in caenorhabditis 
 elegans , attenuation 
 of the insulin / igf-1 signaling ( iis ) pathway during adulthood leads 
 to significant changes in worm physiology , including extended life - span , 
 increased innate immunity and stress - resistance , and extensively altered 
 metabolism . these phenotypic adaptations rely , for the most part , on the activation 
 and nuclear translocation of the forkhead box o ( foxo ) transcription 
 factor daf-16 , which triggers an extensive genetic program that is 
 normally associated with an alternate developmental arrest state called 
 the dauer diapause . in mammals , disruption of the 
 insulin receptor in the brain is associated with expression of endocrine 
 neuropeptides that stimulate the uptake of food , leading to obesity 
 and symptoms of type ii diabetes along with reduced reproductive competence . in this respect , iis in c. elegans appears remarkably similar to mammalian insulin signaling in that 
 the nematode nervous system ( together with the intestine ) was identified 
 as a central regulator of daf-2-mediated energy metabolism 
 and longevity . although disruption of iis in 
 nematodes does not lead to any clear pathological conditions , the 
 conservation of this pathway in the regulation of fat and carbohydrate 
 metabolism as well as in life - span determination from nematodes to 
 mammals is at least striking . therefore , a thorough understanding of daf-2-specific 
 metabolism and its regulation , including species - specific peculiarities , 
 may lead to the discovery of novel metabolic pathways involved in 
 insulin - dependent pathologies or other fundamental processes such 
 as aging in humans . whole - genome transcript 
 profiling of daf-2 mutants 
 and/or dauers , using either microarray or sage analysis , has proven 
 to be successful at identifying differentially expressed gene sets 
 dependent on daf-16 , some of which are involved in energy metabolism . however , reported 
 transcript profiles , especially with regard to energy metabolism , 
 were sometimes inconsistent between studies , complicating the formulation 
 of straightforward statements about iis - mediated metabolism . moreover , the poor correlation between mrna transcript levels and 
 actual protein abundances necessitates great caution when drawing 
 conclusions based solely on interpreting transcript profiles . proteomics analysis by dong et al . of daf-2(e1370 ) mutants has identified a limited set of enzymes in carbohydrate 
 metabolism ( glycolysis / gluconeogenesis ) as well as the tca and glyoxylate 
 cycles that are differentially expressed in daf-2 mutants and showed that these enzymes are important determinants 
 of nematode life span . recently , several groups have published the 
 first metabolite profiles of the daf-2 mutant , revealing 
 significant shifts in carbohydrate , amino acid , and lipid metabolism , 
 several of which were not apparent from previously published transcriptomics 
 studies . although these incentives contribute significantly 
 to the identification of altered metabolic pathways upon impaired 
 iis , an integrated understanding of the metabolic restructuring and 
 its impact on worm physiology and longevity is still lacking . using lc ms / ms - based proteomics analysis , we found that 
 activation of daf-16 in the nucleus results in over - representation 
 of many enzymes of major , often reciprocally regulated , metabolic 
 pathways involved in the synthesis and utilization of internal carbohydrate 
 ( glycolysis and gluconeogenesis ) , lipid stores ( lipogenesis and lipolysis ) , 
 amino acid metabolism , and respiration ( aerobic and anaerobic ) . in 
 the following sections , we integrate our proteomic data set together 
 with a selection of enzymatic activity assays , available literature , 
 and metabolomics data in an attempt to reconstruct and resolve the 
 intricacies of altered daf-2 metabolism . the 
 following strains were used in this study : nlga154 glp-4(bn2ts)i;daf-2(e1370)iii ( long - lived ) and ga153 glp-4(bn2ts)i daf-16(mgdf50)i;daf-2(e1370)iii ( reference strain ) , which were kindly 
 provided by david gems at the university college of london . the glp-4(bn2 ) background allele 
 results in germline - deficient worms when grown at the nonpermissive 
 temperature of 24 c . this allows for focusing on the proteome 
 of aging somatic cells , excluding the gonadal and complex embryonic 
 material . and tekipe and alballay demonstrated that no increase 
 in lifespan results from the presence of the glp-4(bn2 ) allele in otherwise wild - type or daf-16;daf-2 mutants grown on live escherichia coli . however , glp-4(bn2 ) causes 
 a small but significant daf-16-dependent extension of lifespan of daf-2 mutants and nematodes grown on killed e. coli . previous ms proteome 
 profiling of glp-4(bn2 ) after n metabolic 
 labeling revealed only modest changes compared to the n2 wild - type . the rnai - hypersensitive nl2099 rrf-3(pk1426 ) ii strain was used for rnai lifespan analyses . the initiation 
 and culturing of large age - synchronized populations 
 have been described in depuydt et al . to prevent glp-4daf-16;daf-2 animals from becoming dauer larvae , worms were grown 
 at 16 c until the third larval stage ( l3 ) and were then shifted 
 to 24 c for the remainder of the experiment . although shifting 
 temperature at the l3 stage rendered glp-4;daf-2 animals sterile , we noticed that by day 2 of adulthood glp-4daf-16;daf-2 mutants 
 exhibited gonads with incomplete morphogenesis , yet they were able 
 to produce 24 eggs per animal . therefore , we opted to add 
 5-fluoro-2-deoxyuridine ( fudr , 75 m fc . , acros organics ) 
 to all mass cultures in all biological replicates in order to maintain 
 complete sterility . we note that the use of fudr in light of 
 recent reports warrants caution , especially with regard to insulin / igf-1 
 mutants . however , we argue that our data reflect 
 bona fide metabolic changes in response to the daf-2 mutation . our results are in line and corroborate with the reported 
 metabolomics findings by fuchs et al . who used both the daf-2(e1370 ) and daf-2(m41 ) alleles without the use of fudr 
 in their cultures . also , the daf-2(e1370 ) proteomic fingerprint made by dong et al . in 
 the absence of fudr identified a limited number of differentially 
 expressed enzymes in carbohydrate , fatty acid , and amino acid metabolism 
 whose expression pattern was confirmed in our study . a wealth of transcriptomics 
 data is available that show extensive changes in metabolic gene expression 
 upon daf-16 activation in the absence of fudr . although there is poor concordance 
 between these studies , our data correspond reasonably well with reported 
 microarray profiles ( but it clashes with sage - generated transcript 
 profiles ) . we also note that the fudr concentration used by the davies 
 study is very high ( 400 m , compared to the more - than - sufficient 
 75 m used in our study ) , and the dose - dependency of fudr was 
 not been taken into account . this 400 
 m concentration , in our hands , is borderline toxic for c. elegans . for all experiments , samples were 
 collected at day 2 of adulthood 
 and freed from dead animals , debris , and bacteria through percoll 
 ( sigma - aldrich ) washing and flotation on a 60% w / w sucrose solution . animals were immediately flash frozen in liquid nitrogen and stored 
 at 80 c . for comparing 
 the metabolic 
 profile between long - lived glp-4(bn2);daf-2(e1370 ) and reference glp-4(bn2 ) daf-16(mgdf50);daf-2(e1370 ) c. elegans worm 
 strains , the accurate mass and time ( amt ) lc  ms proteomics 
 data set was used , which was published earlier by our group in depuydt 
 et al . and is freely available through 
 peptideatlas at http://www.peptideatlas.org/pass/pass00308 ( see supporting information file 1 for 
 details ) . the same criteria as in depuydt et al . were used to determine relative protein abundances , with 
 the exception of the following proteins that included redundant peptides 
 arising from different expression isoforms of the same gene : tps-1 , 
 r11a5.4 , w05g11.6 , t22f3.3 , mdh-1 , h24k24.3 , gpd-1,-2,-3,-4 , gpi-1 , 
 and enol-1 . proteomics data was visualized with mev ( multiexperiment 
 viewer ) , part of the tm4 microarray software suite . pavlidis template matching ( ptm ) was 
 used to rank proteins conforming to an expression pattern of interest . the ptm algorithm allows a data set to be searched for proteins of 
 which the abundance profile matches a user - defined template profile , 
 which is based on the pearson correlation between the template and 
 the proteins in the data set.the kyoto 
 encyclopedia of genes and genomes ( kegg ) pathway and uniprot knowledgebase were 
 used in conjunction with the process of reconstructing the c. elegans metabolic network . gene set - enrichment 
 analysis ( gsea ) was used to determine whether metabolic pathways show 
 statistically significant differential expression as described in 
 detail in subramanian et al . in short , 
 a ranked list of proteins is generated according to their differential 
 abundance level between two experimental groups . an enrichment score 
 ( es ) test statistic is calculated that reflects the degree to which 
 a defined set of proteins is overrepresented at one of the extremes 
 of the entire ranked list . positive es values correspond with enrichment 
 for enzymes ranked to have higher expression levels in daf-2 mutants , whereas negative es values correspond with enrichment for 
 enzymes ranked to have lower expression levels . gene sets eligible 
 for gsea are reported with their nominal p value 
 and false discovery rate ( fdr ) , calculated from 1000 permutations 
 of the experimental group labels . the nominal p value 
 estimates the statistical significance of the es for a single set . the fdr is the estimated probability that an enriched set represents 
 a false positive finding . subramanian et al . have suggested a fdr 
 cutoff of 25% as appropriate to signify biologically significant results . the gsea - p software used for gsea can be found 
 at the broad institute s web site http://www.broadinstitute.org/gsea/index.jsp . elegans nematodes were fixed , and transverse sections 
 were taken according to fonderie et al . electron microscopy was performed using a jeol jem 1010 ( jeol , tokyo , 
 japan ) operating at 60 kv . digitizing of images was performed using 
 a ditabis system ( pforzheim , germany ) . determination 
 of relative fat content in individual worms by oil red o staining 
 was performed according to the protocol described in orourke 
 et al . briefly , age - synchronized worms 
 were sampled at different ages and washed in s - buffer ( 0.05 m k2hpo4 , 0.05 m kh2po4 , and 
 100 mm nacl , ph 7.4 ) . nematodes were fixed in mrwb - buffer ( 160 mm 
 kcl , 40 mm nacl , 14 mm na2egta , 1 mm spermidine - hcl , 0.4 
 mm spermine , 30 mm na - pipes , ph 7.4 , and 0.2% -mercaptoethanol ) 
 containing 1% paraformaldehyde ( pfa ) for 1 h at room temperature with 
 gentle stirring . nematodes were then washed with s - buffer ( ph 7.4 ) 
 to remove pfa and incubated in 60% isopropanol for 15 min at room 
 temperature to dehydrate . isopropanol was removed , and samples were 
 stained overnight in 60% oil red o stain ( 0.5 g/100 ml isopropanol 
 stock solution ) with gentle stirring at room temperature . after the 
 dye was removed by washing in s - buffer containing 0.01% triton , animals 
 were mounted and observed with a reichert - jung polyvar light microscope 
 and imaged with an olympus camedia c-5050 digital camera . corel photopaint 
 ( corel corporation , ottawa , canada ) and fiji were used for image processing and analysis . in short , the red channel 
 of rgb images was converted to 8-bit grayscale , and the resulting 
 pixel values were inverted . the density per worm surface was then determined 
 by calculating the integrated density for each worm , normalized to 
 the total worm surface . equal amounts of 
 worms were flash 
 frozen in liquid nitrogen and stored at 80 c . worms 
 were homogenized by bead beating ( glass beads 0.2480.318 m ) 
 in 50 mm sodium / potassium phosphate buffer , ph 7.0 , at 5000 strokes / min 
 for 30 s. chaps was added at a 1% final concentration to the resulting 
 homogenate , and the mixture was bead - beated again for another 30 s. 
 the mixture was kept on ice for 15 min and centrifuged at 20  000 
 rcf for 8 min at 4 c . the enzymatic activities were assayed spectrophotometrically 
 against the appropriate blanks at 25 c in microtiter plate wells 
 using an infinite m200 multiplate reader ( tecan , mnnedorf , 
 switzerland ) . isocitrate lyase , pyruvate kinase , aconitase , and phosphenolpyruvate 
 carboxykinase activities were assayed as described previously by castelein 
 et al . and references therein . 6-phosphogluconic 
 dehydrogenase activity was assayed according to the method of bergmeyer 
 et al . following the manufacturer s 
 instructions ( sigma no . p4553 ) , with the exception that only half 
 of the described concentrations were used for nadp and 6-phosphogluconate . fumarase activity was assayed following the protocol of racker , with the exception that only 8 mm sodium malate 
 was used ( final concentration ) instead of the 50 mm in racker s 
 work . activity of 3-hydroxyacyl - coa dehydrogenase , catalyzing the 
 conversion of s - acetoacetyl - coa to -hydroxybutyryl - coa with 
 consequent nadh consumption , was monitored according to the method 
 by lynen et al . all enzyme activities 
 were scaled to the protein concentration of the worm extracts , estimated 
 by the bicinchoninic acid protein assay kit ( pierce , rockford , il , 
 usa ) . functional annotation clustering of the proteomics 
 data set showed a significant enrichment 
 of proteins involved in intermediary metabolism among long - lived glp-4(bn2);daf-2(e1370 ) compared to reference glp-4(bn2 ) daf-16(mgdf50);daf-2(e1370 ) c. elegans worm strains ( throughout 
 the text , we will refer to both strains as daf-2 and daf-16;daf-2 , respectively ) . during culturing 
 of the worms , because the use of fudr in c. elegans aging studies warrants caution , we discuss this issue in more detail 
 in the materials and methods ( c. elegans culturing and sampling ) . table 1 shows the result of functional annotation clustering for entries in the kegg database category , which deals mainly with 
 annotation of intermediary metabolism genes . by searching the uniprot protein knowledgebase and on the basis of the available literature , we were able to reconstruct many of the pathways in c. elegans intermediary metabolism from our data 
 set , which is the focus of this article . we detected increased protein abundance of many glycolytic 
 enzymes in daf-2 mutants , including hexokinase ( h25p06.1 ) 
 and phosphofructokinase ( y71h10a.1 ) , two major regulatory sites of 
 glycolysis that catalyze irreversible reactions ( figure 1 ) . the only exception occurs in the final ( irreversible ) step 
 in glycolysis catalyzed by the pyruvate kinase enzymes pyk-1 and pyk-2 , 
 which remain unaltered in daf-2 mutants . we also 
 find increased protein expression of enzymes in gluconeogenesis : fructose-1,6-bisphosphatase 
 ( fbp-1 ) , pyruvate carboxylase ( pyc-1 ) , and two isoforms of phosphoenolpyruvate 
 carboxykinase ( pepck ) , r11a5.4 and w05g11.6 . pepck constitutes a key 
 activator of gluconeogenesis , and elevated 
 levels of pepck in daf-2 mutants confirm earlier 
 reports on pepck transcript and protein levels in insulin / igf-1 signaling - defective 
 mutants . simultaneous activation of glycolysis and 
 gluconeogenesis would lead to a futile cycle , generating heat at the 
 cost of atp hydrolysis . this seems unlikely because daf-2 mutants have lower mass - specific thermogenesis and both pathways 
 are reciprocally regulated . to investigate the 
 relative activities of glycolysis / gluconeogenesis , we measured in 
 vitro enzymatic activities of pepck ( gluconeogenesis ) and pyruvate 
 kinase ( glycolysis ) ( figure 1 ) . surprisingly , 
 in stark contrast with the more than doubled pepck enzyme levels , 
 relative pepck enzymatic activity in daf-2 mutants 
 was only 50% compared to the daf-2;daf-16 reference , indicating that gluconeogenesis in young adult daf-2 mutants is repressed . in contrast , pyruvate kinase 
 activity remains unchanged in the daf-2 mutant , suggesting 
 normal glycolysis activity . ( left ) 
 heat map of proteins 
 that are part of carbohydrate and fermentative metabolism ( base-2 
 logarithmic scale ) . red and green colors indicate a relative 
 increase and decrease in protein content for a particular protein , 
 respectively ( row ) . numbers next to each heat map row denote fold 
 change ( linear ) in abundance for statistically significant proteins . proteins with significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; and * * * , p < 0.0005 . proteins are grouped according to whether 
 protein profiles comply with a preset template as defined by the pavlidis 
 template ( pt ) matching algorithm ( see materials and 
 methods and ref ( 51 ) ) . green and red pt bars indicate lower and higher protein levels 
 among strains , respectively . gray pt bars indicate protein profiles 
 that do not match any obvious expression pattern ( see materials and methods for details ) . gene set enrichment analysis 
 ( gsea ) allows for the assessment of whether a group of proteins ( e.g. , 
 the set of genes that are part of carbohydrate metabolism in our data 
 set ) shows statistically significant differential expression as a 
 whole ( see materials and methods and ref ( 54 ) for more details ) . es , 
 enrichment score from gene set enrichment analysis of the metabolic 
 pathway ; p , nominal p value of the 
 es ; fdr , false discovery rate . the color of protein names matches their 
 expression profile ( green , down ; red , up ; and black , not significant ) . green arrows depict the possible route of malate production via the 
 reverse action of phosphoenolpyruvate carboxykinase and malate dehydrogenase 
 ( see results and discussion ) . inset bar graphs 
 represent relative enzyme activity of phosphoenolpyruvate carboxykinase 
 ( pepck ) and pyruvate kinase ( pk ) . ( 1 ) glycogen synthase , ( 2 ) glycogen 
 phosphorylase , ( 3 ) hexokinase / glucokinase , ( 4 ) phosphohexoseisomerase , 
 ( 5 ) phosphofructokinase , ( 6 ) fructose-1,6-bisphosphate , ( 7 ) aldolse , 
 ( 8) triose phosphate isomerase , ( 9 ) glyceraldehyde-3-phosphate dehydrogenase , 
 ( 10 ) phosphoglycerate kinase , ( 11 ) phosphoglycerate mutase , ( 12 ) enolase , 
 ( 13 ) pyruvate kinase , ( 14 ) pyruvate dehydrogenase complex ( e1 , pyruvate 
 dehydrogenase ; e2 , dihydrolipyl transacetylase ; and e3 , dihydrolipoyl 
 dehydrogenase ) , ( 15 ) pyruvate carboxylase , ( 16 ) mitochondrial / cytosolic 
 malate dehydrogenase , ( 17 ) malate--ketoglutarate antiporter 
 ( malate shuttle ) , ( 18 ) phosphoenolpyruvate carboxykinase , ( 19 ) phosphoglycerate 
 dehydrogenase , ( 20 ) lactate dehydrogenase , ( 21 ) alcohol dehydrogenase , 
 ( 22 ) aldehyde dehydrogenase , ( 23 ) acetyl - coa synthetase , and ( 24 ) 
 trehalose-6-phosphate synthase . glu-6p , glucose-6-phosphate ; fru-6p , 
 fructose-6-phosphate ; fru-1,6-bp , fructose 1,6-bisphosphate ; dhap , 
 dihydroxyacetone phosphate ; g3p , glyceraldehyde-3-phosphate ; 3pg , 
 3-phosphoglycerate ; pep , phosphoenolpyruvate ; and ooa , oxaloacetate . we also found significantly elevated 
 protein levels of both glycogen 
 synthase-1 ( gsy-1/y46g5a.31 , glycogenesis ) and the orthologue of human 
 glycogen phosphorylase ( t22f3.3 , catalyzing the first step in glycogenolysis ) 
 in daf-2 adults ( figure 1 ) . ultrastructural and biochemical studies showed daf-2 mutants and dauers store significantly more glycogen in their intestinal 
 and hypodermal cells compared to wild - type nematodes . electron micrographs taken from our samples also confirmed the presence 
 of high amounts of glycogen in hypodermal cells and body - wall muscles 
 of daf-2 mutants ( figure 2 ) , consistent with increased glycogen synthase activity . the fermentation 
 of stored glycogen constitutes a major source of energy during anoxia / hypoxia 
 and is determinate for nematode survival under these conditions . consistently , the increased survival of daf-2 mutants 
 during anoxic insult is dependent on its high glycogen levels . in addition , in dauer larvae , high glycogen reserves likely serve 
 to maintain their high degree of motility during the first few weeks 
 after dauer formation , which is in agreement 
 with the expression of glycogen phosphorylase in c. 
 elegans muscles . we found significantly increased protein levels of tps-1 , one of 
 two c. elegans trehalose-6-phosphate 
 synthase genes responsible for trehalose biosynthesis , consistent 
 with increased trehalose levels in iis - defective c. 
 elegans mutants and dauers and daf-16-dependent transcription regulation . importantly , tps-1 and tps-2 were found to be required , in part , for daf-2 longevity . besides its 
 role in carbohydrate storage and transport , trehalose also has extensive cytoprotective functions , most probably 
 by stabilizing the proteome and lipid membranes under various stress 
 conditions , including cold , heat , dehydration , hypoxic , and oxidative 
 insult . elevated levels of trehalose are associated with 
 life - span extension in c. elegans(74 ) and also render daf-2 dauers 
 resistant to extreme desiccation . transmission 
 electron microscopy images of transversal midbody 
 cross sections of day 2 adult nematodes . ( left ) daf-2 mutants exhibit relatively large amounts of glycogen ( delineated 
 in green ) and fat droplets ( orange arrowhead ) stored inside hypodermal 
 and intestinal cells compared to the reference strain . daf-2 mutants also show a smaller cross - sectional diameter compared to 
 the daf-16;daf-2 reference . quantitative 
 determination of worm length , diameter , and volume of both daf-2 and daf-2;daf-16 worms is shown in supporting information file 
 2 . ( right ) high - magnification image ( 25  000 ) of 
 a small section of daf-2 striated muscle tissue showing 
 the tight organization of glycogen and mitochondria close to the myofilament 
 lattice . our data show a significant upregulation 
 in the expression of three 
 enzymes predicted to be part of the pentose phosphate pathway , including 
 gspd-1 , the c. elegans orthologue of 
 glucose-6-phosphate dehydrogenase ( g6pdh ) , which catalyzes the first 
 step in the pentose phosphate pathway and converts nadp into nadph ( figure 3 ) . however , we found 
 no significant difference in the activity of this pathway by measuring 
 the enzymatic activity of 6-phosphogluconate dehydrogenase ( figure 3 ) . nonetheless , temporal activation of this pathway 
 could be involved in the increased oxidative stress resistance of 
 the daf-2 mutant because the rerouting of carbohydrate 
 intermediates from glycolysis to the pentose phosphate shunt has been 
 shown to be part of a coordinated response to oxidative stress by 
 maintaining high cytoplasmatic nadph / nadp ratios in c. elegans , drosophila , and mammals . ( right ) heat map ( base-2 logarithmic 
 scale ) and ( left ) schematic overview of proteins that are part of 
 the pentose phosphate pathway . numbers next to each heat map row denote 
 fold change ( linear ) in abundance for statistically significant proteins . proteins with significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; * * * , p < 0.0005 . ( 1 ) glucose-6-phosphate degydrogenase , ( 2 ) 
 gluconolactone hydrolase , ( 3 ) 6-phosphogluconate dehydrogenase , ( 4 ) 
 ribulose-5-phosphate-3-epimerase , ( 5 ) ribose-5-phosphate - ketoisomerase , 
 ( 6 ) transketolase , ( 7 ) transaldolase , and ( 8) ribokinase . inset bar 
 graph represents relative enzyme activity of 6-phosphogluconate dehydrogenase 
 ( 6pgdh ) . a strong upregulation was 
 found of several putative alcohol 
 dehydrogenases and aldehyde dehydrogenases involved in alcohol metabolism 
 ( figure 1 ) . alcohol dehydrogenases include 
 the predicted sorbitol dehydrogenases sodh-1 and its homologue r04b5.5 , 
 the ribitol - dehydrogenase domain containing protein f20g2.2 . the actual substrate of these alcohol dehydrogenases is unknown 
 at present , but sodh-1 appears capable of metabolizing 
 ethanol . sodh-1 is known to be activated 
 by daf-16 , and higher mrna levels of sodh-1 were also found 
 in long - lived worms cultured axenically . furthermore , sodh-1 was the most upregulated ( 1200% ) spot in 2d - dige 
 gels from staphylococcus aureus - treated 
 worms but not in aeromonas hydrophila - treated worms . therefore , the biological role 
 of this enzyme may reach further than fermentative metabolism . indeed , 
 some alcohol dehydrogenase enzymes are also capable of neutralizing 
 cytotoxic aldehydes originating from lipid peroxidation , including 
 4-hydroxynonenal ( 4-hne ) . alh-1 is an orthologue 
 of mitochondrial aldehyde dehydrogenase 2 in humans that possibly 
 catalyzes the conversion of acetaldehyde , resulting from the oxidation 
 of ethanol , into acetate . although we found strongly increased abundance 
 of alh-1 in daf-2 mutants , metabolomic profiles of daf-2 mutants indicate a significant drop in acetate content 
 in iis mutants . alh-1 is also involved 
 in the detoxification of toxic aldehyde byproducts of alcohol metabolism 
 or lipid peroxidation . more specifically , c. elegans alh-1 was shown capable to oxidize 4-hne 
 further to the lesser reactive 4-hna ( 4-hydroxynon-2-enoic acid ) , 
 similar to mammalian aldehyde dehydrogenase . on the basis of these reports , the activation of alcohol - fermenting 
 enzymes in daf-2 mutants may well be part of a coordinated 
 stress response system that boosts the nematodes resistance to toxic 
 byproducts of lipid and intrinsic alcohol metabolism . a general increase was found of the citric acid 
 cycle enzymes , including citrate synthase , the isocitrate dehydrogenase 
 complex , and the -ketoglutarate dehydrogenase complex , which 
 was the main sites of regulation within this cycle ( figure 4 ) . this increase is accompanied with higher activity 
 of fumarase and aconitase in vitro ( figure 4 ) . transcript profiles of daf-2 mutants did not 
 show any significant change in mrna levels for citric acid cycle enzymes . expression of these genes therefore may be regulated post - transcriptionally 
 or be the result of lowered turnover of these enzymes or the mitochondria 
 in which they reside . here , daf-2 energy metabolism 
 appears to differ from that of the dauer larva , which is characterized 
 by both reduced gene expression and enzymatic activity of citric acid 
 cycle genes . however , aco-1 is thought to be a cytosolic aconitase involved 
 in the regulation of cellular iron concentrations and is therefore 
 not part of the citric acid cycle . similarly , 
 idh-1 is a predicted cytosolic nadp - dependent isocitrate dehydrogenase 
 ( idh ) and shows no upregulation in daf-2 . we note here that cytosolic nadp - dependent idh 
 activity is strongly increased in the daf-2 mutant 
 ( data not shown ) . ( left ) heat map 
 ( base-2 
 logarithmic scale ) and ( right ) schematic overview of enzymes in mitochondrial 
 intermediary metabolism . numbers next to each heat map row denote 
 fold change ( linear ) in abundance for statistically significant proteins . proteins with significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; and * * * , p < 0.0005 . inset bar graphs represent relative enzymatic 
 activities of aconitase , fumarase , and isocitrate lyase . ( 1 ) pyruvate 
 dehydrogenase complex , ( 2 ) citrate synthase , ( 3 ) aconitase , ( 4 ) isocitrate 
 dehydrogenase complex , ( 5 ) -ketoglutarate dehydrogenase complex 
 ( e1 , oxoglutarate dehydrogenase ; e2 , dihydrolipoyl succinyl transferase ; 
 and e3 , dihydrolipoyl dehydrogenase ) , ( 6 ) succinate thiokinase , ( 7 ) 
 succinate dehydrogenase , ( 8) fumarase , ( 9 ) malate dehydrogenase , ( 10 ) 
 pyruvate carboxylase , ( 11 ) isocitrate lyase , ( 12 ) malate synthase , 
 ( 13 ) mitochondrial tri / dicarboxylate carrier protein , ( 14 ) gdh-1 , 
 glutamate dehydrogenase ; w07e11.1 , glutamate synthase , ( 15 ) malate--ketoglutarate 
 antiporter ( malate shuttle ) , and ( 16 ) aspartate aminotransferase . 
 succ , succinate ; succ - coa , succinyl - coa ; aco , aconitase ; and fum , 
 fumarase . the glyoxylate shunt allows the synthesis of carbohydrates 
 via gluconeogenesis from acetyl - coa obtained from fatty acid -oxidation 
 and is generally known in plant and microorganism biochemistry . nematodes 
 also have been shown to contain an active glyoxylate shunt , which is required for the full longevity phenotype of daf-2 and several etc - defective mutants ( mit mutants ) as well as the mitochondrial 
 mutant clk-1(qm30 ) . in c. elegans , the 
 key glyoxylate cycle enzymes , isocitrate lyase and malate synthase , we observed a very strong increase 
 in icl-1 abundance , consistent with previous transcriptional evidence . consistently , in vitro activity 
 of both isocitrate lyase and malate synthase is increased in daf-2 ( figure 4 ) and age-1(98 ) mutants , respectively . one - carbon 
 metabolism involves the storage and utilization 
 of one - carbon moieties in anabolic and transmethylation - based regulatory 
 pathways . the carrier of activated one - carbon 
 fragments is tetrahydrofolate ( thf ) , an essential water - soluble derivative 
 of vitamin b9 involved in the formation of nucleotides for dna synthesis , 
 the metabolism of certain amino acids , and the source of methyl groups 
 for s - adenosyl methionine ( sam ) . a particularly 
 strong increase was found in the protein levels of the putative c. elegans aicar formyltransferase ( c55f2.1 ) and 
 c1-thf synthase ( k07e3.4 ) in daf-2 , both of which 
 are involved in the interconversion of one - carbon derivates of thf 
 ( figure 5 ) . serine is the major donor of one - carbon 
 in the form of n , n - ch2-thf through 
 its catabolism via glycine . we found 
 increased levels in the daf-2 mutant of phosphoglycerate 
 dehydrogenase ( c31c9.2 ) , which catalyzes the first step in glucose - derived 
 serine biosynthesis , and f25b4.1 , the c. elegans orthologue of the glycine cleavage system t - protein , an aminomethyltransferase 
 that catalyzes the actual transfer of methylene carbon from the decarboxylated 
 glycine to tetrahydrofolate ( thf ) . taken together , our results suggest 
 changes in thf - based anabolism in daf-2 mutants , 
 possibly fueled via de novo synthesis of serine and subsequent oxidation 
 over glycine to supply one - carbon units . ( right ) heat map ( base-2 logarithmic 
 scale ) of enzymes part of c. elegans one - carbon metabolism . ( left ) shematic overview of one - carbon metabolism 
 involving both the single - carbon carriers tetrahydrofolate and s - adenosylmethionine . 
 numbers next to each heat map row denote fold change ( linear ) in abundance 
 for statistically significant proteins . proteins with significantly 
 changed abundance levels are denoted * , p < 0.05 ; 
 * * , p < 0.005 ; and * * * p < 
 0.0005 . ( 1 ) phosphoglycerate dehydrogenase ( c31c9.2 ) , phosphoserine 
 aminotransferase ( f26h9.6 ) , ( 2 ) serine hydroxymethyl transferase , 
 ( 3 ) glycine cleavage system t - protein ( aminomethyltransferase ) , ( 4 ) 
 aicar formyltransferase ( c55f2.1 ) , c1-thf - synthase ( k07e3.4 ) , ( 5 ) 
 s - adenosyl methionine synthase , ( 6 ) methionine synthase , ( 7 ) s - adenosylhomocysteinase , 
 ( 8 and 10 ) cystathionine -synthase / cysteine synthase , and ( 9 ) 
 cystathionine -lyase . in contrast to thf - metabolism , the biosynthetic 
 pathway of sam , 
 the other main carrier for single - carbon units , appears repressed 
 in daf-2 mutants ( figure 5 ) . s - adenosylmethionine synthase activity produces sam by catalyzing 
 the condensation of methionine with atp . protein levels of this enzyme 
 ( sams-1 ) are strongly reduced in the daf-2 mutant . demethylation of sam results in the production of s - adenosylhomocysteine 
 ( sah ) , which is a strong inhibitor of all sam - dependent transmethylation 
 reactions . s - adenosylhomocysteinase 
 catalyzes the hydrolytic cleavage of sah into homocysteine plus adenosine , 
 but expression of this enzyme ( ahcy-1 ) at the protein level also appears 
 significantly reduced in daf-2 mutants . homocysteine 
 lies at the cross - point of two competing pathways : it is either used 
 to form methionine ( for sam synthesis ) via the remethylation pathway 
 or to form cysteine via the transsulfuration pathway . transsulfuration of homocysteine to cysteine 
 is catalyzed by two enzymes : cystathione -synthase ( cbs ) , which 
 forms cystathionine from homocysteine and serine , and cystathionine 
 -lyase , which converts cystathionine to cysteine , -ketobutyrate , 
 and ammonia . the putative c. elegans orthologue of cbs ( k10h10.2 ) is strongly upregulated in daf-2 , suggesting increased funneling of homocysteine toward 
 cysteine synthesis , away from remethylation of homocysteine into methionine . 
 because cysteine is normally readily available from diet , the major 
 role of the transsulfuration pathway in animals is thought to be the 
 regulation of sam levels by controlling methionine / homocysteine degradation 
 and not cysteine biosynthesis per se thus , high cbs activity in daf-2 mutants is an 
 additional strong indication of repressed sam synthesis . in contrast , 
 the putative c. elegans cystathionine 
 -lyase orthologue cth-1 is downregulated , and its paralogue , 
 cth-2 , shows no change in expression . because there is no apparent 
 alternate route for the further processing of cystathionine , it seems 
 that reduced cth-1 expression aims to temper the increased flux through 
 cbs . taken together , our data strongly suggest a reduced flux through 
 the methyl cycle in daf-2 mutants . in mammals , fatty acid synthesis is 
 initiated by citrate transport across the mitochondrial membranes 
 into the cytoplasm via tricarboxylate carriers ( tic ) in exchange with 
 malate . once in the cytosplasm , citrate 
 is cleaved by atp - citrate lyase ( acl ) into acetyl - coa and oxaloacetate . this enzyme is the main source for cytosolic 
 acetyl - coa for fatty acid synthesis and represents a major link between 
 the citric acid cycle and lipogenesis . in mammals , expression of this mitochondrial citrate carrier is 
 activated by insulin signaling , and inhibition 
 of acl was shown to reduce fatty acid synthesis significantly and 
 to increase fatty acid -oxidation . we found strongly increased abundance of the c. elegans mitochondrial tic ( k11h3.3 ) and acl ( d1005.1 ) in daf-2 mutants compared to the reference strain , suggesting increased lipogenesis 
 ( figure 6b ) . paradoxically , we found a strong 
 suppression of the predicted c. elegans fatty acid synthase , fasn-1 , in daf-2 adults . this 
 suggests that in adult daf-2 worms fatty acid synthesis 
 is attenuated even in the presence of plenty of substrates . we note 
 that the c. elegans genome contains 
 at least three other putative fatty acid synthase genes ( c41a3.1 , 
 f10g8.9 , and f32h2.6 ) , which could also be important determinants 
 of fatty acid synthesis . fatty acid metabolism . ( right ) heat 
 map and ( left ) schematic overview of enzymes implicated in fatty acid 
 synthesis . ( 1 ) citrate synthase , ( 2 ) mitochondrial tricarboxylate / dicarboxylate 
 carrier protein ( citrate transport protein ) , ( 3 ) atp - citrate lyase , 
 and ( 4 ) fasn-1 , fatty acid synthase ; elo-1 , long - chain fatty acid 
 elongase ; dhs-25 , mitochondrial beta - ketoacyl - acp reductase ; fat-6 
 and fat-2 encode fatty acyl desaturases . 3-hadh , 3-hydroxyacyl - coa 
 dehydrogenase . ( right ) heat map and 
 ( left ) schematic overview of detected fatty acid -oxidation 
 enzymes . numbers next to each heat map row denote fold change ( linear ) 
 in abundance for statistically significant proteins . proteins with 
 significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; and * * * , p < 0.0005 . ( 1 ) acyl - coa synthetase , ( 2 ) carnitine 
 o - acyltransferase , ( 3 ) acyl - coa dehydrogenase , ( 4 ) trans-2-enoyl - coa 
 hydratase , ( 5 ) 3-hydroxyacyl - coa dehydrogenase , ( 6 ) thiolase , ( 7 ) 
 acetyl - coa acetyltransferase ( thiolase ) , ( 8) 3-hydroxy-3-methylglutaryl - coa 
 ( hmg - coa ) lyase , and ( 9 ) succinyl - coa - acetoacetate coa transferase . at the same time , we found increased 
 expression in daf-2 adults of many fatty acid -oxidation 
 proteins ( figure 6a ) as well as an enzyme involved 
 in glycerol catabolism 
 ( t24g3.4 ) and a choline / carnitine palmitoyl transferase ( b0395.3 ) . the latter is possibly responsible for the translocation of long - chain 
 fatty acids into the mitochondrial matrix prior to oxidation . we also 
 found increased protein levels of maoc-1 , a predicted peroxisomal 
 fatty acid -oxidase enzyme . to assess whether fatty acid -oxidation 
 is increased in the daf-2 mutant , we measured the 
 in vitro activity of 3-hydroxyacyl coa dehydrogenase , which catalyzes 
 the oxidation of l-3-hydroxyacyl coa , and found that its activity 
 was unchanged ( figure 6 ) . nonetheless , the 
 coordinated upregulation of most -oxidation enzymes clearly 
 indicates that daf-2(e1370 ) worms tend to rely more 
 on internal fat stores compared to wild type to fuel their energy 
 needs . the formed acetyl - coa is either further oxidized to co2 and water in the citric acid cycle or , alternatively , acetyl - coa 
 is converted into succinate and malate via the upregulated glyoxylate 
 cycle for use in gluconeogenesis . in addition , glyceraldehyde-3-phosphate 
 derived from the catabolism of glycerol can be fed into the glycolytic 
 pathway for energy generation . we note that the relative increase 
 in -oxidation proteins found in our study appears not to be 
 reflected at the transcript level , as both sage- and microarray - based 
 transcript profiling detected no change in the mrna expression of 
 -oxidation enzymes in daf-2 mutants . on the basis of our results , we hypothesize that daf-2 mutants switch from fat synthesis and storage during development 
 and early adulthood to controlled lipid breakdown for the remainder 
 of life , mimicking the dauer larva . to 
 test this hypothesis , we determined the age - dependent alterations 
 in fat content of daf-2 mutants and daf16;daf-2 reference worms by staining with the fat - soluble 
 dye oil red o ( figure 7 ) . we combined these results with data on the feeding behavior 
 of the same iis mutants ( but lacking the glp-4 mutation ) 
 collected over many aging series that were run independently in liquid 
 cultures in our lab . as expected , our data show that daf-2 mutants accumulate significantly more fat than the daf-16;daf-2 reference at day 2 of young adulthood . although 
 we noticed a sudden drop in oil red o intensity at day 6 of adulthood , daf-2 mutants were able to maintain relatively high fat 
 content , whereas in reference worms , fat levels decreased steadily 
 with age . this result is remarkable given that daf-2(e1370 ) mutants exhibit a clear eat phenotype ( reduced food uptake ) that 
 manifests itself during early adulthood ( figure 7 ) . therefore , daf-2 fat metabolism is reminiscent 
 to the nonfeeding dauer larva , which depends on the slow and controlled 
 release of energy and anabolic intermediates via -oxidation 
 of internal fat stores for its survival . nematodes 
 were stained with oil red o to determine the relative fat content 
 in individual worms . for each time point , the amount of e. coli in grams required each day to maintain a constant turbidity ( od550 = 1.8 ) in culture medium ( liquid cultures ) was used as 
 a measure to assess the feeding behavior as a function of age . we 
 note that zero values for oil red o intensity do not indicate that 
 these animals are devoid of fat but merely indicate the detection 
 limit of this dye for quantifying fat levels . the carbon atoms of propionate are recycled into the citric acid 
 cycle component succinyl - coa via an evolutionary conserved multienzyme 
 pathway that was shown to be fully functional in c. 
 elegans ( figure 8) . we detected significantly increased abundance of the propionate 
 catabolic enzymes methylmalonyl - coa racemase ( mce-1 ) , methylmalonyl - coa 
 mutase ( mmcm-1 ) , and the alpha ( pcca-1 ) and beta ( pccb-1 ) subunits 
 of propionyl - coa decarboxylase in daf-2 adult animals 
 ( figure 8) . ( right ) heat map 
 and ( left ) schematic overview 
 of propionate metabolism enzymes . numbers next to each heat map row 
 denote fold change ( linear ) in abundance for statistically significant 
 proteins . proteins with significantly changed abundance levels are 
 denoted * , p < 0.05 ; * * , p < 
 0.005 ; and * * * , p < 0.0005 . ( 2 ) propionyl - coa 
 carboxylase , ( 3 ) methylmalonyl - coa racemase , and ( 4 ) methylmalonyl - coa 
 isomerase . propionate is either activated 
 to propionyl - coa by an appropriate 
 acyl - coa synthetase or originates directly from the catabolism of 
 the branched amino acids valine and isoleucine or -oxidation 
 of odd - chain - length fatty acids . activation 
 of this catabolic pathway would thus indicate increased catabolism 
 of fatty acids or branched chain amino acids in the daf-2 mutant . recent metabolomic profiling studies 
 of the daf-2 mutant have revealed an increase in 
 the concentration of free amino acids , in particular of branched - chain 
 amino acids ( bcaas ) , suggesting altered amino acid metabolism in this 
 mutant . in accordance , we report an increase in 
 the level of glutamate dehydrogenase ( gdh-1 ) , an enzyme essential 
 in amino acid breakdown that catalyzes the deamination of glutamate 
 into -ketoglutarate ( figure 4 ) . we also 
 find a dramatic increase ( 16.6-fold ) in the putative glutamate synthase 
 w07e11.1 , which catalyzes the formation of glutamate from glutamine . 
 in mammals , glutamine serves as a universal transporter of nitrogen 
 and is the most common free amino acid in human blood plasma . interestingly , 
 martin et al . showed that the level of glutamine and glutamate were 
 increased and decreased , respectively , in the daf-2 mutant . we speculate that the strong 
 increase of glutamate synthase in combination with glutamate dehydrogenase 
 is indicative of increased amino acid catabolism in the daf-2 mutant , fueling the tca cycle . also consistent with increased 
 amino acid catabolism is the clear increase of most tyrosine catabolic 
 enzymes , including hpd-1 ( figure 9a ) . our result is consistent with an earlier proteomics 
 report where fumarylacetoacetate hydrolase ( k10c2.4 ) was found to 
 be significantly upregulated in the daf-2 mutant . moreover , the transcript level of cytosolic 
 tyrosine aminotransferase ( tat , f24d1.2 ) , which catalyzes the first 
 step in tyrosine catabolism , was found to be highly enriched in dauers . ( right ) heat map and ( left ) schematic 
 overview of tyrosine catabolism enzymes . numbers next to each heat 
 map row denote fold change ( linear ) in abundance for statistically 
 significant proteins . proteins with significantly changed abundance 
 levels are denoted * , p < 0.05 ; * * , p < 0.005 ; and * * * , p < 0.0005 . ( 1 ) ( tyrosine / aspartate ) 
 transaminase , ( 2 ) p - hydroxyphenylpyruvate hydroxylase , 
 ( 3 ) homogentisate oxidase , ( 4 ) maleylacetoacetate isomerase , and ( 5 ) 
 fumarylacetoacetate hydrolase . ( 1 ) branched - chain 
 aminotransferase , ( 2 ) branched - chain -keto acid decarboxylase 
 complex , and ( 3 ) ivd-1 , isovaryl - coa dehydrogenase ; b0250.5 , 3-hydroxyisobutyrate 
 dehydrogenase . the level of cellular 
 tyrosine is thought to be determined by insulin 
 signaling through modulation of tyrosine catabolism . like in mammals , c. elegans tat and 
 the gluconeogenic enzyme pepck both contain an insulin - responsive 
 element ( ire ) in the promoter region , conferring insulin - induced inhibition 
 of expression . thus , rather surprisingly and in contrast with our 
 and others proteome data , reduced insulin 
 signaling in c. elegans has been associated 
 with the transcriptional repression of tyrosine catabolic enzyme hpd-1 . this discrepancy between 
 transcript and protein levels indicates complex regulation of tyrosine 
 catabolism enzymes . in contrast to tyrosine , we found reduced 
 concentrations of bcaa 
 catabolic enzymes , including the branched - chain aminotransferase enzyme 
 ( bcat-1 ) , in the daf-2 mutant ( figure 9b ) , in line with transcriptional evidence and paralleled by 
 increased levels of bcaas as reported previously by us and others . finally , 
 we also found increased abundance of the aspartate aminotransferases 
 t01c8.5 and c14f11.1 ( figure 4 ) , which catalyze 
 the interconversion of aspartate and -ketoglutarate into oxaloacetate 
 and glutamate . an increased protein level of 
 several subunits of complexes i and ii and to a lesser extent complexes 
 iii and v was found in daf-2 mutants ( figure 10 ) . remarkably , protein expression of cytochrome 
 c oxidase ( complex iv ) was repressed in daf-2 . cytochrome 
 c oxidase catalyzes the terminal reduction of one o2 molecule 
 to two molecules of h2o with the additional translocation 
 of four protons to the intermembrane space . if complex iv activity 
 is reduced in daf-2 animals , then it can be expected 
 that less oxygen is consumed compared to wild type . a slightly reduced 
 mass - specific metabolic rate , as measured by carbon dioxide gas respirometry , 
 has been reported in daf-2 mutants . however , no decrease in oxygen consumption between daf-2(e1370 ) and wild - type or daf-16(m26);daf-2(e1370 ) has been observed in locomotory active 
 worms . carbon dioxide 
 gas respirometry is expected to leave the worms practically undisturbed 
 on a spot of e. coli bacteria , whereas direct oxygen consumption measurements 
 in liquid require extensive stirring , stimulating worm movement and 
 thus metabolic activity , which could explain the discrepancy in reported daf-2(e1370 ) metabolic rates . in addition , adult daf-2 mutants cultured on undisturbed 
 agar plates appear to be rather inactive when compared to the reference 
 strain , but they retain the ability to move freely 
 in response to , for example , mechanical stimulation , similar to dauers.daf-2 mutants are also characterized 
 by reduced food uptake ( as was discussed earlier ) and low heat output . therefore , we suggest that daf-2 standard metabolism , like dauers , is hypometabolic , but the metabolic 
 rate is readily raised when necessary , and this is ensured by the 
 availability of large amounts of fat and glycogen and the increased 
 levels of enzymes of core intermediary metabolism . schematic representation of the 
 respiratory chain ( complexes i  iv ) together with heat maps 
 of detected subunits . numbers next to each heat map row denote fold 
 change ( linear ) in abundance for statistically significant proteins . proteins with significantly changed abundance levels are denoted * , p < 0.05 ; * * , p < 0.005 ; and * * * , p < 0.0005 . green arrows indicate the possible alternative 
 flux of electrons via rhodoquinone ( rq ) ( instead of ubiquinone , uq ) 
 over complex ii using fumarate as the final electron acceptor . we also found elevated 
 protein levels of the electron - transferring 
 flavoproteins ( etf ) f27d4.1 and etf - dehydrogenase ( let-721 ) in daf-2 mutants , although the latter upregulation was not 
 statistically significant . these proteins transfer the electron pair 
 of fadh2 , resulting from the -oxidation of acyl - coa , 
 to the flavo - iron  sulfur protein etf - dehydrogenase , which in 
 turn reduces coenzyme q in the electron transport chain in the mitochondrial 
 inner membrane . this is in line with 
 the enhanced fatty acid oxidation pathway in daf-2 mutants , as described above . interestingly , mai-2 , a member 
 of the f1 atpase inhibitor ( if1 ) 
 family , showed a modest but significant upregulation in daf-2 adults . if1 inhibitor proteins prevent the deleterious hydrolytic 
 consumption of atp by complex v under anoxic conditions when the electrochemical 
 gradient across the inner membrane collapses . this finding is consistent with an earlier report of elevated 
 transcriptional expression of if1 inhibitors in daf-2 mutants and dauers . increased expression 
 of mai-2 may , in part , explain the ability of daf-2 to withstand prolonged hypoxic insult better . we 
 found increased levels of most glycolytic enzymes ( with the exception 
 of pyruvate kinase ) and gluconeogenic enzymes , including pepck . together 
 with an experimentally confirmed increased flux of carbon through 
 the glyoxylate shunt and high levels of the storage sugars glycogen 
 and trehalose , it was assumed gluconeogenic activity is high in the daf-2 mutant . however , despite high pepck enzyme levels , 
 a significant decrease of in vitro pepck activity was detected for 
 the young adult daf-2 , implying reduced gluconeogenesis . in contrast , pyruvate kinase activity was similar between daf-2 and the daf-16;daf-2 reference , suggesting glycolytic flux is not drastically altered 
 in daf-2 . in the event pyruvate kinase activity would 
 be a limiting factor , the conversion of pep into pyruvate is prevented , 
 favoring glucose production via gluconeogenesis . alternatively , as was suggested by oriordan and burnell , 
 pepck potentially acts in reverse , driving co2 fixation 
 via pep and oxaloacetate with the subsequent formation of malate via 
 malate dehydrogenase , as is seen in parasitic helminthes ( green arrows in figure 1 ) . although increased levels of malate have been found in the daf-2 mutant , malate synthesis 
 is most likely also increased via the upregulated glyoxylate shunt . helminths such as fasciola hepatica and ascaris suum are able to survive 
 under anaerobic conditions in their host environment by generating 
 atp via the malate dismutation pathway . the malate dismutation pathway appears to be conserved in c. elegans and is thought to play an essential role 
 in dauer energy metabolism and survival . furthermore , rea and johnson hypothesized that a shift from aerobic 
 to mitochondrial anaerobic respiration underlies daf-2 longevity and possibly most , if not all , other life - span extending 
 mutations , including mit mutants . in 
 this pathway , glycolytic phosphoenolpyruvate is converted into oxaloacetate 
 by pepck , which is then further reduced by cytosolic malate dehydrogenase 
 to produce malate . once malate is transported into ( partially ) anaerobically 
 functioning mitochondria , it is further catabolized to generate atp 
 from the proton gradient generated across the inner mitochondrial 
 membrane by electron transport , but fumarate is utilized as the terminal 
 electron acceptor instead of o2 , producing succinate instead 
 of h2o . we speculate the strong increased abundance of 
 the putative mitochondrial tricarboxylate carrier k11h3.3 could be 
 responsible for the increased import of cytosolic malate into the 
 mitochondrial matrix in exchange for citrate ( figure 6b ) . as was noted earlier , strong activation of the glyoxylate 
 shunt in daf-2 mutants could also constitute a considerable 
 source of intramitochondrial malate to drive anaerobic respiration . although we were able to quantify several enzymes involved in malate 
 dismutation , including malic enzyme ( y48b6a.12 ) , asc ( c05c10.3 ) , and 
 stk subunits ( c50f7.4 , f47b10.1 , and f23h11.3 ) , none of them showed 
 any significant alteration in protein expression ( data not shown ) . however , we detected a clear upregulation of all propionate catabolism 
 enzymes in daf-2 . propionate is one of the principal 
 waste products ( along with acetate and succinate ) of malate dismutation and was previously found to be present 
 in higher levels in the daf-2 mutant . lastly , the conspicuous downregulation of complex 
 iv components but increases in complexes i  iii could indicate 
 that mitochondrial respiration in daf-2 relies less 
 on oxygen as the terminal electron acceptor to maintain the proton 
 gradient . taken together , these results support activation of the 
 malate dismutation pathway in daf-2 , a hypothesis 
 that merits further study . protein expression 
 of enzymes in thf anabolism and the sam biosynthetic pathway were 
 increased and decreased in the daf-2 mutant , respectively . interestingly , previous proteomic and metabolomic analysis of the pept-1 mutant revealed a very similar decrease in the abundance 
 of one - carbon metabolism enzymes , including s - adenosylmethionine synthase 
 ( sams-1 ) and s - adenosylhomocysteine hydrolase ( ahcy-1 ) , and changes 
 in methionine ( decreased ) and homocysteine ( increased).pept-1 encodes an intestinal 
 peptide transporter required for the absorption of amino acids in 
 form of di- and tripeptides from the environment , and its mutation 
 further extends daf-2 lifespan by approximately 60% . in contrast with our findings , however , the 
 same group was unable to find these changes for the daf-2 mutant . nevertheless , reduced sams-1 protein levels in daf-2 have been reported before , and daf-2 is characterized as a strong transcriptional repression 
 of pept-1 . sams-1 was 
 previously discovered to be a mediator of the dr response , possibly 
 downstream of tor signaling , and is required for a normal protein 
 synthesis rate . in addition , metformin - induced 
 lifespan extension in c. elegans was 
 recently shown to result from methionine restriction following disruption 
 of folate metabolism in the e. coli food source . therefore , one attractive 
 hypothesis is that alterations in the methionine cycle results in 
 reduced protein synthesis in the daf-2 mutant , the 
 latter of which has been experimentally confirmed by us and others . by 
 extension , we postulate decreased protein synthesis in the pept-1 mutant and metformin - treated c. elegans , which could conceivably underlie their longevity phenotype . moreover , because rnai knockdown of pept-1 results 
 in a significant decrease in the intracellular pool of free amino 
 acids , a concomitant decrease in protein synthesis is to be expected . several metabolomic profiling studies have revealed changes in 
 the metabolism of amino acids in the daf-2 mutant . in particular , changes in bcaa metabolism have been linked to daf-2 , but it remains unclear how these changes contribute 
 to daf-2 longevity , if at all . in mammals , bcaas 
 ( leucine in particular ) are well - known to stimulate protein synthesis 
 through activation of tor ( target of rapamycin ) kinase . however , because we recently showed that protein 
 synthesis is repressed in the daf-2 mutant , such 
 action by bcaas would thus have to be restricted to certain tissues , 
 such as body - wall muscles , which we have shown previouslty are preserved 
 from degradation in daf-2 . more recently , increased levels of aromatic amino acids ( phe and 
 tyr ) and especially bcaa degradation products , such as glu , ala , and 
 c3 and c5 acylcarnitines , were found to be strongly associated with 
 insulin resistance and type 2 diabetes in humans . it was suggested that accumulation of these incompletely 
 oxidized intermediates causes mitochondrial stress , leading to impaired 
 insulin action . it is therefore conceivable 
 that the rationale for the inhibition of bcaa catabolism in the daf-2 mutant is a ( futile ) attempt to restore normal insulin / igf-1 
 signaling levels . in parallel , tyrosine was identified in c. elegans as a potent antagonist of insulin signaling , 
 promoting dauer formation and inducing longevity ( a. ferguson , p. hu , d. kim , and a. fisher , personal communication ) , and enzymes of 
 this pathway have been associated with c. elegans longevity before . similarly , the increased catabolism of tyrosine is 
 conceivably part of a feedback mechanism for restoration of normal 
 insulin / igf-1 signaling . finally , c3 and c5 acylcarnitines mainly 
 arise from the incomplete catabolism of propionate ( following bcaa 
 degradation , figure 8) . thus , increased expression 
 of propionate catabolism enzymes should further prevent accumulation 
 of these intermediates . interestingly , attenuation of intermediary 
 metabolism ( glycolysis , the tca cycle , and gluconeogenesis ) and the electron transport chain has been reported to extend life - span of daf-2 mutants 
 significantly further . this is unexpected because expression of all 
 these enzymes is increased in response to reduced iis . it has been 
 suggested that activation of metabolism is part of a compensatory 
 mechanism triggered by reduced iis that antagonizes full life - span 
 potential of daf-2 mutants . we additionally propose that further attenuation of an already reduced 
 metabolic rate could have an additive effect on daf-2 lifespan , perhaps at the cost of reduced metabolic responsiveness 
 to environmental triggers . note , however , that activation of the glyoxylate 
 shunt ( isocitrate lyase / malate synthase , icl-1 ) has been shown to 
 be required for full life - span extension in the daf-2 mutant as well as in ubiquinone - defective clk-1 , suggesting that increased 
 activity of this pathway is indispensable for iis- or etc - induced 
 longevity . in summary , our data suggests daf-2 mutant cells are equipped with highly bioenergetic competent mitochondria 
 and a proficient , partly rerouted metabolic network that makes economical 
 use of internal energy stores to maintain energy homeostasis during 
 its long life . a major challenge for the field will be to unravel 
 the multiplicity of tissue - specific metabolic changes and how these 
 tissues in turn interact to achieve the extensive physiological transformation 
 induced by mutation in daf-2 . ultimately , such detailed 
 understanding of the altered metabolism of c. elegans iis mutants could also lead to important insights into various insulin - related 
 disease pathologies in humans such as diabetes and obesity as well 
 as aging .
the 
 insulin / igf-1 receptor is a major known determinant of dauer 
 formation , stress resistance , longevity , and metabolism in caenorhabditis elegans . in the past , whole - genome 
 transcript profiling was used extensively to study differential gene 
 expression in response to reduced insulin / igf-1 signaling , including 
 the expression levels of metabolism - associated genes . taking advantage 
 of the recent developments in quantitative liquid chromatography mass 
 spectrometry ( lc  ms)-based proteomics 
 , we profiled the proteomic 
 changes that occur in response to activation of the daf-16 transcription 
 factor in the germline - less glp-4(bn2);daf-2(e1370 ) receptor mutant . strikingly , the daf-2 profile 
 suggests extensive reorganization of intermediary metabolism , characterized 
 by the upregulation of many core intermediary metabolic pathways . 
 these include glycolysis / gluconeogenesis , glycogenesis , pentose phosphate 
 cycle , citric acid cycle , glyoxylate shunt , fatty acid -oxidation , 
 one - carbon metabolism , propionate and tyrosine catabolism , and complexes 
 i , ii , iii , and v of the electron transport chain . 
 interestingly , 
 we found simultaneous activation of reciprocally regulated metabolic 
 pathways , which is indicative of spatiotemporal coordination of energy 
 metabolism and/or extensive post - translational regulation of these 
 enzymes . 
 this restructuring of daf-2 metabolism is 
 reminiscent to that of hypometabolic dauers , allowing the efficient 
 and economical utilization of internal nutrient reserves and possibly 
 also shunting metabolites through alternative energy - generating pathways 
 to sustain longevity .