A bstract The complex interplay of enzymatic processes involving nicotinamide adenine dinucleotide (NAD+) is crucial for the maintenance of metabolic equilibrium and genomic stability. As an essential cofactor, NAD+ orchestrates key metabolic reactions, including glycolysis and the citric acid cycle. Both ADP-ribosyltransferases (PARPs) and sirtuins depend on NAD+ for the post-translational modification of proteins. The activation of PARP1 following DNA damage results in a rapid reduction of NAD+ levels, thereby compromising cellular viability. Thus, precise control over NAD+ concentrations is imperative. Our findings demonstrate that exogenously supplied NAD+, unlike its precursors, directly influences mitochondrial functions. Short-term exposure to NAD+ enhances the activities of the citric acid cycle and the electron transport chain, and it boosts pyrimidine biosynthesis. Prolonged exposure leads to pyrimidine depletion, purine accumulation, activation of the replication stress response, and subsequent cell cycle arrest. Furthermore, the co-administration of NAD+ and 5-fluorouridine selectively eradicates cancer cells dependent on de novo pyrimidine synthesis. We present a comprehensive model illustrating how NAD+ modulates nucleotide metabolism, which has significant implications for understanding healthspan, aging, and the therapeutic targeting of cancer.
NAD+, a vital metabolite and co-enzyme, exists in two primary states within cells: oxidized (NAD+) and reduced (NADH), both crucial for metabolic processes like glycolysis, the tricarboxylic acid (TCA) cycle, and the electron transport chain (ETC). NAD+ is essential for the enzymatic activities that drive post-translational modifications, including those catalyzed by sirtuins such as SIRT1 and ADP-ribosyltransferases (PARPs). Particularly, SIRT1 regulates basal metabolism by deacetylating key metabolic regulators, while PARP1, under DNA damage, consumes NAD+ significantly, impacting cellular viability due to ATP depletion.
Aging triggers chronic DNA damage and subsequent PARP activation, leading to NAD+ depletion and mitochondrial dysfunction, which can be counteracted by NAD+ precursors like nicotinamide mononucleotide (NMN). Additionally, PARP1 inhibitors can enhance mitochondrial function by raising cellular NAD+ levels. Cellular NAD+ is maintained through three synthesis pathways: the de novo pathway from tryptophan in the liver, the Preiss–Handler pathway from nicotinic acid, and the salvage pathway from nicotinamide riboside (NR) or nicotinamide (NAM). Fluctuations in NAD+ levels have significant cellular impacts, with emerging evidence suggesting NAD+ can traverse cellular membranes via specific transport proteins, impacting mitochondrial function and DNA synthesis directly.
In experimental studies, altering NAD+ levels through inhibition of PARP1, SIRT1, and NAMPT affected DNA synthesis across various cell models. PARP inhibition consistently reduced DNA synthesis, while NAMPT inhibition had a pronounced effect in HeLa cells, significantly reducing intracellular NAD(H) levels. The study extended to measuring DNA synthesis and DNA damage response (DDR) activation, finding that NAMPT inhibition decreased DNA synthesis in HeLa cells without affecting U2OS cells, and that PARP1 inhibition reduced DNA synthesis globally and triggered DDR. Analyses using the DNA fibre technique further quantified replication fork dynamics, revealing that PARP1 inhibition could enhance fork progression and activate DDR. This comprehensive study underscores the complex role of NAD+ in regulating mitochondrial function, DNA replication, and cell proliferation, proposing a nuanced model of NAD+ action within cellular and genomic contexts.
