Nicotinamide adenine dinucleotide (NAD+) levels in blood and tissues are widely proposed to decline with age, yet evidence in human blood is inconsistent. Using a rigorously validated ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry system that accounts for real-world analytical variability, we quantify NAD+ across seven independent human cohorts. We find that whole-blood NAD+ levels remain remarkably stable with age and across lifestyle interventions, but change in response to nicotinamide riboside supplementation, as expected. Our results challenge the utility of blood NAD+ levels as a biomarker of ageing or lifestyle factors.

Chronic kidney disease (CKD) is projected to become the fifth leading cause of mortality by 2040. Tubular senescence drives kidney fibrosis, but current treatments do not target senescent cells. Here, we identify nicotinamide-N-methyltransferase (NNMT) as a critical mediator of tubular senescence and kidney fibrosis. Human CKD microarrays link NNMT to senescence and fibrosis transcriptomic signatures, and diabetic kidney disease (DKD) biopsies show NNMT protein associating with p21, fibrosis, and kidney function decline. Spatial transcriptomics in human biopsies demonstrates that NNMT-positive tubules are senescent, fibrotic, and surrounded by a pro-inflammatory microenvironment. Importantly, this pattern is conserved in aged and DKD mice, mimicking early-stage CKD features. Mechanistically, NNMT overexpression in tubular epithelial cells exacerbates senescence and partial epithelial-to-mesenchymal transition, while selective NNMT inhibition in senescent kidney cells, organoids, and in vivo is protective. Altogether, these findings position NNMT as a promising therapeutic target to reduce tubular senescence and fibrosis in early CKD.

Modulation of cellular metabolism is crucial in pluripotent stem cell (PSC) development and differentiation. Glycine was shown to promote liver cell maturation, boosting Cytochrome P450 (CYP) isoform 3A4 activity, a key enzyme in phase I metabolism. This study examines the remodeling of central and xenobiotic metabolism during the glycine-supplemented differentiation of induced PSCs (iPSCs) into hepatocyte-like cells (HLCs) and HepG2 into a metabolically active form (mHepG2). In these cell systems, glycine promoted oxidative metabolism and mitochondrial function, collagen, glycogen, bile acid anabolism, one-carbon metabolism, and heme biosynthesis, typical of a hepatocyte phenotype. While the metabolic effects of glycine were divergent in mHepG2 and HLCs, in both cases, heme synthesis was boosted by glycine incorporation, a vital feature in supporting xenobiotic metabolism through the heme-containing enzymes CYPs. From this study, a link between glycine supplementation, carbohydrate metabolism, and enhancement of the xenobiotic machinery is established through metabolic plasticity in the maturing hepatocyte.

Mitochondria-derived acyl-coenzyme A (acyl-CoA) species chemically modify proteins, causing damage when acylation reactions are not adequately detoxified by enzymatic removal or protein turnover. Defects in genes encoding the mitochondrial respiratory complex and TCA cycle enzymes have been shown to increase acyl-CoA levels due to reduced enzymatic flux and result in proteomewide hyperacylation. How pathologically elevated acyl-CoA levels contribute to bioenergetics failure in mitochondrial diseases is not well understood. Here, we demonstrate that bulk succinylation from succinyl-CoA excess consumes the enzymatic cofactor NAD+ and propagates mitochondrial respiratory defects in a zebrafish model of succinyl-CoA ligase deficiency, a childhood-onset encephalomyopathy. To explore this mechanism as a therapeutic target, we developed a workflow to monitor behavioral defects in sucla2-/-zebrafish and show that hypersuccinylation is associated with reduced locomotor behavior and impaired ability to execute food hunting patterns. Postembryonic NAD+ precursor supplementation restores NAD+ levels and improves locomotion and survival of sucla2-/-zebrafish. Mechanistically, nicotinamide and nicotinamide riboside require the NAD+-dependent desuccinylase Sirt5 to enhance oxidative metabolism and nitrogen elimination through the urea cycle. Collectively, NAD+ supplementation activates Sirt5 to protect against damage to mitochondria and locomotor circuits caused by protein succinylation.

NAD+ is a crucial metabolic cofactor whose intracellular levels can influence the progression of multiple metabolic and age-related complications. There is therefore a strong interest in using NAD+ precursors (vitamin B3s) as therapeutic tools, but most current precursors exhibit either poor bioavailability or adverse effects. This study examines the metabolic impact of chronic dietary supplementation with a newly described NAD+ precursor, dihydronicotinamide riboside (NRH), in mice using a comprehensive approach including phenotyping tests, RNA sequencing in different tissues and microbiome analyses. We show that chronic NRH administration at 100 mg/(kg*day) is well tolerated, yet has minimal metabolic effects in mice on a regular diet. However, NRH mitigates high-fat diet-induced metabolic complications when used as a preventive or as a treatment strategy, including improvements in glucose tolerance, increased hepatic expression of lipid catabolism genes and fat redistribution. These results highlight the potential of NRH as a therapeutic agent, although further studies are needed to optimize its use, as higher doses reveal signs of toxicity.

Central carbon metabolism (CCM) is the primary metabolic hub of the cell, governing energy production and providing precursors essential for a myriad of biosynthetic pathways. Developing analytical tools that can identify and quantify intermediates of these metabolic reactions is crucial for studying cell metabolism in biomedical and biotechnological applications. This study proposes a liquid chromatography (LC)-high-resolution (HR) mass spectrometry (MS) method, covering the CCM of mammalian cell systems. Cells were extracted using a one-step liquid extraction, recovering the hydrophilic metabolites. A stable isotope dilution approach was employed, utilizing a U-13C-yeast internal standard (IS). A LC-HRMS metabolomics method using hydrophilic interaction liquid chromatography (HILIC) coupled to a Zeno-time-of-flight (ZenoTOF) MS was implemented for metabolite semi-quantification. A total of 82 CCM metabolites is reported, of which 77 were confirmed with authentic standards, and for 63 , linearity ranges were obtained. IS normalization enhanced overall robustness, from sample preparation to metabolite semi-quantification. To study the effects on CCM by 5 chemical inhibitors (2-deoxy-d-glucose, etomoxir, UK-5099, rotenone, and 3-nitropropionic acid), our HILIC-HR-TOF-MS method was used. The approach proved efficient in capturing altered metabolite concentrations, within implicated metabolic reactions, as a consequence of inhibitor exposure. Our HILIC-HR-TOF-MS metabolome method is efficient in mapping changes in metabolic intermediates of the CCM in mammalian cells. This approach holds potential for analysing a variety of biological samples across a range of applications, from drug development to biomedicine.

To drastically reduce the carbon footprint of the food production chain, a major shift towards alternatives to conventional meat and dairy products is required. The use of plant-based proteins is a promising route, but it also comes with challenges: Plant-based proteins often contain antinutritional factors and off-flavors, which can negatively impact consumer acceptance. Fermentation is broadly used to improve the quality of these products. However, how these unwanted molecules are synthesized and degraded is poorly understood, but this knowledge is essential for fermentation-based strategies to improve the sensory and nutritional value of plant-based products. This review provides a comprehensive overview of synthesis and degradation pathways of key antinutritional factors and off-flavor compounds in plant-based substrates, including aldehydes, furans, sulfur compounds, pyrazines, glycoalkaloids (GAs), pyrimidine glycosides, polyphenols, saponins, glucosinolates (GSLs), phytic acid (PA), oxalates, lectins, and protease and amylase inhibitors. With this we identified the research gaps in the field, which can be divided into three types: (i) degradation pathways that are unknown (furans, alkyl-methoxypyrazines, and dimethyl trisulfide), (ii) well-characterized pathways but typically not found in food-grade organisms (dimethyl sulfide, dimethyl disulfide, and isothiocyanates derived from GSLs), and (iii) pathways that are only described partially (GAs, saponins, polyphenols, PA, and pyrimidine glycosides). Other molecule classes, like aldehydes, alcohols, and oxalate, have well-characterized degradation pathways in food-grade organisms. Focusing future research on compounds with poorly understood degradation pathways will help to accelerate the development of more rationally designed cultures for producing healthy and sustainable plant-based foods.

Chronic kidney disease (CKD) is a major global health issue, projected to become the fifth leading cause of mortality by 2040. Renal tubular cell senescence is a key driver of kidney fibrosis, the final manifestation of CKD. However, current treatment strategies, do not target senescent cells, as the underlying mechanisms driving this dysfunctional phenotype remain poorly described. Here, we identify nicotinamide-N-methyltransferase (NNMT), as a critical mediator of tubular senescence and fibrosis in CKD. Using human RNAseq profiles of CKD, we show that NNMT expression in the renal tubulointerstitium is strongly associated with CKD pathology and transcriptional signatures of cellular senescence. In human diabetic kidney disease biopsies, NNMT levels correlate with the senescence marker p21, kidney function decline, and fibrosis. Spatial transcriptomics further highlights that NNMT-positive tubules are senescent, fibrotic, and surrounded by a pro-inflammatory microenvironment. Preclinical models of early-stage CKD, show upregulation of NNMT and association with senescence. Overexpression of NNMT in TGF-beta-stimulated tubular epithelial cells promotes senescence and partial epithelial-to-mesenchymal transition (EMT), while inhibition of NNMT in kidney cells and organoids is protective. Altogether, we identify NNMT as a novel therapeutic target in the early stages of CKD with the potential to reduce tubular senescence, fibrosis and significantly slow disease progression. ### Competing Interest Statement L.C., L.T., S.C., S.K., S.L., G.L., J.L.S.-G., S.M., J.A.H., M.J.S., J.N.F., and V.S. are or were employees of Nestle Research which is part of the Societe des Produits Nestle SA. D.H.v.R. has participated in advisory boards for AstraZeneca, Boehringer Ingelheim-Eli Lilly Alliance, MSD, Novo Nordisk, and Sanofi, and has received research grants from AstraZeneca, Boehringer Ingelheim-Eli Lilly Alliance, MSD, and Sanofi; all honoraria are paid to the employer, Amsterdam University Medical Centres. The other authors declare no competing interests.

Drug biotransformation and bioactivation play a pivotal role in drug discovery, driving the development of analytical methods to investigate xenobiotic metabolism. However, the identification of drug metabolism products (i.e., drug metabolites) remains challenging. Drug metabolites are difficult to predict, and they are often missed without prior knowledge of the drug’s metabolic fate. Untargeted approaches overcome this requirement, but demand strategies for metabolite identification. In this study, we developed a computational workflow, using high resolution LC–MS/MS metabolomics data, integrating BioTransformer and SIRIUS for the prediction and putative identification of drug metabolite structures. We challenged our workflow to the analysis of human metabolites from 6 well-known drugs, administered to primary human hepatocytes and human liver microsomes: amitriptyline (10 µM), carbamazepine (12.5 µM), cyclophosphamide (20 µM), fipronil (20 µM), phenytoin (50 µM), and verapamil (6 µM). Of the drugs’ metabolites, 62–100

Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD + ) levels are hallmarks of skeletal muscle ageing and sarcopenia 1 – 3 , but it is unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we report a functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid 4 , NAD + levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally occurring and isotopically labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD + pool and increases NAD + levels in Caenorhabditis elegans , mice and primary myotubes from healthy individuals and individuals with sarcopenia. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the nicotinate phosphoribosyltransferase/Preiss–Handler pathway 5 , 6 across models. In C. elegans , trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD + -dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during ageing. Collectively, we identify nutritional supplementation of trigonelline as an NAD + -boosting strategy with therapeutic potential for age-associated muscle decline.

EDITORIAL article Front. Mol. Biosci., 18 December 2023Sec. Metabolomics Volume 10 - 2023 | https://doi.org/10.3389/fmolb.2023.1337566

Metabolomics is a continuously dynamic field of research that is driven by demanding research questions and technological advances alike. In this review we highlight selected recent and ongoing developments in the area of mass spectrometry-based metabolomics. The field of view that can be seen through the metabolomics lens can be broadened by adoption of separation techniques such as hydrophilic interaction chromatography and ion mobility mass spectrometry (going broader). For a given biospecimen, deeper metabolomic analysis can be achieved by resolving smaller entities such as rare cell populations or even single cells using nano-LC and spatially resolved metabolomics or by extracting more useful information through improved metabolite identification in untargeted metabolomic experiments (going deeper). Integration of metabolomics with other (omics) data allows researchers to further advance in the understanding of the complex metabolic and regulatory networks in cells and model organisms (going further). Taken together, diverse fields of research from mechanistic studies to clinics to biotechnology applications profit from these technological developments.

Through evolution, eukaryote organisms have developed the ability to use different molecules as independent precursors to generate nicotinamide adenine dinucleotide (NAD+), an essential molecule for life. However, whether these different precursors act in an additive or complementary manner is not truly well understood. Here, we have evaluated how combinations of different NAD+ precursors influence intracellular NAD+ levels. We identified dihydronicotinic acid riboside (NARH) as a new NAD+ precursor in hepatic cells. Second, we demonstrate how NARH, but not any other NAD+ precursor, can act synergistically with nicotinamide riboside (NR) to increase NAD+ levels in cultured cells and in mice. Finally, we demonstrate that the large increase in NAD+ prompted by the combination of these two precursors is due to their chemical interaction and conversion to dihydronicotinamide riboside (NRH). Altogether, this work demonstrates for the first time that NARH can act as a NAD+ precursor in mammalian cells and how different NAD+ precursors can interact and influence each other when co-administered.

Introduction and aims:Dietary polyphenols have long been associated with health benefits, including the prevention of obesity and related chronic diseases. Overfeeding was shown to rapidly induce weight gain and fat mass, associated with mild insulin resistance in humans, and thus represents a suitable model of the metabolic complications resulting from obesity. We studied the effects of a polyphenol-rich grape extract supplementation on the plasma metabolome during an overfeeding intervention in adults, in two randomized parallel controlled clinical trials. Methods:Blood plasma samples from 40 normal weight to overweight male adults, submitted to a 31-day overfeeding (additional 50% of energy requirement by a high calorie-high fructose diet), given either 2 g/day grape polyphenol extract or a placebo at 0, 15, 21, and 31 days were analyzed (Lyon study). Samples from a similarly designed trial on females (20 subjects) were collected in parallel (Lausanne study). Nuclear magnetic resonance (NMR)-based metabolomics was conducted to characterize metabolome changes induced by overfeeding and associated effects from polyphenol supplementation. The clinical trials are registered under the numbers NCT02145780 and NCT02225457 at ClinicalTrials.gov. Results:Changes in plasma levels of many metabolic markers, including branched chain amino acids (BCAA), ketone bodies and glucose in both placebo as well as upon polyphenol intervention were identified in the Lyon study. Polyphenol supplementation counterbalanced levels of BCAA found to be induced by overfeeding. These results were further corroborated in the Lausanne female study. Conclusion:Administration of grape polyphenol-rich extract over 1 month period was associated with a protective metabolic effect against overfeeding in adults.

During the past few decades, the direct analysis of metabolic intermediates in biological samples has greatly improved the understanding of metabolic processes. The most used technologies for these advances have been mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. NMR is traditionally used to elucidate molecular structures and has now been extended to the analysis of complex mixtures, as biological samples: NMR-based metabolomics. There are however other areas of small molecule biochemistry for which NMR is equally powerful. These include the quantification of metabolites (qNMR); the use of stable isotope tracers to determine the metabolic fate of drugs or nutrients, unravelling of new metabolic pathways, and flux through pathways; and metabolite-protein interactions for understanding metabolic regulation and pharmacological effects. Computational tools and resources for automating analysis of spectra and extracting meaningful biochemical information has developed in tandem and contributes to a more detailed understanding of systems biochemistry. In this review, we highlight the contribution of NMR in small molecule biochemistry, specifically in metabolic studies by reviewing the state-of-the-art methodologies of NMR spectroscopy and future directions.

Nicotinamide riboside kinases (NRKs) control the conversion of dietary Nicotinamide Riboside (NR) to NAD+, but little is known about their contribution to endogenous NAD+ turnover and muscle plasticity during skeletal muscle growth and remodeling. Using NRK1/2 double KO (NRKdKO) mice, we investigated the influence of NRKs on NAD+ metabolism and muscle homeostasis, and on the response to neurogenic muscle atrophy and regeneration following muscle injury. Muscles from NRKdKO animals have altered nicotinamide (NAM) salvage and a decrease in mitochondrial content. In single myonuclei RNAseq of skeletal muscle, NRK2 mRNA expression is restricted to type IIx muscle fibers, and perturbed NAD+ turnover and mitochondrial metabolism shifts the fiber type composition of NRKdKO muscle to fast glycolytic IIB fibers. NRKdKO does not influence muscle atrophy during denervation but alters muscle repair after myofiber injury. During regeneration, muscle stem cells (MuSCs) from NRKdKO animals hyper-proliferate but fail to differentiate. NRKdKO also alters the recovery of NAD+ during muscle regeneration as well as mitochondrial adaptations and extracellular matrix remodeling required for tissue repair. These metabolic perturbations result in a transient delay of muscle regeneration which normalizes during myofiber maturation at late stages of regeneration via over-compensation of anabolic IGF1-Akt signaling. Altogether, we demonstrate that NAD+ synthesis controls mitochondrial metabolism and fiber type composition via NRK1/2 and is rate-limiting for myogenic commitment and mitochondrial maturation during skeletal muscle repair.

Polyphenols, natural products present in plant-based foods, play a protective role against several complex diseases through their antioxidant activity and by diverse molecular mechanisms. Here we develop a network medicine framework to uncover mechanisms for the effects of polyphenols on health by considering the molecular interactions between polyphenol protein targets and proteins associated with diseases. We find that the protein targets of polyphenols cluster in specific neighbourhoods of the human interactome, whose network proximity to disease proteins is predictive of the molecule’s known therapeutic effects. The methodology recovers known associations, such as the effect of epigallocatechin-3- O -gallate on type 2 diabetes, and predicts that rosmarinic acid has a direct impact on platelet function, representing a novel mechanism through which it could affect cardiovascular health. We experimentally confirm that rosmarinic acid inhibits platelet aggregation and α-granule secretion through inhibition of protein tyrosine phosphorylation, offering direct support for the predicted molecular mechanism. Our framework represents a starting point for mechanistic interpretation of the health effects underlying food-related compounds, allowing us to integrate into a predictive framework knowledge on food metabolism, bioavailability and drug interaction.