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

HO 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 HO removal under selected conditions. Surprisingly, SMM from Nnt mice removed exogenous HO at wild-type levels and exhibited a maintained or even decreased net emission of endogenous HO 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 HO removal and their implications for an evolving concept in the literature are discussed. We concluded that NNT is capable of meaningfully sustaining NADPH-dependent HO removal in intact SMM. Nonetheless, if the available substrates favor non-NNT sources of NADPH, the HO removal by SMM is maintained in Nnt mice SMM.