A metabolic signature of long life in Caenorhabditis elegans

Abstract

Background: Many Caenorhabditis elegans mutations increase longevity and much evidence suggests that they do so at least partly via changes in metabolism. However, up until now there has been no systematic investigation of how the metabolic networks of long-lived mutants differ from those of normal worms. Metabolomic technologies, that permit the analysis of many untargeted metabolites in parallel, now make this possible. Here we use one of these, 1H nuclear magnetic resonance spectroscopy, to investigate what makes long-lived worms metabolically distinctive.

Results: We examined three classes of long-lived worms: dauer larvae, adult Insulin/IGF-1 signalling (IIS)-defective mutants, and a translation-defective mutant. Surprisingly, these ostensibly different long-lived worms share a common metabolic signature, dominated by shifts in carbohydrate and amino acid metabolism. In addition the dauer larvae, uniquely, had elevated levels of modified amino acids (hydroxyproline and phosphoserine). We interrogated existing gene expression data in order to integrate functional (metabolite-level) changes with transcriptional changes at a pathway level.

Conclusions: The observed metabolic responses could be explained to a large degree by upregulation of gluconeogenesis and the glyoxylate shunt as well as changes in amino acid catabolism. These responses point to new possible mechanisms of longevity assurance in worms. The metabolic changes observed in dauer larvae can be explained by the existence of high levels of autophagy leading to recycling of cellular components.See associated minireview: http://jbiol.com/content/9/1/7.

Figure 1

The insulin-signalling mutant daf-2(m41) has a distinctive metabolic profile. In this experiment m41 and N2 wild-type worms were initially raised at 15°C, transferred to 22.5°C at L4, and assayed at 240 hours post-bleaching. A. PCA (above) on the binned spectra shows that daf-2(m41) are clearly separable from N2 along PC1 (Means and 95% CI are given by ellipses); cluster analysis (below) separates the samples into two groups, one predominantly wild-type (8/9), the other predominantly m41 (10/13). The first three PCs respectively account for 50, 11 and 7% of the variance. B. Heatmap (middle) showing the standardized relative concentration of 34 bins out of 179 with a substantial (>0.1 and <-0.1) loading on PC1, their loads (below) and position on the ⁺¹H NMR spectrum (above). The spectrum is the median of five N2 samples; intensities >5.0 ppm are scaled by a factor of 10.

Figure 2

Longevity mutants and dauer larvae have distinctive metabolic profiles. A. PCA of four IIS mutants and a long-lived translation-defective mutant, ife-2(ok306); cluster analysis separates the mutants into distinct groups. daf-2(e1370) is the most similar to wild-type. ife-2(ok306), while distinctive relative to wild-type and all other mutants, is not distinctive relative to the IIS mutants as a class. In this experiment mutant and wild-type worms were initially raised at 15°C, transferred to 25°C at L4, and assayed at 240 hours post bleaching. The first three PCs respectively account for 40, 20 and 16% of the variance. B. PCA shows that daf-2(m41) has a distinctive profile even at L1 (15 hours); and appears to become successively more distinctive as a middle-aged adult (144 hours) and old (240 hours) adult. The first three PCs explain, respectively, 35, 26 and 11% of the variance. C. PCA of dauers and wild-type worms (240 hours) raised at two temperatures, 20°C and 25°C. The PCA and cluster analysis shows that dauers and adults clearly have distinct metabolomes as do worms raised at 20°C rather than 25°C. The first three PCs explain, respectively, 48, 16 and 11% of the variance, with PC1 distinguishing stages and PCs 2 and 3 distinguishing temperature. Note that the relative position of the samples raised at the two temperatures is reversed along PC2, a consequence of strong temperature × stage interaction.

Figure 3

The metabolic signature of long-life. A. Relative concentrations of 26 metabolites in worms sampled at 25°C and 240 hours post-hatching. B. Summary of metabolic responses. Here we show, for all experiments, the observed response in long-lived worms relative to appropriate controls and rank them by their consistency; p values for individual experiment are given in each cell: * P < 0.05; ** <0.01; *** <0.001; **** <0.0001. For all data and statistics see Additional file 1.

Figure 4

DAF-16 dependence of metabolites. A. PCA of metabolic profiles of wild-type, single mutant daf-2(e1370) and daf-16(m26) and double-mutant daf-2(e1370);daf-16(m26) samples. PCA and cluster analysis shows that daf-2(e1370) have the most divergent metabolism of the four genotypes, implying that some of the distinctive features of the daf-2 metabolome is daf-16 dependent. The first three PCs explain, respectively, 31, 21 and 13% of the variance; most of the separation between daf-2 and other genotypes is along PC2. In this experiment mutant and wildype worms were initially raised at 15°C, transferred to 22.5°C at L4, and assayed at 144 hours post bleaching. B. Relative concentrations of 11 metabolites in worms sampled at 22.5°C and 144 hours post hatch. Four metabolites - isoleucine, valine, leucine and phosphocholine - show classical DAF-16 dependence, the rest show more complex patterns of epistasis or none.

Figure 5

Carbohydrate metabolism in daf-2 worms. Five signature metabolites - malate, acetate, succinate, glucose and trehalose - are linked by three major pathways: the glyoxylate shunt, gluconeogenesis, and starch metabolism. Expression data [20] shows that these three pathways (blue lines) are upregulated in daf-2(e1370) worms. Glycolysis and citric acid cycle genes (brown lines) are, by contrast, generally downregulated or unregulated. In this model, carbon from acetate or fatty acid metabolism enters the glyoxylate pathway mediated by increased expression isocitrate lyase (4.1.3.1) and malate synthase (4.1.3.2) which are encoded by a single gene, gei-7. The product of this pathway, malate, is then converted to oxaloacetate by cytosolic malate dehydrogenase (1.1.1.37) which then results, via gluconeogenesis, to the production of carbohydrates. The irreversible steps of gluconeogenesis are catalysed by phosphoenolpyruvate carboxykinase (4.1.1.32, PEPCK), pyruvate carboxylase (6.4.1.1), and fructose 1,6-biphosphatase (3.1.3.11). Most of the genes encoding components of these enzymes are upregulated in daf-2(-) worms. In most animals, glucose is synthesized from glucose-6-phosphate by glucose-6-phosphatase (3.1.3.9) but C. elegans does not contain a homologue of this gene. We suppose, then, that glucose is produced by the metabolism of trehalose by trehalase (3.2.1.28). Several trehalase genes are downregulated in daf-2 worms implying a reduced flux to glucose. However, glucose demand is also probably reduced since two genes encoding hexokinase (2.7.1.1), responsible for a irreversible reaction in glycolysis, are repressed in daf-2 worms as are many of the genes encoding the pyruvate dehydrogenase complex that links glycolysis to the citric acid cycle via acetyl-CoA. In contrast to the glyoxylate pathway genes, TCA genes are not generally regulated in daf-2(-) worms. Genes and metabolites are taken to be regulated if P = 0.05 (Additional file 2).