What is NAD+?
Nicotinamide adenine dinucleotide (NAD+) was discovered more than a century ago by Sir Arthur Harden as a low molecular weight substance present in a boiled yeast extract, which could stimulate fermentation and alcohol production in vitro. Subsequent studies over the next several decades determined that the structure of NAD+ comprised two covalently joined mononucleotides (nicotinamide mononucleotide or NMN, and AMP), and identified the keystone function of NAD+ and NADH as enzyme cofactors mediating hydrogen transfer in oxidative or reductive metabolic reactions.
From single-cell organisms like bacteria to sophisticated multicellular ones like primates, NAD+ is one of the most abundant and crucial molecules. Basically, without NAD+, we would be on the fast track to death. The molecule is a linchpin to the function of the generators of cells, mitochondria. NAD+ not only helps convert food to energy but also plays a crucial role in maintaining DNA integrity and ensures proper cellular function to protect our bodies from aging and disease.
How does NAD+ work?
NAD+ works as a shuttle bus, transferring electrons from one molecule to another within cells to carry out all sorts of reactions and processes. With its molecular counterpart, NADH, this vital molecule participates in various metabolic reactions that generate our cell’s energy. Without sufficient NAD+ levels, our cells wouldn’t be able to generate any energy to survive and carry out their functions. Other functions of NAD+ include regulating our circadian rhythm, which controls our body’s sleep/wake cycle.
NAD+ Levels Decline with Age
As we age, NAD+ levels fall, suggesting important implications in metabolic function and age-related diseases. DNA damage accumulates and snowballs with aging. See image below.
The damage to our genetic blueprint activates several proteins, including enzymes called PARPs. By consuming NAD+, PARPs can perform DNA repair functions. The depletion of NAD+ through PARP activation during aging appears to contribute to various diseases. Out of all these functions that require NAD+, many scientists believe that PARPs contribute the most.
Enzymes in our immune system consume NAD+, too. The more active the immune system is the more NAD+ the enzyme consumes. The level of enzymes in our immune system increases as we age, depleting the NAD+ levels in the body.
Another class of enzymes that use NAD+ are called sirtuins. These proteins, which are linked to healthy aging and longevity, use NAD+ to regulate metabolism, maintain stable chromosomes, and repair damaged DNA. As DNA damage and chromosome instability accumulate with age, sirtuins consume more NAD+.
What have Research Studies Shown?
Scientific research has revealed that NAD+ can do the following:
• Aid in the aging process
• Increase muscle function
• Improve metabolic disorders
• Improve heart function
• Neuroprotective capabilites
Known as the “guardians of genomes,” sirtuins are genes that protect organisms, from plants to mammals, against deterioration and diseases. When the genes sense the body is under physical stress, such as exercising or hunger, it sends out troops to defend the body. Sirtuins sustain genome integrity, promote DNA repair and have shown anti-aging related properties in model animals like increasing lifespan.
Decreased levels of NAD+ are associated with the hallmarks of aging as well as several age-related diseases, including metabolic disorders, cancer and neurodegenerative diseases. Replenishment of NAD+ levels via administration of its precursors have been demonstrated to display beneficial effects against aging and age-related diseases. Importantly, boosting NAD+ levels have been shown to extend lifespan of various laboratory animal models including worms, flies, and rodents.
NAD+ and Sirtuins in Aging and Disease.
“Nicotinamide adenine dinucleotide (NAD+) is a classical coenzyme mediating many redox reactions. NAD+ also plays an important role in the regulation of NAD+-consuming enzymes, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38/157 ectoenzymes. NAD+ biosynthesis, particularly mediated by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 function together to regulate metabolism and circadian rhythm. NAD+ levels decline during the aging process and may be an Achilles’ heel, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies. Restoring NAD+ by supplementing NAD+ intermediates can dramatically ameliorate these age-associated functional defects, counteracting many diseases of aging, including neurodegenerative diseases. Thus, the combination of sirtuin activation and NAD+ intermediate supplementation may be an effective anti-aging intervention, providing hope to aging societies worldwide.”
Various uses of NAD+ for canonical redox and NAD+-consuming enzymatic reactions. Whereas NAD+ is converted to NADH by many metabolic enzymes (a), it is also used as a cosubstrate for NAD+-consuming enzymes, such as poly-ADP-ribose polymerases (PARPs) (b), sirtuins (c), and CD38/157 ectoenzymes (d).
From the above study, we know that NAD+ plays a key role in regulating metabolism and circadian rhythm through sirtuins, NAD+ becomes limiting during aging, affecting sirtuin’s activities. NAD+ declines due to an NAD +biosynthesis defect and increased depletion, and supplementing key NAD+ intermediates can restore NAD+ levels and ameliorate age-associated pathophysiology’s.
Muscle Function
NAD+ is a major player in skeletal muscle development, regeneration, aging, and disease. The vast majority of studies indicate that lower NAD+ levels are deleterious for muscle health and higher NAD+ levels augment muscle health.
Increasing NAD+ levels in muscle can improve its mitochondria and fitness in mice. Other studies also show that mice that take NAD+ boosters are leaner and can run farther on the treadmill, showing a higher exercise capacity. Aged animals that have a higher level of NAD+ outperforms its peers.
A need for NAD+ in muscle development, homeostasis, and aging.
In a review study, researchers discuss the recent data that document conserved roles for NAD+ in skeletal muscle development, regeneration, aging, and disease as well as interventions targeting skeletal muscle and affecting NAD+ that suggest promising therapeutic benefits. The researchers also highlight gaps in our knowledge and propose avenues of future investigation to better understand why and how NAD+ regulates skeletal muscle biology.
Compartmentalization of NAD+ pools in skeletal muscle. Diagram of a striated skeletal muscle fiber. NAD+ is localized to mitochondrial, nuclear, cytosolic, and membrane proximal pools in muscle cells. Additional NAD+ compartments not diagrammed here include vesicular compartments. The NAD+/NADH ratio is higher in the nuclear and cytosolic compartments compared to the mitochondrial compartment. The ratio is unknown in the membrane proximal compartment in muscle. Enzymes that consume NAD+ and their relative subcellular localizations are found within black or white boxes. Integrin receptors and membrane channels that transport NAD+ and calcium across the sarcolemma can be seen in the diagram.
“It is clear that NAD+ plays a beneficial role in muscle health. The mechanisms underlying promotion of muscle development and homeostasis by NAD+ are best understood in the context of sirtuin regulation in the nucleus, mitochondria, and cytosol. The role of NAD+ in other cellular compartments—particularly the vesicular and membrane proximal compartments—in muscle health is currently understudied. Future research will likely delve into not only the mechanisms of NAD+ action in these different compartments but also the interplay and signaling between NAD+ pools within and between cells.”
Metabolic Disorders
Declared as an epidemic by the World Health Organization (WHO), obesity is one of the most common diseases in modern society. Obesity can lead to other metabolic disorders such as diabetes, which killed 1.6 million people around the globe in 2016.
Aging and a high-fat diet reduce the level of NAD+ in the body. Studies have shown that taking NAD+ boosters can alleviate diet-associated and age-associated weight gain in mice and improve their exercise capacity, even in aged mice. Other studies even reversed the diabetes effect in female mice, showing new strategies to fight metabolic disorders.
Implications of altered NAD metabolism in metabolic disorders.
NAD metabolism has a potential protective effect against various metabolic diseases through redox reactions, sirtuins, and possibly PARPs. NAD is a co-enzyme that mediates various redox reactions in glycolysis, the TCA cycle, fatty acid oxidation, and oxidative phosphorylation. It also serves as a substrate for PARPs and sirtuins and regulates various biological pathways, including energy metabolism, gene expression, DNA repair, and cellular stress response. See the image to the right.
“NAD metabolism is spotlighted as a therapeutic target for metabolic disorders, such as obesity, diabetes, dyslipidemia, and fatty liver. The genetic manipulation of NAD synthesis or catabolizing enzymes has established that reduction in NAD levels causes metabolic disorders in mice. Furthermore, mounting evidence has demonstrated that complementing NAD with NAD precursors ameliorates various metabolic diseases. Recently, several human clinical trials have been reported. Overall, NR administration is safe, well tolerated, and can efficiently increase NAD levels in healthy volunteers. However, efficacy in patients with metabolic disorders remains unclear, and further studies are awaited. Moreover, some small molecules boosting NAD levels have been reported. Outcomes of these molecules against metabolic diseases in patients should be clarified in future studies. It is also demonstrated that NMN and NR are contained in natural foods, including cow milk, broccoli, cucumber, avocado, and beef. Thus, NAD metabolism is considered a practical target for a nutritional intervention.”
Heart Function
The elasticity of the arteries acts as a buffer between pressure waves sent out by heartbeats. But arteries stiffen as we age, contributing to high blood pressure, the most important risk factors for cardiovascular disease. One person dies from cardiovascular disease every 37 seconds in the United States alone, CDC reports.
High blood pressure can cause an enlarged heart and blocked arteries that lead to strokes. Boosting NAD+ levels give protection to the heart, improving cardiac functions. In mice, NAD+ boosters have replenished NAD+ levels in the heart to baseline levels and prevented injuries to the heart caused by a lack of blood flow. Other studies have shown that NAD+ boosters can protect mice from abnormal heart enlargement.
NAD+ Metabolism as an Emerging Therapeutic Target for Cardiovascular Diseases Associated with Sudden Cardiac Death.
Here, researchers review the basics of NAD+ homeostasis, the molecular physiology and new advances in ischemic-reperfusion injury, heart failure, and arrhythmias, all of which are associated with increased risks for sudden cardiac death. They also summarize the progress of NAD+-boosting therapy in human cardiovascular diseases and the challenges for future studies.
Mechanisms of cardio protection from NAD+ repletion. Pathological stimuli that predispose the heart to SCD-associated CVD may result in reduced activity of NAMPT and increased activities of CD38 and PARP, which leads to NAD+ depletion and disease progression. Repletion of NAD+ by supplementation of NAD+ boosters confer cardioprotective effects through multiple signaling pathways. NAD+ repletion enhances catabolism and stimulates ATP production resulting in downregulation of AMPK, which prevents overactivation of autophagy, promotes the PI3K/AKT and ERK1/2 pro-survival kinase pathways (RISK pathways), and inhibits the mPTP-induced cell death. NAD+ repletion also activates SIRT1/FOXO1/MnSOD pathway that increases the clearance of ROS, SIRT3-dependent mitochondrial protein deacetylation that restores mitochondrial function and SIRT7-dependent deacetylation of GATA4 which inhibits hypertrophy-related gene expression. These mechanisms lead to reduced myocardial injury and hypertrophic remodeling as well as improved cardiac function. In addition, NAD+ repletion activates CD38-mediated Ca2+ signaling and PKC-dependent phosphorylation which improves the function of cardiac sodium channel NaV1.5 and lowers the risk for arrhythmia.
“In summary, current human studies have shown that NAD+-boosting therapy can reduce mortality (Carlson and Rosenhamer, 1988; Berge and Canner, 1991; Brown et al., 2001; Landray et al., 2014) and provide moderate clinical benefits for patients with CAD. However, conflicting results on critical clinical outcomes such as incidence of composite mortality and major vascular events have raised the concern that whether NAD+-boosting therapy can ultimately become a primary treatment for CAD and other CVD. Several important aspects may help overcome these hurdles. First, it is critical to determine the effective dose of NAD+ boosters for each individual patient. Direct measurement for NAD+ level or NAD+ metabolome from accessible samples [i.e., plasma (Grant et al., 2019)] should be considered. It is possible to achieve the effective therapeutic level, novel NAD+ precursors or novel pharmaceutical formulations are required. Second, the optimal time window for NAD+ booster supplementation remains to be established in human subjects. NAD+-boosting therapy should coordinate with the intrinsic circadian oscillation of NAD+ level in human body so that maximal beneficial effects can be achieved. With a more nuanced understanding of NAD+ biology in the heart and clinical studies designed with more sophistication, we anticipate that NAD+-boosting therapy would ultimately harness its potential for SCD-associated CVD.”
Neurodegeneration
By 2050, the world’s population aged 60 and older is projected to total 2 billion, nearly double the number of 2015, according to WHO. People worldwide are living longer. However, aging is the main risk factor for many neurodegenerative diseases including Parkinson’s disease and Alzheimer’s, causing cognitive impairment.
In mice with Alzheimer’s, raising the NAD+ level can decrease protein build up that disrupts cell communication and increases cognitive function. Boosting NAD+ levels also protects brain cells from dying when there’s insufficient blood flow to the brain. Many studies in animal models present new prospects of helping the brain age healthy and defend against neurodegeneration.
NAD + in Brain Aging and Neurodegenerative Disorders.
“NAD+ is a pivotal metabolite involved in cellular bioenergetics, genomic stability, mitochondrial homeostasis, adaptive stress responses, and cell survival. Multiple NAD+-dependent enzymes are involved in synaptic plasticity and neuronal stress resistance. Here, we review emerging findings that reveal key roles for NAD+ and related metabolites in the adaptation of neurons to a wide range of physiological stressors and in counteracting processes in neurodegenerative diseases, such as those occurring in Alzheimer’s, Parkinson’s, and Huntington diseases, and amyotrophic lateral sclerosis. Advances in understanding the molecular and cellular mechanisms of NAD+-based neuronal resilience will lead to novel approaches for facilitating healthy brain aging and for the treatment of a range of neurological disorders.”
Relationships between NAD+ and the Ten Hallmarks of Brain Aging.
The 10 hallmarks of brain aging include mitochondrial dysfunction; accumulation of oxidative damage; impaired waste disposal including autophagy, mitophagy, and proteostasis; Ca2+ deregulation; compromised adaptive stress responses; dysfunctional neuronal network; impaired DNA repair; inflammation; impaired neurogenesis; and senescence and telomere attrition. Evidence from cell culture, C. elegans, and mouse studies shows that NAD+ augmentation counteracts the adversities of the hallmarks of brain aging. See the text for details. iPSCs, induced pluripotent stem cells; NAD+, nicotinamide adenine dinucleotide; UPRmt, mitochondrial unfolded protein response.
NAD+ Depletion and Impaired Mitophagy Are Pivotal Events in Common Neurodegenerative Diseases.
Based on the evidence summarized in the current review, we propose a hypothesis that may explain, in part, why age is the primary driver of the common neurodegenerative diseases. At a younger age, sufficient cellular NAD+ maintains mitochondrial quality to sustain normal neuronal function via the NAD+-dependent regulations of autophagy/mitophagy, UPRmt, proteasome degradation, and mitochondrial biogenesis. As we age, increased NAD+ consumption drives NAD+ depletion, leading to impaired mitochondrial homeostasis and neuronal function. Depending on the disease-related pathogenesis in the patient, age-dependent NAD+ depletion and impaired mitophagy may exacerbate the disease progression. This hypothesis explains why age makes people susceptible to neurodegenerative diseases, but it is not sufficient. Center: the mitochondrion in the center exhibits the various mitochondrial deficiencies observed in the four neurodegenerative diseases AD, PD, HD, and ALS. These include impairment of Complex I (CI) in the ETC utilizing NADH as an electron donor, creating NAD+. Impairments of both CI and ATP production create reactive oxygen species (ROS), which increase the level of oxidative stress, likely affecting both the ETC itself and the TCA cycle, and increasing the amount of oxidative damage. This can again affect the flux of Ca2+ and the membrane potential, resulting in dysfunctional mitochondria. Furthermore, compromised autophagy and/or mitophagy has been linked to several neurodegenerative diseases, resulting in accumulation of dysfunctional mitochondria. The four panels surrounding the mitochondrion illustrate the suggested explanations for NAD+ depleted and the downstream effects of NAD+ depletion in AD, PD, HD, and ALS. See the text for detailed explanations and references. Kynurenine P, kynurenine pathway.
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