS-pathway flux"? - The NS-pathway is defined as the pathway with convergent electron input into the Q-junction from N- and S-substrates. N+S is the algebraic sum of the separate N- and S-pathway fluxes. The combined and summed pathway capacities are equal (NS = N+S) only in the special case of complete additivity (Gnaiger 2020, Chapters 6 and 7).
Abstract: 'under S- and some N+S pathway states, rotenone elicits a paradoxical increase in JO2, ..'
  • In the S-pathway state (without rotenone), an increase of JO2 elicited by rotenone is by no means paradoxical, but is classically expected, if oxaloacetate (inhibitor of CII) — accumulating in mitochondria without malate-anaplerotic capacity — can leak out of the mt-matrix such that its concentration is lowered after inhibiting its production by rotenone through redox-coupled NADH (product) inhibition of MDH.
Introduction: 'monitoring the effect of selective CI or CII substrates ..'
  • NADH is the substrate for CI. Monitoring its selective effect requires application of NADH autofluorescence, which is not addressed in the present manuscript.
Introduction: 'High-resolution respirometry (HRR) enables stepwise evaluation of oxygen consumption rates (JO2) in mitochondrial preparations to titrations of anaplerotic substrates that feed the TCA cycle at different sites (Gnaiger 2020).'
  • The term 'anaplerotic substrates' is too narrow to describe the stepwise evaluation of JO2. For instance, succinate is not an anaplerotic substrate when it is oxidized to fumarate and malate, and malate is - in the presence of rotenone - transported out of the mt-matrix. This has been shown most rigorously by a calorimetric enthalpy balance study in rat liver mitochondria (Gnaiger et al 2000).
Introduction: 'NAD-dependent enzymes'
  • Replace by 'NAD+-dependent enzymes'
Introduction and Figure 1: 'A simplistic view of this convergence predicts that electron flow from N-pathway substrates sum with succinate (S-pathway) electrons to account for 100 % of the integrated JO2 resulting from TCA cycle flux and related oxidation reactions upstream (Figure 1B).'
  • It is difficult to interpret this sentence with its emphasis on 'TCA cycle flux and related oxidation reactions upstream'. The integrated NS-pathway flux may be controlled upstream (of Q) and downstream (of Q). If 'electron flow from N-pathway substrates sum with succinate (S-pathway) electrons' has the meaning of the two entry contributions to the integrated pathway flux, then this sentence is not a simplistic view but a definition of convergent (integrated) NS-JO2. However, if 'electron flow from N-pathway substrates sum with succinate (S-pathway) electrons' has the meaning of the algebraic sum of the separate N-JO2 and S-JO2 (= N+S), then this view (explicit in Figure 1C) contradicts most experimental results (see Table 7.2 summarizing muscle mitochondria; Gnaiger 2020). Figure 1C should be replaced by a figure that shows the prevailing incomplete additivity. A more recent account of incomplete additivity is given in Komlodi et al (2021).
Figure 1:
  • There is no reference to the coupling state - is it LEAK, OXPHOS, or ET? The coupling state matters. In case of ET excess capacity, additivity in the OXPHOS state is different from the additivity in the ET state. Additivity in the LEAK state is an entirely different topic. Is the 'simplistic' view simply a view without concern of the basics of mitochondrial physiology?
Introduction: 'substrate-control factors to describe the relative contributions of N- and S-pathway across a range of tissue and conditions (Gnaiger 2020).
  • Substrate control factors do not provide information on the relative contributions of the N- and S-pathways to NS-JO2, except in the singular case of complete additivity. It is not clear how this statement can be derived from Gnaiger (2020).
TCA (text) and CAC (Figures 1 and 2).
  • What is the difference? Why use two different abbreviations?
Abbreviations: 'N-pathway = NADH-producing pathway'
  • It is important to distinguish a single step from the pathway. The dehydrogenase step produces NADH, the CI-catalyzed step consumes NADH. The pathway produces (in case of complete oxidation) CO2 and H2O. Respiratory N-pathway capacities are measured at (pseudo-) steady states, when O2 flux is constant over time. Under these conditions, the N-pathway keeps the redox sate of the NADH + NAD+ pool constant. As such, the N-pathway is not a NADH-producing pathway.

Reply by Adam Chicco

As for the abstract, it has been difficult for me to weave together the ‘Rotenone paradox’ with the malate and malonate studies given the strict word limit – but if we are allowed some flexibility here, I have presented a revised version below my responses for your review.
1. It is trivial that the S-pathway – particularly with GM and PGM – is not fully substrate-deprived, and malonate inhibits the contribution of the S-pathway which is supported by incomplete TCA-intermediate depletion. Does malonate inhibition of P(G)M-supported respiration address the rotenone paradox?
It is important to realize that in our manuscript we only begin with presentation of the “rotenone paradox” in liver mitochondria (which we can all agree involves oxaloacetate inhibition of CII) as a means of introducing the broader question of how succinate production/oxidation interacts with N-pathway substrate oxidation to influence JO2 in common SUIT protocols. We extend the “paradox” observation in liver mt by showing that rotenone elicits a less-than-expected inhibition of NS-supported JO2 in muscle and cardiac mitochondria (based on the N-supported JO2 prior addition of S). We hypothesize that this involves an interaction of malate concentration (and downstream oxaloacetate) on succinate oxidation even in the absence of exogenous succinate (i.e., during N-supported JO2). This leads to our malate and malonate experiments, which further extend the observations of the tissue-specific interactions of malate concentration (via fumarate and oxaloacetate) with succinate oxidation in the N-pathway state. These interactions are at the heart of the “rotenone paradox”, and extend its relevance beyond a curious observation in liver mitochondria.
While these concepts may be obvious to us, I don’t think it they trivial for the majority of HRR users in the field. I know many scientists who assume that either: 1) JO2 supported by N-substrates exclusively involves electron flow to Q through CI (due to fully inhibitory effects of malate-fumarate on CII), or 2) JO2 supported by N-substrates involves full TCA cycle flux, just like the text books show. Even if we acknowledge that the S-pathway is “not fully substrate deprived with P(G)M”, I think the variation of this effect across tissues and its dependence on small mM changes in malate concentration merit broad publication and discussion in our field.
1. Increasing external malate inhibits the S-pathway by product inhibition (fumarate) of the S-pathway. With Zuzana Sumbalova we presented in several MiPevents the data (unfortunately still not fully published) showing that increasing malate concentrations increase the PM- and GM-pathway capacity while inhibiting the S-pathway contribution (in the absence of malonate). This was published by John Lemasters long ago. This S-pathway inhibition is competitive and is prevented by high succinate concentrations. But then we switch from the N-pathway to the NS-pathway.
Yes, I have seen some of these abstracts (I have cited Komlodi 2017 in the MS, but please others so I can include them all in the revised MS and Bioblast talk), and I am aware of these observations extending at least as far back as Harris and Manger in 1968 (also cited in the MS). I would never argue that we are uncovering a new biochemical phenomenon here - only attempting to bring it to light for further consideration and discussion among current and future HRR users.- and what better platform for this than BEC and Bioblast 2022! In addition, we have many new experiments that I have not presented that extend these studies by targeting oxaloacetate handling more directly (e.g., via mPEPCK inhibition), which are alluded to in the manuscript as potential areas of future experimentation.
I hope these responses make sense and provide a better foundation for understanding my revised abstract.
Best wishes, Adam