Glycine promotes longevity in Caenorhabditis elegans in a methionine cycle-dependent fashion
[['Aging is characterized by a progressive deterioration of the functional capacity of tissues and organs .', 'Pioneering studies in the nematode C . elegans have identified longevity-associated genes and provided us with great insights into the plasticity of aging [1–3] .', 'In the last few decades , genetic and nutritional interventions have been employed in multiple organisms including Saccharomyces cerevisiae , C . elegans , Drosophila melanogaster , rodents , and more recently fish [4–6] .', 'These models have set the stage for characterizing the genetic basis of physiological aging and for developing efficient strategies to control the rate of aging .', 'To date , metabolic pathways including the mTOR , insulin/IGF-1 , and AMP-activated protein kinase ( AMPK ) signaling pathways have emerged as playing a critical role in aging [reviewed in [7]] .', 'Several studies demonstrate that the levels of specific amino acids effectively influence lifespan by affecting these pathways .', 'For example , the branched-chain amino acids valine , leucine , and isoleucine when administered to C . elegans can function as signaling metabolites that mediate a mTOR-dependent neuronal-endocrine signal that in turn promotes a longer lifespan [8] .', 'Moreover , inhibition of threonine and tryptophan degradation also contributes to lifespan extension by enhancing protein homeostasis in C . elegans [9 , 10] .', 'Additionally , restriction of methionine extends the lifespan of flies in a mTOR-dependent manner [11] .', 'However , supplementation of other amino acids such as methionine , serine , glycine , histidine , arginine , and lysine have been shown to promote lifespan in C . elegans by mechanisms that are to date not known [12] .', 'In addition to the mTOR signaling pathway , alterations in one-carbon metabolism involving the folate and methionine cycles couple amino acid metabolism to the regulation of human health and disease [13] .', 'Glycine , as one of the input amino acids that feeds into one-carbon metabolism , provides a single carbon unit to the folate cycle to yield a variety of one-carbon bound tetrahydrofolates ( THFs ) [14] .', 'These function as coenzymes in methylation reactions including the production of methionine through methionine synthase ( METR-1 in C . elegans ) as well as the universal methyl donor , S-adenosylmethionine ( SAMe ) through S-adenosyl methionine synthetase ( SAMS-1 in C . elegans ) [14] .', 'These output metabolites of one-carbon metabolism support a range of biological functions [14] .', 'In C . elegans , mutations in the metabolic gene sams-1 and the levels of SAMe and S-adenosylhomocysteine ( SAH ) have been implicated in the regulation of aging [15 , 16] .', 'Although the underlying mechanism of how SAMe/SAH status influences aging needs further investigation , studies in vivo have provided evidences that the level of SAMe couples with the trimethylation status of lysine 4 on histone H3 ( H3K4me3 ) and affects gene regulation [17] .', 'Another study in mouse pluripotent stem cells demonstrates that threonine catabolism contributes one carbon to SAMe synthesis and histone methylation through glycine cleavage pathway [18] .', 'Of particular note , several histone methyl-transferases and de-methyltransferases in C . elegans have been identified as longevity regulators [19–21] .', 'Taken together , these studies all suggest that altering one-carbon metabolism is a mechanism that controls the aging process .', 'We recently showed that glycine accumulates with age in a large scale metabolomics study profiling levels of fatty acids , amino acids , and phospholipids across the lifespan of C . elegans , [22] .', 'Another study in human fibroblasts suggested that epigenetic suppression of two nuclear-coded genes , glycine C-acetyltransferase ( GCAT ) and serine hydroxymethyltransferase 2 ( SHMT2 ) which are both involved in glycine synthesis in mitochondria , was partly responsible for aging-associated mitochondrial respiration defects [23] .', 'This study went on to report that glycine treatment rejuvenated the respiration capacity of fibroblasts derived from elderly individuals [23] .', 'However , to date the role of glycine has not been systematically defined in animal models of longevity .', 'In this study , we build upon our previous observations that suggest glycine accumulation in aging animals may play a unique and as-of-yet unexplored role in the regulation of eukaryote lifespan .'], ['We previously measured amino acid levels throughout the life of C . elegans , including four larval phases ( L1-L4 ) and ten days of adulthood from young worms to aged ones ( days 1–10 ) [22] .', 'We reported that the concentrations of most amino acids peaked at the later larval stage or early adult phase and then began declining at different adult stages , reaching low levels by the latest stages of the animals’ life [22] .', 'One stark exception was glycine , which continued to accumulate in aged worms [22] .', 'To determine if the accumulation of glycine with age is due to increased synthesis or reduced degradation , we measured the expression levels of genes directly involved in glycine metabolism in worms collected at different ages ( Fig 1A ) .', 'Specifically , we observed that the expression levels of most genes in glycine degradation and consumption pathways including glycine decarboxylase ( gldc-1 ) , glycine cleavage system H protein ( gcsh-1 ) , phosphoribosylamine-glycine ligase ( F38B6 . 4 ) , and D-amino acid oxidase ( daao-1 ) were dramatically lower at day 9 of adulthood ( D9 ) ( Fig 1B ) .', 'In contrast , the expression levels of most genes involved in glycine synthesis including threonine aldolase ( R102 . 4 ) , serine hydroxymethyltransferase ( mel-32 ) , alanine-glyoxylate aminotransferase 2 ( T09B4 . 8 ) , and alanine-glyoxylate amino transferase ( agxt-1 ) remained unchanged in aged worms ( Fig 1C ) .', 'These data suggest that the accumulation of glycine observed in aged worms is predominantly due to a reduction in the expression of genes required for its degradation .', 'To gain a better understanding of the significance of glycine accumulation in aged animals , we next asked whether this phenomenon is prevalent among long-lived worms such as daf-2 ( e1370 ) and eat-2 ( ad465 ) , the C . elegans models of impaired insulin signalling [2] and dietary restriction [24] , respectively .', 'To characterize the changes in the levels of glycine during aging in the daf-2 ( e1370 ) and eat-2 ( ad465 ) mutant lines , we measured glycine levels in long-lived worms at young and old stages , specifically L3 ( larval stage 3 ) and day 10 ( D10 ) of adulthood .', 'Interestingly , the levels of glycine in daf-2 ( e1370 ) and eat-2 ( ad465 ) at D10 were significantly increased relative to both their levels at L3 ( S1 Fig ) .', 'These results suggest that there might be a generic regulatory mechanism mediating the level of glycine during aging in both wild type and long-lived C . elegans .', 'To confirm the metabolic branch points of glycine metabolism in the control of glycine levels in C . elegans , we subjected worms to RNAi against the genes in glycine metabolism ( Fig 1A ) at the time of hatching , and then measured the level of glycine in D1 worms .', 'Worms treated with RNAi against the genes in glycine synthesis pathways showed no effect on the levels of glycine ( Fig 1D ) .', 'This is perhaps because glycine from bacteria may compensate for the reduction of glycine synthesis in worms .', 'Interestingly , a notable exception to this pattern was found for the knockdown of mel-32 , encoding a worm homologue of mammalian SHMT1 and SHMT2 , which acts to interconvert serine and glycine in one-carbon pathway ( Fig 1A ) [25] .', 'mel-32 RNAi led to a strong increase in the level of glycine ( Fig 1D ) and a concomitant slight decrease in the level of serine ( S2 Fig ) .', 'Thus , these results indicate that MEL-32 is prone to synthesize serine from glycine in C . elegans ( Fig 1D ) .', 'Likewise , RNAi of T25B9 . 1 also led to a subtle increase of glycine in worms , suggesting that T25B9 . 1 tends to degrade glycine in C . elegans ( Fig 1D ) .', 'RNAi knockdown of genes in glycine catabolic pathways including daao-1 , gss-1 , gcsh-1 , glycine cleavage system T-protein ( gcst-1 ) , and dihydrolipoamide dehydrogenase ( dld-1 ) , all significantly increased endogenous glycine compared to control ( Fig 1E ) .', 'Surprisingly , RNAi of phosphoribosylformylglycinamidine synthase ( pfas-1 ) , which is thought to block glycine being used in purine synthesis , was found to lower the level of glycine ( Fig 1E ) .', 'This implies that there are complex metabolic consequences from the perturbation of de novo purine synthesis .', 'Collectively , these data suggest that the majority of glycine in C . elegans is influenced by two metabolic branches , namely one-carbon metabolism via glycine cleavage complex and serine synthesis via MEL-32 .', 'We next verified the effects of glycine on lifespan by administering various concentrations of glycine to worms .', 'To avoid influences of glycine on bacterial metabolism and vice versa , we killed E . coli OP50 with a combination of ultraviolet ( UV ) -irradiation and antibiotic ( carbenicillin ) supplementation .', 'In line with a previously reported observation [26] , worms being fed UV- and carbenicillin-killed E . coli OP50 ( referred to hereafter as “killed bacteria” ) live significantly longer compared to those being fed live E . coli OP50 ( S3 Fig ) .', 'Therefore , to confirm if glycine still accumulates in aged worms after switching to a killed bacteria diet , we again quantified amino acids levels in worms at different stages of C . elegans lifespan including L3 ( larval stage 3 ) , day 1 ( D1 ) , day 3 ( D3 ) , day 6 ( D6 ) and day 9 ( D9 ) of adulthood .', 'We found that most amino acids remained unchanged from L3 to D9 , including valine , tryptophan , lysine , isoleucine , glutamine , asparagine , aspartate , arginine , serine , and proline ( S4A Fig ) .', 'Some amino acids change with age such as leucine , methionine , ornithine , and glutamate ( S4A Fig ) .', 'The levels of these either peak at the L3 stage or at D3 , then decrease with age ( S4A Fig ) .', 'Interestingly , the level of tyrosine peaked at both L3 and D9 , and the level of alanine peaked at D3 , then maintained stable from D3 to D9 ( S4A Fig ) .', 'Although the levels of some of these amino acids in worms fed killed bacteria were more stable with age compared to those in worms fed live OP50 [22] , we consistently found that the levels of leucine , methionine , ornithine , and glutamate decreased and the level of glycine increased in aged worms ( Fig 2A and S4A Fig ) .', 'The results suggest that the changes of these amino acids during aging are robust phenotypes that are independent of the worms being fed live or killed bacteria .', 'On killed bacteria we tested how a range of glycine concentrations from 5 μM to 10 mM affects the lifespan of C . elegans .', 'We observed a significant increase in median lifespan at concentrations of 5 μM , 50 μM , and 500 μM of dietary glycine as compared to untreated controls , with a 7 . 7% ( p < 0 . 0001 ) , 19 . 2% ( p < 0 . 0001 ) , and 19 . 2% ( p < 0 . 0001 ) extension observed , respectively ( Fig 2B ) .', 'Higher concentrations , however , including 5 mM and 10 mM , failed to extend worm lifespan suggesting a dose response where only low concentrations of glycine between 5–500 μM are beneficial to lifespan ( Fig 2B , S4B Fig ) .', 'Our findings are in agreement with previous studies suggesting a dose-effect of amino acids on worm lifespan [12] .', 'Next , we measured amino acid levels in C . elegans at D1 of the adult stage to investigate how glycine supplementations at different doses affect glycine levels in vivo .', 'We did not detect obvious changes of glycine abundance itself or of the other amino acids at any concentration of supplementations such as serine and threonine , the substrates for glycine synthesis ( S5A–S5E Fig ) .', 'These results suggest that in worms , glycine immediately fuels metabolic pathways , likely through glycine cleavage complex and MEL-32 ( Fig 1D and 1E ) .', 'Moreover , the results suggest that the levels of amino acids are tightly controlled in the early adult stage when glycine metabolic genes are actively expressed ( Fig 1B and 1C ) .', 'To test whether a specific timing is required for the beneficial effect of glycine on C . elegans lifespan , we administered glycine to worms at different times in the animal’s life .', 'These times included', '( a ) the development phase from the time of hatching to L4 ,', '( b ) the early adult period from D1 to D3 ,', '( c ) the adulthood starting from D1 until death , and', '( d ) during the entire life ( Fig 2C ) .', 'Intriguingly , the longevity-promotion function of glycine was observed to act exclusively during adulthood , especially the early adult phase , as supplementation from D1 to D3 was sufficient to prolong lifespan by 19 . 0% ( p < 0 . 0001 ) ( Fig 2D ) .', 'In contrast , glycine supplementation during development did not exert a beneficial effect on lifespan ( Fig 2D ) .', 'These data suggest that the first three days of adulthood are important for glycine to confer its longevity effects in C . elegans .', 'RNAi knockdown of mel-32 or the components in glycine cleavage complex ( e . g . gcst-1 ) resulted in pronounced increases of the endogenous levels of glycine in C . elegans ( Fig 1D and 1E ) .', 'As such , we wanted to determine whether such increase by blocking two distinct metabolic branches also impacts lifespan in C . elegans .', 'We hence assayed the lifespan of animals fed RNAi targeting mel-32 and gcst-1 from the time of hatching .', 'Intriguingly , we found that while RNAi of gcst-1 increased the endogenous level of glycine 6 . 9-fold ( Fig 1E ) , it failed to increase lifespan in C . elegans ( Fig 2E ) .', 'In contrast , mel-32 RNAi not only increased the endogenous level of glycine robustly ( 6 . 7-fold ) ( Fig 1D ) , but also improved the survival of wild-type N2 C . elegans significantly ( Fig 2F ) .', 'Thus , these results suggest that elevated endogenous or exogenous glycine levels drive lifespan extension , and that an intact glycine cleavage complex is likely required for this extension .', 'As glycine is an important one-carbon donor and an essential substrate for de novo purine synthesis , we next sought to understand how one-carbon and purine metabolism are influenced by aging .', 'We therefore quantified the expression levels of genes involved in one-carbon metabolism ( Fig 3A ) at five developmental distinct time points ( L3 , D1 , D3 , D6 , and D9 ) in the worm lifespan .', 'We found that with the exception of tyms-1 and dhfr-1 ( thymidylate synthetase and dihydrofolate reductase ) which were upregulated in aged worms , the expression of genes participating in transferring the one-carbon moiety of glycine to form SAMe including gcst-1 ( glycine cleavage system T-protein ) , dld-1 ( dihydrolipoamide dehydrogenase ) , gcsh-1 ( glycine cleavage system H-protein ) , mthf-1 ( methylene tetrahydrofolate reductase ) , metr-1 ( methionine synthase ) , and sams-1 ( S-adenosyl methionine synthetase ) , dropped dramatically in aged animals ( Fig 3B ) [27] .', 'Furthermore , the de novo purine synthesis genes atic-1 ( 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase ) ( Fig 3B ) and F38B6 . 4 ( Fig 1C ) were markedly reduced in aged worms compared to their expression levels in worms at D1 .', 'Together these data suggest a downregulation of both one-carbon and purine metabolism during aging in C . elegans .', 'To resolve in greater detail how glycine supplementation counteracts the age-related changes in one-carbon and purine metabolic genes , we performed next generation RNA-sequencing on D1 worms which were fed control diet ( UV-killed E . coli OP50 ) and 500 μM glycine-supplemented diet from the time of hatching , respectively .', 'In contrast to the changes occurring with age , glycine induced a marked increase in the expression of genes in purine metabolic pathways ( Fig 3C ) .', 'Concomitantly , several genes in one-carbon metabolism ( indicated in red in S6 Fig ) were differentially expressed , including upregulation of atic-1 , F38B6 . 4 , dhfr-1 , tyms-1 , mel-32 , and gcsh-1 , and downregulation of sams-1 , mthf-1 , and gldc-1 ( S6 Fig ) .', 'These results suggest that the expression of purine metabolism genes is subjected to an overall upregulation by exogenous supplementation of dietary glycine , while genes in one-carbon metabolism are under complex regulations upon glycine supplementation .', 'Having determined one of the effects of glycine on worm lifespan and its ability to partly counteract age-related declines in gene expressions in one-carbon and purine metabolic pathways , we next aimed to investigate whether similar gene regulatory events are also present in long-lived mutant worms .', 'To test this , we turned to microarray datasets from three long-lived worm models , daf-2 ( e1370 ) and eat-2 ( ad465 ) [28] or mrps-5 RNAi worms ( reported here ) .', 'We specifically looked at glycine-associated metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes ( KEGG ) database including ‘glycine serine and threonine metabolism’ , ‘one carbon pool by folate’ , ‘cysteine and methionine metabolism’ , and ‘purine metabolism’ .', 'Strikingly , although distinct longevity pathways are known to be active in these long-lived worms [2 , 29 , 30] , all these longevity worm models consistently showed a transcriptional activation of glycine metabolism , folate-dependent one-carbon metabolism , and methionine metabolism ( S7A–S7C Fig ) .', 'This suggests that transcriptional activation of glycine and one-carbon metabolism is a prominent signature of longevity shared by these long-lived worms .', 'In contrast , overall purine metabolism including de novo synthesis , the salvage pathways , and purine degradation was only mildly deactivated across the three long-lived strains ( S7A–S7C Fig ) .', 'To identify prominent transcriptional features in the three long-lived strains , we next examined the expression profile of individual genes belonging to “glycine , serine and threonine metabolism” , as well as folate-mediated one-carbon metabolism and purine metabolism .', 'We found that genes involved in glycine anabolism including T09B4 . 8 , agxt-1 , C15B12 . 1 , R102 . 4 , and T25B9 . 1 were upregulated in daf-2 ( e1370 ) , eat-2 ( ad465 ) [28] and mrps-5 RNAi worms ( S7D–S7F Fig ) .', 'Moreover , a concomitant rise in the expression levels of genes in glycine catabolism and de novo purine synthesis occurred , including gldc-1 , gcst-1 , gcsh-1 , F38B6 . 4 , and atic-1 ( S7D–S7I Fig ) , suggesting a stimulation of metabolic activity of glycine-associated processes in these long-lived worms .', 'To query the expression of genes in the production of SAMe in the long-lived worm models , we specifically checked the expression of five homologues of SAMe synthetases in C . elegans from the microarray data including sams-1 , sams-2 , sams-3 , sams-4 , and sams-5 ( S8 Fig ) .', 'Interestingly , the expression of sams-1 , which encodes the enzyme accounting for the majority of overall SAMe production in C . elegans [31] , was significantly upregulated in all these long-lived worms ( S8 Fig ) .', 'Moreover , sams-5 was increased in daf-2 ( e1370 ) and eat-2 ( ad465 ) [28] , while sams-2 , sams-3 , and sams-4 were suppressed in mrps-5 RNAi and daf-2 ( e1370 ) ( S8 Fig ) .', 'Collectively , the data point to elevated methylation activities in these long-lived worms .', 'In contrast to an overall upregulation of purine metabolism genes in response to dietary glycine treatment , all three longevity worm models specifically induced the expression of genes in de novo purine synthesis compared to control , including F38B6 . 4 , F10F2 . 2 ( pfas-1 , phosphoribosylformylglycinamidine synthase ) , B0286 . 3 ( pacs-1 , phosphoribosylaminoimidazole succinocarboxamide synthetase ) , and atic-1 ( S9A–S9C Fig ) .', 'Taken together , our data reveal that transcriptional activations of glycine and glycine-associated pathways , including one-carbon and de novo purine synthesis , are present in three distinct longevity models .', 'To understand the mechanism of glycine-mediated lifespan extension on a more global scale , we returned to our next-generation RNA-sequencing dataset and performed unsupervised Principle Component Analysis ( PCA ) on the individual libraries .', 'We found a clear separation between glycine-treated versus untreated samples ( Fig 4A ) , corresponding to a large difference in gene expression ( Fig 4B ) .', 'Interestingly , more genes were transcriptionally repressed in response to glycine treatment in which 2629 genes were differentially down-regulated , and 983 genes were up-regulated compared to control ( Fig 4B ) .', 'This suggests an inhibition propensity of glycine on gene expression .', 'To probe the processes changed upon glycine supplementation , we performed gene ontology ( GO ) term enrichment analysis on the differentially expressed genes using the Database for Annotation , Visualization and Integrated Discovery ( DAVID ) bioinformatics resource [32] .', 'A larger number of GO terms were found enriched in the downregulated gene list , among which were GO terms for body morphogenesis , growth regulation , post-embryonic development , molting cycle , cuticle development , multicellular organism growth , and positive regulation of growth rate ( Fig 4C ) .', 'These enrichments are all related to growth control , suggesting a decelerating effect of glycine upon development and growth which is a phenomenon known to be associated to longevity [33] .', 'Additionally , unlike some mutations that confer longevity to the soma at the cost of a reduction in fecundity [34] , supplementation of glycine mildly enhanced the expression of genes in reproduction-related biological processes such as the GO terms of vitellogenesis , meiosis cell cycle , and gamete generation ( Fig 4D ) .', 'While we did not observe clear differences in the number or the size of embryos , the progenies from glycine- and serine-supplemented worms seem to be healthy and normal .', 'Overall , with the observations of the lifespan extending effects of glycine , these data suggest that the beneficial role of glycine slows down pathways that are traditionally ameliorated in healthy aging models .', 'MEL-32 and glycine cleavage complex are major gatekeepers responsible for the flux of glycine into metabolic pathways ( Fig 1A ) .', 'Particularly , although suppression of mel-32 or the components in glycine cleavage complex in C . elegans dramatically elevated the level of endogenous glycine ( Fig 1D and 1E ) , only mel-32 RNAi showed a potent lifespan extension effect on worms likely by favoring the flux of glycine into one-carbon metabolism ( Fig 2E and 2F ) .', 'Additionally , by fueling the one-carbon metabolic network through glycine cleavage complex , glycine contributes to the synthesis of SAMe ( Fig 3A ) , the availability of which has been implicated in the regulation of histone methylation patterns and subsequently gene expressions [35] .', 'Collectively , this led us to hypothesize that the methionine cycle may be required for the lifespan-extending effect of glycine .', 'To test if the methionine cycle is necessary for the longevity effect of glycine , we performed lifespan analyses with the methionine cycle-deficient mutants metr-1 ( ok521 ) and sams-1 ( ok3033 ) .', 'In these mutants , 500 μM glycine failed to promote lifespan ( Fig 5A–5C ) , demonstrating that the effects of glycine on worm longevity depend on the methionine cycle .', 'Serine is another important one-carbon donor and the major precursor for glycine synthesis in vivo [14] .', 'Thus , serine and glycine are closely related to each other in one-carbon metabolism .', 'Given their similarities , we next investigated whether serine can also exert beneficial effects on worm lifespan .', 'Serine supplementation at a concentration from 1 mM to 10 mM has been shown previously to extend worm lifespan [12] .', 'We therefore administered 5 mM serine to worms and measured lifespan .', 'Similar to glycine , we confirmed that serine prolonged the lifespan of worms ( +20 . 8% in median lifespan ) ( Fig 6A ) .', 'Given that the adulthood is important for glycine-mediated lifespan extension , we investigated whether serine increases lifespan in the same fashion as glycine .', 'Similarly , we treated worms with serine at different times in worm’s lifespan including', '( a ) developmental stage ,', '( b ) the beginning of adulthood from D1 to D3 ,', '( c ) adulthood from D1 until death , and', '( d ) during the whole lifetime .', 'Similar to the effects of glycine on lifespan , serine treatment from D1 to D3 is sufficient to recapitulate the beneficial effects on lifespan as did the treatment throughout the entire life or during adulthood only ( Fig 6B ) .', 'In contrast , supplementation during the developmental stage failed to increase lifespan ( Fig 6B ) .', 'This further implies that serine acts on the same downstream longevity signalling pathways to influence aging as does glycine .', 'We further investigated whether the anti-aging effects of serine also rely on the methionine cycle .', 'In agreement with the results observed with glycine supplementation , the lifespan extending effect of serine was also ablated by mutations of metr-1 and sams-1 ( Fig 6C and 6D ) .', 'These results further suggest that serine and glycine prolong C . elegans lifespan via a similar mechanism .', 'To determine the common regulators in both glycine and serine-mediated longevity , we performed RNA-sequencing on serine-supplemented worms .', 'As expected , PCA analysis showed a clear separation between worms treated with either of the amino acids when compared to non-treated worms , and a strong similarity between glycine- and serine-treated worm groups ( Fig 6E ) .', 'Statistical analysis found one significantly differentially expressed gene , F38B6 . 4 ( S10A Fig ) , an enzyme that consumes glycine for purine synthesis .', 'In addition , visualizing the data as a volcano plot showed again a greater number of genes repressed by serine treatment ( 2865 downregulated genes vs 973 upregulated genes ) , in line with the same gene expression suppression pattern of glycine-supplemented worms ( S10B Fig , Fig 4B ) .', 'Likewise , we found a strong overlap between glycine- and serine-treated worms when looking at the up- and downregulated genes , as shown in the Venn diagrams where 82 . 8% ( 2335 ) of the downregulated and 72 . 3% ( 804 ) of the upregulated genes are shared ( Fig 6E–6G ) .', 'Taken together , we suggest a model whereby both glycine and serine supplementation stimulate longevity in a methionine cycle-dependent fashion and through common signaling pathways .', 'Moreover , this seems to be dependent on the expression of sams-1 and metr-1 .', 'This model is illustrated in Fig 6H .'], ['Our work sheds light onto the means by which the amino acid glycine can increase C . elegans lifespan when supplemented to the diet .', 'Using a metabolomics approach , we found that glycine steadily and significantly accumulates in aging C . elegans [22] .', 'Furthermore , we demonstrated that this accumulation is mainly coupled to a decrease in the expression levels of genes in glycine cleavage pathway which control the majority of glycine breakdown in C . elegans .', 'We found that mel-32 RNAi causes a marked rise in the endogenous level of glycine which in turn extends lifespan .', 'Moreover , supplementing dietary glycine extends lifespan at concentrations between 5–500 μM , while mutations in methionine synthase [metr-1 ( ok521 ) ] and S-adenosyl methionine synthetase [sams-1 ( ok3033 ) ] , two enzymes involved in methionine cycle , can fully abrogate this lifespan extension .', 'Furthermore , we found that serine , another amino acid that feeds into one-carbon metabolism , shows similar transcriptional changes , metr-1 and sams-1 dependency , and lifespan extension upon dietary supplementation as does glycine .', 'These results confirm an important role for the methionine cycle in the longevity effects of glycine .', 'Our work reveals a timing requirement of glycine supplementation in the promotion of longevity in C . elegans .', 'Specifically , the first three days of adulthood ( from D1 to D3 ) are crucial for glycine to confer the benefits on longevity .', 'Given that the DAF-2 pathway also acts exclusively during adulthood and throughout the reproductive period to affect lifespan in C . elegans [36] , further investigation is warranted to see if this classical longevity pathway is fully or only partially required for the beneficial effects of glycine .', 'Furthermore , the first three days of adulthood coincide with the reproductive period of worms , raising an interesting question for future studies about the crosstalk between the reproductive system and glycine-activated longevity pathways .', 'Our work identified a counterintuitive biological phenomenon , whereby glycine accumulation was observed during the aging process in worms while supplementation of glycine was nonetheless able to prolong worm lifespan .', 'However , it is not uncommon for changes that occur with age to also benefit lifespan when artificially induced .', 'For example , suppression of IGF1 signaling may extend lifespan in many model organisms [2 , 37 , 38] , while IGF1 levels themselves have been observed to decline with age [39 , 40] .', 'Moreover , methionine restriction is beneficial to lifespan in a variety of model organisms [11 , 41] , while methionine abundance in vivo has been observed to decline with age [observed in this study ( S4A Fig ) and [22]] .', 'Similar to these phenomena , glycine supplementation may activate protective cellular pathways that promote longevity when exogenously applied , while a natural glycine accumulation with age may reflect the organism’s need to upregulate these same cytoprotective pathways to deal with the damage and detrimental changes occurring during aging .', 'Studies in rodents have suggested glycine supplementation to have pro-longevity effects [42 , 43] , anti-inflammatory effects [44] , to be cytoprotective [45] , and to ameliorate metabolic disorders [46] .', 'In humans , glycine supplementation in patients with metabolic disorders has a protective effect against oxidative stress and inflammation [47–49] .', 'In line with these observations in mammalian systems , our data demonstrated that glycine promotes longevity in C . elegans .', 'Furthermore , we show this benefit to occur in a metr-1 and sams-1 dependent manner , implicating the methionine cycle in longevity regulation .', 'Finally , we show glycine supplementation induces widespread suppression of genes including many that are hallmarks of the aging process .', 'Taken together , our findings suggest that dietary glycine is an effective strategy to increase lifespan and warrant further investigation for life- and healthspan studies in humans .'], ['C . elegans strains N2 Bristol , RB2204 sams-1 ( ok3033 ) X , RB755 metr-1 ( ok521 ) II , eat-2 ( ad465 ) , and daf-2 ( e1370 ) were obtained from the Caenorhabditis Genetics Center ( CGC , University of Minnesota ) .', 'Nematodes were grown and maintained on Nematode growth media ( NGM ) agar plates at 20°C as previously described [50] .', 'E . coli OP50 and E . coli HT115 ( DE3 ) with the empty vector L4440 was obtained from the CGC .', 'Bacterial feeding RNAi experiments were carried out as described [51] .', 'RNAi E . coli feeding clones used were mrps-5 ( E02A10 . 1 ) , T09B4 . 8 , agxt-1 ( T14D7 . 1 ) , C15B12 . 1 , R102 . 4 , T25B9 . 1 , gss-1 ( M176 . 2 ) , gcsh-1 ( D1025 . 2 ) , gcst-1 ( F25B4 . 1 ) , pfas-1 ( F10F2 . 2 ) , and dld-1 ( LLC1 . 3 ) .', 'Clones of agxt-1 ( T14D7 . 1 ) , C15B12 . 1 , R102 . 4 , T25B9 . 1 , gss-1 ( M176 . 2 ) , gcsh-1 ( D1025 . 2 ) , gcst-1 ( F25B4 . 1 ) , and pfas-1 ( F10F2 . 2 ) were derived from the Ahringer RNAi library [52]; Clones of mrps-5 ( E02A10 . 1 ) , T09B4 . 8 , and dld-1 ( LLC1 . 3 ) were derived from the Vidal RNAi library [53]; Worms were fed RNAi bacteria from the time of hatching unless otherwise indicated .', 'Glycine and serine were purchased from Merck Millipore ( no . 8 . 1603 . 0250 ) and Sigma ( no . S4500 ) , respectively .', 'A stock of concent
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['The deregulation of metabolism is a hallmark of aging .', 'As such , changes in the expression of metabolic genes and the profiles of amino acid levels are features associated with aging animals .', 'We previously reported that the levels of most amino acids decline with age in Caenorhabditis elegans ( C . elegans ) .', 'Glycine , in contrast , substantially accumulates in aging C . elegans .', 'In this study we show that this is coupled to a decrease in gene expression of enzymes important for glycine catabolism .', 'We further show that supplementation of glycine significantly prolongs C . elegans lifespan , and early adulthood is important for its salutary effects .', 'Moreover , supplementation of glycine ameliorates specific transcriptional changes that are associated with aging .', 'Glycine feeds into the methionine cycle .', 'We find that mutations in components of this cycle , methionine synthase ( metr-1 ) and S-adenosylmethionine synthetase ( sams-1 ) , completely abrogate glycine-induced lifespan extension .', 'Strikingly , the beneficial effects of glycine supplementation are conserved when we supplement with serine , which also feeds into the methionine cycle .', 'RNA-sequencing reveals a similar transcriptional landscape in serine- and glycine-supplemented worms both demarked by widespread gene repression .', 'Taken together , these data uncover a novel role of glycine in the deceleration of aging through its function in the methionine cycle .']
['There is a growing number of studies showing that amino acids function as signal metabolites that influence aging and health .', 'Although contemporary -OMICs studies have uncovered various associations between metabolite levels and aging , in many cases the directionality of the relationships is unclear .', 'In a recent metabolomics study , we found that glycine accumulates in aged C . elegans while other amino acids decrease .', 'The present study shows that glycine supplementation increases lifespan and drives a genome-wide inhibition effect on C . elegans gene expression .', 'Glycine as a one-carbon donor fuels the methyl pool of one-carbon metabolism composed of the folate and methionine cycles .', 'We find that the glycine-mediated longevity effect is fully dependent on the methionine cycle , and that all of our observations are conserved with supplementation of the other one-carbon amino acid , serine .', 'These results provide a novel role for glycine as a promoter of longevity and bring new insight into the role of one-carbon amino acids in the regulation of aging that may ultimately be beneficial for humans .']
['invertebrates', 'rna interference', 'chemical compounds', 'aliphatic amino acids', 'caenorhabditis', 'organic compounds', 'animals', 'animal models', 'caenorhabditis elegans', 'model organisms', 'serine', 'experimental organism systems', 'amino acids', 'epigenetics', 'research and analysis methods', 'amino acid metabolism', 'glycine', 'purine metabolism', 'animal studies', 'proteins', 'genetic interference', 'gene expression', 'chemistry', 'methionine', 'sulfur containing amino acids', 'biochemistry', 'rna', 'eukaryota', 'organic chemistry', 'nucleic acids', 'genetics', 'hydroxyl amino acids', 'biology and life sciences', 'nematoda', 'physical sciences', 'metabolism', 'organisms']