A hallmark of aging is a decline in metabolic homeostasis, which is attenuated by dietary restriction (DR). However, the interaction of aging and DR with the metabolome is not well understood. We report that DR is a stronger modulator of the rat metabolome than age in plasma and tissues. A comparative metabolomic screen in rodents and humans identified circulating sarcosine as being similarly reduced with aging and increased by DR, while sarcosine is also elevated in long-lived Ames dwarf mice. Pathway analysis in aged sarcosine-replete rats identify this biogenic amine as an integral node in the metabolome network. Finally, we show that sarcosine can activate autophagy in cultured cells and enhances autophagic flux in vivo, suggesting a potential role in autophagy induction by DR. Thus, these data identify circulating sarcosine as a biomarker of aging and DR in mammalians and may contribute to age-related alterations in the metabolome and in proteostasis.

When screening for metabolites with similar responses between species, we identified circulating sarcosine, a biogenic amine involved in methionine (Met), glycine, and folate metabolism, as decreased with aging per se in rodents and humans and increased by DR in both species. These shifts correlated with changes in rat liver glycine-N-methyltransferase (GNMT) content, which is a known sarcosine-generating enzyme. Long-lived Ames dwarf mice demonstrate significantly elevated sarcosine levels across age, while correlation analysis of metabolites following sarcosine refeeding in old rats prominently places this metabolite as an integral node linking amines, amino acids, glycerophospholipids, and sphingolipids. We also show that sarcosine feeding reduces Met levels in old animals and is a strong activator of macroautophagy in vitro and in vivo. Taken together, these data identify sarcosine as a potentially important biomarker of diet and aging in mammals and suggest that this metabolite plays a previously unappreciated role in mediating at least some of the beneficial effects attributed to DR on proteostasis.

Here, we have characterized changes in the metabolome with aging and DR using established techniques in plasma and multiple tissues from a well-characterized hybrid rat model of aging. We have also interrogated the metabolome for shared changes in a set of human samples obtained from a cohort of younger and older subjects consuming a Western or DR diet (). We report on some unique shifts in the metabolome, including alterations in glycerophospholipids, biogenic amines, and amino acids with diet and age. In addition, statistical analyses revealed that DR is a stronger driver of the circulating and tissue rat metabolomic phenotype than age.

The metabolomic profile of aging has also been assessed in a number of other species, including Drosophila melanogaster (), naked mole rat (), marmoset (), and humans (). Tissue-specific metabolomic signatures were reported to correlate with body mass and lifespan across a diverse number of species, and some tissue metabolites were found that discriminated long-lived rodents from controls (). Although data on metabolomic shifts with aging and diet are rapidly accumulating, replication across studies has been limited, which has slowed progress toward ascertaining consensus hallmark candidates and signatures that define the aging metabolome across sex, strain, and species. Furthermore, to what extent these metabolomic shifts are merely a consequence of aging per se, as opposed to playing a causal role in the aging process, has been difficult to discern from what has largely been observational data.

A longitudinal analysis of the effects of age on the blood plasma metabolome in the common marmoset, Callithrix jacchus.

An early metabolomic study using 4- and 22-month-old male C57BL/6 mice reported that aging was characterized by altered glucose and fatty acid metabolism, including a lower respiratory exchange ratio (RER), increased plasma fatty acids, and reductions in triglycerides (TGs), various amino acids, and acylcarnitines (). In another study using older female C57BL/6 mice, it was reported that DR opposed a significant number of identified age-related changes in lipid metabolism, fatty acid metabolism, and bile acid biosynthesis (), suggesting that at least some age-related shifts in the metabolome can be prevented.

An overall decline in metabolic homeostasis is a hallmark of aging across mammalian species (). We and others have shown that visceral and ectopic fat accrual, subcutaneous fat depletion, hyperlipidemia, and insulin resistance are characteristic age-related changes in rodents and humans (). In spite of these well-recognized gross metabolic manifestations, our understanding of how shifts in specific metabolites and pathways are affected by aging, as well as by age-delaying strategies such as dietary restriction (DR), has only recently begun to be elucidated.

Finally, we determined whether sarcosine can activate in vivo autophagy in the liver of old rats fed a standard chow diet or a sarcosine-supplemented diet for 10 days. Sarcosine-treated animals displayed lower levels of the autophagic cargo p62 and showed increased autophagic flux (measured as both clearance of LC3-II and p62) in the liver ( Figure 7 A). Furthermore, electron microscopy and morphometric analysis revealed similar numbers of autophagic vacuoles in the livers from both groups of rats but a significantly higher abundance of autolysosomes and reduced autophagosome content in sarcosine-supplemented animals ( Figures 7 B and S7 E). In fact, autophagosomes containing high-density lipofuscin pigment (indicative of poor degradation) were highly abundant in the control group but rarely observed in the sarcosine-fed animals ( Figure S7 E), while the percentage of vacuoles that matured into autolysosomes increased from <30% to >70% after only 10 days of sarcosine supplementation ( Figure 7 B). Thus, induction of macroautophagy by sarcosine also occurs in vivo and does not seem restricted to a specific cell type. Supporting that the beneficial effect of sarcosine treatment was coming to a large extent through serum, incubation of 3T3 cells with heat-inactivated serum from old sarcosine-treated rats led to less inhibition of autophagy, as compared to old control rat serum ( Figure 7 C). Moreover, while sarcosine levels per se do not appear to demonstrate a discernible diurnal pattern, the ability of sarcosine to activate autophagy was highly dependent on time of day, because less inhibitory effects were limited to samples obtained at 9 a.m. and not at later time points taken throughout the day ( Figure S7 F). Furthermore, sarcosine treatment tended to activate a similar signaling network in liver, as observed in 3T3 cells, evidenced by a strong induction in pS6 and numerical increase in phospho-AMPK (pAMPK) ( Figures 7 D and 7E).

All results were obtained from a minimum of 3 independent experiments unless otherwise stated. Lines and bars indicate means ± SEMs. Significantly different from controls: ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

(D and E) Evaluation of signaling pathway activation in old rat liver following a short-term (10 days) dietary sarcosine treatment, as demonstrated by representative western blots (D) and the corresponding densitometry measurements (E), revealed that sarcosine increased pS6 in vivo, while a tendency toward increased activation of Akt and AMPK was observed (n = 8 per group).

(C) Effect of old control and sarcosine-treated rat serum on autophagy. Heat-inactivated serum collected from old control or sarcosine-treated rats at 9 a.m. was added to the culture media of NIH 3T3 cells stably expressing the autophagy reporter mCherry-GFP-LC3. Cells were imaged, and the number per cell of AVs (mCherry + vesicles), APGs (mCherry + and GFP + vesicles), and AUTs (ALs, mCherry + GFP − vesicles) were quantified using high-content microscopy (n > 2,500 cells). Differences with controls (supplemented with serum from untreated rats) were significant at most serum concentrations tested.

(B) Effect of a short-term (10 days) dietary sarcosine treatment on the autophagic compartments in aged rat liver. Low-magnification images (left) and examples of the autophagic compartments more abundant in each of the groups (APGs in untreated and autolysosomes [AUTs] in sarcosine treated). Red arrows indicate AUTs, and yellow arrows indicate APGs. Quantification of the number of AVs, APGs, AUTs and lysosomes (LYSs) per section (left) and the percentage of AVs that display characteristics of APGs or AUTs (right) are shown. Data reveal improved maturation of APGs into AUTs after sarcosine treatment, which is indicative of increased autophagic flux (n = 20 sections from 3 different animals). More examples of each compartment are shown in Figure S7 E.

(A) Autophagic flux increases in rat liver after short-term (10 days) dietary sarcosine feeding. Treatment with the inhibitors of lysosomal proteolysis (20 mM ammonium chloride and 100 μm leupeptin; N/L) revealed that degradation of LC3 and p62 in lysosomes was accelerated in the sarcosine-treated group. Representative immunoblot (left) and quantification of the levels and flux of LC3 and p62 (right) are both shown. n = 3 per group.

Although identification of the mechanism by which sarcosine stimulates autophagy is beyond the scope of the present study, considering the close connections between nutrition and autophagy and the key role that the mechanistic target of rapamycin (mTOR) plays in both processes, we next analyzed the status of mTOR signaling ( Figures 6 I and 6J). In treated cells, we found a trend toward increased mTOR phosphorylation and TOR complex 1 (TORC1) signaling (using pS6 as a readout), without noticeable changes in TORC2 (pAktSer473 levels). TORC1 is a well-established macroautophagy inhibitor during nutrient sufficiency, in part through direct phosphorylation of the autophagy effector Ulk1 at Ser 757, thus preventing its interaction with the activating kinase 5′ adenosine monophosphate-activated protein kinase (AMPK) (). Despite higher TORC1 activity, sarcosine-treated cells displayed significantly higher phosphorylation of Ulk at Ser 555, one of the AMPK sites ( Figures 6 I and 6J). These results suggest that sarcosine is able to stimulate macroautophagy over a background of enhanced mTOR signaling, likely by potentiating the stimulatory effect of AMPK on macroautophagy.

To determine whether elevated sarcosine may serve as more than simply a biomarker of aging and DR, we next investigated whether sarcosine may have direct effects on cellular processes implicated in aging. Given the tight connection between metabolism and proteostasis, including the reported inhibitory effect of SAM levels on autophagy (), we decided to determine whether sarcosine could serve to activate autophagy. We first used NIH 3T3 mouse fibroblasts and two different fluorescent reporters to quantify the activity of the two types of mammalian autophagy known to decline with age—macroautophagy (mcherry-GFP-LC3) and chaperone-mediated autophagy (CMA, KFERQ-PS-Dendra2) (). The mCherry-GFP-LC3 reporter is used to label autophagosomes. As they fuse with lysosomes, GFP fluorescence is quenched due to the low lysosomal pH resulting in mCherry-only labeled autolysosomes. Sarcosine stimulated basal macroautophagy in a dose-dependent manner and to a lesser extent macroautophagy induced by serum removal ( Figures 6 A and 6B ). A separate analysis of changes in the different vesicular compartments involved in autophagy revealed that sarcosine enhanced macroautophagy induction (quantified by autophagic vesicle number) as well as increased efficiency of lysosomal clearance of autophagosomes, as shown by an increased number of autolysosomes without a concomitant increase in autophagosomes ( Figures 6 C–6E). Analysis of autophagic flux by quantification of the degradation of the well-established autophagic cargo p62 also confirmed a dose-dependent increase in p62 clearance in cells treated with sarcosine ( Figure 6 F). The effect of sarcosine seems to be selective on macroautophagy rather than a general improvement of all lysosomal pathways, since analysis of a second form of autophagy, CMA, in the same cells did not reveal significant changes in basal CMA or CMA induced by serum deprivation or oxidative stress following increasing concentrations of sarcosine ( Figures S7 A–S7D). Induction of autophagy by sarcosine was more discrete than was observed by known inducers such as rapamycin and spermidine, but it was more effective than metformin ( Figure 6 G; p < 0.05). Sarcosine (500 μM) showed partially additive effects on induction of macroautophagy by serum removal and oxidative stress, suggesting alternative mechanisms of autophagic activation through sarcosine, compared to these stressors ( Figure 6 H). However, sarcosine had no additional effect upon macroautophagy activation through endoplasmic reticulum (ER) stress or lipid stimuli.

All results were obtained from a minimum of 3 independent experiments unless otherwise stated. Bars and lines indicate means ± SEMs (n = 3–4 per treatment). Significantly different from control: ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

(I and J) Representative immunoblots (I) and densitometry analysis (J) in 3T3 cells demonstrate that sarcosine activates the mTOR signaling pathway in cells, but that this occurs in concert with Ulk and LAMP1 activation, suggesting that AMPK activity is also increased, thereby permitting increased autophagy in spite of mTOR activation.

(H) Effect of the indicated treatments alone or in combination with 500 μM sarcosine on autophagic flux in cultured mouse fibroblasts. Several methods of autophagic induction were investigated, including oxidative damage (paraquat [PQ]), ER stress (thapsigargin [TG]) and lipotoxicity (oleic) in addition to serum-starved induction. Sarcosine showed an additive effect to serum starvation and paraquat, suggesting alternate mechanisms of activation, but not to thapsigargin or lipotoxicity (n > 2,500 cells).

(G) A comparative analysis of several well-known inducers of autophagy with sarcosine. Sarcosine is more effective than metformin at inducing autophagy but less effective than rapamycin and spermidine (n > 2,500 cells). Quantification was done using high-content microscopy. Differences with untreated (“none”) are indicated.

(F) Degradation of the autophagic cargo p62 in cells treated with increasing concentrations of sarcosine. Top: representative immunoblot. Bottom: quantification of the changes in p62 upon addition of lysosomal inhibitors ammonium chloride and leupeptin ammonium chloride and leupeptin (N/L) (n > 4 cells per condition).

(C–E) Number of autophagic vacuoles (AVs; C), autophagosomes (APGs; D) and autolysosomes (AUTs; E) in cells treated with sarcosine (500 μM) show increased induction (vesicle count) and efficient clearance (AUT/APGs) of APGs (n > 2,500 cells).

(B) Quantification of autophagic flux (number of autophagosomes matured into autolysosomes) at the indicated sarcosine concentrations shows a dose response to sarcosine (n > 2,500 cells).

(A) Mouse fibroblasts in culture (NIH 3T3 cells) expressing the tandem reporter mCherry-GFP-LC3 were exposed to the indicated concentrations of sarcosine for 16 hr in the absence or presence of other autophagy inducers. Representative images of both controls and cells treated with 500 μM sarcosine in the presence or absence of serum are shown.

To better understand to what extent sarcosine levels are dynamically regulated, including circadian effects and nutrient availability, we collected serial blood samples from an indwelling catheter at 4-hr intervals throughout the day in young rats. As can be observed in Figure S6 B, plasma sarcosine levels were relatively unchanged on average in a 12-hr period, although some variability was observed across time points in individual animals ( Figure S6 C). Furthermore, when samples were collected under fasted and refed conditions, respectively, there was no significant effect of nutrient status on sarcosine concentrations ( Figures S6 D and S6E). Thus, these data suggest that the ability of DR to maintain “youthful” sarcosine levels in old animals is likely adaptive rather than dynamically regulated by nutrient availability per se.

To more specifically examine the relation of sarcosine and age, as well as the impact of sarcosine replacement on one-carbon metabolism in old animals, we performed a second refeeding experiment in aged FBN rats for 4 weeks. Using a fluorescent-based sarcosine assay in young (4 mo; n = 8), middle-aged (12 mo; n = 8), old (25–26 mo; n = 10), and old sarcosine-supplemented male FBN rats (n = 10), we confirmed that plasma sarcosine was similar between young and middle-aged animals but significantly decreased at 25 mo, and this age-related reduction was restored by sarcosine feeding ( Figure 5 I; p < 0.05). We next performed targeted metabolomics in plasma from old control (n = 10) and old sarcosine-supplemented rats (n = 10) for intermediates involved in folate, Met, glycine, and sarcosine metabolism. We observed that sarcosine led to a significant reduction in plasma Met levels ( Figure 5 J; p = 0.04), but it did not alter the SAM:SAH ratio in plasma or liver ( Figure 5 K). Sarcosine feeding did tend to increase plasma glycine (p = 0.10) and N-acetyl glycine levels (p = 0.06; Figure S6 A), but no effects were observed on other related plasma metabolites, such as choline, serine, betaine, or dimethylglycine ( Figure S6 A).

Next, we aimed to determine whether restoring sarcosine to more youthful levels could alter the aging metabolome and recapitulate at least some of the DR effects on plasma metabolites. Old FBN rats were provided either regular chow (control) or a matched diet supplemented with sarcosine (1,250 mg/kg) for 8 weeks (n = 8 per group). A metabolomic analysis confirmed that plasma sarcosine was increased nearly 60% by the diet ( Figure 5 A; p < 0.05). However, no effect was observed on body weight, composition, food intake, and glucose or insulin levels in old sarcosine-replete rats ( Figures 5 B–5F). However, PCA revealed that the metabolome from old sarcosine-fed rats was distinct from controls ( Figure 5 G) and that global differences were well visualized by heat cluster map ( Figure S5 A), with a shift in a minority of metabolites, including Glu, Trp, Met-sulfoxide (Met-SO), and several glycerophospholipids, although these effects were no longer significant after adjusting for the FDR. Furthermore, using correlations between metabolites to construct a sarcosine network, a high “betweenness” score was identified for sarcosine in the linkage of amines, amino acids, and lipids ( Figures 5 H and S5 B). Glutamate was most strongly correlated with sarcosine (p < 0.001; Figures 5 H, S5 B, and S5C), while sarcosine was also significantly and positively correlated with the polyamines spermidine (p = 0.034) and spermine (p = 0.034) (FDR α = 0.12; Figures S5 B and S5C).

Box and whisker plots represent the lower and upper quartile ranges and highest and lowest observations, respectively, and heavy white lines indicate the median. Bars and lines represent means ± SEMs. Dot plots overlaid on boxes represent individual data points. Unless otherwise stated, brackets with asterisks indicate a significant difference between groups: ∗ p ≤ 0.05, ∗∗∗ p < 0.001.

(I–K) Examination of sarcosine levels across multiple ages confirms that levels do not decline until older age. Furthermore, 4 weeks of sarcosine feeding raised sarcosine levels in old animals (I), and correspondingly reduced Met levels (J), without effects on the plasma or liver SAM:SAH ratio (K) (n = 8–10 per group). Sarcosine, liver GNMT levels, and plasma SAM and SAH were measured by single detection, while GNMT activity and liver SAM and SAH were measured in duplicate.

(H) Representative simplified correlation map of sarcosine levels with other metabolites. See Figure S5 B for the corresponding detailed correlation map. Sarcosine is shown in green, and other metabolites/nodes are shown in blue. The size of the nodes is representative of the number of correlated metabolites at a given node. Blue interconnecting lines indicate a positive correlation, and red lines indicate a negative correlation. Double lines indicate that a strong correlation exists between metabolites, as was found with glutamate (p < 0.001). Sarcosine was also positively related to spermine and spermidine (see Figure S5 C) and demonstrated a high “betweenness” score among metabolites.

(G) Despite no effect on gross phenotypic characteristics, PCA confirmed that 8 weeks of sarcosine supplementation in old rats (red) resulted in distinct clustering of metabolites, as compared to controls (blue), which can be further visualized by heat cluster map (see Figure S5 A).

We next examined the rat and human metabolome for metabolites demonstrating conserved changes with aging and diet. Among nearly 400 detected metabolites, only sarcosine was found to be similarly altered with aging and DR, as determined by the Biocrates AbsoluteIDQ p180 kit. Specifically, sarcosine was reduced in circulation with aging per se in rats (p = 0.0002; FDR α = 0.05) and humans (p = 0.0056; FDR α = 0.15), while DR per se increased levels in rats (p = 0.0001; FDR α = 0.05) and humans (p = 0.0059; FDR α = 0.08). Furthermore, DR prevented the age-related decline in rat plasma sarcosine, maintaining levels comparable to young animals (interaction p = 0.021) ( Figures 4 A and 4B ). We further observed that both young and old Ames dwarf mice had markedly elevated levels of sarcosine in serum ( Figure 4 C), a finding that is in agreement with the enhanced liver GNMT activity previously documented in these mice (). Metscape analysis of metabolite networks confirmed that sarcosine is integral to metabolite shifts with aging and DR in rats and humans ( Figures 4 D–4G). In liver, sarcosine levels were relatively unchanged with aging, but were reduced by DR ( Figure 4 H). Meanwhile, GNMT, which is the most abundant methyltransferase in liver and thought to be a major source of circulating sarcosine, via the conversion of S-adenosyl Met (SAM) and glycine to S-adenosyl homocysteine (SAH) and sarcosine, demonstrated reduced expression with aging. Meanwhile, GNMT content ( Figure 4 I; p < 0.05) and activity were increased by DR per se ( Figure 4 J; age p = 0.396, diet p = 0.021, interaction p = 0.147).

Box and whisker plots represent lower and upper quartile ranges and highest and lowest observations, respectively, and heavy black lines indicate the median. Dot plots overlaid on boxes represent individual data points. Metabolomic detection of sarcosine and liver GNMT levels were measured by single detection, and GNMT activity was measured in duplicate. Bars represent means ± SEMs, n = 8 per group. Brackets with asterisks indicate a significant difference between groups: ∗ p ≤ 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Different letters denote a significant difference between groups, p ≤ 0.05.

(H–J) Liver sarcosine is reduced with DR, but not with age (H). Meanwhile, liver GNMT expression decreases with age but is increased by DR (I), while GNMT activity was numerically decreased with aging and increased by DR (J) (age p = 0.276, diet p = 0.039, age × diet p = 0.232).

(D–G) Metscape analysis identifies sarcosine as similarly modulated within the metabolite network by aging (blue circle; downregulated) and DR (red circle; upregulated) in rats (D and E) and humans (F and G).

(A and B) Among all of the detected metabolites, only sarcosine levels were similarly reduced with aging and increased by DR in rats (A) and humans (B) (n = 8 per group for rats; n = 6–7 per group for humans).

Sarcosine Is Uniquely Modulated and Integral to the Aging and DR Effects on the Rat and the Human Metabolome

Figure 4 Sarcosine Is Uniquely Modulated and Integral to the Aging and DR Effects on the Rat and the Human Metabolome

In liver, the metabolites that make up PC2, which were almost entirely glycerophospholipids, revealed a strong effect of age on the metabolome, although a diet × age interaction was evident due to the high overlap of DR groups on the PCA plot, irrespective of age ( Figure S4 A). A small minority of amino acids (Cit, Thr, isoleucine [Ile], valine [Val], phenylalanine [Phe], and Pro) were affected by age per se in the liver, and these changes were largely unaffected by DR ( Figure S4 B; Table S5 ). Meanwhile, the effect of aging on other lipids was more complex, with glycerophospholipids either significantly increased or decreased, respectively, while nearly 60% of measured sphingolipids were increased with age. DR failed to counteract age-related changes in liver sphingolipids but was able to attenuate changes in several glycerophospholipids ( Figures S4 C and S4D; Table S5 ).

In rat gastrocnemius muscle (SkM), PC3 revealed a strong effect of age on the metabolome, particularly glycerophospholipids and lysine (Lys), Arg, carnosine, and C4, but DR attenuated much of the age effect on the metabolomic profile in this tissue ( Figure S3 A). A significant effect of age was observed for several amino acids analyzed, including Arg, asparagine (Asn), His, Lys, ornithine (Orn), Pro, and serine (Ser) (FDR α = 0.05), as were the biogenic amines carnosine (p = 0.001), kynurenine (p = 0.007), and sarcosine (p = 0.014) ( Figure S3 B; Table S4 ). Several glycerophospholipids were also perturbed in aged SkM, with some metabolites, including lysoPCaC16:0 (p = 0.001; FDR α = 0.05) and PCaaC34.3 (p = 0.014), generally increased with age. However, several longer, more unsaturated glycerophospholipids, including PCaaC36:5 (p = 0.005), PCaaC36:6 (p = 0.001), PCaaC38:6 (p = 1.82 × 10), and PCaaC40:6 (p = 3.7 × 10) were reduced with age. In contrast, age-related changes in some acylcarnitines were characterized by an increase in metabolites (C14, C16, C18:2), while several shorter acylcarnitines, including C3 (p = 0.028; FDR α = 0.2), C4 (p = 0.001), and C5 (p = 0.001), were decreased. Lifelong DR protected against some of the perturbations to amino acids, including Arg and Lys, and the biogenic amine carnosine ( Table S2 ). Similar to BAT, DR attenuated age-related changes in SkM for some glycerophospholipids altered by age, while the effect of DR closely resembled the effect of age on other metabolite levels, such as PCaaC38:0, PCaaC40:6, and PCaeC40:6 ( Figures S3 C and S3D).

We next assessed the metabolome in rat BAT. Similar to plasma, PC1 demonstrates a very strong effect of diet on the BAT metabolome, clearly separating the AL and DR groups. As with plasma, there is a smaller effect of age in BAT, although the effect of age is more clearly visualized in AL versus DR groups ( Figure S2 A). In contrast to plasma, robust reductions were noted for some amino acids (citrulline [Cit], Gly, Gln, and histidine [His]) and several glycerophospholipids, sphingolipids, and biogenic amines, all of which characterized the age-related metabolomic footprint in this tissue, although very few acylcarnitines were affected (p < 0.05; Figure S2 B; Table S3 ). DR robustly counteracted age-related changes in several BAT metabolites, particularly glycerophospholipids, and tended to preserve Cit and His levels (FDR α = 0.2), but not other amino acids. DR per se also resulted in marked effects on some metabolites, which resembled changes observed with age per se, including elevated levels of PCaaC34:1, PCaa36:3, and SM.C24:0 and lower levels of spermidine ( Figure S2 Table S3 ).

In humans, only a limited number of effects were congruent with those found in rodents ( Figure S1 Table S2 ). For instance, glutamic acid, which did not change with age in either species, was similarly reduced by DR in rodents (p = 0.0006; FDR α = 0.05) and humans (p = 0.0001; FDR α = 0.05). However, a number of other metabolites identified as changing in rodents, were either not altered in humans or presented with disparate results. While circulating glycerophospholipids were robustly and consistently elevated in OAL rat plasma, a more modest number of glycerophospholipids were affected by age in human serum (lysoPCaC17:0, lysoPCaC18:1, lysoPCaC18:2, lysoPCaC20:3, lysoPCaC20:4, lysoPCaC28:0, and lysoPCaC32:0), and these metabolites tended to be reduced rather than increased with age. Likewise, the acylcarnitine C14:1 was reduced with human aging (interaction p = 0.001; FDR α = 0.2) and tended to be maintained by DR, while C14:1 levels in rodents increased with aging and were reduced by DR. Discrepant effects were also observed with DR per se between species. The longer-chain glycerophospholipids (PCaeC42:1, PCaeC42:2, and PCaeC42:3) tended to be increased in humans engaged in long-term DR, but the same metabolites in rodents were increased with age rather than with DR.

We next determined to what extent the observed shifts in metabolites were an inevitable consequence of aging or whether these alterations were modifiable by lifelong DR. While comparing the slopes of age and diet on all of the metabolites did not suggest a strong effect of DR on age ( Figure 2 A), PCA clearly demonstrates that YDR and ODR metabolomic profiles cluster together, irrespective of age ( Figure 3 A). DR opposed the age-related increase in nearly half of the detected glycerophospholipids and sphingolipids (interaction FDR α = 0.05 for the majority of metabolites) and a minority of amino acids (glutamine [Gln] [interaction p = 0.009; FDR α = 0.05] and arginine [Arg] [p = 0.02]) and acylcarnitines (C14:1 [p = 0.0009] and C3 [p = 0.0014]) ( Figures 3 C and 3D). In addition, a small metabolite screen by gas chromatography-mass spectrometry (GC-MS) determined that a minority of other metabolites found to increase in rat plasma with aging, including ribose 5-phosphate (interaction p = 9.13 × 10; FDR α = 0.05), dehydroepiandrosterone (interaction p = 1.77 × 10), and glycerolphosphate (interaction p = 0.0018), were maintained at youthful levels by lifelong DR ( Table S1 ). However, DR did not significantly prevent age-related changes in 3-hydroxybutyric acid, pyrophosphate, or tetradecanoic acid.

Reductions in various amino acids, acylcarnitines, and other fatty acids were previously reported to define the aging mouse blood metabolome (); thus, we evaluated these metabolites in our experimental model. Principal-component analysis (PCA) in rat plasma illustrated that the metabolomic profiles for YAL and OAL are distinct. PC1, which separates YAL and OAL samples, is heavily influenced by changes in glycerophospholipids ( Figure 3 A). Indeed, we observed a generalized, significant increase in many glycerophospholipids; amino acids such as proline (Pro) (p = 0.007; false discovery rate [FDR] α = 0.05), threonine (Thr) (p = 1.35 × 10), glycine (Gly) (p = 0.028), and alanine (Ala) (p = 0.024); the acylcarnitine C14:1 (p < 0.001); and approximately half of the detected sphingolipids in rat plasma with aging per se ( Figure 3 B; Table S1 ). A similar increase in the sphingolipid SM.C16:1 (p = 0.0008), the acylcarnitine C16:1 (p = 0.05, but non-significant [NS] after FDR correction), and lysoPCaC16:0 (p = 0.039) were observed with age per se in humans, while lysoPCaC17:0 (p = 0.0016), lysoPCaC18:2 (p = 0.011; FDR α = 0.2), lysoPCaC20:3 (p = 0.0002; FDR α = 0.05), and lysoPCaC20:4 (p = 0.0014) were downregulated with age per se in human serum ( Table S2 ).

Box and whisker plots represent lower and upper quartile ranges and highest and lowest observations, respectively, and heavy black lines indicate the median. Dot plots overlaid on boxes represent individual data points. The asterisk indicates significantly different from other experimental groups, p ≤ 0.05.

(D) Representative sphingolipids in which an age × diet interaction was observed. Post hoc comparisons detected an age-related increase in the levels of these metabolites, but DR only opposed the increase in SM.C16:0 and SM.C16:1, while DR led to a similar increase in SM.C18:1 levels as age (n = 8 per group). Samples were measured by single detection, and no coefficient of variation (CV) was calculated for this dataset because a low number of technical replicates (2) were included in the run. See Table S7 for CVs of the same individual metabolites with the Biocrates assay from 6 technical replicates in a separate run on FBN plasma samples.

(C) Representative glycerophospholipid metabolites in which a significant age × diet interaction was observed. Post hoc comparisons for these metabolites detected an increase in levels for OAL rats, which were completely attenuated by lifelong DR (n = 8 per group).

(A) PC1 metabolites reveal that while shifts in the plasma metabolome can be discriminated by age, metabolites cluster in similar quadrants with DR, regardless of age. These features of age and diet were largely driven by glycerophosholipids and to a lesser extent by amino acids and sphingolipids.

We next assessed changes at the level of the metabolome with aging and DR in rat plasma, brown adipose tissue (BAT), skeletal muscle (SkM), and liver as well as human serum. Specifically, we compared the slopes of age and diet on all metabolite levels, as determined by the β values obtained from Equation 1 (see Method Details ) to specifically test whether diet could attenuate the effect of age on each metabolite. While a few weak associations were observed, no strong relations were found for any of these analyses ( Figure 2 A). However, we observed that the majority of significant changes in individual metabolites were attributed to DR alone ( Figure 2 A, blue dots), with far fewer metabolites affected by only age ( Figure 2 A, red dots) or by both diet and age ( Figure 2 A, green dots). We also asked whether the sign and strength of the effect of diet or of age on each of the metabolites measured in one tissue was associated with the effect in other tissues. We found that the effect of diet on metabolites, as measured by β values, was highly correlated across tissues. In contrast, the effect of age on metabolite levels appears to be highly tissue specific ( Figure 2 B). We also found that the effect of diet on metabolite levels in human serum and rat plasma was significantly and positively correlated (p < 0.01).

(B) Correlation plots between β values of individual metabolites across sample type for plasma, serum, and tissue analytes, as measured via the Biocrates AbsoluteIDQ p180 kit. Between-tissue comparisons are given for diet (lower diagonal) and age (upper diagonal). Strong correlations were observed among rat plasma and tissues for the effects of diet but not age. Likewise, a significant correlation was observed in the response to DR between human serum and rat plasma (p < 0.01).

(A) x and y axes indicate the value of β for the effect of age and diet, respectively, on each metabolite, shown as individual points. The β values are taken from the linear model,(see Method Details ), with statistical significance (p ≤ 0.05) indicated by color. A negative correlation across metabolites suggests that the effect of DR on metabolite levels acts opposite to the effect of age on metabolite levels. Metabolites were detected via the Biocrates AbsoluteIDQ p180 and GC-MS small metabolite screen modules. The figures indicate a slight but significant negative correlation between age and diet effects for rat plasma and human serum (GC-MS screen), but not in rat tissues. Furthermore, we generally observed more significant effects on metabolite levels for DR alone (blue) versus age alone (red) or for both DR and age (green).

We first characterized the metabolic phenotype of young and old ad libitum (AL) and DR Fischer 344 × Brown Norway (FBN) male rats (abbreviated YAL, OAL, YDR, and ODR, respectively) ( Figure 1 ). OAL animals had increased body mass with age and weighed more than age-matched DR rats ( Figure 1 A; p < 0.05), along with a tendency toward greater amounts of lean body mass ( Figure 1 B; age p = 0.025, diet p < 0.001, interaction p = 0.098) and adiposity ( Figure 1 C; p < 0.05). By design, DR animals consumed 40% fewer calories than AL animals ( Figure 1 D; age p = 0.18, diet p < 0.001, interaction p = 0.18) and had a lower absolute energy expenditure (EE) ( Figure 1 E; age p = 0.082, diet p < 0.001, interaction p = 0.69), while 24 hr EE was maintained at youthful levels after adjusting for lean body mass (LBM) ( Figure 1 F; interaction p = 0.029). Substrate utilization in FBN rats was modulated over time by aging (age × time p = 0.019) and diet (diet × time p < 0.001), as demonstrated by disparate substrate utilization between AL and DR animals, particularly during the dark phase ( Figure 1 G, red box). In response to an overnight fast, YAL and OAL animals demonstrated a rapid and significant fall in RER (<0.7), suggesting a severe shift away from carbohydrate utilization and heavy reliance on fat and ketone metabolism, while DR animals were better able to handle this provocation, irrespective of age ( Figure 1 G, green box; p < 0.05). No difference in spontaneous activity ( Figure 1 H) or plasma glucose ( Figure 1 I) was observed, but OAL animals tended to be hyperinsulinemic ( Figure 1 J; age p < 0.001, diet p < 0.001, interaction p = 0.40) and had increased plasma TGs ( Figure 1 K; p < 0.05). Free fatty acid (FFA) levels were similar ( Figure 1 L). However, glycerol was increased in YDR as compared to YAL animals, while ODR animals demonstrated lower levels than controls ( Figure 1 M; p < 0.05).

(L and M) Plasma FFA levels did not vary among groups (L), but free glycerol was markedly elevated in YDR and OAL, respectively (M) (n = 8 per group). Glucose and insulin were measured in duplicate, and FFA, TG, and glycerol were assessed in triplicate. Bars and lines represent means ± SEMs. Different letters denote a significant difference between groups, p ≤ 0.05.

(I–K) Fasting glucose levels (I) were similar among groups, but OAL animals tended to have elevated insulin levels (J) and significantly increased TG levels (K), which were prevented by lifelong DR (n = 8 per group).

(G and H) Substrate utilization was measured during a 24-hr light and dark photoperiod, as well as in response to an overnight fast. As indicated in the red box, DR per se led to a lower RER, particularly during the dark photoperiod, a time in which DR mice are fasted, while RER did not vary with age in AL-fed groups (G). However, an overnight fast resulted in a more severe reduction in RER for AL animals than those on DR. Furthermore, no difference was observed in spontaneous activity among groups (H) (n = 8 per group).

(E and F) Energy expenditure tended to be reduced in young and old DR rats (E), but adjusting for LBM revealed a preservation in metabolic rate with DR (F) (n = 8 per group).

(A–D) Total body mass (A), but not LBM (B), were increased with aging, while fat mass was also increased with age (C), which was prevented by lifelong DR to ∼60% of AL intake (D) (n = 8 per group).

Discussion

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Wei I.H. Antidepressant-like effects of long-term sarcosine treatment in rats with or without chronic unpredictable stress. While sarcosine has been associated with various diseases and disorders, it is less clear whether sarcosine per se is a cause or simply a consequence in these instances. In support of the former possibility, sarcosine supplementation in humans has produced promising results as a primary therapy for Parkinson's disease with dementia () and as an adjuvant treatment for patients suffering from schizophrenia (). Likewise, beneficial effects of sarcosine have been found for learning and memory deficits () and depression in rats (). While sarcosine feeding in this study did not alter energy balance or endocrine status in aged rats, it did lead to discrete changes in the rat metabolome, including a reduction in plasma Met and a tendency toward boosting glycine pools. Sarcosine was also positively correlated with glutamate as well as spermidine and spermine, and SAM is involved in the biosynthesis of these polyamines. However, because no effect on the SAM:SAH ratio was detected, it is not entirely clear whether the reduction in Met by sarcosine feeding was in fact due to alterations in SAM metabolism or less homocysteine being recycled back to Met. However, the lack of changes in cysteine, betaine, and dimethylglycine levels does not support the latter possibility.

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et al. Methionine and S-adenosylmethionine levels are critical regulators of PP2A activity modulating lipophagy during steatosis. Sutter et al., 2013 Sutter B.M.

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Tu B.P. Methionine inhibits autophagy and promotes growth by inducing the SAM-responsive methylation of PP2A. In addition to our identification of sarcosine as a biomarker of aging and DR, our data also demonstrate that this metabolite can impinge on pathways that are critical to proteostasis and directly modulate macroautophagy in vitro and in vivo. Autophagy and proteostasis decline with aging in several tissues () and can be modulated by related metabolites such as Met and SAM (), which have been shown to inhibit autophagy (). We observed that sarcosine can directly stimulate basal macroautophagy in a dose-dependent manner in vitro and that this effect was recapitulated in vivo by sarcosine feeding, as demonstrated by an increase in autophagic flux and the clearance of autophagosomes and autophagic-like structures filled with lipofuscin-like dense material that accumulates in aged livers. Although a complete dissection of the mechanism behind sarcosine-induced autophagy requires further investigation, it was of interest that mTOR signaling and the AMPK pathway were both increased in response to sarcosine feeding. These findings suggest that sarcosine may be able to sustain enhanced autophagic flux in the context of mTOR activation by acting on AMPK, a well-known enhancer of autophagy. This activation of macroautophagy could contribute to improve proteostasis and increase resistance to stress, as previously described in DR. Ultimately, whether sarcosine supplementation can be harnessed to target some of the deleterious manifestations of aging through stimulation of autophagy is an intriguing possibility to explore in future studies.

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Pamplona R. Human aging is a metabolome-related matter of gender. Beyond sarcosine, another important observation was the overall increase in glycerophospholipids and sphingolipids in rat plasma with aging, although changes in glycerophospholipid levels among examined tissues were more complex. Studies have shown that increased incorporation of unsaturated phospholipids in the membrane lipid bilayer increases with age and that long-lived species have correspondingly lower levels of unsaturated fatty acids and a lower peroxidation index than their shorter-lived counterparts (). However, DR prevented the age-related increase of several glycerophospholipids and sphingolipids in rat plasma, and to a lesser extent, changes to glycerophospholipids in BAT, SkM, and liver, respectively. In addition, lower concentrations of sphingolipids have been linked to healthy aging, because high levels found in plasma have been associated with disease, senescence, and reduced physical function (). Consistent with this observation, our data show that several sphingolipids in plasma and tissue increased with aging in rodents and were substantially opposed by DR, although little effect of diet or age was observed in humans. Furthermore, conserved changes in metabolites with diet or age in humans were only detected for a minority of metabolites, while serum glycerophospholipids tended to decline rather than increase with age, as was the case in rodents. Thus, there is a clear heterogeneity that exists in the metabolomic response to diet and aging across species, strain, and, likely, sex that will need to be accounted for in future efforts ().

In summary, using a rat model of aging, we have shown that DR is a robust modulator of the metabolome in plasma and tissues and effectively opposes a significant number of age-related changes in amino acids, glycerophospholipids, acylcarnitines, and sphingolipids in plasma and tissue. We have identified circulating sarcosine as a metabolite that is similarly and uniquely modulated by aging and DR in rats and humans and is dramatically elevated in the serum of long-lived Ames dwarf mice. Furthermore, supplementation studies in old animals identified sarcosine as a critical node linking amino acid and lipid metabolism, while mechanistic studies demonstrate sarcosine as a potent stimulator of basal macroautophagy, implicating an interesting link among aging, growth factors, metabolism, and proteostasis. Thus, these data identify sarcosine, which sits at the nexus of folate, Met, and glycine metabolism, as a potential functional biomarker of the aging and DR metabolic phenotype.