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. 2023 Dec 12;13(12):1196.
doi: 10.3390/metabo13121196.

Metabolic Alterations in NADSYN1-Deficient Cells

Nils W F Meijer  1 , Johan Gerrits  1 , Susan Zwakenberg  2 , Fried J T Zwartkruis  2 , Nanda M Verhoeven-Duif  1 , Judith J M Jans  1
Affiliations

Metabolic Alterations in NADSYN1-Deficient Cells

Nils W F Meijer et al. Metabolites. .

Abstract

NAD synthetase 1 (encoded by the gene NADSYN1) is a cytosolic enzyme that catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) from tryptophan and nicotinic acid. NADSYN1 deficiency has recently been added to the spectrum of congenital NAD+ deficiency disorders. To gain insight into the metabolic consequences of NADSYN1 deficiency, the encoding gene was disrupted in A549 and HEK293T cells, and the metabolome was profiled in the presence of different NAD+ precursors, including tryptophan, nicotinamide and nicotinic acid. We demonstrate that when precursors of the NAD+ salvage pathway in the form of nicotinamide become limiting, NADSYN1 deficiency results in a decline in intracellular NAD+ levels even in the presence of other potential NAD+ sources such as tryptophan and nicotinic acid. As a consequence, alterations in 122 and 69 metabolites are observed in NADSYN1-deficient A549 and HEK293T cells compared to the wild-type cell line (FC > 2 and p < 0.05). We thus show that NADSYN1 deficiency results in a metabolic phenotype characterized by alterations in glycolysis, the TCA cycle, the pentose phosphate pathway, and the polyol pathway.

Keywords: NAD+ deficiency; NAD+ salvage pathway; NADSYN1; Preiss–Handler pathway; de novo NAD+ synthesis pathway; glycolysis; metabolomics; pentose phosphate pathway; polyol pathway.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Graphical depiction of NAD+ synthesis, including the de novo synthesis pathway, the Preiss–Handler pathway, the nicotinamide ribose kinase pathway, and the salvage pathway. Adapted from “Overview of BCAA Catabolic Enzymes” by BioRender.com (2023). Retrieved from https://app.biorender.com/biorender-templates (accessed on 24 November 2023). (B) Total (labeled and unlabeled) concentration of NAD+ in A549 and HEK293T cells cultured in the presence of either 8.4 µM nicotinamide (NAM) or 0.5 mM tryptophan (Tryp) or 0.5 mM 2H5tryptophan (TD5) or 0.5 mM quinolinic acid (Quin) or 0.5 mM nicotinic acid (NA) or 0.5 mM 2H4nicotinic acid (D4-NA) (mean ± SD, n = 3, t-test with *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001). (C) Total (labeled and unlabeled) concentration of tryptophan, n’-formylkynurenine, and kynurenine after 48 h incubation with 0.5 mM tryptophan (Tryp) or 0.5 mM 2H5tryptophan (TD5) in A549 and HEK293T cells (mean ± SD, n = 3). Isotopologues are described as follows: unlabeled (M + 0), containing 1 deuterium 2H atom (M + 1), etc.
Figure 2
Figure 2
Total concentration of NAAD+ and NAD+ in wild-type and NADSYN1-deficient A549 and HEK293T cells cultured in the presence of either 8.4 µM nicotinamide (NAM) or 0.5 mM nicotinic acid (NA) for 48 h (mean ± SD, n = 3, t-test with *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001).
Figure 3
Figure 3
(A) Unsupervised principal component analysis of wild-type and NADSYN1-deficient A549 and HEK293T cells cultured in the presence of 0.5 mM nicotinic acid (n = 3). (B) Heatmap analysis (based on t-test p < 0.05) of most significantly changed metabolites in wild type and NADSYN1-deficient A549 and HEK293T cells cultured in the presence of 0.5 mM nicotinic acid. Blue areas represent a reduced intensity of the respective metabolite, while red areas represent an increased intensity (n = 3). (C,D) Total concentration of fructose 1,6-biphosphate, DHAP, glyceraldehyde 3-phosphate (C), and sedoheptulose 7-phosphate (D) in wild-type and NADSYN1-deficient A549 and HEK293T cells cultured in the presence of 8.4 µM nicotinamide (NAM) or 0.5 mM nicotinic acid (NA) for 48 h (mean ± SD, n = 3, t-test with *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001).
Figure 4
Figure 4
(A) Graphical representation of the polyol pathway. Adapted from “Overview of BCAA Catabolic Enzymes” by BioRender.com (2023). Retrieved from https://app.biorender.com/biorender-templates (accessed on 1 December 2023). (B) Total peak area of sorbitol in wild-type and NADSYN1-deficient A549 and HEK293T cells cultured in the presence of 8.4 µM nicotinamide (NAM) or 0.5 mM nicotinic acid (NA) for 48 h (mean ± SD, n = 3, t-test with *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001).
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