Annemie De Vreese

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.

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.

Apparent uncoupling of energy production and consumption in long-lived Clk mutants of Caenorhabditis elegans

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. We tested this hypothesis by measuring respiration rates, light production capacities (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.

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.

Rate of aerobic metabolism and superoxide production rate potential in the nematode Caenorhabditis elegans

We have monitored oxygen consumption as a measure of the rate of aerobic metabolism during the lifetime of Caenorhabditis elegans. We have also developed a chemiluminescent technique which measures exogenous NADPH-stimulated superoxide anion production by freeze-thawed worms. In this assay light production depends on the combined activities of all of the enzymes involved in superoxide production, both directly and indirectly, thus reflecting their activity levels immediately prior to freeze fixation. We have designated this parameter the superoxide production rate potential. The superoxide production rate potential is controlled by the longevity determining gene age-1 and varies in a life cycle-dependent fashion. The metabolic rate generally follows these fluctuations, but additionally shows specific alterations as a response to environmental factors. Metabolic rate and superoxide production rate potential increase by 1.3- and 3-fold, respectively, in reproducing adults. This increase is not due to the contribution of embryonating eggs, however. Culture conditions have a large effect on metabolic rate, but not on the superoxide production rate potential. The energetic cost of movement, measured as consumed oxygen, is low relative to the costs of maintenance and reproduction. Identical superoxide production rate potentials are scored in paralyzed and motile worms, as would be expected.

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 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.

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 by growth in axenic medium induces discrete changes in the transcriptional output of genes involved in energy metabolism in Caenorhabditis elegans

Dietary restriction increases life span in a wide range of species, including the nematode worm Caenorhabditis elegans. The mechanism by which it does so remains largely unknown, although it is commonly thought that a reduction of reactive oxygen species (ROS) plays a pivotal role. More specifically, for C. elegans, it has been proposed that food restriction reduces energy expenditure, possibly in conjunction with an anaerobic shift in energy production, with consequent reduction in the formation of ROS. We have measured differential transcript abundance of 49 genes known to play roles in energy metabolism in axenic culture medium, which causes a nutritional deficit and leads to a substantial increase of life span. We found no evidence for a reduction in metabolic rate or a shift to anaerobic metabolism in axenic culture. Major changes induced by growth in axenic medium include down-regulation of lipid degradation and up-regulation of glyoxylate cycle activity glyceroneogenesis and, possibly, gluconeogenesis. The activities determined in worm extracts for pyruvate kinase, phosphoenolpyruvate carboxykinase and isocitrate lyase followed a similar trend. We conclude that growth in axenic culture is marked by a general up-regulation of replenishing pathways.