The Impact of NAD Bioavailability on DNA Double-Strand Break Repair Capacity in Human Dermal Fibroblasts after Ionizing Radiation.

Nicotinamide adenine dinucleotide (NAD) serves as a substrate for protein deacetylases sirtuins and poly(ADP-ribose) polymerases, which are involved in the regulation of DNA double-strand break (DSB) repair molecular machinery by various mechanisms. However, the impact of NAD bioavailability on DSB repair remains poorly characterized. Herein, using immunocytochemical analysis of γH2AX, a marker for DSB, we investigated the effect of the pharmacological modulation of NAD levels on DSB repair capacity in human dermal fibroblasts exposed to moderate doses of ionizing radiation (IR). We demonstrated that NAD boosting with nicotinamide riboside did not affect the efficiency of DSB elimination after the exposure of cells to IR at 1 Gy. Moreover, even after irradiation at 5 Gy, we did not observe any decrease in intracellular NAD content. We also showed that, when the NAD pool was almost completely depleted by inhibition of its biosynthesis from nicotinamide, cells were still able to eliminate IR-induced DSB, though the activation of ATM kinase, its colocalization with γH2AX and DSB repair capacity were reduced in comparison to cells with normal NAD levels. Our results suggest that NAD-dependent processes, such as protein deacetylation and ADP-ribosylation, are important but not indispensable for DSB repair induced by moderate doses of IR.

Purine nucleoside phosphorylase controls nicotinamide riboside metabolism in mammalian cells.

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 reports questioning the efficacy of NR supplementation. Indeed, the mechanisms of its utilization by cells are not fully understood. Herein, we investigated the role of purine nucleoside phosphorylase (PNP) in NR metabolism in mammalian cells. Using both PNP overexpression and genetic knockout, we show that after being imported into cells by members of the equilibrative nucleoside transporter family, NR is predominantly metabolized by PNP, resulting in nicotinamide (Nam) accumulation. Intracellular cleavage of NR to Nam is prevented by the potent PNP inhibitor Immucillin H in various types of mammalian cells. In turn, suppression of PNP activity potentiates NAD synthesis from NR. Combining pharmacological inhibition of PNP with NR supplementation in mice, we demonstrate that the cleavage of the riboside to Nam is strongly diminished, maintaining high levels of NR in blood, kidney, and liver. Moreover, we show that PNP inhibition stimulates Nam mononucleotide and NAD+ synthesis from NR in vivo, in particular, in the kidney. Thus, we establish PNP as a major regulator of NR metabolism in mammals and provide evidence that the health benefits of NR supplementation could be greatly enhanced by concomitant downregulation of PNP activity.

Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells.

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells.

Nicotinamide adenine dinucleotide (NAD) is an essential redox carrier, whereas its degradation is a key element of important signaling pathways. Human cells replenish their NAD contents through NAD biosynthesis from extracellular precursors. These precursors encompass bases nicotinamide (Nam) and nicotinic acid and their corresponding nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR), now collectively referred to as vitamin B3. In addition, extracellular NAD+ and nicotinamide mononucleotide (NMN), and potentially their deamidated counterparts, nicotinic acid adenine dinucleotide (NAAD) and nicotinic acid mononucleotide (NAMN), may serve as precursors of intracellular NAD. However, it is still debated whether nucleotides enter cells directly or whether they are converted to nucleosides and bases prior to uptake into cells. Here, we studied the metabolism of extracellular NAD+ and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD+ intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD+ and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD+ and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam. Our results demonstrate that both the cultured cells and the culture medium mediate a rather active conversion of NAD+ intermediates. Consequently, in studies of precursor supplementation and uptake, the culture conditions need to be carefully defined.

NAD Metabolome Analysis in Human Cells Using ¹H NMR Spectroscopy.

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide is also used to generate second messengers, such as cyclic ADP-ribose, and serves as substrate for protein modifications including ADP-ribosylation and protein deacetylation by sirtuins. The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells accomplish their NAD supply through biosynthesis using different forms of vitamin B3: Nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN, which are adenylylated to the dinucleotides NAD and NAAD, respectively. Here, we have developed an NMR-based experimental approach to detect and quantify NAD(P) and its biosynthetic intermediates in human cell extracts. Using this method, we have determined NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in standard culture medium containing Nam as the only NAD precursor. When cells were grown in the additional presence of both NAR and NR, intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD) were also detectable. We have also tested this method to quantify NAD+ in human platelets and erythrocytes. Our results demonstrate that ¹H NMR spectroscopy provides a powerful method for the assessment of the cellular NAD metabolome.

DNA double-strand break repair is impaired in presenescent Syrian hamster fibroblasts.

BACKGROUND: Studies of DNA damage response are critical for the comprehensive understanding of age-related changes in cells, tissues and organisms. Syrian hamster cells halt proliferation and become presenescent after several passages in standard conditions of cultivation due to what is known as "culture stress". Using proliferating young and non-dividing presenescent cells in primary cultures of Syrian hamster fibroblasts, we defined their response to the action of radiomimetic drug bleomycin (BL) that induces DNA double-strand breaks (DSBs). RESULTS: The effect of the drug was estimated by immunoblotting and immunofluorescence microscopy using the antibody to phosphorylated histone H2AX (gH2AX), which is generally accepted as a DSB marker. At all stages of the cell cycle, both presenescent and young cells demonstrated variability of the number of gH2AX foci per nucleus. gH2AX focus induction was found to be independent from BL-hydrolase expression. Some differences in DSB repair process between BL-treated young and presenescent Syrian hamster cells were observed: (1) the kinetics of gH2AX focus loss in G0 fibroblasts of young culture was faster than in cells that prematurely stopped dividing; (2) presenescent cells were characterized by a slower recruitment of DSB repair proteins 53BP1, phospho-DNA-PK and phospho-ATM to gH2AX focal sites, while the rate of phosphorylated ATM/ATR substrate accumulation was the same as that in young cells. CONCLUSIONS: Our results demonstrate an impairment of DSB repair in prematurely aged Syrian hamster fibroblasts in comparison with young fibroblasts, suggesting age-related differences in response to BL therapy.