Nicotinamide Adenine Dinucleotide (NAD): Difference between revisions

Latest revision as of 00:40, 3 February 2024

Nicotinamide Adenine Dinucleotide (NAD) is a vital coenzyme found in every cell of our bodies and has become a focal point in the field of longevity and aging research. NAD+ plays a central role in energy metabolism and is essential for the function of several enzymes that are associated with aging and DNA repair.

NAD exists in two main forms: NAD+ and NADH. NAD+ is the oxidized form of the compound and is essential for various cellular processes, including DNA repair, gene expression, and calcium signaling. When NAD+ accepts electrons during metabolic reactions, it becomes reduced and transforms into NADH. NADH, the reduced form, primarily functions in the production of ATP, the cell's primary energy currency, through the electron transport chain. The dynamic interconversion between these two forms, NAD+ and NADH, is fundamental to the cell's energy production and overall function.

The NAD+/NADH ratio is an important cellular indicator, reflecting the cell's metabolic state. A healthy balance between NAD+ and NADH is required for optimal function of several key enzymes, including those involved in energy production, DNA repair, and cell signaling.

The Role of NAD+ in the Cell

NAD+ is involved in several crucial biological processes:

Energy Production: NAD+ helps in converting nutrients into energy within the mitochondria, the powerhouse of cells.
DNA Repair: It's essential for the function of enzymes like PARPs and sirtuins, which are involved in DNA repair and have links to longevity.
Cell Signaling: As a substrate for various enzymes, it plays a role in cellular communication and adaptations to stress.

CD38/NADase increases during aging, and causes NAD decline and subsequent mitochondrial dysfunction.

NAD+ Decline with Age

A significant finding in the field of aging research is that NAD+ levels naturally decline as we age. This reduction has been associated with:

A decrease in mitochondrial function, leading to reduced energy output.
Reduced activity of sirtuins, proteins linked to lifespan extension in various organisms.
Enhanced vulnerability of DNA to damage.
Increased susceptibility to age-related diseases such as diabetes, cardiovascular diseases, and neurodegenerative diseases.

A gradual increase in CD38 has been implicated in the decline of NAD+ with age.^[1]^[2] Treatment of old mice with a specific CD38 inhibitor, 78c, prevents age-related NAD+ decline.^[3] CD38 knockout mice have twice the levels of NAD+ and are resistant to age-associated NAD+ decline,^[4] with dramatically increased NAD+ levels in major organs (liver, muscle, brain, and heart).^[5] On the other hand, mice overexpressing CD38 exhibit reduced NAD+ and mitochondrial dysfunction.^[4]

Boosting NAD+ Levels

Given the importance of NAD+ in various cellular functions and its decline with age, researchers have been exploring ways to replenish or boost NAD+ levels in the body. Several methods are under investigation:

Nicotinamide Mononucleotide (NMN): A precursor to NAD+ that, when supplemented, has shown potential in increasing NAD+ levels in various studies, mainly in animals.
Nicotinamide Riboside (NR): Another NAD+ precursor that can elevate NAD+ levels in the body.
Caloric Restriction: It has been observed to enhance NAD+ levels and activate sirtuins.
NAD+ Infusions: Direct infusion of NAD+ is being explored as a method, although it's still in the early stages of research.

⇒ see NAD+ Booster

Safety and Implications for Longevity

While initial studies, primarily on animal models, have shown promise in boosting NAD+ levels for promoting health and extending lifespan, it's essential to approach the findings with caution. Comprehensive human trials are needed to understand:

The long-term effects of boosting NAD+.
The effective dosages and potential side effects.
The real impact on human longevity.

NAD+ and Its Role in Aging

Nicotinamide adenine dinucleotide (NAD+) is a crucial molecule in our bodies, involved in turning nutrients into energy, adapting to stress, and maintaining our daily biological rhythms. As we age, the amount of NAD+ in our bodies decreases. This is partly due to the action of an enzyme called CD38, which breaks down NAD+, leading to lower levels in older individuals^[6]. Keeping a balance of NAD+ is important for our cells to work properly, and enzymes that use NAD+ are being studied for their potential to slow down aging processes. These enzymes include CD38, sirtuins (SIRT), which are involved in cell regulation, PARP1, important for DNA repair, and SARM1, linked to nerve cell health^[7]^[8].

Our bodies can make NAD+ in two ways: either from scratch using certain nutrients like nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN), or by recycling components from chemical reactions in our cells^[9]. An enzyme on the surface of our cells, called CD73, also helps to keep NAD+ levels up by converting NMN to NR^[10].

The Role of NNMT in NAD+ Levels

An enzyme named nicotinamide N-methyltransferase (NNMT) plays a key role in managing NAD+ levels. It changes nicotinamide, a form of vitamin B3, into a substance called methylnicotinamide (MNT). This change affects how much nicotinamide is available to make NAD+ in our cells. NNMT is linked to conditions like obesity and type two diabetes^[11]. Interestingly, NNMT also helps stabilize SIRT1, an enzyme that protects cells from stress and can increase lifespan^[12]^[13]^[14]. Researchers are looking into NNMT inhibitors as potential treatments for diseases like cancer, obesity, and liver diseases related to alcohol use^[11]^[15]^[16]^[17]^[18]. The balance between NNMT, MNT, and NAD+ is important for our health, especially as we age.

NAD+, Sirtuins and Longevity-Promoting Pathway

The CD38/NAD+/SIRT1 Axis. NAD+ levels in the body can be influenced by the supplementation of precursors nicotinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN). NAD+ levels decrease with age and are further metabolized by the activation of SIRT1, PARP1, SARM1, and CD38. Restoring NAD+ levels allows for an increase in SIRT1 activity due to increased substrate availability, resulting in the inhibition of age-promoting pathways and activation of adaptive and protective transcription factors and processes. The central lineage may be described as the CD38/NAD+/SIRT1 axis, and targeting this access with nutraceutical interventions may prevent the age-related decline of NAD+ levels in the body. Black lines indicate conversion or activation. Red lines indicate inhibitors or destroyers of the indicated target.^[19]

Maintaining the right levels of NAD+ and the activity of sirtuin proteins is crucial in the fight against aging^[20]. Taking supplements that are NAD+ precursors, like Nicotinamide Riboside (NR) and Nicotinamide Mononucleotide (NMN), has shown promise in combating the natural decline of NAD+ that comes with aging and related diseases^[21]^[22]^[23]. The decrease in NAD+ as we age is primarily due to its reduction in our bodies, not an increase in its counterpart, NADH^[24]. Adding more NAD+ can help fix issues in mitochondria, the energy factories of our cells, that happen because of this decline^[25].

SIRT1, a member of the sirtuin protein family involved in cellular response to stress, has been linked to longer life spans, though the results vary depending on the situation. For example, elite athletes, who have higher SIRT1 levels, tend to have longer telomeres (a sign of cellular aging) and are less likely to develop insulin resistance^[26]. SIRT1 works by turning on certain genes, like FoxO and PGC1α, which are important for managing stress, controlling cell growth, and preventing tumors. These genes are known to contribute to longer lifespans in some animals^[27]^[28]^[29]^[30]. The IIS pathway, which influences growth, metabolism, and longevity, also promotes longer life under certain conditions by activating these genes^[31]^[32]. PGC1α, in particular, is key in creating mitochondria and has been linked to better insulin sensitivity in muscles^[33]^[34]^[35]. Furthermore, AMPK, which is involved in energy management in the body, interacts with SIRT1 and can inhibit mTOR, another aging-related process. AMPK also helps increase NAD+ levels, thus boosting SIRT1 activity^[36]. Additionally, SIRT1 can slow down NF-κB signaling, which is part of the immune response, helping to reduce long-term inflammation^[37]. Having enough NAD+ to keep SIRT1 active is essential in manipulating the aging process and promoting longevity^[22]^[38]^[39]^[40]. Keeping NAD+ at healthy levels is key for making sure SIRT1 can do its job effectively as we age.

NAD+ and Its Influence on the Body's Biological Clock

NAD+, a crucial molecule in our body, plays an important role in keeping our biological clock, or circadian rhythm, in check. Studies involving older mice have shown that when they have less NAD+, they experience more disruptions in their biological clock compared to younger mice with more NAD+^[41]. This leads to problems in how their cells handle energy and time. By increasing NAD+ levels in mice with circadian rhythm issues, researchers have been able to fix these problems, particularly through the action of a protein called SIRT3^[42]. Having enough NAD+ and ensuring that sirtuin proteins (like SIRT3) are active is key to keeping our internal clocks working properly. Taking supplements that increase NAD+ levels might help fix age-related issues with these biological clocks^[43]. Low NAD+ levels are a common problem in many age-related diseases, so treatments that increase NAD+ are being researched as a way to help with these issues^[44]^[45]^[46]^[47].

Todo

2021, NAD⁺ metabolism and its roles in cellular processes during ageing ^[48]
2022, Efficient Assay and Marker Significance of NAD⁺ in Human Blood ^[49]
2021, Age-Dependent Decline of NAD⁺-Universal Truth or Confounded Consensus? ^[50]
2018, Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence ^[51]

References

↑ Camacho-Pereira J, Tarragó MG, Chini CC, Nin V, Escande C, Warner GM, Puranik AS, Schoon RA, Reid JM, Galina A, Chini EN (June 2016). "CD38 Dictates Age-Related NAD Decline and Mitochondrial Dysfunction through an SIRT3-Dependent Mechanism". Cell Metabolism. 23 (6): 1127–1139. doi:10.1016/j.cmet.2016.05.006. PMC 4911708. PMID 27304511.
↑ Schultz MB, Sinclair DA (June 2016). "Why NAD(+) Declines during Aging: It's Destroyed". Cell Metabolism. 23 (6): 965–966. doi:10.1016/j.cmet.2016.05.022. PMC 5088772. PMID 27304496.
↑ Tarragó MG, Chini CC, Kanamori KS, Warner GM, Caride A, de Oliveira GC, Rud M, Samani A, Hein KZ, Huang R, Jurk D, Cho DS, Boslett JJ, Miller JD, Zweier JL, Passos JF, Doles JD, Becherer DJ, Chini EN (May 2018). "A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD+ Decline". Cell Metabolism. 27 (5): 1081–1095.e10. doi:10.1016/j.cmet.2018.03.016. PMC 5935140. PMID 29719225.
↑ ^4.0 ^4.1 Cambronne XA, Kraus WL (2020). "Location, Location, Location: Compartmentalization of NAD + Synthesis and Functions in Mammalian Cells". Trends in Biochemical Sciences. 45 (10): 858–873. doi:10.1016/j.tibs.2020.05.010. PMC 7502477. PMID 32595066.
↑ Kang BE, Choi J, Stein S, Ryu D (2020). "Implications of NAD + boosters in translational medicine". European Journal of Clinical Investigation. 50 (10): e13334. doi:10.1111/eci.13334. PMID 32594513. S2CID 220254270.
↑ Schultz MB & Sinclair DA: Why NAD(+) Declines during Aging: It's Destroyed. Cell Metab 2016. (PMID 27304496) [PubMed] [DOI] [Full text] NAD(+) is required not only for life but for a long life. In this issue, Camacho-Pereira et al. (2016) implicate CD38 in the decline of NAD(+) during aging, with implications for combating age-related diseases.
↑ Ray Chaudhuri A & Nussenzweig A: The multifaceted roles of PARP1 in DNA repair and chromatin remodelling. Nat Rev Mol Cell Biol 2017. (PMID 28676700) [PubMed] [DOI] [Full text] Cells are exposed to various endogenous and exogenous insults that induce DNA damage, which, if unrepaired, impairs genome integrity and leads to the development of various diseases, including cancer. Recent evidence has implicated poly(ADP-ribose) polymerase 1 (PARP1) in various DNA repair pathways and in the maintenance of genomic stability. The inhibition of PARP1 is therefore being exploited clinically for the treatment of various cancers, which include DNA repair-deficient ovarian, breast and prostate cancers. Understanding the role of PARP1 in maintaining genome integrity is not only important for the design of novel chemotherapeutic agents, but is also crucial for gaining insights into the mechanisms of chemoresistance in cancer cells. In this Review, we discuss the roles of PARP1 in mediating various aspects of DNA metabolism, such as single-strand break repair, nucleotide excision repair, double-strand break repair and the stabilization of replication forks, and in modulating chromatin structure.
↑ Gerdts J et al.: SARM1 activation triggers axon degeneration locally via NAD⁺ destruction. Science 2015. (PMID 25908823) [PubMed] [DOI] [Full text] Axon degeneration is an intrinsic self-destruction program that underlies axon loss during injury and disease. Sterile alpha and TIR motif-containing 1 (SARM1) protein is an essential mediator of axon degeneration. We report that SARM1 initiates a local destruction program involving rapid breakdown of nicotinamide adenine dinucleotide (NAD(+)) after injury. We used an engineered protease-sensitized SARM1 to demonstrate that SARM1 activity is required after axon injury to induce axon degeneration. Dimerization of the Toll-interleukin receptor (TIR) domain of SARM1 alone was sufficient to induce locally mediated axon degeneration. Formation of the SARM1 TIR dimer triggered rapid breakdown of NAD(+), whereas SARM1-induced axon destruction could be counteracted by increased NAD(+) synthesis. SARM1-induced depletion of NAD(+) may explain the potent axon protection in Wallerian degeneration slow (Wld(s)) mutant mice.
↑ Katsyuba E et al.: NAD⁺ homeostasis in health and disease. Nat Metab 2020. (PMID 32694684) [PubMed] [DOI] [Full text] The conceptual evolution of nicotinamide adenine dinucleotide (NAD+) from being seen as a simple metabolic cofactor to a pivotal cosubstrate for proteins regulating metabolism and longevity, including the sirtuin family of protein deacylases, has led to a new wave of scientific interest in NAD+. NAD+ levels decline during ageing, and alterations in NAD+ homeostasis can be found in virtually all age-related diseases, including neurodegeneration, diabetes and cancer. In preclinical settings, various strategies to increase NAD+ levels have shown beneficial effects, thus starting a competitive race to discover marketable NAD+ boosters to improve healthspan and lifespan. Here, we review the basics of NAD+ biochemistry and metabolism, and its roles in health and disease, and we discuss current challenges and the future translational potential of NAD+ research.
↑ Mateuszuk Ł et al.: Reversal of endothelial dysfunction by nicotinamide mononucleotide via extracellular conversion to nicotinamide riboside. Biochem Pharmacol 2020. (PMID 32389638) [PubMed] [DOI] BACKGROUND: Nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) are effective substrates for NAD synthesis, which may act as vasoprotective agents. Here, we characterize the effects of NMN and NR on endothelial inflammation and dysfunction and test the involvement of CD73 in these effects. MATERIALS AND METHODS: The effect of NMN and NR on IL1β- or TNFα-induced endothelial inflammation (ICAM1 and vWF expression), intracellular NAD concentration and NAD-related enzyme expression (NAMPT, CD38, CD73), were studied in HAECs. The effect of NMN and NR on angiotensin II-induced impairment of endothelium-dependent vasodilation was analyzed in murine aortic rings. The involvement of CD73 in NMN and NR effects was tested using CD73 inhibitor-AOPCP, or CD73-/- mice. RESULTS: 24 h-incubation with NMN and NR induced anti-inflammatory effects in HAEC stimulated by IL1β or TNFα, as evidenced by a reduction in ICAM1 and vWF expression. Effects of exogenous NMN but not NR was abrogated in the presence of AOPCP, that efficiently inhibited extracellular endothelial conversion of NMN to NR, without a significant effect on the metabolism of NMN to NA. Surprisingly, intracellular NAD concentration increased in HAEC stimulated by IL1β or TNFα and this effect was associated with upregulation of NAMPT and CD73, whereas changes in CD38 expression were less pronounced. NMN and NR further increased NAD in IL1β-stimulated HAECs and AOPCP diminished NMN-induced increase in NAD, without an effect on NR-induced response. In ex vivo aortic rings stimulated with angiotensin II for 24 h, NO-dependent vasorelaxation induced by acetylcholine was impaired. NMN and NR, both prevented Ang II-induced endothelial dysfunction in the aorta. In aortic rings taken from CD73-/- mice NMN effect was lost, whereas NR effect was preserved. CONCLUSION: NMN and NR modulate intracellular NAD content in endothelium, inhibit endothelial inflammation and improve NO-dependent function by CD73-dependent and independent pathways, respectively. Extracellular conversion of NMN to NR by CD73 localized in the luminal surface of endothelial cells represent important vasoprotective mechanisms to maintain intracellular NAD.
↑ ^11.0 ^11.1 Kannt A et al.: A small molecule inhibitor of Nicotinamide N-methyltransferase for the treatment of metabolic disorders. Sci Rep 2018. (PMID 29483571) [PubMed] [DOI] [Full text] Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that catalyzes the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) onto the substrate, nicotinamide (NA) to form 1-methyl-nicotinamide (MNA). Higher NNMT expression and MNA concentrations have been associated with obesity and type-2 diabetes. Here we report a small molecule analog of NA, JBSNF-000088, that inhibits NNMT activity, reduces MNA levels and drives insulin sensitization, glucose modulation and body weight reduction in animal models of metabolic disease. In mice with high fat diet (HFD)-induced obesity, JBSNF-000088 treatment caused a reduction in body weight, improved insulin sensitivity and normalized glucose tolerance to the level of lean control mice. These effects were not seen in NNMT knockout mice on HFD, confirming specificity of JBSNF-000088. The compound also improved glucose handling in ob/ob and db/db mice albeit to a lesser extent and in the absence of weight loss. Co-crystal structure analysis revealed the presence of the N-methylated product of JBSNF-000088 bound to the NNMT protein. The N-methylated product was also detected in the plasma of mice treated with JBSNF-000088. Hence, JBSNF-000088 may act as a slow-turnover substrate analog, driving the observed metabolic benefits.
↑ Campagna R et al.: Nicotinamide N-methyltransferase in endothelium protects against oxidant stress-induced endothelial injury. Biochim Biophys Acta Mol Cell Res 2021. (PMID 34153425) [PubMed] [DOI] Nicotinamide N-methyltransferase (NNMT, EC 2.1.1.1.) plays an important role in the growth of many different tumours and is also involved in various non-neoplastic disorders. However, the presence and role of NNMT in the endothelium has yet to be specifically explored. Here, we characterized the functional activity of NNMT in the endothelium and tested whether NNMT regulates endothelial cell viability. NNMT in endothelial cells (HAEC, HMEC-1 and EA.hy926) was inhibited using two approaches: pharmacological inhibition of the enzyme by NNMT inhibitors (5-amino-1-methylquinoline - 5MQ and 6-methoxynicotinamide - JBSF-88) or by shRNA-mediated silencing. Functional inhibition of NNMT was confirmed by LC/MS/MS-based analysis of impaired MNA production. The effects of NNMT inhibition on cellular viability were analyzed in both the absence and presence of menadione. Our results revealed that all studied endothelial lines express relatively high levels of functionally active NNMT compared with cancer cells (MDA-MB-231). Although the aldehyde oxidase 1 enzyme was also expressed in the endothelium, the further metabolites of N1-methylnicotinamide (N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide) generated by this enzyme were not detected, suggesting that endothelial NNMT-derived MNA was not subsequently metabolized in the endothelium by aldehyde oxidase 1. Menadione induced a concentration-dependent decrease in endothelial viability as evidenced by a decrease in cell number that was associated with the upregulation of NNMT and SIRT1 expression in the nucleus in viable cells. The suppression of the NNMT activity either by NNMT inhibitors or shRNA-based silencing significantly decreased the endothelial cell viability in response to menadione. Furthermore, NNMT inhibition resulted in nuclear SIRT1 expression downregulation and upregulation of the phosphorylated form of SIRT1 on Ser47. In conclusion, our results suggest that the endothelial nuclear NNMT/SIRT1 pathway exerts a cytoprotective role that safeguards endothelial cell viability under oxidant stress insult.
↑ Hong S et al.: Nicotinamide N-methyltransferase regulates hepatic nutrient metabolism through Sirt1 protein stabilization. Nat Med 2015. (PMID 26168293) [PubMed] [DOI] [Full text] Nicotinamide N-methyltransferase (Nnmt) methylates nicotinamide, a form of vitamin B3, to produce N(1)-methylnicotinamide (MNAM). Nnmt has emerged as a metabolic regulator in adipocytes, but its role in the liver, the tissue with the strongest Nnmt expression, is not known. In spite of its overall high expression, here we find that hepatic expression of Nnmt is highly variable and correlates with multiple metabolic parameters in mice and humans. Further, we find that suppression of hepatic Nnmt expression in vivo alters glucose and cholesterol metabolism and that the metabolic effects of Nnmt in the liver are mediated by its product MNAM. Supplementation of high-fat diet with MNAM decreases serum and liver cholesterol and liver triglycerides levels in mice. Mechanistically, increasing Nnmt expression or MNAM levels stabilizes sirtuin 1 protein, an effect that is required for their metabolic benefits. In summary, we describe here a novel regulatory pathway for vitamin B3 that could provide a new opportunity for metabolic disease therapy.
↑ Schmeisser K et al.: Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide. Nat Chem Biol 2013. (PMID 24077178) [PubMed] [DOI] [Full text] Sirtuins, a family of histone deacetylases, have a fiercely debated role in regulating lifespan. In contrast with recent observations, here we find that overexpression of sir-2.1, the ortholog of mammalian SirT1, does extend Caenorhabditis elegans lifespan. Sirtuins mandatorily convert NAD(+) into nicotinamide (NAM). We here find that NAM and its metabolite, 1-methylnicotinamide (MNA), extend C. elegans lifespan, even in the absence of sir-2.1. We identify a previously unknown C. elegans nicotinamide-N-methyltransferase, encoded by a gene now named anmt-1, to generate MNA from NAM. Disruption and overexpression of anmt-1 have opposing effects on lifespan independent of sirtuins, with loss of anmt-1 fully inhibiting sir-2.1-mediated lifespan extension. MNA serves as a substrate for a newly identified aldehyde oxidase, GAD-3, to generate hydrogen peroxide, which acts as a mitohormetic reactive oxygen species signal to promote C. elegans longevity. Taken together, sirtuin-mediated lifespan extension depends on methylation of NAM, providing an unexpected mechanistic role for sirtuins beyond histone deacetylation.
↑ van Haren MJ et al.: Macrocyclic peptides as allosteric inhibitors of nicotinamide N-methyltransferase (NNMT). RSC Chem Biol 2021. (PMID 34704059) [PubMed] [DOI] [Full text] Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide (MNA) using S-adenosyl-l-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly being elucidated and provides an indication that NNMT may be an interesting therapeutic target for a variety of diseases including cancer, diabetes, and obesity. Most inhibitors of NNMT described to date are structurally related to one or both of its substrates. In the search for structurally diverse NNMT inhibitors, an mRNA display screening technique was used to identify macrocyclic peptides which bind to NNMT. Several of the cyclic peptides identified in this manner show potent inhibition of NNMT with IC50 values as low as 229 nM. The peptides were also found to downregulate MNA production in cellular assays. Interestingly, substrate competition experiments reveal that these cyclic peptide inhibitors are noncompetitive with either SAM or NA indicating they may be the first allosteric inhibitors reported for NNMT.
↑ van Haren MJ et al.: Esterase-Sensitive Prodrugs of a Potent Bisubstrate Inhibitor of Nicotinamide N-Methyltransferase (NNMT) Display Cellular Activity. Biomolecules 2021. (PMID 34572571) [PubMed] [DOI] [Full text] A recently discovered bisubstrate inhibitor of Nicotinamide N-methyltransferase (NNMT) was found to be highly potent in biochemical assays with a single digit nanomolar IC50 value but lacking in cellular activity. We, here, report a prodrug strategy designed to translate the observed potent biochemical inhibitory activity of this inhibitor into strong cellular activity. This prodrug strategy relies on the temporary protection of the amine and carboxylic acid moieties of the highly polar amino acid side chain present in the bisubstrate inhibitor. The modification of the carboxylic acid into a range of esters in the absence or presence of a trimethyl-lock (TML) amine protecting group yielded a range of candidate prodrugs. Based on the stability in an aqueous buffer, and the confirmed esterase-dependent conversion to the parent compound, the isopropyl ester was selected as the preferred acid prodrug. The isopropyl ester and isopropyl ester-TML prodrugs exhibit improved cell permeability, which also translates to significantly enhanced cellular activity as established using assays designed to measure the enzymatic activity of NNMT in live cells.
↑ Neelakantan H et al.: Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice. Biochem Pharmacol 2018. (PMID 29155147) [PubMed] [DOI] [Full text] There is a critical need for new mechanism-of-action drugs that reduce the burden of obesity and associated chronic metabolic comorbidities. A potentially novel target to treat obesity and type 2 diabetes is nicotinamide-N-methyltransferase (NNMT), a cytosolic enzyme with newly identified roles in cellular metabolism and energy homeostasis. To validate NNMT as an anti-obesity drug target, we investigated the permeability, selectivity, mechanistic, and physiological properties of a series of small molecule NNMT inhibitors. Membrane permeability of NNMT inhibitors was characterized using parallel artificial membrane permeability and Caco-2 cell assays. Selectivity was tested against structurally-related methyltransferases and nicotinamide adenine dinucleotide (NAD+) salvage pathway enzymes. Effects of NNMT inhibitors on lipogenesis and intracellular levels of metabolites, including NNMT reaction product 1-methylnicotianamide (1-MNA) were evaluated in cultured adipocytes. Effects of a potent NNMT inhibitor on obesity measures and plasma lipid were assessed in diet-induced obese mice fed a high-fat diet. Methylquinolinium scaffolds with primary amine substitutions displayed high permeability from passive and active transport across membranes. Importantly, methylquinolinium analogues displayed high selectivity, not inhibiting related SAM-dependent methyltransferases or enzymes in the NAD+ salvage pathway. NNMT inhibitors reduced intracellular 1-MNA, increased intracellular NAD+ and S-(5'-adenosyl)-l-methionine (SAM), and suppressed lipogenesis in adipocytes. Treatment of diet-induced obese mice systemically with a potent NNMT inhibitor significantly reduced body weight and white adipose mass, decreased adipocyte size, and lowered plasma total cholesterol levels. Notably, administration of NNMT inhibitors did not impact total food intake nor produce any observable adverse effects. These results support development of small molecule NNMT inhibitors as therapeutics to reverse diet-induced obesity and validate NNMT as a viable target to treat obesity and related metabolic conditions. Increased flux of key cellular energy regulators, including NAD+ and SAM, may potentially define the therapeutic mechanism-of-action of NNMT inhibitors.
↑ Gao Y et al.: Potent Inhibition of Nicotinamide N-Methyltransferase by Alkene-Linked Bisubstrate Mimics Bearing Electron Deficient Aromatics. J Med Chem 2021. (PMID 34424711) [PubMed] [DOI] [Full text] Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide (vitamin B3) to generate 1-methylnicotinamide (MNA). NNMT overexpression has been linked to a variety of diseases, most prominently human cancers, indicating its potential as a therapeutic target. The development of small-molecule NNMT inhibitors has gained interest in recent years, with the most potent inhibitors sharing structural features based on elements of the nicotinamide substrate and the S-adenosyl-l-methionine (SAM) cofactor. We here report the development of new bisubstrate inhibitors that include electron-deficient aromatic groups to mimic the nicotinamide moiety. In addition, a trans-alkene linker was found to be optimal for connecting the substrate and cofactor mimics in these inhibitors. The most potent NNMT inhibitor identified exhibits an IC50 value of 3.7 nM, placing it among the most active NNMT inhibitors reported to date. Complementary analytical techniques, modeling studies, and cell-based assays provide insights into the binding mode, affinity, and selectivity of these inhibitors.
↑ Sharma A et al.: Potential Synergistic Supplementation of NAD+ Promoting Compounds as a Strategy for Increasing Healthspan. Nutrients 2023. (PMID 36678315) [PubMed] [DOI] [Full text] Disrupted biological function, manifesting through the hallmarks of aging, poses one of the largest threats to healthspan and risk of disease development, such as metabolic disorders, cardiovascular ailments, and neurodegeneration. In recent years, numerous geroprotectors, senolytics, and other nutraceuticals have emerged as potential disruptors of aging and may be viable interventions in the immediate state of human longevity science. In this review, we focus on the decrease in nicotinamide adenine dinucleotide (NAD+) with age and the supplementation of NAD+ precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), in combination with other geroprotective compounds, to restore NAD+ levels present in youth. Furthermore, these geroprotectors may enhance the efficacy of NMN supplementation while concurrently providing their own numerous health benefits. By analyzing the prevention of NAD+ degradation through the inhibition of CD38 or supporting protective downstream agents of SIRT1, we provide a potential framework of the CD38/NAD+/SIRT1 axis through which geroprotectors may enhance the efficacy of NAD+ precursor supplementation and reduce the risk of age-related diseases, thereby potentiating healthspan in humans.
↑ Mendelsohn AR & Larrick JW: The NAD+/PARP1/SIRT1 Axis in Aging. Rejuvenation Res 2017. (PMID 28537485) [PubMed] [DOI] NAD+ levels decline with age in diverse animals from Caenorhabditis elegans to mice. Raising NAD+ levels by dietary supplementation with NAD+ precursors, nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN), improves mitochondrial function and muscle and neural and melanocyte stem cell function in mice, as well as increases murine life span. Decreased NAD+ levels with age reduce SIRT1 function and reduce the mitochondrial unfolded protein response, which can be overcome by NR supplementation. Decreased NAD+ levels cause NAD+-binding protein DBC1 to form a complex with PARP1, inhibiting poly(adenosine diphosphate-ribose) polymerase (PARP) catalytic activity. Old mice have increased amounts of DBC1-PARP1 complexes, lower PARP activity, increased DNA damage, and reduced nonhomologous end joining and homologous recombination repair. DBC1-PARP1 complexes in old mice can be broken by increasing NAD+ levels through treatment with NMN, reducing DNA damage and restoring PARP activity to youthful levels. The mechanism of declining NAD+ levels and its fundamental importance to aging are yet to be elucidated. There is a correlation of PARP activity with mammalian life span that suggests that NAD+/SIRT1/PARP1 may be more significant than the modest effects on life span observed for NR supplementation in old mice. The NAD+/PARP1/SIRT1 axis may link NAD+ levels and DNA damage with the apparent epigenomic DNA methylation clocks that have been described.
↑ Fang EF et al.: NAD⁺ in Aging: Molecular Mechanisms and Translational Implications. Trends Mol Med 2017. (PMID 28899755) [PubMed] [DOI] [Full text] The coenzyme NAD+ is critical in cellular bioenergetics and adaptive stress responses. Its depletion has emerged as a fundamental feature of aging that may predispose to a wide range of chronic diseases. Maintenance of NAD+ levels is important for cells with high energy demands and for proficient neuronal function. NAD+ depletion is detected in major neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, cardiovascular disease and muscle atrophy. Emerging evidence suggests that NAD+ decrements occur in various tissues during aging, and that physiological and pharmacological interventions bolstering cellular NAD+ levels might retard aspects of aging and forestall some age-related diseases. Here, we discuss aspects of NAD+ biosynthesis, together with putative mechanisms of NAD+ action against aging, including recent preclinical and clinical trials.
↑ ^22.0 ^22.1 Yaku K et al.: NAD metabolism: Implications in aging and longevity. Ageing Res Rev 2018. (PMID 29883761) [PubMed] [DOI] Nicotinamide adenine dinucleotide (NAD) is an important co-factor involved in numerous physiological processes, including metabolism, post-translational protein modification, and DNA repair. In living organisms, a careful balance between NAD production and degradation serves to regulate NAD levels. Recently, a number of studies have demonstrated that NAD levels decrease with age, and the deterioration of NAD metabolism promotes several aging-associated diseases, including metabolic and neurodegenerative diseases and various cancers. Conversely, the upregulation of NAD metabolism, including dietary supplementation with NAD precursors, has been shown to prevent the decline of NAD and exhibits beneficial effects against aging and aging-associated diseases. In addition, many studies have demonstrated that genetic and/or nutritional activation of NAD metabolism can extend the lifespan of diverse organisms. Collectively, it is clear that NAD metabolism plays important roles in aging and longevity. In this review, we summarize the basic functions of the enzymes involved in NAD synthesis and degradation, as well as the outcomes of their dysregulation in various aging processes. In addition, a particular focus is given on the role of NAD metabolism in the longevity of various organisms, with a discussion of the remaining obstacles in this research field.
↑ Chini CCS et al.: NAD and the aging process: Role in life, death and everything in between. Mol Cell Endocrinol 2017. (PMID 27825999) [PubMed] [DOI] [Full text] Life as we know it cannot exist without the nucleotide nicotinamide adenine dinucleotide (NAD). From the simplest organism, such as bacteria, to the most complex multicellular organisms, NAD is a key cellular component. NAD is extremely abundant in most living cells and has traditionally been described to be a cofactor in electron transfer during oxidation-reduction reactions. In addition to participating in these reactions, NAD has also been shown to play a key role in cell signaling, regulating several pathways from intracellular calcium transients to the epigenetic status of chromatin. Thus, NAD is a molecule that provides an important link between signaling and metabolism, and serves as a key molecule in cellular metabolic sensoring pathways. Importantly, it has now been clearly demonstrated that cellular NAD levels decline during chronological aging. This decline appears to play a crucial role in the development of metabolic dysfunction and age-related diseases. In this review we will discuss the molecular mechanisms responsible for the decrease in NAD levels during aging. Since other reviews on this subject have been recently published, we will concentrate on presenting a critical appraisal of the current status of the literature and will highlight some controversial topics in the field. In particular, we will discuss the potential role of the NADase CD38 as a driver of age-related NAD decline.
↑ Clement J et al.: The Plasma NAD⁺ Metabolome Is Dysregulated in "Normal" Aging. Rejuvenation Res 2019. (PMID 30124109) [PubMed] [DOI] [Full text] Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as an electron carrier in cellular metabolism and plays a crucial role in the maintenance of balanced redox homeostasis. Quantification of NAD+:NADH and NADP+:NADPH ratios are pivotal to a wide variety of cellular processes, including intracellular secondary messenger signaling by CD38 glycohydrolases, DNA repair by poly(adenosine diphosphate ribose) polymerase (PARP), epigenetic regulation of gene expression by NAD-dependent histone deacetylase enzymes known as sirtuins, and regulation of the oxidative pentose phosphate pathway. We quantified changes in the NAD+ metabolome in plasma samples collected from consenting healthy human subjects across a wide age range (20-87 years) using liquid chromatography coupled to tandem mass spectrometry. Our data show a significant decline in the plasma levels of NAD+, NADP+, and other important metabolites such as nicotinic acid adenine dinucleotide (NAAD) with age. However, an age-related increase in the reduced form of NAD+ and NADP+-NADH and NADPH-and nicotinamide (NAM), N-methyl-nicotinamide (MeNAM), and the products of adenosine diphosphoribosylation, including adenosine diphosphate ribose (ADPR) was also reported. Whereas, plasma levels of nicotinic acid (NA), nicotinamide mononucleotide (NMN), and nicotinic acid mononucleotide (NAMN) showed no statistically significant changes across age groups. Taken together, our data cumulatively suggest that age-related impairments are associated with corresponding alterations in the extracellular plasma NAD+ metabolome. Our future research will seek to elucidate the role of modulating NAD+ metabolites in the treatment and prevention of age-related diseases.
↑ Gomes AP et al.: Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging. Cell 2013. (PMID 24360282) [PubMed] [DOI] [Full text] Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD(+) and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD(+) levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.
↑ Aguiar SS et al.: Telomere Length, SIRT1, and Insulin in Male Master Athletes: The Path to Healthy Longevity?. Int J Sports Med 2022. (PMID 34256387) [PubMed] [DOI] Lower SIRT1 and insulin resistance are associated with accelerated telomere shortening. This study investigated whether the lifestyle of master athletes can attenuate these age-related changes and thereby slow aging. We compared insulin, SIRT1, and telomere length in highly trained male master athletes (n=52; aged 49.9±7.2 yrs) and age-matched non-athletes (n=19; aged 47.3±8.9 yrs). This is a cross-sectional study, in which all data were collected in one visit. Overnight fasted SIRT1 and insulin levels in whole blood were assessed using commercial kits. Relative telomere length was determined in leukocytes through qPCR analyses. Master athletes had higher SIRT1, lower insulin, and longer telomere length than age-matched non-athletes (p<0.05 for all). Insulin was inversely associated with SIRT1 (r=-0.38; p=0.001). Telomere length correlated positively with SIRT1 (r=0.65; p=0.001), whereas telomere length and insulin were not correlated (r=0.03; p=0.87). In conclusion, master athletes have higher SIRT1, lower insulin, and longer telomeres than age-matched non-athletes. Furthermore, SIRT1 was negatively associated with insulin and positively associated with telomere length. These findings suggest that in this sample of middle-aged participants reduced insulin, increased SIRT1 activity, and attenuation of biological aging are connected.
↑ Tang BL: Sirt1 and the Mitochondria. Mol Cells 2016. (PMID 26831453) [PubMed] [DOI] [Full text] Sirt1 is the most prominent and extensively studied member of sirtuins, the family of mammalian class III histone deacetylases heavily implicated in health span and longevity. Although primarily a nuclear protein, Sirt1's deacetylation of Peroxisome proliferator-activated receptor Gamma Coactivator-1α (PGC-1α) has been extensively implicated in metabolic control and mitochondrial biogenesis, which was proposed to partially underlie Sirt1's role in caloric restriction and impacts on longevity. The notion of Sirt1's regulation of PGC-1α activity and its role in mitochondrial biogenesis has, however, been controversial. Interestingly, Sirt1 also appears to be important for the turnover of defective mitochondria by mitophagy. I discuss here evidences for Sirt1's regulation of mitochondrial biogenesis and turnover, in relation to PGC-1α deacetylation and various aspects of cellular physiology and disease.
↑ Brunet A et al.: Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 2004. (PMID 14976264) [PubMed] [DOI] The Sir2 deacetylase modulates organismal life-span in various species. However, the molecular mechanisms by which Sir2 increases longevity are largely unknown. We show that in mammalian cells, the Sir2 homolog SIRT1 appears to control the cellular response to stress by regulating the FOXO family of Forkhead transcription factors, a family of proteins that function as sensors of the insulin signaling pathway and as regulators of organismal longevity. SIRT1 and the FOXO transcription factor FOXO3 formed a complex in cells in response to oxidative stress, and SIRT1 deacetylated FOXO3 in vitro and within cells. SIRT1 had a dual effect on FOXO3 function: SIRT1 increased FOXO3's ability to induce cell cycle arrest and resistance to oxidative stress but inhibited FOXO3's ability to induce cell death. Thus, one way in which members of the Sir2 family of proteins may increase organismal longevity is by tipping FOXO-dependent responses away from apoptosis and toward stress resistance.
↑ Greer EL & Brunet A: FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 2005. (PMID 16288288) [PubMed] [DOI] A wide range of human diseases, including cancer, has a striking age-dependent onset. However, the molecular mechanisms that connect aging and cancer are just beginning to be unraveled. FOXO transcription factors are promising candidates to serve as molecular links between longevity and tumor suppression. These factors are major substrates of the protein kinase Akt. In the presence of insulin and growth factors, FOXO proteins are relocalized from the nucleus to the cytoplasm and degraded via the ubiquitin-proteasome pathway. In the absence of growth factors, FOXO proteins translocate to the nucleus and upregulate a series of target genes, thereby promoting cell cycle arrest, stress resistance, or apoptosis. Stress stimuli also trigger the relocalization of FOXO factors into the nucleus, thus allowing an adaptive response to stress stimuli. Consistent with the notion that stress resistance is highly coupled with lifespan extension, activation of FOXO transcription factors in worms and flies increases longevity. Emerging evidence also suggests that FOXO factors play a tumor suppressor role in a variety of cancers. Thus, FOXO proteins translate environmental stimuli into changes in gene expression programs that may coordinate organismal longevity and tumor suppression.
↑ Zhao Y & Liu YS: Longevity Factor FOXO3: A Key Regulator in Aging-Related Vascular Diseases. Front Cardiovasc Med 2021. (PMID 35004893) [PubMed] [DOI] [Full text] Forkhead box O3 (FOXO3) has been proposed as a homeostasis regulator, capable of integrating multiple upstream signaling pathways that are sensitive to environmental changes and counteracting their adverse effects due to external changes, such as oxidative stress, metabolic stress and growth factor deprivation. FOXO3 polymorphisms are associated with extreme human longevity. Intriguingly, longevity-associated single nucleotide polymorphisms (SNPs) in human FOXO3 correlate with lower-than-average morbidity from cardiovascular diseases in long-lived people. Emerging evidence indicates that FOXO3 plays a critical role in vascular aging. FOXO3 inactivation is implicated in several aging-related vascular diseases. In experimental studies, FOXO3-engineered human ESC-derived vascular cells improve vascular homeostasis and delay vascular aging. The purpose of this review is to explore how FOXO3 regulates vascular aging and its crucial role in aging-related vascular diseases.
↑ Kaletsky R et al.: The C. elegans adult neuronal IIS/FOXO transcriptome reveals adult phenotype regulators. Nature 2016. (PMID 26675724) [PubMed] [DOI] [Full text] Insulin/insulin-like growth factor signalling (IIS) is a critical regulator of an organism's most important biological decisions from growth, development, and metabolism to reproduction and longevity. It primarily does so through the activity of the DAF-16 transcription factor (forkhead box O (FOXO) homologue), whose global targets were identified in Caenorhabditis elegans using whole-worm transcriptional analyses more than a decade ago. IIS and FOXO also regulate important neuronal and adult behavioural phenotypes, such as the maintenance of memory and axon regeneration with age, in both mammals and C. elegans, but the neuron-specific IIS/FOXO targets that regulate these functions are still unknown. By isolating adult C. elegans neurons for transcriptional profiling, we identified both the wild-type and IIS/FOXO mutant adult neuronal transcriptomes for the first time. IIS/FOXO neuron-specific targets are distinct from canonical IIS/FOXO-regulated longevity and metabolism targets, and are required for extended memory in IIS daf-2 mutants. The activity of the forkhead transcription factor FKH-9 in neurons is required for the ability of daf-2 mutants to regenerate axons with age, and its activity in non-neuronal tissues is required for the long lifespan of daf-2 mutants. Together, neuron-specific and canonical IIS/FOXO-regulated targets enable the coordinated extension of neuronal activities, metabolism, and longevity under low-insulin signalling conditions.
↑ Slack C et al.: dFOXO-independent effects of reduced insulin-like signaling in Drosophila. Aging Cell 2011. (PMID 21443682) [PubMed] [DOI] [Full text] The insulin/insulin-like growth factor-like signaling (IIS) pathway in metazoans has evolutionarily conserved roles in growth control, metabolic homeostasis, stress responses, reproduction, and lifespan. Genetic manipulations that reduce IIS in the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the mouse have been shown not only to produce substantial increases in lifespan but also to ameliorate several age-related diseases. In C. elegans, the multitude of phenotypes produced by the reduction in IIS are all suppressed in the absence of the worm FOXO transcription factor, DAF-16, suggesting that they are all under common regulation. It is not yet clear in other animal models whether the activity of FOXOs mediate all of the physiological effects of reduced IIS, especially increased lifespan. We have addressed this issue by examining the effects of reduced IIS in the absence of dFOXO in Drosophila, using a newly generated null allele of dfoxo. We found that the removal of dFOXO almost completely blocks IIS-dependent lifespan extension. However, unlike in C. elegans, removal of dFOXO does not suppress the body size, fecundity, or oxidative stress resistance phenotypes of IIS-compromised flies. In contrast, IIS-dependent xenobiotic resistance is fully dependent on dFOXO activity. Our results therefore suggest that there is evolutionary divergence in the downstream mechanisms that mediate the effects of IIS. They also imply that in Drosophila, additional factors act alongside dFOXO to produce IIS-dependent responses in body size, fecundity, and oxidative stress resistance and that these phenotypes are not causal in IIS-mediated extension of lifespan.
↑ Brenmoehl J & Hoeflich A: Dual control of mitochondrial biogenesis by sirtuin 1 and sirtuin 3. Mitochondrion 2013. (PMID 23583953) [PubMed] [DOI] In this review, we discuss the dual control of mitochondrial biogenesis and energy metabolism by silent information regulator-1 and -3 (SIRT1 and SIRT3). SIRT1 activates the peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α)-mediated transcription of nuclear and mitochondrial genes encoding for proteins promoting mitochondria proliferation, oxidative phosphorylation and energy production, whereas SIRT3 directly acts as an activator of proteins important for oxidative phosphorylation, tricarboxylic acid (TCA) cycle and fatty-acid oxidation and indirectly of PGC-1α and AMP-activated protein kinase (AMPK). The complex network involves different cellular compartments, transcriptional activation, post-translational modification and a plethora of secondary effectors. Overall, the mode of interaction between both sirtuin family members may be considered as a prominent case of molecular job-sharing.
↑ Chan MC & Arany Z: The many roles of PGC-1α in muscle--recent developments. Metabolism 2014. (PMID 24559845) [PubMed] [DOI] [Full text] Skeletal muscle is the largest organ in the body and contributes to innumerable aspects of organismal biology. Muscle dysfunction engenders numerous diseases, including diabetes, cachexia, and sarcopenia. At the same time, skeletal muscle is also the main engine of exercise, one of the most efficacious interventions for prevention and treatment of a wide variety of diseases. The transcriptional coactivator PGC-1α has emerged as a key driver of metabolic programming in skeletal muscle, both in health and in disease. We review here the many aspects of PGC-1α function in skeletal muscle, with a focus on recent developments.
↑ Summermatter S et al.: PGC-1α improves glucose homeostasis in skeletal muscle in an activity-dependent manner. Diabetes 2013. (PMID 23086035) [PubMed] [DOI] [Full text] Metabolic disorders are a major burden for public health systems globally. Regular exercise improves metabolic health. Pharmacological targeting of exercise mediators might facilitate physical activity or amplify the effects of exercise. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) largely mediates musculoskeletal adaptations to exercise, including lipid refueling, and thus constitutes such a putative target. Paradoxically, forced expression of PGC-1α in muscle promotes diet-induced insulin resistance in sedentary animals. We show that elevated PGC-1α in combination with exercise preferentially improves glucose homeostasis, increases Krebs cycle activity, and reduces the levels of acylcarnitines and sphingosine. Moreover, patterns of lipid partitioning are altered in favor of enhanced insulin sensitivity in response to combined PGC-1α and exercise. Our findings reveal how physical activity improves glucose homeostasis. Furthermore, our data suggest that the combination of elevated muscle PGC-1α and exercise constitutes a promising approach for the treatment of metabolic disorders.
↑ Cantó C et al.: AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature 2009. (PMID 19262508) [PubMed] [DOI] [Full text] AMP-activated protein kinase (AMPK) is a metabolic fuel gauge conserved along the evolutionary scale in eukaryotes that senses changes in the intracellular AMP/ATP ratio. Recent evidence indicated an important role for AMPK in the therapeutic benefits of metformin, thiazolidinediones and exercise, which form the cornerstones of the clinical management of type 2 diabetes and associated metabolic disorders. In general, activation of AMPK acts to maintain cellular energy stores, switching on catabolic pathways that produce ATP, mostly by enhancing oxidative metabolism and mitochondrial biogenesis, while switching off anabolic pathways that consume ATP. This regulation can take place acutely, through the regulation of fast post-translational events, but also by transcriptionally reprogramming the cell to meet energetic needs. Here we demonstrate that AMPK controls the expression of genes involved in energy metabolism in mouse skeletal muscle by acting in coordination with another metabolic sensor, the NAD+-dependent type III deacetylase SIRT1. AMPK enhances SIRT1 activity by increasing cellular NAD+ levels, resulting in the deacetylation and modulation of the activity of downstream SIRT1 targets that include the peroxisome proliferator-activated receptor-gamma coactivator 1alpha and the forkhead box O1 (FOXO1) and O3 (FOXO3a) transcription factors. The AMPK-induced SIRT1-mediated deacetylation of these targets explains many of the convergent biological effects of AMPK and SIRT1 on energy metabolism.
↑ Kauppinen A et al.: Antagonistic crosstalk between NF-κB and SIRT1 in the regulation of inflammation and metabolic disorders. Cell Signal 2013. (PMID 23770291) [PubMed] [DOI] Recent studies have indicated that the regulation of innate immunity and energy metabolism are connected together through an antagonistic crosstalk between NF-κB and SIRT1 signaling pathways. NF-κB signaling has a major role in innate immunity defense while SIRT1 regulates the oxidative respiration and cellular survival. However, NF-κB signaling can stimulate glycolytic energy flux during acute inflammation, whereas SIRT1 activation inhibits NF-κB signaling and enhances oxidative metabolism and the resolution of inflammation. SIRT1 inhibits NF-κB signaling directly by deacetylating the p65 subunit of NF-κB complex. SIRT1 stimulates oxidative energy production via the activation of AMPK, PPARα and PGC-1α and simultaneously, these factors inhibit NF-κB signaling and suppress inflammation. On the other hand, NF-κB signaling down-regulates SIRT1 activity through the expression of miR-34a, IFNγ, and reactive oxygen species. The inhibition of SIRT1 disrupts oxidative energy metabolism and stimulates the NF-κB-induced inflammatory responses present in many chronic metabolic and age-related diseases. We will examine the molecular mechanisms of the antagonistic signaling between NF-κB and SIRT1 and describe how this crosstalk controls inflammatory process and energy metabolism. In addition, we will discuss how disturbances in this signaling crosstalk induce the appearance of chronic inflammation in metabolic diseases.
↑ Mao K & Zhang G: The role of PARP1 in neurodegenerative diseases and aging. FEBS J 2022. (PMID 33460497) [PubMed] [DOI] Neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by progressive memory loss and motor impairment. Aging is a major risk factor for neurodegenerative diseases. Neurodegenerative diseases and aging often develop in an irreversible manner and cause a significant socioeconomic burden. When considering their pathogenesis, many studies usually focus on mitochondrial dysfunction and DNA damage. More recently, neuroinflammation, autophagy dysregulation, and SIRT1 inactivation were shown to be involved in the pathogenesis of neurodegenerative diseases and aging. In addition, studies uncovered the role of poly (ADP-ribose)-polymerase-1 (PARP1) in neurodegenerative diseases and aging. PARP1 links to a cluster of stress signals, including those originated by inflammation and autophagy dysregulation. In this review, we summarized the recent research progresses on PARP1 in neurodegenerative diseases and aging, with an emphasis on the relationship among PARP1, neuroinflammation, mitochondria, and autophagy. We discussed the possibilities of treating neurodegenerative diseases and aging through targeting PARP1.
↑ Amjad S et al.: Role of NAD⁺ in regulating cellular and metabolic signaling pathways. Mol Metab 2021. (PMID 33609766) [PubMed] [DOI] [Full text] BACKGROUND: Nicotinamide adenine dinucleotide (NAD+), a critical coenzyme present in every living cell, is involved in a myriad of metabolic processes associated with cellular bioenergetics. For this reason, NAD+ is often studied in the context of aging, cancer, and neurodegenerative and metabolic disorders. SCOPE OF REVIEW: Cellular NAD+ depletion is associated with compromised adaptive cellular stress responses, impaired neuronal plasticity, impaired DNA repair, and cellular senescence. Increasing evidence has shown the efficacy of boosting NAD+ levels using NAD+ precursors in various diseases. This review provides a comprehensive understanding into the role of NAD+ in aging and other pathologies and discusses potential therapeutic targets. MAJOR CONCLUSIONS: An alteration in the NAD+/NADH ratio or the NAD+ pool size can lead to derailment of the biological system and contribute to various neurodegenerative disorders, aging, and tumorigenesis. Due to the varied distribution of NAD+/NADH in different locations within cells, the direct role of impaired NAD+-dependent processes in humans remains unestablished. In this regard, longitudinal studies are needed to quantify NAD+ and its related metabolites. Future research should focus on measuring the fluxes through pathways associated with NAD+ synthesis and degradation.
↑ Sedlackova L & Korolchuk VI: The crosstalk of NAD, ROS and autophagy in cellular health and ageing. Biogerontology 2020. (PMID 32124104) [PubMed] [DOI] [Full text] Cellular adaptation to various types of stress requires a complex network of steps that altogether lead to reconstitution of redox balance, degradation of damaged macromolecules and restoration of cellular metabolism. Advances in our understanding of the interplay between cellular signalling and signal translation paint a complex picture of multi-layered paths of regulation. In this review we explore the link between cellular adaptation to metabolic and oxidative stresses by activation of autophagy, a crucial cellular catabolic pathway. Metabolic stress can lead to changes in the redox state of nicotinamide adenine dinucleotide (NAD), a co-factor in a variety of enzymatic reactions and thus trigger autophagy that acts to sequester intracellular components for recycling to support cellular growth. Likewise, autophagy is activated by oxidative stress to selectively recycle damaged macromolecules and organelles and thus maintain cellular viability. Multiple proteins that help regulate or execute autophagy are targets of post-translational modifications (PTMs) that have an effect on their localization, binding affinity or enzymatic activity. These PTMs include acetylation, a reversible enzymatic modification of a protein's lysine residues, and oxidation, a set of reversible and irreversible modifications by free radicals. Here we highlight the latest findings and outstanding questions on the interplay of autophagy with metabolic stress, presenting as changes in NAD levels, and oxidative stress, with a focus on autophagy proteins that are regulated by both, oxidation and acetylation. We further explore the relevance of this multi-layered signalling to healthy human ageing and their potential role in human disease.
↑ Levine DC et al.: NAD⁺ Controls Circadian Reprogramming through PER2 Nuclear Translocation to Counter Aging. Mol Cell 2020. (PMID 32369735) [PubMed] [DOI] [Full text] Disrupted sleep-wake and molecular circadian rhythms are a feature of aging associated with metabolic disease and reduced levels of NAD+, yet whether changes in nucleotide metabolism control circadian behavioral and genomic rhythms remains unknown. Here, we reveal that supplementation with the NAD+ precursor nicotinamide riboside (NR) markedly reprograms metabolic and stress-response pathways that decline with aging through inhibition of the clock repressor PER2. NR enhances BMAL1 chromatin binding genome-wide through PER2K680 deacetylation, which in turn primes PER2 phosphorylation within a domain that controls nuclear transport and stability and that is mutated in human advanced sleep phase syndrome. In old mice, dampened BMAL1 chromatin binding, transcriptional oscillations, mitochondrial respiration rhythms, and late evening activity are restored by NAD+ repletion to youthful levels with NR. These results reveal effects of NAD+ on metabolism and the circadian system with aging through the spatiotemporal control of the molecular clock.
↑ Peek CB et al.: Circadian clock NAD+ cycle drives mitochondrial oxidative metabolism in mice. Science 2013. (PMID 24051248) [PubMed] [DOI] [Full text] Circadian clocks are self-sustained cellular oscillators that synchronize oxidative and reductive cycles in anticipation of the solar cycle. We found that the clock transcription feedback loop produces cycles of nicotinamide adenine dinucleotide (NAD(+)) biosynthesis, adenosine triphosphate production, and mitochondrial respiration through modulation of mitochondrial protein acetylation to synchronize oxidative metabolic pathways with the 24-hour fasting and feeding cycle. Circadian control of the activity of the NAD(+)-dependent deacetylase sirtuin 3 (SIRT3) generated rhythms in the acetylation and activity of oxidative enzymes and respiration in isolated mitochondria, and NAD(+) supplementation restored protein deacetylation and enhanced oxygen consumption in circadian mutant mice. Thus, circadian control of NAD(+) bioavailability modulates mitochondrial oxidative function and organismal metabolism across the daily cycles of fasting and feeding.
↑ Rehan L et al.: SIRT1 and NAD as regulators of ageing. Life Sci 2014. (PMID 24657895) [PubMed] [DOI] The recent research on ageing processes in mammals throws new light on the biochemistry of circadian clock. The already known regulatory pathways for biological rhythms and metabolism, combined with newly discovered functions of sirtuins, unveil a perspective for new hypotheses, regarding possible links between ageing and circadian rhythms. The NAD World hypothesis - postulated as a systemic regulatory network for the metabolism and ageing, linked with mammalian, NAD+ dependent Sirtuin 1 - conceptually involves two critical elements. One is the systemic, Nampt-controlled NAD+ (nicotinamide phosphoribosyltransferase) biosynthesis, where Nampt (nicotinamide phosphoribosyltransferase) acts as "propulsion" for metabolism and the other is NAD+ dependent deacetylase (SIRT1) - a regulator responsible for various biological effects, depending on its localisation in organism. In this approach, the role of sirtuins, which are evolutionary conservative, NAD+ dependent histone deacetylases, may be very important for the mammalian metabolic clock. This paper is a review of current research on possible links among SIRT1 (Sirtuin 1), metabolism and ageing with particular consideration of the NAD World hypothesis.
↑ Chu X & Raju RP: Regulation of NAD⁺ metabolism in aging and disease. Metabolism 2022. (PMID 34743990) [PubMed] [DOI] [Full text] More than a century after discovering NAD+, information is still evolving on the role of this molecule in health and diseases. The biological functions of NAD+ and NAD+ precursors encompass pathways in cellular energetics, inflammation, metabolism, and cell survival. Several metabolic and neurological diseases exhibit reduced tissue NAD+ levels. Significantly reduced levels of NAD+ are also associated with aging, and enhancing NAD+ levels improved healthspan and lifespan in animal models. Recent studies suggest a causal link between senescence, age-associated reduction in tissue NAD+ and enzymatic degradation of NAD+. Furthermore, the discovery of transporters and receptors involved in NAD+ precursor (nicotinic acid, or niacin, nicotinamide, and nicotinamide riboside) metabolism allowed for a better understanding of their role in cellular homeostasis including signaling functions that are independent of their functions in redox reactions. We also review studies that demonstrate that the functional effect of niacin is partially due to the activation of its cell surface receptor, GPR109a. Based on the recent progress in understanding the mechanism and function of NAD+ and NAD+ precursors in cell metabolism, new strategies are evolving to exploit these molecules' pharmacological potential in the maintenance of metabolic balance.
↑ Gilmour BC et al.: Targeting NAD⁺ in translational research to relieve diseases and conditions of metabolic stress and ageing. Mech Ageing Dev 2020. (PMID 31953124) [PubMed] [DOI] Nicotinamide adenine dinucleotide (NAD+) plays a fundamental role in life and health through the regulation of energy biogenesis, redox homeostasis, cell metabolism, and the arbitration of cell survival via linkages to apoptosis and autophagic pathways. The importance of NAD+ in ageing and healthy longevity has been revealed from laboratory animal studies and early-stage clinical testing. While basic researchers and clinicians have investigated the molecular mechanisms and translation potential of NAD+, there are still major gaps in applying laboratory science to design the most effective trials. This mini-review was based on the programme and discussions of the 3rd NO-Age Symposium held at the Akershus University Hospital, Norway on the 28th October 2019. This symposium brought together leading basic researchers on NAD+ and clinicians who are leading or are going to perform NAD+ augmentation-related clinical studies. This meeting covered talks about NAD+ synthetic pathways, subcellular homeostasis of NAD+, the benefits of NAD+ augmentation from maternal milk to offspring, current clinical trials of the NAD+ precursor nicotinamide riboside (NR) on Ataxia-Telangiectasia (A-T), Parkinson's disease (PD), post-sepsis fatigue, as well as other potential NR-based clinical trials. Importantly, a consensus is emerging with respect to the design of clinical trials in order to measure meaningful parameters and ensure safety.
↑ Zhang M & Ying W: NAD⁺ Deficiency Is a Common Central Pathological Factor of a Number of Diseases and Aging: Mechanisms and Therapeutic Implications. Antioxid Redox Signal 2019. (PMID 29295624) [PubMed] [DOI] Increasing evidence has indicated critical roles of nicotinamide adenine dinucleotide, oxidized form (NAD+) in various biological functions. NAD+ deficiency has been found in models of a number of diseases such as cerebral ischemia, myocardial ischemia, and diabetes, and in models of aging. Applications of NAD+ or other approaches that can restore NAD+ levels are highly protective in these models of diseases and aging. NAD+ produces its beneficial effects by targeting at multiple pathological pathways, including attenuating mitochondrial alterations, DNA damage, and oxidative stress, by modulating such enzymes as sirtuins, glyceraldehyde-3-phosphate dehydrogenase, and AP endonuclease. These findings have suggested great therapeutic and nutritional potential of NAD+ for diseases and senescence. Recent Advances: Approaches that can restore NAD+ levels are highly protective in the models of such diseases as glaucoma. The NAD+ deficiency in the diseases and aging results from not only poly(ADP-ribose) polymerase-1 (PARP-1) activation but also decreased nicotinamide phosphoribosyltransferase (Nampt) activity and increased CD38 activity. Significant biological effects of extracellular NAD+ have been found. Increasing evidence has suggested that NAD+ deficiency is a common central pathological factor in a number of diseases and aging. Critical Issues and Future Directions: Future studies are required for solidly establishing the concept that "NAD+ deficiency is a common central pathological factor in a number of disease and aging." It is also necessary to further investigate the mechanisms underlying the NAD+ deficiency in the diseases and aging. Preclinical and clinical studies should be conducted to determine the therapeutic potential of NAD+ for the diseases and aging.
↑ Zhou FQ: NAD⁺, Senolytics, or Pyruvate for Healthy Aging?. Nutr Metab Insights 2021. (PMID 34720589) [PubMed] [DOI] [Full text] In last decades, healthy aging has become one of research hotspots in life science. It is well known that the nicotinamide adenine dinucleotide oxidized form (NAD+) level in cells decreases with aging and aging-related diseases. Several years ago, one of NAD+ precursors was first demonstrated with its new role in DNA damage repairing in mice, restoring old mice to their physical state at young ones. The finding encourages extensive studies in animal models and patients. NAD+ and its precursors have been popular products in nutrition markets. Alternatively, it was also evidenced that clearance of cellular senescence by senolytics preserved multiorgan (kidney and heart) function and extended healthy lifespan in mice. Subsequent studies confirmed findings in elderly patients subjected with idiopathic pulmonary fibrosis. The senolytic therapy is now focused on various diseases in animal and clinical studies. However, pyruvate, as both a NAD+ substitute and a new senolytic, may be advantageous, on the equimolar basis, over current products above in preventing and treating diseases and aging. Pyruvate-enriched fluids, particularly pyruvate oral rehydration salt, may be a novel intervention for diseases and aging besides critical care. Albeit the direct evidence that benefits healthy aging is still limited to date, pyruvate, as both NAD+ provider and senolytic agent, warrants intensive research to compare NAD+ or senolytics for healthy aging, specifically on the equimolar basis, in effective blood levels. This review briefly discussed the recognition of healthy aging by comparing NAD+ and Senolytics with sodium pyruvate from the clinical point of view.
↑ Covarrubias AJ et al.: NAD⁺ metabolism and its roles in cellular processes during ageing. Nat Rev Mol Cell Biol 2021. (PMID 33353981) [PubMed] [DOI] [Full text] Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for redox reactions, making it central to energy metabolism. NAD+ is also an essential cofactor for non-redox NAD+-dependent enzymes, including sirtuins, CD38 and poly(ADP-ribose) polymerases. NAD+ can directly and indirectly influence many key cellular functions, including metabolic pathways, DNA repair, chromatin remodelling, cellular senescence and immune cell function. These cellular processes and functions are critical for maintaining tissue and metabolic homeostasis and for healthy ageing. Remarkably, ageing is accompanied by a gradual decline in tissue and cellular NAD+ levels in multiple model organisms, including rodents and humans. This decline in NAD+ levels is linked causally to numerous ageing-associated diseases, including cognitive decline, cancer, metabolic disease, sarcopenia and frailty. Many of these ageing-associated diseases can be slowed down and even reversed by restoring NAD+ levels. Therefore, targeting NAD+ metabolism has emerged as a potential therapeutic approach to ameliorate ageing-related disease, and extend the human healthspan and lifespan. However, much remains to be learnt about how NAD+ influences human health and ageing biology. This includes a deeper understanding of the molecular mechanisms that regulate NAD+ levels, how to effectively restore NAD+ levels during ageing, whether doing so is safe and whether NAD+ repletion will have beneficial effects in ageing humans.
↑ Balashova NV et al.: Efficient Assay and Marker Significance of NAD⁺ in Human Blood. Front Med (Lausanne) 2022. (PMID 35665345) [PubMed] [DOI] [Full text] Oxidized nicotinamide adenine dinucleotide (NAD+) is a biological molecule of systemic importance. Essential role of NAD+ in cellular metabolism relies on the substrate action in various redox reactions and cellular signaling. This work introduces an efficient enzymatic assay of NAD+ content in human blood using recombinant formate dehydrogenase (FDH, EC 1.2.1.2), and demonstrates its diagnostic potential, comparing NAD+ content in the whole blood of control subjects and patients with cardiac or neurological pathologies. In the control group (n = 22, 25-70 years old), our quantification of the blood concentration of NAD+ (18 μM, minimum 15, max 23) corresponds well to NAD+ quantifications reported in literature. In patients with demyelinating neurological diseases (n = 10, 18-55 years old), the NAD+ levels significantly (p < 0.0001) decrease (to 14 μM, min 13, max 16), compared to the control group. In cardiac patients with the heart failure of stage II and III according to the New York Heart Association (NYHA) functional classification (n = 24, 42-83 years old), the blood levels of NAD+ (13 μM, min 9, max 18) are lower than those in the control subjects (p < 0.0001) or neurological patients (p = 0.1). A better discrimination of the cardiac and neurological patients is achieved when the ratios of NAD+ to the blood creatinine levels, mean corpuscular volume or potassium ions are compared. The proposed NAD+ assay provides an easy and robust tool for clinical analyses of an important metabolic indicator in the human blood.
↑ Peluso A et al.: Age-Dependent Decline of NAD⁺-Universal Truth or Confounded Consensus?. Nutrients 2021. (PMID 35010977) [PubMed] [DOI] [Full text] Nicotinamide adenine dinucleotide (NAD+) is an essential molecule involved in various metabolic reactions, acting as an electron donor in the electron transport chain and as a co-factor for NAD+-dependent enzymes. In the early 2000s, reports that NAD+ declines with aging introduced the notion that NAD+ metabolism is globally and progressively impaired with age. Since then, NAD+ became an attractive target for potential pharmacological therapies aiming to increase NAD+ levels to promote vitality and protect against age-related diseases. This review summarizes and discusses a collection of studies that report the levels of NAD+ with aging in different species (i.e., yeast, C. elegans, rat, mouse, monkey, and human), to determine whether the notion that overall NAD+ levels decrease with aging stands true. We find that, despite systematic claims of overall changes in NAD+ levels with aging, the evidence to support such claims is very limited and often restricted to a single tissue or cell type. This is particularly true in humans, where the development of NAD+ levels during aging is still poorly characterized. There is a need for much larger, preferably longitudinal, studies to assess how NAD+ levels develop with aging in various tissues. This will strengthen our conclusions on NAD metabolism during aging and should provide a foundation for better pharmacological targeting of relevant tissues.
↑ Rajman L et al.: Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence. Cell Metab 2018. (PMID 29514064) [PubMed] [DOI] [Full text] Nicotinamide adenine dinucleotide (NAD), the cell's hydrogen carrier for redox enzymes, is well known for its role in redox reactions. More recently, it has emerged as a signaling molecule. By modulating NAD+-sensing enzymes, NAD+ controls hundreds of key processes from energy metabolism to cell survival, rising and falling depending on food intake, exercise, and the time of day. NAD+ levels steadily decline with age, resulting in altered metabolism and increased disease susceptibility. Restoration of NAD+ levels in old or diseased animals can promote health and extend lifespan, prompting a search for safe and efficacious NAD-boosting molecules that hold the promise of increasing the body's resilience, not just to one disease, but to many, thereby extending healthy human lifespan.

[1] Camacho-Pereira J, Tarragó MG, Chini CC, Nin V, Escande C, Warner GM, Puranik AS, Schoon RA, Reid JM, Galina A, Chini EN (June 2016). "CD38 Dictates Age-Related NAD Decline and Mitochondrial Dysfunction through an SIRT3-Dependent Mechanism". Cell Metabolism. 23 (6): 1127–1139. doi:10.1016/j.cmet.2016.05.006. PMC 4911708. PMID 27304511.

[2] Schultz MB, Sinclair DA (June 2016). "Why NAD(+) Declines during Aging: It's Destroyed". Cell Metabolism. 23 (6): 965–966. doi:10.1016/j.cmet.2016.05.022. PMC 5088772. PMID 27304496.

[3] Tarragó MG, Chini CC, Kanamori KS, Warner GM, Caride A, de Oliveira GC, Rud M, Samani A, Hein KZ, Huang R, Jurk D, Cho DS, Boslett JJ, Miller JD, Zweier JL, Passos JF, Doles JD, Becherer DJ, Chini EN (May 2018). "A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD+ Decline". Cell Metabolism. 27 (5): 1081–1095.e10. doi:10.1016/j.cmet.2018.03.016. PMC 5935140. PMID 29719225.

[pmid32595066-4] 4.0 ^4.1 Cambronne XA, Kraus WL (2020). "Location, Location, Location: Compartmentalization of NAD + Synthesis and Functions in Mammalian Cells". Trends in Biochemical Sciences. 45 (10): 858–873. doi:10.1016/j.tibs.2020.05.010. PMC 7502477. PMID 32595066.

[pmid32594513-5] Kang BE, Choi J, Stein S, Ryu D (2020). "Implications of NAD + boosters in translational medicine". European Journal of Clinical Investigation. 50 (10): e13334. doi:10.1111/eci.13334. PMID 32594513. S2CID 220254270.

[pmid27304496-6] Schultz MB & Sinclair DA: Why NAD(+) Declines during Aging: It's Destroyed. Cell Metab 2016. (PMID 27304496) [PubMed] [DOI] [Full text] NAD(+) is required not only for life but for a long life. In this issue, Camacho-Pereira et al. (2016) implicate CD38 in the decline of NAD(+) during aging, with implications for combating age-related diseases.

[pmid28676700-7] Ray Chaudhuri A & Nussenzweig A: The multifaceted roles of PARP1 in DNA repair and chromatin remodelling. Nat Rev Mol Cell Biol 2017. (PMID 28676700) [PubMed] [DOI] [Full text] Cells are exposed to various endogenous and exogenous insults that induce DNA damage, which, if unrepaired, impairs genome integrity and leads to the development of various diseases, including cancer. Recent evidence has implicated poly(ADP-ribose) polymerase 1 (PARP1) in various DNA repair pathways and in the maintenance of genomic stability. The inhibition of PARP1 is therefore being exploited clinically for the treatment of various cancers, which include DNA repair-deficient ovarian, breast and prostate cancers. Understanding the role of PARP1 in maintaining genome integrity is not only important for the design of novel chemotherapeutic agents, but is also crucial for gaining insights into the mechanisms of chemoresistance in cancer cells. In this Review, we discuss the roles of PARP1 in mediating various aspects of DNA metabolism, such as single-strand break repair, nucleotide excision repair, double-strand break repair and the stabilization of replication forks, and in modulating chromatin structure.

[pmid25908823-8] Gerdts J et al.: SARM1 activation triggers axon degeneration locally via NAD⁺ destruction. Science 2015. (PMID 25908823) [PubMed] [DOI] [Full text] Axon degeneration is an intrinsic self-destruction program that underlies axon loss during injury and disease. Sterile alpha and TIR motif-containing 1 (SARM1) protein is an essential mediator of axon degeneration. We report that SARM1 initiates a local destruction program involving rapid breakdown of nicotinamide adenine dinucleotide (NAD(+)) after injury. We used an engineered protease-sensitized SARM1 to demonstrate that SARM1 activity is required after axon injury to induce axon degeneration. Dimerization of the Toll-interleukin receptor (TIR) domain of SARM1 alone was sufficient to induce locally mediated axon degeneration. Formation of the SARM1 TIR dimer triggered rapid breakdown of NAD(+), whereas SARM1-induced axon destruction could be counteracted by increased NAD(+) synthesis. SARM1-induced depletion of NAD(+) may explain the potent axon protection in Wallerian degeneration slow (Wld(s)) mutant mice.

[pmid32694684-9] Katsyuba E et al.: NAD⁺ homeostasis in health and disease. Nat Metab 2020. (PMID 32694684) [PubMed] [DOI] [Full text] The conceptual evolution of nicotinamide adenine dinucleotide (NAD+) from being seen as a simple metabolic cofactor to a pivotal cosubstrate for proteins regulating metabolism and longevity, including the sirtuin family of protein deacylases, has led to a new wave of scientific interest in NAD+. NAD+ levels decline during ageing, and alterations in NAD+ homeostasis can be found in virtually all age-related diseases, including neurodegeneration, diabetes and cancer. In preclinical settings, various strategies to increase NAD+ levels have shown beneficial effects, thus starting a competitive race to discover marketable NAD+ boosters to improve healthspan and lifespan. Here, we review the basics of NAD+ biochemistry and metabolism, and its roles in health and disease, and we discuss current challenges and the future translational potential of NAD+ research.

[pmid32389638-10] Mateuszuk Ł et al.: Reversal of endothelial dysfunction by nicotinamide mononucleotide via extracellular conversion to nicotinamide riboside. Biochem Pharmacol 2020. (PMID 32389638) [PubMed] [DOI] BACKGROUND: Nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) are effective substrates for NAD synthesis, which may act as vasoprotective agents. Here, we characterize the effects of NMN and NR on endothelial inflammation and dysfunction and test the involvement of CD73 in these effects. MATERIALS AND METHODS: The effect of NMN and NR on IL1β- or TNFα-induced endothelial inflammation (ICAM1 and vWF expression), intracellular NAD concentration and NAD-related enzyme expression (NAMPT, CD38, CD73), were studied in HAECs. The effect of NMN and NR on angiotensin II-induced impairment of endothelium-dependent vasodilation was analyzed in murine aortic rings. The involvement of CD73 in NMN and NR effects was tested using CD73 inhibitor-AOPCP, or CD73-/- mice. RESULTS: 24 h-incubation with NMN and NR induced anti-inflammatory effects in HAEC stimulated by IL1β or TNFα, as evidenced by a reduction in ICAM1 and vWF expression. Effects of exogenous NMN but not NR was abrogated in the presence of AOPCP, that efficiently inhibited extracellular endothelial conversion of NMN to NR, without a significant effect on the metabolism of NMN to NA. Surprisingly, intracellular NAD concentration increased in HAEC stimulated by IL1β or TNFα and this effect was associated with upregulation of NAMPT and CD73, whereas changes in CD38 expression were less pronounced. NMN and NR further increased NAD in IL1β-stimulated HAECs and AOPCP diminished NMN-induced increase in NAD, without an effect on NR-induced response. In ex vivo aortic rings stimulated with angiotensin II for 24 h, NO-dependent vasorelaxation induced by acetylcholine was impaired. NMN and NR, both prevented Ang II-induced endothelial dysfunction in the aorta. In aortic rings taken from CD73-/- mice NMN effect was lost, whereas NR effect was preserved. CONCLUSION: NMN and NR modulate intracellular NAD content in endothelium, inhibit endothelial inflammation and improve NO-dependent function by CD73-dependent and independent pathways, respectively. Extracellular conversion of NMN to NR by CD73 localized in the luminal surface of endothelial cells represent important vasoprotective mechanisms to maintain intracellular NAD.

[pmid29483571-11] 11.0 ^11.1 Kannt A et al.: A small molecule inhibitor of Nicotinamide N-methyltransferase for the treatment of metabolic disorders. Sci Rep 2018. (PMID 29483571) [PubMed] [DOI] [Full text] Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that catalyzes the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) onto the substrate, nicotinamide (NA) to form 1-methyl-nicotinamide (MNA). Higher NNMT expression and MNA concentrations have been associated with obesity and type-2 diabetes. Here we report a small molecule analog of NA, JBSNF-000088, that inhibits NNMT activity, reduces MNA levels and drives insulin sensitization, glucose modulation and body weight reduction in animal models of metabolic disease. In mice with high fat diet (HFD)-induced obesity, JBSNF-000088 treatment caused a reduction in body weight, improved insulin sensitivity and normalized glucose tolerance to the level of lean control mice. These effects were not seen in NNMT knockout mice on HFD, confirming specificity of JBSNF-000088. The compound also improved glucose handling in ob/ob and db/db mice albeit to a lesser extent and in the absence of weight loss. Co-crystal structure analysis revealed the presence of the N-methylated product of JBSNF-000088 bound to the NNMT protein. The N-methylated product was also detected in the plasma of mice treated with JBSNF-000088. Hence, JBSNF-000088 may act as a slow-turnover substrate analog, driving the observed metabolic benefits.

[pmid34153425-12] Campagna R et al.: Nicotinamide N-methyltransferase in endothelium protects against oxidant stress-induced endothelial injury. Biochim Biophys Acta Mol Cell Res 2021. (PMID 34153425) [PubMed] [DOI] Nicotinamide N-methyltransferase (NNMT, EC 2.1.1.1.) plays an important role in the growth of many different tumours and is also involved in various non-neoplastic disorders. However, the presence and role of NNMT in the endothelium has yet to be specifically explored. Here, we characterized the functional activity of NNMT in the endothelium and tested whether NNMT regulates endothelial cell viability. NNMT in endothelial cells (HAEC, HMEC-1 and EA.hy926) was inhibited using two approaches: pharmacological inhibition of the enzyme by NNMT inhibitors (5-amino-1-methylquinoline - 5MQ and 6-methoxynicotinamide - JBSF-88) or by shRNA-mediated silencing. Functional inhibition of NNMT was confirmed by LC/MS/MS-based analysis of impaired MNA production. The effects of NNMT inhibition on cellular viability were analyzed in both the absence and presence of menadione. Our results revealed that all studied endothelial lines express relatively high levels of functionally active NNMT compared with cancer cells (MDA-MB-231). Although the aldehyde oxidase 1 enzyme was also expressed in the endothelium, the further metabolites of N1-methylnicotinamide (N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide) generated by this enzyme were not detected, suggesting that endothelial NNMT-derived MNA was not subsequently metabolized in the endothelium by aldehyde oxidase 1. Menadione induced a concentration-dependent decrease in endothelial viability as evidenced by a decrease in cell number that was associated with the upregulation of NNMT and SIRT1 expression in the nucleus in viable cells. The suppression of the NNMT activity either by NNMT inhibitors or shRNA-based silencing significantly decreased the endothelial cell viability in response to menadione. Furthermore, NNMT inhibition resulted in nuclear SIRT1 expression downregulation and upregulation of the phosphorylated form of SIRT1 on Ser47. In conclusion, our results suggest that the endothelial nuclear NNMT/SIRT1 pathway exerts a cytoprotective role that safeguards endothelial cell viability under oxidant stress insult.

[pmid26168293-13] Hong S et al.: Nicotinamide N-methyltransferase regulates hepatic nutrient metabolism through Sirt1 protein stabilization. Nat Med 2015. (PMID 26168293) [PubMed] [DOI] [Full text] Nicotinamide N-methyltransferase (Nnmt) methylates nicotinamide, a form of vitamin B3, to produce N(1)-methylnicotinamide (MNAM). Nnmt has emerged as a metabolic regulator in adipocytes, but its role in the liver, the tissue with the strongest Nnmt expression, is not known. In spite of its overall high expression, here we find that hepatic expression of Nnmt is highly variable and correlates with multiple metabolic parameters in mice and humans. Further, we find that suppression of hepatic Nnmt expression in vivo alters glucose and cholesterol metabolism and that the metabolic effects of Nnmt in the liver are mediated by its product MNAM. Supplementation of high-fat diet with MNAM decreases serum and liver cholesterol and liver triglycerides levels in mice. Mechanistically, increasing Nnmt expression or MNAM levels stabilizes sirtuin 1 protein, an effect that is required for their metabolic benefits. In summary, we describe here a novel regulatory pathway for vitamin B3 that could provide a new opportunity for metabolic disease therapy.

[pmid24077178-14] Schmeisser K et al.: Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide. Nat Chem Biol 2013. (PMID 24077178) [PubMed] [DOI] [Full text] Sirtuins, a family of histone deacetylases, have a fiercely debated role in regulating lifespan. In contrast with recent observations, here we find that overexpression of sir-2.1, the ortholog of mammalian SirT1, does extend Caenorhabditis elegans lifespan. Sirtuins mandatorily convert NAD(+) into nicotinamide (NAM). We here find that NAM and its metabolite, 1-methylnicotinamide (MNA), extend C. elegans lifespan, even in the absence of sir-2.1. We identify a previously unknown C. elegans nicotinamide-N-methyltransferase, encoded by a gene now named anmt-1, to generate MNA from NAM. Disruption and overexpression of anmt-1 have opposing effects on lifespan independent of sirtuins, with loss of anmt-1 fully inhibiting sir-2.1-mediated lifespan extension. MNA serves as a substrate for a newly identified aldehyde oxidase, GAD-3, to generate hydrogen peroxide, which acts as a mitohormetic reactive oxygen species signal to promote C. elegans longevity. Taken together, sirtuin-mediated lifespan extension depends on methylation of NAM, providing an unexpected mechanistic role for sirtuins beyond histone deacetylation.

[pmid34704059-15] van Haren MJ et al.: Macrocyclic peptides as allosteric inhibitors of nicotinamide N-methyltransferase (NNMT). RSC Chem Biol 2021. (PMID 34704059) [PubMed] [DOI] [Full text] Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide (MNA) using S-adenosyl-l-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly being elucidated and provides an indication that NNMT may be an interesting therapeutic target for a variety of diseases including cancer, diabetes, and obesity. Most inhibitors of NNMT described to date are structurally related to one or both of its substrates. In the search for structurally diverse NNMT inhibitors, an mRNA display screening technique was used to identify macrocyclic peptides which bind to NNMT. Several of the cyclic peptides identified in this manner show potent inhibition of NNMT with IC50 values as low as 229 nM. The peptides were also found to downregulate MNA production in cellular assays. Interestingly, substrate competition experiments reveal that these cyclic peptide inhibitors are noncompetitive with either SAM or NA indicating they may be the first allosteric inhibitors reported for NNMT.

[pmid34572571-16] van Haren MJ et al.: Esterase-Sensitive Prodrugs of a Potent Bisubstrate Inhibitor of Nicotinamide N-Methyltransferase (NNMT) Display Cellular Activity. Biomolecules 2021. (PMID 34572571) [PubMed] [DOI] [Full text] A recently discovered bisubstrate inhibitor of Nicotinamide N-methyltransferase (NNMT) was found to be highly potent in biochemical assays with a single digit nanomolar IC50 value but lacking in cellular activity. We, here, report a prodrug strategy designed to translate the observed potent biochemical inhibitory activity of this inhibitor into strong cellular activity. This prodrug strategy relies on the temporary protection of the amine and carboxylic acid moieties of the highly polar amino acid side chain present in the bisubstrate inhibitor. The modification of the carboxylic acid into a range of esters in the absence or presence of a trimethyl-lock (TML) amine protecting group yielded a range of candidate prodrugs. Based on the stability in an aqueous buffer, and the confirmed esterase-dependent conversion to the parent compound, the isopropyl ester was selected as the preferred acid prodrug. The isopropyl ester and isopropyl ester-TML prodrugs exhibit improved cell permeability, which also translates to significantly enhanced cellular activity as established using assays designed to measure the enzymatic activity of NNMT in live cells.

[pmid29155147-17] Neelakantan H et al.: Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice. Biochem Pharmacol 2018. (PMID 29155147) [PubMed] [DOI] [Full text] There is a critical need for new mechanism-of-action drugs that reduce the burden of obesity and associated chronic metabolic comorbidities. A potentially novel target to treat obesity and type 2 diabetes is nicotinamide-N-methyltransferase (NNMT), a cytosolic enzyme with newly identified roles in cellular metabolism and energy homeostasis. To validate NNMT as an anti-obesity drug target, we investigated the permeability, selectivity, mechanistic, and physiological properties of a series of small molecule NNMT inhibitors. Membrane permeability of NNMT inhibitors was characterized using parallel artificial membrane permeability and Caco-2 cell assays. Selectivity was tested against structurally-related methyltransferases and nicotinamide adenine dinucleotide (NAD+) salvage pathway enzymes. Effects of NNMT inhibitors on lipogenesis and intracellular levels of metabolites, including NNMT reaction product 1-methylnicotianamide (1-MNA) were evaluated in cultured adipocytes. Effects of a potent NNMT inhibitor on obesity measures and plasma lipid were assessed in diet-induced obese mice fed a high-fat diet. Methylquinolinium scaffolds with primary amine substitutions displayed high permeability from passive and active transport across membranes. Importantly, methylquinolinium analogues displayed high selectivity, not inhibiting related SAM-dependent methyltransferases or enzymes in the NAD+ salvage pathway. NNMT inhibitors reduced intracellular 1-MNA, increased intracellular NAD+ and S-(5'-adenosyl)-l-methionine (SAM), and suppressed lipogenesis in adipocytes. Treatment of diet-induced obese mice systemically with a potent NNMT inhibitor significantly reduced body weight and white adipose mass, decreased adipocyte size, and lowered plasma total cholesterol levels. Notably, administration of NNMT inhibitors did not impact total food intake nor produce any observable adverse effects. These results support development of small molecule NNMT inhibitors as therapeutics to reverse diet-induced obesity and validate NNMT as a viable target to treat obesity and related metabolic conditions. Increased flux of key cellular energy regulators, including NAD+ and SAM, may potentially define the therapeutic mechanism-of-action of NNMT inhibitors.

[pmid34424711-18] Gao Y et al.: Potent Inhibition of Nicotinamide N-Methyltransferase by Alkene-Linked Bisubstrate Mimics Bearing Electron Deficient Aromatics. J Med Chem 2021. (PMID 34424711) [PubMed] [DOI] [Full text] Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide (vitamin B3) to generate 1-methylnicotinamide (MNA). NNMT overexpression has been linked to a variety of diseases, most prominently human cancers, indicating its potential as a therapeutic target. The development of small-molecule NNMT inhibitors has gained interest in recent years, with the most potent inhibitors sharing structural features based on elements of the nicotinamide substrate and the S-adenosyl-l-methionine (SAM) cofactor. We here report the development of new bisubstrate inhibitors that include electron-deficient aromatic groups to mimic the nicotinamide moiety. In addition, a trans-alkene linker was found to be optimal for connecting the substrate and cofactor mimics in these inhibitors. The most potent NNMT inhibitor identified exhibits an IC50 value of 3.7 nM, placing it among the most active NNMT inhibitors reported to date. Complementary analytical techniques, modeling studies, and cell-based assays provide insights into the binding mode, affinity, and selectivity of these inhibitors.

[pmid36678315-19] Sharma A et al.: Potential Synergistic Supplementation of NAD+ Promoting Compounds as a Strategy for Increasing Healthspan. Nutrients 2023. (PMID 36678315) [PubMed] [DOI] [Full text] Disrupted biological function, manifesting through the hallmarks of aging, poses one of the largest threats to healthspan and risk of disease development, such as metabolic disorders, cardiovascular ailments, and neurodegeneration. In recent years, numerous geroprotectors, senolytics, and other nutraceuticals have emerged as potential disruptors of aging and may be viable interventions in the immediate state of human longevity science. In this review, we focus on the decrease in nicotinamide adenine dinucleotide (NAD+) with age and the supplementation of NAD+ precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), in combination with other geroprotective compounds, to restore NAD+ levels present in youth. Furthermore, these geroprotectors may enhance the efficacy of NMN supplementation while concurrently providing their own numerous health benefits. By analyzing the prevention of NAD+ degradation through the inhibition of CD38 or supporting protective downstream agents of SIRT1, we provide a potential framework of the CD38/NAD+/SIRT1 axis through which geroprotectors may enhance the efficacy of NAD+ precursor supplementation and reduce the risk of age-related diseases, thereby potentiating healthspan in humans.

[pmid28537485-20] Mendelsohn AR & Larrick JW: The NAD+/PARP1/SIRT1 Axis in Aging. Rejuvenation Res 2017. (PMID 28537485) [PubMed] [DOI] NAD+ levels decline with age in diverse animals from Caenorhabditis elegans to mice. Raising NAD+ levels by dietary supplementation with NAD+ precursors, nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN), improves mitochondrial function and muscle and neural and melanocyte stem cell function in mice, as well as increases murine life span. Decreased NAD+ levels with age reduce SIRT1 function and reduce the mitochondrial unfolded protein response, which can be overcome by NR supplementation. Decreased NAD+ levels cause NAD+-binding protein DBC1 to form a complex with PARP1, inhibiting poly(adenosine diphosphate-ribose) polymerase (PARP) catalytic activity. Old mice have increased amounts of DBC1-PARP1 complexes, lower PARP activity, increased DNA damage, and reduced nonhomologous end joining and homologous recombination repair. DBC1-PARP1 complexes in old mice can be broken by increasing NAD+ levels through treatment with NMN, reducing DNA damage and restoring PARP activity to youthful levels. The mechanism of declining NAD+ levels and its fundamental importance to aging are yet to be elucidated. There is a correlation of PARP activity with mammalian life span that suggests that NAD+/SIRT1/PARP1 may be more significant than the modest effects on life span observed for NR supplementation in old mice. The NAD+/PARP1/SIRT1 axis may link NAD+ levels and DNA damage with the apparent epigenomic DNA methylation clocks that have been described.

[pmid28899755-21] Fang EF et al.: NAD⁺ in Aging: Molecular Mechanisms and Translational Implications. Trends Mol Med 2017. (PMID 28899755) [PubMed] [DOI] [Full text] The coenzyme NAD+ is critical in cellular bioenergetics and adaptive stress responses. Its depletion has emerged as a fundamental feature of aging that may predispose to a wide range of chronic diseases. Maintenance of NAD+ levels is important for cells with high energy demands and for proficient neuronal function. NAD+ depletion is detected in major neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, cardiovascular disease and muscle atrophy. Emerging evidence suggests that NAD+ decrements occur in various tissues during aging, and that physiological and pharmacological interventions bolstering cellular NAD+ levels might retard aspects of aging and forestall some age-related diseases. Here, we discuss aspects of NAD+ biosynthesis, together with putative mechanisms of NAD+ action against aging, including recent preclinical and clinical trials.

[pmid29883761-22] 22.0 ^22.1 Yaku K et al.: NAD metabolism: Implications in aging and longevity. Ageing Res Rev 2018. (PMID 29883761) [PubMed] [DOI] Nicotinamide adenine dinucleotide (NAD) is an important co-factor involved in numerous physiological processes, including metabolism, post-translational protein modification, and DNA repair. In living organisms, a careful balance between NAD production and degradation serves to regulate NAD levels. Recently, a number of studies have demonstrated that NAD levels decrease with age, and the deterioration of NAD metabolism promotes several aging-associated diseases, including metabolic and neurodegenerative diseases and various cancers. Conversely, the upregulation of NAD metabolism, including dietary supplementation with NAD precursors, has been shown to prevent the decline of NAD and exhibits beneficial effects against aging and aging-associated diseases. In addition, many studies have demonstrated that genetic and/or nutritional activation of NAD metabolism can extend the lifespan of diverse organisms. Collectively, it is clear that NAD metabolism plays important roles in aging and longevity. In this review, we summarize the basic functions of the enzymes involved in NAD synthesis and degradation, as well as the outcomes of their dysregulation in various aging processes. In addition, a particular focus is given on the role of NAD metabolism in the longevity of various organisms, with a discussion of the remaining obstacles in this research field.

[pmid27825999-23] Chini CCS et al.: NAD and the aging process: Role in life, death and everything in between. Mol Cell Endocrinol 2017. (PMID 27825999) [PubMed] [DOI] [Full text] Life as we know it cannot exist without the nucleotide nicotinamide adenine dinucleotide (NAD). From the simplest organism, such as bacteria, to the most complex multicellular organisms, NAD is a key cellular component. NAD is extremely abundant in most living cells and has traditionally been described to be a cofactor in electron transfer during oxidation-reduction reactions. In addition to participating in these reactions, NAD has also been shown to play a key role in cell signaling, regulating several pathways from intracellular calcium transients to the epigenetic status of chromatin. Thus, NAD is a molecule that provides an important link between signaling and metabolism, and serves as a key molecule in cellular metabolic sensoring pathways. Importantly, it has now been clearly demonstrated that cellular NAD levels decline during chronological aging. This decline appears to play a crucial role in the development of metabolic dysfunction and age-related diseases. In this review we will discuss the molecular mechanisms responsible for the decrease in NAD levels during aging. Since other reviews on this subject have been recently published, we will concentrate on presenting a critical appraisal of the current status of the literature and will highlight some controversial topics in the field. In particular, we will discuss the potential role of the NADase CD38 as a driver of age-related NAD decline.

[pmid30124109-24] Clement J et al.: The Plasma NAD⁺ Metabolome Is Dysregulated in "Normal" Aging. Rejuvenation Res 2019. (PMID 30124109) [PubMed] [DOI] [Full text] Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as an electron carrier in cellular metabolism and plays a crucial role in the maintenance of balanced redox homeostasis. Quantification of NAD+:NADH and NADP+:NADPH ratios are pivotal to a wide variety of cellular processes, including intracellular secondary messenger signaling by CD38 glycohydrolases, DNA repair by poly(adenosine diphosphate ribose) polymerase (PARP), epigenetic regulation of gene expression by NAD-dependent histone deacetylase enzymes known as sirtuins, and regulation of the oxidative pentose phosphate pathway. We quantified changes in the NAD+ metabolome in plasma samples collected from consenting healthy human subjects across a wide age range (20-87 years) using liquid chromatography coupled to tandem mass spectrometry. Our data show a significant decline in the plasma levels of NAD+, NADP+, and other important metabolites such as nicotinic acid adenine dinucleotide (NAAD) with age. However, an age-related increase in the reduced form of NAD+ and NADP+-NADH and NADPH-and nicotinamide (NAM), N-methyl-nicotinamide (MeNAM), and the products of adenosine diphosphoribosylation, including adenosine diphosphate ribose (ADPR) was also reported. Whereas, plasma levels of nicotinic acid (NA), nicotinamide mononucleotide (NMN), and nicotinic acid mononucleotide (NAMN) showed no statistically significant changes across age groups. Taken together, our data cumulatively suggest that age-related impairments are associated with corresponding alterations in the extracellular plasma NAD+ metabolome. Our future research will seek to elucidate the role of modulating NAD+ metabolites in the treatment and prevention of age-related diseases.

[pmid24360282-25] Gomes AP et al.: Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging. Cell 2013. (PMID 24360282) [PubMed] [DOI] [Full text] Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD(+) and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD(+) levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.

[pmid34256387-26] Aguiar SS et al.: Telomere Length, SIRT1, and Insulin in Male Master Athletes: The Path to Healthy Longevity?. Int J Sports Med 2022. (PMID 34256387) [PubMed] [DOI] Lower SIRT1 and insulin resistance are associated with accelerated telomere shortening. This study investigated whether the lifestyle of master athletes can attenuate these age-related changes and thereby slow aging. We compared insulin, SIRT1, and telomere length in highly trained male master athletes (n=52; aged 49.9±7.2 yrs) and age-matched non-athletes (n=19; aged 47.3±8.9 yrs). This is a cross-sectional study, in which all data were collected in one visit. Overnight fasted SIRT1 and insulin levels in whole blood were assessed using commercial kits. Relative telomere length was determined in leukocytes through qPCR analyses. Master athletes had higher SIRT1, lower insulin, and longer telomere length than age-matched non-athletes (p<0.05 for all). Insulin was inversely associated with SIRT1 (r=-0.38; p=0.001). Telomere length correlated positively with SIRT1 (r=0.65; p=0.001), whereas telomere length and insulin were not correlated (r=0.03; p=0.87). In conclusion, master athletes have higher SIRT1, lower insulin, and longer telomeres than age-matched non-athletes. Furthermore, SIRT1 was negatively associated with insulin and positively associated with telomere length. These findings suggest that in this sample of middle-aged participants reduced insulin, increased SIRT1 activity, and attenuation of biological aging are connected.

[pmid26831453-27] Tang BL: Sirt1 and the Mitochondria. Mol Cells 2016. (PMID 26831453) [PubMed] [DOI] [Full text] Sirt1 is the most prominent and extensively studied member of sirtuins, the family of mammalian class III histone deacetylases heavily implicated in health span and longevity. Although primarily a nuclear protein, Sirt1's deacetylation of Peroxisome proliferator-activated receptor Gamma Coactivator-1α (PGC-1α) has been extensively implicated in metabolic control and mitochondrial biogenesis, which was proposed to partially underlie Sirt1's role in caloric restriction and impacts on longevity. The notion of Sirt1's regulation of PGC-1α activity and its role in mitochondrial biogenesis has, however, been controversial. Interestingly, Sirt1 also appears to be important for the turnover of defective mitochondria by mitophagy. I discuss here evidences for Sirt1's regulation of mitochondrial biogenesis and turnover, in relation to PGC-1α deacetylation and various aspects of cellular physiology and disease.

[pmid14976264-28] Brunet A et al.: Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 2004. (PMID 14976264) [PubMed] [DOI] The Sir2 deacetylase modulates organismal life-span in various species. However, the molecular mechanisms by which Sir2 increases longevity are largely unknown. We show that in mammalian cells, the Sir2 homolog SIRT1 appears to control the cellular response to stress by regulating the FOXO family of Forkhead transcription factors, a family of proteins that function as sensors of the insulin signaling pathway and as regulators of organismal longevity. SIRT1 and the FOXO transcription factor FOXO3 formed a complex in cells in response to oxidative stress, and SIRT1 deacetylated FOXO3 in vitro and within cells. SIRT1 had a dual effect on FOXO3 function: SIRT1 increased FOXO3's ability to induce cell cycle arrest and resistance to oxidative stress but inhibited FOXO3's ability to induce cell death. Thus, one way in which members of the Sir2 family of proteins may increase organismal longevity is by tipping FOXO-dependent responses away from apoptosis and toward stress resistance.

[pmid16288288-29] Greer EL & Brunet A: FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 2005. (PMID 16288288) [PubMed] [DOI] A wide range of human diseases, including cancer, has a striking age-dependent onset. However, the molecular mechanisms that connect aging and cancer are just beginning to be unraveled. FOXO transcription factors are promising candidates to serve as molecular links between longevity and tumor suppression. These factors are major substrates of the protein kinase Akt. In the presence of insulin and growth factors, FOXO proteins are relocalized from the nucleus to the cytoplasm and degraded via the ubiquitin-proteasome pathway. In the absence of growth factors, FOXO proteins translocate to the nucleus and upregulate a series of target genes, thereby promoting cell cycle arrest, stress resistance, or apoptosis. Stress stimuli also trigger the relocalization of FOXO factors into the nucleus, thus allowing an adaptive response to stress stimuli. Consistent with the notion that stress resistance is highly coupled with lifespan extension, activation of FOXO transcription factors in worms and flies increases longevity. Emerging evidence also suggests that FOXO factors play a tumor suppressor role in a variety of cancers. Thus, FOXO proteins translate environmental stimuli into changes in gene expression programs that may coordinate organismal longevity and tumor suppression.

[pmid35004893-30] Zhao Y & Liu YS: Longevity Factor FOXO3: A Key Regulator in Aging-Related Vascular Diseases. Front Cardiovasc Med 2021. (PMID 35004893) [PubMed] [DOI] [Full text] Forkhead box O3 (FOXO3) has been proposed as a homeostasis regulator, capable of integrating multiple upstream signaling pathways that are sensitive to environmental changes and counteracting their adverse effects due to external changes, such as oxidative stress, metabolic stress and growth factor deprivation. FOXO3 polymorphisms are associated with extreme human longevity. Intriguingly, longevity-associated single nucleotide polymorphisms (SNPs) in human FOXO3 correlate with lower-than-average morbidity from cardiovascular diseases in long-lived people. Emerging evidence indicates that FOXO3 plays a critical role in vascular aging. FOXO3 inactivation is implicated in several aging-related vascular diseases. In experimental studies, FOXO3-engineered human ESC-derived vascular cells improve vascular homeostasis and delay vascular aging. The purpose of this review is to explore how FOXO3 regulates vascular aging and its crucial role in aging-related vascular diseases.

[pmid26675724-31] Kaletsky R et al.: The C. elegans adult neuronal IIS/FOXO transcriptome reveals adult phenotype regulators. Nature 2016. (PMID 26675724) [PubMed] [DOI] [Full text] Insulin/insulin-like growth factor signalling (IIS) is a critical regulator of an organism's most important biological decisions from growth, development, and metabolism to reproduction and longevity. It primarily does so through the activity of the DAF-16 transcription factor (forkhead box O (FOXO) homologue), whose global targets were identified in Caenorhabditis elegans using whole-worm transcriptional analyses more than a decade ago. IIS and FOXO also regulate important neuronal and adult behavioural phenotypes, such as the maintenance of memory and axon regeneration with age, in both mammals and C. elegans, but the neuron-specific IIS/FOXO targets that regulate these functions are still unknown. By isolating adult C. elegans neurons for transcriptional profiling, we identified both the wild-type and IIS/FOXO mutant adult neuronal transcriptomes for the first time. IIS/FOXO neuron-specific targets are distinct from canonical IIS/FOXO-regulated longevity and metabolism targets, and are required for extended memory in IIS daf-2 mutants. The activity of the forkhead transcription factor FKH-9 in neurons is required for the ability of daf-2 mutants to regenerate axons with age, and its activity in non-neuronal tissues is required for the long lifespan of daf-2 mutants. Together, neuron-specific and canonical IIS/FOXO-regulated targets enable the coordinated extension of neuronal activities, metabolism, and longevity under low-insulin signalling conditions.

[pmid21443682-32] Slack C et al.: dFOXO-independent effects of reduced insulin-like signaling in Drosophila. Aging Cell 2011. (PMID 21443682) [PubMed] [DOI] [Full text] The insulin/insulin-like growth factor-like signaling (IIS) pathway in metazoans has evolutionarily conserved roles in growth control, metabolic homeostasis, stress responses, reproduction, and lifespan. Genetic manipulations that reduce IIS in the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the mouse have been shown not only to produce substantial increases in lifespan but also to ameliorate several age-related diseases. In C. elegans, the multitude of phenotypes produced by the reduction in IIS are all suppressed in the absence of the worm FOXO transcription factor, DAF-16, suggesting that they are all under common regulation. It is not yet clear in other animal models whether the activity of FOXOs mediate all of the physiological effects of reduced IIS, especially increased lifespan. We have addressed this issue by examining the effects of reduced IIS in the absence of dFOXO in Drosophila, using a newly generated null allele of dfoxo. We found that the removal of dFOXO almost completely blocks IIS-dependent lifespan extension. However, unlike in C. elegans, removal of dFOXO does not suppress the body size, fecundity, or oxidative stress resistance phenotypes of IIS-compromised flies. In contrast, IIS-dependent xenobiotic resistance is fully dependent on dFOXO activity. Our results therefore suggest that there is evolutionary divergence in the downstream mechanisms that mediate the effects of IIS. They also imply that in Drosophila, additional factors act alongside dFOXO to produce IIS-dependent responses in body size, fecundity, and oxidative stress resistance and that these phenotypes are not causal in IIS-mediated extension of lifespan.

[pmid23583953-33] Brenmoehl J & Hoeflich A: Dual control of mitochondrial biogenesis by sirtuin 1 and sirtuin 3. Mitochondrion 2013. (PMID 23583953) [PubMed] [DOI] In this review, we discuss the dual control of mitochondrial biogenesis and energy metabolism by silent information regulator-1 and -3 (SIRT1 and SIRT3). SIRT1 activates the peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α)-mediated transcription of nuclear and mitochondrial genes encoding for proteins promoting mitochondria proliferation, oxidative phosphorylation and energy production, whereas SIRT3 directly acts as an activator of proteins important for oxidative phosphorylation, tricarboxylic acid (TCA) cycle and fatty-acid oxidation and indirectly of PGC-1α and AMP-activated protein kinase (AMPK). The complex network involves different cellular compartments, transcriptional activation, post-translational modification and a plethora of secondary effectors. Overall, the mode of interaction between both sirtuin family members may be considered as a prominent case of molecular job-sharing.

[pmid24559845-34] Chan MC & Arany Z: The many roles of PGC-1α in muscle--recent developments. Metabolism 2014. (PMID 24559845) [PubMed] [DOI] [Full text] Skeletal muscle is the largest organ in the body and contributes to innumerable aspects of organismal biology. Muscle dysfunction engenders numerous diseases, including diabetes, cachexia, and sarcopenia. At the same time, skeletal muscle is also the main engine of exercise, one of the most efficacious interventions for prevention and treatment of a wide variety of diseases. The transcriptional coactivator PGC-1α has emerged as a key driver of metabolic programming in skeletal muscle, both in health and in disease. We review here the many aspects of PGC-1α function in skeletal muscle, with a focus on recent developments.

[pmid23086035-35] Summermatter S et al.: PGC-1α improves glucose homeostasis in skeletal muscle in an activity-dependent manner. Diabetes 2013. (PMID 23086035) [PubMed] [DOI] [Full text] Metabolic disorders are a major burden for public health systems globally. Regular exercise improves metabolic health. Pharmacological targeting of exercise mediators might facilitate physical activity or amplify the effects of exercise. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) largely mediates musculoskeletal adaptations to exercise, including lipid refueling, and thus constitutes such a putative target. Paradoxically, forced expression of PGC-1α in muscle promotes diet-induced insulin resistance in sedentary animals. We show that elevated PGC-1α in combination with exercise preferentially improves glucose homeostasis, increases Krebs cycle activity, and reduces the levels of acylcarnitines and sphingosine. Moreover, patterns of lipid partitioning are altered in favor of enhanced insulin sensitivity in response to combined PGC-1α and exercise. Our findings reveal how physical activity improves glucose homeostasis. Furthermore, our data suggest that the combination of elevated muscle PGC-1α and exercise constitutes a promising approach for the treatment of metabolic disorders.

[pmid19262508-36] Cantó C et al.: AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature 2009. (PMID 19262508) [PubMed] [DOI] [Full text] AMP-activated protein kinase (AMPK) is a metabolic fuel gauge conserved along the evolutionary scale in eukaryotes that senses changes in the intracellular AMP/ATP ratio. Recent evidence indicated an important role for AMPK in the therapeutic benefits of metformin, thiazolidinediones and exercise, which form the cornerstones of the clinical management of type 2 diabetes and associated metabolic disorders. In general, activation of AMPK acts to maintain cellular energy stores, switching on catabolic pathways that produce ATP, mostly by enhancing oxidative metabolism and mitochondrial biogenesis, while switching off anabolic pathways that consume ATP. This regulation can take place acutely, through the regulation of fast post-translational events, but also by transcriptionally reprogramming the cell to meet energetic needs. Here we demonstrate that AMPK controls the expression of genes involved in energy metabolism in mouse skeletal muscle by acting in coordination with another metabolic sensor, the NAD+-dependent type III deacetylase SIRT1. AMPK enhances SIRT1 activity by increasing cellular NAD+ levels, resulting in the deacetylation and modulation of the activity of downstream SIRT1 targets that include the peroxisome proliferator-activated receptor-gamma coactivator 1alpha and the forkhead box O1 (FOXO1) and O3 (FOXO3a) transcription factors. The AMPK-induced SIRT1-mediated deacetylation of these targets explains many of the convergent biological effects of AMPK and SIRT1 on energy metabolism.

[pmid23770291-37] Kauppinen A et al.: Antagonistic crosstalk between NF-κB and SIRT1 in the regulation of inflammation and metabolic disorders. Cell Signal 2013. (PMID 23770291) [PubMed] [DOI] Recent studies have indicated that the regulation of innate immunity and energy metabolism are connected together through an antagonistic crosstalk between NF-κB and SIRT1 signaling pathways. NF-κB signaling has a major role in innate immunity defense while SIRT1 regulates the oxidative respiration and cellular survival. However, NF-κB signaling can stimulate glycolytic energy flux during acute inflammation, whereas SIRT1 activation inhibits NF-κB signaling and enhances oxidative metabolism and the resolution of inflammation. SIRT1 inhibits NF-κB signaling directly by deacetylating the p65 subunit of NF-κB complex. SIRT1 stimulates oxidative energy production via the activation of AMPK, PPARα and PGC-1α and simultaneously, these factors inhibit NF-κB signaling and suppress inflammation. On the other hand, NF-κB signaling down-regulates SIRT1 activity through the expression of miR-34a, IFNγ, and reactive oxygen species. The inhibition of SIRT1 disrupts oxidative energy metabolism and stimulates the NF-κB-induced inflammatory responses present in many chronic metabolic and age-related diseases. We will examine the molecular mechanisms of the antagonistic signaling between NF-κB and SIRT1 and describe how this crosstalk controls inflammatory process and energy metabolism. In addition, we will discuss how disturbances in this signaling crosstalk induce the appearance of chronic inflammation in metabolic diseases.

[pmid33460497-38] Mao K & Zhang G: The role of PARP1 in neurodegenerative diseases and aging. FEBS J 2022. (PMID 33460497) [PubMed] [DOI] Neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by progressive memory loss and motor impairment. Aging is a major risk factor for neurodegenerative diseases. Neurodegenerative diseases and aging often develop in an irreversible manner and cause a significant socioeconomic burden. When considering their pathogenesis, many studies usually focus on mitochondrial dysfunction and DNA damage. More recently, neuroinflammation, autophagy dysregulation, and SIRT1 inactivation were shown to be involved in the pathogenesis of neurodegenerative diseases and aging. In addition, studies uncovered the role of poly (ADP-ribose)-polymerase-1 (PARP1) in neurodegenerative diseases and aging. PARP1 links to a cluster of stress signals, including those originated by inflammation and autophagy dysregulation. In this review, we summarized the recent research progresses on PARP1 in neurodegenerative diseases and aging, with an emphasis on the relationship among PARP1, neuroinflammation, mitochondria, and autophagy. We discussed the possibilities of treating neurodegenerative diseases and aging through targeting PARP1.

[pmid33609766-39] Amjad S et al.: Role of NAD⁺ in regulating cellular and metabolic signaling pathways. Mol Metab 2021. (PMID 33609766) [PubMed] [DOI] [Full text] BACKGROUND: Nicotinamide adenine dinucleotide (NAD+), a critical coenzyme present in every living cell, is involved in a myriad of metabolic processes associated with cellular bioenergetics. For this reason, NAD+ is often studied in the context of aging, cancer, and neurodegenerative and metabolic disorders. SCOPE OF REVIEW: Cellular NAD+ depletion is associated with compromised adaptive cellular stress responses, impaired neuronal plasticity, impaired DNA repair, and cellular senescence. Increasing evidence has shown the efficacy of boosting NAD+ levels using NAD+ precursors in various diseases. This review provides a comprehensive understanding into the role of NAD+ in aging and other pathologies and discusses potential therapeutic targets. MAJOR CONCLUSIONS: An alteration in the NAD+/NADH ratio or the NAD+ pool size can lead to derailment of the biological system and contribute to various neurodegenerative disorders, aging, and tumorigenesis. Due to the varied distribution of NAD+/NADH in different locations within cells, the direct role of impaired NAD+-dependent processes in humans remains unestablished. In this regard, longitudinal studies are needed to quantify NAD+ and its related metabolites. Future research should focus on measuring the fluxes through pathways associated with NAD+ synthesis and degradation.

[pmid32124104-40] Sedlackova L & Korolchuk VI: The crosstalk of NAD, ROS and autophagy in cellular health and ageing. Biogerontology 2020. (PMID 32124104) [PubMed] [DOI] [Full text] Cellular adaptation to various types of stress requires a complex network of steps that altogether lead to reconstitution of redox balance, degradation of damaged macromolecules and restoration of cellular metabolism. Advances in our understanding of the interplay between cellular signalling and signal translation paint a complex picture of multi-layered paths of regulation. In this review we explore the link between cellular adaptation to metabolic and oxidative stresses by activation of autophagy, a crucial cellular catabolic pathway. Metabolic stress can lead to changes in the redox state of nicotinamide adenine dinucleotide (NAD), a co-factor in a variety of enzymatic reactions and thus trigger autophagy that acts to sequester intracellular components for recycling to support cellular growth. Likewise, autophagy is activated by oxidative stress to selectively recycle damaged macromolecules and organelles and thus maintain cellular viability. Multiple proteins that help regulate or execute autophagy are targets of post-translational modifications (PTMs) that have an effect on their localization, binding affinity or enzymatic activity. These PTMs include acetylation, a reversible enzymatic modification of a protein's lysine residues, and oxidation, a set of reversible and irreversible modifications by free radicals. Here we highlight the latest findings and outstanding questions on the interplay of autophagy with metabolic stress, presenting as changes in NAD levels, and oxidative stress, with a focus on autophagy proteins that are regulated by both, oxidation and acetylation. We further explore the relevance of this multi-layered signalling to healthy human ageing and their potential role in human disease.

[pmid32369735-41] Levine DC et al.: NAD⁺ Controls Circadian Reprogramming through PER2 Nuclear Translocation to Counter Aging. Mol Cell 2020. (PMID 32369735) [PubMed] [DOI] [Full text] Disrupted sleep-wake and molecular circadian rhythms are a feature of aging associated with metabolic disease and reduced levels of NAD+, yet whether changes in nucleotide metabolism control circadian behavioral and genomic rhythms remains unknown. Here, we reveal that supplementation with the NAD+ precursor nicotinamide riboside (NR) markedly reprograms metabolic and stress-response pathways that decline with aging through inhibition of the clock repressor PER2. NR enhances BMAL1 chromatin binding genome-wide through PER2K680 deacetylation, which in turn primes PER2 phosphorylation within a domain that controls nuclear transport and stability and that is mutated in human advanced sleep phase syndrome. In old mice, dampened BMAL1 chromatin binding, transcriptional oscillations, mitochondrial respiration rhythms, and late evening activity are restored by NAD+ repletion to youthful levels with NR. These results reveal effects of NAD+ on metabolism and the circadian system with aging through the spatiotemporal control of the molecular clock.

[pmid24051248-42] Peek CB et al.: Circadian clock NAD+ cycle drives mitochondrial oxidative metabolism in mice. Science 2013. (PMID 24051248) [PubMed] [DOI] [Full text] Circadian clocks are self-sustained cellular oscillators that synchronize oxidative and reductive cycles in anticipation of the solar cycle. We found that the clock transcription feedback loop produces cycles of nicotinamide adenine dinucleotide (NAD(+)) biosynthesis, adenosine triphosphate production, and mitochondrial respiration through modulation of mitochondrial protein acetylation to synchronize oxidative metabolic pathways with the 24-hour fasting and feeding cycle. Circadian control of the activity of the NAD(+)-dependent deacetylase sirtuin 3 (SIRT3) generated rhythms in the acetylation and activity of oxidative enzymes and respiration in isolated mitochondria, and NAD(+) supplementation restored protein deacetylation and enhanced oxygen consumption in circadian mutant mice. Thus, circadian control of NAD(+) bioavailability modulates mitochondrial oxidative function and organismal metabolism across the daily cycles of fasting and feeding.

[pmid24657895-43] Rehan L et al.: SIRT1 and NAD as regulators of ageing. Life Sci 2014. (PMID 24657895) [PubMed] [DOI] The recent research on ageing processes in mammals throws new light on the biochemistry of circadian clock. The already known regulatory pathways for biological rhythms and metabolism, combined with newly discovered functions of sirtuins, unveil a perspective for new hypotheses, regarding possible links between ageing and circadian rhythms. The NAD World hypothesis - postulated as a systemic regulatory network for the metabolism and ageing, linked with mammalian, NAD+ dependent Sirtuin 1 - conceptually involves two critical elements. One is the systemic, Nampt-controlled NAD+ (nicotinamide phosphoribosyltransferase) biosynthesis, where Nampt (nicotinamide phosphoribosyltransferase) acts as "propulsion" for metabolism and the other is NAD+ dependent deacetylase (SIRT1) - a regulator responsible for various biological effects, depending on its localisation in organism. In this approach, the role of sirtuins, which are evolutionary conservative, NAD+ dependent histone deacetylases, may be very important for the mammalian metabolic clock. This paper is a review of current research on possible links among SIRT1 (Sirtuin 1), metabolism and ageing with particular consideration of the NAD World hypothesis.

[pmid34743990-44] Chu X & Raju RP: Regulation of NAD⁺ metabolism in aging and disease. Metabolism 2022. (PMID 34743990) [PubMed] [DOI] [Full text] More than a century after discovering NAD+, information is still evolving on the role of this molecule in health and diseases. The biological functions of NAD+ and NAD+ precursors encompass pathways in cellular energetics, inflammation, metabolism, and cell survival. Several metabolic and neurological diseases exhibit reduced tissue NAD+ levels. Significantly reduced levels of NAD+ are also associated with aging, and enhancing NAD+ levels improved healthspan and lifespan in animal models. Recent studies suggest a causal link between senescence, age-associated reduction in tissue NAD+ and enzymatic degradation of NAD+. Furthermore, the discovery of transporters and receptors involved in NAD+ precursor (nicotinic acid, or niacin, nicotinamide, and nicotinamide riboside) metabolism allowed for a better understanding of their role in cellular homeostasis including signaling functions that are independent of their functions in redox reactions. We also review studies that demonstrate that the functional effect of niacin is partially due to the activation of its cell surface receptor, GPR109a. Based on the recent progress in understanding the mechanism and function of NAD+ and NAD+ precursors in cell metabolism, new strategies are evolving to exploit these molecules' pharmacological potential in the maintenance of metabolic balance.

[pmid31953124-45] Gilmour BC et al.: Targeting NAD⁺ in translational research to relieve diseases and conditions of metabolic stress and ageing. Mech Ageing Dev 2020. (PMID 31953124) [PubMed] [DOI] Nicotinamide adenine dinucleotide (NAD+) plays a fundamental role in life and health through the regulation of energy biogenesis, redox homeostasis, cell metabolism, and the arbitration of cell survival via linkages to apoptosis and autophagic pathways. The importance of NAD+ in ageing and healthy longevity has been revealed from laboratory animal studies and early-stage clinical testing. While basic researchers and clinicians have investigated the molecular mechanisms and translation potential of NAD+, there are still major gaps in applying laboratory science to design the most effective trials. This mini-review was based on the programme and discussions of the 3rd NO-Age Symposium held at the Akershus University Hospital, Norway on the 28th October 2019. This symposium brought together leading basic researchers on NAD+ and clinicians who are leading or are going to perform NAD+ augmentation-related clinical studies. This meeting covered talks about NAD+ synthetic pathways, subcellular homeostasis of NAD+, the benefits of NAD+ augmentation from maternal milk to offspring, current clinical trials of the NAD+ precursor nicotinamide riboside (NR) on Ataxia-Telangiectasia (A-T), Parkinson's disease (PD), post-sepsis fatigue, as well as other potential NR-based clinical trials. Importantly, a consensus is emerging with respect to the design of clinical trials in order to measure meaningful parameters and ensure safety.

[pmid29295624-46] Zhang M & Ying W: NAD⁺ Deficiency Is a Common Central Pathological Factor of a Number of Diseases and Aging: Mechanisms and Therapeutic Implications. Antioxid Redox Signal 2019. (PMID 29295624) [PubMed] [DOI] Increasing evidence has indicated critical roles of nicotinamide adenine dinucleotide, oxidized form (NAD+) in various biological functions. NAD+ deficiency has been found in models of a number of diseases such as cerebral ischemia, myocardial ischemia, and diabetes, and in models of aging. Applications of NAD+ or other approaches that can restore NAD+ levels are highly protective in these models of diseases and aging. NAD+ produces its beneficial effects by targeting at multiple pathological pathways, including attenuating mitochondrial alterations, DNA damage, and oxidative stress, by modulating such enzymes as sirtuins, glyceraldehyde-3-phosphate dehydrogenase, and AP endonuclease. These findings have suggested great therapeutic and nutritional potential of NAD+ for diseases and senescence. Recent Advances: Approaches that can restore NAD+ levels are highly protective in the models of such diseases as glaucoma. The NAD+ deficiency in the diseases and aging results from not only poly(ADP-ribose) polymerase-1 (PARP-1) activation but also decreased nicotinamide phosphoribosyltransferase (Nampt) activity and increased CD38 activity. Significant biological effects of extracellular NAD+ have been found. Increasing evidence has suggested that NAD+ deficiency is a common central pathological factor in a number of diseases and aging. Critical Issues and Future Directions: Future studies are required for solidly establishing the concept that "NAD+ deficiency is a common central pathological factor in a number of disease and aging." It is also necessary to further investigate the mechanisms underlying the NAD+ deficiency in the diseases and aging. Preclinical and clinical studies should be conducted to determine the therapeutic potential of NAD+ for the diseases and aging.

[pmid34720589-47] Zhou FQ: NAD⁺, Senolytics, or Pyruvate for Healthy Aging?. Nutr Metab Insights 2021. (PMID 34720589) [PubMed] [DOI] [Full text] In last decades, healthy aging has become one of research hotspots in life science. It is well known that the nicotinamide adenine dinucleotide oxidized form (NAD+) level in cells decreases with aging and aging-related diseases. Several years ago, one of NAD+ precursors was first demonstrated with its new role in DNA damage repairing in mice, restoring old mice to their physical state at young ones. The finding encourages extensive studies in animal models and patients. NAD+ and its precursors have been popular products in nutrition markets. Alternatively, it was also evidenced that clearance of cellular senescence by senolytics preserved multiorgan (kidney and heart) function and extended healthy lifespan in mice. Subsequent studies confirmed findings in elderly patients subjected with idiopathic pulmonary fibrosis. The senolytic therapy is now focused on various diseases in animal and clinical studies. However, pyruvate, as both a NAD+ substitute and a new senolytic, may be advantageous, on the equimolar basis, over current products above in preventing and treating diseases and aging. Pyruvate-enriched fluids, particularly pyruvate oral rehydration salt, may be a novel intervention for diseases and aging besides critical care. Albeit the direct evidence that benefits healthy aging is still limited to date, pyruvate, as both NAD+ provider and senolytic agent, warrants intensive research to compare NAD+ or senolytics for healthy aging, specifically on the equimolar basis, in effective blood levels. This review briefly discussed the recognition of healthy aging by comparing NAD+ and Senolytics with sodium pyruvate from the clinical point of view.

[pmid33353981-48] Covarrubias AJ et al.: NAD⁺ metabolism and its roles in cellular processes during ageing. Nat Rev Mol Cell Biol 2021. (PMID 33353981) [PubMed] [DOI] [Full text] Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for redox reactions, making it central to energy metabolism. NAD+ is also an essential cofactor for non-redox NAD+-dependent enzymes, including sirtuins, CD38 and poly(ADP-ribose) polymerases. NAD+ can directly and indirectly influence many key cellular functions, including metabolic pathways, DNA repair, chromatin remodelling, cellular senescence and immune cell function. These cellular processes and functions are critical for maintaining tissue and metabolic homeostasis and for healthy ageing. Remarkably, ageing is accompanied by a gradual decline in tissue and cellular NAD+ levels in multiple model organisms, including rodents and humans. This decline in NAD+ levels is linked causally to numerous ageing-associated diseases, including cognitive decline, cancer, metabolic disease, sarcopenia and frailty. Many of these ageing-associated diseases can be slowed down and even reversed by restoring NAD+ levels. Therefore, targeting NAD+ metabolism has emerged as a potential therapeutic approach to ameliorate ageing-related disease, and extend the human healthspan and lifespan. However, much remains to be learnt about how NAD+ influences human health and ageing biology. This includes a deeper understanding of the molecular mechanisms that regulate NAD+ levels, how to effectively restore NAD+ levels during ageing, whether doing so is safe and whether NAD+ repletion will have beneficial effects in ageing humans.

[pmid35665345-49] Balashova NV et al.: Efficient Assay and Marker Significance of NAD⁺ in Human Blood. Front Med (Lausanne) 2022. (PMID 35665345) [PubMed] [DOI] [Full text] Oxidized nicotinamide adenine dinucleotide (NAD+) is a biological molecule of systemic importance. Essential role of NAD+ in cellular metabolism relies on the substrate action in various redox reactions and cellular signaling. This work introduces an efficient enzymatic assay of NAD+ content in human blood using recombinant formate dehydrogenase (FDH, EC 1.2.1.2), and demonstrates its diagnostic potential, comparing NAD+ content in the whole blood of control subjects and patients with cardiac or neurological pathologies. In the control group (n = 22, 25-70 years old), our quantification of the blood concentration of NAD+ (18 μM, minimum 15, max 23) corresponds well to NAD+ quantifications reported in literature. In patients with demyelinating neurological diseases (n = 10, 18-55 years old), the NAD+ levels significantly (p < 0.0001) decrease (to 14 μM, min 13, max 16), compared to the control group. In cardiac patients with the heart failure of stage II and III according to the New York Heart Association (NYHA) functional classification (n = 24, 42-83 years old), the blood levels of NAD+ (13 μM, min 9, max 18) are lower than those in the control subjects (p < 0.0001) or neurological patients (p = 0.1). A better discrimination of the cardiac and neurological patients is achieved when the ratios of NAD+ to the blood creatinine levels, mean corpuscular volume or potassium ions are compared. The proposed NAD+ assay provides an easy and robust tool for clinical analyses of an important metabolic indicator in the human blood.

[pmid35010977-50] Peluso A et al.: Age-Dependent Decline of NAD⁺-Universal Truth or Confounded Consensus?. Nutrients 2021. (PMID 35010977) [PubMed] [DOI] [Full text] Nicotinamide adenine dinucleotide (NAD+) is an essential molecule involved in various metabolic reactions, acting as an electron donor in the electron transport chain and as a co-factor for NAD+-dependent enzymes. In the early 2000s, reports that NAD+ declines with aging introduced the notion that NAD+ metabolism is globally and progressively impaired with age. Since then, NAD+ became an attractive target for potential pharmacological therapies aiming to increase NAD+ levels to promote vitality and protect against age-related diseases. This review summarizes and discusses a collection of studies that report the levels of NAD+ with aging in different species (i.e., yeast, C. elegans, rat, mouse, monkey, and human), to determine whether the notion that overall NAD+ levels decrease with aging stands true. We find that, despite systematic claims of overall changes in NAD+ levels with aging, the evidence to support such claims is very limited and often restricted to a single tissue or cell type. This is particularly true in humans, where the development of NAD+ levels during aging is still poorly characterized. There is a need for much larger, preferably longitudinal, studies to assess how NAD+ levels develop with aging in various tissues. This will strengthen our conclusions on NAD metabolism during aging and should provide a foundation for better pharmacological targeting of relevant tissues.

[pmid29514064-51] Rajman L et al.: Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence. Cell Metab 2018. (PMID 29514064) [PubMed] [DOI] [Full text] Nicotinamide adenine dinucleotide (NAD), the cell's hydrogen carrier for redox enzymes, is well known for its role in redox reactions. More recently, it has emerged as a signaling molecule. By modulating NAD+-sensing enzymes, NAD+ controls hundreds of key processes from energy metabolism to cell survival, rising and falling depending on food intake, exercise, and the time of day. NAD+ levels steadily decline with age, resulting in altered metabolism and increased disease susceptibility. Restoration of NAD+ levels in old or diseased animals can promote health and extend lifespan, prompting a search for safe and efficacious NAD-boosting molecules that hold the promise of increasing the body's resilience, not just to one disease, but to many, thereby extending healthy human lifespan.

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-Nicotinamide Adenine Dinucleotide (NAD+) is a vital coenzyme found in every cell of our bodies and has become a focal point in the field of longevity and aging research. NAD+ plays a central role in energy metabolism and is essential for the function of several enzymes that are associated with aging and DNA repair.
+[[File:NAD oxidation reduction.svg|right|frameless]]
+'''Nicotinamide Adenine Dinucleotide (NAD)''' is a vital coenzyme found in every cell of our bodies and has become a focal point in the field of longevity and aging research. NAD+ plays a central role in energy metabolism and is essential for the function of several enzymes that are associated with aging and DNA repair.
-=== The Role of NAD+ in the Cell ===
+NAD exists in two main forms: NAD+ and NADH. NAD+ is the oxidized form of the compound and is essential for various cellular processes, including DNA repair, gene expression, and calcium signaling. When NAD+ accepts electrons during metabolic reactions, it becomes reduced and transforms into NADH. NADH, the reduced form, primarily functions in the production of ATP, the cell's primary energy currency, through the electron transport chain. The dynamic interconversion between these two forms, NAD+ and NADH, is fundamental to the cell's energy production and overall function.
+The NAD+/NADH ratio is an important cellular indicator, reflecting the cell's metabolic state. A healthy balance between NAD+ and NADH is required for optimal function of several key enzymes, including those involved in energy production, DNA repair, and cell signaling.
+== The Role of NAD+ in the Cell ==
 NAD+ is involved in several crucial biological processes:
-# Energy Production: NAD+ helps in converting nutrients into energy within the mitochondria, the powerhouse of cells.
+# '''Energy Production''': NAD+ helps in converting nutrients into energy within the mitochondria, the powerhouse of cells.
-# DNA Repair: It's essential for the function of enzymes like PARPs and sirtuins, which are involved in DNA repair and have links to longevity.
+# '''DNA Repair''': It's essential for the function of enzymes like PARPs and sirtuins, which are involved in DNA repair and have links to longevity.
-# Cell Signaling: As a substrate for various enzymes, it plays a role in cellular communication and adaptations to stress.
+# '''Cell Signaling''': As a substrate for various enzymes, it plays a role in cellular communication and adaptations to stress.
-[[File:nihms790132f7.jpg|thumb|CD38/NADase increases during aging, and causes NAD decline and subsequent mitochondrial dysfunction.]]
+[[File:nihms790132f7.jpg|thumb|[[CD38]]/[[NADase]] increases during aging, and causes NAD decline and subsequent mitochondrial dysfunction.]]
-=== NAD+ Decline with Age ===
+== NAD+ Decline with Age ==
-A significant finding in the field of aging research is that NAD+ levels naturally decline as we age. This reduction has been associated with:
+A significant finding in the field of aging research is that [[NAD+]] levels naturally decline as we age. This reduction has been associated with:
 * A decrease in mitochondrial function, leading to reduced energy output.
-* Reduced activity of sirtuins, proteins linked to lifespan extension in various organisms.
+* Reduced activity of [[Sirtuins|sirtuins]], proteins linked to lifespan extension in various organisms.
 * Enhanced vulnerability of DNA to damage.
 * Increased susceptibility to age-related diseases such as diabetes, cardiovascular diseases, and neurodegenerative diseases.
-A gradual increase in CD38 has been implicated in the decline of [[wikipedia:NAD+|NAD+]] with age.<ref>Camacho-Pereira J, Tarragó MG, Chini CC, Nin V, Escande C, Warner GM, Puranik AS, Schoon RA, Reid JM, Galina A, Chini EN (June 2016). "CD38 Dictates Age-Related NAD Decline and Mitochondrial Dysfunction through an SIRT3-Dependent Mechanism". ''Cell Metabolism''. '''23''' (6): 1127–1139. doi:10.1016/j.cmet.2016.05.006. PMC&nbsp;4911708. PMID&nbsp;27304511.</ref><ref>Schultz MB, Sinclair DA (June 2016). "Why NAD(+) Declines during Aging: It's Destroyed". ''Cell Metabolism''. '''23''' (6): 965–966. doi:10.1016/j.cmet.2016.05.022. PMC&nbsp;5088772. PMID&nbsp;27304496.</ref> Treatment of old mice with a specific CD38 inhibitor, [[wikipedia:CD38-IN-78c|78c]], prevents age-related NAD+ decline.<ref>Tarragó MG, Chini CC, Kanamori KS, Warner GM, Caride A, de Oliveira GC, Rud M, Samani A, Hein KZ, Huang R, Jurk D, Cho DS, Boslett JJ, Miller JD, Zweier JL, Passos JF, Doles JD, Becherer DJ, Chini EN (May 2018). "A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD+ Decline". ''Cell Metabolism''. '''27''' (5): 1081–1095.e10. doi:10.1016/j.cmet.2018.03.016. PMC&nbsp;5935140. PMID&nbsp;29719225.</ref> CD38 [[wikipedia:Knockout_mouse|knockout mice]] have twice the levels of NAD+ and are resistant to age-associated NAD+ decline,<ref name="pmid32595066">Cambronne XA, Kraus WL (2020). "Location, Location, Location: Compartmentalization of NAD + Synthesis and Functions in Mammalian Cells". ''Trends in Biochemical Sciences''. '''45''' (10): 858–873. doi:10.1016/j.tibs.2020.05.010. PMC&nbsp;7502477. PMID&nbsp;32595066.</ref> with dramatically increased NAD+ levels in major organs (liver, muscle, brain, and heart).<ref name="pmid32594513">Kang BE, Choi J, Stein S, Ryu D (2020). "Implications of NAD + boosters in translational medicine". ''European Journal of Clinical Investigation''. '''50''' (10): e13334. doi:10.1111/eci.13334. PMID&nbsp;32594513. S2CID&nbsp;220254270.</ref> On the other hand, mice [[wikipedia:Gene_expression|overexpressing]] CD38 exhibit reduced NAD+ and [[wikipedia:Mitochondrial_disease|mitochondrial dysfunction]].<ref name="pmid32595066" />
+A gradual increase in CD38 has been implicated in the decline of NAD+ with age.<ref>Camacho-Pereira J, Tarragó MG, Chini CC, Nin V, Escande C, Warner GM, Puranik AS, Schoon RA, Reid JM, Galina A, Chini EN (June 2016). "CD38 Dictates Age-Related NAD Decline and Mitochondrial Dysfunction through an SIRT3-Dependent Mechanism". ''Cell Metabolism''. '''23''' (6): 1127–1139. doi:10.1016/j.cmet.2016.05.006. PMC&nbsp;4911708. PMID&nbsp;27304511.</ref><ref>Schultz MB, Sinclair DA (June 2016). "Why NAD(+) Declines during Aging: It's Destroyed". ''Cell Metabolism''. '''23''' (6): 965–966. doi:10.1016/j.cmet.2016.05.022. PMC&nbsp;5088772. PMID&nbsp;27304496.</ref> Treatment of old mice with a specific [[CD38]] inhibitor, [[78c]], prevents age-related NAD+ decline.<ref>Tarragó MG, Chini CC, Kanamori KS, Warner GM, Caride A, de Oliveira GC, Rud M, Samani A, Hein KZ, Huang R, Jurk D, Cho DS, Boslett JJ, Miller JD, Zweier JL, Passos JF, Doles JD, Becherer DJ, Chini EN (May 2018). "A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD+ Decline". ''Cell Metabolism''. '''27''' (5): 1081–1095.e10. doi:10.1016/j.cmet.2018.03.016. PMC&nbsp;5935140. PMID&nbsp;29719225.</ref> CD38 knockout mice have twice the levels of NAD+ and are resistant to age-associated NAD+ decline,<ref name="pmid32595066">Cambronne XA, Kraus WL (2020). "Location, Location, Location: Compartmentalization of NAD + Synthesis and Functions in Mammalian Cells". ''Trends in Biochemical Sciences''. '''45''' (10): 858–873. doi:10.1016/j.tibs.2020.05.010. PMC&nbsp;7502477. PMID&nbsp;32595066.</ref> with dramatically increased NAD+ levels in major organs (liver, muscle, brain, and heart).<ref name="pmid32594513">Kang BE, Choi J, Stein S, Ryu D (2020). "Implications of NAD + boosters in translational medicine". ''European Journal of Clinical Investigation''. '''50''' (10): e13334. doi:10.1111/eci.13334. PMID&nbsp;32594513. S2CID&nbsp;220254270.</ref> On the other hand, mice overexpressing CD38 exhibit reduced NAD+ and mitochondrial dysfunction.<ref name="pmid32595066" />
-=== Boosting NAD+ Levels ===
+== Boosting NAD+ Levels ==
 Given the importance of NAD+ in various cellular functions and its decline with age, researchers have been exploring ways to replenish or boost NAD+ levels in the body. Several methods are under investigation:
-# [[Nicotinamid Mononukleotid (NMN)|NMN (Nicotinamide Mononucleotide)]]: A precursor to NAD+ that, when supplemented, has shown potential in increasing NAD+ levels in various studies, mainly in animals.
+# [[Nicotinamide Mononucleotide (NMN)|'''Nicotinamide Mononucleotide (NMN)''']]: A precursor to NAD+ that, when supplemented, has shown potential in increasing NAD+ levels in various studies, mainly in animals.
-# [[Nicotinamide Riboside (NR)|NR (Nicotinamide Riboside)]]: Another NAD+ precursor that can elevate NAD+ levels in the body.
+# [[Nicotinamide Riboside (NR)|'''Nicotinamide Riboside (NR)''']]: Another NAD+ precursor that can elevate NAD+ levels in the body.
-# [[Caloric Restriction]]: It has been observed to enhance NAD+ levels and activate sirtuins.
+# [[Caloric Restriction|'''Caloric Restriction''']]: It has been observed to enhance NAD+ levels and activate sirtuins.
-# NAD+ Infusions: Direct infusion of NAD+ is being explored as a method, although it's still in the early stages of research.
+# [[NAD+ Infusions|'''NAD+ Infusions''']]: Direct infusion of NAD+ is being explored as a method, although it's still in the early stages of research.
+{{See|NAD+ Booster}}
 === Safety and Implications for Longevity ===
@@ Line 33: / Line 40: @@
 * The effective dosages and potential side effects.
 * The real impact on human longevity.
+== NAD+ and Its Role in Aging ==
+Nicotinamide adenine dinucleotide (NAD+) is a crucial molecule in our bodies, involved in turning nutrients into energy, adapting to stress, and maintaining our daily biological rhythms. As we age, the amount of NAD+ in our bodies decreases. This is partly due to the action of an enzyme called CD38, which breaks down NAD+, leading to lower levels in older individuals{{pmid|27304496}}. Keeping a balance of NAD+ is important for our cells to work properly, and enzymes that use NAD+ are being studied for their potential to slow down aging processes. These enzymes include CD38, sirtuins (SIRT), which are involved in cell regulation, PARP1, important for DNA repair, and SARM1, linked to nerve cell health{{pmid|28676700}}{{pmid|25908823}}.
+Our bodies can make NAD+ in two ways: either from scratch using certain nutrients like nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN), or by recycling components from chemical reactions in our cells{{pmid|32694684}}. An enzyme on the surface of our cells, called CD73, also helps to keep NAD+ levels up by converting NMN to NR{{pmid|32389638}}.
+=== The Role of NNMT in NAD+ Levels ===
+An enzyme named nicotinamide N-methyltransferase (NNMT) plays a key role in managing NAD+ levels. It changes nicotinamide, a form of vitamin B3, into a substance called methylnicotinamide (MNT). This change affects how much nicotinamide is available to make NAD+ in our cells. NNMT is linked to conditions like obesity and type two diabetes{{pmid|29483571}}. Interestingly, NNMT also helps stabilize SIRT1, an enzyme that protects cells from stress and can increase lifespan{{pmid|34153425}}{{pmid|26168293}}{{pmid|24077178}}. Researchers are looking into NNMT inhibitors as potential treatments for diseases like cancer, obesity, and liver diseases related to alcohol use{{pmid|29483571}}{{pmid|34704059}}{{pmid|34572571}}{{pmid|29155147}}{{pmid|34424711}}. The balance between NNMT, MNT, and NAD+ is important for our health, especially as we age.
+== NAD+, Sirtuins and Longevity-Promoting Pathway ==
+[[File:CD38-NAD+-SIRT1 Axis.png|thumb|The CD38/NAD+/SIRT1 Axis. NAD+ levels in the body can be influenced by the supplementation of precursors nicotinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN). NAD+ levels decrease with age and are further metabolized by the activation of SIRT1, PARP1, SARM1, and CD38. Restoring NAD+ levels allows for an increase in SIRT1 activity due to increased substrate availability, resulting in the inhibition of age-promoting pathways and activation of adaptive and protective transcription factors and processes. The central lineage may be described as the CD38/NAD+/SIRT1 axis, and targeting this access with nutraceutical interventions may prevent the age-related decline of NAD+ levels in the body. Black lines indicate conversion or activation. Red lines indicate inhibitors or destroyers of the indicated target.{{pmid|36678315}}|450x450px]]
+Maintaining the right levels of NAD+ and the activity of sirtuin proteins is crucial in the fight against aging{{pmid|28537485}}. Taking supplements that are NAD+ precursors, like Nicotinamide Riboside (NR) and Nicotinamide Mononucleotide (NMN), has shown promise in combating the natural decline of NAD+ that comes with aging and related diseases{{pmid|28899755}}{{pmid|29883761}}{{pmid|27825999}}. The decrease in NAD+ as we age is primarily due to its reduction in our bodies, not an increase in its counterpart, NADH{{pmid|30124109}}. Adding more NAD+ can help fix issues in mitochondria, the energy factories of our cells, that happen because of this decline{{pmid|24360282}}.
+SIRT1,  a member of the sirtuin protein family involved in cellular response to stress, has been linked to longer life spans, though the results vary depending on the situation. For example, elite athletes, who have higher SIRT1 levels, tend to have longer telomeres (a sign of cellular aging) and are less likely to develop insulin resistance{{pmid|34256387}}. SIRT1 works by turning on certain genes, like FoxO and PGC1α, which are important for managing stress, controlling cell growth, and preventing tumors. These genes are known to contribute to longer lifespans in some animals{{pmid|26831453}}{{pmid|14976264}}{{pmid|16288288}}{{pmid|35004893}}. The IIS pathway, which influences growth, metabolism, and longevity, also promotes longer life under certain conditions by activating these genes{{pmid|26675724}}{{pmid|21443682}}. PGC1α, in particular, is key in creating mitochondria and has been linked to better insulin sensitivity in muscles{{pmid|23583953}}{{pmid|24559845}}{{pmid|23086035}}. Furthermore, AMPK, which is involved in energy management in the body, interacts with SIRT1 and can inhibit mTOR, another aging-related process. AMPK also helps increase NAD+ levels, thus boosting SIRT1 activity{{pmid|19262508}}. Additionally, SIRT1 can slow down NF-κB signaling, which is part of the immune response, helping to reduce long-term inflammation{{pmid|23770291}}. Having enough NAD+ to keep SIRT1 active is essential in manipulating the aging process and promoting longevity{{pmid|29883761}}{{pmid|33460497}}{{pmid|33609766}}{{pmid|32124104}}. Keeping NAD+ at healthy levels is key for making sure SIRT1 can do its job effectively as we age.
+== NAD+ and Its Influence on the Body's Biological Clock ==
+NAD+, a crucial molecule in our body, plays an important role in keeping our biological clock, or circadian rhythm, in check. Studies involving older mice have shown that when they have less NAD+, they experience more disruptions in their biological clock compared to younger mice with more NAD+{{pmid|32369735}}. This leads to problems in how their cells handle energy and time. By increasing NAD+ levels in mice with circadian rhythm issues, researchers have been able to fix these problems, particularly through the action of a protein called SIRT3{{pmid|24051248}}. Having enough NAD+ and ensuring that sirtuin proteins (like SIRT3) are active is key to keeping our internal clocks working properly. Taking supplements that increase NAD+ levels might help fix age-related issues with these biological clocks{{pmid|24657895}}. Low NAD+ levels are a common problem in many age-related diseases, so treatments that increase NAD+ are being researched as a way to help with these issues{{pmid|34743990}}{{pmid|31953124}}{{pmid|29295624}}{{pmid|34720589}}.
+== See also ==
+* [[NAD+ Booster]]
+* [[NAD+ Precursor]]
+* [[NADase]]
+* {{SeeWikipedia|Nicotinamide adenine dinucleotide}}
+== Todo ==
+*{{pmid text|33353981}}
+*{{pmid text|35665345}}
+*{{pmid text|35010977}}
+*{{pmid text|29514064}}
 == References ==
 <references />
-[[Category:Other Longevity Molecules]]
+[[Category:Other Longevity Compounds]]
+[[Category:Molecular and Cellular Biology]]