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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:36-47 (2006)
© 2006 The Gerontological Society of America

The Effect of Dietary Restriction on Mitochondrial Protein Density and Flight Muscle Mitochondrial Morphology in Drosophila

Tapiwanashe Magwere1, Steve Goodall1, Jeremy Skepper2, William Mair1, Martin D. Brand3 and Linda Partridge1,

1 Department of Biology, University College London, United Kingdom.
2 Department of Anatomy, University of Cambridge, United Kingdom.
3 MRC Dunn Human Nutrition Unit, Cambridge, United Kingdom.

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


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Dietary restriction (DR) extends life span in diverse organisms and may do so by attenuating production of mitochondrial reactive oxygen species (ROS). However, measurements of ROS production from isolated mitochondria of organisms subjected to DR have produced inconsistent results. In the fruit fly Drosophila, DR does not reduce production of ROS from isolated mitochondria. In this study, we used Drosophila to test whether DR lowered mitochondrial density. We assessed mitochondrial densities of flies on DR and Control diets using (a) the activities of mitochondrial enzymes and (b) electron microscopy. Both methods showed no overall effect of DR on mitochondrial density; however, mitochondrial enzyme activities and morphology differed significantly between DR and Control flies. We concluded that life-span extension by DR in Drosophila is not mediated through a reduction in mitochondrial density. If DR in Drosophila extends life span by reducing ROS production, then it does so through mechanisms that operate only in vivo.

The free radical (mitochondrial) theory of aging (1,2) proposes that mitochondria play a central role in the aging process by virtue of being a major source of reactive oxygen species (ROS). If this theory were correct, then variation of ROS production or antioxidant defense systems would be predicted to modulate life span. Correlational support for the mitochondrial theory of aging comes mainly from the finding that rates of mitochondrial ROS production vary inversely with maximum life-span potential of several animal species (3–7). Comparative studies of ROS production and maximal life span between birds and mammals of similar body size have shown that birds are longer-lived possibly because they exhibit lower rates of mitochondrial ROS production compared to mammals (7,8). Studies of Drosophila have also shown that longer lived strains contain higher levels of cytosolic copper-zinc superoxide dismutase (CuZnSOD) compared to shorter lived strains (9). Genetic manipulations of various antioxidant enzymes have provided additional support for the role of ROS in limiting life span. Knockout of the mitochondrial manganese superoxide dismutase (MnSOD) gene in mice leads to severely shortened life span (10), and silencing of the same gene in Drosophila leads to early adult-onset mortality (11). However, gene knockouts are known also to introduce novel pathologies to the organism, but it is inconclusive whether they normally limit life span. Overexpression of antioxidant defenses has, however, produced variable effects. Whereas targeted overexpression of CuZnSOD in Drosophila motor neurons was shown to extend life span by up to 40% (12,13), other studies involving overexpression of the same enzyme in flies have shown that life-span extension can be influenced by genetic background (14).

Dietary restriction (DR) is an environmental intervention whose life-prolonging effects have been observed in diverse animal species (15,16). One of the suggested mechanisms of action of DR is through decreasing mitochondrial ROS production and the associated oxidative damage (17–20). However, the effect of DR on limiting ROS production in isolated mitochondria appears inconsistent given the disparate findings from different researchers. In rodents, DR was shown to decrease mitochondrial generation of hydrogen peroxide (21–24) as well as the accumulation of oxidative damage to protein (25,26), lipids (27), and DNA (24,28,29). However, other researchers have observed no effect of DR on mitochondrial ROS production. In Drosophila, Miwa and colleagues (30) did not find any differences in mitochondrial ROS production between DR and fully fed controls, and similar observations were later made with cells isolated from rats (31). The inconsistent effects of DR in rats on mitochondrial ROS production measured in isolated mitochondria in vitro in state 4 resting condition (in the absence of adenosine 5'-diphosphate [ADP]) and in cells suggest that conditions for isolated mitochondria may not adequately mimic mitochondrial ROS production in vivo where ADP and other factors such as a regulated fuel supply are present.

Another way in which DR could lower ROS production in vivo is by reducing the mitochondrial density (MD). This would lower the number of ROS-producing sites and make each mitochondrion carry out oxidative phosphorylation at a higher rate, lowering its proton motive force and therefore its ROS production (32,33). In this study, we tested whether DR decreases MD in vivo using the Drosophila model. Drosophila is an organism well suited for this purpose because it shows life-span extension in response to DR (34,35) similar to rodents, and is easier to handle and manipulate under laboratory conditions. Determination of MD was carried out using two methods: 1) indirectly, by measuring the activities of mitochondrial enzymes (matrix: citrate synthase [CTS] and MnSOD; inner membrane: cytochrome c oxidase [COX] and adenine nucleotide translocase [ANT]) in isolated mitochondria compared to homogenates; and 2) directly, by using electron microscopy (EM) of flight muscle mitochondria. The enzyme method relies on the assumption that cell populations (hence mitochondrial populations) harvested from the two groups of flies are the same, whereas the stereological method used in EM can be prone to bias (36). Because each method has its own strengths and weaknesses, it is advantageous to use both. The enzyme method also gives additional information about intrinsic properties of the mitochondria such as the activities of oxidative phosphorylation and antioxidant enzymes, whereas the EM method can additionally provide information on mitochondrial morphology such as shape and volume. The aim of this study therefore was to investigate whether life-span extension by DR involved changes in MD or other properties of the mitochondria.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Chemical Reagents
The following reagents and biochemicals from Sigma Chemical Company (St. Louis, MO) were used: imidazole; ethylenediamine tetraacetate (EDTA); 5,5'-dithiobis-2-nitrobenzoic acid (DTNB); acetyl-coenzyme A (acetyl-CoA); oxaloacetate, oxidized cytochrome c; Tween-80; Triton X-100; potassium cyanide (KCN); rotenone; oligomycin; carbonyl cyanide p-(trifluoromethoxy) phenyl-hydrazone (FCCP); P1,P5-di(adenosine-5') pentaphosphate; L-{alpha}-glycerophosphate; bovine serum albumin (BSA); ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA); xanthine; nitroblue tetrazolium (NBT); sodium cyanide; diethylenetriaminepentaacetic acid (DTPA); catalase; bathocuproine disulphonic acid; and xanthine oxidase. Carboxyatractyloside (CAT) was obtained from Calbiochem (Nottingham, U.K., Ltd). All other chemicals and reagents were of pure analytical grade.

Experimental Flies
Male Drosophila melanogaster were used for enzyme measurements to avoid complications from ovarian activity, while female flies were used for flight muscle EM because of their larger size and greater ease of handling. All flies were of the wild type Dahomey base stock, and both sexes increase life span in response to DR (35). For the enzyme experiments, flies were cultured for two generations on normal strength 10% sucrose–yeast (SY) medium in 189-ml bottles at standard density (37). Flies of uniform age were collected for the experiments from the third generation using light CO2 anesthesia. A total of 6000 flies (3000 Control and 3000 DR) were set up for each of the four enzyme assays.

For EM, eggs were collected from stock cages by introducing petri dishes containing grape agar medium (30% grape juice, 2.5% agar, 0.2% nipagin) seeded with a small amount of live yeast. The plates were retrieved 4 hours later and incubated at 25°C overnight. The next day, first instar larvae were picked from the plates and placed in vials of 10% SY medium at 50 larvae per vial. When the flies had emerged, they were placed in vials of fresh SY food for 48 hours. Females were then collected under CO2 anesthesia and placed on experimental food.

DR
The concentrations of yeast and sugar in the DR medium was 4% for enzyme assays and 6.5% for EM [the concentrations that maximize Dahomey male and female life span, respectively (35,38)] and that in the Control medium was 16% and 15%, respectively. Flies were randomly allocated to each of the DR and Control regimens in groups of 100 flies per 189 ml bottle (10 flies per 7 ml vial for EM), and were transferred to fresh food once every 3 days (3 times per week for EM). Deaths were scored daily.

Flies for the enzyme assays and EM studies were collected from randomly selected bottles or vials, and the required numbers were counted under CO2 anesthesia. Sampling of flies was done at comparable chronological and physiological ages for the DR and Control groups. The time points for the enzyme assays were those corresponding to survivorship of 95%, 65%, and 35% for the Control flies and additional sampling points were introduced when survivorship for the DR flies had reached 65% and 35%. For the EM experiments, samples were taken for dissection at 80% and 60% survivorship for the Control group as well as at time points when DR survivorship had reached 80% and 60% survivorship.

Enzyme Assays
Whole-fly homogenates were prepared from 10 flies in the appropriate buffer for each of the enzymes CTS, COX, and MnSOD. For the CTS assay, the buffer was 50 mM imidazole containing 2 mM EDTA and 0.5% Triton X-100, pH 7.1 (Buffer A); for the COX assay, it was 200 mM Tris-HCl containing 0.03% Tween 80 pH 7.5 (Buffer B); for MnSOD, it was 50 mM potassium phosphate, pH 7.8 (Buffer C). The flies were chilled on ice, weighed, and homogenized in 1 ml of buffer on ice by using three 20-second bursts of a T8 Ultra-Turrax homogenizer (Jencons-PLS) given at 1-minute intervals. The homogenates were centrifuged twice at 2000 g for 15 seconds in a refrigerated centrifuge (4°C), and the resultant supernatant were assayed immediately for enzyme activity.

Mitochondria were isolated according to procedures described earlier (33) in freshly prepared STE buffer (250 mM sucrose, 5 mM Tris, 2 mM EGTA, and 1% BSA, pH 7.1). Mitochondrial pellets for the CTS, COX, and MnSOD assays were dissolved in the appropriate assay buffer to about 30 mg/ml. For the ANT assay, mitochondrial pellets were gently resuspended in 0.5 ml of STE buffer (25–30 mg protein/ml) taking care to avoid rupturing the mitochondria. Fly mitochondria become uncoupled quickly (33), thus all procedures were carried out speedily on ice (or 4°C) and the mitochondria were used within 1 hour of isolation.

Protein determinations in homogenates and mitochondrial fractions were carried out using the Pierce BCA Protein Assay Kit (Perbio Science, UK Ltd., Cheshire). Enzyme activities for CTS, COX, and MnSOD were assayed under conditions of enzyme saturation with substrate in a HITACHI model U-2001 spectrophotometer (Hitachi High-Technologies Corporation,Wokingham, U.K.) at 25°C, and ANT activity was measured using a Clarke-type Electrode (Rank Brothers, Cambridge, U.K.). The enzyme activities were expressed as both activity per mg protein and as activity per fly. The concentrations of reagents given for all assays are final concentrations unless stated otherwise.

CTS activity was measured in fresh whole-fly homogenates and fresh mitochondrial fractions using the procedure described by Garciá-Esquivel and colleagues (39) with modifications. The reaction mixture contained 50 mM Buffer A, 0.1 mM DTNB, 0.3 mM acetyl-CoA, 0.05 mM oxaloacetate, and 10 µL of whole-fly homogenate or mitochondrial fraction. The reaction was initiated by addition of the oxaloacetate (omitted from the blank), and the reduction of DTNB was monitored at 412 nm.

COX activity was measured according to the method of Smith (40) with modifications. Enzyme activity was measured in a cuvette containing Buffer B, 0.03% Tween-80, 100 µM reduced cytochrome c, and 10 µl of whole-fly homogenate or mitochondrial protein. The oxidation of reduced cytochrome c was followed at 520 nm in a spectrophotometer at 25°C. The background (nonenzymatic) rate was determined by adding 200 µM cyanide, and did not exceed 2%–5% of the enzymatic rate under these assay conditions.

MnSOD activity was determined using the procedure described by Mockett and colleagues (41) with some modifications. The whole-fly homogenates and mitochondrial fractions were first passed through a Sephadex G-25 column to remove small-molecular-weight antioxidants that could interfere with the assay (42). The eluate was incubated with 5 mM KCN at room temperature for 45 minutes to completely inhibit the cytosolic CuZnSOD. The ability of the whole-fly homogenate or mitochondrial fractions to inhibit the xanthine/xanthine oxidase-driven autoxidation of NBT in Buffer C was monitored at 560 nm (41). The unit of MnSOD activity was calculated as the amount of protein that gave half-maximal inhibition of NBT oxidation under the assay conditions.

ANT activity was measured as follows: To the thermoregulated (25°C) Clarke-type electrode chamber were added 1.95 ml of KHE buffer (120 mM KCl, 5 mM KH2PO4, 3 mM HEPES, 1 mM EGTA, 1 mM MgCl2, and 0.2% BSA [pH 7.2]), 10 µl of 1 mM rotenone, 20 µl of 6 mM P1,P5-di(adenosine-5')pentaphosphate, and whole-fly homogenate or mitochondrial fraction at about 0.1–0.2 mg/ml; then the chamber was sealed to exclude atmospheric air. While measuring respiration, 20 µl of 2 M {alpha}-glycerophosphate and 10 µl of 50 mM ADP were added to obtain states II and III respiration rates, respectively. The reaction was then titrated with aliquots of 10 µM stock of CAT until the respiration rate decreased to the state II rate. Finally, 20 µl of 5 mM FCCP were added to ensure that the mitochondria were still coupled; otherwise, fresh mitochondrial isolates had to be prepared. ANT activity was calculated as the extrapolated minimum amount of CAT needed to fully return respiration to state II from a plot of respiration rate versus CAT concentration, and was expressed as picomoles of CAT titer per mg protein or per fly.

Mitochondrial Protein Density
The MD of DR and Control flies was calculated indirectly from the activities of enzymes in whole-fly homogenates and mitochondrial fractions per mg protein. The whole-fly homogenate enzyme activity was divided by the mitochondrial enzyme activity to give mg mitochondrial protein per mg whole-fly homogenate.

EM and Stereology
Flies were dissected for examination of flight muscle by transmission EM in Drosophila Ringers solution (43). The indirect flight muscles and the dorsal longitudinal muscles (DLMs), were examined [reviewed in (44)]. Heads and abdomens were removed, and the thoraces were bisected longitudinally along the midline to expose the flight muscle. The thorax halves were then placed in primary fix (3% gluteraldehyde, 2.5 mM CaCl2, 100 mM HEPES (pH 7.2), H2O2 added to 0.33% just before use) at 4°C for 3 hours (45) after which they were washed with a solution containing 2.5 mM CaCl2 and 100 mM HEPES (pH 7.2) four times, each wash lasting 15 minutes. They were then put in secondary fix [1% osmium tetroxide, 1.5 % ferricyanate, 100 mM HEPES (pH 7.2) (46)] for 1.5 hours and then washed in distilled water four times for 15 minutes each time before storage at 4°C.

Dehydration of samples was done by first washing several times with increasing concentrations of ethanol, then incubating in two changes of acetonitrile, and finally embedding in Spurr's resin. The samples were then cured at 60°C for 24 hours. Sections were cut at 50 nm in the transverse plane for volume fraction estimates or rotated to produce a vertical section plane for surface-density estimates with a Leica Ultracut UCT microtome (Leica, Vienna, Austria). They were mounted on 300 mesh copper grids, stained with uranyl acetate and lead citrate, and were then viewed on a Philips CM-100 transmission electron microscope operated at 80 Ky (Philips, Eindhoven, The Netherlands).

The mitochondrial volume density (Vv (mt,c)) was measured according to the stereological methods described (36). A square 16-point lattice with a spacing of 17.6 µm was overlaid on the display monitor connected to a Gatan 673 CCD camera (Gatan, U.K.) at a final magnification of x2800. Fields of view were randomly sampled by identifying the first grid square containing flight muscle, positioning the lattice in the upper right hand corner of the grid (36), and repeating this in subsequent grid squares until 50 fields of view had been counted. Lattice intersections overlying mitochondria and myoplasm (including all other components of the myofibers) were counted. Mitochondrial volume density was calculated by the ratio of points counted within the mitochondrial outer membrane to those outside mitochondria.

For the mitochondrial inner membrane surface density (Sv (mt,mt)) determination, five individual mitochondria from each group were randomly selected by point counting as above and were photographed at a magnification of x35,000. The negatives from these photographs were scanned and counts were done by digitally overlaying the quadratic lattice with an effective line spacing of 314.2 nm in Adobe Photoshop 6.0.1 (Copyright © 1989–2001 Adobe Systems Incorporated). Inner membrane surface density was calculated by the formula 2 · I/(l/p · P), where I is the number of intersections with the line matrix for the membrane of interest, P is the number of points within the area of interest (reference volume), and l/p is the line length per point (47).

The outer membrane surface densities of mitochondria per cubic micrometer of mitochondria (Sv (mt,mt)) or per cubic micrometer of myofiber (Svmitochondrial outer membrane/mitochondria or Svmitochondrial outer membrane/myofiber, respectively), were also estimated and expressed as square micrometers of outer membrane per cubic micrometer of mitochondria or myofiber. Ten fields of view were randomly selected as above and viewed at a final magnification of x4500. They were overlain with a 16-point quadratic lattice with a spacing of 3.4 µm. Linear intercepts with mitochondrial outer membranes were also counted and the formula Sv = 2i/L was used to estimate surface density of outer mitochondrial membrane, where i = number of intercepts and L = total test line length overlying either mitochondria or myofiber (36,47).

Statistical Analyses
Data sets were compared using JMP statistical data package (version 4.05; [Academic] Copyright © 1989–2001, SAS Institute Inc.). Survival data were analyzed using log rank tests, and MD, enzyme activities, and microscopy data sets were all compared using one- and two-way analyses of variance. P values less than.05 were considered significant.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Survival and Mortality Rates
The survivorship of the DR flies exceeded that of the Control flies by approximately 50%–60% for both the flies used for enzyme assays (log rank test, {chi}2 = 138.43, p =.0000) and EM studies (log rank test, {chi}2 = 307.58, p <.0001).

Protein Content
There was no significant effect of age on whole-fly homogenate protein content (Figure 1a; F ratio = 3.0, p =.089). Control flies had significantly higher protein content than did DR flies at all ages (F ratio = 39.5, p <.0001), and even when flies of similar survival stages (Control days 22 and 35 vs DR days 47 and 53) were compared.


Figure 01
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  		Figure 1. Effect of dietary restriction (DR) and Control feeding on (a) whole-fly homogenate protein and (b) mitochondrial protein content. The sampling time points are as described in text. Each point is mean ± standard deviation of triplicate determinations for N = 3 samples. *Values are significantly different from Control group (p <.0001)

 
Mitochondrial protein per fly (Figure 1b) for DR flies increased significantly with age up to day 35 (F ratio = 87.4, p <.001) after which it then decreased. Mitochondrial protein content did not differ significantly with age between DR and Control groups (F ratio = 0.92, p =.346). However, when similar survival stages were compared (Control days 22 and 35 vs DR days 47 and 53), Control flies had significantly higher mitochondrial protein content than did DR flies. To summarize, these results (Figure 1) indicate that DR lowers total protein content in flies but does not alter mitochondrial protein content except at very young ages.

Enzyme Activities
The enzyme activities from whole-fly homogenates and mitochondrial fractions were expressed both as activity per fly and activity per mg protein for two main reasons: (1) to provide more information about the metabolic capacity of flies in the DR and Control groups, and (2) to ascertain that any observed differences in MD were not just a reflection of differences in protein content between DR and Control flies.

Whole-fly homogenate activities.-- Enzyme activities per mg protein for the four enzymes are shown in Figure 2. The inner membrane enzymes' (COX and ANT) activities both declined significantly with age (COX: F ratio = 10.8, p =.0016; ANT: F ratio = 48.23, p <.0001, respectively) for DR and Control flies with no significant difference between the two groups at any age. However, when similar survival stages were compared (Control days 20 and 29 vs DR days 35 and 43), COX enzyme activities were significantly higher for Control than for DR flies (F ratio = 36.6, p <.0001), but ANT activity remained unaffected by survival stage (F ratio = 0.005, p =.94).


Figure 02
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  		Figure 2. Enzyme activity per mg protein for the four enzymes cytochrome c oxidase (COX), adenine nucleotide translocase (ANT), citrate synthase (CTS), and manganese superoxide dismutase (MnSOD) in whole-fly homogenates. The sampling time points are as described in text. Each point is mean ± standard error of triplicate determinations for N = 3 samples. *Values are significantly different between dietary restriction (DR) and Control groups (p <.0001)

 
Matrix enzymes (CTS and MnSOD) did not show any uniform pattern: CTS activity peaked at an intermediate age and then declined thereafter, whereas MnSOD activity significantly increased with age for both Control and DR flies (F ratio = 9.6, p <.02). When same age flies were compared, CTS activity was higher for Control than for DR flies on day 20 but much lower on day 29 (p <.0001). However, there was no effect of survival stage on both CTS and MnSOD enzyme activities between Control and DR flies. When expressed in terms of activity per fly, all four enzyme activities mirrored the activity patterns observed earlier (Figure 2), indicating that any differences in enzyme activities between DR and Control flies were independent of the protein concentrations.

Mitochondrial activities.-- Mitochondrial activities per mg protein (Figure 3) declined with age for both inner membrane enzymes (COX: [Control F ratio = 73.4, p <.0001 vs DR F ratio = 17.8, p <.0001] and ANT [Control F ratio = 6.1, p <.043 vs DR F ratio = 48.2, p <.0001]). When flies of similar ages were compared, there were no significant differences in COX activity between DR and Control groups at any age. ANT activity was, however, significantly higher for DR than for Control flies of similar age on day 8 (p <.0001). Comparison of both COX and ANT activities at similar survival stages (Control days 20 and 29 vs DR days 35 and 43) showed that neither were affected by survival stage of the flies.


Figure 03
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  		Figure 3. Enzyme activity per mg protein for the four enzymes cytochrome c oxidase (COX), adenine nucleotide translocase (ANT), citrate synthase (CTS), and manganese superoxide dismutase (MnSOD) in mitochondrial fractions. The sampling time points are as described in text. Each point is mean ± standard error of triplicate determinations for N = 3 samples. *Values are significantly different between dietary restriction (DR) and Control groups (p <.0001)

 
CTS activity per mg protein (Figure 3) was higher for Control than DR flies on days 8 and 20 (p <.0001), but decreased sharply thereafter and was lower than DR on day 29 (p <.0001). When equivalent survival stages were compared (Figure 3; Control day 20 vs DR day 35 and Control day 29 vs DR day 43) there were no significant differences in enzyme activity per mg protein between the two groups suggesting that DR only delayed the age-dependent decrease in CTS activity. MnSOD activity per mg protein (Figure 3) declined with age for both Control (F ratio = 9.65, p <.017) and DR (F ratio = 43.5, p <.0001) flies; however, Control flies had higher MnSOD activity on days 20 (p <.023) and 29 (p <.021). When similar survival stages were compared (Control days 20 and 29 vs DR days 35 and 43), MnSOD activity was still lower for DR flies suggesting than DR did not seem to prevent the age-dependent decrease for MnSOD activity.

The mitochondrial activities per mg protein for all four enzymes were also reflected in terms of activities per fly, again indicating that enzyme activities were not influenced by differences in protein content between DR and Control flies. Therefore, to summarize these results, DR appeared to delay the age-dependent decline in COX, ANT, and CTS activities, but did not seem to have any effect on MnSOD activity.

Mitochondrial Protein Density
MD calculated using the inner membrane enzymes (COX and ANT) showed a different picture from that calculated using the matrix enzymes (CTS and MnSOD) (Figure 4). The COX and ANT measurements both showed MD decreasing with age for DR and Control groups (COX: F ratio = 8.17, p <.0058; ANT: F ratio = 12.58, p <.009), a trend that was identical to that of the enzyme activities (Figure 3). The ANT graph (Figure 4) shows that MD was significantly higher for the Control group on days 8 (p <.01) and 20 (p <.05). However, comparison of MD between DR and Control flies at equivalent survival stages (ANT) shows that the difference was not significant.


Figure 04
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  		Figure 4. Mitochondrial protein density calculated from the specific activities of inner membrane (cytochrome c oxidase [COX] and adenine nucleotide translocase [ANT]) and matrix (citrate synthase [CTS] and manganese superoxide dismutase [MnSOD]) enzymes as described in the text. Each point is mean ± standard error of triplicate determinations. *Values significantly different from Control are ANT: day 8 (p <.01) and day 20 (p <.002); CTS: days 10, 20, and 29 (p <.01); MnSOD: days 20 and 29 (p <.001)

 
The matrix enzymes CTS and MnSOD (Figure 4) showed that MD increased with age (CTS: Control F ratio = 51.41, p <.0002; MnSOD: Control F ratio = 61.57, p <.0001 vs DR F ratio = 122.96, p <.0001). For CTS, MD was higher in the DR group compared to Control on days 8 and 20 (p <.01), but was much lower than DR on day 29 (p <.001). Comparison of equivalent survival stages (MnSOD: Control days 20 and 29 vs DR days 35 and 43) showed that MD was still higher in DR flies, suggesting that the difference in MD between DR and Control flies was not affected by the age of the flies.

MD obtained by the enzyme methods should be independent of method used; therefore, averaging all four panels would give an unbiased and more representative effect of DR on this parameter. The average MD plot (Figure 4) shows an effect of neither age (F ratio = 0.54, p =.47) nor food (F ratio = 0.05, p =.83) on MD in flies. Comparison of equivalent survival stages (Control days 20 and 29 vs DR days 35 and 43) also showed no significant effect of DR on MD.

EM Results
Typical electron micrographs of flight muscle mitochondria from Control and DR flies are shown in Figure 5, a and b, respectively. The mitochondria from DR flies (Figure 5b) appear larger than those from Control flies (Figure 5a). The myofiber diameters from DR flies also appear larger than those from Controls.


Figure 05
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  		Figure 5. Typical electron micrographs of flight muscle mitochondria from dietary restriction (DR) and Control flies. a and b, Low magnification images (x3500) of flight muscle showing arrangement of mitochondria within tissue. a is from a Control fly and b is from a DR fly. c–f, High magnification (x35,000) images of flight muscle mitochondria from day 12 individuals; c and f are from Control flies; d and e are from DR flies. a and b are representative of at least six different samples taken from either group of flies on day 12. Double arrows point to the mitochondria, single arrows show glycogen granules, and m = flight muscle myofibers

 
Mitochondrial volume density (Figure 6a) decreased with age in both Control and DR groups (F ratio = 17.66, df = 2, p <.0001), and was lower in Control flies (F ratio = 13.20, df = 1, p =.0013). When comparisons are made between the DR and Control flies at equivalent survival stages, i.e., days 24 and 28 for Control flies versus days 48 and 53 for DR flies, Control flies had lower mitochondrial volume density. DR flies showed a slight increase in volume density with survival stage, whereas the Controls showed a sizeable decrease.


Figure 06
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  		Figure 6. The effect of diet and age on stereological parameters of flight muscle mitochondria: (a) mitochondrial volume density (Vv [mt, c]), (b) mitochondrial inner membrane surface density (Sv [mt,mt]), (c) mitochondrial outer membrane surface density per mitochondrion (Sv (mt,mt)), (d) mitochondrial outer membrane surface density per cell (Sv [mt,c]), and (e) mitochondrial inner membrane surface density per cell. Data shown are mean ± standard error of the mean for N = 5–6 different samples. Open circles represent dietary restriction (DR) animals; closed circles represent Control animals

 
Mitochondrial inner membrane surface density (Figure 6b) did not differ significantly between DR and Control flies of comparable age and survival stages (F ratio = 0.2677, df = 2, p =.7669). When flies at equivalent survival stages were compared, DR flies had significantly lower inner membrane surface density (F ratio = 6.5827, df = 1, p =.0195) compared to Control flies but there was no significant effect of survival stage (Figure 6b: F ratio = 0.7785, df = 1, p =.3892).

The mitochondrial outer membrane surface density per mitochondrion (Sv (mt,mt)) (Figure 6c) decreased with age for DR and Control flies (F ratio = 4.0879, df = 2, p =.0286) and was much higher for DR than Control on day12. When Sv (mt,mt) from equivalent survival stages was compared, no significant differences were observed between the two groups (F ratio = 1.9233, df = 1, p =.1845).

Mitochondrial outer membrane surface density per cell (Sv (mt,c)) (Figure 6d) was significantly lower in Control than in DR flies of comparable age (F ratio = 38.4411, df = 1, p <.0001), and this difference was not affected by age. When Sv (mt,c) was compared between DR and Control flies at comparable survival stages, the effect of DR was not significant (F ratio = 0.8134, df = 1, p =.7377), suggesting that DR only affects Sv(mt,c) at younger ages.

Figure 6e shows the mitochondrial inner membrane surface density per cell (Sv(mt;c)) or mitochondrial inner membrane surface area as a ratio of tissue volume for the two groups of flies. Inner membrane surface density was calculated by multiplying the mitochondrial volume density (Figure 6a) by the inner membrane surface density per mitochondria (Figure 6b). It is a more appropriate measure of mitochondrial density because: (1) it reflects the actual ROS-generating capacity per cell and (2) it gives the most comparable measurement to mg mitochondrial protein per mg whole-homogenate protein (MD) shown in Figure 4. Figure 6e shows that there was no significant effect of age on inner membrane surface density for DR (F ratio = 1.0845, df = 1, p =.3742) or Control (F ratio = 0.0155, df = 1, p =.9212) groups; neither were there any significant differences between the two groups of flies with age (F ratio = 0.2608, df = 1, p =.6313).

In summary, the EM results collectively show that, on average, mitochondrial density did not significantly differ with age between Control and DR flies (Figure 6e), although other parameters such as size (Figure 5) and morphology of mitochondria could be altered with age and show significant differences between DR and Control groups (Figure 6, a–d).


   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The overall aim of this study was to test if the effects of DR on life span were mediated through changes in MD. Our working hypothesis was that, if DR lowers ROS production by decreasing the number of ROS-producing organelles, then MD should decrease, rather than increase, in DR flies compared to Controls. The enzyme method showed differences in mitochondrial activities between DR and Control flies (Figures 2 and 3), but there were no associated changes in mitochondrial protein density. The EM method also showed morphological differences between mitochondria from DR and Control flies, but again no differences in inner membrane surface density per cell volume. Taken together, both enzyme and EM results suggest that DR only alters mitochondrial morphology (size, shape, and number) as well as specific enzyme activities, but does not alter mitochondrial content per cell. Support for this suggestion comes from the mitochondrial protein content per fly (Figure 1b), which did not differ significantly between DR and Control flies to the same extent as did total protein content per fly (Figure 1a). Whole-fly homogenate protein content (Figure 1a) was, however, much higher for Control flies indicating that the Control diet also increased protein content of other cellular organelles apart from the mitochondria. Genome-wide profiling of genes associated with aging in Drosophila showed that genes involved in cell growth and metabolism were overly expressed in fully fed (Control) compared to DR flies (38), and may possibly be related to the increase in whole-fly homogenate protein content for Control flies from this study.

MD calculated using the enzyme methods should be independent of method used, hence the opposite trends between inner membrane and matrix enzymes (Figure 4) suggest either that: (1) the inner membrane and matrix enzymes were assessing different properties of the mitochondria or (2) there were methodological or technical errors associated with the use of enzymes from the two mitochondrial compartments. The first possibility is highly unlikely because any of the measurements cancel out in the calculation of MD and the final result becomes independent of the enzyme used. However, the second possibility is more likely given the observed trends in the activities of the four enzymes. Fluctuations in CTS activity as well as the opposite trends shown by MnSOD activities from whole-fly homogenates and mitochondrial fractions (Figures 2 and 3) strongly suggest loss of enzyme activity from the matrix possibly due to leakage during isolation of mitochondria. For example, 10%–15% of the CTS activity could be detected in the supernatants of the mitochondrial fractions from 8-day-old flies (result not shown) confirming this supposition. Loss of enzyme from the matrix would give a progressive underestimate of MD using the matrix enzymes (because the mitochondrial preparation has lost matrix contents but not membranes to the same extent), and a corresponding overestimate using the membrane enzymes (because there will be some membranes that lack matrix, so more membranes per mg protein in the mitochondrial fractions).

The EM results may support earlier findings that mitochondrial numbers in vivo decrease with age (48,49); however, mechanisms through which mitochondrial numbers fluctuate in vivo are still obscure. Mitochondrial biogenesis in biological systems is a poorly understood process (50), and processes of mitochondrial fusion (51) and fission (52) that may be involved in regulating mitochondrial numbers in vivo are still poorly understood. In rodents it has been shown that chronic energy deprivation (caloric restriction) induces mitochondrial biogenesis through metabolic adaptations that involve both AMP (adenosine 5'-monophosphate) kinase-dependent (53) and -independent (54) pathways. However, it has not been determined whether similar metabolic adaptations do occur in Drosophila under DR. The finding from this study that DR alters mitochondrial morphology (Figure 6, a–d) without altering mitochondrial protein density (Figure 4) or inner membrane density per cell volume in Drosophila (Figure 6e) suggests that DR may influence mitochondrial fusion and/or fission events in vivo, but this has yet to be demonstrated.

Mitochondrial populations in tissues are not homogeneous and can be separated into heavy, medium, or light fractions according to their degree of maturation and size (55). It has been shown for brown adipose tissue from rats that chronic interventions such as overfeeding or prolonged fasting produce changes mainly detected in the heavy mature mitochondrial populations, whereas acute short-term fasting or cold exposure produces adaptive changes in light immature mitochondrial fractions (56,57). Apparently lifelong (chronic) DR provides a different physiological stimulus from full feeding and may alter mitochondrial populations differently between DR and Control animals. Hence the assumption that the cell population from which mitochondria are harvested is the same in both DR and Control flies may be incorrect, and determinations of MD using the enzyme method may always need confirmation by at least one other method, as we did here using EM.

Summary
DR did not significantly decrease mitochondrial density (mitochondrial protein density or mitochondrial inner membrane surface density per cell) in Drosophila. DR, however, appeared to delay the age-dependent decreases in some mitochondrial enzyme activities and morphological changes seen in the Control group of flies. We therefore conclude that life-span extension by DR in Drosophila may not be mediated through a reduction in mitochondrial density and, if DR in Drosophila extends life span by reducing ROS production, then it does so through mechanisms that operate only in vivo.


   Acknowledgments
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We gratefully acknowledge funding from the Biotechnology and Biological Sciences Research Council (BBSRC), Medical Research Council (MRC) Leverhulme Trust, and the Wellcome Trust.

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


   Footnotes
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Decision Editor: James R. Smith, PhD

Received June 14, 2005

Accepted August 8, 2005


   References
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  1. Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol. 1956;11:298-300.
  2. Harman D. The biologic clock: the mitochondria? J Am Geriatr Soc. 1972;20:145-147.[Medline]
  3. Barja G. Rate of generation of oxidative stress-related damage and animal longevity. Free Radic Biol Med. 2002;33:1167-1172.[Medline]
  4. Holmes DJ, Fluckiger R, Austad SN. Comparative biology of aging in birds: an update. Exp Gerontol. 2001;36:869-883.[Medline]
  5. Sohal RS, Arnold LA, Sohal BH. Age-related changes in antioxidant enzymes and pro xidant generation in tissues of the rat with special reference to parameters in two insect species. Free Radic Biol Med. 1990;9:495-500.[Medline]
  6. 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]
  7. Barja G. Mitochondrial free radical production and aging in mammals and birds. Ann N Y Acad Sci. 1998;854:224-238.[Medline]
  8. Barja G, Cadenas S, Rojas C, Perez-Campo R, Lopez-Torres M. Low mitochondrial free radical production per unit O2 consumption can explain the simultaneous presence of high longevity and high aerobic metabolic rate in birds. Free Radic Res. 1994;21:317-327.[Medline]
  9. Hari R, Burde V, Arking R. Immunological confirmation of elevated levels of CuZn superoxide dismutase protein in an artificially selected long-lived strain of Drosophila melanogaster. Exp Gerontol. 1998;33:227-237.[Medline]
  10. Lebovitz RM, Zhang H, Vogel H, et al. Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice. Proc Natl Acad Sci U S A. 1996;93:9782-9787.[Abstract/Free Full Text]
  11. Kirby K, Hu J, Hilliker AJ, Phillips JP. RNA interference-mediated silencing of Sod2 in Drosophila leads to early adult-onset mortality and elevated endogenous oxidative stress. Proc Natl Acad Sci U S A. 2002;99:16162-16167.[Abstract/Free Full Text]
  12. Parkes TL, Elia AJ, Dickinson D, Hilliker AJ, Phillips JP, Boulianne GL. Extension of Drosophila lifespan by overexpression of human SOD1 in motorneurons. Nat Genet. 1998;19:171-174.[Medline]
  13. Phillips JP, Parkes TL, Hilliker AJ. Targeted neuronal gene expression and longevity in Drosophila. Exp Gerontol. 2000;35:1157-1164.[Medline]
  14. Sun J, Tower J. FLP recombinase-mediated induction of Cu/Zn-superoxide dismutase transgene expression can extend the life span of adult Drosophila melanogaster flies. Mol Cell Biol. 1999;19:216-228.[Abstract/Free Full Text]
  15. Masoro EJ. Caloric restriction and aging: an update. Exp Gerontol. 2000;35:299-305.[Medline]
  16. McCay CM, Cromwell MF, Maynard LA. The effect of retarded growth upon the length of lifespan and upon the ultimate body size. J Nutr. 1935;10:63-79.[Abstract/Free Full Text]
  17. Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science. 1996;273:59-63.[Abstract]
  18. Yu BP, Lim BO, Sugano M. Dietary restriction downregulates free radical and lipid peroxide production: plausible mechanism for elongation of life span. J Nutr Sci Vitaminol (Tokyo). 2002;48:257-264.[Medline]
  19. Sohal RS, Ku HH, Agarwal S, Forster MJ, Lal H. Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse. Mech Ageing Dev. 1994;74:121-133.[Medline]
  20. Barja G. Aging in vertebrates, and the effect of caloric restriction: a mitochondrial free radical production-DNA damage mechanism? Biol Rev Camb Philos Soc. 2004;79:235-251.[Medline]
  21. Bevilacqua L, Ramsey JJ, Hagopian K, Weindruch R, Harper ME. Effects of short- and medium-term calorie restriction on muscle mitochondrial proton leak and reactive oxygen species production. Am J Physiol Endocrinol Metab. 2004;286:E852-E861.[Abstract/Free Full Text]
  22. Gredilla R, Barja G, Lopez-Torres M. Effect of short-term caloric restriction on H2O2 production and oxidative DNA damage in rat liver mitochondria and location of the free radical source. J Bioenerg Biomembr. 2001;33:279-287.[Medline]
  23. Lass A, Sohal BH, Weindruch R, Forster MJ, Sohal RS. Caloric restriction prevents age-associated accrual of oxidative damage to mouse skeletal muscle mitochondria. Free Radic Biol Med. 1998;25:1089-1097.[Medline]
  24. Sanz A, Caro P, Barja G. Protein restriction without strong caloric restriction decreases mitochondrial oxygen radical production and oxidative DNA damage in rat liver. J Bioenerg Biomembr. 2004;36:545-552.[Medline]
  25. Forster MJ, Sohal BH, Sohal RS. Reversible effects of long-term caloric restriction on protein oxidative damage. J Gerontol Biol Sci Med Sci. 2000;55A:B522-B529.[Abstract/Free Full Text]
  26. Youngman LD, Park JY, Ames BN. Protein oxidation associated with aging is reduced by dietary restriction of protein or calories. Proc Natl Acad Sci U S A. 1992;89:9112-9116.[Abstract/Free Full Text]
  27. Wu A, Sun X, Wan F, Liu Y. Modulations by dietary restriction on antioxidant enzymes and lipid peroxidation in developing mice. J Appl Physiol. 2003;94:947-952.[Abstract/Free Full Text]
  28. Gredilla R, Sanz A, Lopez-Torres M, Barja G. Caloric restriction decreases mitochondrial free radical generation at complex I and lowers oxidative damage to mitochondrial DNA in the rat heart. FASEB J. 2001;15:1589-1591.[Free Full Text]
  29. Sohal RS, Agarwal S, Candas M, Forster MJ, Lal H. Effect of age and caloric restriction on DNA oxidative damage in different tissues of C57BL/6 mice. Mech Ageing Dev. 1994;76:215-224.[Medline]
  30. Miwa S, Riyahi K, Partridge L, Brand MD. Lack of correlation between mitochondrial reactive oxygen species production and life span in Drosophila. Ann N Y Acad Sci. 2004;1019:388-391.[Medline]
  31. Lambert AJ, Merry BJ. Lack of effect of caloric restriction on bioenergetics and reactive oxygen species production in intact rat hepatocytes. J Gerontol Biol Sci Med Sci. 2005;60A:175-180.[Abstract/Free Full Text]
  32. Miwa S, Brand MD. Mitochondrial matrix reactive oxygen species production is very sensitive to mild uncoupling. Biochem Soc Trans. 2003;31:1300-1301.[Medline]
  33. 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]
  34. 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]
  35. Magwere T, Chapman T, Partridge L. Sex differences in the effect of dietary restriction on lifespan and mortality rates in female and male Drosophila melanogaster. J Gerontol Biol Sci Med Sci. 2004;59A:B3-B9.[Abstract/Free Full Text]
  36. Weibel ER. Stereological Methods. New York: Academic Press; 1979.
  37. Clancy DJ, Kennington WJ. A simple method to achieve consistent larval density in bottle cultures. Drosoph Inf Serv. 2001;84:168-169.
  38. Pletcher SD, Macdonald SJ, Marguerie R, et al. Genome-wide transcript profiles in aging and calorically restricted Drosophila melanogaster. Curr Biol. 2002;12:712-723.[Medline]
  39. Garciá-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]
  40. Smith L. Bacterial cytochromes; difference spectra. Arch Biochem Biophys. 1954;50:299-314.[Medline]
  41. Mockett RJ, Bayne AC, Sohal BH, Sohal RS. Biochemical assay of superoxide dismutase activity in Drosophila. Methods Enzymol. 2002;349:287-292.[Medline]
  42. Segura-Aguilar J. A new direct method for determining superoxide dismutase activity by measuring hydrogen peroxide formation. Chem Biol Interact. 1993;86:69-78.[Medline]
  43. Ashburner M. Drosophila: A Laboratory Handbook. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989.
  44. Dickinson MH, Tu MS. The function of dipteran flight muscle. Comp Biochem Physiol A Physiol. 1997;116:223-238.
  45. Perachia C, Mittler BS. Fixation by means of glutaraldehyde-hydrogen peroxide reaction mixtures. J Cell Biol. 1972;53:234.[Free Full Text]
  46. de Bruijn WC. Gycogen: its chemistry and morphological appearance in the electron microscope I. a modified OsO4 fixative that selectively stains glycogen. J Ultrastruct Res. 1973;42:29.[Medline]
  47. Howard CV, Reed MG. Unbiased Stereology: Three-Dimensional Measurement in Microscopy. Oxford, U.K.: Bios Scientific Publishers: 1998.
  48. Fleming JE, Miquel J, Bensch KG. Age dependent changes in mitochondria. Basic Life Sci. 1985;35:143-156.[Medline]
  49. Sohal RS, McCarthy JL, Allison VF. The formation of ‘giant’ mitochondria in the fibrillar flight muscles of the house fly, Musca domestica L. J Ultrastruct Res. 1972;39:484-495.[Medline]
  50. Nisoli E, Clementi E, Moncada S, Carruba MO. Mitochondrial biogenesis as a cellular signaling framework. Biochem Pharmacol. 2004;67:1-15.[Medline]
  51. Westermann B. Merging mitochondria matters: cellular role and molecular machinery of mitochondrial fusion. EMBO Rep. 2002;3:527-531.[Medline]
  52. Bossy-Wetzel E, Barsoum MJ, Godzik A, Schwarzenbacher R, Lipton SA. Mitochondrial fission in apoptosis, neurodegeneration and aging. Curr Opin Cell Biol. 2003;15:706-716.[Medline]
  53. Zong H, Ren JM, Young LH, et al. AMP kinase is required for mitochondrial biogenesis in skeletal muscle in response to chronic energy deprivation. Proc Natl Acad Sci U S A. 2002;99:15983-15987.[Abstract/Free Full Text]
  54. Gonzalez AA, Kumar R, Mulligan JD, Davis AJ, Weindruch R, Saupe KW. Metabolic adaptations to fasting and chronic caloric restriction in heart, muscle, and liver do not include changes in AMPK activity. Am J Physiol Endocrinol Metab. 2004;287:E1032-E1037.[Abstract/Free Full Text]
  55. Justo R, Oliver J, Gianotti M. Brown adipose tissue mitochondrial subpopulations show different morphological and thermogenic characteristics. Mitochondrion. 2005;5:45-53.[Medline]
  56. Matamala JC, Gianotti M, Pericas J, et al. Changes induced by fasting and dietetic obesity in thermogenic parameters of rat brown adipose tissue mitochondrial subpopulations. Biochem J. 1996;319:(Pt 2): 529-534.[Medline]
  57. Gianotti M, Clapes J, Llado I, Palou A. Effect of 12, 24 and 72 hours fasting in thermogenic parameters of rat brown adipose tissue mitochondrial subpopulations. Life Sci. 1998;62:1889-1899.[Medline]




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