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.publishedVersio

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.publishedVersio

Processed caspase-2 can induce mitochondria-mediated apoptosis independently of its enzymatic activity

The mechanism by which caspase-2 executes apoptosis remains obscure. Recent findings indicate that caspase-2 is activated early in response to DNA-damaging antineoplastic agents and may be important for the engagement of the mitochondrial apoptotic pathway. We demonstrate here that fully processed caspase-2 stimulates mitochondrial release of cytochrome c and Smac/DIABLO, but not apoptosis-inducing factor (AIF). This event occurs independently of several Bcl-2 family proteins, including Bax, Bak and Bcl-2, and inactivation experiments reveal that the proteolytic activity of caspase-2 is not required for the effect. Further, functional studies of mitochondria indicate that processed caspase-2 stimulates state 4 respiration and decreases the respiratory control ratio as a result of, in large part, an uncoupling effect. Combined, our data suggest that caspase-2 retains a unique ability to engage directly the mitochondrial apoptotic pathway, an effect that requires processing of the zymogen but not the associated catalytic activity

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

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

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

MOESM5 of DNA double-strand break repair is impaired in presenescent Syrian hamster fibroblasts

Additional file 5: Table S1. Colocalization of gH2AX with 53BP1, DNA-PK, pATM and p-ATM/ATR substrates in Syrian hamster fibroblasts at two time points after BL treatment

MOESM2 of DNA double-strand break repair is impaired in presenescent Syrian hamster fibroblasts

Additional file 2: Figure S2. Proliferation is arrested in Syrian hamster fibroblasts at the 5th passage. EdU was incorporated for 30 min in untreated Syrian hamster cells at the 1st (p 1) and the 5th (p 5) passages. Proliferating cells at the 1st passage are Ki-67-positive, S-phase cells incorporate EdU, G0 cells are Ki-67- and EdU-negative. All cells at the 5th passage are Ki-67- and EdU-negative. DNA in cell nuclei is counterstained with DAPI. Bar is 10 Âľm