Abstract
The forward reaction of nicotinamide nucleotide transhydrogenase (NNT) reduces NADP(+) at the expense of NADH oxidation and H(+) movement down the electrochemical potential across the inner mitochondrial membrane, establishing an NADPH/NADP(+) ratio severalfold higher than the NADH/NAD(+) ratio in the matrix. In turn, NADPH drives processes, such as peroxide detoxification and reductive biosynthesis. In this study, we generated a congenic mouse model carrying a mutated Nnt(C57BL/6J) allele from the C57BL/6J substrain. Suspensions of isolated mitochondria from Nnt(+/+), Nnt(+/-), and Nnt(-/-) mouse liver were biochemically evaluated and challenged with exogenous peroxide under different respiratory states. The respiratory substrates were also varied, and the participation of concurrent NADPH sources (i.e. isocitrate dehydrogenase-2, malic enzymes, and glutamate dehydrogenase) was assessed. The principal findings include the following: Nnt(+/-) and Nnt(-/-) exhibit ∼50% and absent NNT activity, respectively, but the activities of concurrent NADPH sources are unchanged. The lack of NNT activity in Nnt(-/-) mice impairs peroxide metabolism in intact mitochondria. The contribution of NNT to peroxide metabolism is decreased during ADP phosphorylation compared with the non-phosphorylating state; however, it is accompanied by increased contributions of concurrent NADPH sources, especially glutamate dehydrogenase. NNT makes a major contribution to peroxide metabolism during the blockage of mitochondrial electron transport. Interestingly, peroxide metabolism in the Nnt(+/-) mitochondria matched that in the Nnt(+/+) mitochondria. Overall, this study demonstrates that the respiratory state and/or substrates that sustain energy metabolism markedly influence the relative contribution of NNT (i.e. varies between nearly 0 and 100%) to NADPH-dependent mitochondrial peroxide metabolism.
Highlights
The coenzymes nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) are soluble electron carriers that are reduced in oxidative reactions during the catabolism of energy substrates in the cytosol and mitochondria
Despite their structural similarity, reduced NAD and NADP are used to drive rather different processes in the cells [1]. Given their roles as specific electron donors in diverse metabolic pathways, it is interesting that the redox states of NAD and NADP are linked to each other in the mitochondria of many organisms because the enzyme nicotinamide nucleotide transhydrogenase (NNT)4 catalyzes the transfer of redox potential between these two coenzymes, reducing one at the expense of the oxidation of the other [2]
The mating between C57BL/6/JUnib and C57BL/6J mice, which are, respectively, homozygous wild type and mutated for Nnt alleles [16], followed by successive backcrossing of the heterozygous mutated Nnt mice with C57BL/6/JUnib provided a congenic experimental model to study the relative contribution of NNT to mitochondrial peroxide metabolism at the expense of NADPH
Summary
The mating between C57BL/6/JUnib and C57BL/6J mice, which are, respectively, homozygous wild type and mutated for Nnt alleles [16], followed by successive backcrossing of the heterozygous mutated Nnt mice with C57BL/6/JUnib provided a congenic experimental model to study the relative contribution of NNT to mitochondrial peroxide metabolism at the expense of NADPH. Based on pilot studies and the data shown, a bolus addition of 30 M t-BOOH (a load of 30 nmol/mg) was used in most experiments Using this concentration of t-BOOH, wild-type liver mitochondria generally recover the fully reduced state of NAD(P)H in ϳ2.5 min and are capable of withstanding repeated t-BOOH challenges when respiring on malate/pyruvate as substrates. NAD(P)H fluorescence decay in NntϪ/Ϫ mitochondria is an indication that the rate of t-BOOH metabolism may not be limited by NAD(P) redox state when exogenous isocitrate is supporting a high NADPH supply via IDH2. In this condition, the peroxidase/reductase systems involved in t-BOOH metabolism could become a limiting factor. The respiratory control is the ratio between ADP-stimulated and the non-phosphorylating respiratory rates
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