Deletion of the Intestinal Peptide Transporter Affects Insulin and TOR Signaling in Caenorhabditis elegans
2004; Elsevier BV; Volume: 279; Issue: 35 Linguagem: Inglês
10.1074/jbc.m403415200
ISSN1083-351X
AutoresBarbara Meissner, Michael Boll, Hannelore Daniel, Ralf Baumeister,
Tópico(s)GDF15 and Related Biomarkers
ResumoThe mammalian intestinal peptide transporter PEPT1 mediates the uptake of di- and tripeptides from the gut lumen into intestinal epithelial cells and acts in parallel with amino acid transporters. Here we address the importance of the PEPT1 orthologue PEP-2 for the assimilation of dietary protein and for overall protein nutrition in Caenorhabditis elegans. pep-2 is expressed specifically along the apical membrane of the intestinal cells, and in pep-2 deletion mutant animals, uptake of intact peptides from the gut lumen is abolished. The consequences are a severely retarded development, reduced progeny and body size, and increased stress tolerance. We show here that pep-2 cross-talks with both the C. elegans target of rapamycin (TOR) and the DAF-2/insulin-signaling pathways. The pep-2 mutant enhances the developmental and longevity phenotypes of daf-2, resulting, among other effects, in a pronounced increase in adult life span. Moreover, all aspects of a weak let-363/TOR RNA interference phenotype are intensified by pep-2 deletion, indicating that pep-2 function upstream of TOR-mediated nutrient sensing. Our findings provide evidence for a predominant role of the intestinal peptide transporter for the delivery of bulk quantities of amino acids for growth and development, which consequently affects signaling pathways that regulate metabolism and aging. The mammalian intestinal peptide transporter PEPT1 mediates the uptake of di- and tripeptides from the gut lumen into intestinal epithelial cells and acts in parallel with amino acid transporters. Here we address the importance of the PEPT1 orthologue PEP-2 for the assimilation of dietary protein and for overall protein nutrition in Caenorhabditis elegans. pep-2 is expressed specifically along the apical membrane of the intestinal cells, and in pep-2 deletion mutant animals, uptake of intact peptides from the gut lumen is abolished. The consequences are a severely retarded development, reduced progeny and body size, and increased stress tolerance. We show here that pep-2 cross-talks with both the C. elegans target of rapamycin (TOR) and the DAF-2/insulin-signaling pathways. The pep-2 mutant enhances the developmental and longevity phenotypes of daf-2, resulting, among other effects, in a pronounced increase in adult life span. Moreover, all aspects of a weak let-363/TOR RNA interference phenotype are intensified by pep-2 deletion, indicating that pep-2 function upstream of TOR-mediated nutrient sensing. Our findings provide evidence for a predominant role of the intestinal peptide transporter for the delivery of bulk quantities of amino acids for growth and development, which consequently affects signaling pathways that regulate metabolism and aging. In all organisms the uptake of amino acids is mediated by a multitude of integral cell membrane carriers that transport amino acids either in free or in peptide-bound form. The two mammalian peptide transporters PEPT1 and PEPT2 are proton-dependent rheogenic carriers and have been grouped into the POT (proton-coupled oligopeptide transporter) superfamily, which is also called peptide transporter family (1Steiner H.Y. Naider F. Becker J.M. Mol. Microbiol. 1995; 16: 825-834Crossref PubMed Scopus (203) Google Scholar). They transport short-chain peptides but also a variety of pharmacologically important compounds including selected β-lactam antibiotics and angiotensin-converting enzyme inhibitors, as well as antiviral and antineoplastic drugs (2Rubio-Aliaga I. Boll M. Daniel H. Biochem. Biophys. Res. Commun. 2000; 276: 734-741Crossref PubMed Scopus (47) Google Scholar, 3Bai J.P. Amidon G.L. Pharm. Res. 1992; 9: 969-978Crossref PubMed Scopus (147) Google Scholar). In contrast, these transporters cannot transport larger peptides or free amino acids (4Daniel H. J. Membr. Biol. 1996; 154: 197-203Crossref PubMed Scopus (80) Google Scholar). PEPT1 is localized to apical membranes of intestinal epithelial cells where it mediates the uptake of di- and tripeptides following the digestion of dietary proteins. In kidney, PEPT1 is also found in epithelial cells of the proximal tubule where it may contribute to the reabsorption of peptides after glomerular filtration (5Ganapathy M.E. Brandsch M. Prasad P.D. Ganapathy V. Leibach F.H. J. Biol. Chem. 1995; 270: 25672-25677Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The Caenorhabditis elegans genome contains two homologues of the human pept1 and pept2 genes, designated pep-2 and pep-1 (aka opt-2 and opt-1, encoding CPTB and CPTA, respectively) (6Fei Y.J. Fujita T. Lapp D.F. Ganapathy V. Leibach F.H. Biochem. J. 1998; 332: 565-572Crossref PubMed Scopus (38) Google Scholar). Although the deduced transporter proteins reveal only modest amino acid similarities with the mammalian proteins, the in vitro transport characteristics of the C. elegans and mammalian orthologues in Xenopus laevis oocytes are very similar (6Fei Y.J. Fujita T. Lapp D.F. Ganapathy V. Leibach F.H. Biochem. J. 1998; 332: 565-572Crossref PubMed Scopus (38) Google Scholar). The C. elegans pep-2 gene encodes a protein with 36.9% amino acid sequence identity compared with the human PEPT1 and represents the low affinity/high capacity isoform of proton-coupled peptide transporters. Based on its role in intestinal absorption of peptide-bound amino acids, the PEP-2 protein may be important for overall protein nutrition of the organism. Whole body protein nutrition is linked to the available amino acid pool that regulates metabolic and reproductive adaptations through the partially interconnected insulin/IGF 1The abbreviations used are: IGF, insulin-like growth factor; TOR, target of rapamycin; AMCA, N(ϵ)-7-amino-4-methylcoumarin-3-acetic acid; S6K, ribosomal protein S6 kinase; GFP, green fluorescent protein; RNAi, RNA interference.1The abbreviations used are: IGF, insulin-like growth factor; TOR, target of rapamycin; AMCA, N(ϵ)-7-amino-4-methylcoumarin-3-acetic acid; S6K, ribosomal protein S6 kinase; GFP, green fluorescent protein; RNAi, RNA interference. and TOR/S6K signaling pathways. TOR senses the cellular amino acid pool and contributes to a signaling cascade that regulates transcription, translation, and protein degradation (7Chou M.M. Blenis J. Curr. Opin. Cell Biol. 1995; 7: 806-814Crossref PubMed Scopus (245) Google Scholar). A partial loss of TOR function in Drosophila reduces growth, and flies deficient in the p70S6K gene (DS6K, a downstream target of TOR) are extremely delayed in development and are smaller in size (8Montagne J. Stewart M.J. Stocker H. Hafen E. Kozma S.C. Thomas G. Science. 1999; 285: 2126-2129Crossref PubMed Scopus (624) Google Scholar). More recently, let-363, a C. elegans homologue of TOR, was identified and characterized (9Long X. Spycher C. Han Z.S. Rose A.M. Muller F. Avruch J. Curr. Biol. 2002; 12: 1448-1461Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). let-363 loss-of-function mutants exhibit a developmental arrest and death at the L3 larval stage (9Long X. Spycher C. Han Z.S. Rose A.M. Muller F. Avruch J. Curr. Biol. 2002; 12: 1448-1461Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Furthermore, it was shown that TOR function affects the life span and may interact with the DAF-2/insulin-signaling pathway in C. elegans (10Vellai T. Takacs-Vellai K. Zhang Y. Kovacs A.L. Orosz L. Muller F. Nature. 2003; 426: 620Crossref PubMed Scopus (819) Google Scholar). TOR has been implicated in the insulin/IGF network based on cell culture experiments (11Scott P.H. Brunn G.J. Kohn A.D. Roth R.A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (409) Google Scholar, 12Raught B. Gingras A.C. Gygi S.P. Imataka H. Morino S. Gradi A. Aebersold R. Sonenberg N. EMBO J. 2000; 19: 434-444Crossref PubMed Scopus (226) Google Scholar), and in Drosophila TOR is required for the growth-stimulating effect of the phosphatidylinositol 3-kinase pathway (13Zhang H. Stallock J.P. Ng J.C. Reinhard C. Neufeld T.P. Genes Dev. 2000; 14: 2712-2724Crossref PubMed Scopus (507) Google Scholar). In both Drosophila and mammals, the levels of dietary protein or amino acid availability also affect the insulin-signaling pathway to control metabolism and growth (14Takenaka A. Oki N. Takahashi S.I. Noguchi T. J. Nutr. 2000; 130: 2910-2914Crossref PubMed Scopus (56) Google Scholar, 15Gems D. Partridge L. Curr. Opin. Genet. Dev. 2001; 11: 287-292Crossref PubMed Scopus (142) Google Scholar, 16Britton J.S. Lockwood W.K. Li L. Cohen S.M. Edgar B.A. Dev. Cell. 2002; 2: 239-249Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar). In addition, the aging process was shown to be regulated hormonally by this evolutionarily conserved signaling pathway (17Bluher M. Kahn B.B. Kahn C.R. Science. 2003; 299: 572-574Crossref PubMed Scopus (1057) Google Scholar, 18Holzenberger M. Dupont J. Ducos B. Leneuve P. Geloen A. Even P.C. Cervera P. Le Bouc Y. Nature. 2003; 421: 182-187Crossref PubMed Scopus (1608) Google Scholar, 19Tatar M. Bartke A. Antebi A. Science. 2003; 299: 1346-1351Crossref PubMed Scopus (1035) Google Scholar). In C. elegans, the daf-2 gene encodes an insulin/IGF receptor, and the downstream components include the AGE-1/phosphatidylinositol 3-kinase, PDK-1/PDK1, the AGC kinases AKT-1/-2 and SGK-1, PKB, DAF-18/PTEN, and the forkhead transcription factor DAF-16/FKHRL1/FOXO (20Hertweck M. Gobel C. Baumeister R. Dev. Cell. 2004; 6: 577-588Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Extensive studies revealed that the DAF-2 pathway regulates aging, reproduction, lipid metabolism, and dauer formation independently of one another (15Gems D. Partridge L. Curr. Opin. Genet. Dev. 2001; 11: 287-292Crossref PubMed Scopus (142) Google Scholar, 21Dillin A. Crawford D.K. Kenyon C. Science. 2002; 298: 830-834Crossref PubMed Scopus (340) Google Scholar, 22Guarente L. Kenyon C. Nature. 2000; 408: 255-262Crossref PubMed Scopus (1098) Google Scholar, 23Wolkow C.A. Kimura K.D. Lee M.S. Ruvkun G. Science. 2000; 290: 147-150Crossref PubMed Scopus (525) Google Scholar). Here we describe the phenotypic alterations in C. elegans caused by the deletion of the intestinal peptide transporter pep-2. Our analysis clearly identifies intestinal peptide absorption as a key process in body protein homeostasis that affects both the TOR and the insulin-signaling pathways. C. elegans Strains—The strains used were as follows: N2 Bristol (wild type), DA453: eat-2(ad453)II, CB1370: daf-2(e1370)III, DR26: daf-16(m26)I, DH1033: bIs1[vit-2::gfp,pFR4,sqt-1(sc103)], BR2746: bIs1-[vit-2::GFP,pFR4,sqt-1(sc103)]; pep-2(lg601)X, BR2688: daf-2(e1370)III; pep-2(lg601)X, BR2689: daf-16(m26)I;pep-2(lg601)X, BR3061: daf-16-(m26)I;daf-2(e1370)III;pep-2(lg601)X, DR1808: mIs6[rol-6(su1006), daf-7p::GFP], BR3062: mIs6[rol-6(su1006),daf-7p::gfp];pep-2(lg601)X, BR2743: pep-1(lg501)IV and BR2744: pep-1(lg501)IV; pep-2(lg601)X.To isolate the pep-1 and pep-2 deletion mutants, a UV/trimethyl psoralen mutagenized C. elegans library was screened by PCR with gene-specific primers. The mutant allele lg601 was back-crossed with N2 wild type animals seven times before analysis. pep-2(lg601) is recessive and represent a strong loss-of-function allele that could be rescued by a wild type transgene. In the pep-1 mutant allele lg501, a 2.5-kb deletion removes 1269 bp of the promoter sequence, the translational start codon, and the first six exons of the gene (bp 18289–20838 on cosmid C06G8). β-Ala-Lys-AMCA Staining—Mixed staged animals were washed off of agar plates with M9 buffer. Equal amounts of worms were incubated in a 1 mm β-Ala-Lys-AMCA solution (in M9) for 2–3 h followed by at least four additional washing steps with M9. As a control, worms were incubated in M9 buffer for the same time period. Pictures were taken with an AxioPlan 2 (Zeiss) using AxioVision 3.0 software. Body Length Measurements—Synchronized wild type and pep-2(lg601) animals were collected 0–5 days after L4 moult. Pictures of 20 individual worms were taken with Axioplan 2, and the precise body length was measured with AxioVision 3.0 software. Postembryonic Development—5–15 adult hermaphrodites were placed on fresh plates for egg laying. After 2–3 h they were removed, and at least 16 worms of the progeny were singled onto individual plates. The F1 animals were monitored every 2 h until they laid the first egg. Yolk Protein Distribution—Yolk protein distribution was analyzed by visual inspection of GFP expression in DH1033 bIs1[vit-2::gfp,-pFR4,sqt-1(sc103)] (24Grant B. Hirsh D. Mol. Biol. Cell. 1999; 10: 4311-4326Crossref PubMed Scopus (450) Google Scholar) BR2746 bIs1[vit-2::GFP,pFR4,sqt-1(sc103)]; pep-2(lg601). Life Span—Life span assays were performed as described (25Lakowski B. Hekimi S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13091-13096Crossref PubMed Scopus (724) Google Scholar), except that adult hermaphrodites were allowed to lay eggs for 8–10 h. Animals were grown at 20 °C. For assays at 25 °C, the animals were grown at 15 °C until the L4 moult and then shifted to 25 °C. We used the L4 moult as t = 0 for life span analysis. Stress Resistance and Other Assays—The assays for heat stress (35 °C) and oxidative stress (paraquat) resistance were performed as described previously (20Hertweck M. Gobel C. Baumeister R. Dev. Cell. 2004; 6: 577-588Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), except that on plates containing 150 mm paraquat animals were scored once a day. Self-brood size, embryonic development, defecation, and pharyngeal pumping assays were performed as described previously (26Wong A. Boutis P. Hekimi S. Genetics. 1995; 139: 1247-1259Crossref PubMed Google Scholar). All analyses used animals fed with Escherichia coli OP50. For amino acid supplementation, 300 μl of amino acids (mixture 1:1 of minimum Eagle's medium (100×) nonessential amino acids and minimum Eagle's medium amino acids (50×) without l-glutamine (Invitrogen)) were added on top of the agar (35-mm plates) seeded with E. coli. Fresh plates were prepared each day during the experiment. RNAi through-feeding experiments were performed as described previously (27Kamath R.S. Martinez-Campos M. Zipperlen P. Fraser A.G. Ahringer J. Genome. Biol. 2001; 2 (RESEARCH0002)PubMed Google Scholar). Two independent sources of TOR(RNAi) vectors were used. The partial cDNA from yk18c10 was cloned into vector pPD129.36, resulting in a plasmid (gift of A. Gartner, Max-Planck-Institute for Biochemistry, Martinsried, Munich) that induced only a weak let-363 phenotype. A plasmid inducing a strong let-363/TOR phenotype had already been described previously (9Long X. Spycher C. Han Z.S. Rose A.M. Muller F. Avruch J. Curr. Biol. 2002; 12: 1448-1461Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 10Vellai T. Takacs-Vellai K. Zhang Y. Kovacs A.L. Orosz L. Muller F. Nature. 2003; 426: 620Crossref PubMed Scopus (819) Google Scholar). For developmental and intestinal phenotypes, animals at the L4 stage were placed on RNAi-inducing plates and allowed to lay progeny. Adults were removed or transferred to new RNAi-producing plates. First and third generation progeny grown on RNAi plates were scored for a TOR phenotype and yielded identical results. Life span experiments were performed according to Vellai et al. (10Vellai T. Takacs-Vellai K. Zhang Y. Kovacs A.L. Orosz L. Muller F. Nature. 2003; 426: 620Crossref PubMed Scopus (819) Google Scholar). Hermaphrodites of wild type and mutants were grown on NGM agar plates seeded with E. coli OP50. Eggs were collected by sodium hypochlorite treatment and allowed to hatch by being incubated overnight at 20 °C in M9 buffer. Newly hatched L1 larvae were cultured on 150-mm agar plates. To prevent progeny production, 5-fluoro-2′deoxyuridine (dFUR, Sigma) was added to the agar at a final concentration of 40 μm after the animals had reached adulthood. Worms were washed off of the agar plates with M9 buffer. Living animals were collected because they floated on sucrose, washed several times with M9 buffer, resuspended with 5 mm EDTA, and frozen at –80 °C until use. Protein extracts were made by pounding and sonification. Their protein carbonyl content was measured as previously described (28Yasuda K. Adachi H. Fujiwara Y. Ishii N. J. Gerontol. A Biol. Sci. Med. Sci. 1999; 54: B47-B51Crossref PubMed Scopus (61) Google Scholar). Protein concentration was determined by the Bio-Rad protein assay (Bio-Rad Laboratories). Three to nine separate determinations were used to calculate the mean ± S.E. for each different age group. PEP-2 Functions as the Peptide Transporter in Intestinal Cells—We characterized a 1.7-kb deletion mutant, pep-2(lg601), which lacks 257 bp of the promoter, the translational start codon, and the first six exons of the pep-2 gene (Fig. 1A). Even if PEP-2 were expressed from this mutant allele, it would lack the N-terminal six transmembrane domains required for substrate binding and transport (29Doring F. Martini C. Walter J. Daniel H. J. Membr. Biol. 2002; 186: 55-62Crossref PubMed Scopus (30) Google Scholar) and therefore pep-2(lg601) most likely represents a strong loss-of-function or null allele. To demonstrate that the pep-2(lg601) mutant strain has lost its capability for transport of di- and tripeptides, animals were exposed to the fluorescent dipeptide conjugate β-Ala-Lys-AMCA that was previously shown to be a representative substrate of PEPT1 (30Groneberg D.A. Doring F. Eynott P.R. Fischer A. Daniel H. Am. J. Physiol. 2001; 281: G697-G704Crossref PubMed Google Scholar). Efficient uptake of the reporter molecule into intestinal epithelial cells of wild type animals was indicated by a strong fluorescence of all intestinal cells, whereas the gut lumen lacked staining suggesting complete and rapid intestinal peptide absorption (Fig. 1B). When pep-2(lg601) animals were exposed to β-Ala-Lys-AMCA, the fluorescence was detectable only in the gut lumen, indicative of a normal ingestion but lack of absorption; we never observed fluorescence (other than the normal gut epifluorescence) inside of the epithelial cells (Fig. 1B). We conclude that PEP-2 is the only functional transporter in the intestine of C. elegans that is capable of transporting the dipeptide-conjugate β-Ala-Lys-AMCA representative for di-/tripeptides (30Groneberg D.A. Doring F. Eynott P.R. Fischer A. Daniel H. Am. J. Physiol. 2001; 281: G697-G704Crossref PubMed Google Scholar) across the apical membrane into the intestinal cells. Loss of PEP-2 Function Affects Growth and Development— Deletion of the pep-2 gene results in a decreased body size and a markedly reduced brood size (Fig. 2, A and D, and Table I), a similar phenotype to that reported for animals treated with opt-2 RNAi (31Nehrke K. J. Biol. Chem. 2003; 278: 44657-44666Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Moreover, the postembryonic development, in which the animals depend on external food supply, is delayed 1.8-fold in pep-2 mutants at 15 and 20 °C (Fig. 2B and Table I), whereas embryonic development that takes place in a protective eggshell is not significantly affected (Fig. 2C). In addition, the reproductive life span is extended from 5 days (wild type) to 9 days (Fig. 2E). Unlike in Egl (egg-laying-defective) animals, the eggs are laid at approximately the same developmental stage as in wild type animals (28–56 cell stage). This suggests that the production, rather than the retention, of eggs in the uterus appears to be the limiting factor in pep-2 animals. Fertilized eggs in the uterus of pep-2(lg601) displayed the same level of vitellogenin::gfp (vit-2::gfp) fluorescence as those of wild type animals (Fig. 2F), indicating a similar yolk concentration in mature eggs. These data indicate that receptor-mediated endocytosis of yolk is not affected in pep-2 mutant animals. However, because the production of individual eggs is severely retarded, the reduced availability of amino acid nitrogen most likely results in a delay of de novo protein synthesis. Deletion of pep-2, however, does not cause lethality, and the additional deletion of pep-1 (orthologue of hPepT2) did not enhance the pep-2 phenotype (Fig. 2G). Transgenic expression of pep-2 rescued both β-Ala-Lys-AMCA uptake (Fig. 1B) and the developmental defects (Fig. 2B). Therefore, the expression of the pep-2 genomic region in mutant animals is sufficient to restore the function of the transporter. This data provide further support for PEP-2 being the only uptake system for di-/tripeptides in the intestine.Table IDevelopmental phenotypes in pep-2 and daf-2 mutantsGenotypePostembryonic developmentaValues are means ± S.E. with number of animals shown in parentheses.N2No. of progenyaValues are means ± S.E. with number of animals shown in parentheses.N2hours%%Wild type67.1 ± 1.6 (48)100316.6 ± 4.1 (54)100daf-16(m26)74.1 ± 0.8 (37)110292.6 ± 7.3 (28)92pep-2(lg601)117.5 ± 2.6 (64)175109.3 ± 1.9 (79)35daf-16(m26);pep-2(lg601)114.0 ± 2.1 (16)17070.9 ± 4.6 (10)22daf-2(e1370)108.8 ± 4.9 (20)162210.0 ± 5.4 (30)66daf-16(m26);daf-2(e1370)78.7 ± 1.4 (37)117260.8 ± 4.2 (18)82daf-2(e1370);pep-2(lg601)148.7 ± 6.1 (21)22251.6 ± 1.1 (32)16daf-16(m26);daf-2(e1370); pep-2(lg601)125.1 ± 2.2 (24)186113.6 ± 2.6 (20)36a Values are means ± S.E. with number of animals shown in parentheses. Open table in a new tab Loss of PEP-2 Cannot Be Compensated by Amino Acid Supplementation of the Food Source—The developmental defects observed in pep-2 mutants could be caused by impaired food intake, a limited availability of amino acids as energy substrates resulting in caloric restriction, or a more specific effect by an insufficient supply of amino acids for maintaining protein homeostasis. We have measured pharyngeal pumping and defecation as two of the behavioral parameters recognized as valid indicators of sufficient food consumption (32Avery L. Thomas J.H. Riddle D.L. Blumenthal T. Meyer B.I. Priess J. Feeding and Defecation, C. elegans II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997Google Scholar). Both parameters are indistinguishable in pep-2 and wild type animals (data not shown; Ref. 31Nehrke K. J. Biol. Chem. 2003; 278: 44657-44666Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Therefore, malabsorption of dipeptides and the associated lower intake of amino acids appear to be the sole determinants of the developmental defects. To assess whether amino acid deprivation in the pep-2 mutants can be overcome by the supplementation of free amino acids in the food, an amino acid mixture was added to the standard E. coli food source. In wild type animals, this amino acid supplementation did not affect brood size (Fig. 2D). In contrast, in pep-2(lg601) animals, it resulted in a partial but significant suppression of the brood size defect (Fig. 2D) and only a modest reduction of generation time (data not shown). These data suggest that amino acids from the supplement delivered via the amino acid transporters cannot compensate for the loss of PEP-2 mediated peptide transport under normal feeding conditions. Therefore, PEP-2 activity is critical for amino acid availability and homeostasis in the organism. pep-2(lg601) Interacts with the DAF-2 Insulin-signaling Pathway—C. elegans animals with reduced activity of the insulin/IGF receptor (DAF-2) also display developmental and reproductive defects (33Gems D. Sutton A.J. Sundermeyer M.L. Albert P.S. King K.V. Edgley M.L. Larsen P.L. Riddle D.L. Genetics. 1998; 150: 129-155Crossref PubMed Google Scholar). In our experiments, both daf-2(e1370) and pep-2(lg601) mutants show a similarly delayed postembryonic development and reduced brood sizes (Table I). In daf-2;pep-2 double mutants, these phenotypic aspects are even more pronounced than in either of the single mutants (Table I). Only the phenotype caused by daf-2(e1370) was suppressed by daf-16(m26). In the daf-16;pep-2 double and daf-16;daf-2;pep-2 triple mutants, the phenotype of pep-2(lg601) was not affected by daf-16(m26). Thus, neither growth retardation nor reduced brood size of the pep-2 animals can be suppressed by daf-16 (Table I). These results suggest that pep-2 acts in parallel to daf-2 in a pathway that does not depend on DAF-16 function. The consequences of a restricted amino acid intake in adult worms have not been addressed previously, but it is known that a reduced caloric intake, or the perturbation of the underlying signaling pathways that sense energy availability, prolongs life span (34Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Crossref PubMed Scopus (1473) Google Scholar). The reduced dietary availability of amino acids in pep-2 mutants may be interpreted as a reduced intake of calories, and therefore we tested whether the adult life span is altered in this mutant. As a control we used eat-2(ad453) (35Raizen D.M. Lee R.Y. Avery L. Genetics. 1995; 141: 1365-1382Crossref PubMed Google Scholar), a calorically restricted mutant that exhibits an extended life span as compared with the wild type (25Lakowski B. Hekimi S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13091-13096Crossref PubMed Scopus (724) Google Scholar). eat-2 was shown to also act in parallel to the insulin-like signaling pathway, a major contributor to the regulation of life span in C. elegans. Whereas we observed that both eat-2(ad453) and daf-2(e1370) animals showed a longevity phenotype at 20 or 25 °C as reported previously (25Lakowski B. Hekimi S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13091-13096Crossref PubMed Scopus (724) Google Scholar, 36Kenyon C. Chang J. Gensch E. Rudner A. Tabtiang R. Nature. 1993; 366: 461-464Crossref PubMed Scopus (2464) Google Scholar), the life span of pep-2(lg601) animals was not altered at 20 °C and was even slightly shorter at 25 °C (Table II, Fig. 3A). Similar results have been reported recently after RNAi treatment of pep-2 (31Nehrke K. J. Biol. Chem. 2003; 278: 44657-44666Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 37Murphy C.T. McCarroll S.A. Bargmann C.I. Fraser A. Kamath R.S. Ahringer J. Li H. Kenyon C. Nature. 2003; 424: 277-283Crossref PubMed Scopus (1684) Google Scholar). Therefore, the reduced amino acid availability does not account for a caloric restriction that is severe enough to alter the adult life span substantially. In addition, unlike food deprivation, which can induce a dauer phenotype, pep-2(lg601) at 25 °C did not show an increase in dauer formation in the presence of food as compared with wild type.Table IIAdult life spanGenotypeMean life spanaValues are means ± S.E.Maximum life spanNbTotal number of animals.daysdays20 °CWild type13.6 ± 0.225500eat-2(ad453)18.3 ± 0.93650pep-2(lg601)14.2 ± 0.226250daf-2(e1370)21.8 ± 1.052150daf-16(m26)10.4 ± 0.220300daf-2(e1370);pep-2(lg601)31.9 ± 1.371150daf-16(m26);pep-2(lg601)9.3 ± 0.220300let-363(RNAi_weak)18.3 ± 0.630100let-363(RNAi_weak); pep-2(lg601)22.2 ± 0.43410025 °CWild type11.0 ± 0.321200pep-2(lg601)9.0 ± 0.318200daf-2(e1370)24.3 ± 0.746197daf-16(m26)8.9 ± 0.317100daf-16(m26);daf-2(e1370)10.9 ± 0.523100daf-16(m26);pep-2(lg601)7.4 ± 0.317100daf-2(e1370);pep-2(lg601)38.3 ± 0.977187daf-16(m26);daf-2(e1370);pep-2(lg601)9.8 ± 0.526100let-363(RNAi_strong)15.4 ± 0.422100let-363(RNAi_strong); pep-2(lg601)15.8 ± 0.323100a Values are means ± S.E.b Total number of animals. Open table in a new tab To assess whether there exists a cross-talk between the dietary intake of amino acids and daf-2-controlled longevity, we analyzed life span alterations in the daf-2(e1370);pep-2(lg601) double mutant strains, in which both pathways are perturbed. Surprisingly, the life span of daf-2(e1370) can be extended drastically in a pep-2 mutant background by around 60% when compared with the daf-2 single mutant (Fig. 3A, Table II). In addition, this longevity effect of pep-2(lg601) was completely suppressed in a daf-16;daf-2;pep-2 triple mutant (Fig. 3B, Table II). Thus, pep-2 deletion affects both daf-16-dependent and -independent outputs. Stress Resistance Is Enhanced in pep-2(lg601)—All long lived mutants in the daf-2 signaling pathway are hyperresistant to oxidative stress, heat, or UV stress (38Lithgow G.J. White T.M. Hinerfeld D.A. Johnson T.E. J. Gerontol. 1994; 49: B270-B276Crossref PubMed Scopus (167) Google Scholar, 39Lithgow G.J. White T.M. Melov S. Johnson T.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7540-7544Crossref PubMed Scopus (674) Google Scholar, 40Vanfleteren J.R. Biochem. J. 1993; 292: 605-608Crossref PubMed Scopus (332) Google Scholar). To assess whether there is an increased stress resistance in the pep-2(lg601) mutant background, we tested the animals for heat tolerance (35 °C) and resistance to oxidative stress (paraquat) (Fig. 3, C and D). Both assays showed an increased stress resistance of pep-2(lg601) mutant animals, which cannot be suppressed by daf-16(m26). In addition, the stress resistance of daf-2(e1370) mutant animals was significantly increased in the pep-2 mutant background. Most strikingly, under conditions (150 mm paraquat) that kill 100% of the pep-2(lg601) animals within 7 days and 90% of the daf-2(e1370) mutants within 10 days, no lethality was observed among daf-2;pep-2 mutants (Fig. 3D). Oxidative stress is known to damage proteins by the introduction of carbonyl groups, and the age-dependent accumulation of protein carbonyl appears to mirror the life span as shown in a variety of C. elegans mutants (28Yasuda K. Adachi H. Fujiwara Y. Ishii N. J. Gerontol. A Biol. Sci. Med. Sci. 1999; 54: B47-B51Crossref PubMed Scopus (61) Google Scholar). The relevance of dietary amino acid supply on the protein oxidation level during the aging process has also been demonstrated in mammals (rats), where a low protei
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