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Reply to Figueira et al.: Can NAD(P)+ transhydrogenase (NNT) mediate a physiologically meaningful increase in energy expenditure by mitochondria during H2O2 removal?

  • Cody D. Smith
    Affiliations
    East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina, USA

    Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, USA
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  • Cameron A. Schmidt
    Affiliations
    East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina, USA

    Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, USA
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  • Kelsey H. Fisher-Wellman
    Affiliations
    East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina, USA

    Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, USA
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  • P. Darrell Neufer
    Correspondence
    For correspondence: P. Darrell Neufer
    Affiliations
    East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina, USA

    Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, USA
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Open AccessPublished:March 15, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100378
      In our recent publication (
      • Smith C.D.
      • Schmidt C.A.
      • Lin C.T.
      • Fisher-Wellman K.H.
      • Neufer P.D.
      Flux through mitochondrial redox circuits linked to nicotinamide nucleotide transhydrogenase generates counterbalance changes in energy expenditure.
      ), as well as an earlier publication from our lab (
      • Fisher-Wellman K.H.
      • Lin C.T.
      • Ryan T.E.
      • Reese L.R.
      • Gilliam L.A.
      • Cathey B.L.
      • Lark D.S.
      • Smith C.D.
      • Muoio D.M.
      • Neufer P.D.
      Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy-consuming redox circuit.
      ), we demonstrate under specific experimental conditions designed to increase flux through redox buffering circuits in mitochondria, and thus NADPH demand, that at least a portion of the accompanying increase in JO2 (i.e., proton conductance) is directly and reproducibly attributed to nicotinamide nucleotide transhydrogenase (NNT; e.g., Fig. 3D in (
      • Smith C.D.
      • Schmidt C.A.
      • Lin C.T.
      • Fisher-Wellman K.H.
      • Neufer P.D.
      Flux through mitochondrial redox circuits linked to nicotinamide nucleotide transhydrogenase generates counterbalance changes in energy expenditure.
      )). In their letter to the editor, Figueira et al. question whether flux through redox circuits linked to NNT can mediate a meaningful increase in energy expenditure. Their critique is based on two main points: 1) recent structural studies on NNT indicating a 1:1 stoichiometry of hydride transfer to NADPH generation, and 2) two prior studies that failed to show any difference in respiration in mitochondria with and without functional NNT (
      • Ronchi J.A.
      • Francisco A.
      • Passos L.A.
      • Figueira T.R.
      • Castilho R.F.
      The contribution of nicotinamide nucleotide transhydrogenase to peroxide detoxification is dependent on the respiratory state and counterbalanced by other sources of NADPH in liver mitochondria.
      ,
      • Parker N.
      • Crichton P.G.
      • Vidal-Puig A.J.
      • Brand M.D.
      Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria.
      ). Regarding the first point, we agree that recent structural studies of NNT (
      • Zhang Q.
      • Padayatti P.S.
      • Leung J.H.
      Proton-Translocating nicotinamide nucleotide transhydrogenase: A structural perspective.
      ), including a 2019 paper by Kampjut and Sazanov (
      • Kampjut D.
      • Sazanov L.A.
      Structure and mechanism of mitochondrial proton-translocating transhydrogenase.
      ) (a citation we regrettably omitted), provide compelling evidence of an H+ to hydride transfer reaction stoichiometry of 1:1, which is considerably different than the apparent stoichiometry observed in our functional studies. We fully acknowledged this point in the discussion of our paper. However, NNT function cannot be determined solely from structural data. There are a number of plausible mechanisms that could explain a different apparent stoichiometry in respiring mitochondria, including a “permissible” transfer of protons by NNT under certain conditions, akin to the proton conductance of ANT that is independent of ATP/ADP exchange. On the technical side, it is also possible that JNADPH production was in excess of that required for H2O2 detoxification and/or that the rate of H2O2 emission as measured underestimated that produced. We attempted to control for the latter using inhibitors to both glutathione (BCNU) and thioredoxin reductase (auranofin), but such inhibitors have their own caveats, including slightly inhibiting respiration (as pointed out by Figueira et al. with respect to Fig. 4D). Regarding the apparent inconsistencies with the previous studies referenced, respiration was assessed in the study by Ronchi et al. (
      • Ronchi J.A.
      • Francisco A.
      • Passos L.A.
      • Figueira T.R.
      • Castilho R.F.
      The contribution of nicotinamide nucleotide transhydrogenase to peroxide detoxification is dependent on the respiratory state and counterbalanced by other sources of NADPH in liver mitochondria.
      ) without accounting for potential differences in membrane potential. Identifying potential differences in proton conductance between experimental conditions requires comparing respiration at the same defined membrane potential. Parker et al. (
      • Parker N.
      • Crichton P.G.
      • Vidal-Puig A.J.
      • Brand M.D.
      Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria.
      ) did perform proton conductance assays in mitochondria with and without functional NNT, but not under conditions that would increase NNT activity (i.e., elevate NADPH demand). While any increase in proton conductance back into the mitochondrial matrix does represent, by definition, an increase in energy expenditure, we agree with Figueira et al. that more research is needed to define the contribution of flux through NNT-linked redox circuits to energy expenditure in vivo.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

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