NAD+ metabolism is one of the most important reactions in the body. NAD+ is an energy-providing molecule found in every body cell, used for metabolism, fighting free radicals, DNA damage, and cell signaling. Coenzyme NAD+ is naturally found in all body cells, but its presence decreases with aging. A therapeutic effect is achieved on various diseases and conditions associated with aging by boosting its levels. This anti-aging molecule binds to other enzymes in the body and, by activating them contributes to better organ function. The body absorbs the maximum possible amount of additional NAD+ coenzyme and converts it into molecular energy.
What is NAD+?
Nicotinamide adenine dinucleotide (NAD) is a cofactor crucial for metabolism. NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains the nucleobase adenine and the other nicotinamide. NAD exists in two forms: an oxidized and a reduced form, abbreviated as NAD+ and NADH (H for hydrogen), respectively. In metabolism, NAD plays an important role in electron transfer in various enzyme-catalyzed redox reactions.
In addition to NAD’s important function in electron transfer during redox reactions, NAD serves as an ADP-ribose donor in a post-translational modification of some proteins called ADP-ribosylation. In processes of deacetylation of some proteins, hydrolysis of NAD to ADP-ribose and nicotinamide allows removal of the acetyl group from the protein and attachment to ADP-ribose, forming O-acetyl-ADP-ribose.
Popular NAD+ precursors
People get NAD+ from their diet through foods made up of amino acids, which are also precursors of NAD+. However, NMN and NR are very efficient precursors of NAD+. If you can get NAD+ precursors through different routes, NMN and NR are often considered the best available route to NAD+. In addition to NMN and NR, Nicotinic Acid (NA), Nicotinamide (Nam), and Tryptophan (Trp) are also popular precursors of NAD+.
Tryptophan is the least effective NAD+ precursor because it has to move along various pathways to become NAD+.
How is NAD+ maintained in our body?
NAD+ levels in our bodies are maintained by three independent pathways, including:
Preiss-Handler pathway (dietary NA to NAD+)
This pathway begins with the conversion of nicotinic acid, whether from food or in a supplement form. The enzyme included in this reaction is nicotinic acid phosphoribosyltransferase (NaPRT), which converts NA into nicotinic acid mononucleotide (NaMN), its mononucleotide form. As a cosubstrate in this reaction is used 5-phosphoribosyl-1-pyrophosphate (PRPP).
In this reaction, as a starting point, NaMN is converted in its dinucleotide form nicotinic acid-adenine dinucleotide (NaAD), with the help of nicotinate mononucleotide adenylyltransferase (NMNAT). NMNAT can limit its activity during aging because NaMN has the same amount in the young and elderly, while NaAD is decreased in the old. In other words, NAD+ starts to decrease with aging, that’s why NMNAT activity can have some limitations.
In the final step of this process, NaAD is amidated into NAD+ with the help of glutamine-dependent NAD+ synthase (NADS).
De Novo biosynthesis pathway, also known as the Kynurenine pathway
In this process, Tryptophan is used for making NAD+. Quinolinic acid (QA) is used either from tryptophan or aspartic acid, and it is transformed into nicotinic acid mononucleotide (NaMN). Unstable α-amino-β-carboxymuconate-ε-semialdehyde (ACMS) is formed, which undergoes cyclization to form QA. The formation of ACMS into α-amino-β-muconate-ε-semialdehyde (AMS) is enabled by an enzyme called α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD). The production of ACMS is spontaneous. Furthermore, the amount of ACMS is determined for cyclization to produce NAD+.
Salvage pathway (NR, NMN to NAD+)
Salvage pathways use three precursors for NAD+, including NR, NMN, and Nam. These precursors can be obtained from the diet and produced within cells. This metabolic way of producing NAD+ is most known due to the high efficacy of the precursors. NAMPT (nicotinamide phosphoribosyltransferase) is an enzyme that converts NAM to NMN. In mammals, it is a rate-limiting enzyme, which is adelytaed to NAD with the help of NMNATs. NAMPT and NAPRT (nicotinate phosphoribosyltransferase) are the two crucial enzymes in the salvage pathway because they help NAD+ synthesize as the main energy source.
Covarrubias AJ, Perrone R, Grozio A, Verdin E. NAD+ metabolism and its roles in cellular processes during ageing. Nat Rev Mol Cell Biol. 2021 Feb;22(2):119-141. doi: 10.1038/s41580-020-00313-x. Epub 2020 Dec 22. PMID: 33353981; PMCID: PMC7963035.
(NAD+ metabolism, including de novo synthesis, Preiss-Handler pathway, and the Salvage pathway.)
How is NMN converted to NAD+?
NMN is a precursor to NAD+, and it is more efficiently and rapidly converted to NAD+ with the help of NMNAT. It is more efficient compared to NR because NMN can be directly converted into NAD+, and NR is an indirect precursor, which means it has to be converted into NMN first and then to NAD+ or it must undergo five pathways to NAD+ (NR → Nam → NAMN+ → NAAD+ → NAD+).
When the body signalizes a low level of NAD+, the cells try to produce more SLC12A8 transporters. One study found that NMN has a specific transporter gene called SLC12A8. This gene is found in more tissues and abundant in the small intestine, where the gut quickly absorbs NMN, converting it to NAD+ within 20-30 minutes. This study was conducted on mice research.
Also another way to boost NAD+ levels is for NMN to be converted to NR outside of the cells. This conversion is helped by the CD73 enzyme, where NR is converted to NMN back by NRKs in the cell.
Wu LE, Sinclair DA. The elusive NMN transporter is found. Nat Metab. 2019 Jan;1(1):8-9. doi: 10.1038/s42255-018-0015-6. PMID: 32694812.
(NMN cell transport mechanisms.)
Three main classes of NAD+– consuming enzymes
Since NAD+ is a very important molecule in the body, it can be divided by three NAD+-consuming enzymes, including the sirtuins, Poly(ADP-ribose) polymerases (PARP1–PARP3), and CD38.
Sirtuins
Sirtuins genes are responsible for many processes, including repairing damaged DNA, reducing inflammatory processes, maintaining healthy mitochondria, and more. There are seven types of sirtuins SIRT1-SIRT7, localized in different places in the body. The changes in NAD+ levels are correlated to these NAD+-consuming enzymes because they can change the organelle-specific sirtuin functions. Sirtuins are always actively doing their specific functions. One-third of total NAD+ consumption belongs to SIRT1 and SIRT2. The NAD+ levels will significantly increase under a calorie-restricted diet and fasting. Also, it is worth mentioning that sirtuin activity is correlated with the circadian clock, where SIRT1 and NAMPT take place in the circadian regulation of NAD+ levels.
The sirtuins remove the acetyl group from the lysine residuals with NAD+ as a cofactor. NAD+ is split, producing nicotinamide (NAM) and ADP-ribose. What is known about the function of these genes is that they are sensors for energy consumption and energy flow in cells. They regulate homeostasis, mitochondrial metabolism, and quality, which can impact NAD+ levels and improve age-related conditions. Due to these characteristics in the cellular processes, the sirtuins can be used as a possible treatment against aging.
Poly(ADP-ribose) polymerases (PARP1–PARP3)
Poly(ADP ribose) (PAR) is a post-translational modification of proteins from NAD+ synthesized by proteins from the poly(ADP-ribosyl) polymerase (PARP) family. PARylation is a reversible reaction, and specialized enzymes such as poly(ADP-ribosyl) glycohydrolase (PARG) hydrolyze PAR polymers. PAR participates in regulating numerous cellular processes such as transcription, replication, senescence, .intracellular transport, cell death, and repair of DNA.
The first discovered PARP is PARP1, a protein that participates in several processes (repair DNA damage, transcription control, and chromatin remodeling) and accounts for the majority of PARylation activities in the cell. 17 different proteins from the PARP family have been discovered that differ according to the composition of additional non-catalytic protein domains and specific roles in the cell. According to common biochemical characteristics and roles in the cell, they are divided into 6 subgroups. For example, PARP proteins from subgroup 1, which includes PARP1 and PARP2 and PARP5aand PARP5b from subgroup 4 catalyze the synthesis of PAR chains with long ADP ribose chains, while most other PARPs possess primarily mono(ADPribosyl)transferase activity. For this reason, PARPs are often called ADP-ribosyl transferases (ARTDs).
PARP2 is similar to PAP1, and it can impact NAD+ bioavailability. In one mice study, PARP2 shows a big influence on sirtuins activation. PARP3 is also responsible for repairing DNA damage. Further investigation into the other PARP proteins is needed, but they are believed to be less important in regulating NAD+ levels.
CD38
CD38, together with CD157, are multifunctional proteins that serve not only as antigens but also as enzymes. CD38 is found on the surface of the immune cells, such as white blood cells, B lymphocytes, CD4, CD8, and more. It can act in calcium signaling, cell adhesion, immune response, and more. CD38 is mainly used in inflammatory processes and antimicrobial resistance. The antimicrobial resistance is linked to the NAD+ metabolites from bacteria that need NAD+ for growth and survival. CD38 can break down NMN to NAM and ribose monophosphate (RMP). CD38 has ADP-ribosyl cyclase activity, generating NAM and cADPR from NAD+. Because CD38 takes place in a base-exchange reaction, it can switch the NAM of NAD(P)+ for nicotinic acid (NA), producing nicotinic acid adenine dinucleotide (phosphate) (NAAD(P)).
The apigenin powder is a popular CD38 enzyme inhibitor, and works with NMN to improve your NAD+ levels.
Covarrubias AJ, Perrone R, Grozio A, Verdin E. NAD+ metabolism and its roles in cellular processes during ageing. Nat Rev Mol Cell Biol. 2021 Feb;22(2):119-141. doi: 10.1038/s41580-020-00313-x. Epub 2020 Dec 22. PMID: 33353981; PMCID: PMC7963035.
(Three main classes of NAD+– consuming enzymes, including sirtuins, Poly(ADP-ribose) polymerases (PARP1–PARP3), and CD38.)
Cellular roles of NAD+
NAD + plays several important roles in metabolism, including:
Metabolic dysfunction
NAD+ directly impacts the activity of metabolic enzymes in many energy production pathways. The most common metabolic disease is obesity. People with obesity are more likely to develop diabetes type 2, high blood pressure, high blood glucose levels, and more symptoms. According to a study done in yeast extract, it is known that NAD+ can regulate metabolic rates. The longevity gene SIRT2 was discovered to be correlated with metabolic status. Time-restricted diet, calorie restriction, and the keto diet boost the NAD+ levels, thus activating the sirtuins genes. In addition, increasing cellular NAD+ levels have been linked with metabolic reactions because it can reduce stress and activate deacetylase and deacylase activity, in both, SIRT1 (in the nucleus) and SIRT 3 (in the mitochondria).
Immune function
Inflammation has been recognized as the main factor for aging and the development of age-related diseases. The innate and adaptive immune system can respond properly during aging due to reduced cell functions called immunosenescence. Chronic inflammation increases certain inflammatory cytokines such as IL-6, IL-1β, and TNF (tumor necrosis factor), which are recognized to play a crucial role in developing metabolic disease. Certain inflammatory signals lead to a switch in macrophage polarization states, which in aging the polarization state is altered, and the macrophage is present in abundance. Increased levels of NAD+ have been shown to impact macrophage function and reduce the levels of TNF and other proinflammatory interleukins. In the adaptive immune system, taking NAD+ precursor or CD38 inhibitors may be used as therapeutical agents to boost NAD+ levels in aging.
Neurodegeneration
Nicotinamide acts on neurogenesis by accelerating the embryonic differentiation of stem cells or neural progenitor cells into post-mitotic neurons. NAD+ proved to be an important item in extending the lifespan of nerve cells, which is most evident in neurotransmission, learning, and memory. Dysfunction has been found in the early stages of Alzheimer’s disease, as well as in aging mitochondria and reduced NAD+ concentrations. In mice, it has been proven that increasing the concentration of NAD+ can improve mitochondrial functions because NAD+ serves as an antagonist to cognitive decline. In patients with Parkinson’s disease, a decrease in the concentration of NAD+ is visible. NAD+ supplement diet prevents the degeneration of neurons, which suggests that neurotoxicity is associated with mitochondrial defects and can be prevented by including the NAD+ salvation path to increase the NAD+ bioavailability.
NAD+ metabolism in aging
The higher the level of NAD+, the more activated sirtuins in the body are, which in turn means that we have healthier mitochondria. This prolongs longevity, thus fighting the signs of aging. The NAD+ enzyme is said to potentially slow down the aging process. NAD+ is found in all body cells and is primarily responsible for DNA repair. NAD+ also works with proteins to maintain good health, especially under stressful conditions. NAD+ is also known as the primary component responsible for cellular energy generation. Although NAD+ cannot stop aging, studies show that NAD+ can at least slow down the aging process.
References
- Katsyuba, E., Mottis, A., Zietak, M., De Franco, F., van der Velpen, V., Gariani, K., Ryu, D., Cialabrini, L., Matilainen, O., Liscio, P., Giacchè, N., Stokar-Regenscheit, N., Legouis, D., de Seigneux, S., Ivanisevic, J., Raffaelli, N., Schoonjans, K., Pellicciari, R., & Auwerx, J. (2018). De novo NAD+ synthesis enhances mitochondrial function and improves health. In Nature (Vol. 563, Issue 7731, pp. 354–359). Springer Science and Business Media LLC. https://doi.org/10.1038/s41586-018-0645-6
- Li, X., Lei, J., Mao, L., Wang, Q., Xu, F., Ran, T., Zhou, Z., & He, S. (2019). NAMPT and NAPRT, Key Enzymes in NAD Salvage Synthesis Pathway, Are of Negative Prognostic Value in Colorectal Cancer. In Frontiers in Oncology (Vol. 9). Frontiers Media SA. https://doi.org/10.3389/fonc.2019.00736
- Covarrubias, A. J., Perrone, R., Grozio, A., & Verdin, E. (2020). NAD+ metabolism and its roles in cellular processes during ageing. In Nature Reviews Molecular Cell Biology (Vol. 22, Issue 2, pp. 119–141). Springer Science and Business Media LLC. https://doi.org/10.1038/s41580-020-00313-x
