NAD(+) Metabolism and the Control of Energy Homeostasis: A Balancing Act between Mitochondria and the Nucleus

Abstract

NAD(+) has emerged as a vital cofactor that can rewire metabolism, activate sirtuins, and maintain mitochondrial fitness through mechanisms such as the mitochondrial unfolded protein response. This improved understanding of NAD(+) metabolism revived interest in NAD(+)-boosting strategies to manage a wide spectrum of diseases, ranging from diabetes to cancer. In this review, we summarize how NAD(+) metabolism links energy status with adaptive cellular and organismal responses and how this knowledge can be therapeutically exploited.

Figure 1. NAD⁺ precursor metabolism and NAD⁺ consuming enzymes

Tryptophan (Trp), nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR) are utilized through distinct metabolic pathways to form NAD⁺. A. NAD⁺ synthesis from NA, also known as the Preiss-Handler pathway, is initiated by the NA phosphoribosyltransferase (NAPRT), which uses phosphoribosyl pyrophosphate (PRPP) to form NAMN. Together with ATP, NAMN is then converted into NAAD by the NMN adenylyl transferase (NMNAT1-3) enzymes. Finally, NA adenine dinucleotide (NAAD) is transformed to NAD⁺ through an amidation reaction catalyzed by the NAD⁺ synthase (NADSYN) enzyme. B. The de novo biosynthesis of NAD⁺ from tryptophan (Trp) starts with the conversion of Trp to N-formylkynurenine by either indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO). After four reaction steps, N-formylkynurenine can be subsequently converted to the unstable α-amino-β-carboxymuconate-ε-semialdehyde (ACMS), which can undergo nonenzymatic cyclization to quinolinic acid. The last step of the de novo biosynthesis component is comprised of the quinolinate phosphoribosyltransferase (QPRT)-catalyzed formation of NAMN, using PRPP as a co-substrate, which is converted to NAD⁺ via the remaining pathway described in panel A. C. ACMS can also be diverted away from NAD⁺ synthesis, by ACMS decarboxylase (ACMSD), to form α-amino-β-muconate-ε-semialdehyde (AMS) and can then be oxidized via the glutarate pathway and TCA cycle to CO₂ and water, or nonenzymatically converted to picolinic acid. D. The synthesis of NAD⁺ from NAM or NR is more direct and relies on only 2 steps each. NAM is converted by the rate-limiting nicotinamide phosphoribosyltransferase (NAMPT) to form NMN, using PRPP as cosubstrate. NMN is also the product of phosphorylation of NR by the NR kinases (NRK1-2). The subsequent conversion of NMN to NAD⁺ is catalyzed by the NMNAT enzymes. The blue boxes depict the 3 families of NAD⁺ consuming enzymes and some of the key processes to which they have been linked. NMN, NAM mononucleotide; NAMN, NA mononucleotide; NAAD, NA adenine dinucleotide; NRK, NR kinase; NMNAT, NMN adenylyltransferase; NADSYN, NAD⁺ synthetase.

Figure 2. Central nodes for cellular NAD⁺ metabolism

In normal circumstances, most NAD⁺ or NMN in blood is converted to NR, which enters the cell through specific transporters and is metabolized into NMN through NRK activity. Similarly, circulating NAM can be metabolized to NMN extracellulary by the extracellular NAMPT or enter the cell and be metabolized into NMN by the intracellular NAMPT. Extracellular NA can also enter the cell and be converted to NAD⁺ via a three-step reaction that is reliant on NAPRT, NMNAT and NADSYN. NMN and, possibly, NAM are potentially transported into the mitochondrial and nuclear compartments. In those compartments, NMN can lead to NAD⁺ synthesis via NMNAT activity. In each subcellular compartment, NAD⁺ and NADH equilibriums will be determined by their unique redox states. In the mitochondria, the electron transport chain is a major contributor to NADH oxidation into NAD⁺, coupling this action to ATP synthesis. In addition, the mitochondria and the cytosol can exchange redox equivalents through the malate/aspartate (M/A) and glyceraldehyde 3-phosphate (G3P) shuttles. In all compartments, the activity of NAD⁺ consuming enzymes, such as sirtuins or PARPs, lead to NAM production, which can be salvaged for NAD⁺ synthesis via NAMPT activity. Dashed arrows indicate pathways that need further validation.

Figure 3. The reciprocal relationship between SIRT1 and PARPs during NAD⁺ homeostasis and metabolic signaling in the cell

A. NAD⁺ is an essential coenzyme for sirtuin, PARP and CD38 activity, all of which metabolize NAD⁺ into NAM. Glycolysis and the TCA cycle also consume available NAD⁺ for the production of NADH, providing reducing equivalents for either lactate dehydrogenase (LDH) or the electron transport chain (ETC). Red font indicates environmental or physiological stimuli that activate sirtuins by increasing NAD⁺ while blue font indicates a reduction in NAD⁺, thereby diminishing sirtuin activity. NAM can be shunted away from NAD⁺ production following methylation by NNMT, a pathway activated by a HFD or with long term or high doses of NAM, which can favor the development of a fatty liver, due to reductions in available methyl groups. In contrast, NNMT depletion by NNMT-antisense oligonucleotides in animals, or mNAM supplementation in cells reduces NAM methylation. With a HFD, NAD⁺ can be reduced by elevating energy availability and NADH production, while exercise, fasting and CR reverses this process providing more NAD⁺ for sirtuin activation and protein deacetylation. NR supplementation or intraperitoneal NMN increases NAD⁺ availability via the NAD⁺ salvage pathway in mice. Ultimately, SIRT1 induces mitochondrial biogenesis, energy expenditure, antioxidant defenses, and lifespan extension by a mechanism that involves the mitochondrial unfolded protein response (UPR^mt). PARPs consume NAD⁺, reducing SIRT1 activity, by increasing PARylation of DNA and proteins during aging, cancer, neurodegeneration, and mitochondrial diseases. B. SIRT1 negatively regulates PARP1 through the inhibition of transcription and possibly through deacetylation. Reciprocally, PARP1 inhibits SIRT1 by limiting NAD⁺ levels, while PARP2 directly inhibits SIRT1 transcription. Interestingly, PARP1 is required for the transcriptional co-activation of NF-κB, while SIRT1 inhibits NF-κB activity through the deacetylation of RelA/p65. In addition, PARP1 and SIRT1 oppositely regulate p53 nuclear accumulation and activation following cytotoxic stress. Since the K_m of PARP1 for NAD⁺ is lower than that of SIRT1, as NAD⁺ levels drop following cell stress or senescence SIRT1 becomes less effective at regulating PARP1, and inhibiting inflammation or cell death through the inactivation of NF-κB and p53. Dashed arrows indicate pathways that need further validation.

Figure 4. Energy stress, NAD⁺-dependent UPR^mt signaling and mitochondrial health

The aging process and associated metabolic diseases, including obesity and mitochondrial diseases can be improved in mice and C. elegans using NAD⁺ boosters or PARP/CD38 inhibitors (PARPi/CD38i) in much the same way as has been demonstrated by calorie restriction (CR). Part of the metabolic decline during aging is due to a PARP-directed reduction in NAD⁺ levels, attenuating SIRT1 and FOXOA3 activities and leading to the activation of HIF1α and an increased reliance on glycolysis. Recently, a mechanism has been proposed for these NAD⁺-mediated improvements that include the induction of UPR^mt, which is triggered by SIRT1 and SIRT3 induced mitochondrial biogenesis, creating an imbalance in mitochondrial- versus nuclear-encoded mitochondrial proteins. This mitonuclear imbalance activates the UPR^mt, a retrograde signal that induces a mitohormetic and adaptive nuclear response, ultimately repairing and improving mitochondrial function. These mitohormetic signals can attenuate the impact of aging, mitochondrial diseases or a high-fat diet on metabolism.