PMC

(A) x and y axes indicate the value of b for the effect of age and diet, respectively, on each metabolite, shown as individual points. The β values are taken from the linear model, Ŷ_i=α+β₁age +β₂diet +ε (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).
(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 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).
(C) Sarcosine is significantly elevated in young and old long-lived Ames dwarf mice (n = 8 per group).
(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).
(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).
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.

(A) PC1 metabolites reveal that while shifts in the plasma metabolome can be discriminated by age, metabolites cluster in similar quadrants with DR, regardless ofage. These features of age and diet were largely driven by glycerophosholipids and to a lesser extent by amino acids and sphingolipids.
(B) Heatmap of all metabolites analyzed illustrating hierarchical clustering of plasma levels with age and diet.
(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).
(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 for CVs of the same individual metabolites with the Biocrates assay from 6 technical replicates in a separate run on FBN plasma samples.
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.

(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).
(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).
(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).
(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).
(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.

(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.
(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 .
(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.
(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).
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.

(A) Mouse fibroblasts in culture (NIH 3T3 cells) expressing the tandem reporter mCherry-GFP-LC3 were exposed to the indicated concentrations of sarcosine for16 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.
(B) Quantification of autophagic flux (number of autophagosomes matured into autolysosomes) at the indicated sarcosine concentrations shows a dose responseto sarcosine (n > 2,500 cells).
(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).
(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).
(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.
(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).
(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.
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.

(A) Sarcosine feeding in aged rats for 8 weeks was able to raise plasma sarcosine levels (n = 8 per group).
(B–F) Sarcosine-fed rats had no significant change in body weight (B), food intake (C), fat mass, lean mass (D), glucose (E), or insulin levels (F) (n = 8 per group). (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 ).
(H) Representative simplified correlation map of sarcosine levels with other metabolites. See 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 ) and demonstrated a high “betweenness” score among metabolites.
(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.
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.