Accounting for NAD Concentrations in Genome-Scale Metabolic Models Captures Important Metabolic Alterations in NAD-Depleted Systems.

Nicotinamide adenine dinucleotide (NAD) is a ubiquitous molecule found within all cells, acting as a crucial coenzyme in numerous metabolic reactions. It plays a vital role in energy metabolism, cellular signaling, and DNA repair. Notably, NAD levels decline naturally with age, and this decline is associated with the development of various age-related diseases. Despite this established link, current genome-scale metabolic models, which offer powerful tools for understanding cellular metabolism, do not account for the dynamic changes in NAD concentration. This impedes our understanding of a fluctuating NAD level's impact on cellular metabolism and its contribution to age-related pathologies. To bridge this gap in our knowledge, we have devised a novel method that integrates altered NAD concentration into genome-scale models of human metabolism. This approach allows us to accurately reflect the changes in fatty acid metabolism, glycolysis, and oxidative phosphorylation observed experimentally in an engineered human cell line with a compromised level of subcellular NAD.

Early Evolutionary Selection of NAD Biosynthesis Pathway in Bacteria.

Bacteria use two alternative pathways to synthesize nicotinamide adenine dinucleotide (NAD) from nicotinamide (Nam). A short, two-step route proceeds through nicotinamide mononucleotide (NMN) formation, whereas the other pathway, a four-step route, includes the deamidation of Nam and the reamidation of nicotinic acid adenine dinucleotide (NAAD) to NAD. In addition to having twice as many enzymatic steps, the four-step route appears energetically unfavourable, because the amidation of NAAD includes the cleavage of ATP to AMP. Therefore, it is surprising that this pathway is prevalent not only in bacteria but also in yeast and plants. Here, we demonstrate that the considerably higher chemical stability of the deamidated intermediates, compared with their amidated counterparts, might compensate for the additional energy expenditure, at least at elevated temperatures. Moreover, comprehensive bioinformatics analyses of the available >6000 bacterial genomes indicate that an early selection of one or the other pathway occurred. The mathematical modelling of the NAD pathway dynamics supports this hypothesis, as there appear to be no advantages in having both pathways.

Tryptophan metabolism is inversely regulated in the tumor and blood of patients with glioblastoma.

Tryptophan (Trp)-catabolic enzymes (TCEs) produce metabolites that activate the aryl hydrocarbon receptor (AHR) and promote tumor progression and immunosuppression in glioblastoma. As therapies targeting TCEs or AHR become available, a better understanding of Trp metabolism is required. Methods: The combination of LC-MS/MS with chemical isobaric labeling enabled the simultaneous quantitative comparison of Trp and its amino group-bearing metabolites in multiple samples. We applied this method to the sera of a cohort of 43 recurrent glioblastoma patients and 43 age- and sex-matched healthy controls. Tumor volumes were measured in MRI data using an artificial neural network-based approach. MALDI MSI visualized Trp and its direct metabolite N-formylkynurenine (FK) in glioblastoma tissue. Analysis of scRNA-seq data was used to detect the presence of Trp metabolism and AHR activity in different cell types in glioblastoma. Results: Compared to healthy controls, glioblastoma patients showed decreased serum Trp levels. Surprisingly, the levels of Trp metabolites were also reduced. The decrease became smaller with more enzymatic steps between Trp and its metabolites, suggesting that Trp availability controls the levels of its systemic metabolites. High tumor volume associated with low systemic metabolite levels and low systemic kynurenine levels associated with worse overall survival. MALDI MSI demonstrated heterogeneity of Trp catabolism across glioblastoma tissues. Analysis of scRNA-seq data revealed that genes involved in Trp metabolism were expressed in almost all the cell types in glioblastoma and that most cell types, in particular macrophages and T cells, exhibited AHR activation. Moreover, high AHR activity associated with reduced overall survival in the glioblastoma TCGA dataset. Conclusion: The novel techniques we developed could support the identification of patients that may benefit from therapies targeting TCEs or AHR activation.

Natural isotope correction improves analysis of protein modification dynamics.

Stable isotope labelling in combination with high-resolution mass spectrometry approaches are increasingly used to analyze both metabolite and protein modification dynamics. To enable correct estimation of the resulting dynamics, it is critical to correct the measured values for naturally occurring stable isotopes, a process commonly called isotopologue correction or deconvolution. While the importance of isotopologue correction is well recognized in metabolomics, it has received far less attention in proteomics approaches. Although several tools exist that enable isotopologue correction of mass spectrometry data, the majority is tailored for the analysis of low molecular weight metabolites. We here present PICor which has been developed for isotopologue correction of complex isotope labelling experiments in proteomics or metabolomics and demonstrate the importance of appropriate correction for accurate determination of protein modifications dynamics, using histone acetylation as an example.

Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes-a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC-MS.

Histone acetylation is an important, reversible post-translational protein modification and a hallmark of epigenetic regulation. However, little is known about the dynamics of this process, due to the lack of analytical methods that can capture site-specific acetylation and deacetylation reactions. We present a new approach that combines metabolic and chemical labeling (CoMetChem) using uniformly 13C-labeled glucose and stable isotope-labeled acetic anhydride. Thereby, chemically equivalent, fully acetylated histone species are generated, enabling accurate relative quantification of site-specific lysine acetylation dynamics in tryptic peptides using high-resolution mass spectrometry. We show that CoMetChem enables site-specific quantification of the incorporation or loss of lysine acetylation over time, allowing the determination of reaction rates for acetylation and deacetylation. Thus, the CoMetChem methodology provides a comprehensive description of site-specific acetylation dynamics.

Welcome to the Family: Identification of the NAD+ Transporter of Animal Mitochondria as Member of the Solute Carrier Family SLC25.

Subcellular compartmentation is a fundamental property of eukaryotic cells. Communication and metabolic and regulatory interconnectivity between organelles require that solutes can be transported across their surrounding membranes. Indeed, in mammals, there are hundreds of genes encoding solute carriers (SLCs) which mediate the selective transport of molecules such as nucleotides, amino acids, and sugars across biological membranes. Research over many years has identified the localization and preferred substrates of a large variety of SLCs. Of particular interest has been the SLC25 family, which includes carriers embedded in the inner membrane of mitochondria to secure the supply of these organelles with major metabolic intermediates and coenzymes. The substrate specificity of many of these carriers has been established in the past. However, the route by which animal mitochondria are supplied with NAD+ had long remained obscure. Only just recently, the existence of a human mitochondrial NAD+ carrier was firmly established. With the realization that SLC25A51 (or MCART1) represents the major mitochondrial NAD+ carrier in mammals, a long-standing mystery in NAD+ biology has been resolved. Here, we summarize the functional importance and structural features of this carrier as well as the key observations leading to its discovery.

Hypoxia Routes Tryptophan Homeostasis Towards Increased Tryptamine Production.

The liver is the central hub for processing and maintaining homeostatic levels of dietary nutrients especially essential amino acids such as tryptophan (Trp). Trp is required not only to sustain protein synthesis but also as a precursor for the production of NAD, neurotransmitters and immunosuppressive metabolites. In light of these roles of Trp and its metabolic products, maintaining homeostatic levels of Trp is essential for health and well-being. The liver regulates global Trp supply by the immunosuppressive enzyme tryptophan-2,3-dioxygenase (TDO2), which degrades Trp down the kynurenine pathway (KP). In the current study, we show that isolated primary hepatocytes when exposed to hypoxic environments, extensively rewire their Trp metabolism by reducing constitutive Tdo2 expression and differentially regulating other Trp pathway enzymes and transporters. Mathematical modelling of Trp metabolism in liver cells under hypoxia predicted decreased flux through the KP while metabolic flux through the tryptamine branch significantly increased. In line, the model also revealed an increased accumulation of tryptamines under hypoxia, at the expense of kynurenines. Metabolic measurements in hypoxic hepatocytes confirmed the predicted reduction in KP metabolites as well as accumulation of tryptamine. Tdo2 expression in cultured primary hepatocytes was reduced upon hypoxia inducible factor (HIF) stabilisation by dimethyloxalylglycine (DMOG), demonstrating that HIFs are involved in the hypoxic downregulation of hepatic Tdo2. DMOG abrogated hepatic luciferase signals in Tdo2 reporter mice, indicating that HIF stability also recapitulates hypoxic rewiring of Trp metabolism in vivo. Also in WT mice HIF stabilization drove homeostatic Trp metabolism away from the KP towards enhanced tryptamine production, leading to enhanced levels of tryptamine in liver, serum and brain. As tryptamines are the most potent hallucinogens known, the observed upregulation of tryptamine in response to hypoxic exposure of hepatocytes may be involved in the generation of hallucinations occurring at high altitude. KP metabolites are known to activate the aryl hydrocarbon receptor (AHR). The AHR-activating properties of tryptamines may explain why immunosuppressive AHR activity is maintained under hypoxia despite downregulation of the KP. In summary our results identify hypoxia as an important factor controlling Trp metabolism in the liver with possible implications for immunosuppressive AHR activation and mental disturbances.

G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling.

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.

Systems biology: current status and challenges.

We put together a special issue on current approaches in systems biology with a focus on mathematical modeling of metabolic networks. Mathematical models have increasingly been used to unravel molecular mechanisms of complex dynamic biological processes. We here provide a short introduction into the topics covered in this special issue, highlighting current developments and challenges.

Multi-scale modeling of drug binding kinetics to predict drug efficacy.

Optimizing drug therapies for any disease requires a solid understanding of pharmacokinetics (the drug concentration at a given time point in different body compartments) and pharmacodynamics (the effect a drug has at a given concentration). Mathematical models are frequently used to infer drug concentrations over time based on infrequent sampling and/or in inaccessible body compartments. Models are also used to translate drug action from in vitro to in vivo conditions or from animal models to human patients. Recently, mathematical models that incorporate drug-target binding and subsequent downstream responses have been shown to advance our understanding and increase predictive power of drug efficacy predictions. We here discuss current approaches of modeling drug binding kinetics that aim at improving model-based drug development in the future. This in turn might aid in reducing the large number of failed clinical trials.

Hypoxia Inducible Factor 1α Inhibits the Expression of Immunosuppressive Tryptophan-2,3-Dioxygenase in Glioblastoma.

Abnormal circulation in solid tumors results in hypoxia, which modulates both tumor intrinsic malignant properties as well as anti-tumor immune responses. Given the importance of hypoxia in glioblastoma (GBM) biology and particularly in shaping anti-tumor immunity, we analyzed which immunomodulatory genes are differentially regulated in response to hypoxia in GBM cells. Gene expression analyses identified the immunosuppressive enzyme tryptophan-2,3-dioxygenase (TDO2) as the second most downregulated gene in GBM cells cultured under hypoxic conditions. TDO2 catalyses the oxidation of tryptophan to N-formyl kynurenine, which is the first and rate-limiting step of Trp degradation along the kynurenine pathway (KP). In multiple GBM cell lines hypoxia reduced TDO2 expression both at mRNA and protein levels. The downregulation of TDO2 through hypoxia was reversible as re-oxygenation rescued TDO2 expression. Computational modeling of tryptophan metabolism predicted reduced flux through the KP and lower intracellular concentrations of kynurenine and its downstream metabolite 3-hydroxyanthranilic acid under hypoxia. Metabolic measurements confirmed the predicted changes, thus demonstrating the ability of the mathematical model to infer intracellular tryptophan metabolite concentrations. Moreover, we identified hypoxia inducible factor 1α (HIF1α) to regulate TDO2 expression under hypoxic conditions, as the HIF1α-stabilizing agents dimethyloxalylglycine (DMOG) and cobalt chloride reduced TDO2 expression. Knockdown of HIF1α restored the expression of TDO2 upon cobalt chloride treatment, confirming that HIF1α controls TDO2 expression. To investigate the immunoregulatory effects of this novel mechanism of TDO2 regulation, we co-cultured isolated T cells with TDO2-expressing GBM cells under normoxic and hypoxic conditions. Under normoxia TDO2-expressing GBM cells suppressed T cell proliferation, while hypoxia restored the proliferation of the T cells, likely due to the reduction in kynurenine levels produced by the GBM cells. Taken together, our data suggest that the regulation of TDO2 expression by HIF1α may be involved in modulating anti-tumor immunity in GBM.

Identification of evolutionary and kinetic drivers of NAD-dependent signaling.

Nicotinamide adenine dinucleotide (NAD) provides an important link between metabolism and signal transduction and has emerged as central hub between bioenergetics and all major cellular events. NAD-dependent signaling (e.g., by sirtuins and poly-adenosine diphosphate [ADP] ribose polymerases [PARPs]) consumes considerable amounts of NAD. To maintain physiological functions, NAD consumption and biosynthesis need to be carefully balanced. Using extensive phylogenetic analyses, mathematical modeling of NAD metabolism, and experimental verification, we show that the diversification of NAD-dependent signaling in vertebrates depended on 3 critical evolutionary events: 1) the transition of NAD biosynthesis to exclusive usage of nicotinamide phosphoribosyltransferase (NamPT); 2) the occurrence of nicotinamide N-methyltransferase (NNMT), which diverts nicotinamide (Nam) from recycling into NAD, preventing Nam accumulation and inhibition of NAD-dependent signaling reactions; and 3) structural adaptation of NamPT, providing an unusually high affinity toward Nam, necessary to maintain NAD levels. Our results reveal an unexpected coevolution and kinetic interplay between NNMT and NamPT that enables extensive NAD signaling. This has implications for therapeutic strategies of NAD supplementation and the use of NNMT or NamPT inhibitors in disease treatment.

The evolution of the plastid phosphate translocator family.

MAIN CONCLUSION: The plastid phosphate translocators evolved in algae but diversified into several groups, which adopted different physiological functions by extensive gene duplications and losses in Streptophyta. The plastid phosphate translocators (pPT) are a family of transporters involved in the exchange of metabolites and inorganic phosphate between stroma and cytosol. Based on their substrate specificities, they were divided into four subfamilies named TPT, PPT, GPT and XPT. To analyse the occurrence of these transporters in different algae and land plant species, we identified 652 pPT genes in 101 sequenced genomes for phylogenetic analysis. The first three subfamilies are found in all species and evolved before the split of red and green algae while the XPTs were derived from the duplication of a GPT gene at the base of Streptophyta. The analysis of the intron-exon structures of the pPTs corroborated these findings. While the number and positions of introns are conserved within each subfamily, they differ between the subfamilies suggesting an insertion of the introns shortly after the three subfamilies evolved. During angiosperm evolution, the subfamilies further split into different groups (TPT1-2, PPT1-3, GPT1-6). Angiosperm species differ significantly in the total number of pPTs, with many species having only a few, while several plants, especially crops, have a higher number, pointing to the importance of these transporters for improved source-sink strength and yield. The differences in the number of pPTs can be explained by several small-scale gene duplications and losses in plant families or single species, but also by whole genome duplications, for example, in grasses. This work could be the basis for a comprehensive analysis of the molecular and physiological functions of this important family of transporters.

The PI3K and MAPK/p38 pathways control stress granule assembly in a hierarchical manner.

All cells and organisms exhibit stress-coping mechanisms to ensure survival. Cytoplasmic protein-RNA assemblies termed stress granules are increasingly recognized to promote cellular survival under stress. Thus, they might represent tumor vulnerabilities that are currently poorly explored. The translation-inhibitory eIF2α kinases are established as main drivers of stress granule assembly. Using a systems approach, we identify the translation enhancers PI3K and MAPK/p38 as pro-stress-granule-kinases. They act through the metabolic master regulator mammalian target of rapamycin complex 1 (mTORC1) to promote stress granule assembly. When highly active, PI3K is the main driver of stress granules; however, the impact of p38 becomes apparent as PI3K activity declines. PI3K and p38 thus act in a hierarchical manner to drive mTORC1 activity and stress granule assembly. Of note, this signaling hierarchy is also present in human breast cancer tissue. Importantly, only the recognition of the PI3K-p38 hierarchy under stress enabled the discovery of p38's role in stress granule formation. In summary, we assign a new pro-survival function to the key oncogenic kinases PI3K and p38, as they hierarchically promote stress granule formation.

Keeping the balance in NAD metabolism.

Research over the last few decades has extended our understanding of nicotinamide adenine dinucleotide (NAD) from a vital redox carrier to an important signalling molecule that is involved in the regulation of a multitude of fundamental cellular processes. This includes DNA repair, cell cycle regulation, gene expression and calcium signalling, in which NAD is a substrate for several families of regulatory proteins, such as sirtuins and ADP-ribosyltransferases. At the molecular level, NAD-dependent signalling events differ from hydride transfer by cleavage of the dinucleotide into an ADP-ribosyl moiety and nicotinamide. Therefore, non-redox functions of NAD require continuous biosynthesis of the dinucleotide. Maintenance of cellular NAD levels is mainly achieved by nicotinamide salvage, yet a variety of other precursors can be used to sustain cellular NAD levels via different biosynthetic routes. Biosynthesis and consumption of NAD are compartmentalised at the subcellular level, and currently little is known about the generation and role of some of these subcellular NAD pools. Impaired biosynthesis or increased NAD consumption is deleterious and associated with ageing and several pathologies. Insults to neurons lead to depletion of axonal NAD and rapid degeneration, partial rescue can be achieved pharmacologically by administration of specific NAD precursors. Restoring NAD levels by stimulating biosynthesis or through supplementation with precursors also produces beneficial therapeutic effects in several disease models. In this review, we will briefly discuss the most recent achievements and the challenges ahead in this diverse research field.

Upregulation of tryptophanyl-tRNA synthethase adapts human cancer cells to nutritional stress caused by tryptophan degradation.

Tryptophan (Trp) metabolism is an important target in immuno-oncology as it represents a powerful immunosuppressive mechanism hijacked by tumors for protection against immune destruction. However, it remains unclear how tumor cells can proliferate while degrading the essential amino acid Trp. Trp is incorporated into proteins after it is attached to its tRNA by tryptophanyl-tRNA synthestases. As the tryptophanyl-tRNA synthestases compete for Trp with the Trp-catabolizing enzymes, the balance between these enzymes will determine whether Trp is used for protein synthesis or is degraded. In human cancers expression of the Trp-degrading enzymes indoleamine-2,3-dioxygenase-1 (IDO1) and tryptophan-2,3-dioxygenase (TDO2) was positively associated with the expression of the tryptophanyl-tRNA synthestase WARS. One mechanism underlying the association between IDO1 and WARS identified in this study is their joint induction by IFNγ released from tumor-infiltrating T cells. Moreover, we show here that IDO1- and TDO2-mediated Trp deprivation upregulates WARS expression by activating the general control non-derepressible-2 (GCN2) kinase, leading to phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) and induction of activating transcription factor 4 (ATF4). Trp deprivation induced cytoplasmic WARS expression but did not increase nuclear or extracellular WARS levels. GCN2 protected the cells against the effects of Trp starvation and enabled them to quickly make use of Trp for proliferation once it was replenished. Computational modeling of Trp metabolism revealed that Trp deficiency shifted Trp flux towards WARS and protein synthesis. Our data therefore suggest that the upregulation of WARS via IFNγ and/or GCN2-peIF2α-ATF4 signaling protects Trp-degrading cancer cells from excessive intracellular Trp depletion.

Human long intrinsically disordered protein regions are frequent targets of positive selection.

Intrinsically disordered regions occur frequently in proteins and are characterized by a lack of a well-defined three-dimensional structure. Although these regions do not show a higher order of structural organization, they are known to be functionally important. Disordered regions are rapidly evolving, largely attributed to relaxed purifying selection and an increased role of genetic drift. It has also been suggested that positive selection might contribute to their rapid diversification. However, for our own species, it is currently unknown whether positive selection has played a role during the evolution of these protein regions. Here, we address this question by investigating the evolutionary pattern of more than 6600 human proteins with intrinsically disordered regions and their ordered counterparts. Our comparative approach with data from more than 90 mammalian genomes uses a priori knowledge of disordered protein regions, and we show that this increases the power to detect positive selection by an order of magnitude. We can confirm that human intrinsically disordered regions evolve more rapidly, not only within humans but also across the entire mammalian phylogeny. They have, however, experienced substantial evolutionary constraint, hinting at their fundamental functional importance. We find compelling evidence that disordered protein regions are frequent targets of positive selection and estimate that the relative rate of adaptive substitutions differs fourfold between disordered and ordered protein regions in humans. Our results suggest that disordered protein regions are important targets of genetic innovation and that the contribution of positive selection in these regions is more pronounced than in other protein parts.

SBMLmod: a Python-based web application and web service for efficient data integration and model simulation.

BACKGROUND: Systems Biology Markup Language (SBML) is the standard model representation and description language in systems biology. Enriching and analysing systems biology models by integrating the multitude of available data, increases the predictive power of these models. This may be a daunting task, which commonly requires bioinformatic competence and scripting. RESULTS: We present SBMLmod, a Python-based web application and service, that automates integration of high throughput data into SBML models. Subsequent steady state analysis is readily accessible via the web service COPASIWS. We illustrate the utility of SBMLmod by integrating gene expression data from different healthy tissues as well as from a cancer dataset into a previously published model of mammalian tryptophan metabolism. CONCLUSION: SBMLmod is a user-friendly platform for model modification and simulation. The web application is available at http://sbmlmod.uit.no , whereas the WSDL definition file for the web service is accessible via http://sbmlmod.uit.no/SBMLmod.wsdl . Furthermore, the entire package can be downloaded from https://github.com/MolecularBioinformatics/sbml-mod-ws . We envision that SBMLmod will make automated model modification and simulation available to a broader research community.

Suppression of indoleamine-2,3-dioxygenase 1 expression by promoter hypermethylation in ER-positive breast cancer.

Kynurenine formation by tryptophan-catabolic indoleamine-2,3-dioxygenase 1 (IDO1) plays a key role in tumor immune evasion and inhibition of IDO1 is efficacious in preclinical models of breast cancer. As the response of breast cancer to immune checkpoint inhibitors may be limited, a better understanding of the expression of additional targetable immunomodulatory pathways is of importance. We therefore investigated the regulation of IDO1 expression in different breast cancer subtypes. We identified estrogen receptor α (ER) as a negative regulator of IDO1 expression. Serum kynurenine levels as well as tumoral IDO1 expression were lower in patients with ER-positive than ER-negative tumors and an inverse relationship between IDO1 and estrogen receptor mRNA was observed across 14 breast cancer data sets. Analysis of whole genome bisulfite sequencing, 450k, MassARRAY and pyrosequencing data revealed that the IDO1 promoter is hypermethylated in ER-positive compared with ER-negative breast cancer. Reduced induction of IDO1 was also observed in human ER-positive breast cancer cell lines. IDO1 induction was enhanced upon DNA demethylation in ER-positive but not in ER-negative cells and methylation of an IDO1 promoter construct reduced IDO1 expression, suggesting that enhanced methylation of the IDO1 promoter suppresses IDO1 in ER-positive breast cancer. The association of ER overexpression with epigenetic downregulation of IDO1 appears to be a particular feature of breast cancer as IDO1 was not suppressed by IDO1 promoter hypermethylation in the presence of high ER expression in cervical or endometrial cancer.

Dynamics of NAD-metabolism: everything but constant.

NAD, as well as its phosphorylated form, NADP, are best known as electron carriers and co-substrates of various redox reactions. As such they participate in approximately one quarter of all reactions listed in the reaction database KEGG. In metabolic pathway analysis, the total amount of NAD is usually assumed to be constant. That means that changes in the redox state might be considered, but concentration changes of the NAD moiety are usually neglected. However, a growing number of NAD-consuming reactions have been identified, showing that this assumption does not hold true in general. NAD-consuming reactions are common characteristics of NAD(+)-dependent signalling pathways and include mono- and poly-ADP-ribosylation of proteins, NAD(+)-dependent deacetylation by sirtuins and the formation of messenger molecules such as cyclic ADP-ribose (cADPR) and nicotinic acid (NA)-ADP (NAADP). NAD-consuming reactions are thus involved in major signalling and gene regulation pathways such as DNA-repair or regulation of enzymes central in metabolism. All known NAD(+)-dependent signalling processes include the release of nicotinamide (Nam). Thus cellular NAD pools need to be constantly replenished, mostly by recycling Nam to NAD(+). This process is, among others, regulated by the circadian clock, causing complex dynamic changes in NAD concentration. As disturbances in NAD homoeostasis are associated with a large number of diseases ranging from cancer to diabetes, it is important to better understand the dynamics of NAD metabolism to develop efficient pharmacological invention strategies to target this pathway.