The secret life of NAD+: an old metabolite controlling new metabolic signaling pathways

Abstract

A century after the identification of a coenzymatic activity for NAD(+), NAD(+) metabolism has come into the spotlight again due to the potential therapeutic relevance of a set of enzymes whose activity is tightly regulated by the balance between the oxidized and reduced forms of this metabolite. In fact, the actions of NAD(+) have been extended from being an oxidoreductase cofactor for single enzymatic activities to acting as substrate for a wide range of proteins. These include NAD(+)-dependent protein deacetylases, poly(ADP-ribose) polymerases, and transcription factors that affect a large array of cellular functions. Through these effects, NAD(+) provides a direct link between the cellular redox status and the control of signaling and transcriptional events. Of particular interest within the metabolic/endocrine arena are the recent results, which indicate that the regulation of these NAD(+)-dependent pathways may have a major contribution to oxidative metabolism and life span extension. In this review, we will provide an integrated view on: 1) the pathways that control NAD(+) production and cycling, as well as its cellular compartmentalization; 2) the signaling and transcriptional pathways controlled by NAD(+); and 3) novel data that show how modulation of NAD(+)-producing and -consuming pathways have a major physiological impact and hold promise for the prevention and treatment of metabolic disease.

Figure 1

De novo NAD⁺ biosynthesis from tryptophan. The de novo biosynthesis of NAD⁺ starts with the conversion of tryptophan to N-formylkynurenine catalyzed by either IDO or TDO. N-Formylkynurenine is subsequently converted in four individual steps to the unstable ACMS, which can undergo either enzymatic conversion directed to total oxidation or nonenzymatic cyclization to quinolinic acid. The final step of the dedicated de novo biosynthesis of NAD⁺ is comprised of the QPRT-catalyzed formation of NAMN. NAMN is subsequently converted to NAAD by one of the NMNAT enzymes. The final step in the biosynthesis of NAD⁺ is the amidation of NAAD by the NAD synthase enzyme.

Figure 2

Mammalian NAD⁺ salvage pathway. NAD⁺ is synthesized in the NAD⁺ salvage pathway from its precursors NA, NAM, or NR. The initial step in NAD⁺ synthesis from NA, the so-called Preiss-Handler pathway, is catalyzed by NAPT and results in the formation of NAMN, which can also be derived from de novo NAD⁺ biosynthesis. In an identical reaction, but catalyzed by a different enzyme, NAM is converted by NAMPT forming NMN, which is also the product of phosphorylation of NR by NRK. The subsequent conversion of both NAMN and NMN is catalyzed by the same enzyme, i.e., NMNAT. In the case of NAMN, this reaction is followed by amidation, finally producing NAD⁺. NADS, NAD synthase.

Figure 3

Redox potentials of the mitochondrial and cytosolic NAD(H) and NADP(H) systems in the liver. c, Cytosolic; m, mitochondrial.

Figure 4

Metabolite indicators for NAD⁺/NADH. A, Lactate dehydrogenase as specific metabolite indicator reaction for cytosolic NAD⁺/NADH. B, Mitochondrial 3-hydroxybutyrate dehydrogenase as a specific metabolite indicator reaction for mitochondrial NAD⁺/NADH.

Figure 5

The role of the energy-linked transhydrogenase for the redox state. Schematic representation of the mitochondrial respiratory chain system, including complex VI, which is the energy-linked transhydrogenase catalyzing the reaction: NADH + NADP⁺ ↔ NAD⁺ + NADPH. The fact that complex VI is driven by the proton gradient across the mitochondrial membrane (just like the F₁F₀-ATP-ase) drives the transhydrogenase reaction far to the site of NADP reduction so that the transhydrogenase reaction is virtually unidirectional (NADH + NADP⁺ → NAD⁺ + NADPH). MIM, Mitochondrial inner membrane; cytc, cytochrome c; IMS, intermembrane space.

Figure 6

Redox shuttles. A, Schematic description of the NADP(H) redox shuttle: the NADPH as produced in the transhydrogenase (TH) reaction is transduced to the cytosol via the 2-oxoglutarate (2OG)–isocitrate redox shuttle in which mitochondrial NADPH first reacts with 2-oxoglutarate and bicarbonate as catalyzed by the mitochondrial NADP-linked isocitrate dehydrogenase (mIDH^NADP) to produce isocitrate and NADP⁺, followed by the export of isocitrate to the cytosolic in exchange for 2-oxoglutarate. The isocitrate now present in the cytosol space is then converted back into 2-oxoglutarate and bicarbonate via the cytosolic NADPH-linked isocitrate dehydrogenase (cIDH^NADP), followed by the uptake of 2-oxoglutarate into the mitochondrial space in exchange for isocitrate. TCC, Tricarboxylate. B, Schematic description of the malate/aspartate NAD(H)-redox shuttle. The NADH that is produced in the cytosol, for instance during glycolysis, is first converted into malate via cytosolic NAD⁺-linked malate dehydrogenase (mMDH^NAD), after which the malate is transported into the mitochondria in exchange for 2-oxoglutarate (20G). Intramitochondrial malate is then converted into oxaloacetate (OAA), during which NADH is generated, which can now be used in the respiratory chain (RC) with formation of ATP. The oxaloacetate is not very permeable to mitochondrial membranes and is therefore first converted into aspartate via the glutamate oxaloacetate transaminase (GOT; cGOT, cytosolic GOT; mGOT, mitochondrial GOT), followed by the export of aspartate in exchange for glutamate. Cytosolic aspartate is subsequently converted back into cytosolic oxaloacetate, thus completing the cycle. Note that cytosolic NADH can also be reoxidized via other redox shuttles including the α-glycerol-3-phosphate shuttle in which the NADH first reacts with dihydroxyacetonephosphate to generate glycerol-3-phosphate plus NAD⁺, after which glycerol-3-phosphate directly transfers its electrons to the respiratory chain at the level of ubiquinone via the membrane-bound enzyme α-glycerol-3-phosphate dehydrogenase, thus completing the cycle. DCC, Dicarboxylate carrier; AGC, aspartate-glutamate carrier.

Figure 7

Regulation of mitochondrial and cytosolic redox state. A, Anaerobic metabolism of glucose into lactate with NADH produced in the glyceraldehyde-3-phosphate dehydrogenase (GADPH) reaction and with lactate dehydrogenase (LDH) reconverting NADH back into NAD⁺. B, Schematic representation of the aerobic oxidation of glucose with particular attention for the sites at which NADH is formed including: 1) the GADPH reaction during glycolysis; 2) the pyruvate dehydrogenase (PDH) complex; and 3) the citric acid cycle, followed by the reoxidation of NADH back to NAD⁺ by the respiratory chain (RC) with the concomitant formation of ATP from ADP + phosphate.

Figure 8

Sirtuin enzymatic activities. Sirtuins display at least two different NAD⁺-consuming activities, both of which render NAM as a product. In mammals, SIRT1, SIRT2, SIRT3, SIRT5, and SIRT7 act as deacetylase enzymes, using NAD⁺ to cleave acetyl groups from ε-acetyl lysine residues of target proteins in a reaction that, in addition to NAM, generates 2′-O-acetyl-ADP-ribose. SIRT4 and SIRT6, rather, act as mono-ADP-ribosyl transferases, in a reaction where the ADP-ribosyl moiety of NAD⁺ is transferred to a substrate protein. Despite their predominant deacetylase activity, SIRT2 and SIRT3 also display ADP-ribosyl transferase activity.

Figure 9

PARPs and cADP-ribose synthases as NAD⁺-consuming enzymes. Intracellular NAD⁺ content largely depends on the activities of two different families of enzymes: PARPs and cADP-ribose synthases (CD38 and CD157), both of which render NAM as an end-product. PARPs catalyze a reaction in which the ADP-ribose moiety is transferred to a substrate protein. PARPs can transfer multiple ADP-ribosyl groups and form long chains and branches of ADP-ribosyl polymers. cADP-ribose synthases use NAD⁺ to generate cADP-ribose, which acts as an intracellular secondary messenger.

Figure 10

Therapeutical strategies associated with NAD⁺ metabolism. A, Several therapeutical strategies have been described to either increase (blue) or decrease (red) NAD⁺ content. B, Various enzymes or metabolites linked to NAD⁺ metabolism were implicated in longevity and/or its related health issues establishing the link between NAD⁺ and healthspan. The dashed arrow indicates a metabolic conversion that is not present in mammals. 1-MT, 1-Methyltryptophan.