The energetic cost of NNT-dependent ROS removal

Abstract

Under conditions of high nutrient availability and low ATP synthesis, mitochondria generate reactive oxygen species (ROS) that must be removed to avoid cell injury. Among the enzymes involved in this scavenging process, peroxidases play a crucial role, using NADPH provided mostly by nicotinamide nucleotide transhydrogenase (NNT). However, scarce information is available on how and to what extent ROS formation is linked to mitochondrial oxygen consumption. A new study by Smith et al. shows that NNT activity maintains low ROS levels by means of a fine modulation of mitochondrial oxygen utilization.

The rate of human energy expenditure fluctuates, increasing during periods of weight gain and decreasing during weight loss, to prevent large swings in body weight. Central to this ability are mitochondrial redox circuits responsible for nutrient oxidation and reactive oxygen species (ROS) generation. The redox circuits coupling the partial reduction of oxygen with ROS removal are linked with the major redox circuit represented by the complete reduction of oxygen into water at the level of the electron transport chain (ETC). This link enables cells to respond to changes in nutrient availability and energy demand. A better understanding of how these circuits are intertwined could lead to new therapeutic avenues for the treatment of metabolic disorders. New work by Smith et al. (1) reveals a mechanism by which mitochondria sense excess energy supply—particularly when energy demand is low—via ETC. This mechanism dependent on nicotinamide nucleotide transhydrogenase (NNT) couples the maintenance of low ROS levels with an increased oxygen consumption (i.e. energy expenditure).

Within the inner mitochondrial membrane (IMM), the energetically favorable flow of electrons from NADH(H⁺) and FADH₂ to oxygen allows proton pumping from the matrix to the intermembrane space. The resulting proton gradient (Δp) is utilized for ATP synthesis, as well as for other mitochondrial processes, such as ion homeostasis, protein import, etc.

A minor fraction of the electrons flowing through the ETC is diverted, causing the partial reduction of O₂ into superoxide, which is subsequently converted to H₂O₂ (2, 3). This electron detour is favored when flow is slowed down by a decrease in ATP demand. The carnitine-dependent β-oxidation of fatty acids exacerbates this situation, as it can increase NADH(H⁺) and FADH₂ availability, promoting mitochondrial ROS formation. This potentially deleterious process is counterbalanced by efficacious ROS removal systems. In particular, H₂O₂ reduction into water is catalyzed by peroxidases using reduced GSH and thioredoxin (Trx). The resulting oxidized forms of GSH and Trx are reduced by the reductases catalyzing NADPH(H⁺) oxidation. Various cytosolic and mitochondrial enzymes are involved in restoring the high NADPH(H⁺)/NADP⁺ ratio required not only for an optimal redox balance, but also for many anabolic processes. NNT, a ubiquitously expressed integral protein of the IMM (4), plays a major role among the enzymes contributing to NADP⁺ reduction.

NNT functions as a redox-driven proton pump catalyzing the reversible reduction of NADP⁺ to NADPH(H⁺) at the expense of NADH(H⁺) oxidation into NAD⁺. Much of our current knowledge on the role of NNT derives from studies on the mouse strain C57BL/6J (B6J) displaying a markedly lower NNT protein expression as compared with the control B6N strain (5). The lack of NNT curtails NADPH(H⁺) availability and thus peroxidase activities leading to oxidative stress. A severe increase in mitochondrial ROS levels has been linked to various pathologies. On the other hand, a slight increase in mitochondrial ROS formation appears to contribute to endogenous defense mechanisms against cell injury (6). Because NNT is relevant for maintaining NADPH availability necessary for the peroxidase activities required for buffering H₂O₂, the following question arises: To what extent does NNT activity link mitochondrial ROS production resulting from excess substrate availability with mitochondrial oxygen consumption?

Dr. Neufer's group investigated whether an increase in H₂O₂ production due to high rates of substrate oxidation under resting conditions is counterbalanced by a corresponding increase in NNT-mediated oxygen consumption (1). Experiments in mitochondria isolated from hind limb skeletal muscle from B6N mice under conditions mimicking a resting state (i.e. in the absence of ADP) demonstrated that increasing carnitine from 25 μm to 5 mm to maximize palmitoyl-CoA oxidation resulted in a 3-fold increase in the rate of H₂O₂ emission. The combined inhibition of Trx reductase by auranofin (AF) and GSH reductase by carmustine (BCNU) increased H₂O₂ emission by almost 4-fold, demonstrating that the activity of mitochondrial peroxidases buffers >70% of H₂O₂ production driven by β-oxidation. In addition, H₂O₂ formation was shown to depend on a complete fatty acid oxidation, including acetyl-CoA buffering by carnitine acetyltransferase and/or acetyl CoA utilization by the TCA cycle. Notably, the highest rate of fatty acid oxidation obtained at 5 mm carnitine caused an increase in proton conductance that was prevented by AF/BCNU treatment. This interesting finding suggests that GSH and Trx reductases utilize NADPH(H⁺) produced by NNT, which in turn uses Δp generated by mitochondrial respiration. The authors validated this hypothesis comparing permeabilized fibers from B6J and B6N mice. Indeed, the increase in proton conductance was absent in B6J fibers that also were not affected by AF/BCNU addition. Notably, oxygen consumption was 18.6% lower in B6J samples. Therefore, a significant fraction of mitochondrial respiration supports NNT activity in mediating an optimal rate of ROS removal (Fig. 1).

Figure 1.

Schematic of the pathways involved in NNT coupling of mitochondrial ROS formation with oxygen consumption under resting conditions (i.e. no ATP synthesis). ROS removal requires NADPH(H⁺) provided by NNT in a process coupled with the utilization of the proton gradient generated by oxygen consumption. For the sake of simplicity, flavin nucleotides and thioredoxin are omitted, as well as the utilization of the proton gradient for ATP synthesis. FAO, fatty acid oxidation; GPX, GSH peroxidase; GR, GSH reductase; LCACoA, long-chain acyl-CoA; SOD, superoxide dismutase(s).

The work conducted by Smith et al. suggests that NNT performs direct and indirect coupling activities that are tightly linked. NNT directly couples NADPH(H⁺) formation from NADH(H⁺) with mitochondrial proton uptake, as is well-established. Smith et al. (1) demonstrate that Δp is maintained by oxygen consumption, such that NNT-mediated ROS removal is physiologically coupled with mitochondrial respiration. However, it is worth noting that the current study does not include in situ or in vivo experiments, perhaps because the carnitine titration of β-oxidation along with the use of reductase and respiration inhibitors could not be applied to intact cells or organs. Thus, it will be important to extend this work to more intact models. Moreover, a word of caution should be mentioned for the use of B6N mice as control strain. A recent study demonstrated that B6N hearts are more prone to contractile failure because of the absence of MYLK3, a protein kinase required for actin assembly (7). However, this defect is unlikely to impact the findings of Smith et al. (1). Nevertheless, a proteomic analysis of BJ6 mitochondria is lacking. Future studies should investigate whether the lack of NNT is compensated by changes in mitochondrial proteins involved in substrate oxidation and ROS removal.