Nicotinamide (nam) adenine dinucleotide [NAD+; initially known as diphosphopyradine nucleotide (DPN+)], is a ubiquitous cellular coenzyme that was first discovered by Arthur Harden and William Young, when they identified a heat-labile fraction of cell-free glucose fermentation containing ATP, Mg2+ and NAD+, which they coined, “cozymase” (78).
Our understanding of the role of NAD+ and its reduced form, NADH, in cellular function and metabolism was subsequently expanded by a “who’s who” of biochemistry, with researchers such as Hans von Euler-Chelpin, Otto Warburg, Conrad Elvehjem, Arthur Kornberg, Albert Lehninger, and Britton Chance, all making substantial contributions. Four of the aforementioned researchers were awarded the Nobel Prize, with Harden and von Euler-Chelpin sharing the Nobel Prize in 1929 for their work on the fermentation of sugar and fermentative enzymes, which included the identification of the “nucleotide sugar phosphate” NAD+. Subsequently, Warbug demonstrated that NAD+ acted as a carrier of hydrogen and transferred it from one molecule to another, which was key to understanding the metabolic function of NAD+ (128). Ultimately, it was work by Freidkin and Lehninger (55) that showed that NADH was an integral component of ATP production via oxidative phosphorylation. Thus, for many years, the primary cellular function of NAD+ was considered to be its ability to harness energy from glucose, fatty acids, and amino acids in pathways such as glycolysis, β-oxidation, and the citric acid cycle.
In recent years, however, the importance of NAD+ as a central signaling molecule and substrate that can impact numerous fundamental biological processes has come to the fore. Indeed, a remarkable number of regulatory pathways that utilize NAD+ in signaling reactions have been identified, and these cover broad aspects of cellular homeostasis, including functions in energy metabolism, lifespan regulation, DNA repair, apoptosis, and telomere maintenance (11, 12, 84, 97, 190). Thus, while the tissue NAD+/NADH ratio was once thought to be “simply” a balance of the redox state, the complexity of NAD+ metabolism has evolved considerably with the discovery of highly integrated networks of NAD+-consuming pathways and NAD+ biosynthetic and salvage pathways (11, 12, 84, 97, 128, 144, 190). Part of the reason for the renaissance of NAD+ has been the discovery of NAD+-consuming enzymes, particularly sirtuins (SIRT). SIRT1 is the best-described of the seven mammalian sirtuins, and, based on its dependence for NAD+ as a substrate (and therefore its sensitivity to perturbations in NAD+),
SIRT1 has been put forth as a key regulator of acute and chronic exercise-mediated mitochondrial adaptations in skeletal muscle (40, 70, 72, 76, 174, 185, 193). In addition, SIRT3 and poly(ADP-ribose) (PAR) polymerases (PARPs), which also use NAD+ as a substrate, have been proposed as important regulators of mitochondrial function and/or biogenesis (40, 76, 125, 174, 185, 193). In this review, our aim is to provide an overview of NAD+ metabolism in skeletal muscle and the changes that occur in NAD+, NADH, and the NAD+/NADH ratio in response to acute and chronic endurance exercise. Our intention is not to discuss the impact of the redox state and NAD+/NADH ratio on cellular bioenergetics and substrate utilization, which is covered in highly informative reviews by others (9, 26, 106, 109, 110). Rather, our goal is to discuss the changes in pyridine nucleotide redox state that occur with exercise in the context of what we know and do not know about the effects of SIRT1, SIRT3, the PARPs, and carboxyl-terminal binding protein (CtBP) on mitochondrial adaptations to exercise in skeletal muscle. It is, of course, difficult to extrapolate the findings from one cell line or tissue type to another, and we acknowledge that we do not discuss many important studies that have contributed to our understanding of NAD+ metabolism and SIRT1, SIRT3, and PARP biology in cell lines and tissue types other than skeletal muscle and muscle cell lines. For a more general and encompassing discussion on NAD+ metabolism and its potential clinical implications, readers are encouraged to read some excellent and comprehensive reviews (see Refs. 11, 12, 84, 97, 128, 144, and 190).
Where in the Cell Is NAD+?
It is broadly accepted that NAD+ is primarily found in three distinct cellular pools: 1) the cytosolic, 2) the mitochondrial, and 3) the nuclear pools. A general overview of the compartmentation of NAD+ and NADH is provided in Fig. 1 and provides a point of reference for the ensuing discussion on NAD+(H) “compartmentation” and their movement into the mitochondria and nucleus. Initial studies used differential centrifugation methods, cell disruption methods, and compounds to modulate mitochondrial NAD+(H) metabolism in order to determine NAD+(H) location. More recently, the compartmentation of NAD+, which was originally suggested by Ragland and Hackett (146), has been extrapolated from the localization of enzymes in the NAD+ consuming, biosynthetic, and salvage pathways and the use of innovative molecular biology techniques (11, 12, 84, 97, 144, 190). Thus, Dölle et al. (43) used the novel PAR Assisted Protein Localization AssaY (PARAPLAY) in HeLa S3 cells, in which they targeted the catalytic domain of PARP1 (which consumes NAD+) to various cellular compartments.
The idea behind this method is that if NAD+ is present in the compartment to which PARP1 is targeted, then PAR will accumulate and can be detected by immunocytochemistry (43). Using PARAPLAY, NAD+ was found in mitochondria (specifically the matrix but not intermembrane space) and peroxisomes, and surprisingly to the endoplasmic reticulum (ER) and Gogli complex (43, 112). Cytosolic NAD+ was not detected in that study, most likely due to the fact that PAR glycohydrolase (PARG), which consumes PAR, is most abundant in the cytosol. Little is known about the role of NAD+ and NADH in regulating Golgi complex and ER function, and certainly its function in skeletal muscle is unknown. Furthermore, surprisingly very little is known about nuclear NAD+ levels in general, and to our knowledge nuclear NAD+(H) levels have not been measured in skeletal muscle.
Overall, the free cytosolic and nuclear NAD+(H) compartments are traditionally thought to be in equilibrium, with NAD+(H) being able to freely pass through pore complexes in the nuclear membrane (46, 98–103, 187, 190). In Cos7 cells, the free nuclear NAD+ concentration is estimated to be ∼10–100 μM (53, 188), which is comparable to the estimations for the cytosol (∼150 μM) of muscle (42, 119). Thus, in response to exercise, it would be expected that the pyridine redox state in the nucleus reflects changes that occur in the cytosol. The relevance of nuclear NAD+(H) to adaptations to exercise will be covered when discussing SIRT1, PARPs, and CtBP.
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