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.

Two-parameter logistic and Weibull equations provide better fits to survival data from isogenic populations of Caenorhabditis elegans in axenic culture than does the Gompertz model.

We have fitted Gompertz, Weibull, and two- and three-parameter logistic equations to survival data obtained from 77 cohorts of Caenorhabditis elegans in axenic culture. Statistical analysis showed that the fitting ability was in the order: three-parameter logistic > two-parameter logistic = Weibull > Gompertz. Pooled data were better fit by the logistic equations, which tended to perform equally well as population size increased, suggesting that the third parameter is likely to be biologically irrelevant. Considering restraints imposed by the small population sizes used, we simply conclude that the two-parameter logistic and Weibull mortality models for axenically grown C. elegans generally provided good fits to the data, whereas the Gompertz model was inappropriate in many cases. The survival curves of several short- and long-lived mutant strains could be predicted by adjusting only the logistic curve parameter that defines mean life span. We conclude that life expectancy is genetically determined; the life span-altering mutations reported in this study define a novel mean life span, but do not appear to fundamentally alter the aging process.

Age-specific modulation of light production potential, and alkaline phosphatase and protein tyrosine kinase activities in various age mutants of Caenorhabditis elegans.

Previous work has shown that reduction-of-function mutations in the genes daf-2 and age-1 can increase adult life (Age phenotype) of Caenorhabditis elegans and that certain daf-12 alleles considerably amplify this effect in daf-2; daf-12 doubles. We have measured the light production potential (LPP) and alkaline phosphatase (ALP) and protein tyrosine kinase (PTK) activity levels as suitable biochemical markers to further investigate genetic interactions between these genes. The light production assay measures superoxide anion production by freeze-thawed worms in assay medium containing sufficient amounts of nicotineamide adenine dinucleotide, reduced form (NADH) and nicotineamide adenine dinucleotide phosphate, reduced form (NADPH) to drive the chemiluminescent reaction at maximal speed, and 5 mM cyanide to fully repress cytosolic superoxide dismutase (SOD). This assay thus provides an estimate of the maximum output of the metabolic pathways involved at the instant of freeze-fixation, and under the condition of the assay. LPP and PTK activities decreased similarly in daf-12(m20), and a control strain that had wild-type alleles of daf-12, age-1, and daf-2. The age-dependent decrease of LPP and PTK was reduced in age-1(hx542) and age-1(hx542); daf-2(e1370), and virtually absent in daf-2(e1370) and daf-2(e1370); daf-12(m20) mutant worms. ALP activity increased with age in non-Age genotypes and showed little, if any, age-dependent alteration in daf-2(e1370) and daf-2(e1370); daf-12(m20) mutant worms. Mutation in both age-1 and daf-2 caused no stronger phenotype than a single mutation as estimated by LPP, PTK, and ALP. We propose that (a) daf-2 is the major effector of metabolic activity during adult life, (b) daf-2 downregulates metabolic activity with increasing age, and (c) daf-12 stimulates oxygen consumption independently of daf-2.

Modulation of kinase activities in dauers and in long-lived mutants of Caenorhabditis elegans.

Mutant alleles of the genes age-1 and daf-2 that lengthen life span (Age phenotype) of Caenorhabditis elegans cause higher protein kinase (PKA, PKC, PTK) activity levels in senescing worms relative to wild-type. Elevated levels of PKA and PTK were also present in dauer larvae, developmentally arrested juveniles specialized for long-term survival, relative to L3 larvae, the alternative developmental stage. PKC activity was downregulated in dauers of a non-Age control strain and in age-1 mutant dauers, compared to L3 larvae, but similar activities were measured in dauers and L3 larvae of a daf-2 mutant strain. Thus, age-1 and daf-2 mutant worms may express distinct elements of a dauer-specific survival program during adult life.

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.

The gerontogenes age-1 and daf-2 determine metabolic rate potential in aging Caenorhabditis elegans.

Mutations in the genes age-1 and daf-2 extend life span of Caenorhabditis elegans by 100 and 200%, respectively, in axenic culture. Adult worms that are mutant in either of these genes have higher metabolic capacities, called metabolic rate potentials, at all ages and the extension of their life expectancies are positively correlated with the increases of metabolic rate potential. The activities of catalase, superoxide dismutase, isocitrate dehydrogenase, isocitrate lyase, and malate synthase are all higher relative to those in worms that are wild type for these genes, but acid phosphatase is down-regulated and alkaline phosphatase activity is lowered to 10% of the activity measured in age-1(+) and daf-2(+) worms. These results suggest that genes that regulate metabolic activity may play central roles in longevity and senescence.

Analysis of the proteins of aging Caenorhabditis elegans by high resolution two-dimensional gel electrophoresis.

The metabolic rate decreases dramatically as a function of age in the nematode worm Caenorhabditis elegans. Superoxide anion production, which is tightly linked to oxygen consumption, and thus to metabolic rate, drops to a 20-fold lower level in 10-day-old, senescent worms, as compared to 4-day-old young adults. In a long-lived mutant strain of the same species metabolic activity is much better preserved. High resolution two-dimensional gel electrophoresis was employed to study alterations in the protein profile, correlating with changes of metabolic activity. Surprisingly, few proteins show age- or age- and strain-specific variations of spot intensity. The abundance of the huge majority of proteins displayed on these gels remains unaltered, irrespective of age and strain differences. These results imply that there are no major age-related alterations of proteins due to faulty protein synthesis or free radical attack, and that age-related changes in the rate of protein synthesis and breakdown must be strictly coordinated throughout the aging process.