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Pharmacological or genetic suppression of PNP potentiates the NAD synthesis from NR in human cells. A-D, HEK293, HeLa or THP1 cells were treated with NR and the inhibitor of PNP, Immucillin H (ImmH) as indicated. To inhibit NAD synthesis from Nam, cells were treated with FK866. E,F, Wild type (wt) or PNP knockout (ko) HEK293 cells were treated with NR and FK866, as indicated. Twenty-four hours after the treatment, intracellular NAD levels were measured by NMR spectroscopy (A,E) and relative metabolic activity was assessed using the MTT-assay (B,F). Forty-eight hours after the treatment the fraction of dead cells was determined by flow cytometry (C,G). Twenty-four hours after the treatment, the extent of α-tubulin (K40) acetylation was estimated by immunoblotting (D); right panel, densitometric quantification of immunoblots as in left panel. Data are presented as mean ± S.D. (n = 3). nd, not detected. ns, not significant. Statistical analysis of differences between the groups was carried out by one-way ANOVA with post hoc comparisons using the Tukey test. Panels A,C-G, * indicates statistical significance at p < 0.05, ** indicates statistical significance at p < 0.01, *** indicates statistical significance at p < 0.001. Panel B, * indicates statistical difference at p < 0.001 vs the FK866-treated cells, # indicates statistical difference at p < 0.001 and ## -statistical difference at p < 0.05
Source publication
Nicotinamide riboside (NR) is an effective precursor of nicotinamide adenine dinucleotide (NAD) in human and animal cells. NR supplementation can increase the level of NAD in various tissues and thereby improve physiological functions that are weakened or lost in experimental models of aging or various human pathologies. However, there are also rep...
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... and cell death (39). NR was added to the medium at a concentration of 1, 10, or 100 μM. To inhibit PNP, cells were additionally treated with Immucillin H. 24 h after the treatment we estimated the concentration of intracellular NAD using NMR spectroscopy. Treatment with FK866 depleted NAD to undetectable levels in HEK293, HeLa and THP1 cells (Fig. 5A). NAD in extracts obtained from cells additionally treated with 10 µM NR was still below the detection limit, while co-treatment with Immucillin H was able to recover NAD to up to 50% of the control level. Consequently, PNP inhibition considerably improved the ability of cells to synthesize NAD from NR. When NR was added to HEK293 ...
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... to recover NAD to up to 50% of the control level. Consequently, PNP inhibition considerably improved the ability of cells to synthesize NAD from NR. When NR was added to HEK293 cells at a concentration of 100 µM, the cellular NAD concentration could be maintained at normal levels, even in the presence of FK866, but only when PNP was inhibited (Fig. 5A, left panel). We additionally assessed the NAD(P)H-dependent cell metabolic activity using the MTT test (Fig. 5B). FK866 led to a significant decrease in the metabolic activity of HEK293 and HeLa cells. NR, at a concentration of 100 μM, completely restored metabolic activity of the cells to control level. However, NR at a concentration ...
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... of cells to synthesize NAD from NR. When NR was added to HEK293 cells at a concentration of 100 µM, the cellular NAD concentration could be maintained at normal levels, even in the presence of FK866, but only when PNP was inhibited (Fig. 5A, left panel). We additionally assessed the NAD(P)H-dependent cell metabolic activity using the MTT test (Fig. 5B). FK866 led to a significant decrease in the metabolic activity of HEK293 and HeLa cells. NR, at a concentration of 100 μM, completely restored metabolic activity of the cells to control level. However, NR at a concentration of 10 μM was less effective and restored metabolic activity only up to 30-40%. Co-treatment with NR (10 μM) and ...
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... and HeLa cells. NR, at a concentration of 100 μM, completely restored metabolic activity of the cells to control level. However, NR at a concentration of 10 μM was less effective and restored metabolic activity only up to 30-40%. Co-treatment with NR (10 μM) and Immucillin H led to complete recovery of the metabolic activity of NAD-depleted cells (Fig. ...
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... we analyzed how pharmacological inhibition of PNP may affect the viability of HeLa cell in the presence of FK866 and NR using flow cytometry. Following treatment with FK866 for 48 hours the number of dead cells, as evaluated by staining with propidium iodide, increased significantly in comparison with the control untreated cells (Fig. 5C). Co-treatment with 1 μM NR led to a partial recovery of cell viability. Importantly, when cells were treated with both NR and Immucillin H, cell viability increased almost up to control level observed in population of cells grown in standard medium (Fig. ...
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... propidium iodide, increased significantly in comparison with the control untreated cells (Fig. 5C). Co-treatment with 1 μM NR led to a partial recovery of cell viability. Importantly, when cells were treated with both NR and Immucillin H, cell viability increased almost up to control level observed in population of cells grown in standard medium (Fig. ...
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... also used these experimental conditions to characterize the possible functional consequences of the suppression of PNP-dependent cleavage of NR to Nam. As shown in Fig. 5D, the treatment of HeLa cells with FK866 for 24 hours led to the accumulation of acetylated α-tubulin K40 -the target of NAD + -dependent cytoplasmic protein deacetylase SIRT2 (40,41). Cotreatment with 1 μM NR had no effect on the acetylation status of this lysine, however, when J o u r n a l P r e -p r o o f Immucillin H was ...
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... α-tubulin K40 -the target of NAD + -dependent cytoplasmic protein deacetylase SIRT2 (40,41). Cotreatment with 1 μM NR had no effect on the acetylation status of this lysine, however, when J o u r n a l P r e -p r o o f Immucillin H was simultaneously added to cells, we observed a significant decrease in the level of acetylated α-tubulin K40 (Fig. 5D) indicating that, under this condition, more cytosolic NAD+ is ...
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... using PNP knockout cells under similar experimental conditions, NR at a concentration of 10 µM was sufficient to maintain NAD synthesis in PNP knockout cells in the presence of FK866 (Fig. 5E). Likewise, knocking out the PNP gene increased NAD(P)Hdependent metabolic activity in cells treated with FK866 and NR (Fig. 5F). Moreover, NR supplementation counteracted FK866-induced cell death more efficiently in PNP knockout in comparison to wild type HEK293 cells (Fig. ...
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... using PNP knockout cells under similar experimental conditions, NR at a concentration of 10 µM was sufficient to maintain NAD synthesis in PNP knockout cells in the presence of FK866 (Fig. 5E). Likewise, knocking out the PNP gene increased NAD(P)Hdependent metabolic activity in cells treated with FK866 and NR (Fig. 5F). Moreover, NR supplementation counteracted FK866-induced cell death more efficiently in PNP knockout in comparison to wild type HEK293 cells (Fig. ...
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... maintain NAD synthesis in PNP knockout cells in the presence of FK866 (Fig. 5E). Likewise, knocking out the PNP gene increased NAD(P)Hdependent metabolic activity in cells treated with FK866 and NR (Fig. 5F). Moreover, NR supplementation counteracted FK866-induced cell death more efficiently in PNP knockout in comparison to wild type HEK293 cells (Fig. ...
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... (39). NR was added to the medium at a concentration of 1, 10, or 100 μM. To inhibit PNP, cells were additionally treated with Immucillin H. Twenty-four hours after the treatment, we estimated the concentration of intracellular NAD using NMR spectroscopy. Treatment with FK866 depleted NAD to undetectable levels in HEK293, HeLa, and THP1 cells (Fig. 5A). NAD in extracts obtained from cells additionally treated with 10 μM NR was still below the detection limit, while cotreatment with Immucillin H was able to recover NAD to up to 50% of the control level. Consequently, PNP inhibition considerably improved the ability of cells to synthesize NAD from NR. When NR was added to HEK293 cells ...
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... to recover NAD to up to 50% of the control level. Consequently, PNP inhibition considerably improved the ability of cells to synthesize NAD from NR. When NR was added to HEK293 cells at a concentration of 100 μM, the cellular NAD concentration could be maintained at normal levels, even in the presence of FK866, but only when PNP was inhibited (Fig. 5A, left panel). We additionally assessed the NAD(P)H-dependent cell metabolic activity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (Fig. 5B). FK866 led to a significant decrease in the metabolic activity of HEK293 and HeLa cells. NR, at a concentration of 100 μM, completely restored metabolic activity of ...
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... to HEK293 cells at a concentration of 100 μM, the cellular NAD concentration could be maintained at normal levels, even in the presence of FK866, but only when PNP was inhibited (Fig. 5A, left panel). We additionally assessed the NAD(P)H-dependent cell metabolic activity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (Fig. 5B). FK866 led to a significant decrease in the metabolic activity of HEK293 and HeLa cells. NR, at a concentration of 100 μM, completely restored metabolic activity of the cells to control level. However, NR at a concentration of 10 μM was less effective and restored metabolic activity only up to 30 to 40%. Cotreatment with NR (10 μM) and ...
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... HeLa cells. NR, at a concentration of 100 μM, completely restored metabolic activity of the cells to control level. However, NR at a concentration of 10 μM was less effective and restored metabolic activity only up to 30 to 40%. Cotreatment with NR (10 μM) and Immucillin H led to complete recovery of the metabolic activity of NAD-depleted cells (Fig. ...
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... we analyzed how pharmacological inhibition of PNP may affect the viability of HeLa cell in the presence of FK866 and NR using flow cytometry. Following treatment with FK866 for 48 h, the number of dead cells, as evaluated by staining with propidium iodide, increased significantly in comparison with the control untreated cells (Fig. 5C). Cotreatment with 1 μM NR led to a partial recovery of cell viability. Importantly, when cells were treated with both NR and Immucillin H, cell viability increased almost up to control level observed in population of cells grown in standard medium (Fig. ...
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... propidium iodide, increased significantly in comparison with the control untreated cells (Fig. 5C). Cotreatment with 1 μM NR led to a partial recovery of cell viability. Importantly, when cells were treated with both NR and Immucillin H, cell viability increased almost up to control level observed in population of cells grown in standard medium (Fig. ...
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... also used these experimental conditions to characterize the possible functional consequences of the suppression of PNP-dependent cleavage of NR to Nam. As shown in Figure 5D, the treatment of HeLa cells with FK866 for 24 h led to the accumulation of acetylated α-tubulin K40-the target of NAD + -dependent cytoplasmic protein deacetylase SIRT2 (40,41). Cotreatment with 1 μM NR had no effect on the acetylation status of this lysine; however, when Immucillin H was simultaneously added to cells, we observed a significant decrease in the level of acetylated α-tubulin K40 (Fig. D) indicating that, under this condition, more cytosolic NAD+ is available. ...
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... media were analyzed by NMR spectroscopy. Levels of NR and Nam in culture medium are presented. E, HEK293 cell extracts derived from wt or PNP ko cells were When using PNP knockout cells under similar experimental conditions, NR at a concentration of 10 μM was sufficient to maintain NAD synthesis in PNP knockout cells in the presence of FK866 (Fig. 5E). Likewise, knocking out the PNP gene increased NAD(P)H-dependent metabolic activity in cells treated with FK866 and NR (Fig. 5F). Moreover, NR supplementation counteracted FK866-induced cell death more efficiently in PNP knockout in comparison to wild type HEK293 cells (Fig. ...
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... from wt or PNP ko cells were When using PNP knockout cells under similar experimental conditions, NR at a concentration of 10 μM was sufficient to maintain NAD synthesis in PNP knockout cells in the presence of FK866 (Fig. 5E). Likewise, knocking out the PNP gene increased NAD(P)H-dependent metabolic activity in cells treated with FK866 and NR (Fig. 5F). Moreover, NR supplementation counteracted FK866-induced cell death more efficiently in PNP knockout in comparison to wild type HEK293 cells (Fig. ...
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... NAD synthesis in PNP knockout cells in the presence of FK866 (Fig. 5E). Likewise, knocking out the PNP gene increased NAD(P)H-dependent metabolic activity in cells treated with FK866 and NR (Fig. 5F). Moreover, NR supplementation counteracted FK866-induced cell death more efficiently in PNP knockout in comparison to wild type HEK293 cells (Fig. ...
Citations
... [3][4][5] Purine nucleosides showed a relatively low permeability and poor uptake into cells. [6][7][8] In order to overcome these limitations, it is a common strategy to convert one or more of the hydroxyl (OH) units in purine nucleosides to the corresponding ester groups to prepare purine nucleoside prodrugs for better performance. [9][10][11][12][13] Valganciclovir, the monoester derivative of ganciclovir, has better efficacy and bioavailability as an antiviral alternative. ...
Purine nucleoside ester is one of the derivatives of purine nucleoside, which has antiviral and anticancer activities. In this work, a continuous flow synthesis of purine nucleoside esters catalyzed by lipase TL IM from Thermomyces lanuginosus was successfully achieved. Various parameters including solvent, reaction temperature, reaction time/flow rate and substrate ratio were investigated. The best yields were obtained with a continuous flow microreactor for 35 min at 50 °C with the substrate ratio of 1 : 5 (nucleosides to vinyl esters) in the solvent of tert-amyl alcohol. 12 products were efficiently synthesized with yields of 78–93%. Here we reported for the first time the use of lipase TL IM from Thermomyces lanuginosus in the synthesis of purine nucleoside esters. The significant advantages of this methodology are a green solvent and mild conditions, a simple work-up procedure and the highly reusable biocatalyst. This research provides a new technique for rapid synthesis of anticancer and antiviral nucleoside drugs and is helpful for further screening of drug activity.
... Moreover, in mammalian cells, PNP was recently shown to be a major regulator of NR metabolism. For example, administration of NR at doses of 250 and 500 mg/kg leads to a significant increase in the NAM pool in plasma, liver (Giroud-Gerbetant et al., 2019) and heart , and when PNP activity is suppressed high levels of riboside are maintained in the blood, kidneys and liver, which generally reduces the accumulation of NAM (Kropotov et al., 2022). ...
The article discusses the metabolism of niacin, also known as vitamin B3 or PP, and the mechanisms of its receptor-induced functions in the human body. Niacin exists as a several molecular compounds that act as the nicotinamide coenzymes precursors. These coenzymes being electron donors or acceptors in redox reactions catalyzed by various enzymes play a crucial role in metabolism. Maintenance of the intracellular niacin pool is vital not only for redox metabolism, but also for the NAD-dependent pathways functioning. At the same time, pathophysiological situations and changes in enzyme activity can affect the necessity for various niacin forms. In addition to indirect effects via nicotinamide coenzymes, it also has a number of direct effects, including anti-lipolytic, vasodilatory, and neuroprotective functions, the exact mechanism of which has not been studied fully up to date. Overall, niacin plays a vital role in maintaining the efficient cell functioning, and further study of its influence on various physiological aspects, including the gut microbiome and epigenetic regulation, could lead to new discoveries and treatments for various diseases.
... Nicotinamide riboside (NR), which is delivered into cells, is broken down by PNP, causing a buildup of NAM. When PNP activity is inhibited, NR can be used to synthesize NAD, and cancer is linked to dysregulation of NAD+-dependent metabolism and signaling (Sethi, Zhang et al. 2018, Kropotov, Kulikova et al. 2022. As a result, it is thought that PNP may slow the development of cancer via influencing NAD+ production. ...
Background
Nicotinamide (NAM) regulates redox and metabolic activities in the mitochondria. The intention of the research was to identify key genes that relate to nicotinamide in hepatocellular carcinoma (HCC).
Methods
Relevant clinical information were collected as well as RNA-seq data using the Cancer Genome Atlas (TCGA) database. Differential analysis was used to discover the genes that were differently expressed. On the key genes associated with NAM, functional enrichment analysis were carried out. Next, receiver operating characteristic (ROC) and prognosis Kaplan-Meier (K-M) curve analyses were used to evaluate the importance of important gene expression, respectively. The immune cell signatures were estimated using the CIBERSORT algorithm. The key genes were validated using clinical RT-qPCR finally, and experiments were performed to verify inhibitory effect of NAM on HCC.
Results
Six prognostic key genes (NAXE, NADSYN1, NT5C, NT5C3A, PNP and NT5E) were identified. There is an association between the level of key gene expression and the clinical prognosis. Four key genes (NAXE, NADSYN1, NT5C and NT5C3A) have statistical significance of survival prognosis. Finally, the expression of NAM-related genes and the inhibitory effect of NAM on HCC were verified by experiments.
Conclusion
The study first found some Nicotinamide metabolism-related differentially expressed genes (NMRDEGs) that are related to HCC can contribute to predicting survival and monitoring the treatment.
... Previously, we showed that NR is imported into cultured human cells by members of the equilibrative transporter family (Nikiforov et al., 2011;Kropotov et al., 2021). The imported NR, in addition to phosphorylation to NMN, is intensively cleaved to nicotinamide by cytosolic purine nucleoside phosphorylase (PNP) (Kropotov et al., 2022). We also demonstrated that PNP-dependent NR cleavage to nicotinamide is completely suppressed by specific PNP protein inhibitor forodesine, in mESC E14 cells also (Kropotov et al., 2022). ...
... The imported NR, in addition to phosphorylation to NMN, is intensively cleaved to nicotinamide by cytosolic purine nucleoside phosphorylase (PNP) (Kropotov et al., 2022). We also demonstrated that PNP-dependent NR cleavage to nicotinamide is completely suppressed by specific PNP protein inhibitor forodesine, in mESC E14 cells also (Kropotov et al., 2022). In order to enhance the effect of nucleoside NR as a NAD + precursor, E14 cells were cultured for 1 day in the presence of NR (150 μM) and 5 μM forodesine. ...
Nicotinamide adenine dinucleotide (NAD+) plays a key role in cell metabolism and signaling. In recent years, evidence has accumulated that NAD+-dependent processes are involved in the regulation of pluripotency and differentiation of mammalian embryonic stem cells. The major means to maintain NAD+ levels in mammalian cells is through its biosynthesis from various forms of vitamin B3. In this study, we elucidated how stimulation and suppression of NAD+ biosynthesis affect the maintenance of the pluripotency of E14 Tg2a mouse embryonic stem cells (E14 cells). The pluripotency status of E14 cells was assessed by immunocytochemical analysis and immunoblotting using antibodies to the pluripotency factor Oct4, as wellas by staining for alkaline phosphatase. Using NMR spectroscopy, we have found that the concentration of NAD+ in pluripotent E14 cells cultured in the presence of the leukemia inhibitory factor is about 4 nmol/mg and it remains unchanged after the induction of differentiation with retinoic acid. We have also shown that pharmacological stimulation of NAD+ biosynthesis with nicotinamide riboside increases the level of intra-cellular NAD+ by 20%, but it does not affect the maintenance of pluripotency of E14 cells. Moreover, under conditions of critical depletion of the intracellular NAD+ pool, when its synthesis from nicotinamide was suppressed by nicotinamide phosphoribosyltransferase inhibitor (FK866), E14 cells retained pluripotency,while the Oct4 protein level was reduced.
... NMN and NR metabolism in mammalian cells is predominantly controlled by NRK1 and purine nucleoside phosphorylase (PNP). Simultaneous PNP downregulation could improve the health advantages of NRS [14,238]. Kulikova et al. (2015) studied the generation, release, and uptake of the NAD + precursor NAR by human cells. ...
Many studies have suggested that the oxidized form of nicotinamide adenine dinucleotide (NAD+) is involved in an extensive spectrum of human pathologies, including neurodegenerative disorders, cardiomyopathy, obesity, and diabetes. Further, healthy aging and longevity appear to be closely related to NAD+ and its related metabolites, including nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). As a dietary supplement, NR appears to be well tolerated, having better pharmacodynamics and greater potency. Unfortunately, NR is a reactive molecule, often unstable during its manufacturing, transport, and storage. Recently, work related to prebiotic chemistry discovered that NR borate is considerably more stable than NR itself. However, immediately upon consumption, the borate dissociates from the NR borate and is lost in the body through dilution and binding to other species, notably carbohydrates such as fructose and glucose. The NR left behind is expected to behave pharmacologically in ways identical to NR itself. This review provides a comprehensive summary (through Q1 of 2023) of the literature that makes the case for the consumption of NR as a dietary supplement. It then summarizes the challenges of delivering quality NR to consumers using standard synthesis, manufacture, shipping, and storage approaches. It concludes by outlining the advantages of NR borate in these processes.
... NMN and NMNH have also been shown to increa in both cells and animal models although some controversy remains as t of nucleotide transport across cellular membranes [54][55][56]. In addition to numerous phosphatases that convert nucleotides to nucleosides [57], the e cleoside transporters (Figure 1) regulate the levels of nucleoside supplem their way to the intracellular space [58], while expression of kinases and hy by purine nucleoside phosphorylase [59] and BST-1 [60] control the path NAD + precursors truly affect NAD + and NADH intracellular levels of a ce Challenges of measuring levels of precursors and NAD(P)(H) in b should be noted that NRH is readily oxidized in the presence of riboflavin flavin cofactors and other oxidants are readily present in cells and tissue e cesses that do not require enzymatic catalysis [62]. This oxidation process a ily upon storage, even at low-temperature, and can also convert NMNH to to NAD + , and NADPH to NADP + ( Figure 2). ...
Dietary vitamin B3 components, such as nicotinamide and nicotinic acid, are precursors to the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD+). NAD+ levels are thought to decline with age and disease. While the drivers of this decline remain under intense investigation, strategies have emerged seeking to functionally maintain NAD+ levels through supplementation with NAD+ biosynthetic intermediates. These include marketed products, such as nicotinamide riboside (NR) and its phosphorylated form (NMN). More recent developments have shown that NRH (the reduced form of NR) and its phosphorylated form NMNH also increases NAD+ levels upon administration, although they initially generate NADH (the reduced form of NAD+). Other means to increase the combined levels of NAD+ and NADH, NAD(H), include the inhibition of NAD+-consuming enzymes or activation of biosynthetic pathways. Multiple studies have shown that supplementation with an NAD(H) precursor changes the profile of NAD(H) catabolism. Yet, the pharmacological significance of NAD(H) catabolites is rarely considered although the distribution and abundance of these catabolites differ depending on the NAD(H) precursor used, the species in which the study is conducted, and the tissues used for the quantification. Significantly, some of these metabolites have emerged as biomarkers in physiological disorders and might not be innocuous. Herein, we review the known and emerging catabolites of the NAD(H) metabolome and highlight their biochemical and physiological function as well as key chemical and biochemical reactions leading to their formation. Furthermore, we emphasize the need for analytical methods that inform on the full NAD(H) metabolome since the relative abundance of NAD(H) catabolites informs how NAD(H) precursors are used, recycled, and eliminated.
... The majority of NAD + synthesis following oral intake of NR or NMN has been attributed to the breakdown of these precursors to nicotinamide, which is then salvaged via nicotinamide phosphoribosyltransferase (Nampt) to form NMN and later converted to NAD + via the actions of adenylyltransferases (Nmnat1,2,3) [6,7,21]. Recent works have also shown that a significant proportion of NR is cleaved by purine nucleoside phosphorylase (PNP) intracellularly, leading to the accumulation of nicotinamide [22]. Additionally, NR is mainly processed via nicotinamide riboside kinase 1 (Nrk1) [6,7,21], which phosphorylates the 5 ′ -OH group of NR and produces NMN as the intracellular substrate for NAD + synthesis. ...
Numerous efforts in basic and clinical studies have explored the potential anti-aging and health-promoting effects of NAD+-boosting compounds such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). Despite these extensive efforts, our understanding and characterization of their whole-body pharmacodynamics, impact on NAD+ tissue distribution, and mechanism of action in various tissues remain incomplete. In this study, we administered NMN via intraperitoneal injection or oral gavage and conducted a rigorous evaluation of NMN's pharmacodynamic effects on whole-body NAD+ homeostasis in mice. To provide more confident insights into NMN metabolism and NAD+ biosynthesis across different tissues and organs, we employed a novel approach using triple-isotopically labeled [18O-phosphoryl-18O-carbonyl-13C-1-ribosyl] NMN. Our results provide a more comprehensive characterization of the NMN impact on NAD+ concentrations and absolute amounts in various tissues and the whole body. We also demonstrate that mice primarily rely on the nicotinamide and NR salvage pathways to generate NAD+ from NMN, while the uptake of intact NMN plays a minimal role. Overall, the tissue-specific pharmacodynamic effects of NMN administration through different routes offer novel insights into whole-body NAD+ homeostasis, laying a crucial foundation for the development of NMN as a therapeutic supplement in humans.
... NMN and NAR metabolism in mammalian cells is predominantly controlled by NRK1 and purine nucleoside phosphorylase (PNP). Simultaneous PNP downregulation could improve the health advantages of NARS [14,238]. Kulikova et al. (2015) studied the generation, release, and uptake of the NAD + precursor NAcR by human cells. ...
NAD+ is known classically as a metabolite that participates in catabolic and anabolic pathways throughout the metabolism that is taught to students in introductory biochemistry courses. However, non-classical studies starting over a decade ago found that NAD+ is also involved in higher order functions, in part because of its involvement in the activation of SIRTs and the support of the mitochondrial unfolded protein response. Many studies since have suggested that NAD+ is involved in an extensive spectrum of human pathologies, including neurodegenerative disorders, cardiomyopathy, obesity, and diabetes, Further, healthy aging and longevity appear to be closely related to NAD+ and its related metabolites, including NAR and NMN. Together, these studies show that this system has value as a dietary supplement to improve general health overall, as well as mitigating specific disease conditions. Accordingly, many are now recommending the consumption of materials in this system as dietary supplements. Nicotinamide riboside (NAR) appears to have special value in this regard. It appears to be better tolerated than other molecules in this system, as well as better pharmacodynamics and greater potency. Unfortunately, NAR is a reactive molecule, often unstable during its manufacturing, transport, and storage. Indeed, HPLC analyses of many commercial samples of NAR shows that they contain substantial amounts of material that are not, in fact, NAR. In some of these commercial preparations, NAR is a minority component. Therefore, more stable derivatives of NAR that are easily converted upon consumption into NAR are therefore desired. Recently work related to prebiotic chemistry provided the borate derivative of NAR. NARB is considerably more stable than NAR itself. However, immediately upon consumption, the borate dissociates from NARB, and is lost in the body through dilution and binding to other species, notably carbohydrates such as fructose and glucose. The NAR left behind is expected to behave pharmacologically in ways identical to NAR itself. This review provides a comprehensive summary (through Q1 of 2023) of literature that makes the case for the consumption of NAR as a dietary supplement. It then summarizes the challenges of delivering quality NAR to consumers using standard synthesis, manufacture, shipping, and storage approaches. It concludes by outlining the advantages of NAR-borate in these processes.
Nicotinamide adenine dinucleotide (NAD+) plays a key role in cellular metabolism and signaling. In recent years, evidence has accumulated that NAD+-dependent processes are involved in the regulation of pluripotency and differentiation of mammalian embryonic stem cells. The major means to maintain NAD+ levels in mammalian cells is through its biosynthesis from various forms of vitamin B3. In this study, we examined how stimulation and inhibition of NAD+ biosynthesis affect the maintenance of the pluripotency of mouse embryonic stem cells E14 Tg2a (E14 cells). The pluripotency status of E14 cells was assessed by immunocytochemical and immunoblotting analysis using antibodies to the pluripotency factor Oct4, as well as by staining for alkaline phosphatase. Using NMR spectroscopy, we have found that the concentration of NAD+ in pluripotent E14 cells cultured in the presence of LIF is about 4 nmol/mg, and it remains unchanged after induction of differentiation with retinoic acid. We have also demonstrated that pharmacological stimulation of NAD+ biosynthesis by nicotinamide riboside increases the level of intracellular NAD+ by 20%, but it does not affect the maintenance of pluripotency in E14 cells. Moreover, under conditions of critical depletion of NAD+ pool by Nampt inhibition with FK866 E14 cells maintained pluripotency, though the expression level of Oct4 was decreased.
