NAD+

NAD＋ Research

What is NAD+?

NAD+ (Nicotinamide Adenine Dinucleotide) is a crucial coenzyme widely present in living organisms. It is formed by the connection of adenosine ribonucleotide and nicotinamide ribonucleotide through a phosphate group. As a core coenzyme in redox reactions, NAD+ plays an important role in cellular metabolism. It can convert between the oxidized state (NAD+) and the reduced state (NADH), participating in energy metabolism processes such as glycolysis, the citric acid cycle, and oxidative phosphorylation, helping cells convert food into energy (ATP). In addition, NAD+ serves as a necessary cofactor for various enzymes (such as PARP and Sirtuins), participating in processes related to DNA repair, cell signaling, and anti-aging.

What is the research background of NAD+?

Essential Cofactor in Multiple Reactions: 

NAD+ is an essential cofactor in multiple redox reactions (Shats I, 2020). In cells, it is involved in many cellular processes such as energy metabolism, genomic stability, and immune response. For example, in energy metabolism, NAD+ acts as an electron carrier in processes such as glycolysis and the tricarboxylic acid cycle, participating in redox reactions to convert the chemical energy in nutrients such as glucose into an energy form that cells can utilize.

Interaction with Multiple Enzymes: 

NAD+ also interacts with multiple enzymes, such as the DNA repair enzyme poly-(adenosine diphosphate-ribose) polymerase (PARP), the protein deacylase SIRTUINS, and the cyclic ADP ribose enzyme CD38. These enzymes regulate cellular processes, such as DNA repair, gene expression, and cell cycle regulation, by consuming NAD+.

What is the mechanism of action of NAD+?

As a Coenzyme in Redox Reactions

Maintaining Cellular Redox Homeostasis: 

"NAD" usually refers to the chemical backbone of nicotinamide adenine dinucleotide, while "NAD+" and "NADH" refer to its oxidized and reduced forms, respectively. NAD+ plays a key role in controlling many biochemical processes, and the NAD+/NADH ratio is crucial for maintaining cellular redox homeostasis^[1]. The intracellular redox balance is essential for normal cellular functions, including energy metabolism, antioxidant defense, etc. NAD+ acts as an electron acceptor or donor in redox reactions, participating in the intracellular energy production process, such as the tricarboxylic acid cycle and oxidative phosphorylation.

Regulating Energy Metabolism: 

NAD+ is involved in multiple key energy metabolism processes. For example, in glycolysis and the tricarboxylic acid cycle, NAD+ accepts hydrogen atoms and is converted into NADH. NADH then transfers electrons to oxygen through the electron transport chain on the inner mitochondrial membrane to produce ATP. The regulation of this energy metabolism is essential for the survival and function of cells, especially in tissues with high energy demands such as the heart and brain ^[1].

Participating in Enzymatic Reactions

The Role with Poly(ADP-ribose) Polymerase 1 (PARP1): 

NAD+ acts as a sensing or consuming enzyme for PARP1 and is involved in multiple key processes. PARP1 plays an important role in DNA damage repair. When cells suffer DNA damage, PARP1 is activated and uses NAD+ to synthesize poly ADP-ribose (PAR) chains, which are then attached to proteins, thus promoting the DNA repair process. However, excessive activation of PARP1 will consume a large amount of NAD+, leading to a decrease in intracellular NAD+ levels, which in turn affects the energy metabolism and viability of cells ^{[1, 2]}.

The Role with Cyclic ADP-ribose (cADPR) Synthases: 

Cyclic ADP-ribose synthases such as CD38 and CD157 are also NAD+ consuming enzymes. These enzymes use NAD+ to synthesize cADPR. cADPR acts as a second messenger to participate in calcium signaling, regulating the intracellular calcium ion concentration, and thus affecting various cellular functions, such as muscle contraction and neurotransmitter release.

The Role with Sirtuin Protein Deacetylases: 

Sirtuin protein deacetylases (SIRTs) also rely on NAD+ to function. SIRTs regulate gene expression, cellular metabolism, and stress responses by catalyzing the deacetylation of proteins. At high NAD+ levels, the activity of SIRTs is enhanced, promoting the health and survival of cells. For example, under conditions such as calorie restriction, the intracellular NAD+ level increases, activating SIRTs, thereby extending lifespan and improving metabolic health^[2].

The Role in Axonal Degeneration

The Interaction between NMNAT2 and SARM1: 

During the process of axonal degeneration, the NAD+ synthase NMNAT2 and the pro-degeneration factor SARM1 play crucial roles. NMNAT2 is an axonal survival factor, while SARM1 has NADase and related activities and is a pro-degeneration factor. The interaction between the two is essential for maintaining axonal integrity. In many cases, axonal degeneration is caused by a central signaling pathway, which is mainly regulated by these two key proteins with opposite effects. For example, in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, axons degenerate before the death of neuronal cell bodies, and this axonal degeneration is also common in axonal lesions such as hereditary spastic paraplegia. In these diseases, the activation of this signaling pathway may lead to axonal pathological changes ^{[3, 4]}.

The NAD+-Mediated Self-Inhibition Mechanism of SARM1: 

Studies have found that NAD+ is an unexpected ligand for the armadillo/heat repeat motifs (ARM) domain of SARM1. The binding of NAD+ to the ARM domain inhibits the NADase activity of the Toll/interleukin-1 receptor (TIR) domain of SARM1 through the domain interface. Disrupting the NAD+ binding site or the ARM-TIR interaction will lead to the constitutive activation of SARM1, resulting in axonal degeneration. This indicates that NAD+ mediates the self-inhibition of this pro-neurodegenerative protein^[5].

The Role in Cardiovascular Diseases

Protecting Cardiovascular Health: 

NAD+ has a protective effect in cardiovascular diseases. For example, NAD+ can protect the heart from diseases such as metabolic syndrome, heart failure, ischemia-reperfusion injury, arrhythmia, and hypertension. Its protective mechanism may involve multiple aspects such as regulating energy metabolism, maintaining redox balance, and inhibiting the inflammatory response. With aging or under stress, the intracellular NAD+ level decreases, leading to changes in the metabolic state and increasing the susceptibility to diseases. Therefore, maintaining the NAD+ level in the heart or reducing its loss is crucial for cardiovascular health^[1].

The Role in Tuberculosis

The Impact on Mycobacterium tuberculosis (Mtb): 

In Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis, the terminal enzyme of NAD synthesis, NAD synthetase (NadE), and the terminal enzyme of NADP biosynthesis, NAD kinase (PpnK), have different metabolic and microbiological effects. The inactivation of NadE leads to a parallel decrease in the NAD and NADP pools and a decline in the viability of Mtb, while the inactivation of PpnK selectively depletes the NADP pool but only stops growth. The inactivation of each enzyme is accompanied by metabolic changes specific to the affected enzyme and the related microbiological phenotype. Bacteriostatic levels of NAD depletion can cause a compensatory remodeling of NAD-dependent metabolic pathways without affecting the NADH/NAD ratio, while bactericidal levels of NAD depletion can disrupt the NADH/NAD ratio and inhibit oxygen respiration. These findings reveal previously unrecognized physiological specificities related to the necessity of two evolutionarily ubiquitous cofactors, suggesting that NAD biosynthesis inhibitors should be prioritized in the development of anti-tuberculosis drugs^[6].

The Role in Aging and Diseases

Decrease in Cellular NAD Levels Related to Aging: 

With aging, the intracellular NAD+ level gradually decreases. This decrease in NAD+ level is related to the change in the metabolic state of aging cells and may increase the susceptibility to diseases. Many pathological conditions, including cardiovascular diseases, obesity, neurodegenerative diseases, cancer, and aging, are related to the direct or indirect impairment of intracellular NAD+ levels^{[2, 7]}.

The Relationship between NAD+ Biosynthesis and Consuming Enzymes and Diseases: 

NAD+ biosynthesis and consuming enzymes are involved in several key biological pathways, affecting gene transcription, cell signaling, and cell cycle regulation. Therefore, many diseases are related to the abnormal functions of these enzymes. For example, in neurodegenerative diseases, NAD+-dependent mechanisms involve proteins such as WLDs, NMNAT2, and SARM1, indicating that neurodegenerative diseases are inherently related to NAD+ and energy metabolism ^[4]

Source:PubMed^[7]

What are the application fields of NAD+?

Applications in Cardiovascular Diseases

Protective Effect: 

NAD+ plays an important role in cardiovascular diseases, and it can protect the heart from a variety of diseases. For example, NAD+ can protect the heart from diseases such as metabolic syndrome, heart failure, ischemia-reperfusion injury, arrhythmia, and hypertension^[1]. This is because NAD+ acts as a sensing or consuming enzyme for enzymes such as poly(ADP-ribose) polymerase 1 (PARP1), cyclic ADP-ribose (cADPR) synthases (CD38 and CD157), and sirtuin protein deacetylases (Sirtuins, SIRTs), and is involved in several key processes in cardiovascular diseases.

Maintaining Redox Balance: 

The NAD+/NADH ratio is crucial for maintaining the redox homeostasis of cells and regulating energy metabolism ^[1]. Therefore, maintaining the NAD+ level in the heart or reducing its loss is crucial for cardiovascular health.

Applications in Anti-aging

Extending Lifespan: 

The causes of molecular aging and longevity interventions have witnessed a surge in the past decade. Nicotinamide adenine dinucleotide (NAD) and its precursors, such as nicotinamide riboside, nicotinamide mononucleotide, nicotinamide, and nicotinic acid, have attracted interest as potentially interesting molecules in the application of small molecules as potential geroprotectors and/or pharmacogenomics. These compounds have shown that they can improve aging-related conditions after supplementation and may prevent the death of model organisms^[8].

Influencing Lifespan Regulation: 

In model organisms such as yeast, studies have shown that NAD precursors play an important role in aging and longevity. Through the study of the chronological lifespan (CLS) and replicative lifespan (RLS) of yeast, we can better understand the mechanism of NAD metabolism and its regulatory role in aging and longevity^[8].

Potential Applications in the Treatment of Tuberculosis

Drug Target: 

The inactivation of the terminal enzyme of NAD synthesis, NAD synthetase (NadE), in Mycobacterium tuberculosis (Mtb) leads to a parallel decrease in the NAD and NADP pools and a decline in the viability of Mtb, while the inactivation of the terminal enzyme of NADP biosynthesis, NAD kinase (PpnK), selectively depletes the NADP pool but only stops growth (Sharma R, 2023). This indicates that NAD synthesis inhibitors have priority in the development of anti-tuberculosis drugs, because NAD deficiency is bactericidal, while NADP deficiency is bacteriostatic.

Metabolic Changes and Microbial Phenotypes: 

The inactivation of each enzyme is accompanied by metabolic changes specific to the affected enzyme and the related microbial phenotype. Bacteriostatic levels of NAD depletion cause a compensatory remodeling of NAD-dependent metabolic pathways without affecting the NADH/NAD ratio, while bactericidal levels of NAD depletion lead to the disruption of the NADH/NAD ratio and the inhibition of oxygen respiration ^[6].

The Role in Cellular Metabolism

Multiple Important Functions: 

NAD(H) and NADP(H) have traditionally been regarded as cofactors involved in countless redox reactions, including electron transfer in mitochondria. However, NAD pathway metabolites have many other important functions, including roles in signaling pathways, post-translational modifications, epigenetic changes, and regulating RNA stability and function through NAD capping of RNA^[9].

Dynamic Metabolic Process: 

Non-oxidative reactions ultimately lead to the net catabolism of these nucleotides, indicating that NAD metabolism is an extremely dynamic process. In fact, recent studies clearly show that in some tissues, the half-life of NAD is about a few minutes^[9].

The Role in Cell Biology

Extracellular NAD Metabolism: 

Extracellular NAD is a key signaling molecule under different physiological and pathological conditions. It acts directly by activating specific purinergic receptors or indirectly as a substrate for exonucleases (such as CD73, nucleotide pyrophosphatase/phosphodiesterase 1, CD38 and its paralog CD157, and ecto-ADP-ribosyltransferases). These enzymes determine the availability of extracellular NAD by hydrolyzing NAD, thus regulating its direct signaling effect (Gasparrini M, 2021). In addition, they can generate smaller signaling molecules from NAD, such as the immunomodulator adenosine, or use NAD to ADP-ribosylate various extracellular proteins and membrane receptors, having a significant impact on immune control, inflammatory response, tumorigenesis, and other diseases. The extracellular environment also contains nicotinamide phosphoribosyltransferase and nicotinic acid phosphoribosyltransferase, which catalyze key reactions in the NAD salvage pathway intracellularly. The extracellular forms of these enzymes act as cytokines with pro-inflammatory functions^[10].

In conclusion, NAD+ has become a key molecule connecting health and disease by regulating energy metabolism, delaying aging, regulating immunity, and providing protection for multiple systems. Supplementing its precursors can improve mitochondrial function and slow down the progression of metabolic and neurodegenerative diseases. It shows potential in the fields of cardiovascular protection, anti-infection, and anti-aging, providing innovative therapeutic targets for aging-related diseases.

Relevant Citations

[1] Lin Q, Zuo W, Liu Y, et al. NAD and cardiovascular diseases[J]. Clinica Chimica Acta, 2021,515:104-110.DOI:10.1016/j.cca.2021.01.012.

[2] Shats I, Li X. Bacteria boost host NAD metabolism[J]. Aging-Us, 2020,12(23):23425-23426.DOI:10.18632/aging.104219.

[3] Hopkins E L, Gu W, Kobe B, et al. A Novel NAD Signaling Mechanism in Axon Degeneration and its Relationship to Innate Immunity[J]. Frontiers in Molecular Biosciences, 2021,8.DOI:10.3389/fmolb.2021.703532.

[4] Cao Y, Wang Y, Yang J. NAD+-dependent mechanism of pathological axon degeneration.[J]. Cell Insight, 2022,1(2):100019.DOI:10.1016/j.cellin.2022.100019.

[5] Jiang Y F, Liu T T, Lee C, et al. The NAD⁺-mediated self-inhibition mechanism of pro-neurodegenerative SARM1[J]. Nature, 2020,588(7839):658.DOI:10.1038/s41586-020-2862-z.

[6] Sharma R, Hartman T E, Beites T, et al. Metabolically distinct roles of NAD synthetase and NAD kinase define the essentiality of NAD and NADP in Mycobacterium tuberculosis[J]. Mbio, 2023,14(4).DOI:10.1128/mbio.00340-23.

[7] Campagna R, Vignini A. NAD⁺ Homeostasis and NAD⁺-Consuming Enzymes: Implications for Vascular Health[J]. Antioxidants, 2023,12(2).DOI:10.3390/antiox12020376.

[8] Odoh C K, Guo X, Arnone J T, et al. The role of NAD and NAD precursors on longevity and lifespan modulation in the budding yeast, Saccharomyces cerevisiae[J]. Biogerontology, 2022,23(2):169-199.DOI:10.1007/s10522-022-09958-x.

[9] Chini C C S, Zeidler J D, Kashyap S, et al. Evolving concepts in NAD⁺ metabolism[J]. Cell Metabolism, 2021,33(6):1076-1087.DOI:10.1016/j.cmet.2021.04.003.

[10] Gasparrini M, Sorci L, Raffaelli N. Enzymology of extracellular NAD metabolism[J]. Cellular and Molecular Life Sciences, 2021,78(7):3317-3331.DOI:10.1007/s00018-020-03742-1.