Methionine metabolism and methyltransferases in the regulation of aging and lifespan extension across species

Abstract Methionine restriction (MetR) extends lifespan across different species and exerts beneficial effects on metabolic health and inflammatory responses. In contrast, certain cancer cells exhibit methionine auxotrophy that can be exploited for therapeutic treatment, as decreasing dietary methionine selectively suppresses tumor growth. Thus, MetR represents an intervention that can extend lifespan with a complementary effect of delaying tumor growth. Beyond its function in protein synthesis, methionine feeds into complex metabolic pathways including the methionine cycle, the transsulfuration pathway, and polyamine biosynthesis. Manipulation of each of these branches extends lifespan; however, the interplay between MetR and these branches during regulation of lifespan is not well understood. In addition, a potential mechanism linking the activity of methionine metabolism and lifespan is regulation of production of the methyl donor S‐adenosylmethionine, which, after transferring its methyl group, is converted to S‐adenosylhomocysteine. Methylation regulates a wide range of processes, including those thought to be responsible for lifespan extension by MetR. Although the exact mechanisms of lifespan extension by MetR or methionine metabolism reprogramming are unknown, it may act via reducing the rate of translation, modifying gene expression, inducing a hormetic response, modulating autophagy, or inducing mitochondrial function, antioxidant defense, or other metabolic processes. Here, we review the mechanisms of lifespan extension by MetR and different branches of methionine metabolism in different species and the potential for exploiting the regulation of methyltransferases to delay aging.

In addition, methionine serves major roles through its metabolism, which fuels a variety of metabolic pathways. Methionine metabolism can be broken into three parts: the methionine cycle, the transsulfuration pathway, and the salvage cycle ( Figure 1).

| Methionine cycle
The first step in methionine metabolism is performed by methionine adenosyltransferase (MAT), an enzyme conserved from Escherichia coli to humans that catalyzes the biosynthesis of S-adenosylmethionine (SAM) from methionine and ATP. SAM is the principal methyl donor and the second most widely used enzyme substrate after ATP (Cantoni, 1975). During substrate methylation, SAM donates its methyl group to acceptor molecules, for example, DNA, RNA, proteins, or other cellular metabolites, generating S-denosylhomocysteinee (SAH). Over 200 known or putative methyltransferases have been identified in the human genome (Petrossian & Clarke, 2011) and 81 in yeast (Petrossian & Clarke, 2009). S-denosylhomocysteine hydrolase (SAHH/AHCY) catalyzes the reversible hydrolysis of SAH to adenosine and l-homocysteine. SAHH/AHCY proteins are tetramers with a NADH/NAD + cofactor bound in the active site of each subunit (Brzezinski, Bujacz, & Jaskolski, 2008). There are also two AHCY-like proteins, AHCYL1 and AHCYL2, which most likely have lost their canonical enzymatic functions due to critical mutations in their AHCY domains. However, via hetero-multimerization, ACHYL1 and AHCYL2 can suppress the enzymatic activity of AHCY and thus act as dominant negative regulators of canonical AHCY (Devogelaere, Sammels, & Smedt, 2008). Cells must maintain low concentrations of SAH, which is a product inhibitor of SAM-dependent methylation reactions. Methyltransferases catalyze a variety of methylation reactions via the transfer of methyl groups on histone proteins as well as to nucleic acids, nonhistone proteins, and metabolites, although different methyltransferases exhibit different sensitivity to inhibition by SAH (Huang et al., 2000). Homocysteine can be remethylated to form methionine and retained in the methylation cycle, or converted to cysteine via the transsulfuration pathway and thus withdrawn from the methylation cycle. Remethylation of homocysteine to form methionine completes the methionine cycle. This process involves either methionine synthase (MS), which requires 5-methyltetrahydrofolate as a methyl donor, or betaine homocysteine methyltransferase (BHMT), which requires betaine as a methyl donor.

| Transsulfuration pathway
Homocysteine from the methionine cycle can also be utilized in the transsulfuration pathway to produce cysteine. Cystathionine-βsynthase is the first and rate-limiting enzyme of the transsulfuration pathway, the primary metabolic pathway for the synthesis of cysteine. Cystathionine-β-synthase synthesizes cystathionine from the condensation of homocysteine and serine. Cystathionine is hydrolyzed by cystathionine-γ-lyase to produce cysteine, which is further used in the synthesis of proteins, glutathione, and taurine.
Cystathionine-γ-lyase and cystathionine-β-synthase also catalyze the production of hydrogen sulfide (H 2 S) from cysteine and homocysteine. H 2 S is a signaling molecule and cytoprotectant with a wide range of physiological functions. H 2 S protects cells from oxidative stress and can modulate neuronal transmission, smooth muscle relaxation, release of insulin, and the inflammatory response.
Putrescine is further converted to spermidine and spermine through the consecutive action of two distinct aminopropyl transferases, spermidine synthase and spermine synthase, which use dcSAM as an aminopropyl donor. dcSAM is converted to MTA after the donation of an aminopropyl group for polyamine synthesis, and MTA is converted via six enzymatic steps back to methionine (Minois et al., 2011;Pegg, 2016). In addition to participating in methylation and synthesis of polyamines, SAM can also be activated by members of the radical SAM superfamily of enzymes that convert SAM to a highly oxidizing 5′-deoxyadenosyl radical intermediate involved in a variety of reactions (Landgraf, McCarthy, & Booker, 2016).
Beside its proteogenic and metabolic roles, methionine can also serve as an antioxidant. Methionine is one of the major targets of reactive oxygen species (ROS). Surface-exposed methionine residues of native proteins can be oxidized by ROS to R-and S-methionine sulfoxide, which can be reduced back to methionine by methionine sulfoxide reductases.
Consistent with the importance of methionine metabolism in cellular physiology, dysregulation of methionine metabolism has been reported in multiple diseases. Moreover, methionine  (Agrawal, Alpini, Stone, Frenkel, & Frankel, 2012;Cavuoto & Fenech, 2012), a feature further exploited in a variety of therapeutic approaches (clini caltr ials.gov). In addition, 11 C-methionine is the most popular amino acid tracer for PET imaging of brain tumors (Glaudemans et al., 2013). Finally, MetR in rodents not only extends lifespan but also protects from visceral fat mass accumulation and from the negative effects of a high-fat diet (Ables, Perrone, Orentreich, & Orentreich, 2012;Malloy et al., 2006;Orentreich, Matias, DeFelice, & Zimmerman, 1993;Wanders et al., 2018). Based on these results, MetR has also been tested in clinical trials of obese adults with metabolic syndrome (Plaisance et al., 2011).
In the following sections, we discuss how the different branches of methionine metabolism-the methionine cycle, the transsulfuration pathway, and polyamine metabolism-regulate lifespan ( Figure 2) and discuss a potential mechanism linking methionine flux and lifespan regulation. We also review mechanisms of lifespan extension by MetR ( Figure 3 and Table 1) with a main focus on the relevance of methionine metabolism and methyltransferases (

| ME THI ONINE ME TABOLIS M AND LIFE S PAN E X TEN S I ON IN YE A S T
The budding yeast Saccharomyces cerevisiae serves as one of the main model organisms for studying evolutionarily conserved mechanisms of aging and age-related diseases. There are two aging models in budding yeast: chronological aging (CLS) and replicative aging (RLS).
CLS is defined as the length of time that a nondividing yeast cell survives. RLS is defined as the number of daughter cells produced by a mother cell prior to senescence (Longo, Shadel, Kaeberlein, & Kennedy, 2012).

| Methionine cycle
Wu et al. tested the effects of amino acids on CLS extension in the S. cerevisiae wild-type BY4742 strain, which is auxotrophic for his/ leu/lys. They found that changing the ratio of nonessential and essential amino acids caused great changes in CLS, whereas increase or decrease in individual amino acids in the culture media had little effect. The exceptions were methionine restriction and addition of glutamic acid, which resulted in CLS extension (Wu, Song, Liu, & Huang, 2013). Methionine restriction can also be achieved by genetically limiting methionine biosynthesis. Yeast can use inorganic sulfur to produce sulfur-containing amino acids. To genetically induce MetR, Ruckenstuhl et al. and Johnson et al. used methionine-auxo-troph strains (genetic MetR) with limited (Δmet15) or no (Δmet2) endogenous methionine biosynthesis. These strains displayed enhanced CLS when grown on complete synthetic medium (i.e., with 30 mg/L methionine). In addition, restricting methionine from the medium increased CLS of the mutant strains but not of the prototrophic strain (Johnson & Johnson, 2014;Ruckenstuhl et al., 2014).
In yeast, MetR is accompanied by activation of autophagy flux, and deletion of autophagy essential genes or decrease in vacuolar acidity

SAM/SAH
Aging via disruption of v-ATPase activity (which is responsible for the maintaining lysosomal pH) abolished MetR-induced CLS extension.
Moreover, MetR-induced CLS extension could not be further extended by TOR inhibition, suggesting that MetR and TOR inhibition function via at least partially overlapping mechanisms . In various organisms including yeast, nuclear-mitochondrial communication is a key player in aging. Induction of the retrograde response pathway, which transmits signals of mitochondrial  Orentreich et al. (1993) stress to the nucleus, can increase replicative lifespan in yeast (Kirchman, Kim, Lai, & Jazwinski, 1999). MetR-induced CLS extension is dependent on the key mediator of retrograde signaling, the transcription factor RTG3, and 20% of differentially expressed genes under genetic MetR are reversed to basal levels in the absence of RTG3 (Johnson & Johnson, 2014).
Interventions that extend lifespan often confer resistance to different stresses. In agreement with this, genetic MetR makes yeast cells more resistant to oxidative damage, heavy metal stresses (Singh & Sherman, 1974), heat stress, and lack of divalent metal cations, which is highly toxic to yeast cells (Johnson & Johnson, 2014

| Nonhistone methylation
In light of the importance of methionine metabolism as a donor of SAM, growth in methionine-deficient media causes tRNA hypomethylation (Fesneau, Robichon-Szulmajster, Fradin, & Feldmann, 1975

| Transsulfuration pathway
The transsulfuration pathway allows interconversion of homocysteine and cysteine via the intermediary formation of cystathionine. S. cerevisiae possess two active transsulfuration pathways (Thomas & Surdin-Kerjan, 1997

| Polyamine metabolism
Methionine metabolism is critical for the production of polyamines because it supplies an aminopropyl group necessary to the process

| Methionine cycle
In an unbiased RNAi screen, Hansen et al. identified 23 new longevity genes in C. elegans that when knocked down, extend lifespan from 10% to 90% (Hansen, Hsu, Dillin, & Kenyon, 2005). One of these genes, sams-1/C49F5.1 encodes methionine adenosyltransferase, which catalyzes the biosynthesis of SAM, the first step in methionine metabolism. In C. elegans, the eat-2 mutant serves as a genetic model for studying DR, as mutation of eat-2 defects disrupts pharyngeal pumping and thus limits food intake. Hansen et al. found that knockdown of sams-1 extended lifespan in a daf-16-independent manner but failed to extend the lifespan of eat-2/ad1116 mutants.
Knockdown of sams-1 did not affect pharyngeal pumping but similar to DR, knockdown of sams-1 resulted in slender worms with reduced brood size and delayed reproduction. Moreover, the level of sams-1 mRNA is reduced threefold in eat-2 mutants (Hansen et al., 2005). Ching, Paal, Mehta, Zhong, and Hsu (2010) showed that overexpression of sams-1 partially suppresses lifespan extension of DR worms and RNAi knockdown of sams-1 reduces the global translation rate.
Lifespan extension via reduced activity of the first enzyme in the methionine cycle suggests that limiting flux through the pathway is beneficial for lifespan, preventing accumulation of harmful metabolites, and is consistent with the beneficial effects observed for MetR and DR. Consistent with this, metformin, a drug widely prescribed to treat type 2 diabetes, increases lifespan in C. elegans co-cultured with E. coli as a food source after treatment with metformin.
Metformin inhibits folate production and methionine metabolism in the bacteria, leading to changes in methionine metabolism and a decrease in the level of SAM in the worms (Cabreiro et al., 2013).
Sirtuins are a family of histone deacetylases that require NAD + as a cosubstrate. During sirtuin-mediated deacetylation of l-lysine residues, NAD + is converted into nicotinamide (

| H3K36 (histone mark, associated with transcriptional elongation and splicing)
Deletion of met-1 H3K36 methyltransferase decreased lifespan in

| Transsulfuration pathway
As noted above, in C. elegans, the eat-2 mutant strain serves as a genetic model for studying DR. Eat-2 mutant worms produced more H 2 S, a product of the transsulfuration pathway, than wild-type worms. CBS-1 is a worm ortholog of cystathionine-β-synthase, the rate-limiting enzyme in the transsulfuration pathway ( Figure 1). RNAi knockdown of cbs-1 decreased the lifespan extension normally associated with eat-2 mutants, and overexpression of CBS-1 in wild-type worms prolonged lifespan (Hine et al., 2015). Supplementation of the diet with the product of the transsulfuration pathway, N-acetyll-cysteine, significantly extended both the mean and maximum lifespan and significantly increased resistance to oxidative stress, heat stress, and UV irradiation in C. elegans (Oh, Park, & Park, 2015).

| Polyamine metabolism
Similar to yeast, supplementation of the food with spermidine in C. elegans induced autophagy and prolonged lifespan by up to 15%, whereas knockdown of Beclin-1, a gene essential for autophagy, abolished the spermidine-mediated increase in lifespan (Eisenberg et al., 2009).

| Methionine cycle
DR is one of the most effective dietary interventions that extends lifespan in diverse organisms (Tatar, Post, & Yu, 2014), but also leads to reduced fecundity. Methionine supplementation alone can increase fecundity in DR animals to levels comparable to full-fed controls without reducing lifespan (Grandison, Piper, & Partridge, 2009).  (Lee et al., 2014). Interestingly, we recently showed that naturally selected long-lived flies, which have twice the lifespan of wild-type strains, have higher levels of endogenous methionine, suggesting that high levels of methionine are not detrimental to lifespan and that flux via methionine metabolism is more critical than the level of methionine itself (Parkhitko et al., 2016). In agreement with this hypothesis, we suggest that methionine metabolism is reprogrammed during aging that leads to the accumulation of SAH and homocysteine. Downregulation of dAhcyL1/dAhcyL2 at the whole-organism and tissue-specific level extends lifespan and healthspan. In our model, downregulation of dAhcyL1/dAhcyL2 activates Ahcy13, which in turn promotes SAH and homocysteine processing, resulting in an increase in methionine flux and an effect reminiscent of methionine restriction (Parkhitko et al., 2016).
In agreement with the importance of flux via methionine metabolism, Obata et al. showed that overexpression of GNMT, which converts glycine to sarcosine (N-methyl-glycine) by methyl group transfer using SAM and functions as a regulator of SAM levels in metabolic organs, suppresses age-dependent SAM increase and extends lifespan (Obata & Miura, 2015). Interestingly, this group also found that, in contrast to C. elegans where sams-1/C49F5.1 (methionine adenosyltransferase, catalyzes the biosynthesis of SAM, the first step in methionine metabolism) extended lifespan, knockdown of Sams in flies significantly shortened lifespan. Notably, there are four Sams genes in C. elegans but only one in Drosophila, such that suppression of Sams in Drosophila, but not in C. elegans, should completely block the methionine cycle. Interestingly, tissue-specific activation of methionine metabolism flux via downregulation of dAhcyL1/L2 in the brain or intestine extends healthspan and lifespan (Parkhitko et al., 2016). In accordance, Obata et al. (2018)

| H3K4 (activation histone mark)
Liu et al. tested the effects on histone methylation of three core enzymes involved in methionine metabolism, Sam-S, Ahcy13, and Cbs. They found that downregulation of Sams led to decreased levels of H3K4me3 and H3K9me2 in Drosophila S2 cells (Liu, Barnes, & Pile, 2015). Consistent with this, we found that activation of flux in methionine metabolism via downregulation of dAhcyL1 significantly suppressed the level of H3K4me3 (Parkhitko et al., 2016). The corepressor SIN3, which controls histone acetylation through association with the histone deacetylase RPD3, binds to the promoter regions of genes involved in methionine metabolism and regulates levels of SAM and H3K4me3 (Liu & Pile, 2017). Interestingly, Liu and Pile (2017) showed that SIN3 regulates crosstalk between histone acetylation and histone methylation via regulation of methionine metabolism.

| H3K27 (repressive histone mark)
The levels of H3K27  (Psc and Su(z)2) promote lifespan. Moreover, they showed that PRCs-deficiency promotes glycolysis and increase in glycolytic genes in wild-type animals extends longevity (Ma et al., 2018).
As mentioned above, suppression of H3K27me3 demethylase (UTX-1) in worms also extends lifespan (different from what is observed for flies); however, overexpression of H3K27me3 demethylase JMJD-3.1 extended lifespan, suggesting that the genes regulated by H3K27me3 are organism-and tissue-specific. Also in contrast to C. elegans, in which suppression of RBR-2 results in elevated lifespan ), Li, Greer, Eisenman, and Secombe (2010 showed that male flies mutant for little imaginal disk (lid), which encodes the Drosophila H3K4me3 demethylase, has a significantly shorter lifespan, and an effect that was not observed in females.

| H3K9 (repressive histone mark)
Whereas euchromatic regions (transcriptionally active) are charac- formation depends on the levels of HP1 and H3K9 methylation (Larson et al., 2012). A significant decrease in both H3K4me3 and H3K36me3 was observed in older flies (Wood et al., 2010). that age-associated increase in specific Drosophila miRNAs isoforms reflected increased 2′-O-methylation and loading of these isoforms into Ago2. They also found that the lack of 29-O-methylation by Hen1 and Ago2 mutations resulted in reduced lifespan and brain degeneration (Abe et al., 2014). Similar to yeast and worms, ubiquitous downregulation of RCM1/dNsun5 in male flies extended the lifespan of flies by 16%-20%, but the effect was abrogated on the diet that was richer in sugar and yeast. Overexpression of dNsun5 reduced mean lifespan by 58% (Schosserer et al., 2015). Lin, Tang, Reddy, and Shen (2005) demonstrated that overexpression of dDnmt2, a methyltransferase, can extend the Drosophila lifespan. Drosophila is thought to have no or low levels of genomic 5-methylcytosine.

| Nonhistone methylation
The human homolog of dDnmt2, TRDMT1/DNMT2, does not display DNA methyltransferase activity but can methylate aspartic acid tRNA (Goll et al., 2006). GSH helps maintain cellular redox homeostasis, acts as a xenobiotic conjugant, facilitating export of xenobiotics from cells, participates in thiolation and dethiolation of proteins, and has additional roles.
Glutamate-cysteine ligase (GCL) is a heterodimeric enzyme consisting of a catalytic subunit, GCLc, and a modulatory subunit, GCLm. Orr et al. (2005) showed that the overexpression of GCLc or GCLm in flies using either global or neuronal drivers of expression led to an increase in the glutathione content observed in fly homogenates and extended lifespan. In agreement with these findings, feeding flies with a cysteine donor for GSH, N-acetylcysteine (NAC), results in a dose-dependent increase in lifespan (Brack, Bechter-Thuring, & Labuhn, 1997).

| Polyamine metabolism
Similar to yeast and worms, supplementation in Drosophila of regular food with 1 mM spermidine was shown to prolong lifespan by up to 30% (Eisenberg et al., 2009).

| Methionine cycle
It was first shown in rats that MetR leads to an increase in lifespan.
The lifelong reduction of a single dietary component, methionine, from 0.86% to 0.17% in Fisher 344 rats resulted in 30% increase in male rat lifespan (Orentreich et al., 1993). In female CB6F1 mice, decrease in methionine from 0.43% by weight to 0.1%-0.15% increased lifespan, slowed immune and lens aging, and decreased levels of serum IGF-I, insulin, glucose, and thyroid hormone, despite an increase in food uptake (Miller et al., 2005). In mice, the ideal range of dietary methionine restriction is from 0.17% to 0.25% (Forney, Wanders, Stone, Pierse, & Gettys, 2017). Restriction of dietary methionine to levels above 0.25% was without effect while restriction to levels below 0.12% produced responses characteristic of essential amino acid deprivation. Although restriction of dietary methionine to 0.12% does not evoke essential amino acid deprivation responses, it provides insufficient methionine to support growth (Forney et al., 2017). In human diploid fibroblasts, reduction of methionine from 30 to 1 mg/L had no significant effect on the rate of cell proliferation in early passage cells but significantly extended their replicative lifespan, postponing cellular senescence. Extended lifespan was associated with reduced oxygen consumption (Koziel et al., 2014). In humans, an 83% reduction in daily methionine uptake for 3 weeks reduced plasma methionine levels and altered circulating metabolism with the most effect on cysteine and methionine metabolism . In addition, in humans, vegan diet is associated with decreased methionine content (McCarty, Barroso-Aranda, & Contreras, 2009). In mice, gene expression profiles of MetR and CR mice do not significantly overlap, suggesting that these two dietary regimens affect longevity through partly independent pathways (Sun, Sadighi Akha, Miller, & Harper, 2009). MetR restores a younger metabolic phenotype in adult mice: MetR in 12-month-old mice (0.172% vs. 0.86% methionine) reversed age-induced alterations in body weight, adiposity, physical activity, and glucose tolerance to levels observed in healthy 2-month-old control-fed mice.
MetR also causes lipid metabolism reprogramming in mice. Shortterm (48 hr) MetR increases hepatic fibroblast growth factor-21 (FGF21) expression/secretion (Lees et al., 2014). In young animals, MetR stunts growth and development, reducing total length, levels of serum insulin-like growth factor 1 (IGF-1), and growth hormone signaling activity. FGF21 is an atypical FGF that is secreted by the liver during fasting and elicits diverse aspects of the adaptive star-  (Zhang et al., 2012). The multiple physiological outcomes of MetR were recently reviewed and will not be further discussed here (Ables, Hens, & Nichenametla, 2016;Cavuoto & Fenech, 2012;Lee, Kaya, & Gladyshev, 2016;McIsaac, Lewis, Gibney, & Buffenstein, 2016;Sanchez-Roman & Barja, 2013;Zhou et al., 2016). Methionine metabolism is also altered in the tissues of long-lived Ames mice (Uthus & Brown-Borg, 2006) and naked mole-rats (Ma et al., 2015); however, whether these changes are causative or correlative is not known. Interestingly, Gu et al. recently demonstrated that SAM disrupts the SAMTOR-GATOR1 complex by binding directly to SAMTOR. GATOR1, the GTPase activating protein for RagA/B, promotes the localization of mTORC1 to the lysosomal surface, its site of activation. MetR reduces SAM levels and promotes the association of SAMTOR with GATOR1, thereby inhibiting mTORC1 signaling in a SAMTOR-dependent fashion (Gu et al., 2017). Because mTORC1 is a central regulator of aging (Parkhitko, Favorova, Khabibullin, Anisimov, & Henske, 2014;, SAMTOR, a SAM sensor, might serve as a critical connection hub between methionine metabolism, mTORC1 signaling, and aging. Another important player in the methionine cycle and aging is homocysteine. In human plasma, total homocysteine is present in four forms: 1%-2% as the thiol form, homocysteine; 82%-83% combined in disulfide linkage with cysteines of proteins (mostly albumin); and the remaining 15%-16% as the free disulfides homocysteine and cysteine-homocysteine disulfide (all these fractions are called total homocysteine, tHCY). One reason homocysteine can be harmful is because homocysteine can be converted to thiolactone as a result of an error-editing function of some aminoacyl-tRNA synthetases.
Thiolactone is chemically reactive and acylates free amino groups in proteins. The amount of thiolactone formation is dependent on methionine flux (Jakubowski, Zhang, Bardeguez, & Aviv, 2000

| DNA, histone, and nonhistone methylation
In general, aging is associated with DNA hypomethylation; however, some DNA regions become hypermethylated. In mammals, DNA methylation patterns change with age (Hannum et al., 2013) and can serve as a marker for chronological age (Horvath, 2013), but it is not known whether these changes are causative or correlative. As mentioned above, methionine metabolism is significantly altered in the tissues of long-lived Ames mice (Uthus & Brown-Borg, 2006 (Zhou et al., 2015). Mitochondrial dysfunction, which is commonly observed with aging, can also alter DNA methylation patterns via effects on methionine metabolism; namely, leading to an increase in the SAM/SAH ratio and an increase in DNA methylation (Lozoya et al., 2018).
In contrast to yeast, worms, and flies, for mice, information about specific methyltransferases capable of extending lifespan is limited.
As discussed earlier, flies expressing PCMT have an extended lifespan (Chavous et al., 2001), whereas Pcmt1−/− mice die at a mean age of 42 days from seizures (Lowenson, Kim, Young, & Clarke, 2001). Depleting the H3K9me3 methyltransferase Suv39h1 in a progeria mouse model reduces H3K9me3 levels, delays senescence in progeroid cells, and extends lifespan, but whether perturbation of Suv39h1 can extend lifespan in healthy mice is not known . Mice lacking functional de novo DNA methyltransferase Dnmt3a are born healthy but degenerate in adulthood and die prematurely (Nguyen, Meletis, Fu, Jhaveri, & Jaenisch, 2007). Mice deficient for NRMT1, N-terminal methyltransferase, which regulates protein-DNA interactions, exhibit phenotypes associated with impaired DNA repair and premature aging (Bonsignore et al., 2015).

| Transsulfuration pathway
Similar to worms and flies, a heterogeneous stock of mice (NIA Interventions Testing Program) fed with N-acetyl-l-cysteine (NAC) had a significantly extended lifespan. Notably, the effect was sex-specific.
In females, NAC treatment did not significantly affect total lifespan.
In males, both high (1,200 mg kg −1 day −1 ) and low (600 mg kg −1 day −1 ) NAC doses increased total lifespan. However, both doses of NAC caused a sudden drop in body weight, followed by a further slow decline (Flurkey, Astle, & Harrison, 2010). A characteristic of aging and a major cause of mortality and morbidity is the age-dependent decline in capillary density and blood flow. Recently, two groups showed that H 2 S, a product of enzymes with roles in transsulfuration pathway, promotes angiogenesis Longchamp et al., 2018).
Longchamp et al. demonstrated that MetR increased VEGF expression in vitro and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway.
This effect was independent of hypoxia or HIF1α. Cystathionine-γlyase was required for VEGF-dependent angiogenesis via increased production of H 2 S that mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport chain activity  NaHS, more potently increased capillary density and reduced the number of apoptotic ECs in vivo and physical endurance of mice than either treatment alone . Interestingly, Tyshkovskiy et al. (2019) found that cystathionine-γ-lyase was one of the most significant commonly upregulated genes among 15 known lifespanextending interventions in mice.

| Polyamine metabolism
Similar to yeast, worms, and flies, in C57BL/6J female mice, supplementation with spermidine and spermine (but not putrescine) significantly extends median lifespan. Spermidine also significantly extends lifespan when supplementation is started late in life (starting at 18-month-old mice). Spermidine supplementation has cardioprotective effects, resulting in reduced cardiac hypertrophy and preserved diastolic function in old mice. These organ-level findings are associated with enhanced cardiac cell autophagy, mitophagy, and mitochondrial respiration and spermidine fails to provide cardioprotection in mice that lack Atg5, which is essential for autophagy in cardiomyocytes (Eisenberg et al., 2016).

| CON CLUS ION
Although the effect of MetR on lifespan extension was reported in 1993, the mechanisms at play are not completely understood. Our recent finding that long-lived flies display higher levels of methionine suggests that regulation of lifespan depends on flux in the methionine cycle and levels of SAH/SAM, rather than absolute methionine levels. Although it is clear that each of the three branches of methionine metabolism-the methionine cycle, the transsulfuration pathway, and polyamine biosynthesis-plays a significant role in lifespan extension, how these branches crosstalk during regulation of lifespan is unknown. Moreover, how activity in these different branches of methionine metabolism change with age in different tissues and organs remains to be elucidated. 13 C-metabolic flux analysis (MFA) is a useful approach to determine cellular metabolic flux (Jin et al., 2004;Sauer, 2006). With this method, cells/organisms are fed isotope-labeled nutrients, the labeling patterns of intracellular metabolites are measured, and computational methods are used to estimate flux (Zamboni, 2011). 13 C-metabolic flux methods to comprehensively quantify metabolic flux through the multiple methionine-related pathways have been developed for mammalian cells (Shlomi, Fan, Tang, Kruger, & Rabinowitz, 2014). To achieve the strongest lifespan extension via manipulations of methionine metabolism, it will be important to quantify how metabolic flux through the multiple methionine-related pathways changes with age in specific tissues and organs. Moreover, a full understanding of MetR is likely to require the identification of specific methyltransferases affected by changes in SAM and SAH. Recently, the set of CpG sites, the so-called "epigenetic clocks," has been used to predict chronological and biological age (Horvath, 2013); it will be interesting to ask if a specific signature of CpG sites predicts the MetR response. Furthermore, it will be of interest to explore whether noninvasive methods similar to Met-PET can be used to evaluate the activity of methionine metabolism in humans and to predict the potential benefits of MetR for lifespan extension. Finally, given the high therapeutic potential, it will be interesting to search for drugs that can mimic the MetR response and extend lifespan via reprogramming of methionine metabolism.

ACK N OWLED G M ENTS
We is an investigator of the Howard Hughes Medical Institute.

CO N FLI C T O F I NTE R E S T
None declared.