HomeLarge Type Edition
HOME ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
PubMed
Right arrow PubMed Citation
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:136-145 (2006)
© 2006 The Gerontological Society of America

Flight Activity, Mortality Rates, and Lipoxidative Damage in Drosophila

Tapiwanashe Magwere, Reinald Pamplona, Satomi Miwa, Pedro Martinez-Diaz, Manuel Portero-Otin, Martin D. Brand and Linda Partridge1,

1 Department of Biology, University College London, United Kingdom.
2 Department of Basic Medical Sciences, Faculty of Medicine, University of Lleida, Spain.
3 MRC Dunn Human Nutrition Unit, Cambridge, United Kingdom.

Address correspondence to Professor Linda Partridge, DPhil, University College London, Department of Biology, Darwin Building, Gower Street, London WC1E 6BT, U.K. E-mail: l.partridge{at}ucl.ac.uk


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
In this study, the effect of flight activity on mortality rates and lipoxidative damage in Drosophila was determined to identify mechanisms through which oxidative damage affects life span. The results showed that flies allowed flying throughout life had higher mortality rates and decreased median and maximum life spans compared to controls. The mortality rate of the flight activity group could be lowered, but not completely reversed by switching to control conditions; and the accrued oxidative damage could not be eliminated. The levels of reactive oxygen species produced by mitochondria isolated from high activity and control flies did not differ significantly. However, the high activity flies had altered membrane fatty acid compositions, which made them prone to increased lipid peroxidation. The effect of flight activity on insect life span differs considerably from the beneficial effects of exercise in mammals; these differences may be caused by physiological differences between the two taxa.

THE benefits of exercise to human health are well-documented (1–3) and, based on demographic and epidemiological studies, various forms of physical activity have been recommended to promote and sustain health (4–6). Regular physical activity involving expenditure of more than 1000 kcal per week was recently shown to be associated with lower mortality rates in humans (7), and endurance training was also shown to improve antioxidant defenses in plasma (8). In rodents, where the effects of exercise have been widely studied experimentally, exercise was shown to increase average life span of female rats despite increased food intake (9), and also to attenuate age-related accumulation of oxidative damage to DNA, lipid, and protein in rat skeletal muscle (10,11). One of the underlying mechanisms behind these health-promoting effects of exercise is postulated to be adaptation of the antioxidant defense systems to exercise-induced oxidative stress, resulting in improved tolerance and resistance to subsequent challenges (11–13).

However, despite all the benefits, the "ideal" form of exercise that maximizes the health benefits, its frequency, as well as its intensity and duration remain largely undefined (14). It is possible that exercise taken to extremes can be harmful, as exemplified by the delayed-onset muscle damage observed in rat skeletal muscle following prolonged acute exercise (15). Markers of muscle damage during high-intensity exercise (16) and severe physical exercise (17) have also been observed in humans. Exercise increases oxygen consumption in skeletal muscle (18–20) and generation of reactive oxygen species (ROS) by the mitochondria thereby exacerbating oxidative damage to tissue (21–23). It has been shown in mammalian (24,25) and some insect species (26) that the rate of mitochondrial ROS production correlates inversely to maximum life-span potential. It is therefore likely that extreme physical activity may adversely affect life span if there are no adaptive effects or adequate antioxidant protection.

Invertebrate organisms such as Drosophila and other insect species may provide a useful model to study the harmful effects of exercise because of their high energy utilization (27). Flight activity in insects and other flying species involves very high energy expenditure (27–29), and metabolic rates can go up 20- to 100-fold during flight compared to other forms of physical activity (28,30,31). Using an insect model that may simulate conditions of high physical activity, other researchers showed that flight activity has life-limiting effects in the housefly Musca domestica (32,33). They observed that: (i) flies that were allowed to fly had life spans only one third of those prevented from flying since adulthood, and (ii) prevention of flight activity abolished age-related increases in oxidative damage to some mitochondrial proteins that were a characteristic of the exercised flies (33).

In this study, we used Drosophila melanogaster as a model to investigate the relationship between physical activity, oxidative damage, and life span. First, we sought to establish whether the exercise phenomenon observed in M. domestica (33) could be mirrored in other dipteran species such as Drosophila, and then went further to determine if the effect of flight activity on mortality rates was reversible. We then tested the hypothesis that flight activity decreases life span in Drosophila through accumulation of oxidative damage by looking at possible mechanisms through which this could be accomplished. It is known, for example, that exercise training increases mitochondrial density in rat (34) and human (35,36) skeletal muscle and that the mitochondrial density of a tissue can be used as a measure of ROS-generating capacity (36). We estimated the capacity for ROS production in exercising and control flies in two ways: (i) by assessing mitochondrial protein densities through measurement of cytochrome c oxidase (COX) and citrate synthase (CTS) activities, and (ii) by measuring rates of hydrogen peroxide (H2O2) production in isolated mitochondria in vitro.

On the basis of recent findings that exercise training, both chronic and acute, can modulate membrane fatty acid composition in rats (37) and humans (38,39), we further hypothesized that flight activity could alter membrane fatty acid profiles in flies in a manner that may predispose to lipoxidative damage. The degree of membrane fatty acid unsaturation has been suggested to play a significant role in the rate of aging in several organisms and was shown to correlate inversely with maximum life span of vertebrates (40–43). We analyzed membrane fatty acid compositions in whole-fly homogenates to find out whether flight exercise altered fatty acid unsaturation, and what influence this could have on peroxidizability, lipoxidative damage, and life span in Drosophila.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Fly Culture
The wild-type Dahomey base stock was cultured in 189 ml bottles for two generations on sucrose-yeast (SY) medium (44) at standard density (45). Experimental flies were taken from the third generation and collected within a 6-hour period and transferred to bottles of SY medium for 24 hours; male flies were then separated under light CO2 anesthesia for use in the experiments.

Manipulation of Flight Activity
Flight activity was manipulated by housing flies in conical flasks of two different sizes, 25 and 500 ml, as shown in Table 1. The container volume per fly was equal in the "exercise" (flight activity) and "control" (low activity) groups, but the control flies (n = 1050) in the 25 ml containers did not fly whereas the exercise flies (n = 900) in the 500 ml containers did so freely. Experimental flies were maintained at 25°C (50% humidity) on a 12-hour light/dark cycle and were transferred every 3 days to fresh SY medium. Deaths were recorded daily.


View this table:
[in this window]
[in a new window]
  		Table 1. Container Sizes and Numbers of Flies Allocated to Each Flask for the "Control" and "Exercise" Groups.

 
To measure flight activity, three flasks from the exercise group were placed on a flat bench top against a white background. After a 10-minute period during which most of the flies had settled on the food surface, the numbers of flies taking to the air in each flask were recorded over a 1-minute period. A total of 50 measurements was recorded for each flask over a 3-hour period starting at 11 AM each day. Flight activity was expressed as the proportion (%) of flies in each flask that flew per minute after adjusting for the number of flies lost through deaths and censors at each time point.

The level of nonflight physical activity (crawling, jumping) in both large (500 ml) and small (25 ml) flasks was measured by a modification of the spontaneous locomotor activity (SLA) described by Minois and colleague (46). Dots were marked on the surface of the flasks, and the fly closest to the dot was observed over a 3-second period. A score of +1 was given if the fly was crawling and a score of 0 if it remained stationary. Flight activity (measured separately above), as well as preening, was not included as SLA. Measurements were made from the large and small flasks on days 3, 13, and 25 of the experiments. The SLA score was given as a number of 25 total observations.

Flight Activity Switch Experiments
Switch experiments were carried out to test whether the mortality rates of exercise (flight activity) and control flies were reversible. In these experiments, 1800 flies were allocated to exercise and 1500 flies to control conditions. After 15 days, half of the surviving flies in the exercise group were switched to control conditions and vice versa, and the mortality rates of the remaining flies were monitored until all were dead.

Mitochondrial Protein Density
Mitochondrial enzymes such as CTS (matrix) and COX (inner membrane) are commonly used for assessing mitochondrial density (36). The specific activity in whole-fly homogenates divided by the specific activity in isolated mitochondria gives a ratio of mitochondrial protein per whole-fly protein, termed the mitochondrial protein density.

Whole-fly homogenates for enzyme assays were prepared from 10 flies in 1 ml of either 50 mM imidazole containing 2 mM EDTA and 0.5% Triton X-100, pH 7.1 (Buffer A) for the CTS assay, or 200 mM Tris-HCl, pH 7.5, containing 0.03% Tween 80, pH 7.5 (Buffer B) for the COX assay. Chilled flies were weighed and homogenized in buffer using three 20-second bursts of a T8 Ultra-Turrax homogenizer (Jencons-PLS, East Sussex, U.K.) given at 1-minute intervals on ice. The homogenates were centrifuged twice at 2000 g for 15 seconds in a refrigerated centrifuge (4°C), and the resultant supernatant was assayed immediately for enzyme activity.

The isolation of fly mitochondria was based on existing protocols (47) with modifications previously described by Miwa and coworkers (48). Fifty flies were used per preparation, and the mitochondrial pellets obtained were dissolved in 0.5 ml of the appropriate buffer for either the CTS or COX assays. Enzyme activities were assayed immediately.

Protein concentrations in the whole-fly homogenates and mitochondrial fractions were determined using the Pierce BCA Protein Assay Kit (Perbio Science UK Ltd., Cheshire).

CTS (E.C.4.1.3.7) activity was measured using the procedure described by García-Esquivel and colleagues (49) with some modifications. The reaction mixture contained Buffer A medium; 0.1 mM 5,5-dithiobis-2-nitrobenzoic acid (DTNB); 0.3 mM acetyl-coenzyme A; 0.05 mM oxaloacetate; and 10 µl of dilution of either whole-fly homogenate or mitochondrial fraction to a total volume of 1 ml. Reduction of DTNB was monitored at 412 nm in a spectrophotometer.

COX (E.C. 1.9.3.1) activity was measured by using a modified method of Smith (50) in a cuvette containing Buffer B (described above), 0.03% Tween 80, 100 µM reduced cytochrome c, and 10 µl of whole-fly homogenate or mitochondrial protein dilution. The oxidation of reduced cytochrome c was followed at 520 nm in a spectrophotometer at 25°C.

Mitochondrial ROS Production
Mitochondria were isolated from 200 flies by using the procedure previously described (48). Determination of the rate of H2O2 production was carried out in the presence and absence of respiratory chain inhibitors (48,51) with sn-glycerol 3-phosphate as substrate. The assay is based on the principle that H2O2 formed by dismutation of superoxide within the mitochondria reacts with homovanillic acid in the presence of horseradish peroxidase (HRP) to form a fluorescent product that can be detected fluorometrically ({lambda}excitation = 312 nm, {lambda}emission = 420 nm) (51). Exogenous superoxide dismutase (50 U/ml) was included in the assay medium. An F-4500 Hitachi fluorescence spectrophotometer (Jencons-PLS) was used to record the fluorescence measurements at 25°C.

Lipid Peroxidation
Lipid peroxidation (assessed as thiobarbituric acid-reactive substances [TBARS]) was determined in exercise, control, and switched flies. A modified method of Buege and Aust (52) was used. Groups of 100 flies each were sampled from exercise and control groups immediately before, and at 6 and 9 days after the switches. Drosophila eye pigment interferes with the TBARS or malondialdehyde (MDA) assays, and the heads of the flies were first removed by sieving after immersion in liquid nitrogen. The bodies of the flies (thoraces and abdomens) were ground to homogeneity in 4 ml of 10 mM HEPES buffer, pH 7.4 (supplemented with 1 µM butylated hydroxytoluene, 1 mM DTPA, and 1 mM phytic acid as antioxidants) using a pestle and mortar on ice, and the rest of the analysis was carried out as described (52).

Lipoxidative Damage to Protein and Membrane Lipid Analysis
Three thousand (3000) flies were allocated to each of exercise and control conditions, and cross-sectional sampling of flies was carried out at time points corresponding to survivorship in the exercise group. For each sample, 100 flies were counted under CO2 anesthesia and homogenized in 5 ml of 10 mM HEPES buffer, pH 7.4 (supplemented with 1 µM butylated hydroxytoluene, 1 mM DTPA, and 1 mM phytic acid as antioxidants) using pestle and mortar on ice. The homogenates were passed through two layers of muslin, aliquoted, snap-frozen in liquid nitrogen, and stored frozen at –80°C until analysis.

Levels of N{varepsilon}-carboxymethyllysine (CML), N{varepsilon}-carboxyethyllysine (CEL), and MDA-lysine (MDAL) in the crude homogenate fractions were quantified by isotope dilution and selected ion-monitoring gas chromatography/mass spectrometry as previously described (53).

Fatty acyl groups were analyzed by gas chromatography as described (54). The following fatty acyl indices were calculated: saturated fatty acids (SFA); unsaturated fatty acids (UFA); monounsaturated fatty acids (MUFA); polyunsaturated fatty acids from n-3 and n-6 series (PUFAn-3 and PUFAn-6); average chain length (ACL) = [({sum}%Total14 x 14) + ({sum}%Total16 x 16) + ({sum}%Total18 x 18)]/100; double bond index (DBI) = [({sum}mol% monoenoic x 1) + ({sum}mol% dienoic x 2) + ({sum}mol% trienoic x 3)]; and peroxidizability index (PI) = [({sum}mol% monoenoic x 0.025) + ({sum}mol% dienoic x 1) + ({sum}mol% trienoic x 2)].

Statistical Analyses
Survival data for exercise and control flies were compared using log-rank tests (55) in JMP statistical software version 5.1 (SAS Institute, Inc., Cary, NC). Mortality rates (the proportion of individuals that enter a time interval that die during it) give a measure of the instantaneous hazard of death at a particular age (56). The mortality rate (µx) at age x is: µx = –ln(px), where px is the probability of an individual alive at age x surviving to age x + 1 (57). The increase of mortality (µx) with age can be expressed using the Gompertz equation as: µx = aebx, where the constant a is the intrinsic or baseline mortality rate, and b is the rate at which mortality rates accelerate with age (58). Values for a and b were calculated using the Gompertz model available in WinModest Statistical Software (59).

Behavioral data (flight activity and SLA) were analyzed by one- and two-way analysis of variance using age as the explanatory factor to test whether there was a significant effect of age on either activity. The chi-square test was used to analyze the mortality rates for the switched flies both before and after the switches.

Differences in ROS production, oxidative damage parameters, and membrane lipid composition between exercise and control flies at each time point were tested for statistical significance using Student's t test.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Survival and Mortality Rates
Flight exercise decreased median and maximum life span by 33% and 31%, respectively (Figure 1a; log-rank test chi-square ({chi}2) = 1005.7, p <.0001). A comparison of the Gompertz parameters a and b for the mortality trajectories (Figure 1b) showed that the intrinsic baseline mortality rate a (intercept) was significantly higher by an order of magnitude in exercise flies {a = 0.00066 (lower, upper 95% confidence intervals [CI]: 0.00048, 0.00092)} compared to control [a = 0.00007 (0.00005, 0.00011), p <.0001]. The rate of increase of mortality with age b (slope) did not differ significantly between exercise [b = 0.17793 (0.16695, 0.18963)] and control flies [b = 0.16980 (0.16055, 0.17959), p >.05].


Figure 01
View larger version (21K):
[in this window]
[in a new window]
  		Figure 1. Cumulative survival (a) and mortality trajectories (b) for "exercise" and "control" flies. Average life span was significantly lower for the exercise group (mean 29 days; 95% confidence intervals: 28-30) compared to controls (43 days; 95 confidence interval: 42-43; p <.0001)

 
Flight and SLA
Flight activity (Figure 2) significantly declined with age from day 2 up to day 30 (F = 124.5, p <.001). It was highest during the first few days of the experiment (days 1–5) but decreased 22% on day 10. Only 60% of the original flight activity in the flasks remained on day 30. Nonflight SLA did not differ significantly between exercise and control flies (Figure 3) between days 3 and 25 of the experiment.


Figure 02
View larger version (12K):
[in this window]
[in a new window]
  		Figure 2. Flight activity of "exercise" flies housed in 500 ml containers. Flight activity is given as the proportion (%) of flies in each flask that fly per minute. Each point shows the mean and standard deviation for three different flasks. Points not connected by the same letter are significantly different, indicating flight activity declined with age (F = 124.5, p <.001)

 

Figure 03
View larger version (23K):
[in this window]
[in a new window]
  		Figure 3. The level of spontaneous locomotor activity (SLA) of "exercise" and "control" flies. The SLA score is the number of instances during which a fly was observed walking or crawling (of a total of 25 observations). Each bar represents the mean and standard deviation of observations taken from seven different containers

 
Flight Activity Switch Experiments
When flies were switched from exercise to control conditions (Figure 4a) the initial baseline mortality rate was higher in exercise flies [a = 0.00693 (0.00588, 0.00817)] than in control flies [a = 0.00170 (0.00131, 0.00219)] (p <.0001) as observed earlier. The mortality rate of the switched flies then slowed down after the switch (day 15), became significantly different from that of the exercise group on day 21 (p <.01), and remained so thereafter. The mortality rate for the switched flies was significantly different from the control group from day 15 (day of switch) until it first became statistically indistinguishable (p >.05) on day 35, a period of 20 days.


Figure 04
View larger version (30K):
[in this window]
[in a new window]
  		Figure 4. Mortality trajectories for flies switched between "exercise" and "control" conditions. Vertical dotted lines: day on which the switch was made. a, Mortality rate of switch to control group first became significantly different from exercise group, *p <.02, and significantly different from control group, +p <.02). b, Mortality rate of switch to exercise group first became statistically significant from control group, *p <.00001, and significantly different from exercise group +p <.03

 
The reciprocal switch from control to exercise (Figure 4b) again showed that the initial baseline mortality rate was higher in exercise flies [a = 0.000546 (0.00464, 0.00644)] than in control flies [a = 0.00061 (0.00047, 0.00080)] (p <.00001). On switching, the mortality rate for switched flies immediately increased, became significantly different from that of the control group on day 20 (p <.000001), and remained so thereafter. The mortality rate for switched flies became indistinguishable from that of the exercise group on day 20 (p =.08) and only became significantly higher on day 30 (p <.03). The change in mortality rate on switching from low exercise to flight exercise happened over a relatively shorter period of time (5 days).

Mitochondrial Protein Density
There were no statistically significant differences in mitochondrial protein density between exercise and control flies with COX as marker (Figure 5), and protein density decreased with age in both groups of flies. However, when CTS was used as marker (Figure 5), mitochondrial protein density was significantly higher for the exercise group on day 12 (p <.01) but was not significantly different on day 29. The graph for CTS also shows that mitochondrial protein density increased with age in both groups of flies, an effect opposite to that obtained with COX.


Figure 05
View larger version (17K):
[in this window]
[in a new window]
  		Figure 5. Changes in mitochondrial protein density with age using cytochrome c oxidase (COX) and citrate synthase (CTS) as markers in "exercise" and "control" flies. Each point represents the mean and standard deviation of replicate determinations for N = 3 samples. *Value significantly different from control (p <.01)

 
Mitochondrial ROS Production
Mitochondrial H2O2 production did not differ significantly between exercise and control flies, and there was no effect of age on H2O2 production in either group (Figure 6). There were also no statistically significant differences in H2O2 produced between the two groups of flies in the presence of the respiratory chain inhibitors rotenone, antimycin A, and myxothiazol.


Figure 06
View larger version (12K):
[in this window]
[in a new window]
  		Figure 6. Native rate of mitochondrial H2O2 production in vitro for "control" and "exercise" flies using sn-glycerol 3-phosphate (GP) as substrate. Day 12 corresponds to 95% survivorship for the exercise flies, whereas days 29 and 43 represent time points when survivorship reached 50% for exercise and control flies, respectively. Concentrations of reagents in the assay mixture were GP (20 mM), rotenone (5 µM), antimycin A (3 µM), myxothiazol (3 µM), and superoxide dismutase (50 U/ml). Each bar represents the mean and standard deviation of replicate determinations for N = 3 samples

 
Oxidative Damage to Lipid and Protein
Exercise flies had higher MDA levels compared to controls of the same chronological age (Figure 7a). MDA levels were 110% higher in the exercise group on day 25 (p <.001) and about 30% higher after day 35 (p <.01). Levels of TBARS were 60% higher in the exercise group compared to the control group before the switches (Figure 7b, p <.01), and remained so to day 9 after the switch. Switching flies from exercise to control conditions and vice versa had no significant effect on levels of TBARS up to day 9 after the switch.


Figure 07
View larger version (18K):
[in this window]
[in a new window]
  		Figure 7. a, Lipid peroxidation levels in "exercise" and "control" flies. Cross-sectional sampling of the flies was done at days 25 and 35, corresponding to 65% and 35% survivorship, respectively, for the exercise flies. b, Levels of thiobarbituric acid-reactive substances (TBARS) before and after switching flies from exercise to control conditions and vice versa. Each bar or point on the two graphs is the mean and standard deviation for N = 4 different samples. Exercise group significantly different from "control" group, *p <.01

 
A common observation in this study was that glycoxidative and lipoxidative damage to protein (CML, CEL, and MDAL markers) increased with age in exercise and control groups of flies (Figure 8). CEL levels were significantly higher for exercised flies on day 35 (p <.01), whereas CML levels were always significantly higher for exercised flies compared to controls at all ages (p <.004). Results for MDAL show that levels were higher in control flies on days 12 and 25, but were much lower on day 35 (p <.0001). In summary, these results show that lipoxidative damage to protein increased with age in flies, and that exercised flies had higher levels of protein modification compared to controls that were prevented from flying.


Figure 08
View larger version (18K):
[in this window]
[in a new window]
  		Figure 8. Levels of glycoxidative and lipoxidative damage markers to protein in "exercise" and "control" flies. Cross-sectional sampling of the flies was done at days 12, 25, and 35, which were at 95%, 65%, and 35% survivorship, respectively, for the exercise group. Additional sampling points were introduced at days 40 and 53 to represent 65% and 35% survivorship for the control flies. Each point represents the mean and standard deviation for N = 5 determinations. CEL = N(epsilon)-(carboxyethyl)lysine; CML = N(epsilon)-(carboxymethyl)lysine; MDAL = N(epsilon)-(malondialdehyde)lysine. *Values significantly different from control, p <.001

 
Lipid Composition of Whole-Fly Homogenates
The membrane fatty acid compositions for exercise and control flies are shown in Table 2 and Figure 9. There were no statistically significant differences in SFA levels between the two groups of flies at ages 12 and 25 days (Figure 9) although SFA levels were higher in control than in exercise flies at age 35 days (p <.001). Exercise flies had significantly higher levels of PUFA at days 12, 25, and 35, compared to controls (Figure 9, p <.0001). The PI (a measure of susceptibility to lipid peroxidation) was also much higher in exercise flies compared to controls (Figure 9). In exercise flies, the PI was higher on days 12, 25, and 35 compared to controls (p <.0001). There was also a close agreement between PI and TBARS (lipid peroxidation levels, Figure 7).


View this table:
[in this window]
[in a new window]
  		Table 2. Whole-Fly Homogenate Fatty Acid Compositions in "Control" and "Exercise" Flies (Expressed as mol% of Total Lipids).

 

Figure 09
View larger version (19K):
[in this window]
[in a new window]
  		Figure 9. Saturated fatty acids (SFA) levels, polyunsaturated fatty acid (PUFA) levels, double bond index (DBI), and peroxidizability index (PI) for "exercise" (filled circle) and "control" (open circle) flies. Cross-sectional sampling of the flies was done at days 12, 25, and 35, which were at 95%, 65%, and 35% survivorship, respectively, for the exercise group. Additional sampling points were also introduced at days 40 and 53 to represent 65% and 35% survivorship for control flies. Points on the graphs are mean and standard deviation for five determinations. *Values significantly different from control, p <.0001

 

   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The results from this study indicate a possible cost of flight activity in Drosophila that is associated with high mortality rates, decreased average and maximum life spans, as well as increased levels of oxidative damage to proteins and lipids. The effect of flight activity on life span and oxidative damage in Drosophila seems to agree with what was first reported in the housefly (33). It appears that the deleterious effects of flight exercise could be similar for dipterans, although the magnitude of the effect differs. The attribution of the observed effects to flight exercise in this study may represent a biased interpretation of the results if we do not discount the influence of other factors such as differences in nutritional intake or differences in food surface area per fly between exercise and control flies. The contribution of these other two factors to the observed effects, however, could be insignificant for the following reasons: (i) The mortality rates of exercise flies could only be lowered, but not completely reversed by switching to control conditions (Figure 4). If differences in nutritional intake between the two groups of flies were a factor, rapid reversals in mortality rates with a change in nutritional status (60) could have been observed, but this was not the case. (ii). If surface area per fly (overcrowding) was also a cause, we could have observed a significant drop in the slope of the mortality trajectory for flies in the large flasks (exercise) with age as fly numbers dwindled due to deaths but, again, that was not the case (Figure 4). A comparison of behavioral data between exercise and control flies strongly suggests that the effects on life span and mortality rates could be attributed to flight activity because all other parameters (SLA activity, fly density) were similar for both groups.

The deleterious effects of flight exercise reported here and elsewhere (33), however, contradict the beneficial effects of exercise reported in humans (61,62) and rodents (9,63), possibly suggesting that the effects of extreme physical activity may differ between species. An explanation for this anomaly could be that flying insects encounter a much greater oxidative challenge as a result of elevated metabolic rates during flight compared to moderate exercise known to promote health in nonflying organisms. Although most vertebrates can adapt relatively easily to modest increases in ROS production in exercise by virtue of their elaborate antioxidant defenses (13,64), it has not been determined whether such adaptive mechanisms operate in invertebrate organisms such as insects that have a high content of postmitotic tissue and very low rates of protein turnover (65).

We tested whether the effect of physical activity on mortality rates of flies was reversible by carrying out a series of switch experiments (Figure 4, a and b). The objectives were to establish two things: first, whether the mortality rates in exercise flies were reversible, and second, whether there was an association between oxidative damage and mortality rates for exercise and control flies. Our hypothesis was that if damage accrued during flight activity was the cause of high mortality rates observed in the exercise group, a reversal of either damage or mortality rate (or both) to lower levels should occur on switching to control conditions, and vice versa. The results (Figure 7b) show that the damage accrued during flight by the exercise group was not reversible even up to 9 days after the switch, although the mortality rate could be slowed down gradually by switching from exercise to control conditions (Figure 4a). The relatively rapid increase in mortality rate on switching from control to exercise conditions (Figure 4b) perhaps highlights the risk that may be associated with imposing extreme physical activity on previously sedentary or low activity individuals. Overall, the switch results suggest one of two things, that either: (i) oxidative damage is not the cause of high mortality rates in flies, or (ii) the damage marker used for this assessment was inappropriate.

We selected lipid peroxidation for assessing oxidative damage before and after the switches because it is considered the first damage to occur following an oxidative stress (66) and would therefore be ideal (compared to other markers) for detecting transient oxidative damage during the switches. Measurement of other markers could potentially yield a different picture because oxidative damage is a highly selective (67) and specific (68) process. However, lipid peroxidation is a very useful damage marker in biological systems because its by-products can cause damage to other biomolecules such as proteins (69,70) and nucleic acids (66,71).

Protein oxidation is considered critical for the aging process because it leads to inactivation of some enzyme systems and damage to structural proteins (72,73) potentially leading to organ failure. Oxidative damage to protein may be even more critical for insect species such as Drosophila because of their high content of postmitotic tissue and low rates of protein turnover (65). A look at nonenzymatic protein modification markers in this study (Figure 8) indicates that glycoxidative and lipoxidative damage were prevalent in the two groups of flies, and increased with age. Although we could not establish a direct link between lipid peroxidation (measured as TBARS) and mortality rates in this study, there is a strong possibility that the higher markers of damage to proteins observed in exercise flies compared to controls could alter some basic physiological processes leading to the high mortality rates. Comparison of our results (Figure 8) with those obtained for other species (74) shows a common trend that lipoxidative damage markers increase with age, although the nature and form of damage appear to differ quantitatively and qualitatively between species, and also between interventions that alter life span.

Our search for mechanisms through which exercise flies accumulated damage more than did control flies showed that there was no significant differences in ROS production potential measured as mitochondrial protein density (Figure 5) or H2O2 production (Figure 6) between the two groups of flies. This is in contrast to reports presented by other researchers studying the effects of flight activity in houseflies (33,75). The rates of superoxide anion (75) and H2O2 (33) production were shown to increase with age in houseflies, although metabolic potential remained the same for both high activity and low activity flies. The lack of correlation between ROS production and oxidative damage that we observed in this study may imply either one of two possibilities: (i) that ROS production in isolated mitochondria in vitro in state 4 (resting condition, in the absence of adenosine diphosphate (ADP) does not adequately mimic mitochondrial ROS production in vivo, or (ii) that there is more than one mechanism through which interventions that decrease life span can cause accrual of oxidative damage in organisms.

An alternative mechanism for explaining the higher oxidative damage parameters in exercise flies is implied by our results of the membrane lipid composition analyses (Table 2 and Figure 9). It can be concluded from these results that flight activity altered lipid composition in flies such that exercise flies had more PUFA than did controls (Figure 9). As the degree of fatty acid unsaturation has been shown to correlate negatively with life span in several animal species (54,76), our present results suggest that flight activity alters fatty acid composition in exercise flies as shown by more PUFA, higher double bond index, and higher PI than in control flies (Figure 9). The alterations to membrane lipid composition could therefore make exercise flies more susceptible to oxidative damage even though they may produce the same amount of ROS as do controls. However, the physiological or biochemical mechanisms through which flight activity alters membrane lipid unsaturation in flies are still unknown.

Conclusion
This study has shown that flight activity in Drosophila is associated with reduced life span, high mortality rates, and increased levels of oxidative damage to lipids and proteins. A comparison of behavioral activity measurements (flight activity and SLA) between exercise and control flies reaffirms that flight activity was the major cause of this phenomenon. The high mortality rates of exercise flies could be slowly reversed when they were prevented from flying; however, the accrued oxidative damage could not be eliminated. A possible explanation for why exercise flies accrued more oxidative damage than did controls is that they contained more unsaturated membrane lipids which may have enhanced their susceptibility to lipoxidative damage. However, the underlying biochemical mechanism through which flight activity alters membrane phospholipid compositions in flies is still unknown. We postulate that the disparity in the effects of exercise on life span between mammals and insects may be due to physiological differences between the two species and mechanisms of coping with exercise-induced oxidative stress.


   Acknowledgments
Top
 Abstract
 Methods
 Results
 Discussion
 References
 
We gratefully acknowledge funding from the following organizations: the Biotechnology and Biological Sciences Research Council (BBSRC); the Wellcome Trust (for funding to M.D.B.); the Spanish Ministry of Science & Technology (BFI2003-01287); the "Marató de TV3" Foundation (990110); and the Generalitat of Catalunya (2001SGR00311) (for grants to R.P. and M.P.O.).

Special thanks go to Dr. B. Wertheim, Department of Biology, University College London, for advice on the statistical analyses and to the anonymous reviewers for their constructive comments.

An abstract of this manuscript was presented at the Liverpool Mini-Symposium on ROS, Muscle & Ageing held at the University of Liverpool, U.K., on May 19–20, 2005.


   Footnotes
Top
 Abstract
 Methods
 Results
 Discussion
 References
 
Decision Editor: James R. Smith, PhD

Received May 24, 2005

Accepted August 8, 2005


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Bauman AE. Updating the evidence that physical activity is good for health: an epidemiological review 2000-2003. J Sci Med Sport. 2004;7:6-19.[Medline]
  2. Deschenes MR, Kraemer WJ. Performance and physiologic adaptations to resistance training. Am J Phys Med Rehabil. 2002;81:S3-S16.[Medline]
  3. Speakman JR, Selman C. Physical activity and resting metabolic rate. Proc Nutr Soc. 2003;62:621-634.[Medline]
  4. Blair SN, Cooper KH. Dose of exercise and health benefits. Arch Intern Med. 1997;157:153-154.[Abstract/Free Full Text]
  5. Pate RR, Pratt M, Blair SN, et al. Physical activity and public health. A recommendation from the Centers for Disease Control and Prevention and the American College of Sports Medicine. JAMA. 1995;273:402-407.[Abstract/Free Full Text]
  6. Blair SN, Kohl HW, III, Barlow CE, Paffenbarger RS, Jr, Gibbons LW, Macera CA. Changes in physical fitness and all-cause mortality. A prospective study of healthy and unhealthy men. JAMA. 1995;273:1093-1098.[Abstract/Free Full Text]
  7. Lee IM, Sesso HD, Oguma Y, Paffenbarger RS, Jr. The "weekend warrior" and risk of mortality. Am J Epidemiol. 2004;160:636-641.[Abstract/Free Full Text]
  8. Ookawara T, Haga S, Ha S, et al. Effects of endurance training on three superoxide dismutase isoenzymes in human plasma. Free Radic Res. 2003;37:713-719.[Medline]
  9. Holloszy JO. Exercise increases average longevity of female rats despite increased food intake and no growth retardation. J Gerontol Biol Sci. 1993;48:B97-B100.
  10. Radak Z, Kaneko T, Tahara S, et al. The effect of exercise training on oxidative damage of lipids, proteins, and DNA in rat skeletal muscle: evidence for beneficial outcomes. Free Radic Biol Med. 1999;27:69-74.[Medline]
  11. Radak Z, Naito H, Kaneko T, et al. Exercise training decreases DNA damage and increases DNA repair and resistance against oxidative stress of proteins in aged rat skeletal muscle. Pflugers Arch. 2002;445:273-278.[Medline]
  12. Ji LL. Exercise-induced modulation of antioxidant defense. Ann N Y Acad Sci. 2002;959:82-92.[Medline]
  13. Higuchi M, Cartier LJ, Chen M, Holloszy JO. Superoxide dismutase and catalase in skeletal muscle: adaptive response to exercise. J Gerontol. 1985;40:281-286.
  14. Ashe MC, Khan KM. Exercise prescription. J Am Acad Orthop Surg. 2004;12:21-27.[Abstract/Free Full Text]
  15. Aoi W, Naito Y, Takanami Y, et al. Oxidative stress and delayed-onset muscle damage after exercise. Free Radic Biol Med. 2004;37:480-487.[Medline]
  16. Baker JS, Bailey DM, Hullin D, Young I, Davies B. Metabolic implications of resistive force selection for oxidative stress and markers of muscle damage during 30 s of high-intensity exercise. Eur J Appl Physiol. 2004;92:321-327.[Medline]
  17. Chevion S, Moran DS, Heled Y, et al. Plasma antioxidant status and cell injury after severe physical exercise. Proc Natl Acad Sci U S A. 2003;100:5119-5123.[Abstract/Free Full Text]
  18. Gollnick PD, Bertocci LA, Kelso TB, Witt EH, Hodgson DR. The effect of high-intensity exercise on the respiratory capacity of skeletal muscle. Pflugers Arch. 1990;415:407-413.[Medline]
  19. Holloszy JO. Biochemical adaptations in muscle: effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle. J Biol Chem. 1967;242:2278-2282.[Abstract/Free Full Text]
  20. Matoba H, Gollnick PD. Response of skeletal muscle to training. Sports Med. 1984;1:240-251.[Medline]
  21. Bejma J, Ji LL. Aging and acute exercise enhance free radical generation in rat skeletal muscle. J Appl Physiol. 1999;87:465-470.[Abstract/Free Full Text]
  22. Bejma J, Ramires P, Ji LL. Free radical generation and oxidative stress with ageing and exercise: differential effects in the myocardium and liver. Acta Physiol Scand. 2000;169:343-351.[Medline]
  23. Ji LL, Leeuwenburgh C, Leichtweis S, et al. Oxidative stress and aging. Role of exercise and its influences on antioxidant systems. Ann N Y Acad Sci. 1998;854:102-117.[Medline]
  24. Sohal RS, Arnold LA, Sohal BH. Age-related changes in antioxidant enzymes and prooxidant generation in tissues of the rat with special reference to parameters in two insect species. Free Radic Biol Med. 1990;9:495-500.[Medline]
  25. Barja G. Mitochondrial free radical production and aging in mammals and birds. Ann N Y Acad Sci. 1998;854:224-238.[Medline]
  26. Sohal RS, Sohal BH, Orr WC. Mitochondrial superoxide and hydrogen peroxide generation, protein oxidative damage, and longevity in different species of flies. Free Radic Biol Med. 1995;19:499-504.[Medline]
  27. Wegener G. Flying insects: model systems in exercise physiology. Experientia. 1996;52:404-412.[Medline]
  28. Harrison JF, Roberts SP. Flight respiration and energetics. Annu Rev Physiol. 2000;62:179-205.[Medline]
  29. Sacktor B. Biochemical adaptations for flight in the insect. Biochemical Society Symposium. 1976;41:111-131.
  30. Davis RA, Fraenkel G. The oxygen consumption of flies during flight. J Exp Biol. 1940;17:402-407.[Abstract]
  31. Mercier J, Perez-Martin A, Bigard X, Ventura R. Muscle plasticity and metabolism: effects of exercise and chronic diseases. Mol Aspects Med. 1999;20:319-373.[Medline]
  32. Sohal RS, Buchan PB. Relationship between physical activity and life span in the adult housefly, Musca domestica. Exp Gerontol. 1981;16:157-162.[Medline]
  33. Yan LJ, Sohal RS. Prevention of flight activity prolongs the life span of the housefly, Musca domestica, and attenuates the age-associated oxidative damage to specific mitochondrial proteins. Free Radic Biol Med. 2000;29:1143-1150.[Medline]
  34. McAllister RM, Terjung RL. Training-induced muscle adaptations: increased performance and oxygen consumption. J Appl Physiol. 1991;70:1569-1574.[Abstract/Free Full Text]
  35. MacDougall JD, Sale DG, Moroz JR, Elder GC, Sutton JR, Howald H. Mitochondrial volume density in human skeletal muscle following heavy resistance training. Med Sci Sports. 1979;11:164-166.[Medline]
  36. Leek BT, Mudaliar SR, Henry R, Mathieu-Costello O, Richardson RS. Effect of acute exercise on citrate synthase activity in untrained and trained human skeletal muscle. Am J Physiol Regul Integr Comp Physiol. 2001;280:R441-R447.[Abstract/Free Full Text]
  37. Nikolaidis MG, Petridou A, Matsakas A, Schulz T, Michna H, Mougios V. Effect of chronic wheel running on the fatty acid composition of phospholipids and triacylglycerols in rat serum, skeletal muscle and heart. Acta Physiol Scand. 2004;181:199-208.[Medline]
  38. Andersson A, Sjodin A, Hedman A, Olsson R, Vessby B. Fatty acid profile of skeletal muscle phospholipids in trained and untrained young men. Am J Physiol Endocrinol Metab. 2000;279:E744-E751.[Abstract/Free Full Text]
  39. Helge JW, Wu BJ, Willer M, Daugaard JR, Storlien LH, Kiens B. Training affects muscle phospholipid fatty acid composition in humans. J Appl Physiol. 2001;90:670-677.[Abstract/Free Full Text]
  40. Pamplona R, Barja G, Portero-Otin M. Membrane fatty acid unsaturation, protection against oxidative stress, and maximum life span: a homeoviscous-longevity adaptation? Ann N Y Acad Sci. 2002;959:475-490.[Medline]
  41. Pamplona R, Portero-Otin M, Riba D, et al. Low fatty acid unsaturation: a mechanism for lowered lipoperoxidative modification of tissue proteins in mammalian species with long life spans. J Gerontol Biol Sci. 2000;55A:B286-B291.[Abstract/Free Full Text]
  42. Pamplona R, Portero-Otin M, Ruiz C, Gredilla R, Herrero A, Barja G. Double bond content of phospholipids and lipid peroxidation negatively correlate with maximum longevity in the heart of mammals. Mech Ageing Dev. 2000;112:169-183.[Medline]
  43. Hulbert AJ. On the importance of fatty acid composition of membranes for aging. J Theor Biol. 2005;234:277-288.[Medline]
  44. Chapman T, Partridge L. Female fitness in Drosophila melanogaster: an interaction between the effect of nutrition and of encounter rate with males. Proc R Soc Lond B Biol Sci. 1996;263:755-759.[Medline]
  45. Clancy DJ, Kennington WJ. A simple method to achieve consistent larval density in bottle cultures. Drosoph Inf Serv. 2001;84:168-169.
  46. Minois N, Khazaeli AA, Curtsinger JW. Locomotor activity as a function of age and lifespan in Drosophila melanogaster overexpressing hsp70. Exp Gerontol. 2001;36:1137-1153.[Medline]
  47. Van den Bergh SG. Insect mitochondria. In: Estabrook RW, Pullman ME, eds. Methods in Enzymology. New York: Academic Press;1967:117–122.
  48. Miwa S, St-Pierre J, Partridge L, Brand MD. Superoxide and hydrogen peroxide production by Drosophila mitochondria. Free Radic Biol Med. 2003;35:938-948.[Medline]
  49. García-Esquivel Z, Bricelj VM, Felbeck H. Metabolic depression and whole-body response to enforced starvation by Crassostrea gigas postlarvae. Comp Biochem Physiol A Mol Integr Physiol. 2002;133:63-77.[Medline]
  50. Smith L. Bacterial cytochromes; difference spectra. Arch Biochem Biophys. 1954;50:299-314.[Medline]
  51. Barja G. Kinetic measurement of mitochondrial hydrogen peroxide production. In: Yu BP, ed. Methods in Aging Research. Boca Raton, FL: CRC Press;1999:533–543.
  52. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;52:302-310.[Medline]
  53. Pamplona R, Portero-Otin M, Bellmun MJ, Gredilla R, Barja G. Aging increases Nepsilon-(carboxymethyl)lysine and caloric restriction decreases Nepsilon-(carboxyethyl)lysine and Nepsilon-(malondialdehyde)lysine in rat heart mitochondrial proteins. Free Radic Res. 2002;36:47-54.[Medline]
  54. Pamplona R, Portero-Otin M, Riba D, et al. Mitochondrial membrane peroxidizability index is inversely related to maximum life span in mammals. J Lipid Res. 1998;39:1989-1994.[Abstract/Free Full Text]
  55. Peto R, Peto J. Asymptotically efficient rank invariant procedures. J Roy Stat Soc Series A. 1972;135:185-207.
  56. Carey JR. Longevity: The Biology and Demography of Lifespan. Princeton, NJ: Princeton University Press; 2003.
  57. Carey JR. Applied Demography for Biologists. Oxford: Oxford University Press; 1993.
  58. Promislow DE, Haselkorn TS. Age-specific metabolic rates and mortality rates in the genus Drosophila. Aging Cell. 2002;1:66-74.[Medline]
  59. Pletcher SD. Model fitting and hypothesis testing for age-specific mortality data. J Evol Biol. 1999;12:430-439.
  60. Mair W, Goymer P, Pletcher SD, Partridge L. Demography of dietary restriction and death in Drosophila. Science. 2003;301:1731-1733.[Abstract/Free Full Text]
  61. Eaton SB. An evolutionary perspective on human physical activity: implications for health. Comp Biochem Physiol A Mol Integr Physiol. 2003;136:153-159.[Medline]
  62. Cordain L, Gotshall RW, Eaton SB, Eaton SB 3rd. Physical activity, energy expenditure and fitness: an evolutionary perspective. Int J Sports Med. 1998;19:328-335.[Medline]
  63. Holloszy JO. Effects of exercise on longevity of rats. Fed Proc. 1987;46:1850-1853.[Medline]
  64. Lopez-Torres M, Perez-Campo R, Rojas C, Cadenas S, Barja G. Simultaneous induction of SOD, glutathione reductase, GSH, and ascorbate in liver and kidney correlates with survival during aging. Free Radic Biol Med. 1993;15:133-142.[Medline]
  65. Bailey E. Biochemistry of insect flight. Part 2: Fuel supply. In: Candy DJ, Kilby BA, eds. Insect Biochemistry and Function. London: Chapman and Hall Ltd; 1975:91–176.
  66. Burcham PC. Genotoxic lipid peroxidation products: their DNA damaging properties and role in formation of endogenous DNA adducts. Mutagenesis. 1998;13:287-305.[Abstract/Free Full Text]
  67. Das N, Levine RL, Orr WC, Sohal RS. Selectivity of protein oxidative damage during aging in Drosophila melanogaster. Biochem J. 2001;360:209-216.[Medline]
  68. Yan LJ, Sohal RS. Mitochondrial adenine nucleotide translocase is modified oxidatively during aging. Proc Natl Acad Sci U S A. 1998;95:12896-12901.[Abstract/Free Full Text]
  69. Stadtman ER, Berlett BS. Reactive oxygen-mediated protein oxidation in aging and disease. Drug Metab Rev. 1998;30:225-243.[Medline]
  70. Refsgaard HH, Tsai L, Stadtman ER. Modifications of proteins by polyunsaturated fatty acid peroxidation products. Proc Natl Acad Sci U S A. 2000;97:611-616.[Abstract/Free Full Text]
  71. Box HC, Maccubbin AE. Lipid peroxidation and DNA damage. Nutrition. 1997;13:920-921.[Medline]
  72. Levine RL, Stadtman ER. Oxidative modification of proteins during aging. Exp Gerontol. 2001;36:1495-1502.[Medline]
  73. Stadtman ER. Protein oxidation and aging. Science. 1992;257:1220-1224.[Abstract/Free Full Text]
  74. Pamplona R, Portero-Otin M, Requena J, Gredilla R, Barja G. Oxidative, glycoxidative and lipoxidative damage to rat heart mitochondrial proteins is lower after 4 months of caloric restriction than in age-matched controls. Mech Ageing Dev. 2002;123:1437-1446.[Medline]
  75. Farmer KJ, Sohal RS. Relationship between superoxide anion radical generation and aging in the housefly, Musca domestica. Free Radic Biol Med. 1989;7:23-29.[Medline]
  76. Pamplona R, Portero-Otin M, Requena JR, Thorpe SR, Herrero A, Barja G. A low degree of fatty acid unsaturation leads to lower lipid peroxidation and lipoxidation-derived protein modification in heart mitochondria of the longevous pigeon than in the short-lived rat. Mech Ageing Dev. 1999;106:283-296.[Medline]



This article has been cited by other articles:


Home page
 Mol. Biol. CellHome page
A. Cammarato, C. M. Dambacher, A. F. Knowles, W. A. Kronert, R. Bodmer, K. Ocorr, and S. I. Bernstein
Myosin Transducer Mutations Differentially Affect Motor Function, Myofibril Structure, and the Performance of Skeletal and Cardiac Muscles
Mol. Biol. Cell, February 1, 2008; 19(2): 553 - 562.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
PubMed
Right arrow PubMed Citation

HOME ARCHIVE SEARCH TABLE OF CONTENTS