Metabolomic signature associated with reproduction-regulated aging in Caenorhabditis elegans

Abstract

In Caenorhabditis elegans (C. elegans), ablation of germline stem cells (GSCs) leads to infertility, which extends lifespan. It has been reported that aging and reproduction are both inextricably associated with metabolism. However, few studies have investigated the roles of polar small molecules metabolism in regulating longevity by reproduction. In this work, we combined the nuclear magnetic resonance (NMR) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to profile the water-soluble metabolome in C. elegans. Comparing the metabolic fingerprint between two physiological ages among different mutants, our results demonstrate that aging is characterized by metabolome remodeling and metabolic decline. In addition, by analyzing the metabolic profiles of long-lived germline-less glp-1 mutants, we discovered that glp-1 mutants regulate the levels of many age-variant metabolites to attenuate aging, including elevated concentrations of the pyrimidine and purine metabolism intermediates and decreased concentrations of the citric acid cycle intermediates. Interestingly, by analyzing the metabolome of daf-16;glp-1 double mutants, our results revealed that some metabolic exchange contributing to germline-mediated longevity was mediated by transcription factor FOXO/DAF-16, including pyrimidine metabolism and the TCA cycle. Based on a comprehensive metabolic analysis, we provide novel insight into the relationship between longevity and metabolism regulated by germline signals in C. elegans.

Keywords: Caenorhabditis elegans; UPLC-MS and NMR; aging; metabolome; reproduction.

Figure 1. Age-related comprehensive metabolomics analysis in wide type C. elegans

(A) Scores and loading plots from OPLS-DA model of NMR data for YA (young adult) and 10A (10 days of adulthood) wild-type N2. The region of δ5.0-9.5 in the loading plot was vertically expanded 10 times. NMR metabolites assignment showed in the Table S1 (Supplemental information). (B) Metabolomics analysis from UPLC-MS data for YA and 10A wild-type N2. Heatmap plot showed that 25 most importantly different metabolites from the samples according to their aging status. More differences metabolites were listed in the Table S2 (Supplemental information). Data are presented using hierarchical clustering (Pearson's correlation coefficient). Metabolite abundance level were reflected in the heat-maps using colors, and with blue being lower and red higher when comparing the mean metabolite abundance value. Using the distance function 1-correlation in hierarchical clustering determine the order of metabolite and animal.

Figure 2. Age-related metabolic remodeling in C. elegans

(A) Summary plot for metabolite enrichment analysis(MSEA) (left panel), where metabolite sets were ranked according to Holm p-value, and the cut off of Holm p-value showed with hatched lines (the panel overviews metabolites repeated measured by Mann-Whitney U test, p<0.05). Metabolomics view (right panel) reflects key nodes in metabolic pathways that have been significantly altered with aging, and in which x-axis reflects the increasing metabolic pathway impact according to the between centrality measure. MSEA was performed using package global test and the metabolome view displayed the pathway topological analysis. (B) Model on the aging C. elegans response entails a complex series of metabolic change.

Figure 3. ¹H NMR-based metabolic profile analysis of the long-lived glp-1(e2141) mutants and the daf-16(mu86);glp-1(e2141) double mutants

Scores and loading plots from OPLS-DA model of NMR data for (A) YA and (B) 10A wild-type N2 and glp-1 (e2141), (C) YA and (D) 10A wild-type N2 and daf-16(mu86);glp-1(e2141) double mutants. Detailed information about differences metabolites were summarized in the Table S3 and S4 (supplemental information).

Figure 4. UPLC-MS-based metabolic profile analysis of the long-lived glp-1(e2141) mutants and the daf-16(mu86);glp-1(e2141) double mutants

Metabolomics analysis from UPLC-MS data for (A) YA and (B) 10A wild-type N2 and glp-1 (e2141), and for (C) YA and (D) 10A wild-type N2 and daf-16(mu86);glp-1(e2141) double mutants. Heatmap plot showed that 25 most importantly different metabolites from the comparison of the glp-1 mutants and N2. More information was listed in the Table S3 and S4 (supplemental information). The detailed description of heatmap is as discussed in Figure 1B.

Figure 5. Models of aging-related changes in TCA cycle and pyrimidine metabolism

(A) A summary of the biochemical pathway of TCA cycle metabolism alerted during aging, and glp-1 against WT. In summary, with aging, accumulation of the TCA cycle intermediates such as citrate and malate suggests increased TCA cycle metabolism. Furthermore, in the long-lived glp-1 mutants, the levels of TCA cycle intermediates decreased at stage of the young adults and 10-day adults compared with WT.

Figure 5. Models of aging-related changes in TCA cycle and pyrimidine metabolism

(A) A summary of the biochemical pathway of TCA cycle metabolism alerted during aging, and glp-1 against WT. In summary, with aging, accumulation of the TCA cycle intermediates such as citrate and malate suggests increased TCA cycle metabolism. Furthermore, in the long-lived glp-1 mutants, the levels of TCA cycle intermediates decreased at stage of the young adults and 10-day adults compared with WT.

Figure 6. Aging and reproduction associated with metabolic variations in C. elegans

(A) PCA included young adults and 10-day adults WT, glp-1 and daf-16;glp-1. PC1 and PC2 stand for the first and second principal components, respectively. (B) Model on how germline less signals regulate metabolome of worms to attenuate aging. See detailed explanation in the main text of the discussion.