Restoring NAD(+) Levels with NAD(+) Intermediates,the Second Law of Thermodynamics,and Aging Delay

Aliving organism is a highly ordered low entropy system. However, this order comes at a price and investments of vast amounts of energy are needed to keep the entropy of the system low. Since many natural processes can be viewed in light of the second law of thermodynamics, a hypothesis regarding the role of increased nicotinamide adenine dinucleotide (NAD+) levels with reference to the fundamental concepts of aging and entropy is presented. A living system is not a closed system as organisms can exchange matter and energy with the environment; the systems that can be precisely analyzed using the second law are in general much simpler, and adiabatically isolated. As one author points out, “The ongoing ability to gather free energy from the environment and concentrate it as order within, while discarding entropy as waste is a defining property of living systems.” ¹

The second law of thermodynamics, which refers to the quality of energy and its degradation, states that there is a natural tendency of any isolated system to degenerate into a more disordered state and that “no natural process can occur unless it is accompanied by an increase in the entropy of the universe.”² Organisms, including humans, increase the entropy of their environment by consuming energy-containing substances, by convection, conduction, and heat radiation. Although the organisms increase their level of organization–decrease the entropy by growing, evolving, and becoming more complex, this is done at the expense of the energy investment that will increase entropy in their environment. ³

If entropy is a measure of the “disorder” in a system, aging could be described as an increased state of entropy, ⁴ where randomness and disorder increase along with the susceptibility to age-related disorders. Demetrius has argued that aging is the result of an increase in molecular disorder due to an increase in thermodynamic entropy. ⁵ A lowered energy state is not necessarily a disorder, but if this leads to a limited supply of energy for damage repair and genomic stability maintenance of a cell, then the disorder inside the cell will increase with time. With age, organisms have lower ability to fine-tune the homeostasis to internal and external stressors and, as a consequence, the cellular damage accumulates.

The oxidized form of the coenzyme NAD+ is found in all living cells and is currently an intensely investigated topic in longevity science. ^6,7 As NAD(+) levels decline with age, ^8,9 mitochondrial function is impaired ¹⁰ and the DNA repair activity also declines, ¹¹ resulting in increased oxidative damage. ⁸ All of these events may lead to increased disorder. Indeed, decreased NAD+ influences the hypoxia-inducible factor 1-alpha, which promotes a hypoxic-like state (Warburg effect ¹² ) in the cell ¹³ favoring enhanced glycolysis, which is highly inefficient for generating cellular energy in the form of adenosine triphosphate (ATP). Even under normoxic conditions, glycolysis generates only 2 mols of ATP per 1 mol of glucose, whereas oxidative phosphorylation generates about 36 mols of ATP per 1 mol of glucose. ¹⁴ Therefore, NAD(+) deficiency results in insufficient ATP production, metabolic reprogramming, and limited energy (and substrate) for DNA repair and other cellular processes.

On the other hand, disorder can be manipulated by increasing the available energy in the system. For example, increasing NAD(+) level increases available cellular energy and improves mitochondrial and stem cell function, DNA repair, ¹⁵ and enhances lifespan. ¹⁶ Increased NAD(+) levels would activate Poly (ADP-ribose) polymerases, sirtuins, and regulate the genes involved in the DNA repair and maintenance process, ¹⁷ thus increasing the order of the system. In addition, raising NAD(+) levels would promote oxidative metabolism, which yields more ATP ¹⁸ compared with glycolysis as the bioavailability of NAD(+) is the limiting factor for the maximum oxidative capacity of mitochondria, thus improving the energy efficiency of cells.

If the organism was manipulated by supplementing NAD(+) intermediates, then the metabolic rate (MTR) or/and body temperature or/and ATP levels should be altered since processes that do not increase disorder require and/or provide energy: Δ entropy = ΔMTR/T (by dividing the MTR by body temperature, the number which is a general measure of our entropy production is obtained ¹⁹ ). Slowing down the aging process (according to the second law of thermodynamics) should result in increased stress resistance and exercise performance, uncoupling, and thermogenic activity, as well as increased mitochondrial activity and aerobic capacity.

Several studies have validated the hypothesis that NAD(+) precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), can be used as a nutraceuticals to prevent or lower the entropy. For example, in the study of Yoshino et al., the NMN-administered mice showed that higher body temperature, oxygen consumption, and energy expenditure did not significantly decrease with age in contrast to the control group of tested animals. ²⁰ Mice treated with NMN for 12 months were able to maintain both, oxygen consumption and energy expenditure, close to those of the control mice at 6 months after the NMN administration. NMN was also shown to stimulate mitochondrial oxidative metabolism in mice skeletal muscles. ²⁰ Similarly, treatment of 22-month-old mice for 1 week with NMN increased ATP and enhanced OXPHOS. ²¹ On the other hand, age-related decline in muscle mitochondrial function related to peak oxygen uptake has been observed by several investigators (reviewed in Ref. ²² ).

NR treatment enhanced the oxidative performance of skeletal muscle and brown adipose tissue in mice due to enhanced mitochondrial function. ²³ Unlike white fat, brown fat generates heat by containing large amounts of mitochondria. In a recent article of Zhang et al., ¹⁶ NR treatment of the muscle stem cells (MuSCs) of aged animals increased the expression of genes whose products function in the TCA cycle, OXPHOS, and in the mitochondrial unfolded protein response (UPRmt). Increases in oxidative respiration resulted in higher mitochondrial membrane potential and more ATP in the NR-treated MuSCs of aged animals. ²⁴ One of the factors known to affect thermodynamics is temperature; the NR-fed mice had also enhanced thermogenic capacity, inferred from the ability to maintain body temperature during exposure to cold. ²³

For example, nicotinamide adenine dinucleotide oxidized form (NAD)/nicotinamide adenine dinucleotide, reduced form (NADH) ratio can be increased by NADH quinone oxidoreductase 1 (NQO1) by oxidizing NADH to NAD. Feeding beta-lapachone to tested animals, an exogenous (NQO1) cosubstrate, prevented the age-dependent decline of motor and cognitive function in aged mice. ²³ Additionally, increased energy expenditure was observed as measured by VO2max and by the heat generation. ²⁴ NAD+ can be increased also during caloric restriction or fasting. Indeed, the time-restricted (intermitted fasting) high-fat-fed mice have significantly increased thermogenesis and improved amplitude of circadian rhythms and energy expenditure, leading to many improved health parameters. ²⁵

Women tend to have a slightly higher body temperature and live longer than men (36.4°C ± 0.67°C vs. 36.2°C ± 0.61°C, respectively) and their mean temperature decreases 0.17°C between the ages of 20–30 and 70–80. ²⁶ Also, marsupials have a low body temperature when compared with placental mammals and generally have shorter lifespans, whereas birds tend to have a higher T_b and live longer than similar-sized mammals. ²⁷ On the contrary, some studies show a correlation between lower body temperatures and greater longevity, although there is no proof of a cause-and-effect relationship in humans. Although the view that an increase in the body temperature by itself affects aging is overly simplistic (with many exceptions to the rule in the poikilotherms as reviewed by Keil et al., ²⁸ ); an increased metabolism due to increased temperature and energy efficiency might, at least in theory, result in increased oxidative and/or DNA damage. ^29,30,31 On the other hand, moderate reactive oxygen species (ROS) concentrations might trigger an adaptive stress response (hormesis) and provoke an increased endogenous antioxidant protection and an activation of damage repair processes. ³² Also, mitochondria from females produce fewer ROS than those from males. ³³ Since the reduction state of complex I strongly depends on the NAD+ and NADH levels, more oxygen at rest and increased heat production might be a consequence of the mitochondrial uncoupling effect, ³⁴ which leads to fast electron flow and low leakage and results in decreased susceptibility to degenerative conditions and increased longevity. ³⁴

In the end, it should be stressed that constant elevation of NAD+ in the cells might disturb the delicate circadian clock mechanism, which might be disrupted by changes in NAD+ metabolism. Namely, NAD+ displays a 24-hour circadian rhythmicity due to direct clock transcriptional control of the rate-limiting enzyme in NAD+ biosynthesis, nicotinamide phosphoribosyltransferase, ³⁵ which regulates the cellular metabolism in the circadian oscillation. What is more, recent reports have shown that NAD+ may have deleterious effects on certain cell types. For instance, it was reported that extracellular NAD+ kills naive T cells ³⁶ and incubation of cells with NAD+ might induce cellular death by activation of P2X7 receptor on the cell surface, which triggers apoptosis. ^{28,37 –39}

Considering the second law of thermodynamics, NAD+ seems the appropriate candidate for reversing many aging-associated pathologies, and should be an appropriate candidate for further studies investigating how enhanced NAD+ biosynthesis affects thermogenic capacity, aging delay, and an increase in health span. NAD+ is an essential compound that enables organisms to stay highly organized and well maintained, with a lower entropy inside. However, it is important to bear in mind that organisms are complex systems and even at the cellular level there are several layered homeostatic mechanisms that work over short, medium, and long terms, which interact with one another. Therefore, any modulator must be tested in all these timeframes to deduce its integrated effect on the homeostasis. Only rigorous testing can provide the answers on the optimal physiological levels of NAD+.