Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans.

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.

Effects of resveratrol on lifespan in Drosophila melanogaster and Caenorhabditis elegans.

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.

The longevity effect of dietary restriction in Caenorhabditis 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.

Metabolism, physiology and stress defense in three aging Ins/IGF-1 mutants of the nematode Caenorhabditis elegans.

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.

DAF-2 pathway mutations and food restriction in aging Caenorhabditis elegans differentially affect metabolism.

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.

Dietary restriction in C. elegans: from rate-of-living effects to nutrient sensing pathways.

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.

Alternate metabolism during the dauer stage of the nematode Caenorhabditis 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 alpha-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.

Assessing metabolic activity in aging Caenorhabditis elegans: concepts and controversies.

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.

Dietary restriction in the nematode Caenorhabditis elegans.

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.

Assaying metabolic activity in ageing Caenorhabditis elegans.

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 degrees 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.

No reduction of energy metabolism in Clk mutants.

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.

Life extension via dietary restriction is independent of the Ins/IGF-1 signalling pathway in Caenorhabditis elegans.

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.

Ageing is reversed, and metabolism is reset to young levels in recovering dauer larvae of C. elegans.

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.

No reduction of metabolic rate in food restricted Caenorhabditis elegans.

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.

Axenic growth up-regulates mass-specific metabolic rate, stress resistance, and extends life span in Caenorhabditis elegans.

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.

Alternative oxidase dependent respiration leads to an increased mitochondrial content in two long-lived mutants of the aging model Podospora anserina.

The retrograde response constitutes an important signalling pathway from mitochondria to the nucleus which induces several genes to allow compensation of mitochondrial impairments. In the filamentous ascomycete Podospora anserina, an example for such a response is the induction of a nuclear-encoded and iron-dependent alternative oxidase (AOX) occurring when cytochrome-c oxidase (COX) dependent respiration is affected. Several long-lived mutants are known which predominantly or exclusively respire via AOX. Here we show that two AOX-utilising mutants, grisea and PaCox17::ble, are able to compensate partially for lowered OXPHOS efficiency resulting from AOX-dependent respiration by increasing mitochondrial content. At the physiological level this is demonstrated by an elevated oxygen consumption and increased heat production. However, in the two mutants, ATP levels do not reach WT levels. Interestingly, mutant PaCox17::ble is characterized by a highly increased release of the reactive oxygen species (ROS) hydrogen peroxide. Both grisea and PaCox17::ble contain elevated levels of mitochondrial proteins involved in quality control, i. e. LON protease and the molecular chaperone HSP60. Taken together, our work demonstrates that AOX-dependent respiration in two mutants of the ageing model P. anserina is linked to a novel mechanism involved in the retrograde response pathway, mitochondrial biogenesis, which might also play an important role for cellular maintenance in other organisms.

Intermediary metabolism.

Caenorhabditis elegans has orthologs for most of the key enzymes involved in eukaryotic intermediary metabolism, suggesting that the major metabolic pathways are probably present in this species. We discuss how metabolic patterns and activity change as the worm traverses development and ages, or responds to unfavorable external factors, such as temperature extremes or shortages in food or oxygen. Dauer diapause is marked by an enhanced resistance to oxidative stress and a shift toward microaerobic and anaplerotic metabolic pathways and hypometabolism, as indicated by the increased importance of the malate dismutation and glyoxylate pathways and the repression of citric acid cycle activity. These alterations promote prolonged survival of the dauer larva; some of these changes also accompany the extended lifespan of insulin/IGF-1 and several mitochondrial mutants. We also present a brief overview of the nutritional requirements, energy storage and waste products generated by C. elegans.

Against the oxidative damage theory of aging: superoxide dismutases protect against oxidative stress but have little or no effect on life span in Caenorhabditis elegans.

The superoxide radical (O(2)(-)) has long been considered a major cause of aging. O(2)(-) 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 O(2)(-) is not a major determinant of aging in C. elegans.