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

Abstract

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

“…Nam is released from the cells into the culture medium. These results are in line and extend our previous observations using different experimental conditions [40].…”

“…As shown in this study and a previous study [40], once NR enters the cell, it is actively metabolized to form Nam. The observed formation of Nam can be explained by the fact that NR is metabolized into NAD + , which is then converted to Nam by NAD + -consuming enzymes.…”

Dihydronicotinamide riboside promotes cell-specific cytotoxicity by tipping the balance between metabolic regulation and oxidative stress

“…A far more physiologically relevant supplementation dose is 100 μM, which still showed 15 ± 2.1% cytotoxicity. We selected the 100 μM concentration for further study due to its physiological relevance and published work with NR and NRH, showing the efficient restoration of the intracellular NAD pools and cells' metabolic activity with that concentration [ 37 ]. At 100 μM, NRH is reported to increase neuronal NAD + levels by approximately 5-fold over control [ 32 ]; however, in the present study, NRH is exposed to HepG3 at this concentration resulted in 15 ± 2.1% cytotoxicity ( Fig 2B ).…”

Enzymology of extracellular NAD metabolism

“…However, how these metabolites, as well as those exogenously administered, are able to maintain or even increase intracellular NAD levels is still a matter of investigation. A recent work has clearly established that studies on pyridine supplementation and uptake preformed on cultured cells can be severely affected by the culture conditions, since the serum used in the culture medium, even in a heat-inactivated form, contains enzymes responsible of an efficient degradation of NAD and its intermediates [ 27 ]. Taken into consideration the presence of such activities will allow to get a clearer picture of eNAD metabolism.…”

“…Alternatively, in the extracellular space NR can be converted to Nam by the activity of CD157 [25]. Some studies have shown that NMN needs to be processed to NR prior to enter the cell [23,[26][27][28], whereas other studies support the direct uptake of the mononucleotide [29,30]. The complexity in the detection and quantification of NR and NMN is the major reason why the mechanism of NMN transport into the cell is still under debate [28,31], and why we still lack a clear picture of NR and NMN availability in the extracellular space.…”