These findings underscore the importance of naturally occurring NR as a possible alternative substrate for NAD+ biosynthesis (Bieganowski and Brenner, 2004). Supporting this idea, NR treatment enhances NAD+ levels in all mammalian cells tested (Cantó et al., 2012, Yang et al., 2007b). Interestingly, different lines of evidence suggest that NR is the primary metabolite transported into the cell and metabolized into NAD+, even when cells are cultured in the presence NAD+ or NMN (Lu et al., 2009) (Figure 2). Thus, by using specific inhibitors, it was suggested that free NAD+ or NMN in the medium are metabolized extracellularly to NR, which may be the final metabolite transported to cells for NAD+ biosynthesis (Nikiforov et al., 2011). Interestingly, milk extracts (whey vitamin fraction) rescue the survival of yeast cells defective for QNS1, a necessary enzyme for NA and NAM-triggered NAD+ biosynthesis in yeast (Bieganowski and Brenner, 2004). This extract, however, failed to rescue survival in NRK1-deficient yeast (Bieganowski and Brenner, 2004). The presence of significant NR levels in blood after oral intake is, however, not apparent, as classic reports generally indicate that radiolabelled NR is transformed into NAM at the brush border (Gross and Henderson, 1983). In this sense, it is intriguing that mammalian cultured cells commonly require almost millimolar NR concentrations in order to enhance NAD+ biosynthesis (Cantó et al., 2012, Yang et al., 2007b), which is unlikely to be met in vivo. However, in some microorganisms, such as Haemophilus influenza, only the transport of NR across the membrane allows them to synthesize NAD+ and survive in the host bloodstream, as they are unable to use NA, NAM, or the de novo pathway for this purpose (Cynamon et al., 1988, Herbert et al., 2003). This suggests that either NR or NMN is, in fact, available in the blood. This is partially corroborated by a report indicating that NMN might be present in the bloodstream at concentrations around 50 μM (Revollo et al., 2007). The presence of a circulating extracellular form of the NAMPT enzyme (eNAMPT), to convert NAM to NMN, supports this possibility (Revollo et al., 2007). Recent evidence indicates that eNAMPT activity in the plasma is required to safeguard hypothalamic NAD+ levels (Yoon et al., 2015). However, other labs have failed to detect NMN in plasma (Hara et al., 2011). In addition, the marginal presence of ATP and 5-phosphoribosyl 1-pyrophosphate (PRPP) in blood (Hara et al., 2011), both substrates for the reaction catalyzed by NAMPT, would impede the generation of NMN in the circulation. Furthermore, since NAM plasma levels are also low, it is difficult to substantiate significant NMN synthesis in the bloodstream (Hara et al., 2011).
All the above suggest that plasma levels of most NAD+ precursors are probably unable to systematically sustain high NAD+ production rates. Consequently, it seems that mammalian organisms largely rely on NAD+ salvage from intracellular NAM in order to maintain NAD+ pools. In fact, NAM is an end product of NAD+-consuming activities in the cell (i.e., sirtuins, poly(ADP-ribose) polymerases and cyclic ADP-ribose hydrolases) (Houtkooper et al., 2010a). Accordingly, mice lacking NAMPT are not viable (Revollo et al., 2007). This, however, does not rule out a limited contribution of circulating NR, NMN, NAM, or Trp to NAD+ biosynthesis under basal conditions. However, further technical improvements will be needed, especially for NR, NMN, and NAM determination, to precisely evaluate the contribution of circulating precursors to NAD+ homeostasis.
Cell Compartmentalization of NAD+
In general, intracellular NAD+ levels are maintained between 0.2 and 0.5 mM, depending on the cell type or tissue. However, NAD+ levels can change up to ∼2-fold in response to diverse physiological stimuli. For example, NAD+ levels increase in response to energy stresses, such as glucose deprivation (Fulco et al., 2008), fasting (Cantó et al., 2010, Rodgers et al., 2005), CR (Chen et al., 2008), and exercise (Cantó et al., 2010, Costford et al., 2010), and fluctuate in a circadian fashion (Nakahata et al., 2009, Ramsey et al., 2009). So, where and how do these changes take place in the cell?
The presence of NMNATs in the nucleus, cytosol, and mitochondria suggests that these compartments are fully capable of salvaging NAD+ from NAM (Figure 2). NAD+-degrading enzymes, such as sirtuins, are also present in these compartments. In addition, the presence of different forms of NMNATs in each cellular compartment (e.g., NMNAT1 in the nucleus or NMNAT3 in the mitochondria/cytosol) suggests that NAD+ salvage is tailored according to compartment-specific metabolic needs. However, despite some evidence that NAMPT is localized to the mitochondria (Yang et al., 2007a), there is still some debate as to whether this is really the case (Pittelli et al., 2010). Therefore, further experimental evidence is needed to confirm mitochondrial NAD+ salvage. Nonetheless, it is important to note that NAD+ is not evenly distributed in the cell. Most reports indicate that mitochondrial NAD+ content is ≥250 μM (Nakagawa et al., 2009, Yang et al., 2007a), while according to indirect estimations, nuclear NAD+ levels seem to be much lower, ∼70 μM (Fjeld et al., 2003). To this effect, two-photon microscopy approaches have also been used to indirectly estimate NAD+ levels, confirming that NAD+ content in the nucleus is much lower than in the cytosol (Zhang et al., 2009). In addition, the different NAD+ pools can behave independently. As such, cells treated with methylmethane sulfonate, a genotoxic agent, can survive as long as mitochondrial NAD+ levels are maintained, irrespective of NAD+ depletion in other compartments (Yang et al., 2007a). Given that NAD+ or NADH cannot diffuse through membranes (van Roermund et al., 1995), the maintenance of NAD+ levels in each compartment is reliant on salvaging the NAM produced by NAD+-consuming enzymes (Figure 2). Alternatively, it can be derived from the intermediates NMN or NAMN, generated from NR metabolism or the Preiss-Handler pathway, respectively. It was recently shown that exogenous NAD+ can elevate mitochondrial NAD+ levels more than cytoplasmic levels, indicating that NAD+ precursors or intermediates traverse the mitochondrial membrane (Pittelli et al., 2011). Further, NR treatment was shown to enhance mitochondrial NAD+ levels in cultured cells and in mouse liver (Cantó et al., 2012). However, the mitochondrial compartment lacks NRK activity to initiate NR conversion into NAD+ (Nikiforov et al., 2011). Hence, NR is likely converted to NMN in the cytosol, and NMN may traverse the mitochondrial membrane to produce NAD+ via NMNATs (Figure 2) (Berger et al., 2005, Yang et al., 2007a). This way, both NMN and NAM might act as the main intracellular forms for regulating NAD+ levels between compartments.
The compartmentalization of NAD+ synthesis may have even more layers of complexity than once imagined. For example, NMNAT1 is recruited to target gene promoters by either the NAD+-consuming enzyme SIRT1 (Zhang et al., 2009) or PARP1 (Zhang et al., 2012), which suggests that NAD+ production is regulated at a sub-compartmental level during transcriptional regulation or DNA repair. These observations suggest that SIRT1 and PARP1 may compete for limiting amounts of localized NMNAT1-produced NAD+. Thus, despite estimations indicating that nuclear NAD+ levels are low, NAD+ maintenance is key for survival, as testified by the fact that Nmnat1-KO is embryonically lethal in mice (Conforti et al., 2011).
The Enzymatic Use of NAD+
NAD+ and Redox Reactions in Metabolism
While distinct, the cytosolic/nuclear and mitochondrial pools of NAD+ are interconnected by an intricate set of cellular redox processes. These NAD+ pools can modulate the activity of compartment-specific metabolic pathways such as glycolysis in the cytoplasm and the TCA cycle/oxidative phosphorylation in the mitochondria.
In the cytoplasm, the conversion of glucose to pyruvate by glycolysis requires two NAD+ molecules per molecule of glucose. Following the conversion of glucose to two molecules of glyceraldehyde-3-phosphate (G3P), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reduces NAD+ to NADH to transform G3P into 1-3-biphosphoglycerate. Glycolysis will therefore net two NADH and two pyruvate molecules that can then be transported into to the mitochondrial matrix. Since the outer mitochondrial membrane is very porous, NADH is free to enter the intermembrane space. However, it is the reducing equivalent of NADH that is transported into the mitochondria, via either the malate-aspartate shuttle or the glycerol-3-phosphate shuttle of the inner mitochondrial membrane, rather than NADH itself (Figure 2) (McKenna et al., 2006). As discussed in Cell Compartmentalization of NAD+, cytoplasmic NAD+ levels cannot alter mitochondrial NAD+/NADH ratios directly since NAD+ is not permeable to the mitochondrial membrane (Barile et al., 1996). Therefore, changes in the cytoplasmic NAD+ pool do not acutely alter mitochondrial NAD+ levels (Pittelli et al., 2010, Yang et al., 2007a).
In the mitochondrion, the TCA cycle reduces NAD+ molecules to produce multiple NADH molecules. Mitochondrial NADH, gained from glycolysis or the TCA cycle, are oxidized by Complex I (NADH:ubiquinone oxidoreductase) of the ETC. The subsequent two electrons gained by Complex I are relayed along ubiquinone (Coenzyme Q10), complex III (coenzyme Q-cytochrome c oxidoreductase), cytochrome c, and Complex IV (cytochrome c oxidase). In parallel to the oxidation of NADH to NAD+ by the ETC, the substrate succinate from the mitochondrial TCA cycle provides additional electrons to ubiquinone in parallel with Complex I. Ultimately, the flow of electrons, generated from NADH and succinate, along the ETC is coupled to the pumping of protons from the mitochondrial matrix to the intermembrane space via Complex I, III, and IV, creating a proton gradient. The proton gradient then provides the chemiosmotic gradient to couple the flux of protons back into the matrix via F0F1-ATP synthase with oxidative phosphorylation of ADP to ATP. Overall, the ETC reduces O2 to water and NADH to NAD+ for the purpose of generating ATP. As a result, mitochondrial NAD+ levels are 2-fold greater than the rest of the cell, as measured in mouse skeletal muscle (Pirinen et al., 2014), and 4-fold greater in mouse cardiac myocytes (Alano et al., 2007).
Since NAD+ levels within the cell can be limiting (Bai et al., 2011b, Pirinen et al., 2014, Pittelli et al., 2011), both glycolysis in the cytoplasm and the TCA cycle in the mitochondria can influence metabolic homeostasis by altering cytosolic/nuclear NAD+ and NADH levels. In addition, following DNA damage, NAD+ levels can drop low enough that glycolysis and substrate flux to the mitochondria is blocked, leading to cell death, despite having an excess of available glucose (Alano et al., 2010, Benavente et al., 2009, Ying et al., 2005, Zhang et al., 2014). This finding highlights the need to understand the mechanisms interconnecting subcellular NAD+ pools, as their homeostasis and interactions are essential for the preservation of cell viability and ATP levels.
NAD+-Consuming Enzymes (I): Sirtuins
In mammals, there are seven sirtuin enzymes (SIRT1–SIRT7) based on the presence of a characteristic and evolutionarily conserved catalytic site, comprised of 275 amino acids (Haigis and Sinclair, 2010, Hall et al., 2013, Houtkooper et al., 2010a). Three sirtuins are located in the mitochondria (SIRT3–SIRT5), while SIRT1, SIRT6 and SIRT7 are predominantly located in the nucleus, and SIRT2 is found in the cytoplasm (Michishita et al., 2005, Verdin et al., 2010). However, some sirtuins, such as SIRT1, have been shown to shuttle in and out of the nucleus (Tanno et al., 2007).