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Keywords:

  • aging;
  • Drosophila;
  • insulin/IGF signalling;
  • life span;
  • mortality

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

The insulin/insulin growth factor (IGF)-like signaling (IIS) pathway has a conserved role in regulating lifespan in Caenorhabditis elegans, Drosophila and mice. Extension of lifespan by reduced IIS has been shown in C. elegans to require the key IIS target, forkhead box class O (FOXO) transcription factor, DAF-16. dFOXO, the Drosophila DAF-16 orthologue, is also an IIS target, and its overexpression in adult fat body increases lifespan. In C. elegans, IIS acts exclusively during adulthood to determine adult survival. We show here, using an inducible overexpression system, that in Drosophila continuous dFOXO overexpression in adult fat body reduces mortality rate throughout adulthood. We switched the IIS status of the flies at different adult ages and examined the effects of these switches on dFOXO expression and mortality rates. dFOXO protein levels were switched up or down by the inducible expression system at all ages examined. If IIS status is reversed early in adulthood, similar to the effects of another intervention that reduces adult mortality in Drosophila, dietary restriction (DR), there is a complete switch of subsequent mortality rate to that of flies chronically exposed to the new IIS regime. At this age, IIS thus acts acutely to determine risk of death. Mortality rates continued to respond to a switch in IIS status up to 4 weeks of adult age, but not thereafter. However, unlike DR, as IIS status was altered at progressively later ages, mortality rates showed incomplete switching and responded with progressively smaller changes. These findings indicate that alteration of expression levels of dFOXO may have declining effects on IIS status with age, that there could be some process that prevents or lessens the physiological response to a switch in IIS status or that, unlike DR, this pathway regulates aging-related damage. The decreased mortality and increased lifespan of dFOXO overexpressing flies was uncoupled from any effect on female fecundity and from expression levels of Drosophila insulin-like peptides in the brain.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

The insulin/insulin growth factor (IGF)-like signaling (IIS) pathway has a conserved role in regulating lifespan. Reduced IIS has been shown to increase lifespan in Caenorhabditis elegans, Drosophila and mice (Kimura et al., 1997; Guarente & Kenyon, 2000; Clancy et al., 2001; Tatar et al., 2001; Blüher et al., 2003; Holzenberger et al., 2003). In C. elegans, extension of lifespan by reduced IIS has been shown to require the key IIS target, the forkhead box class O (FOXO) transcription factor, DAF-16 (Ogg et al., 1997). dFOXO, the Drosophila DAF-16 orthologue, is also an IIS target (Jünger et al., 2003; Puig et al., 2003). Overexpression of dFOXO, which mimics reduction of IIS, in the adult fat body, has been shown to increase lifespan (Giannakou et al., 2004; Hwangbo et al., 2004).

Age-specific mortality is a measure of hazard of death for an individual at any given age and, unlike survival, which is a cumulative measure, can be used to independently calculate the risk of death for an individual at any given time (Vaupel et al., 1998). Mortality rates generally increase with age and are characterised by two parameters; the initial baseline mortality rate and the rate at which this mortality increases with age (Finch, 1990). Interventions that increase lifespan can do so either by reducing the rate of increase in mortality with age (which is reducing the slope of the mortality trajectory) or by reducing the initial mortality rate (which is by lowering the Gompertz intercept parameter) or both (Pletcher et al., 2000). For example, dietary restriction (DR) in Drosophila increases both median and maximum lifespan and acts by lowering the initial baseline mortality rate with no effect on the slope of the mortality trajectory (Pletcher et al., 2002). Temperature, on the other hand, affects the slope of the mortality trajectory and flies cultured at a lower temperature exhibit a reduction in the slope of the mortality trajectory compared to flies kept at a higher temperature (Pletcher et al., 2000).

It has been suggested that interventions that reduce the slope of the mortality trajectory change the rate of aging (Finch, 1990). Some confirmation of this idea comes from the results of switching of thermal or dietary regime in Drosophila. Switching flies between dietary regimes causes them to switch their subsequent mortality rate to that characteristic of flies held permanently in the new dietary regime, by acutely reducing the initial mortality (Gompertz intercept parameter) but not the slope of the mortality trajectory (Good & Tatar, 2001; Mair et al., 2003). In contrast, lowered temperature reduces the rate of damage accumulation (i.e. aging), because flies switched between thermal regimes show a permanent effect of their thermal history, and alter the slope rather than the elevation of their mortality trajectory upon switching of thermal regime (Mair et al., 2003). However, there is not a huge amount that can be concluded about implications of shapes of mortality trajectories for underlying mechanisms because data are so limited. The crucial information comes from switching the state of cause of the mortality difference, which reveals the relative roles of risk and damage in producing differences in mortality rate (Mair et al., 2003; Partridge et al., 2005).

In C. elegans, switches in IIS status using double-stranded RNA interference showed that IIS acts specifically during adulthood to influence survival (Dillin et al., 2002). This study was informative about the timing of the effect of IIS status on adult lifespan, but did not include an analysis of mortality rates after switches in IIS status. We have analysed the effects of switches between low and high IIS status during adulthood, by switching dFOXO overexpression on and off and assaying the effect of these switches on subsequent mortality to understand the contribution of risk and damage in the action of IIS on mortality.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

Mortality trajectories of dFOXO overexpressing flies

In order to analyse the dynamics of the effect of IIS on mortality in Drosophila, an inducible system was required where levels of IIS could be changed throughout adulthood and the effect on mortality examined. Two such models have been described, both using inducible Geneswitch drivers, which are induced by the presence of the drug RU486 (Roman et al., 2001), to overexpress dFOXO, which mimics reduction of IIS. One used a driver expressed in adult head fat body, S1-32 (Hwangbo et al., 2004), the other used a driver expressed throughout adult fat body, S1-106 (Giannakou et al., 2004). We assessed these two systems in parallel to determine which had the greater effect on mortality rates, and would therefore be of greatest utility for this study. Surprisingly, the combination of S1-32 driver with upstream activating sequence (UAS)-dFOXO-TM (dFOXO triple mutant, insensitive to insulin signaling) (Hwangbo et al., 2004) had no effect on lifespan in several conditions (Supplementary Fig. S1). This was not due to lack of induction of the driver, because a UAS-lacZ reporter construct showed specific expression in the heads of flies on +RU486 in quantitative β-galactosidase assays, as in the published report (Hwangbo et al., 2004) (Supplementary Fig. S2). We therefore did not investigate this model further. The second system used overexpression of UAS-dFOXO in adult fat body (S1-106 inducible driver) (Giannakou et al., 2004). Age-specific mortality analysis showed that overexpression of dFOXO in the fat body of adult females increased median and maximum lifespan and lowered the age-specific mortality compared to control flies at all ages, in two independent experiments (Fig. 1). Experiments shown in Fig. 1 are representative of 20 independent experiments (with three different transgenes, combinations of backgrounds and food types) that all showed a significant increase in lifespan on +RU486, out of a total of 22 such experiments conducted. The mortality trajectories of the two groups in both experiments shown in Fig. 1 were compared by fitting a Gompertz model over the linear portion of the increase in mortality (Table 1) (Pletcher, 1999). The ‘a’ parameter (initial mortality rate) but not the ‘b’ parameter (rate of increase in mortality with age) was lower in the +RU486 group compared to the –RU486 group, demonstrating that overexpression of dFOXO increased lifespan by lowering the whole mortality trajectory, with no effect on slope. This effect on mortality trajectory has previously been shown in Drosophila to be characteristic of extension of lifespan by DR (Pletcher et al., 2002; Mair et al., 2003) but not by lowered temperature (Mair et al., 2003). Thus, dFOXO overexpression with the S1-106 adult fat body driver gave an age-specific signature that allowed further exploration of the dynamics of the effect.

image

Figure 1. Survival curves and mortality trajectories of S1-106/dFOXO females. Lifespans and mortality trajectories of white Dahomey (wd); S1-106/dFOXO; +/+ and wd; S1-106/+; dFOXO/+ mated females on +200 µm RU486 1.0 SY food since day 3 or maintained on –RU486 food. (A and B) Survival curves. Groups were compared using log-rank tests and maximum lifespans were compared by comparing the average age at death of the last 10% of each group. (A) Survivals of wd; S1-106/dFOXO; +/+ females on +RU486 and on –RU486. Median lifespans are as follows: +RU486 day 3 females, 52 days (33.3% increase over –RU486 control, P < 0.0001, n = 466); –RU486 females, 39 days (n = 536). +RU486 day 3 females have increased maximum lifespans compared to –RU486 controls (P < 0.05). (B) Survival of wd; S1-106/+; dFOXO/+ females on +RU486 and on –RU486. Median lifespans are as follows: +RU486 day 3 females, 57 days (21.2% increase over –RU486 control, P < 0.0001, n = 466); –RU486 females, 47 days (n = 200). +RU486 day 3 females have increased maximum lifespans compared to –RU486 controls (P < 0.05). (C and D) Age-specific mortality analysis. Natural log of the mortality rate (µx) is plotted. Data are smoothed over a 3-day interval. (C) Age-specific mortality analysis wd; S1-106/dFOXO; +/+ females shown in graph A. (D) Age-specific mortality analysis wd; S1-106/+; dFOXO/+ females shown in graph B.

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Table 1.  Estimates of the parameters of the Gompertz mortality model with 95% lower and upper confidence intervals. Overexpression of dFOXO in white Dahomey (wd); S1-106/dFOXO and wd; S1-106/+; dFOXO; +/+ females shown in Fig. 1
GroupaLCIUCIbLCIUCI
  • Mortality models and maximum-likelihood analysis were executed by using WINMODEST (Pletcher, 1999).

  • LCI, lower confidence interval; UCI, upper confidence interval.

  • *

    Indicates significant difference from –RU486 group.

  • †Initial mortality rate.

  • ‡Rate of exponential increase in mortality with age early in life.

+RU486  2 × 10−5*  6 × 10−6  8 × 10−50.190.150.23
S1-106/dFOXO
–RU486  3 × 10−4  2 × 10−4  5 × 10−30.150.130.16
S1-106/dFOXO
+RU4863.8 × 10−41.6 × 10−4  9 × 10−40.090.070.11
S1-106/+; dFOXO/+
–RU486  8 × 10−4  5 × 10−41.2 × 10−30.090.080.1
S1-106/+; dFOXO/+

Expression pattern and induction characteristics of S1-106 driver

To determine the extent of induction of the S1-106 driver by feeding the flies RU486 at different ages, we first determined the expression pattern of the driver, using a UAS-lacZ reporter construct together with quantitative β-galactosidase assays and β-galactosidase staining of tissues. The driver was expressed both in the abdominal and head fat body (Fig. 2A,B). We therefore used whole flies for our analyses. We next examined the effects of inducing UAS-lacZ at different ages and for different lengths of time (Fig. 2C). At all ages examined, the presence of RU486, either chronic from day 3 or for 3 days until the age of sampling, caused a robust, significant increase in β-galactosidase activity, showing that the driver could be induced at all ages examined. However, the increase in β-galactosidase activity upon addition of RU486 tended to decrease with age (over 4 weeks) both with chronic induction and after 3-day induction (at day 28, 3-day induction was significantly reduced compared to 3-day induction at day 13, P < 0.05). These results demonstrate that the S1-106 inducible system could be used for induction at different ages, at least until 4 weeks of age, but that the degree of induction decreased with the age at which induction was initiated.

image

Figure 2. Induction of S1-106 driver. (A) LacZ staining of 7-day-old female S1-106/UAS-lacZ induced by RU486 at day 3 for 4 days. Heads and bodies of S1-106/UAS-lacZ on –RU486 food denoted by –RU486 and heads and bodies of S1-106/UAS-lacZ on +RU486 food denoted by +RU486. (B and C) Induction of S1-106 driver using quantitative β-gal assays. Assays were carried out on female S1-106/UAS-lacZ flies induced by 200 µm ±RU486 1.0 SY food. Data are presented as arbitrary β-gal activity, mean ± SEM, treatments were compared by Welch two sample t-test. Significant differences are denoted by asterisks, * for P < 0.05 and ** for P < 0.0001. (B) Head- and body-specific induction of S1-106 driver. β-Gal activity liquid assays of 7-day-old female S1-106/UAS-lacZ flies induced by RU486 at day 3 for 4 days. n = 12 samples per group, assays on individual heads and bodies. Data are presented as arbitrary β-gal activity/mg wet weight. +RU486 S1-106/UAS-lacZ heads (black) exhibit significantly higher β-galactosidase activity, than –RU486 S1-106/UAS-lacZ heads (white) (P < 0.05, 2.5-fold induction) and +RU486 S1-106/UAS-lacZ bodies exhibit higher β-galactosidase activity than –RU486 S1-106/UAS-lacZ bodies (P < 0.0001, more than 10-fold induction). (C) Induction of S1-106 driver at different ages. Assays on female S1-106/UAS-lacZ flies induced by RU486 at different ages, either chronic since day 3 or for 3 days, sampled at the age indicated on graph. n = 15–25 whole flies per group, assays on individual flies. At days 13, 21 and 28, no significant difference was observed between continuous +RU486 induction from day 3 or 3-day induction only (P = 0.8, P = 0.6 and P = 0.09, respectively). At all time-points, induced conditions, whether continuous or short term, showed significantly higher activity than –RU486 (P < 0.0001 in all cases). Comparing long-term induction, there was no significant difference between +RU486 groups at different ages: between day 13, +RU486 and day 21, +RU486 (P = 0.8); between day 21, +RU486 and day 28, +RU486 (P = 0.4); between day 13, +RU486 and day 28, +RU486 (P = 0.2). Comparing induction for 3 days at different ages, there was no significant difference between: day 13, +3 days RU486 and day 21, +3 days RU486 (P = 0.3) or between day 21, +3 days RU486 and day 28, +3 days RU486 (P = 0.1). There was, however, a significant decrease in 3-day induction between day 13, +3 days RU486 and day 28, +3 days RU486 (P = 0.003).

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dFOXO induction and removal of induction at different ages

We then examined the effects of dFOXO overexpression at different times in adulthood upon levels of dFOXO protein. Western blots of protein extracts from S1-106/UAS-dFOXO females switched onto RU486 at different ages showed increased dFOXO protein levels for the switched groups at all ages examined (Fig. 3A), while protein levels dropped in females switched off RU486 at different ages (Fig. 3B). Native dFOXO protein levels, however, appear to increase with age (relative to total protein), which has implications for the interpretation of the effects of alterations in dFOXO expression upon mortality rates (see Discussion).

image

Figure 3. dFOXO protein levels in white Dahomey (wd); S1-106/+; dFOXO/+ females with dFOXO overexpressed at different ages. (A) Western blots of wd; S1-106/+; dFOXO/+ females put onto +200 µm RU486 1.0 SY food at different ages. Whole fly protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with the dFOXO antibody and tubulin loading control antibody. Flies were put onto RU486 at the age indicated (day 3, day 8, day 15 and day 22) or kept on –RU486 from day 3 (–d3) and flies were sampled at day 8 (+d3, –d3), day 15 (+d3, +d8, –d3), day 22 (+d3, +d8, +d15, –d3) and day 29 (+d3, +d8, +d15, +d22). (B) Western blots of wd; S1-106/+; dFOXO/+ females put onto +200 µm RU486 1.0 SY food at day 3 (d3) and taken off RU486 at different ages. Whole fly protein extracts were subjected to SDS-PAGE and immunoblotted with the dFOXO antibody and tubulin loading control antibody. Flies were put onto +200 µm RU486 at day 3 and taken off RU486 at the age indicated (day 8, day 15 and day 22) or kept on –RU486 from day 3 (–d3) and flies were sampled at day 8 (+d3, –d3), day 15 (+d3, –d8, –d3), day 22 (+d3, –d8, –d15, –d3) and day 29 (+d3, –d8, –d15, –d22, –d3).

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Initiation of dFOXO overexpression at different ages (Fig. 4) increased subsequent lifespan compared to controls, with the magnitude of the increase decreasing as the flies were put on RU486 at older age (see median lifespans, Fig. 4A). Log-rank tests comparing survival from the day of each switch showed that each switched group was subsequently longer lived than its control (P < 0.0001 in all switches). The mortality rate of the flies was thus responsive to switches in IIS status throughout the time period investigated (up to 4 weeks of age). Mortality rates of the switched groups were also compared to those of the chronically induced, +d3 RU486 group (on RU486 from day 3), to determine if induction of dFOXO at later ages could reduce subsequent mortality rates to those characteristic of chronically induced flies. The only group that did not significantly differ from the chronic +RU486 group was the +d7 RU486 group (dFOXO induced from day 7, P = 0.07), whereas the other switched groups (+d14, +d21) were significantly different in mortality rates from both control groups (P < 0.0001, comparing each switched group to each of the control groups). Qualitatively identical data were obtained in four independent experiments for S1-106/dFOXO (two different UAS-dFOXO lines, data not shown). Figure 4B shows the age-specific mortality analysis of S1-106/dFOXO females shown in Fig. 4A. The mortality trajectories of the different groups were compared by fitting of a Gompertz model over the linear portion of the increase in mortality (Table 2) (Pletcher, 1999). The ‘a’ parameter (intercept, initial mortality rate), but not ‘b’ (rate of increase in mortality rate with age), was significantly lower in the +d3 RU486 group compared to the control –d3 RU486 group. This effect was also seen in the groups switched onto RU486 at later ages, day 7, day 14 and day 21, although it was not always significant. This suggests that the dFOXO overexpressing groups started dying later than the controls but that mortality rates subsequently increased with age at the same rate.

image

Figure 4. Lifespans and mortality trajectories of flies with induction of dFOXO initiated at different ages. Lifespans and mortality trajectories of white Dahomey (wd); S1-106/dFOXO; +/+ females switched onto +200 µm RU486 1.0 SY food at different ages. Flies were put onto RU486 at the age indicated (day 3, day 7, day 14 and day 21) or kept on –RU486 from day 3 (–RU486). Survival curves and mortality trajectories are plotted for each switched group from the day of the switch and reflect differences from the day of the switch. (A) Survival curves. Groups were compared using log-rank tests from day of switch. Medians and log-rank P values for switched groups compared to –RU486 group and from the +d3 RU486 group are as follows: –RU486, median = 39 (n = 536); +d3 RU486, median = 53, P < 0.0001 vs. –RU486 (n = 466); +d7 RU486, median = 52, P < 0.0001 vs. –RU486, P = 0.07 vs. +d3 RU486 (n = 182); +d14 RU486, median = 45, P < 0.0001 vs. –RU486 and vs. +d3 RU486 (n = 439); +d21 RU486, median = 49, P < 0.0001 vs. –RU486 and vs. +d3 RU486 (n = 504). (B) Age-specific mortality analysis of wd; S1-106/dFOXO; +/+ females shown in graph A. Natural log of the mortality rate (µx) is plotted. Data are smoothed over a 3-day interval. Analysis of the switch mortality trajectories using the Gompertz mortality model is shown in Table 2.

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Table 2.  Estimates of the parameters of the Gompertz mortality model with 95% lower and upper confidence intervals. Overexpression of dFOXO initiated at different ages in white Dahomey (wd); S1-106/+; dFOXO; +/+ females shown in Fig. 4
Groupa†LCIUCIb‡LCIUCI
  • *

    Indicates significant difference from –RU486 group. Parameters as in Table 1.

+RU486 day 32 × 10−5*5.7 × 10−6  8 × 10−50.190.160.23
+RU486 day 72 × 10−52.9 × 10−61.9 × 10−40.190.130.26
+RU486 day 146 × 10−5*  2 × 10−51.6 × 10−40.180.150.21
+RU486 day 217 × 10−5  3 × 10−51.9 × 10−40.170.140.20
–RU4863 × 10−41.9 × 10−44.9 × 10−40.150.130.16

The effects of removal of dFOXO overexpression at different ages closely mirrored those of induction of expression (Fig. 5A), and produced shorter lifespan compared to +RU486 controls, with the shortest lifespans observed in flies taken off RU486 at the earlier ages (see median lifespans, Fig. 5A). Each switched group was significantly shorter lived than the +d3 RU486 control (P < 0.05). The switched groups were also compared to the chronic –RU486 group, and the only group that did not significantly differ from –d3 RU486 was the –d7 RU486 group (P = 0.21). Qualitatively identical data were obtained in two independent experiments (data not shown). The mortality trajectories of the different groups were compared by fitting of a Gompertz model over the linear portion of the increase in mortality (Table 3) (Pletcher, 1999). Age-specific mortality analysis of S1-106/dFOXO females (Fig. 5B) showed that the ‘a’ parameter (initial mortality rate) but not the ‘b’ parameter (slope of the mortality trajectory) of the +d3 RU486 group was significantly different from that of the –d3, –d7 and –d14 RU486 groups.

image

Figure 5. Lifespans and mortality trajectories of flies with induction of dFOXO removed at different ages. Lifespans and mortality trajectories of white Dahomey (wd); S1-106/+; dFOXO/+ females switched onto +200 µm RU486 1.0 SY food at day 3 and taken off RU486 at different ages. Flies were put onto RU486 at day 3 and taken off RU486 at the age indicated (day 7, day 14 and day 21) or kept on –RU486 from day 3 (–RU486). Survival curves and mortality trajectories are plotted for each switched group from the day of the switch and reflect differences from the day of the switch. (A) Survival curves. Groups were compared using log-rank tests from day of switch. Medians and log-rank P values for switched groups compared to –RU486 group and from the +d3 RU486 group are as follows: –RU486, median = 32 (n = 163); +d3 RU486, median = 56, P < 0.0001 vs. –RU486 (n = 187); –d7 RU486, median = 38, P = 0.21 vs. –RU486, P < 0.0001 vs. +d3 RU486 (n = 195); –d14 RU486, median = 38, P < 0.05 vs. –RU486, P < 0.0001 vs. +d3 RU486 (n = 180); –d21 RU486, median = 40, P < 0.0001 vs. –RU486, P < 0.05 vs. +d3 RU486 (n = 161). (B) Age-specific mortality analysis of wd; S1-106/+; dFOXO/+ females shown in graph A. Natural log of the mortality rate (µx) is plotted. Data are smoothed over a 3-day interval. Analysis of the switch mortality trajectories using the Gompertz mortality model is shown in Table 3.

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Table 3.  Estimates of the parameters of the Gompertz mortality model with 95% lower and upper confidence intervals. dFOXO overexpression turned off at different ages in white Dahomey (wd); S1-106/+; dFOXO/+ females shown in Fig. 5
Groupa†LCIUCIb‡LCIUCI
  • *

    Indicates significant difference from +RU486 group. Parameters as in Table 1.

+RU486 day 3  1 × 10−54.6 × 10−6  5 × 10−50.20.170.23
–RU486 day 71.6 × 10−4*  6 × 10−54.4 × 10−50.180.150.21
–RU486 day 142.3 × 10−4*  9 × 10−56.1 × 10−40.160.130.19
–RU486 day 211.2 × 10−4  3 × 10−5  4 × 10−40.170.140.21
–RU4863.2 × 10−4*1.4 × 10−47.4 × 10−40.170.140.2

Effect of dFOXO overexpression on fecundity

We published previously that adult fat body-specific dFOXO overexpression decreases fecundity (Giannakou et al., 2004), but this result has not proved to be robust (Supplementary Fig. S3). Fecundity of S1-106/dFOXO females on +RU486 was only reduced at one time-point (at day 13, P < 0.05) but at all other time-points and when averaged across the lifespan, no significant difference was observed between the egg-laying rates of S1-106/dFOXO females on ±RU486. RU486 had no effect on fecundity of control females at any age. This finding suggests that decreased fecundity described in other IIS models in Drosophila, such as chico mutants (Clancy et al., 2001), dInR transheterozygotes (Tatar et al., 2001) and long-lived DILP (Drosophila insulin like peptide)-producing cell-ablated flies (Broughton et al., 2005) is either not controlled via fat body dFOXO or is regulated by altered IIS earlier in life, as described in C. elegans (Dillin et al., 2002). CHICO in the ovary alone is sufficient for ovarian maturation and vitellogenesis (Richard et al., 2005), and hence the signal that causes the decreased fecundity of the chico mutants is unlikely to originate from the fat body.

Effect of dFOXO overexpression on dilp expression

A previous study describing increased lifespan of flies overexpressing dFOXO specifically in the head fat body suggested that the increase of lifespan occurred via feedback by dFOXO to the DILP-producing neuroscretory cells, which resulted in reduced dilp2 transcript levels (Hwangbo et al., 2004). Partial ablation of the DILP-producing neurosecretory cells increases lifespan and significantly decreases expression of dilp 2, 3 and 5 (Broughton et al., 2005). However, the long-lived dFOXO overexpressing flies reported here showed no significant effect on dilp 2, 3 or 5 transcript levels (Fig. 6). This result indicates that dilp2 expression is not regulated by dFOXO in this long-lived model, in contrast to other models describing regulation of dilp2 expression by dFOXO (Hwangbo et al., 2004; Wang et al., 2005; Luong et al., 2006).

image

Figure 6. dilp expression levels of S1-106/dFOXO flies.dilp expression levels in heads of once-mated 6-day-old white Dahomey (wd); S1-106/+; dFOXO/+ flies on ±200 µm RU486 1.0 SYA food measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Data are mean ± SEM (n = 8 for each group). Data were analysed by one-way anova. P values are as follows: dilp2, P = 0.9; dilp3, P = 0.12; dilp5, P = 0.12.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

In order to study the dynamics of IIS on mortality in Drosophila an inducible system was required that would permit up- and down-regulation of IIS at different ages and analysis of subsequent mortality rates. We examined both inducible systems available so far for down-regulating Drosophila IIS and increasing lifespan, both based on dFOXO overexpression using different RU486-inducible drivers. The combination of the head fat body Geneswitch driver S1-32 with a constitutively nuclear dFOXO transgene, published by Hwangbo et al. (2004) to increase median lifespan by as much as 35%, had no life-extending effect in our experiments (under a variety of conditions). Indeed, in most experiments, the lifespan of overexpressing females was decreased. This result is surprising and as described above was not due to driver defect as the driver was capable of inducing a UAS-lacZ reporter transgene.

We subsequently focused on the other inducible system for dFOXO overexpression using the fat-body driver S1-106. This system was previously published to extend median lifespan of overexpressing females by 20–50% (Giannakou et al., 2004). In order to study the dynamics of IIS on mortality in Drosophila, we first established whether mortality rates differed throughout adulthood in flies overexpressing fat-body dFOXO. We have shown here that overexpression of two independent UAS-dFOXO transgenes in adult female fat body increases median (by 21–33%) and maximum lifespan (P < 0.05) and lowers age-specific mortality compared to control flies. dFOXO overexpressing flies have a reduced initial mortality rate (intercept) compared to controls but no effect is seen on the slope of the mortality trajectories (rate of increase in mortality with age). Mortality rates between the two groups differ throughout adulthood, and our long-lived model could hence be used to examine the effects on subsequent mortality of IIS switches at different ages.

Increase in lifespan can be a consequence of reducing the slope (rate of mortality increase with age) or the intercept (initial mortality rate) (Pletcher et al., 2000). Increased lifespan as a consequence of reduced IIS can be associated with a reduced initial mortality rate, for instance with ablation of the DILP-producing cells late in development in the Drosophila brain (Broughton et al., 2005) and in long-lived chico1 flies (Clancy et al., 2001; data analysed by SD Pletcher, data not shown). Some effect on slope of the mortality trajectory is also seen in the long-lived chico1 flies (Tu et al., 2002), and in C. elegans, where long-lived age-1 mutants have increased lifespan through reduced slope of the mortality trajectory (Johnson, 1990; Johnson et al., 2001). Nonetheless, it is still unclear whether the initial mortality rate/slope distinction has any implications for reversibility or not, and further experimental work on this point would be valuable. Analysis of intercept (initial mortality rate) and slope (rate of increase in mortality with age) of a mortality trajectory alone does not clearly discriminate between risk and damage (Partridge et al., 2005). The critical experiment is switches in IIS status and analysis of the response of age-specific mortality to this switch.

Mortality rates responded to the change in IIS status, and in early switches (day 7) the subsequent mortality rates switched completely, to become nonsignificantly different from those of chronically induced flies, similar to the effects of DR. Unlike DR, as IIS status was altered at progressively later ages, the effect on mortality declines to become undetectable in older flies. Switches of flies older than 4 weeks of age have no effect on subsequent mortality or lifespan (Supplementary Fig. S4) even though at this age switches have an effect of dFOXO protein levels (Supplementary Fig. S5). The lack of complete reversal of mortality by overexpression of dFOXO in later ages could be due to the accumulation of greater levels of irreversible damage. This would indicate that dFOXO acts acutely to lower mortality, by reducing risk early in life but does so by reducing damage later in adulthood. However, as mentioned previously, there are some limitations to our inducible system that make it difficult to determine precisely the mechanism by which dFOXO overexpression reduces mortality. We have shown that dFOXO overexpression initiated at every age examined increases dFOXO protein levels. However, endogenous dFOXO protein levels appear to increase with age, which makes the interpretation of the overexpression more complex. Another explanation for the lack of effect on mortality in older flies could be that manipulation of dFOXO protein levels may have less of an effect on IIS status in older flies. It is also possible that some other process prevents or lessens the physiological response to a switch in IIS status. In that case, chronically induced flies may have initiated a cascade of downstream events (downstream of dFOXO), which cannot be initiated by dFOXO overexpression initiated in older flies. A recent report on C. elegans suggests that DAF-16 translocates to the cytoplasm in older worms in a manner requiring IIS activity (Weinkove et al., 2006). It is unclear if dFOXO also becomes more cytoplasmic (and therefore inactive) with age. Once-mated flies of 4 weeks of age, as used in these experiments, are near the end of their reproductive period, and it is therefore possible that in flies, as in C. elegans, only switches in IIS status during the reproductive period affect mortality and lifespan (Dillin et al., 2002). It is therefore difficult to distinguish between a model where dFOXO activity becomes less alterable with age and one where, unlike DR, IIS regulates both age-specific risk and accrual of aging-related damage.

Comparison of the mortality trajectories using the Gompertz mortality model showed that the initial mortality rate (intercept) but not the rate of increase with age (slope) was in general altered by the switch in the switched groups, indicating that aging-related mortality in the switched dFOXO overexpressing groups became detectable earlier or later than in the controls but mortality rates subsequently increased with age at the same rate, similar to the switches of status for DR in Drosophila (Mair et al., 2003). The combination for these switches in IIS status of a change in the elevation of the mortality trajectory with incomplete reversal of mortality rates at later ages implies that the pattern of the difference between two mortality trajectories as a result of different experimental treatments may not always be a reliable guide as to whether mortality will reverse with a reversal of the experimental regime.

In our study, unlike other studies on long-lived IIS mutants where increased longevity is accompanied by a decrease in fecundity (chico, dInR and DILP-producing cell ablation) (Clancy et al., 2001; Tatar et al., 2001; Broughton et al., 2005), flies overexpressing dFOXO specifically in the adult fat body show no effect on reproduction, similar to the long-lived flies overexpressing dFOXO-TM in head fat body (Hwangbo et al., 2004). These results suggest that adult fat body dFOXO is not involved in control of reproduction in Drosophila.

In our long-lived model, decreased mortality does not appear to be a consequence of feedback by dFOXO on dilp expression levels as has been suggested by other models of increased dFOXO (Hwangbo et al., 2004; Wang et al., 2005; Luong et al., 2006). The mechanism by which increased fat body dFOXO leads to extended lifespan is still to be determined, but it appears that it is not necessarily by feedback to the DILP-producing cells, with DILP2 acting as a secondary messenger as suggested by Hwangbo et al. (2004).

Our results imply that IIS acts in the adult fat body to kill the fly. It is intriguing to speculate why a pathway that is required for normal growth and development is also responsible for shortening adult lifespan apparently without any counter-balancing benefits to fecundity. It is possible that under specific environmental conditions, elevated IIS does carry fitness benefits to adult flies.

This is the first study to investigate the contribution of risk and damage to the increase in lifespan as a result of reduced IIS. We have shown that IIS reduces mortality at least in part by reducing acute risk of death and, possibly in part, by also reducing accumulation of damage.

Future characterisation of the subcellular localisation of dFOXO with age and in response to IIS status switches is required to distinguish between dFOXO becoming dysregulated with age and IIS influencing the rate of damage accumulation as well age-specific risk.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

Fly stocks and maintenance

The control white Dahomey (wd) stock was derived by backcrossing w1118 into the out-bred wild-type Dahomey background. UAS-dFOXO 2 lines, insert on chromosome 2 and insert on chromosome 3 and RU486-inducible P{Switch} GAL4 driver w; S1-106;+/+ were backcrossed into wd six times and are the same lines described in Giannakou et al.( 2004). UAS-lacZ (1776) was obtained from Bloomington Stock Center. Stocks were maintained and experiments were conducted at 25 °C on a 12 : 12 h light : dark cycle at constant humidity by using standard sugar yeast medium.

Lifespan

Procedures for lifespan studies were as described in Giannakou et al. (2004). Flies, bred at standard density (50 larvae per vial), were transferred 2 days after eclosion (once mated) to experimental vials at a density of ten flies per vial. Food consisted of 1.0 SY food medium [20 g L−1 agar, 100 g L−1 sugar, 100 g L−1 yeast (AYP 34113TT, T. P. Drewitt, UK)], 100 g L−1 nipagin, 3 mL L−1 propionic acid (Giannakou et al., 2004), with or without 200 µm RU486 (mifepristone, Sigma-Aldrich, Dorset, UK), dissolved in 100% ethanol. Deaths were scored almost every day and flies were tipped onto fresh food three times a week.

LacZ staining of tissues and quantitative assays for β-galactosidase

LacZ staining of tissues was carried as described in Ashburner (1981). Photos were taken using ProtoCOL HR software (Synbiosis, Cambridge, UK) on a Nikon C-DSD230 dissecting microscope. β-Galactosidase quantitative assays were carried out as described in Ashburner (1981) on either whole flies, or heads and bodies separately as described in the relevant figure legends.

Antibody generation and Western blots

Polyclonal antibodies against dFOXO were raised in rabbits against peptides ETSRYEKRRGRAKKR (amino acids 192–206) and PTDELDSTKAS-NQQL (amino acids 56–70), using immunisation package AS-DOUB-LX (Eurogentec, Liege, Belgium). The peptides were coupled to keyhole limpet haemocyanin (KLH) and both peptides were injected together into each of two rabbits. The antibodies were purified against the peptides they were raised against using HiTrap NHS-activated HP affinity columns (Amersham plc, Little Chalfont, UK) according to the manufacturer's instructions. Proteins from ten whole flies were extracted by grinding in 80 µL homogenization buffer [25 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 15 mm EGTA (ethylene glycol tetraacetic acid), 75 mm NaCl, 0.1% Nonidet P40, 1 mm DTT (dithiothreitol), protease inhibitor cocktail, 5 µg mL−1 antipain] and the debris pelleted at 10 000 g for 10 min at 4 °C. Protein extracts were quantified using the Bradford protein assay (Bio-Rad protein assay reagent; Bio-Rad Laboratories (UK) Ltd, Hemel Hempstead) according to the manufacturer's instructions. Before loading, protein extracts were incubated with an equal volume of 2× sodium dodecyl sulfate (SDS) sample buffer [125 mm Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 0.01% bromophenol blue, 10%β-mercaptoethanol] and heated at 80 °C for 15 min prior to loading. Equal amounts of protein were loaded for each sample on a 7% SDS-polyacrylamide gel electrophoresis (PAGE) with RainbowTM molecular weight marker (Amersham plc, Little Chalfont, UK). The gels were blotted according to standard protocols and incubated with either anti-dFOXO antibody at 1 : 5000 or antitubulin antibody [rat monoclonal (YL1/2) to tubulin, Abcam, Cambridge, UK] at 1 : 2500. Anti-horseradish peroxidase (HRP) secondary antibodies were used and the signals detected by chemiluminescence using ECL kit (Amersham plc).

Quantitative reverse transcription-polymerase chain reaction

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was carried out on once-mated 6-day-old flies on ±200 µm RU486 1.0 SYA food [15 g L−1 agar, 50 g L−1 sugar, 100 g L−1 yeast (MP Bio, Illkirch, France), 100 g L−1 nipagin, 3 mL L−1 propionic acid] as described in Broughton et al. (2005) using the same primers and SYBR green PCR mastermix (Applied Biosystems, Foster City, CA, USA).

Statistical analyses

Statistical analyses were performed by using JMP (version 4.0.5) software (SAS Institute, Cary, NC, USA) or R (version R.2.2.1) (R Development Team, 2005). Lifespan data were subjected to survival analysis (log-rank tests) and presented as survival curves. Maximum lifespans were compared by t-tests comparing the age at death of the last 10% of each group. Mortality (µx) was estimated as µx = –ln(px), where px is the probability of an individual alive at age x – 1 surviving to age x days (Lee, 1992). Maximum-likelihood methods, executed in WINMODEST (Pletcher, 1999), were used to estimate mortality parameters of the Gompertz model: µx = aebx. Quantitative RT-PCR data were analysed using one-way analysis of variance (anova). Data are presented as means ± SEM, and an asterisk indicates significant difference from control (P = 0.05).

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

This work was supported by a Wellcome Trust Functional Genomic Analysis of Aging Grant, the Biotechnology and Biological Science Research Council and Cancer Research UK. We thank Matthew D. W. Piper for critical reading of the manuscript.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

The following supplementary material is available for this article:

Figure S1 Lifespans of males and females S1-32x UAS-dFOXO TM in single sex vials and mixed sex cages. m6-15, UAS-dFOXO TM line used; m, median; n, number of flies in group. (A and B) Survival curves of females (A) and males (B) in single sex vials on +200 µm RU486 (solid line) and –RU486 (dashed line) food. (A) Median lifespans are as follows: +RU486 females, median = 41, 14% decrease vs. –RU486 females, P < 0.05 (n = 152); –RU486, median = 47 (n = 142). (B) Median lifespans are as follows: +RU486 males, median = 65, P = 0.16 vs. –RU486 males (n = 144); –RU486 males, median = 61 (n = 127). (C and D) Survival curves of females (C) and males (D) in mixed sex population cages on +25 µg mL−1 RU486 (solid line) or –RU486 (dashed line) in yeast paste. (C) Median lifespans are as follows: +RU486 females, median = 41, 17% decrease vs. –RU486 females, P < 0.0001 (n = 100); –RU486 females, median = 48 (n = 100). (D) Median lifespans are as follows: +RU486 males, median = 51, 13.7% decrease vs. –RU486 males, P < 0.0001 (n = 100); –RU486 males, median = 58 (n = 100).

Figure S2 Induction of S1-32 driver using β-gal quantitative assays. β-Gal activity assays of 7-day-old female S1-32/UAS-lacZ flies on ±200 µm RU486 for 3 days. Means and SEMs plotted for heads and bodies separately. n = 20 heads or 20 bodies/assay, assays in groups of two heads or bodies/well. S1-32/UAS-lacZ heads + RU486 exhibit significantly higher β-galactosidase activity than –RU486 S1-32/UAS-lacZ heads (P < 0.0001, more than 10-fold induction) and S1-32/UAS-lacZ bodies +RU486 heads exhibit higher β-galactosidase activity than S1-32/UAS-lacZ bodies –RU486 bodies (P < 0.0001, eight-fold induction).

Figure S3 Fecundity of white Dahomey (wd); S1-106/+; dFOXO/+ and wd flies. Fecundity of wd; S1-106/+; dFOXO/+ and wd females on +/– 200 µm RU486 2.0 SYA food. Data are mean ± SEM. n = 100 females per group, four females per vial. (A) Data are eggs/day/female ± SEM at each time-point. At each time-point, the ±RU486 treatments for each genotype were compared by one-way analysis of variance (anova). Only significant difference was between wd; S1-106/+; dFOXO/+ flies on ±200 µm RU486 at day 13 (P < 0.05). (B) Average number of eggs/day/female for each treatment. Treatments were compared using one-way anova. No significant difference was observed between any of the groups (P = 0.8).

Figure S4 Survival curves and mortality trajectories of S1-106/GFP-dFOXO females switched onto RU486 at different ages. Lifespans and mortality trajectories of white Dahomey/w1118, GFP-dFOXO; S1-106/+; +/+ mated females on +200/400/800 µm RU486 1.0 SY food since day 3 (switched to 400 at day 10, onto 800 at day 17), maintained on –RU486 food or switched at day 31 onto + 800 µm RU486 food. (A) Survival curves. Groups were compared using log-rank tests. Median lifespans are as follows: +200/400/800 RU486 day 3 females, 54 days (38.4% increase over –RU486 control, P < 0.0001, n = 490); –/+800 RU486 day 31 females, 38 days (no increase over –RU486 control, n = 351); –RU486 females, 39 days (n = 628). (B) Age-specific mortality analysis. Natural log of the mortality rate (µx) is plotted. Data are smoothed over a 3-day interval.

Figure S5 dFOXO protein levels in white Dahomey; S1-106/+; dFOXO/+ females with dFOXO overexpression turned on and off at day 29. Western blot of white Dahomey (wd); S1-106/+; dFOXO/+ females put onto +200 µm RU486 1.0 SY food at different ages. Whole fly protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with the dFOXO antibody and tubulin loading control antibody. Flies were put onto RU486 at the age indicated (+d3, +d29), were kept on –RU486 from day 3 (–d3) or taken off RU486 at day 29 (–d29) and flies were sampled at day 35.

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