Disruption of insulin signalling preserves bioenergetic competence of mitochondria in ageing Caenorhabditis elegans

Abstract

Background: The gene daf-2 encodes the single insulin/insulin growth factor-1-like receptor of Caenorhabditis elegans. The reduction-of-function allele e1370 induces several metabolic alterations and doubles lifespan.

Results: We found that the e1370 mutation alters aerobic energy production substantially. In wild-type worms the abundance of key mitochondrial proteins declines with age, accompanied by a dramatic decrease in energy production, although the mitochondrial mass, inferred from the mitochondrial DNA copy number, remains unaltered. In contrast, the age-dependent decrease of both key mitochondrial proteins and bioenergetic competence is considerably attenuated in daf-2(e1370) adult animals. The increase in daf-2(e1370) mitochondrial competence is associated with a higher membrane potential and increased reactive oxygen species production, but with little damage to mitochondrial protein or DNA. Together these results point to a higher energetic efficiency of daf-2(e1370) animals.

Conclusions: We conclude that low daf-2 function alters the overall rate of ageing by a yet unidentified mechanism with an indirect protective effect on mitochondrial function.

Figure 1

Ins/IGF-1 signalling controls age-related changes in aerobic energy production. Left panels: wild-type (WT) versus daf-2(e1370), right panels: WT versus daf-16(mgDf50). (a-b) Respiration rate. (c-d) Metabolic heat production. (e-f) Calorimetric to respirometric ratio. (g-h) Adenosine triphosphate content. (i-j) Adenosine diphosphate content. Displayed values are means ± standard error of mean for three replicate cultures; *, P < 0.05, **, P < 0.01 and ***, P < 0.001.

Figure 2

Effect of age on mitochondrial genome and cytochrome c content and citrate synthase activity. (a) Age-specific mitochondrial genome content of wild-type and daf-2(e1370) animals. Three mitochondrial genes were quantified; the results were normalized to obtain the relative mitochondrial genome content per strain and age cohort. The error bars indicate ± standard error of mean for three mitochondrial genes and three replicate ageing cohorts. (b) Western blots showing age-related changes of cytochrome c abundance in crude worm extract and isolated mitochondria. Results from one representative experiment are shown. The numeric values below each spot denote the abundance of cytochrome c in that spot normalized to the corresponding spot from 1-day-old wild-type (WT) adults. (c-d) Activity levels of citrate synthase in crude worm extract (indicated as 'W') and in isolated mitochondria (indicated as 'M'). Left panel: WT versus daf-2(e1370), right panel: WT versus daf-16(mgDf50). Data represent means ± standard error of mean (bars) for at least three replicate cultures; *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Figure 3

The daf-2(e1370) allele preserves mitochondrial bioenergetic competence throughout the adult life trajectory. (a) State 3 oxygen consumption. (b) State 4 oxygen consumption. (c) Respiratory control ratio. (d) Adenosine diphosphate (ADP)/O ratio. (e) ATP synthesis by isolated mitochondria.

Figure 4

The daf-2(e1370) allele causes a higher mitochondrial membrane potential and enhanced respiratory capacity. (a) Increased accumulation of the fluorescent probe DASPMI inside energized daf-2(e1370) mitochondria. Fluorescence intensity of DASPMI in energized and uncoupled isolated mitochondria of wild-type (WT) and daf-2(e1370). Fluorescence emission over the 2 min interval was averaged for each energetic state. Displayed values are means ± standard error of mean for three replicate cultures. The increase of DASPMI fluorescence is proportional to the amount of dye taken up by the mitochondria which itself is proportional to the membrane potential (Mewes and Rafael [33]). (b) Higher oxygen consumption in the presence of adenosine diphosphate or uncoupler by daf-2(e1370) mitochondria. The assay was performed on two replicate cultures; since no age-dependent differences were noticed, mitochondrial respiration rates were averaged per strain over a 9-day time span. Paired t-tests were performed in within-strain comparisons, unpaired t-tests were performed in between-strain comparisons; * P < 0.05, ** P < 0.01 and *** P < 0.001.

Figure 5

daf-2(e1370) mitochondria generate more H₂O₂ but do not reveal increased carbonyl load. (a) H₂O₂ generation by isolated mitochondria from wild-type and daf-2(e1370). Mitochondria were fuelled with pyruvate, malate and adenosine diphosphate. Cu/Zn SOD from erythrocytes was added to achieve maximal conversion of O₂ to H₂O₂. Data represent means ± standard error of mean (SEM; bars) for mitochondria isolated from four replicate cultures; * P < 0.05 and ** P < 0.01. (b) Carbonyl content of mitochondrial protein derivatized with diphenylhydrazine and detected by Western blotting and a diphenylhydrazone specific antibody. Data represent means ± SEM. Six replicate cultures of each strain were grown but these were sampled at different time intervals, occasionally reducing the number of replicate samples for each time interval to 3; * P < 0.05.