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It was recently reported that the plant polyphenol resveratrol, found, e.g., in grape berry skins, extended lifespan in the fruit fly Drosophila melanogaster and the nematode worm Caenorhabditis elegans. This lifespan extension was dependent on an NAD(+)-dependent histone deacetylase, Sir2 in Drosophila and SIR-2.1 in C. elegans. The extension of lifespan appeared to occur through a mechanism related to dietary restriction (DR), the reduction of available nutrients without causing malnutrition, an intervention that extends lifespan in diverse organisms from yeast to mammals. In Drosophila, lifespan extension by DR is associated with a reduction in fecundity. However, a slight increase in fecundity was reported upon treatment with resveratrol, suggesting a mode of action at least partially distinct from that of DR. To probe this mechanism further, we initiated a new study of the effects of resveratrol on Drosophila. We saw no significant effects on lifespan in seven independent trials. We analysed our resveratrol and found that its structure was normal, with no oxidative modifications. We therefore re-tested the effects of resveratrol in C. elegans, in both wild-type and sir-2.1 mutant worms. The results were variable, with resveratrol treatment resulting in slight increases in lifespan in some trials but not others, in both wild type and sir-2.1 mutant animals. We postulate that the effect of resveratrol upon lifespan in C. elegans could reflect induction of phase 2 drug detoxification or activation of AMP kinase.

Model organisms have been widely used to study the ageing phenomenon in order to learn about human ageing. Although the phylogenetic diversity between vertebrates and some of the most commonly used model systems could hardly be greater, several mechanisms of life extension are public (common characteristic in divergent species) and likely share a common ancestry. Dietary restriction, reduced IGF-signaling and, seemingly, reduced ROS-induced damage are the best known mechanisms for extending longevity in a variety of organisms. In this review, we summarize the knowledge of ageing in the nematode Caenorhabditis elegans and compare the mechanisms of life extension with knowledge from other model organisms.

Summary The insulin/insulin-like growth factor-1 (Ins/IGF-1) pathway regulates the aging rate of the nematode Caenorhabditis elegans. We describe other features of the three Ins/IGF-1 mutants daf-2, age-1 and aap-1. We show that the investigated Ins/IGF-1 mutants all have a reduced body volume, reduced reproductive capacity, increased ATP concentrations and an elevated stress resistance. We also observed that heat production is lower in these mutants, although the respiration rate was similar or higher compared with wild-type individuals, suggesting a metabolic shift in these mutants.

Culture in axenic medium causes two-fold increases in the length of development and adult life span in Caenorhabditis elegans. We asked whether axenic medium imposes dietary restriction (ADR), and causes changes in metabolic activity and stress resistance. Eat mutants, which have a reduced food intake, were studied in parallel with wild-type worms to assess potential synergistic actions of axenic culture and food restriction. We found that axenic culture enhances metabolic activity as assessed by mass-specific oxygen consumption rate and heat production. Axenic culture also caused higher activities of the antioxidant enzymes superoxide dismutase and catalase, and led to increased resistance to high temperature, which was further exacerbated by mutation in eat-2. These results show that axenic medium up-regulates a variety of somatic maintenance functions including oxidative and thermal stress resistance and that food restriction due to axenic growth and to mutation in eat-2 are very similar but not identical.

Dietary restriction (DR) is the most consistent means of extending life span throughout the animal kingdom. Multiple mechanisms by which DR may act have been proposed but none are clearly predominant. We asked whether metabolic rate and stress resistance is altered in Caenorhabditis elegans in response to DR. DR was imposed in two complementary ways: by growing wild-type worms in liquid medium supplemented with reduced concentrations of bacteria and by using eat-2 mutants, which have a feeding defect. Metabolic rate was not reduced when we fed wild-type worms reduced food and was up-regulated in the eat-2 mutants in liquid culture, as assessed by oxygen consumption rate and heat production. The specific activity levels of the antioxidant enzymes superoxide dismutase (SOD) and catalase showed small increases when we reduced food in wild-type worms, but restricted worms acquired no elevated protection against paraquat and hydrogen peroxide. eat-2 mutants showed elevated specific activities of SOD and catalase relative to wild type in liquid culture. These results indicate that the effects imparted by DR and the eat-2 mutation are not identical, and they contradict, at least in C. elegans, the widespread belief that CR acts by lowering the rate of metabolism.

Summary It is widely believed that normal by-products of oxidative metabolism and the subsequent molecular damage inflicted by them couple the aging process to metabolic rate. Accordingly, high metabolic rates would be expected to accelerate aging, and life-extending interventions are often assumed to act by attenuating metabolic rate. Notorious examples in Caenorhabditis elegans are food restriction, mutation in the Clock genes and several genes of the insulin-like signalling pathway. Here we discuss how metabolic rate can be accurately measured and normalized, and how to deal with differences in body size. These issues are illustrated using experimental data of the long-lived mutant strains clk-1(e2519) and daf-2(e1370). Appropriate analysis shows that metabolic rates in wild-type and in the clk-1 mutant are very similar. In contrast, the metabolic rate profiles point to a metabolic shift toward enhanced efficiency of oxidative phosphorylation in the daf-2 worms.

Clk mutants of Caenorhabditis elegans are characterised by an overall slow down of temporal processes and increase in life span. It was hypothesised that Clk mutations slow down the pace of many cellular functions and lower the rate of energy metabolism, possibly resulting in slower production of reactive oxygen species which in turn could result in slower ageing [1–3]. We tested this hypothesis by measuring respiration rates, light production capacities [4] (a measure of metabolic potential) and ATP levels in various strains harbouring mutant alleles of the Clk genes clk-1 and gro-1 and of three other genes that interact with the Clk genes. We found a mild reduction of oxygen consumption rates but little alteration of metabolic capacities in the single Clk mutants during the first 4–5 days of their adult lives, relative to the wild-type strain. This difference tended to fade away with increasing age, however, and aged Clk mutants eventually retained higher metabolic capacities than the wild-type control strain N2. These profiles are suggestive of physiological time being retarded, relative to chronological time in Clk mutants. Ageing clk-1 and gro-1 mutants also retained substantially elevated ATP levels relative to the N2 strain, and the simultaneous presence of mutations in daf-2 or age-1 – genes that affect longevity – boosted this effect. Thus, energy production and consumption appear to be uncoupled in these mutants. Mutation in the transcription factor daf-16 suppressed the Age and ATP phenotypes, but not the reduction of respiration rate imparted by mutation in clk-1 .

At least three mechanisms determine life span in Caenorhabditis elegans. An insulin-like signaling pathway regulates dauer diapause, reproduction and longevity. Reduction-or loss-of-function mutations in this pathway can extend longevity substantially, suggesting that the wild-type alleles shorten life span. The mutations extend life span by activating components of a dauer longevity assurance program in adult life, resulting in altered metabolism and enhanced stress resistance. The Clock (Clk) genes regulate many temporal processes, including life span. Mutation in the Clk genes clk-1 and gro-1 mildly affect energy production, but repress energy consumption dramatically, thereby reducing the rate of anabolic metabolism and lengthening life span. Dietary restriction, either imposed by mutation or by the culture medium increases longevity and uncovers a third mechanism of life span determination. Dietary restriction likely elicits the longevity assurance program. There is still uncertainty as to whether these pathways converge on daf-16 to activate downstream longevity effector genes such as ctl-1 and sod-3. There is overwhelming evidence that the interplay between reactive oxygen species (ROS) and the capacity to resist oxidative stress controls the aging process and longevity. It is as yet not clear whether metabolic homeostasis collapses with age as a direct result of ROS-derived damage or is selectively repressed by longevity-determining genes. The dramatic decline of protein turnover during senescence results in the accumulation of altered enzymes and in a gradual decline of metabolic performance eventually followed by fatal failure of the system.

The superoxide radical (O2−) has long been considered a major cause of aging. O2− in cytosolic, extracellular, and mitochondrial pools is detoxified by dedicated superoxide dismutase (SOD) isoforms. We tested the impact of each SOD isoform in Caenorhabditis elegans by manipulating its five sod genes and saw no major effects on life span. sod genes are not required for daf-2 insulin/IGF-1 receptor mutant longevity. However, loss of the extracellular Cu/ZnSOD sod-4 enhances daf-2 longevity and constitutive diapause, suggesting a signaling role for sod-4. Overall, these findings imply that O2− is not a major determinant of aging in C. elegans.

The nematode Caenorhabditis elegans has proved to be an excellent model organism for the study of development and aging. Many aging mutants have been discovered in the past two decades, and much has been discovered about the physiology of long-lived mutants. It therefore seems surprising that dietary restriction (DR) has not been extensively studied using C. elegans. The main reason for this is the lack of an ideal method to subject C. elegans to DR. However, several authors have tried to study the effect of DR on the metabolism and physiology of C. elegans, and epistasis-type interaction studies have been carried out in order to detect genes that might be involved in DR effects. These studies show that DR life extension is not caused by a reduced metabolic rate, consistent with results in other species. Moreover, the well-known insulin/IGF-1 pathway seems not to mediate life-extending effects. One possibility is that target of rapamycin signaling mediates the effects of DR on life span in C. elegans.

The nematode Caenorhabditis elegans has been subjected to DR by food (Escherichia coli) dilution, growth in axenic medium and using animals having defects in feeding behavior or in specific nutrient transporter proteins. There is evidence that DR causes increased resistance against environmental stressors but no decrease of metabolic rate. The insulin/IGF-1 signaling pathway does not mediate the longevity effect of DR in this species, but TOR signaling may be involved. The metabolic stability-longevity theory offers a plausible explanation of the longevity effect of DR but needs experimental validation.

The nematode Caenorhabditis elegans has proved to be an excellent model organism for the study of development and aging. Many aging mutants have been discovered in the past two decades, and much has been discovered about the physiology of long-lived mutants. It therefore seems surprising that dietary restriction (DR) has not been extensively studied using C. elegans. The main reason for this is the lack of an ideal method to subject C. elegans to DR. However, several authors have tried to study the effect of DR on the metabolism and physiology of C. elegans, and epistasis-type interaction studies have been carried out in order to detect genes that might be involved in DR effects. These studies show that DR life extension is not caused by a reduced metabolic rate, consistent with results in other species. Moreover, the well-known insulin/IGF-1 pathway seems not to mediate life-extending effects. One possibility is that target of rapamycin signaling mediates the effects of DR on life span in C. elegans.

When environmental conditions are unsuitable to support nematode reproduction, Caenorhabditis elegans arrests development before the onset of sexual maturity and specialised ‘dauer’ larvae, adapted for dispersal, and extended diapause are formed. Dauer larvae do not feed and their metabolism is dependent on internal food reserves. Adult worms which express defects in the insulin/insulin-like growth factor receptor DAF-2 also display enhanced longevity. Whole genome mRNA expression profiling has demonstrated that C. elegans dauer larvae and daf-2 adults have similar transcription profiles for a cohort of longevity genes. Important components of this enhanced longevity system are the a-crystallin family of small heat shock proteins, anti-ROS defence systems, increased activity of cellular detoxification processes and possibly also increased chromatin stability and decreased protein turnover. Anaerobic fermentation pathways are upregulated in dauer larvae, while long-lived daf-2 adults appear to have normal oxidative metabolism. Anabolic pathways are down regulated in dauer larvae (and possibly in daf-2 adults as well), and energy consumption appears to be diverted to enhanced cellular maintenance and detoxification processes in both systems.

The insulin/insulin-like growth factor-1 (Ins/IGF-1) pathway regulates the aging rate of the nematode Caenorhabditis elegans. We describe other features of the three Ins/IGF-1 mutants daf-2, age-1 and aap-1. We show that the investigated Ins/IGF-1 mutants all have a reduced body volume, reduced reproductive capacity, increased ATP concentrations and an elevated stress resistance. We also observed that heat production is lower in these mutants, although the respiration rate was similar or higher compared with wild-type individuals, suggesting a metabolic shift in these mutants.

The first observation of the positive effect of reduced food intake on mammalian life span was made 70 years ago. In the decades that followed, researchers successfully applied this method to increase the life span of a very wide range of animals. The nematode Caenorhabditis elegans is an excellent model organism for studying the aging process. However, relatively little effort has been made to study the effects of dietary restriction in C. elegans. In this review we discuss the difficulties of subjecting C. elegans to dietary restriction, the effects of dietary restriction on metabolism and stress defense, and the potential role of different signaling pathways in DR-induced life extension. Recent experiments suggest that the TOR (target of rapamycin) pathway, rather than insulin-like signaling, might be involved in mediating the life-extending effect of dietary restriction.

The nematode Caenorhabditis elegans has been subjected to dietary restriction (DR) by a number of means, with varying results in terms of fecundity and lifespan. Two possible mechanisms by which DR increases lifespan are reduction of metabolic rate and reduction of insulin/IGF-1 signalling. Experimental tests have not supported either possibility. However, interaction studies suggest that DR and insulin/IGF-1 signalling may act in parallel on common regulated processes. In this review, we discuss recent developments in C. elegans DR research, including new discoveries about the biology of nutrient uptake in the gut, and the importance of invasion by the bacterial food source as a determinant of lifespan. The evidence that the effect of DR on lifespan in C. elegans is mediated by the TOR pathway is discussed. We conclude that the effect of DR on lifespan is likely to involve multiple mechanisms, which may differ according to the DR regimen used and the organism under study.

Dietary restriction (DR) increases life span in a wide variety of animals. In Caenorhabditis elegans both reduced bacterial concentration (BDR) and culture on non-bacterial, semi-defined, axenic food sources (ADR) increased longevity. An Ins/IGF-1-like (IIF) signalling pathway has been shown to specify life span in C. elegans and it has been suggested that this IIF signalling pathway mediates life extension via DR. We show that both ADR and BDR act independently with mutations in the IIF pathway to increase longevity, stress resistance, and specific activities of superoxide dismutase and catalase. Moreover, these effects are not dependent on daf-16, which is known to suppress other mutations that act through the IIF pathway. We conclude that DR extends life span by mechanisms distinct from those specified by the IIF pathway.

In Caenorhabditis elegans, metabolism and life expectancy respond to environmental cues of food availability and temperature. Several genes act in a neuroendocrine, DAF-2, insulin/IGF-1 receptor-like pathway in which reduced signaling affects metabolism and increases longevity. Here we describe the effect of reduced DAF-2 signaling on several parameters of metabolism including rates of oxygen consumption and heat output, the calorimetric/respirometric ratio, ATP levels, XTT reduction capacity and accumulation of lipofuscin. We also asked whether the DAF-2 signaling pathway mediates the metabolic and longevity effects of axenic culture medium. We show that both interventions act either antagonistically or in concert, depending on the parameter examined and that axenic culture medium, unlike DAF-2 signaling, does not need DAF-16 for generating these effects. In addition, we provide evidence that DAF-2 signaling controls mitochondrial bioenergetics by adjusting the rate of ATP synthesis to the rate of ATP utilization and by regulating the heat-producing proton leak pathway.

It is widely believed that normal by-products of oxidative metabolism and the subsequent molecular damage inflicted by them couple the aging process to metabolic rate. Accordingly, high metabolic rates would be expected to accelerate aging, and life-extending interventions are often assumed to act by attenuating metabolic rate. Notorious examples in Caenorhabditis elegans are food restriction, mutation in the Clock genes and several genes of the insulin-like signalling pathway. Here we discuss how metabolic rate can be accurately measured and normalized, and how to deal with differences in body size. These issues are illustrated using experimental data of the long-lived mutant strains clk-1(e2519) and daf-2(e1370). Appropriate analysis shows that metabolic rates in wildtype and in the clk-1 mutant are very similar. In contrast, the metabolic rate profiles point to a metabolic shift toward enhanced efficiency of oxidative phosphorylation in the daf-2 worms.

Publisher Summary This chapter discusses the metabolism and life span determination in Caenorhabditis elegans . Several mechanisms can extend longevity of C. elegans , including caloric restriction and single gene mutations notably in the Ins/IGF pathway and in any one of the clk genes. All of them affect metabolism, and it is important to establish the changes consistently associated with aging. Caloric restriction causes increases rather than decreases in metabolic rate, measured as respiration and heat production rates. Reduced Ins/IGF signaling does not affect respiration rates, however lowers heat dissipation and possibly CO 2 production. Mutation in any one of the clk genes deregulates, and on average slows down, a number of temporal processes, however does only mildly, at most, affect respiration and heat production rates. Longevity is generally associated with increased resistance to stress, particularly oxidative stress, which is also reflected by co-ordinated increases in catalase and superoxide dismutase (SOD) activities. Consistent up-regulation of these enzyme activities is not seen in clk mutants. Life extension by dietary restriction is independent of the Ins/IGF signaling pathway in C. elegans , like in the mouse.

Mutation in any of the four clock genes (clk-1, clk-2, clk-3, gro-1) causes an average slowing down of many temporal processes, and an increase of mean life span. The latter effect has been linked to the slow phenotype, and it has been reasoned that any reduction of the rate of living would reduce the load of oxidative damage, which is thought to drive the ageing process. To test this model we measured several parameters describing metabolic output in wild type worms and all four Clk mutants. We found no gross changes in metabolic output, as assessed from oxygen consumption and heat production rates, lucigenin-mediated light production capacity, ATP content, and lipofuscin autofluorescence. Catalase and superoxide dismutase (SOD) were variably altered, but not cooperatively, as would be expected to enhance reactive oxygen species (ROS) scavenging activity. Thus we conclude that the prolonged life span of Clk mutants cannot be attributed to reduced metabolic rate or an increased activity of the major antioxidant enzymes catalase and SOD.

The nematode Caenorhabditis elegans responds to unfavourable environmental conditions by arresting development and entering diapause as a dauer larva. Dauers can survive several times the normal life span and the duration of the dauer state has no effect on postdauer life span. This led to the suggestion that dauers are non-ageing, and that dauers eventually perish as the consequence of depletion of stored nutrients. We have investigated physiological changes associated with long-term diapause survival, and found that dauer larvae slowly develop senescence-like symptoms, including decrease of metabolic capacity, aconitase enzyme activity, and ATP stores, and increase of lipofuscin- and oxidised flavin-specific fluorescence. However, these changes are reversed when the dauers recover. Thus senescence can occur before attainment of reproductive maturity, and furthermore, is reversible. Other life processes, including respiration rate and heat output, remain unaltered over four weeks of diapause at 24 degrees C. Possible determinants of the enhanced life maintenance include increased resistance to oxidative stress provided by enhanced superoxide dismutase and catalase activities, and a shift to a highly reducing redox status.

Accurate measures of physiological and metabolic condition could provide more insight into how longevity genes and signalling pathways affect global metabolic activity and life span. The present study is essentially a methodological treatise in which we describe and evaluate a number of methods to assess changes of metabolic activity in ageing Caenorhabditis elegans . Oxygen consumption and CO 2 production rate assays, and measurement of the heat output by microcalorimetry are performed using live worms. For other assays, frozen (−75 °C) samples can be used. A lucigenin-mediated light production assay provides information on the metabolic capacity (scope for metabolic activity) of the worms just before freezing. Assaying ATP and ADP levels provides a measure of the instantly available energy. The XTT assay measures the activity of enzymes that can reduce XTT. Blue fluorescence emitted at 420–470 nm is a potentially useful biomarker of the rate of ageing. A protein quantification protocol for normalising all data for quantitative comparisons is presented. We illustrate how these methods can validate or disprove models of gene action inferred from molecular identification.

The nervous system acts as a major regulator of the life span of Caenorhabditis elegans. Temperature and chemical stimuli from the environment are integrated with internal signals from the reproductive system to specify adult longevity. An insulin-like signaling cascade acts in neurons and coordinates control of senescence of the entire organism by regulating metabolism and a stress response mechanism. Caloric restriction extends life span, possibly by activation of the stress response program.

The life-span of the nematode Caenorhabditis elegans can be extended by at least six different mechanisms, including calorie restriction, reduced Ins/IGF-1 signaling, germline ablation, food sensing amphid ablation, mitochondrial deficiency, and decreased temperature. Reduced Ins/IGF-1 signaling and calorie restriction can also increase the life-span of flies and mice. The Brevia “Healthy animals with extreme longevity” by N. Arantes-Oliveira et al. (24 Oct. 2003, p. [611][1]) showed that daf-2 RNAi treatment and gonad ablation of worms carrying the daf-2(e1368) hypomorphic mutation in the gene encoding the C. elegans Ins/IGF-1 receptor increases their life-span 6.0-fold. We have found that the average life-span of daf-2(e1370) mutants grown in axenic medium [a sterile liquid medium based on yeast extract, soy peptone, and hemoglobin; see ([1][2])] was 90.9 days, representing a 6.3-fold life extension and a 7.5-fold adult life-span extension relative to wild-type controls grown on plate cultures seeded with live E. coli cells ([1][2]). Arantes-Oliveira et al. also note the health of their long-lived worms. We observed that worms grown in axenic medium appear more vigorous than their monoxenically grown counterparts and that these worms exhibit an increase in metabolic rate ([2][3]), counter to the idea that a reduction of the metabolic rate is associated with a longer life-span. Moreover, both caloric restriction and reduced Ins/IGF-1 signaling increase the resistance to heat and oxidative stressors ([1][2]), and calorically restricted mice are less prone to age-related diseases. Thus, the life of worms can be extended without diminishing health. These results might be important for human aging as well, because both caloric restriction and cell signaling have been shown to regulate the aging rate in organisms ranging from yeast to mammals. 1. 1.[↵][4] 1. K. Houthoofd, 2. B. P. Braeckman, 3. T. E. Johnson, 4. J. R. Vanfleteren , Exp. Gerontol. 38, 947 (2003). [OpenUrl][5][CrossRef][6][PubMed][7][Web of Science][8] 2. 2.[↵][9] 1. K. Houthoofd 2. et al. , Exp. Gerontol. 37, 1371 (2002). [OpenUrl][10][CrossRef][11][PubMed][12][Web of Science][13] 3. 3. K.H. and B.P.B are postdoctoral fellows with the Fund for Scientific Research-Flanders, Belgium. This research was supported by grants from Ghent University and the Fund for Scientific Research-Flanders (J.V.) and from the U.S. National Institutes for Health and the Ellison Medical Foundation (T.E.J.). [1]: /lookup/doi/10.1126/science.1089169 [2]: #ref-1 [3]: #ref-2 [4]: #xref-ref-1-1 "View reference 1. in text" [5]: {openurl}?query=rft.jtitle%253DExperimental%2Bgerontology%26rft.stitle%253DExp%2BGerontol%26rft.aulast%253DHouthoofd%26rft.auinit1%253DK.%26rft.volume%253D38%26rft.issue%253D9%26rft.spage%253D947%26rft.epage%253D954%26rft.atitle%253DLife%2Bextension%2Bvia%2Bdietary%2Brestriction%2Bis%2Bindependent%2Bof%2Bthe%2BIns%252FIGF-1%2Bsignalling%2Bpathway%2Bin%2BCaenorhabditis%2Belegans.%26rft_id%253Dinfo%253Adoi%252F10.1016%252FS0531-5565%252803%252900161-X%26rft_id%253Dinfo%253Apmid%252F12954481%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [6]: /lookup/external-ref?access_num=10.1016/S0531-5565(03)00161-X&link_type=DOI [7]: /lookup/external-ref?access_num=12954481&link_type=MED&atom=%2Fsci%2F305%2F5688%2F1238.3.atom [8]: /lookup/external-ref?access_num=000185622700004&link_type=ISI [9]: #xref-ref-2-1 "View reference 2. in text" [10]: {openurl}?query=rft.jtitle%253DExperimental%2Bgerontology%26rft.stitle%253DExp%2BGerontol%26rft.aulast%253DHouthoofd%26rft.auinit1%253DK.%26rft.volume%253D37%26rft.issue%253D12%26rft.spage%253D1371%26rft.epage%253D1378%26rft.atitle%253DAxenic%2Bgrowth%2Bup-regulates%2Bmass-specific%2Bmetabolic%2Brate%252C%2Bstress%2Bresistance%252C%2Band%2Bextends%2Blife%2Bspan%2Bin%2BCaenorhabditis%2Belegans.%26rft_id%253Dinfo%253Adoi%252F10.1016%252FS0531-5565%252802%252900173-0%26rft_id%253Dinfo%253Apmid%252F12559406%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [11]: /lookup/external-ref?access_num=10.1016/S0531-5565(02)00173-0&link_type=DOI [12]: /lookup/external-ref?access_num=12559406&link_type=MED&atom=%2Fsci%2F305%2F5688%2F1238.3.atom [13]: /lookup/external-ref?access_num=000180112900009&link_type=ISI

Background In the nematode Caenorhabditis elegans the conserved Ins/IGF-1 signaling pathway regulates many biological processes including life span, stress response, dauer diapause and metabolism. Detection of differentially expressed genes may contribute to a better understanding of the mechanism by which the Ins/IGF-1 signaling pathway regulates these processes. Appropriate normalization is an essential prerequisite for obtaining accurate and reproducible quantification of gene expression levels. The aim of this study was to establish a reliable set of reference genes for gene expression analysis in C. elegans. Results Real-time quantitative PCR was used to evaluate the expression stability of 12 candidate reference genes (act-1, ama-1, cdc-42, csq-1, eif-3.C, mdh-1, gpd-2, pmp-3, tba-1, Y45F10D.4, rgs-6 and unc-16) in wild-type, three Ins/IGF-1 pathway mutants, dauers and L3 stage larvae. After geNorm analysis, cdc-42, pmp-3 and Y45F10D.4 showed the most stable expression pattern and were used to normalize 5 sod expression levels. Significant differences in mRNA levels were observed for sod-1 and sod-3 in daf-2 relative to wild-type animals, whereas in dauers sod-1, sod-3, sod-4 and sod-5 are differentially expressed relative to third stage larvae. Conclusion Our findings emphasize the importance of accurate normalization using stably expressed reference genes. The methodology used in this study is generally applicable to reliably quantify gene expression levels in the nematode C. elegans using quantitative PCR.