The discovery of biomarkers in a biological specimen relies on the identification and characterization of unique metabolites, which are statistically distinct among different groups. Table S1, Supporting Information summarizes recent biomarkers of aging discovered using metabolic profiling of biofluids and tissues derived from human and vertebrate models (not focusing on seminal studies performed using invertebrate models) along with the corresponding listing of metabolomics platforms used in their respective studies. For human studies on aging, longitudinal studies have been a transformative resource, 51 of which are in the National Institutes of Aging database.[19] Two of these longitudinal studies have been instrumental in metabolomics based biomarker discoveries, number of which are listed in Table S1, Supporting Information, the Baltimore longitudinal study of aging (https://www.blsa.nih.gov/)[20] and the Leiden longevity study.[21] One of the exciting insights that has emerged from these longevity studies is the association between the APOE4 genotype and aging.[22, 23] Subjects with APOE4 genotype have substantially decreased extreme longevity unlike subjects with E2E3 genotype.[24] Though increased risk of AD pathology conferred by the APOE4 genotype has been studied, the basal metabolomic differences on “normal” aging remain to be elucidated and represent a significant opportunity.

3.1 Hub Metabolites Emerging from Aging: Metabolomics at the Interface Cellular Signaling, Epigenetics, and Metabolism

The important insight that has emerged from metabolomics based studies of aging is the central impact of specific metabolic intermediates on aging. These “hub” metabolites represent nodes in the biochemical network that play a critical role in integrating the information flow in cells between metabolism and signaling pathways, to control aging (Figure 2). There are a number of candidate hub metabolites that might fulfill these criteria, but following is a summary of four conserved emerging hubs in the aging metabolome.

Figure 2 Open in figure viewer PowerPoint Hub metabolites and signaling mechanisms that promote longevity. The aging metabolome can be viewed as two layers comprised of i) hub metabolites (NAD+, NADPH, αKG, and ketone bodies such as βHB) and ii) signaling mechanism involving regulation of aging‐related signaling, epigenetic, and stress responsive pathways. Large green arrows indicate the pro‐longevity effects of their supplementation, red T's indicate inhibitory effects on enzymes/pathways and green arrows indicate activation of enzymes/pathways.

3.1.1 NAD+ Metabolome, at the Hub of Central Carbon Metabolism, DNA Damage, and Protein Deacetylation, and Cellular Senescence Nicotinamide adenine dinucleotide (NAD+) is an essential electron carrier in cellular biochemistry, and the ratio of the oxidized and reduced forms is a readout of the redox status of cells. It is primarily generated via glycolytic and TCA cycle metabolism in cells.[25] Beyond its canonical role as a coenzyme for redox reactions, it serves as a cofactor for three enzymes that are of central interest in aging biology, i) Sirtuins (SIRT), ii) poly(ADP‐ribose) polymerases (PARP), and iii) cyclic ADP‐ribose synthases (e.g., CD38). NAD+ is continually consumed by these three enzymes and organisms to produce nicotinamide. Nicotinamide salvage pathways and biosynthetic pathways maintain stable levels of NAD+ in cells. The salvage pathways is mediated by the enzyme nicotinamide mononucleotide phosphoribosyl transferase (NAMPT) that recycles nicotinamide into nicotinamide mononucleotide (NMN) that is converted to NAD+ by nicotinamide mononucleotide adenylyl‐transferase (NMNAT) family of enzymes. In parallel, NAD+ can be biosynthesized from dietary sources such as nicotinic acid (a form of vitamin B 3 ) via NMNAT enzymes in the Preiss‐Handler pathway.[25] NAD+ can also be synthesized from dietary tryptophan via the kynurenine pathway.[8, 26] The three classes of NAD+ consuming enzymes have emerged as key players in aging. The family of sirtuin enzymes were identified as NAD+ dependent protein deacylases (modifying acetyl, succinyl, malonyl palmitoyl, and glutaryl groups[27-32]) producing nicotinamide and O‐acetyl‐ADP‐ribose as products of the deacylation reaction. Sirtuins serve as NAD+ sensors, and it has been shown that caloric restriction dependent increase in lifespan in SIRT1 dependent fashion.[33] There are seven mammalian sirtuins in the cytoplasm and the mitochondria.[34, 35] The substrates of sirtuins range from acetylated histones to transcription factors (e.g., PGC‐1a, FOXO3a, and NF‐kb).[8] The other NAD+ consuming enzyme PARP is important for the cellular DNA damage sensing and the repair‐response. PARP1 and 2 transfer ADP‐ribose from NAD+ proteins to accepting proteins/nucleic acids generating long poly (ADP‐ribose) chains (PAR). PARP1 in response to DNA damage causes PAR chains at site of damage and leads to a significant decrease in intracellular NAD+ levels.[8, 21, 36] The emerging evidence suggests that levels of NAD+ decrease with aging in both vertebrate and invertebrate systems, and the decline in these levels is associated with aging related pathologies.[37, 38] A recent metabolomics study has comprehensively quantified the NAD+ metabolome in the plasma of human subjects ranging from 20 to 87 years of age.[39] Measurements using LC–MS/MS showed that levels of NAD+ and nicotinic acid adenine dinucleotide (NAAD) decreased significantly with age. This was not the case with the entire NAD+ metabolome as there was no change in nicotinic acid (NA), nicotinamide mononucleotide (NMN), and nicotinic acid mononucleotide (NAMN). An aging‐related increase in the reduced form on NAD, nicotinamide (NAM), and adenosine diphosphate ribose (ADPR) was seen. This suggests an aging‐related alteration in the plasma NAD+ metabolome, but the reasons for this, and specifically for the decrease in NAD+ during aging remains an area of intense study. It is clear that levels of NAD+ will be regulated by the complex interplay of pathways that produce and consume this metabolite. In order to address this, a landmark study has been reported by the Liu et al.,[40] where metabolomics was applied in conjunction with isotope tracer studies to elucidate the fluxes of NAD+ synthesis and breakdown across various tissues in mice. The mass spectrometric analysis was done using HILIC separation and a Q EXACTIVE PLUS mass spectrometer. Isotope abundances were quantified using the freely available AccuCor software (https://github.com/XiaoyangSu/Isotope-Natural-Abundance-Correction).[41] NAD+ precursors that were used was decided by plasma concentrations of at least 0.1 × 10−6 m. The authors selected U‐13C Trp, [2,4,5,6‐2H]NAM and U‐13C NA for in vivo labeling of NAD+. Labeled precursors were infused into C57BL/6 mice in order to quantify biosynthesis of NAD from Trp and NA, flux of the salvage pathway from NAM to NAD+, flux through NAD kinase, and exchange of serum and tissue NAM. An important insight that emerged from this study is that liver is the primary site of de novo synthesis of NAM from Trp, with insignificant fluxes in other tissues. On the other hand, NA seems to be an insignificant contributor to NAM in tissues as judged by infusion of 13C‐labeled NA. Next, the authors measured the incorporation of labeled NAM into NAD in various tissues, this was judged by formation of (M+3) NAM from infused (M+4) NAM. (M+3) NAM is formed by loss of the reducing hydrogen into NAD from the (M+4) NAM and subsequent cleavage to (M+3) NAM. The analysis revealed that liver is the major producer of NAM, which is consumed by peripheral tissues to generated NAD in a tissue‐dependent fashion with the small intestine and spleen having the highest NAD turnover and skeletal muscle with the least. Though these studies were carried out in young mice, nevertheless it emphasizes the need to study fluxes of the NAD metabolome in a tissue specific and temporal fashion to determine the source of the aging‐related dysfunction in the NAD metabolome. There is an intense interest in the role of senescent cells in aging and the emerging possibility of targeting these cells as an anti‐aging therapy.[42, 43] Senescence is a non‐proliferative state of cells that accumulate with aging and at sites of aging‐related pathologies including arthritis, atherosclerosis, obesity, etc.[43] Apart from replicative senescence, this state can also be caused by DNA damage and oncogenes.[42, 43] A number of hallmarks of senescent cells have been recognized (including upregulated expression of p16Ink4A and/or p21)[44] of which the senescence‐associated secretory phenotype (SASP) is a well‐characterized one.[45] It is important to note that depending on the tissues of origin and inducers of senescence, cells display a range of non‐overlapping hallmarks including secreted SASP factors (e.g., TGF‐β, IL‐6, and IL‐8). An important feature of senescent cells is also their dysregulated metabolome. A subtype of senescence is driven by mitochondrial dysfunction that is termed mitochondrial dysfunction‐induced senescence (MIDAS).[46, 47] Studies on how the metabolome of senescent cells contributes to this cell‐state has led to insights on the role of NAD+ as a central metabolic node in senescence. Nacarelli et.al. have recently shown that SASP is controlled by NAD+ metabolism.[48] Specifically, the authors showed that the salvage arm of NAD metabolism, which is controlled by the enzyme nicotinamide phosphoribosyl transferase (NAMPT) controls the proinflammatory SASP. Using an oncogene induced senescence model and LC–MS/MS assays, the authors showed that the senescent cells have significantly increased levels of NAD+, and NAMPT mediated synthesis of NAD+ (using cells with NAMPT inhibition), and was required for the secretion of SASP factors (IL‐10 and TNF‐α). Feeding senescent cells 13C‐labeled glucose followed by metabolic profiling showed that senescent cells had enhanced glycolysis via pyruvate and lactate, and the TCA cycle as a pathway for maintaining NAD+ levels. It was proposed that high NAD+ levels support senescence‐related metabolic changes, and also promote AMPK and NF‐κB signaling pathways that activate the expression of inflammatory cytokines. In contrast to the high NAD+ levels observed in this study, the MIDAS cells were driven by lower NAD+ levels. Wiley et al.[47] showed that mitochondrial dysfunction (by ethidium bromide induced mitochondrial depletion) had low NAD/NADH ratios as expected, and displayed the induction of senescence. This study also showed the importance of NAD sensing mitochondrial sirtuins (SIRT3) in MIDAS, as overexpression of these proteins significantly delayed the induction of senescence. The authors showed that induction of AMPK and its phosphorylation of p53 is the mechanism by which decreased NAD/NADH levels induce MIDAS. An important issue that is underlined by this study is the importance of investigating the cellular compartments which have perturbed NAD/NADH ratios. Unexpectedly the compartment with the altered ratio is the cytoplasm, and not the mitochondria suggesting the important role of NAD+ transport into the cytosol that might be perturbed by mitochondrial dysfunction. Finally, the in vivo significance of this metabolic study was shown using a POLGD257A mouse that has a mutation in the proof reading domain of the mitochondrial DNA polymerase, causing accumulation of mutations in the mitochondrial DNA, leading to mitochondrial dysfunction. The skin and fat tissue of these mice showed a significant accumulation of senescent cells, suggesting that MIDAS could occur in vivo.

3.1.2 NADP Metabolome at the Hub of the Pentose Phosphate Shunt and Redox Homeostasis Compared to NAD+, its phosphorylated cousin, NADP, has received lesser attention until recently in aging biology. It is well known that the reduced form NADPH is the donor of reductive equivalents to glutathione reductase and thioredoxin reductase, in the synthesis of compounds in the oxidative stress response (reduced glutathione and reduced thioredoxin, respectively).[49] NADPH is produced by the oxidative arm of the pentose phosphate shunt, by the enzyme glucose 6‐phosphate dehydrogenase and 6‐phosphogluconate reductase. It can also be produced by the mitochondrial enzymes isocitrate dehydrogenase 1 and 2, NADP dependent malic enzyme, and one carbon cycle enzyme.[49, 50]. Liu et al.[40] have shown that the NAD kinase (a source of NADP) only acts on about 10% of the total NAD+ pool. A comprehensive fluxomics of the NADP metabolome remains to be accomplished and is now necessary to elucidate how pools of this metabolite are maintained in various tissues. In aging biology, NADPH has been recognized in the context of the free radical theory of aging proposed by Hannan in 1950. The theory suggested that free radicals create cumulative damage to macromolecules and nucleic acid causing aging. New biological insights regarding the positive role of free radicals in stem cell activation and are essential signaling factors[51-54] show that the theory needs refinement or revision. None the less molecules that play a critical role in redox homeostasis are expected to play a critical role in aging. It has been shown in D. melanogaster that overexpression of G6PD leads to higher levels of NADPH, higher reduced to oxidized glutathione as expected, and leads to increase in lifespan.[55] A recent study by Wang et al.[56] has used metabolomics in combination with proteomics to suggest a role for this pathway in a long lived mutant fly. In this study, the authors used metabolomics to compare the relative levels of stead state metabolites form the head of flies that were either wild‐type or mutants with one copy of the puc phosphatase resulting in the partial activation of the stress response JNK kinase. It has been previously shown that these puc mutants had a longer lifespan, but the underlying metabolic mechanisms were unclear. Steady state metabolomics revealed a significant increase in glucose 6‐phosphate (G6P) and ribose 5‐phosphate (R5P) in the young puc mutants are compared to wild‐type mutants, suggesting that a metabolic phenotype even in the young state might enable the mutants to live longer. The authors developed an approach to inject U‐13C glucose in the haemocytes of flies to monitor flux of glucose and TCA metabolism in the fly heads. This revealed that the puc mutants had an efficient flux of carbon into the TCA, higher levels of NADPH, and higher mRNA levels of G6PD. Finally, these mutants showed an improved protein homeostasis that was recapitulated by the over expression of G6PD. The two studies suggest that G6PD derived NADPH may be an important node in the metabolic network that can extent lifespan in flies. It is now clear that overexpression of G6PD and resultant increase NADPH can also display benefits in a transgenic mouse.[57] The transgenic mouse with a mild overexpression of G6PD via its endogenous promotor increased the median lifespan of female mice by 14%, including improved glucose tolerance and decreased weight gain. Knowing that NAD+ and NADP can be interconverted with one another, it is possible that there are overlapping benefits of these metabolites in aging, a quantitative fluxomics of both the NAD+ and NADP metabolome in the future will be critical to decouple each of their effects.