NAD+ and Cellular Redox Metabolism: What the Published Research Reports

The NAD+/NADH and NADP+/NADPH couples have been described in the review literature as central to cellular redox homeostasis and energy metabolism, with the two pools maintained separately and with imbalance in either couple reported in association with a range of pathological states [1]. An earlier review of the same field catalogued roles attributed to NAD and NADP in energy metabolism, mitochondrial function, calcium handling, reactive oxygen species generation and removal, gene expression, and cell death pathways including poly(ADP-ribose) polymerase-1 and apoptosis-inducing factor signalling [2]. A further review emphasised that NAD+ serves both as a redox cofactor and as a substrate for signalling enzymes such as sirtuins and poly(ADP-ribose) polymerases, and argued that because these roles occur in distinct compartments, bulk measurements of cellular NAD+ may not reflect the redox landscape encountered by any particular enzyme [3]. A review spanning the biology of pyridine dinucleotides from molecular to human scale noted that fundamental questions remain about what sets the absolute levels of these cofactors and what determines their redox ratios [4]. Much of the mechanistic work in this area rests on genetically encoded reporters. SoNar, a fluorescent sensor of the cytosolic NAD+/NADH state, was developed and characterised in living cells and in vivo, and was used in a high-throughput screen of more than 5,500 compounds that identified KP372-1 as an NQO1-mediated redox cycling agent producing oxidative stress in cancer cells in vitro and reduced tumour growth in an in vivo model [5]. Compartment-targeted versions of that sensor were subsequently reported: mitochondrially and cytosolically directed SoNar responded linearly to physiological NAD+/NADH ratios in live cells, and the two compartments responded rapidly but differently to acute metabolic perturbation, which the authors interpreted as evidence of distinct NAD pools communicating through the malate–aspartate shuttle [6]. The same sensor, alongside the NADPH probe iNap1, was applied in Chinese hamster ovary cell cultures, where a temperature downshift was reported to lower the intracellular NAD+/NADH ratio and raise intracellular NADPH in vitro [7]. Tools for perturbing rather than reading the ratio have also been described. A water-forming NADH oxidase from Lactobacillus brevis (LbNOX) was expressed in human cells to raise the NAD+/NADH ratio in either cytosol or mitochondria, and cytosolic or mitochondrial expression was reported to ameliorate proliferative and metabolic defects caused by an impaired electron transport chain in vitro, which the authors framed as evidence for a role of reductive stress [8]. A complementary tool moving in the opposite direction, a soluble transhydrogenase from Escherichia coli expressed in mammalian cells in untargeted and mitochondrially targeted forms, was reported to raise the NADH/NAD+ ratio in a compartment-specific manner, with metabolic and transcriptomic signatures of reductive stress that differed by cellular background in vitro [9]. Several studies have linked the ratio to cell fate and developmental timing. In an in vitro pluripotent stem cell system recapitulating the roughly twofold difference in developmental rate between mouse and human embryos, inhibiting the electron transport chain slowed the segmentation clock by impairing the NAD+/NADH balance and lowering global protein synthesis, while raising the NAD+/NADH ratio in human cells by LbNOX overexpression increased translation rate and accelerated the clock [10]. Isotopologue and transcriptomic analysis of mammalian preimplantation embryos reported that early embryonic metabolism is rigid in its nutrient requirements and sensitive to reductive stress, with metabolic plasticity increasing later through changes in redox control mechanisms [11]. In human mesenchymal stem cells expanded in vitro, alterations in the NAD+/NADH balance and sirtuin activity accompanied replicative senescence, and treatment with the NAD+ precursor nicotinamide raised intracellular NAD+, shifted the NAD+/NADH ratio, and was associated with increased Sirt-1 activity and partially restored mitochondrial measures in late-passage cells, whereas human fibroblasts showed a comparatively stable ratio across expansion [12]. A multi-enzyme hydride transfer complex assembled from malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase was reported to transfer reducing equivalents from NADH to NADP+, to be repressed in senescent cells and induced by p53 inactivation, and its exogenous expression was sufficient to bypass senescence and to rescue cells from complex I inhibitors in vitro, with high expression of the component enzymes observed in mouse and human prostate cancer models [13]. In cardiovascular and vascular models, a review of cardiac energy metabolism in heart failure described alterations in NAD redox state and metabolite signalling as contributors to post-translational and epigenetic changes affecting energy metabolic gene expression [14]. A related review of diabetic cardiomyopathy described a decreased NAD+/NADH ratio accompanied by an increased acetyl-CoA/CoA ratio, and proposed the mitochondrial redox couple as a target for further investigation [15]. Work combining human patient data, a canine tachycardia model, and human iPSC-derived engineered heart tissue reported that a shift towards anaerobic glycolysis disrupted NAD redox balance and increased global protein acetylation including of SERCA, and that NAD+ supplementation reduced that acetylation and accelerated functional recovery of the engineered tissue after tachycardia [16]. In endothelial cells studied in vitro, ex vivo, and in vivo, limiting NAD did not affect proliferation or migration but prevented cell–cell contact formation and acquisition of quiescence, impaired vascular stabilisation and plexus formation, and was reported to act by permitting mitochondria-derived hydrogen peroxide, with exogenous H2O2 mimicking NAD deficiency and its removal rescuing the phenotype [17]. Human sample work has documented altered NAD handling in disease cohorts. In 28 individuals with RYR1-related myopathies compared with a control dataset of 299, systemic NAD+ deficiency was observed in 19 of 28 and increased NADPH in 22 of 26, with decreased NAD+/NADH and NADP/NADPH ratios in 9 of 28 and 23 of 26 respectively; in patient-derived myotube cultures, nicotinamide riboside increased cellular NAD+ in a dose- and time-dependent manner at 72 hours and modified maximal respiration and ATP production, while redox imbalance was not observed in Ryr1 Y524S mouse specimens [18]. In primary macrophages, TNF was reported to upregulate both NAD+-generating and NAD+-consuming enzymes and to lower cellular NAD+ over time, an effect reversed in macrophages from CD38-null animals [19]. At tissue level, a review of neuroglial coupling proposed a redox switch in which cytosolic NAD+/NADH state governs the selection between glucose and lactate as substrate through inhibition of glyceraldehyde 3-phosphate dehydrogenase, coupling neuronal and astrocytic redox states via monocarboxylate recycling [20].