Dietary restriction of Caenorhabditis elegans by axenic culture reflects nutritional requirement for constituents provided by metabolically active microbes.

In Caenorhabditis elegans, several manipulations that affect nutrition slow development, reduce fecundity, and increase life span. These are viewed as dietary restriction (DR) and include culture in semidefined, nutrient-rich liquid medium that is axenic (i.e., there is no microbial food source). Here we describe convenient ways to exert DR by culture on agar plates containing axenic medium. We used these to explore whether effects of axenic culture really reflect DR. Our results imply that major nutrient components of axenic medium, and overall caloric content, are not limiting for life span. However, adding growth-arrested Escherichia coli as an additional food source rescued the effects of axenic culture. We then sought to identify the component of E. coli that is critical for normal C. elegans nutrition using add-back experiments. Our results suggest that C. elegans has a nutritional requirement for live, metabolically active microbes or, possibly, an unidentified, heat-labile, nonsoluble component present in live microbes.

Adult-limited dietary restriction slows gompertzian aging in Caenorhabditis elegans.

Dietary restriction (DR) delays the onset of age-related deterioration and extends the life span in a variety of model organisms. In many species, age changes in mortality obey the Gompertz equation, which describes an exponential increase with age in age-specific mortality rate. Recently, this model has been used in fruitflies and rodents to investigate the mechanism by which DR reduces adult mortality. We report that food restriction imposed by axenic culture reduces the exponential increase of age-specific mortality of Caenorhabditis elegans. Furthermore, the life span appears largely independent of nutritional status during development, as shown by shifting worms to different food concentrations shortly before adulthood. When DR was exerted after reproduction, a smaller reduction in Gompertzian aging was seen. Thus, the demographic changes exerted by DR in C. elegans resemble those seen in rats, yet are different to those seen in Drosophila and mice.

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.

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.

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.