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Jan 28, 2011 - A global view of the metabolic changes in C. elegans with impaired function of IIS represented by daf-2 and daf-16 and the intestinal d...
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Metabotyping of Caenorhabditis elegans and their Culture Media Revealed Unique Metabolic Phenotypes Associated to Amino Acid Deficiency and Insulin-Like Signaling Francois-Pierre J. Martin,†,q Britta Spanier,q,§ Sebastiano Collino,† Ivan Montoliu,† Carolin Kolmeder,§ Pieter Giesbertz,§ Michael Affolter,† Martin Kussmann,† Hannelore Daniel,§ Sunil Kochhar,† and Serge Rezzi†,* †

Nestle Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland ZIEL Research Center of Nutrition and Food Sciences, Abteilung Biochemie, Technische Universit€at M€unchen, Gregor-Mendel-Strasse 2, 85350 Freising, Germany

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bS Supporting Information ABSTRACT: Insulin/IGF-like signaling (IIS) and nutrient sensing are among the most potent regulators of health status and aging. Here, a global view of the metabolic changes in C. elegans with impaired function of IIS represented by daf-2 and daf-16 and the intestinal di- and tripeptide transport pept-1 was generated using 1H nuclear magnetic resonance spectroscopic analysis of worm extracts and spent culture media. We showed that specific metabolic profiles were significantly associated with each type of mutant. On the basis of the metabonomics data, selected underlying processes were further investigated using proteomic and transcriptomic approaches. The observed changes suggest a decreased activity of the one carbon metabolism in pept-1(lg601) mutants. Higher concentration of branched-chain amino acids (BCAA) and altered transcript levels of genes involved in BCAA metabolism were observed in long-living strains daf-2(e1370) and daf-2(e1370);pept-1(lg601) when compared to wild types and daf-16(m26);daf-2(e1370);pept-1(lg601) C. elegans, suggesting a DAF-16-dependent mechanism. KEYWORDS: Caenorhabditis elegans, metabonomics, nuclear magnetic resonance spectroscopy, proteomics, transcriptomics

’ INTRODUCTION Nutrition research focuses on deciphering the molecular mechanisms involved in the individual response to dietary nutrient intake, by better understanding metabolic disorders and by assessing the efficacy of dietary ingredients.1 Several studies investigated the effects of varying nutrient supply on longevity in humans and animal models, where individual responses were strongly dependent on genotype, age, nutrients, and regulation of nutrient-sensing pathways including IIS.2-6 In model organisms such as Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus, it was shown that the nutrient sensitive IIS pathway is one of the main regulators of lifespan by modulating a variety of metabolic processes and response to oxidative stress, lipids, and adipokine regulators.7-10 One approach to decipher the molecular mechanisms underlying gene-nutrient interactions lies in our ability to define the phenotypes associated with specific gene functions. Such an analysis based on reducing or deleting the function of a single gene provides an efficient way to assign gene contribution to specific metabolic pathways or regulatory mechanisms.11,12 r 2011 American Chemical Society

However, not all genes produce visible phenotypes while one single gene may influence multiple phenotypic traits when inactivated. Therefore, there is a need to perform studies of gene regulation and function in well characterized animal models, such as C. elegans. The nematode C. elegans is a multicellular organism that shares cellular and molecular structures, metabolic pathways and developmental processes with higher organisms.13 Previous works in C. elegans aimed to understand the relationships between nutrient uptake, IIS, and life expectancy.14,15 Suppression of the DAF-2/IIS pathway increases lifespan compared to wild type through the activation of the FOXO transcription factor DAF-16,16 which leads to changes in expression of a wide variety of stress response, antimicrobial and metabolic genes.7,17 Amino acid absorption in form of di- and tripeptides via the intestinal peptide transporter PEPT-1 modulates the systemic amino acid and protein homeostasis, which regulates metabolic and reproductive adaptations through the partially interconnected Received: July 8, 2010 Published: January 28, 2011 990

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Journal of Proteome Research insulin/DAF-2 and TOR (target of rapamycin) signaling pathways.18 Elimination of the intestinal di- and tripeptide transport in pept-1 mutants reduces the pool of intracellular amino acids and induces a modulation of TOR function. The lack of peptide transporter reduces growth, development, reproduction, and oxidatively damaged proteins,18 doubles the body fat content,19 but does not alter lifespan.18 However, daf-2;pept-1 double mutants show a further 60% increase of lifespan and an extremely high resistance to oxidative and heat stress when compared to daf-2 mutants. Focusing on the underlying molecular processes, we recently found that the germline-intestine hormone signaling via DAF-9/DAF-12 is a prime pathway that mediates the extreme longevity of the daf-2;pept-1 double-knockout C. elegans with a strict dependence of DAF-16.20 The pept-1 loss-of-function, that itself leads to amino acid depletion and therefore to a reduced germline activity, further enhances the interaction of both signaling pathways and finally leads to an increased expression of ROS defense and longevity genes. Despite these impressive differences in lifespan and stress resistance, the metabolic processes involved in these C. elegans phenotypes are poorly understood. The analysis of the real end points of physiological regulatory processes through metabolic profiling and biochemical pathways modeling is an approach to further extend the understanding of molecular mechanisms underlying specific biological functions in complex organisms.1,21,22 When associated with a well-defined physiological condition, metabolic profiles provide a snapshot of a functional phenotype, as a result of highly complex metabolic exchanges between diverse biological compartments under the influence of environment, lifestyle, and genetic determinants.23-25 Metabonomics has successfully been applied to study the biological outcome of nutritional interventions,1,25 including caloric restriction-induced metabolic changes in mouse,26 dog,27 and nonhuman primates.28 These studies described changes in regulatory metabolic nodes, such as switches from lipid biosynthesis toward fatty acid catabolism, and modulations of energy metabolism, and, surprisingly, of the gut microbial activity.26-28 Metabonomics has also revealed the latent phenotypes associated with silent mutations in C. elegans in relation to metabolic response to oxidative and heavy metal stress,12,29,30 and more recently increased lifespan.31 In particular, Fuchs et al. described how long-living daf-2(e1370)) mutant worms and dauer larvae share a common metabolic profile, marked by increased metabolic flux through gluconeogenesis, glyoxylate shunt, and amino acid catabolism.31 In the present study, we investigate the metabolic phenotypes associated with alterations of IIS and amino acid absorption, and we discuss the inferences in relation with the previously observed increased lifespan and the higher resistance to oxidative and heat stress. A global view of the metabolic changes in C. elegans induced by combinatorial daf-2, daf-16 and pept-1 gene mutations was generated using 1H nuclear magnetic resonance (NMR) spectroscopic analysis of whole organism extracts and spent culture media. Metabolic profiles were obtained from wild type animals, animals lacking the peptide transporter pept-1, a strain with mutations in the insulin receptor daf-2, a daf-2;pept-1 double mutant, a strain with a mutation in the FOXO-transcription factor daf-16 and a daf-16;daf-2;pept-1 triple mutant. The use of a well-defined axenic liquid medium as food source instead of bacteria enabled the detailed analysis of strain-specific changes in media represented by nutrient uptake and secretion of metabolites defined here as the excretome. Here, different metabolic signatures were associated with each type of mutants, suggesting a specific modulation of one carbon and branched-chain amino

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acid metabolism. These hypotheses were further tested using proteomics and transcriptomics data and their results are discussed hereafter.

’ MATERIAL AND METHODS C. elegans Strains and Sample Preparation

The strains used were as follows: N2 var. Bristol (wild type), BR2742: pept-1(lg601)X, CB1370: daf-2(e1370)III, CF1038: daf16(mu86)I; BR2688: daf-2(e1370)III; pept-1(lg601)X, BR3061: daf-16(m26)I;daf-2(e1370)III;pept-1(lg601)X. The worms were grown at 20 °C on Nematode Growth Medium (NGM) agar plates with Escherichia coli bacteria OP50 as food source.32 A mixed-stage worm culture was washed off the plates with M9 buffer. Eggs were prepared by hypochlorite treatment and were allowed to hatch overnight in M9 buffer. The synchronized L1 larvae were grown on NGM agar plates with E. coli OP50 until the fourth larval state (L4). The L4 larvae were harvested and washed in water. Each L4-larvae pellet was separated into five to ten 0.5 mL samples and each sample was rotated for 6 h in 50 mL Falcon tubes with 9 mL modified axenic CeHR liquid culture medium. After three hours the lids were opened for some minutes to allow air exchange. The modified CeHR culture medium additionally contained 30% DYT (1.6% casein hydrolysate, 1% bacto yeast extract, 85.5 mM sodium chloride, www.lib.umd.edu/drum/bitstream/ 1903/2204/1/CeHR_medium.htm)33 and with these modifications also allowed life-long cultivation of C. elegans.34 The medium contained 0.2 mg/mL gentamycine to avoid bacterial survival. Culture medium samples before and after worm culture and aliquots of 150 μL worms in sterile double-distilled water were shock-frozen and stored at -80 °C. The experiment was performed twice and three technical replicates were analyzed per worm strain and experiment. 1

H NMR Spectroscopic Analysis

Each C. elegans CeHR culture medium was sonicated using an ultrasonic bath for 10 min, and then centrifuged at 17000  g for 10 min. Supernatants (300 μL) were introduced into 5 mm NMR tubes with 250 μL of a phosphate buffer solution containing 90:10 D2O/H2O (v/v) as a field frequency lock. Frozen C. elegans samples were freeze-dried using a Thermo speedvac concentrator (Thermo Fischer Scientific, Switzerland). The freeze-dried C. elegans samples were manually homogenized using a sterile spatula for each individual sample, before being extracted using a phosphate buffer solution containing 90:10 D2O/H2O (v/v). The homogenates were centrifuged at 17 000 g for 10 min and 150 μL of the supernatants were transferred into 1.7 mm NMR tubes. Metabolic profiles were measured on a Bruker Avance III 600 MHz spectrometer equipped with a 5 mm cryogenic inverse probe operating at 300 K (Bruker Biospin, Rheinstetten, Germany). For each C. elegans and culture media sample, 1H NMR spectra were registered using pulse sequences including a standard 1H detection with water suppression.35 The standard spectra were acquired with a relaxation delay of 4 s and a mixing time of 100 ms. For C. elegans samples, an additional spectrum was acquired using a Carr-Purcell-Meiboom-Gill spin-echo sequence with water suppression to attenuate the signals from macromolecules, such as proteins.36 CPMG spin-echo spectra were measured using a spin-echo loop time (2nτ) of 19.2 ms and a relaxation delay of 4 s. For each C. elegans and culture media sample, 256 FIDs were collected into 98 000 data points using a spectral width of 18 315 Hz and an acquisition time of 2.68 s. 991

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The free induction decays were multiplied by an exponential weighting function corresponding to a line broadening of 1 Hz before Fourier transformation for standard and CPMG data sets. The acquired NMR spectra were manually corrected for phase and baseline distortions, and referenced to the chemical shift of R-glucose at δ 5.236 using the TOPSPIN (version 2.1, Bruker Biospin, Rheinstetten, Germany) software package. The metabolite identification was achieved using literature data,12 internal databases of compounds, and confirmed by additional 2D 1H NMR spectroscopy experiments registered on selected samples: 1H-1H correlation spectroscopy (COSY),37 total correlation spectroscopy (TOCSY),38 and 1H-13C heteronuclear single quantum correlation (HSQC).39 For COSY and TOCSY, 128 increments with 64 transients per increment were collected into 4000 data points. The TOCSY NMR spectra were acquired using the MLEV-17 spin-lock scheme for 1H-1H transfers with a spin-lock mixing time of 60 ms. The relaxation delay between successive pulse cycles was 2 s for all pulse sequences. The spectral width in both dimensions was 9615 Hz. The sine-bell (COSY) and sine-bell squared (TOCSY) window functions were applied prior to Fourier transformation. The HSQC NMR spectra were recorded with gradient selection and shaped pulses. Spectra were acquired with 128 transients per increment, 128 increments collected into 4000 data points and a spectral width of 9615 and 22137 Hz in the first and second dimensions, respectively. The coupling constant for HSQC was set to 145 Hz. A sine-bell squared (HSQC) window function was applied prior to Fourier transformation.

of 0.05 was performed on NMR signal areas representative of influential metabolites. Chemometric analysis was performed  using the SIMCA-Pþ (version 12.0, Umetrics AB, Umea, Sweden) software package, the MCR-ALS toolbox (http:// www.ub.edu/mcr/welcome.html), and in-house developed MATLAB routines. Complementary Analyses Performed on Separate Batches of C. elegans

Complementary Transcriptomics Analysis. Transcriptomics analysis was performed on additional cultures of all six C. elegans strains used in the study (n = 5/group). Parts of the data sets have already been published.19,20 The nematode culture, RNA preparation, microarray analysis, data analysis and statistics were done according to the described protocols.20 Complementary Proteomics Analysis. Proteomics analysis was performed on additional cultures of pept-1 and wild type C. elegans (n = 5/group). Biological samples were divided and analyzed using a 2D-PAGE and MALDI-TOF-MS approach and in parallel a shotgun approach. For the 2D-PAGE and MALDI-TOF-MS approach 300 μL of frozen C. elegans samples were cracked under liquid nitrogen using mortar and pestle. The grinded material was solubilized in lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS (w/v), 1% DTT (w/v), EDTA free protease inhibitor (Roche; Mannheim, Germany)). Samples were sonicated on ice (amplitude: 30%, frequency: 0.5 s, duration: 15 s) and centrifuged (4 °C, 40 000 g, 20 min). Overnight, the supernatant was precipitated with 4  vol of acetone. After centrifugation (4 °C, 10 000 g, 40 min), the pellet was solubilized in lysis buffer and the protein content was determined according to Bradford before adding 8% of pharmalytes. For the first dimension separation 500 μg grams of extracted protein were isoelectric focused by using Immobiline Dry Strip stripes (pH range from pH 3 to pH 10) rehydrated in 7 M urea, 2 M thiourea, 4% CHAPS, 4% pharmalytes and a Biosciences Ettan-Dalt II System (Freiburg, Germany). 0.5 V were applied for 30 s, 4000 V for 1.5 h and 8000 V until a total of 25 000 Vh was reached. The IPG stripes, equilibrated in 1.5 M Tris-HCl pH 8.8, 6 M urea, 26% glycerine, 2% SDS, 4% DTT and then in 1.5 M Tris-HCl pH 8.8, 6 M urea, 26% glycerine, 2% SDS, 4% IAA, were loaded on top of 12.5% sodium-dodecylsulfate polyacrylamide gel electrophorese (SDS-PAGE) gels. The gels were run in 25 mM Tris, 192 mM glycerine, 0.1% SDS (v/v) by applying 4 mA per gel for 1 h and 12 mA per gel for 15 h in an Ettan Dalt II System Separation Unit. For four days, the proteins in the gels were stained in 10% (NH4)2SO4 (w/v), 2% H3PO4 (w/v), 25% Metanol (v/v), 0.625% Coomassie brilliant blue G250 (w/w). Gels were destained in bidestilled water until the background was completely clear. After scanning the gels on an Unimax ImageScanner (Amersham Pharmacia Biotechnology, Freiburg, Germany), spots were detected with the software ProteomWeaver 3.1 (DefiniensCognitionware, Munich, Germany). Only spots that had a t test p-value 1.5” in all C. elegans pathways in the WikiPathways database, n = number of genes in the C. elegans BCAA pathway, r = number of genes in the BCAA pathway meeting the criterion of “fold change < -1.5 or fold change >1.5”. Nevertheless, the fact that both daf-2-deficient strains showed a similar expression pattern was significant when compared to wild type and daf-16; daf-2;pept-1 animals (p < 0.0001) (done by an unbalanced twoway analysis of variance using SAS (Statistical Analysis System Software)). These findings bring additional evidence of a modulation of the BCAA pathway in the daf-2-deficient long-living strains with a strict dependence of DAF-16.

respectively (Figure 5A). A separation of the metabolic profiles was achieved along the first PC between initial and spent culture media. Interpretation of the corresponding loadings plots revealed that spent culture media showed relative decreased concentrations of glucose, amino acids, nucleotides, nucleosides, succinate and ethanol and a relative increase in the concentrations of acetate and lactate (Figure 5B). The second PC revealed several subgroups of samples related to C. elegans genetic background (Figure 5A). These metabolic differences were further investigated by PCA of spent culture media. The PCA model was generated using three PCs, which explained 38.8, 28.8, and 10.1% of the total variance, respectively (Figure 5C). The medium obtained from the culture of daf-16 C. elegans was significantly different from all other spent culture media but relatively closer to that obtained from wild type animals. In addition, the spent culture media obtained from the daf-2 and daf-2;pept-1 strains tended to be similar, as well as the strains pept-1 and daf-16;daf-2;pept-1. The spent culture media obtained from daf-16, pept-1 and daf-16;daf2;pept-1 worms were separated from other samples along PC1 due to higher levels of certain nucleosides and nucleotides, glucose, succinate, amino acids and lower levels of acetate and lactate (Figure 5D). Along PC2, culture media obtained from

C. elegans Metabolic Phenotypes Are Reflected in 1H NMR Metabolic Profiles of Spent Culture Media

As an exploratory approach, we applied 1H NMR-based metabonomics to monitor the changes of metabolite composition in the media induced by the culture of C. elegans. Metabolic profiling of spent culture media provided additional insights into both altered utilization of dietary nutrients and the excretome. Due to the relatively limited number of samples collected before and after culture for each strain of C. elegans, data were analyzed using unsupervised chemometric methods. Data analysis was performed using PCA on autoscaled data. PC1-3 explained 39.2, 17.5, and 6.7% of the total variance present in the NMR data, 996

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Figure 3. One carbon metabolism in pept-1(lg601) and wild type C. elegans based on proteomics and metabonomics data. Levels of selected proteins are indicated by histograms. The concentration in wild type is set to one (black bars), the concentration in pept-1 is set relative to wild type (gray bars). Arrows indicate differences in metabolite concentration: down, lower concentration in pept-1 than in wild type; up, higher concentration in pept-1 than in wild type. THF, tetrahydrofolate.

daf-16, daf-2 and daf-2;pept-1 animals were separated from other media, an effect driven by a higher acetate level (Figure 5 D). Moreover, along PC3, samples obtained from daf-16 C. elegans were clustered separately due to higher levels of ethanol and certain nucleotides and nucleosides (Figure 5D).

conserved irrespectively of the developmental stage and the growth temperature in daf-2(e1370) worms. Moreover, this metabolic feature being also dominant in daf-2;pept-1 mutants, the additional knockout of pept-1 in a daf-2 background had only a slight impact on the metabolic phenotypes. This result indicates the dominant role of IIS not only on lifespan regulation but also on the metabolic regulation of BCAAs. The systemic changes in amino acids depend indeed on their rate of appearance (amino acid intake and release by tissues) and on their rate of disappearance through oxidation, protein synthesis and amino acid excretion. Previous work in insulin-deficient animal model demonstrated accelerated muscle proteolysis, more likely caused by a decrease in the activity of phosphatidylinositol 3-kinase and increased ubiquitination.48 Here, the accumulation of BCAA suggests an alteration of overall protein metabolism49 associated with increased protein degradation which was characterized earlier in the double mutants.18 Based on an existing transcriptome data from daf-2 C. elegans,50 Fuchs et al. suggested that the down-regulation of genes coding for the branched-chain ketoacid dehydrogenase complex, a key regulator of the BCAA catabolic pathway, might be responsible for the accumulation of BCAA. In the present study, we conducted complementary transcriptomics analyses of genes involved in the BCAA metabolism on similar cultures of C. elegans. Our observations described a different regulation of the corresponding genes in daf-2 and daf-2;pept-1 mutants when compared to wild type worms (Figure 4). These observations do not provide sufficient evidence to confirm the previous in silico analysis. Nevertheless, the expression of several downstream genes of the pathway was higher in the two long-living strains than in the daf-16; daf-2;pept-1 strain. This effect might be driven by the high leucine concentrations in long-living daf-2 mutant strains. In particular, leucine is a prime regulator of TOR complex 1 (TORC1), an oligomer of the mTOR protein kinase, its substrate-binding subunit raptor, and the polypeptide Lst8/GβL in mammals.51,52 mTORC1

’ DISCUSSION In the present study, 1H NMR-based metabonomics was applied to characterize the metabolic phenotypes associated with alterations of IIS and amino acid absorption induced by combinatorial daf-2, pept-1, and daf-16 gene mutations. On the basis of the metabonomics analysis of C. elegans extracts and spent culture media, strainspecific metabolic fingerprints were found and certain hypotheses on underlying biochemical processes were further tested using complementary proteomics and transcriptomics on selected strains of C. elegans (summarized in Figure 6). Altered BCAA Metabolism is a Dominant Feature of the Metabolic Phenotypes of Long-Lived daf-2 and daf-2;pept-1 C. elegans

There is increasing awareness that IIS is one of the most potent regulators of aging, by modulating glycolytic processes, lipid metabolism, and the metabolic response to oxidative stress.47 A downregulation of IIS in C. elegans induces a dauer larvae-like state and confers extended lifespan and increased stress resistance. Recently, Fuchs et al. reported that 10 days old adult daf-2(e1370) C. elegans were characterized with a complex metabolic profile, marked with increased BCAA levels and decreased levels of acetate and glycerol.31 In the present study, the 1H NMR metabonomic analysis revealed that the metabolic phenotype of both daf-2 and daf-2;pept-1 mutants had characteristic low levels of acetate, choline, glycerol and glutamate and high levels of glutamine and BCAAs. Since our findings were obtained at an early developmental stage (L4 larvae) with a lower growth temperature (20 °C instead of 25 °C), our results provide additional evidence that this metabolic pattern is 997

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Figure 4. Changes in mRNA expression of genes involved in the metabolism of branched-chain amino acids in daf-2, daf-2;pept-1 and daf-16;daf-2;pept-1 C. elegans. The data are based on microarray analysis data in comparison to wild type (n = 5). Each box is separated in three parts: (left) mRNA expression in daf-2; (middle) mRNA expression in daf-2;pept-1; (right) mRNA expression in daf-16;daf-2;pept-1. Genes with a lower expression than in wild type are highlighted in green boxes, and genes expressed in higher levels are highlighted in red boxes. The darker the color, the higher is the change in expression. Genes with changes in expression between 1.5 and -1.5 are not colored. The data were visualized using Pathvisio software (www.pathvisio.org).67

controls cell growth in response to nutrient availability, insulin, growth factors, energy status, and stress conditions. Since these mechanisms are conserved in C. elegans,53 these observations suggest the high concentration of leucine in the long-living strains might promote translation.

in the glycerophospholipid pathways. In accordance with these data, we found that the gene coding for the homologue of the choline transporter chtl-1 was 6- and 3-fold down-regulated at the mRNA level in daf-2 and daf-2;pept-1 mutants, respectively (data not shown). Additionally, the phosphatidylcholine synthesis seems lower in the long-living strains as suggested by lower mRNA expression of various isoforms of the choline kinase, the first enzyme in the phospatidylcholine pathway,54 when compared to wild type, that is, cka-1 (0.7-fold in daf-2 and 0.75-fold in daf-2;pept-1), cka-2 (1.1-fold and 1.3-fold), ckb-2 (0.5-fold and 0.6-fold) and ckc-1 (1.1-fold and

Metabolic Phenotypes of Long-Lived daf-2 and daf-2;pept-1 C. elegans Show a Peculiar Choline and Acetate Metabolism 1

H NMR metabonomics of daf-2 and daf-2;pept-1 animals in comparison to wild type also revealed reduced concentrations of ethanolamine, choline, and betaine, which are closely interconnected 998

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Figure 5. (A) PCA scores and (B) loadings plots for the principal components one and two derived from 1H NMR spectra of culture media and C. elegans spent culture media. (C) PCA scores and (D) loadings plots for the first three principal components derived from 1H NMR spectra of spent culture media obtained from wild type animals, daf-2(e1370), daf-16(mu86), pept-1(lg601), daf-2(e1370);pept-1(lg601) and daf-16(m26);daf-2(e1370); pept-1(lg601) mutants.

1.1-fold). Intriguingly, these features are associated to de novo synthesis of triacylglycerols and phospholipids, which are the predominant mechanism modulating fat storage in daf-2 animals55 and to greater fat content in daf-2;pept-1 animals (data not shown). Therefore, future analyses of the lipidome and choline metabolism would provide further insights into the interrelationships between these metabolic functions with respect to longevity. Moreover, the intracellular acetate concentration in the daf-2; pept-1 double mutant was reduced while the acetate concentration

was higher in the spent culture media when compared to wild type. Anoxic growth conditions, which lead to high levels of acetate, succinate, lactate and propionate,56 can be excluded in the present study by the experimental setup, and the occurrence of concentrations of lactate and succinate similar to those observed in wild type animals. Therefore, the present changes may reflect some compensatory mechanisms in the dauer-like metabolism of this mutant strain involving differential modulation of the endogenous metabolism of glucose and fatty acids.57 999

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Figure 6. pept-1-type, daf-2-type and wild-type like metabolic fingerprints. The lead-compounds with different concentrations in the strain-types in comparison to wild type C. elegans and the corresponding biological processes are indicated. By transcriptomics and proteomics analysis, key-genes and enzymes involved in these metabolic pathways were found to be significantly regulated (p < 0.05). In parallel, the changes of the metabolic composition of the spent culture medium (excretome) reflect the mutation-induced impairment of the metabolism. Arrows indicate the direction of regulation in comparison to wild type C. elegans.

Modulation of One Carbon Metabolism and Proteolysis Characterizes the Metabolic Phenotypes of pept-1 Mutants

In the C. elegans intestine, the elimination of PEPT-1 protein reduces intracellular amino acid levels and affects TOR signaling pathway, with inferred effects on transcription, translation, and protein degradation.18 1H NMR metabonomics analysis of the pept-1 mutants revealed increased concentrations of specific free amino acids, for example, asparagine, threonine, and glutamine and a decrease in glutamate/glutamine ratio, which might result from increased proteolysis.49 These metabolic changes also corroborate previous reports on reduced concentrations of damaged proteins in old pept-1 mutants when compared to wild type at the same age.18 1H NMR and LC-MS analyses of pept-1 mutants also highlighted a particular pattern of reduced concentrations of glycine, methionine and ethanolamine concomitant to an increased concentration of homocysteine (Figure 3, Supplementary Table S1, Supporting Information). Interestingly, this phenotype tended to be conserved in daf-16;daf-2;pept-1 mutants, which describes a dominant effect of the depleted di- and tripeptide transport on the metabolism of these strains. The reciprocal concentrations in methionine and homocysteine suggested a down-regulation of the transmethylation flux between the tetrahydrofolate and methionine cycles. In a wild type situation, methionine can be regenerated from homocysteine by remethylation via the methionine synthase with methyltetrahydrofolate acting as methyl-donor. In pept-1 deficient worms this metabolic step seems to be blocked leading to low methionine and elevated homocysteine concentrations. Furtheron, proteomics analysis showed a down-regulation of S-adenosylmethionine synthase, phosphoethanolamine methyltransferase, and S-adenosylhomocysteine hydrolase. Moreover, the low concentrations of the serine hydroxymethyl transferase and its product glycine suggested a lower carbon flux through the one carbon metabolic pathway, while increased protein level of methylene tetrahydrofolate reductase may corresponds to the activations of compensatory mechanisms concomitant to an overall reduction of flux through the homocysteine cycle. Interestingly, RNA interference for sams-1, the gene coding for S-adenosylmethionine synthetase in

C. elegans, induces a reduced number of progeny, delayed reproductive timing and a smaller body size in the progeny generation, a phenotype also observed in pept-1 C. elegans.58 SAMS-1 was shown to be negatively regulated in eat-2 mutants, suggesting a negative regulation by dietary restriction.58 One of the reactions that quantitatively contributes most to the transmethylation flux in mammals is the methylation of phosphatidylethanolamine (PEA).59 The SAM-dependent methyltransferase homologue PMT-2 is catalyzing this step in C. elegans.60 PMT-2 concentration in pept-1 is 60% lower than in wild type, indicating a reduced methylation of PEA. Interestingly, the observed pattern of one carbone pathway metabolites in pept-1 animals was comparable to the one observed in mice and rats fed on diets deficient in choline, methionine or folate.61,62 In mammals, the reduced availability of methyl groups induced fatty liver diseases ranging from fat accumulation to hepatic steatosis with inflammation.61,62 Since it is the intestine that stores most of the body fat in C. elegans, the obese phenotype of pept-1 mutants19 might not only be induced by an increased uptake of free fatty acids but also by the down-regulation of the one carbon metabolism. Loss of daf-16 Antagonizes the daf-2 but Not the pept-1 Phenotype

By modulating glycolytic processes and the metabolic response to oxidative stress, the IIS is one of the most potent regulator of aging.47 In C. elegans, most of these processes are mediated by the activation of the FOXO transcription factor DAF-16, which is the final effecter in the IIS pathway and a key metabolic regulator to oxidative stress.18,55 Furthermore, metabonomics analyses showed the depth of the metabolic modulations induced by DAF-16 in a daf-2 or a daf-2; pept-1 mutant background. In these mutants, the IIS cascade is not functional, which results in the non-phosphorylation of DAF16 and its translocation into the nucleus,63,64 where it modulates gene expression, and thereby extends lifespan and triggers metabolism.7,17 When daf-16 is impaired as in daf-16;daf-2;pept-1 triple mutants, the animals have a normal lifespan and a low stress 1000

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Journal of Proteome Research resistance. The current metabonomics investigations described how the loss of DAF-16 in the daf-2;pept-1 C. elegans tends to partially normalize metabolite concentrations toward the levels observed in a pept-1 mutant background, indicating a DAF-16 dependent mechanism. In particular, BCAAs and choline showed a DAF-16 dependence (Table 1, Supplementary Figure 3, Supporting Information), while the patterns of acetate, betaine, glycerol, glutamate and lysine were more complex. In addition, the current study shows the concentration of free choline was lower in the long-living strains and concomitant to a downregulation of the chtl-1 choline transporter mRNA. This difference with the reports by Fuchs et al. may be due to the difference in the developmental stage. Moreover, we used water as solvent of extraction, and not methanol, which may be associated with some metabolic differences due to residual enzymatic activities during metabolite extraction. The metabolic phenotypes associated with daf-16;daf-2;pept-1 C. elegans may indicate a specific modulation of the amino acid metabolism for energy production in parallel with a reduced gluconeogenesis (e.g., low lactate and alanine). Here, the single mutation of daf-16 resulted in relatively limited alterations of C. elegans metabolism, which is in agreement with other analyses.31 These findings are not surprising as in well-fed wild type worms the IIS cascade is active and phosphorylated DAF-16 stays in the cytosol.65,66 Furthermore, the metabonomic analysis of the spent culture media described a metabolic similarity between pept-1 and daf-16;daf-2;pept-1 C. elegans. The increased concentration of central metabolites including glucose and amino acids and the reduced excretion of metabolic end products including acetate and lactate suggest a highly efficient metabolic turnover in these strains. All together, our observations on C. elegans extracts and spent culture media suggest that silencing daf-16 in daf-16;daf-2;pept-1 triple mutants antagonizes the effect of the daf-2 mutation, and finally leads to a metabolic profile that is closer to pept-1 than to wild type, in agreement with our previous reports that described a pept-1-like phenotype of the triple mutant worms.18

’ CONCLUSION Our metabonomic investigations demonstrated the feasibility of mapping both profound and subtle metabolic alterations induced by differential nutrient sensing and IIS pathways in C. elegans. In particular, 1H NMR metabonomics showed that silencing daf-2, pept-1, and daf-16 genes in C. elegans leads to a specific modulation of energy flux through various catabolic and anabolic pathways resulting in unique metabolite patterns of amino and organic acids, nucleotides, and osmolytes. For the first time, we also showed that the mutation-induced impairment of the metabolism was reflected in the composition of spent culture media, offering a noninvasive analysis tool for C. elegans. Having demonstrated the potential of metabonomics to decipher the conditional impact of combinatorial gene silencing, it is now feasible to target specific metabolic pathways in combination with proteomics and transcriptomics to get insights into molecular mechanisms associated with specific genotypes. ’ ASSOCIATED CONTENT

bS

Supporting Information Supplementary materials and methods, results, tables, and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION Corresponding Author

*Nestle Research Center, BioAnalytical Sciences, P.O. Box 44, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland. E-mail: [email protected]. Phone: þ41 785 9165. Fax: þ41 (21) 785 9486. Author Contributions ‡

These authors contributed equally to the manuscript.

’ ACKNOWLEDGMENT Some strains included in this work were supplied by the C. elegans Genetics Center (CGC) supported by the National Institute of Health NIH and the University of Minnesota. We acknowledge the excellent technical assistance of Katrin Lasch. ’ ABBREVIATIONS: CPMG, Carr-Purcell-Meiboom-Gill; IIS, Insulin/IGF-like signaling; MS, mass spectrometry; NMR, nuclear magnetic resonance; O-PLS-DA, orthogonal projection to latent structure discriminant analysis; PCA, principal component analysis; PLS-DA, projection to latent structure discriminant analysis. ’ REFERENCES (1) Rezzi, S.; Ramadan, Z.; Fay, L. B.; Kochhar, S. Nutritional metabonomics: applications and perspectives. J. Proteome Res. 2007, 6 (2), 513–525. (2) Piper, M. D.; Mair, W.; Partridge, L. Counting the calories: the role of specific nutrients in extension of life span by food restriction. J. Gerontol. A: Biol. Sci. Med. Sci. 2005, 60 (5), 549–555. (3) Mair, W.; Piper, M. D.; Partridge, L. Calories do not explain extension of life span by dietary restriction in Drosophila. PLoS Biol. 2005, 3 (7), e223. (4) Heilbronn, L. K.; Ravussin, E. Calorie restriction extends life span--but which calories?. PLoS Med. 2005, 2 (8), e231. (5) Sinclair, D. A. Toward a unified theory of caloric restriction and longevity regulation. Mech. Ageing Dev. 2005, 126 (9), 987–1002. (6) Shimokawa, I.; Chiba, T.; Yamaza, H.; Komatsu, T. Longevity genes: insights from calorie restriction and genetic longevity models. Mol. Cells 2008, 26 (5), 427–435. (7) Murphy, C. T.; McCarroll, S. A.; Bargmann, C. I.; Fraser, A.; Kamath, R. S.; Ahringer, J.; Li, H.; Kenyon, C. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 2003, 424 (6946), 277–283. (8) Tatar, M.; Kopelman, A.; Epstein, D.; Tu, M. P.; Yin, C. M.; Garofalo, R. S. A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science 2001, 292 (5514), 107–110. (9) Clancy, D. J.; Gems, D.; Harshman, L. G.; Oldham, S.; Stocker, H.; Hafen, E.; Leevers, S. J.; Partridge, L. Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein. Science 2001, 292 (5514), 104–106. (10) Kaeberlein, M.; Powers, R. W., III; Steffen, K. K.; Westman, E. A.; Hu, D.; Dang, N.; Kerr, E. O.; Kirkland, K. T.; Fields, S.; Kennedy, B. K. Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science 2005, 310 (5751), 1193–1196. (11) Van Raamsdonk, J. M.; Hekimi, S. Deletion of the mitochondrial superoxide dismutase sod-2 extends lifespan in Caenorhabditis elegans. PLoS Genet. 2009, 5 (2), e1000361. (12) Blaise, B. J.; Giacomotto, J.; Elena, B.; Dumas, M. E.; Toulhoat, P.; Segalat, L.; Emsley, L. Metabotyping of Caenorhabditis elegans reveals latent phenotypes. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (50), 19808–19812. 1001

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