NADPH supply and the contribution of NAD(P)+ transhydrogenase (NNT) to H2O2 balance in skeletal muscle mitochondria

Tiago R Figueira; Annelise Francisco; Juliana A Ronchi; Guilherme R R M Dos Santos; William Dos Santos; Jason R Treberg; Roger F Castilho

doi:10.1016/j.abb.2021.108934

NADPH supply and the contribution of NAD(P)⁺ transhydrogenase (NNT) to H₂O₂ balance in skeletal muscle mitochondria

Arch Biochem Biophys. 2021 Aug 15:707:108934. doi: 10.1016/j.abb.2021.108934. Epub 2021 May 25.

Authors

Tiago R Figueira¹, Annelise Francisco², Juliana A Ronchi², Guilherme R R M Dos Santos², William Dos Santos³, Jason R Treberg⁴, Roger F Castilho²

Affiliations

¹ Department of Pathology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Rua Tessália Vieira de Camargo, 126, 13083-887, Campinas, SP, Brazil. Electronic address: figueirat@usp.br.
² Department of Pathology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Rua Tessália Vieira de Camargo, 126, 13083-887, Campinas, SP, Brazil.
³ Department of Biological Sciences, University of Manitoba, General Office 212B Bio-Sci Bldg., R3T 2N2, Winnipeg, MB, Canada.
⁴ Department of Biological Sciences, University of Manitoba, General Office 212B Bio-Sci Bldg., R3T 2N2, Winnipeg, MB, Canada; Centre on Aging, University of Manitoba, Winnipeg, MB, Canada.

PMID: 34043997
DOI: 10.1016/j.abb.2021.108934

Abstract

H₂O₂ is endogenously generated and its removal in the matrix of skeletal muscle mitochondria (SMM) is dependent on NADPH likely provided by NAD(P)⁺ transhydrogenase (NNT) and isocitrate dehydrogenase (IDH2). Importantly, NNT activity is linked to mitochondrial protonmotive force. Here, we demonstrate the presence of NNT function in detergent-solubilized and intact functional SMM isolated from rats and wild type (Nnt^+/+) mice, but not in SMM from congenic mice carrying a mutated NNT gene (Nnt^-/-). Further comparisons between SMM from both Nnt mouse genotypes revealed that the NADPH supplied by NNT supports up to 600 pmol/mg/min of H₂O₂ removal under selected conditions. Surprisingly, SMM from Nnt^-/- mice removed exogenous H₂O₂ at wild-type levels and exhibited a maintained or even decreased net emission of endogenous H₂O₂ when substrates that support Krebs cycle reactions were present (e.g., pyruvate plus malate or palmitoylcarnitine plus malate). These results may be explained by a compensation for the lack of NNT, since the total activities of concurrent NADP⁺-reducing enzymes (IDH2, malic enzymes and glutamate dehydrogenase) were ~70% elevated in Nnt^-/- mice. Importantly, respiratory rates were similar between SMM from both Nnt genotypes despite differing NNT contributions to H₂O₂ removal and their implications for an evolving concept in the literature are discussed. We concluded that NNT is capable of meaningfully sustaining NADPH-dependent H₂O₂ removal in intact SMM. Nonetheless, if the available substrates favor non-NNT sources of NADPH, the H₂O₂ removal by SMM is maintained in Nnt^-/- mice SMM.

Keywords: Antioxidant; C57BL/6J; Krebs cycle; Oxidative stress; Redox balance.

MeSH terms

Animals
Hydrogen Peroxide* / metabolism
Isocitrate Dehydrogenase / genetics
Isocitrate Dehydrogenase / metabolism
Male
Mice
Mice, Inbred C57BL
Mice, Knockout
Mitochondria, Muscle* / enzymology
Mitochondria, Muscle* / metabolism
Mitochondrial Proteins
Muscle, Skeletal* / enzymology
Muscle, Skeletal* / metabolism
NADP Transhydrogenase, AB-Specific / genetics
NADP Transhydrogenase, AB-Specific / metabolism
NADP Transhydrogenases* / genetics
NADP Transhydrogenases* / metabolism
NADP* / metabolism
Rats

Substances

NADP
Hydrogen Peroxide
NADP Transhydrogenases
Nnt protein, mouse
Isocitrate Dehydrogenase
NADP Transhydrogenase, AB-Specific
Mitochondrial Proteins