Age- and calorie-independent life span extension from dietary restriction by bacterial deprivation in Caenorhabditis elegans

Abstract

Background: Dietary restriction (DR) increases life span and delays age-associated disease in many organisms. The mechanism by which DR enhances longevity is not well understood.

Results: Using bacterial food deprivation as a means of DR in C. elegans, we show that transient DR confers long-term benefits including stress resistance and increased longevity. Consistent with studies in the fruit fly and in mice, we demonstrate that DR also enhances survival when initiated late in life. DR by bacterial food deprivation significantly increases life span in worms when initiated as late as 24 days of adulthood, an age at which greater than 50% of the cohort have died. These survival benefits are, at least partially, independent of food consumption, as control fed animals are no longer consuming bacterial food at this advanced age. Animals separated from the bacterial lawn by a barrier of solid agar have a life span intermediate between control fed and food restricted animals. Thus, we find that life span extension from bacterial deprivation can be partially suppressed by a diffusible component of the bacterial food source, suggesting a calorie-independent mechanism for life span extension by dietary restriction.

Conclusion: Based on these findings, we propose that dietary restriction by bacterial deprivation increases longevity in C. elegans by a combination of reduced food consumption and decreased food sensing.

Figure 1

Dietary restriction by bacterial food deprivation (BD) extends life span independent of age or duration. (A) BD increases survival when initiated at 4 (red line, square symbols), 8 (blue line, circles), 14 (green line, triangles), 20 (gray line, plus symbols), or 24 (purple line, astericks) days of adulthood, relative to control fed (CF) animals. Mean life spans ± standard error, statistical significance, and number of animals examined provided in Table 1. (B) Animals subjected to transient BD, from the day 2 of adulthood to day 8 (blue line, square symbols), 14 (purple line, triangles), or 28 (green line, circles) have increased survival relative to animals maintained on CF until death (black line, diamonds). Mean life spans ± standard error, statistical significance, and number of animals examined provided in Table 2. (C) Mean survival of animals in (B) alive when BD-treated animals were transferred back to control fed conditions shows a significant increase in survival of animals exposed to transient BD relative to animals maintained on a control fed diet for life.

Figure 2

Increased thermotolerance from BD is independent of age and maintained upon return to a control diet. (A) BD increases survival at 38°C, relative to control fed (CF) animals at 7, 18, or 22 days of age. Error bars are standard error of the mean. (B) Thermotolerance (survival at 38°C) is significantly increased at the 12th day of adulthood relative to CF animals, regardless of whether BD is initiated at the 4th (BD_4–12) or 8th (BD_8–12) day of adulthood. Transient BD from the 4th to the 8th day of adulthood (BD_4–8) also results in enhanced thermotolerance relative to CF animals.

Figure 3

Bacterial food consumption decreases with age. (A) Fluorescent beads similar in size to bacterial cells are eaten and can be visualized in the intestines of young (day 4 adult) animals (Vis = visible light). (B) The percent of animals consuming a detectable quantity of fluorescent beads decreases with age in control fed (CF) animals. (C) The rate of defecation (average number of defecation cycles per animal in 10 minutes of observation) decreases with age.

Figure 4

BD increases life span of select chemosensory mutants. BD significantly extends the life span of long-lived chemosensory mutants, including (A) che-3(p801) (3 independent trials) (B) che-11(e1810) (two trials), (C) daf-10 (e1387) (2 trials), (D) osm-3(p802) (2 trials), and (E) tax-4(p678) (3 trials), compared to control fed (CF) animals. Number of animals examined, mean life spans ± standard error, median life span, and statistical significance are provided in Table 3. Extended statistical analysis (pair-wise comparisons) for each experimental group is provided in Table 4.

Figure 5

A diffusible bacterial product suppresses life span extension by bacterial deprivation (BD) in C. elegans. (A) Animals cultured on plates treated with cell-free supernatant from an overnight culture of E. coli OP50 (BD + sup) had decreased survival compared to animals maintained on untreated BD plates or BD plates treated with uninoculated LB (BD + LB). The life span of the treated group was still longer than that of control fed (CF) animals. (B) An alternate experimental strategy to exposing animals on BD to bacterial products in the absence of food consumption is to use agar barrier plates, in which a bacterial lawn is separated from the worms by a layer of agar (see Methods for a detailed description). (C) Animals cultured on agar barrier plates had decreased survival compared to animals maintained on BD plates with no bacteria present. Number of animals examined, mean life spans ± standard error, median life span, and statistical significance are provided in Table 5A. Extended statistical analysis (pair-wise comparisons) is provided in Table 5B.

Figure 6

Bacterial deprivation (BD) extends life span through reduced food consumption and reduced food sensing. Animals subjected to BD experience calorie restriction through reduced food consumption, similar to genetic models of DR such as mutation of eat-2. BD animals are also deprived of a longevity-limiting dietary cue(s) produced by the bacterial food source. Genetic epistasis analysis indicates that BD acts in parallel to long-lived chemosensory mutants (yellow box) that influence life span primarily by altering insulin/IGF-1-like signaling (IIS). Thus, we propose that life span extension from BD is a combination of the effects of calorie restriction and reduced food sensing.