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.

中文翻译：

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人 HEK293 细胞培养物中胞外 NAD+ 中间体的降解。


烟酰胺腺嘌呤二核苷酸（NAD）是一种重要的氧化还原载体，而其降解是重要信号通路的关键要素。人体细胞通过细胞外前体的 NAD 生物合成来补充其 NAD 含量。这些前体包括烟酰胺 (Nam) 和烟酸碱及其相应的核苷烟酰胺核苷 (NR) 和烟酸核苷 (NAR)，现在统称为维生素 B3。此外，细胞外 NAD+ 和烟酰胺单核苷酸 (NMN)，以及潜在的脱酰胺对应物烟酸腺嘌呤二核苷酸 (NAAD) 和烟酸单核苷酸 (NAMN)，可能充当细胞内 NAD 的前体。然而，核苷酸是直接进入细胞还是在被细胞吸收之前转化为核苷和碱基仍然存在争议。在这里，我们使用正常和无血清培养基研究了人 HEK293 细胞中细胞外 NAD+ 及其衍生物的代谢。使用含有 10% 胎牛血清 (FBS) 的培养基，单核苷酸和二核苷酸被降解为相应的核苷。反过来，核苷被切割到相应的碱基上。在没有细胞的情况下，在单独的培养基中也观察到降解，表明 FBS 含有降解 NAD+ 中间体的酶活性。令人惊讶的是，在没有 FBS 的情况下，NR 也能相当有效地水解为 Nam。当在无血清培养基中培养时，HEK293 细胞有效地将 NAD+ 和 NAAD 裂解为 NMN 和 NAMN。 NMN在细胞培养中表现出相当高的稳定性，但部分代谢为NR。 使用质膜转运蛋白的药理学抑制剂，我们还表明，NAD+和NMN在细胞外裂解为NR是使用这些核苷酸维持细胞内NAD含量的先决条件。我们还提供证据表明，除了自发水解之外，NR 在细胞培养物中通过细胞内转化为 Nam 进行强烈代谢。我们的结果表明，培养的细胞和培养基均介导 NAD+ 中间体的相当活跃的转化。因此，在前体补充和吸收的研究中，需要仔细确定培养条件。