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.

Exercise is fundamental to healthy aging, yet how it mitigates age-related molecular changes and how fitness level shapes exercise responses remain unclear. To address these questions, we performed transcriptomics, lipidomics and metabolomics on skeletal muscle of young and older adults with differing physical function, both before and after an acute bout of submaximal exercise. At baseline, older adults exhibited reduced expression of genes associated with cellular respiration and energy metabolism compared to young adults with comparable activity levels. Here we found that 50% of these age-related differences were absent in trained older adults, resulting in profiles resembling those of young adults. Although all participants displayed transcriptional immune and stress responses upon acute exercise, the magnitude of these responses in older adults was positively correlated with their physical fitness. Integrated multiomic analyses further revealed links among mitochondrial respiration, lipid metabolism, stress responses and NAD+ biology. These findings demonstrate that sustained physical training transforms age-related molecular profiles and provide a molecular atlas for study of fitness-dependent aging mechanisms.

Nicotinamide adenine dinucleotide (NAD+) is a vital molecule, serving as a redox cofactor and the limiting substrate for numerous enzymes. NAD+ decline is a key feature of aging, while supplementation with NAD+ precursors can efficiently counteract aging traits and prevent age-associated conditions in preclinical models. However, clinical translation remains challenging, likely due to the limited NAD+ boosting capacity of classical precursors, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR). This has brought attention to their reduced forms, reduced NMN (NMNH) and reduced NR (NRH), which are more potent NAD+ boosters but remain poorly characterized. Here, we performed a comprehensive comparative analysis using RNA sequencing, proteomics, and metabolomics on cultured murine hepatocytes treated with NMN, NMNH, NR, or NRH. Global metabolic profiling revealed that NRH and NMNH induced substantially broader metabolic alterations than NR and NMN, with NRH uniquely suppressing metabolites involved in energy metabolism. The pronounced metabolic effects were reflected at a transcriptional level, with reduced precursors triggering a significantly higher number of differentially expressed genes than oxidized ones. Shared differentially expressed genes between NMNH and NRH revealed upregulation of stress-related glutathione-S-transferases (Gsts) which furthermore were reflected in our proteomic profiling. However, the upregulation of Gsts did not cause a depletion of glutathione or oxiglutathione, suggesting a pseudo-stress response to reduced NAD+ precursors. Together, our data demonstrate that reduced NAD+ precursors are unique and distinct from the market-available NAD+ precursors NR and NMN, not only as more potent NAD+ boosters, but also as compounds influencing a broader range of cellular processes.

Epigenetic age acceleration (EAA) is a biological marker of healthy longevity. While diet–EAA associations are documented in high-income countries, data from low- and middle-income populations—where diets can differ substantially—are unavailable. We examined associations between three complementary dietary components (specific food groups, dietary patterns, and plant-based food proportions) and EAA among Ghanaians across the nutrition transition (rural Ghana, urban Ghana, the Netherlands). We analyzed cross-sectional data from 705 adults in the RODAM study (2019–2021). Dietary intake was assessed via a Ghana-adapted Food Frequency Questionnaire (30 food groups; PCA-derived dietary patterns; Satija plant-based diet approach). EAA was estimated using DNA methylation clocks (Horvath, Hannum, PhenoAge, GrimAge) from Illumina EPIC array data. Linear regression models were used adjusted for demographic, lifestyle, health factors, and immune cell composition. Mediation analyses assessed potential biological pathways. In rural Ghana, fish intake was associated with lower Horvath EAA (β= -0.028) and PhenoAge EAA (β= -0.026). In urban Ghana, fermented maize intake was inversely associated with Hannum EAA (β= -0.002) and PhenoAge EAA (β= -0.006). In Amsterdam, condiment intake was positively associated with Hannum EAA (β = 0.010) and PhenoAge EAA (β = 0.012). Dietary patterns and plant-based proportions were not associated with EAA. Dietary vitamins D, B9 and B12 partially mediated associations (11 to 49

Aging entails a progressive decline in physiological functions, elevating the risk of age-related diseases like heart failure or aortic stenosis. Stem cell therapies, especially those that use paracrine signaling, can potentially mitigate the adverse effects of aging. The objective is to explore the potential of small extracellular vesicles (sEVs) derived from young adipose-derived stem cells (ADSC-sEVs) in reversing structural, molecular, and functional changes associated with aging in the heart. Aged C57BL/6J mice were treated intravenously with ADSC-sEVs from young mice or PBS as controls. Young mice were included to identify specific age-associated changes. The impact of sEV treatment on cardiac function was assessed using transthoracic echocardiography and physical endurance tests. Histological and molecular analyses were conducted on heart tissue to evaluate structural changes and markers of senescence, inflammation, and oxidative stress. A comprehensive metabolomic analysis was also performed on heart tissues to identify changes in metabolic profiles associated with aging and treatment status. The administration of ADSC-sEVs significantly improves several aging-associated cardiac parameters, including oxidative stress, inflammation, and cellular senescence reductions. We also report on the age-related reversal of myocardial structure and function changes, highlighted by decreased fibrosis and improved vascularization. Notably, echocardiographic assessments reveal that sEV treatments ameliorate diastolic dysfunction and left ventricle structural alterations typically associated with aging. Furthermore, the treatment shifts the heart metabolome towards a more youthful profile. These results denote the potential of ADSC-sEVs as a novel, noninvasive therapeutic strategy for mitigating cardiac aging-associated functional decline.

Perturbing mitochondrial translation represents a conserved longevity intervention, with proteostasis processes proposed to mediate the resulting lifespan extension. Here, we explore whether other mechanisms may contribute to lifespan extension upon mitochondrial translation inhibition. Using multi-omics and functional in vivo screening, we identify the ethylmalonyl-CoA decarboxylase orthologue C32E8.9 in C. elegans as an essential factor for longevity induced by mitochondrial translation inhibition. Reducing C32E8.9 completely abolishes lifespan extension from mitochondrial translation inhibition, while mitochondrial unfolded protein response activation remains unaffected. We show that C32E8.9 mediates immune responses and lipid remodeling, which play crucial roles in the observed lifespan extension. Mechanistically, sma-4 (a TGF-β co-transcription factor) serves as an effector of C32E8.9, responsible for the immune response triggered by mitochondrial translation inhibition. Collectively, these findings underline the importance of the "immuno-metabolic stress responses" in longevity upon mitochondrial translation inhibition and identify C32E8.9 as a central factor orchestrating these responses.

Complex lipid metabolism plays a crucial role in regulating aging. We recently discovered that the phospholipid bis(monoacylglycero)phosphate (BMP) increases in aged human muscles and many mouse tissues. The phospholipase PLA2G15 is reportedly involved in BMP synthesis, however, its specific role in aging remains unknown. To elucidate the role of PLA2G15 in aging, we used Caenorhabditis elegans as a model. When silencing plag-15, the predicted worm orthologue of PLA2G15, we observed improved healthspan and lifespan extension. Semi-targeted lipidomics highlighted that instead of changes related to BMP, plag-15 RNAi led to lower levels of lysophosphatidic acid, lysophosphatidylcholine, and lysophosphatidylethanolamine. Transcriptome-guided epistasis experiments identified that the lifespan extension of plag-15 RNAi worms is regulated by transcription factors hlh-30 and elt-3, and lysosomal vitamin B12 transporter pmp-5 (human TFEB, GATA, and ABCD4 respectively). Overall, we conclude that targeting phospholipid remodeling through plag-15 could be a promising strategy to promote healthy aging.

Measuring biological age typically requires invasive and costly procedures. To address this, the MoveIt! Age Score was developed: a simple, scalable, and interpretable aging clock that predicts biological age using only wearable-derived steps data. MoveIt! Age was trained on steps data from the United States National Health and Nutrition Examination Survey (NHANES), using chronological age, maximum step count, and step count variability to predict PhenoAge, a blood biochemistry biological age score. MoveIt! Age performance was evaluated in two independent cohorts: Mitochondria and Muscle Health in Elderly (MitoHealth; N = 55; healthy young adults or older adults from the Netherlands) and Restoring Health of Acutely Unwell Adults (RESORT; N = 145; geriatric rehabilitation inpatients from Australia). In RESORT, MoveIt! Age was assessed and compared to SenoClock-BloodAge and PhenoAge (hematological aging clocks). Delta age was the predicted biological age minus chronological age. In the NHANES testing dataset, MoveIt! Age demonstrated high predictive accuracy of chronological age (r = 0.97, RMSE = 5.4 years) and was more significantly associated with mortality than PhenoAge. In MitoHealth, delta MoveIt! Age showed differences between young adults and older adults who were normal, healthy, or health-impaired, with MoveIt! Age more significantly associated with muscle NAD+ levels (r = −0.37, p = 0.023) than chronological age (p = 0.416). Delta MoveIt! Age associated more strongly than other clocks with physical function outcomes, including frailty, handgrip strength, and functional performance. These findings support MoveIt! Age as a practical tool to gain insights into biological age in both clinical and community settings.

The 2021 Aging Report of the European Union projected significant increases in healthcare costs, with some member states expecting up to a 60

Beyond their classical functions as redox cofactors, recent fundamental and clinical research has expanded our understanding of the diverse roles of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) in signaling pathways, epigenetic regulation and energy homeostasis. Moreover, NAD and NADP influence numerous diseases as well as the processes of aging, and are emerging as targets for clinical intervention. Here, we summarize safety, bioavailability and efficacy data from NAD+-related clinical trials, focusing on aging and neurodegenerative diseases. We discuss the established NAD+ precursors nicotinic acid and nicotinamide, newer compounds such as nicotinamide riboside and nicotinamide mononucleotide, and emerging precursors. We also discuss technological advances including in industrial-scale production and real-time detection, which are facilitating NAD+ research and clinical translation. Finally, we emphasize the need for further large-scale studies to determine optimal dose, administration routes and frequency, as well as long-term safety and interindividual variability in response. Levels of the metabolic coenzyme NAD+ decline during aging, which is linked to many age-related diseases. Zhang et al. review recent clinical and translational evidence testing NAD+ supplementation in age-related diseases, highlighting therapeutic challenges and opportunities.

Aging is a major risk factor for disease, and developing effective pharmaceutical interventions to improve healthspan and promote longevity has become a high priority for society. One of the molecular pathways related to longevity in various model organisms revolves around lowering AKT1 levels. This prompted our in silico drug screen for small molecules capable of mimicking the transcriptional effects of AKT1 knockdown. We found topoisomerase inhibitors as a top candidate longevity-drug class. Evaluating multiple compounds from this class in C. elegans revealed that the topoisomerase inhibitor amonafide has the greatest benefit on healthspan and lifespan. Intriguingly, the longevity effect of amonafide was not solely dependent on DAF-16/FOXO, the canonical pathway for lifespan extension via AKT1 inhibition. We performed RNA-seq on amonafide-treated worms and revealed a more youthful transcriptional signature, including the activation of diverse molecular and cellular defense pathways. We found the mitochondrial unfolded protein response (UPRmt) regulator afts-1 to be crucial for both improved healthspan and extended lifespan upon amonafide treatment. Moreover, healthspan was partially dependent on the immune response transcription factor zip-2 and the integrated stress response transcription factor atf-4. We further examined the potential of amonafide in age-related disease. Treating a C. elegans model for Parkinson's disease with amonafide improved mobility. In conclusion, we identified amonafide as a novel geroprotector, which activates mitochondrial-, pathogen-, and xenobiotic-associated defense responses that-though more studies are needed-may serve as a candidate for Parkinson's disease therapy.

Nicotinamide adenine dinucleotide (NAD+) is an essential molecule involved in cellular metabolism, and its decline has been implicated in ageing and age-related disorders. However, evidence for an age-related decline in NAD+ levels in humans has been consistently observed only in a limited number of studies. Similarly, although preclinical studies support the idea that supplementation with NAD+ precursors is a promising therapeutic strategy to promote healthy ageing, human clinical trials have shown limited efficacy. Therefore, an increasing understanding of how NAD+ metabolism is affected in different tissues during disease and following NAD+ precursor supplementation is crucial to defining the therapeutic value of NAD+-targeted therapies. In this Review, we evaluate the clinical evidence supporting the notion that NAD+ levels decline with age, as well as the tissue-specific effects of NAD+ precursor supplementation. Viewed in perspective, the published body of data on NAD+ dynamics in human tissues remains sparse, and the extrapolation of rodent-based data is not straightforward, underscoring the need for more clinical studies to gain deeper insights into systemic and tissue-specific NAD+ metabolism. This Review summarizes existing data, as well as crucial knowledge gaps, emerging from clinical trials involving NAD+ precursor supplementation in humans.

IntroductionGeroprotection is an emerging field of research focused on devising strategies for combating the mechanisms of ageing. This study held three aims: 1) to explore the willingness to utilise five different geroprotective measures (i.e., exercise, supplements, intermittent fasting, metformin, and rapamycin use), 2) to explore whether the willingness differs based on respondents’ sociodemographic characteristics and 3) to explore the association between trust in medical institutions and willingness to utilise different geroprotective measures.MethodsA questionnaire was used to assess the attitudes of a sample of the Dutch population by way of both convenience and snowball sampling (final N = 178). Descriptive data and bivariate correlations were used in the analyses.ResultsRelatively high social acceptance of both exercise (66%) and supplements (82%) was found, whereas intermittent fasting (30%), metformin (26%), and rapamycin (10%) were less supported. Males were significantly more likely to be open to exercise and women to supplement use. Trust in medical institutions correlated significantly with the willingness to start metformin.DiscussionExploratory research can only provide a first step in understanding the social acceptance of geroprotection measures. Nevertheless, this study clearly illustrates more well-known measures promoted by public health policy are also more accepted and used. Public health campaigns could consider the sex differences in the uptake of exercise and supplements, and future research may want to delve deeper into the role of facilitating trust relations between medical institutions and the public in promoting the use of geroprotective drugs.

Complex lipids, essential components in biological processes, exhibit conserved age-related changes that alter membrane properties and cellular functions and are implicated as biomarkers and contributors to longevity and age-related diseases. While physical activity alleviates age-related comorbidities and physical impairments, comprehensive exploration of the underlying biological mechanisms, particularly at the level of complex lipids, remains limited. However, clinical studies suggest that physical activity may counteract these age-related lipidomic changes, presenting a promising avenue for intervention. We performed lipidomic profiling of plasma from an extensively characterized cohort of young and aged individuals. Annotating 1446 unique lipid species across 24 lipid classes, we found the most prominent difference in older adults was an accumulation of triacylglycerols (TGs), with lower physical activity levels associated with higher TG levels in plasma and reduced physical functionality. Remarkably, lipid species in the TG class did not accumulate uniformly. Rather, our study unveiled a negative correlation between higher physical activity levels and TGs with shorter chain lengths and more double bonds in this demographic. Overall, our research highlights that plasma TG length and saturation level can help mark healthy aging groups in humans. These findings deepen our understanding of how aging affects complex lipids and the influence of physical activity on this process.

Studies in preclinical models suggest that complex lipids, such as phospholipids, play a role in the regulation of longevity. However, identification of universally conserved complex lipid changes that occur during aging, and how these respond to interventions, is lacking. Here, to comprehensively map how complex lipids change during aging, we profiled ten tissues in young versus aged mice using a lipidomics platform. Strikingly, from >1,200 unique lipids, we found a tissue-wide accumulation of bis(monoacylglycero)phosphate (BMP) during mouse aging. To investigate translational value, we assessed muscle tissue of young and older people, and found a similar marked BMP accumulation in the human aging lipidome. Furthermore, we found that a healthy-aging intervention consisting of moderate-to-vigorous exercise was able to lower BMP levels in postmenopausal female research participants. Our work implicates complex lipid biology as central to aging, identifying a conserved aging lipid signature of BMP accumulation that is modifiable upon a short-term healthy-aging intervention.

Since its discovery in 1958 in the lens of cows, ophthalmic acid (OPH) has stood in the shadow of its anti-oxidant analog: glutathione (GSH). Lacking the thiol group that gives GSH many of its important properties, ophthalmic acid's function has remained elusive, and it has been widely presumed to be an accidental product of the same enzymes. In this review, we compile evidence demonstrating that OPH is a ubiquitous metabolite found in bacteria, plants, fungi, and animals, produced through several layers of metabolic regulation. We discuss the limitations of the oft-repeated suggestions that aberrations in OPH levels should solely indicate GSH deficiency or oxidative stress. Finally, we discuss the available literature and suggest OPH's role in metabolism as a GSH-regulating tripeptide; controlling both cellular and organelle influx and efflux of GSH, as well as modulating GSH-dependent reactions and signaling. Ultimately, we hope that this review reinvigorates and directs more research into this versatile metabolite.

As the global aging population rises, finding effective interventions to improve aging health is crucial. Drug repurposing, utilizing existing drugs for new purposes, presents a promising strategy for rapid implementation. We explored naltrexone from the Library of Integrated Network-based Cellular Signatures (LINCS) based on several selection criteria. Low-dose naltrexone (LDN) has gained attention for treating various diseases, yet its impact on longevity remains underexplored. Our study on C. elegans demonstrated that a low dose, but not high dose, of naltrexone extended the healthspan and lifespan. This effect was mediated through SKN-1 (NRF2 in mammals) signaling, influencing innate immune gene expression and upregulating oxidative stress responses. With LDN's low side effects profile, our findings underscore its potential as a geroprotector, suggesting further exploration for promoting healthy aging in humans is warranted.

Maintaining mitochondrial function is critical to an improved healthspan and lifespan. Introducing mild stress by inhibiting mitochondrial translation invokes the mitochondrial unfolded protein response (UPR mt ) and increases lifespan in several animal models. Notably, lower mitochondrial ribosomal protein (MRP) expression also correlates with increased lifespan in a reference population of mice. In this study, we tested whether partially reducing the gene expression of a critical MRP, Mrpl54 , reduced mitochondrial DNA-encoded protein content, induced the UPR mt , and affected lifespan or metabolic health using germline heterozygous Mrpl54 mice. Despite reduced Mrpl54 expression in multiple organs and a reduction in mitochondrial-encoded protein expression in myoblasts, we identified few significant differences between male or female Mrpl54 +/− and wild type mice in initial body composition, respiratory parameters, energy intake and expenditure, or ambulatory motion. We also observed no differences in glucose or insulin tolerance, treadmill endurance, cold tolerance, heart rate, or blood pressure. There were no differences in median life expectancy or maximum lifespan. Overall, we demonstrate that genetic manipulation of Mrpl54 expression reduces mitochondrial-encoded protein content but is not sufficient to improve healthspan in otherwise healthy and unstressed mice.

Aging clocks, built from comprehensive molecular data, have emerged as promising tools in medicine, forensics, and ecological research. However, few studies have compared the suitability of different molecular data types to predict age in the same cohort and whether combining them would improve predictions. Here, we explored this at the level of proteins and small RNAs in 103 human blood plasma samples. First, we used a two-step mass spectrometry approach measuring 612 proteins to select and quantify 21 proteins that changed in abundance with age. Notably, proteins increasing with age were enriched for components of the complement system. Next, we used small RNA sequencing to select and quantify a set of 315 small RNAs that changed in abundance with age. Most of these were microRNAs (miRNAs), downregulated with age, and predicted to target genes related to growth, cancer, and senescence. Finally, we used the collected data to build age-predictive models. Among the different types of molecules, proteins yielded the most accurate model (R² = 0.59 ± 0.02), followed by miRNAs as the best-performing class of small RNAs (R² = 0.54 ± 0.02). Interestingly, the use of protein and miRNA data together improved predictions (R2 = 0.70 ± 0.01). Future work using larger sample sizes and a validation dataset will be necessary to confirm these results. Nevertheless, our study suggests that combining proteomic and miRNA data yields superior age predictions, possibly by capturing a broader range of age-related physiological changes. It will be interesting to determine if combining different molecular data types works as a general strategy to improve future aging clocks.

The degenerative processes that occur during aging increase the risk of disease and impaired health. Meanwhile, interventions that target aging to promote healthy longevity are gaining interest, both academically and in the public. While nutritional and physical interventions exist, efficacy is often difficult to determine. It is therefore imperative that an aging score measuring the biological aging process is available to the wider public. However, simple, interpret, and accessible biological aging scores are lacking. Here, we developed PhysiAge, a physiological aging score based on five accessible parameters that have influence on or reflect the aging process: (1) average daily step count, (2) blood glucose, (3) systolic blood pressure, (4) sex, and (5) age. Here, we found that compared to calendar age alone, PhysiAge better predicts mortality, as well as established muscle aging markers such as decrease in NAD(+) levels, increase in oxidative stress, and decline in physical functioning. In order to demonstrate the usefulness of PhysiAge in identifying relevant factors associated with decelerated aging, we calculated PhysiAges for a cohort of aged individuals and obtained mass spectrometry-based blood plasma metabolomic profiles for each individual. Here, we identified a metabolic signature of decelerated aging, which included components of the TCA cycle, including malate, citrate, and isocitrate. Higher abundance of these metabolites was associated with decelerated aging, in line with supplementation studies in model organisms. PhysiAge represents an accessible way for people to track and intervene in their aging trajectories, and identifies a metabolic signature of decelerated aging in human blood plasma, which can be further studied for its causal involvement in human aging.