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Am J Physiol Endocrinol Metab 291: E460-E467, 2006. First published April 18, 2006; doi:10.1152/ajpendo.00258.2005
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Influence of intensity of food restriction on skeletal muscle mitochondrial energy metabolism in rats

Gyasi Johnson, Damien Roussel, Jean-François Dumas, Olivier Douay, Yves Malthièry, Gilles Simard, and Patrick Ritz

Institut National de la Santé et de la Recherche Médicale, Unité 694; Université d'Angers; and Laboratoire de biochimie et biologie moléculaire, Centre Hospitalier Universitaire Angers, Angers, France

Submitted 9 June 2005 ; accepted in final form 24 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Variable durations of food restriction (FR; lasting weeks to years) and variable FR intensities are applied to animals in life span-prolonging studies. A reduction in mitochondrial proton leak is suggested as a putative mechanism linking such diet interventions and aging retardation. Early mechanisms of mitochondrial metabolic adaptation induced by FR remain unclear. We investigated the influence of different degrees of FR over 3 days on mitochondrial proton leak and mitochondrial energy metabolism in rat hindlimb skeletal muscle. Animals underwent 25, 50, and 75% and total FR compared with control rats. Proton leak kinetics and mitochondrial functions were investigated in two mitochondrial subpopulations, intermyofibrillar (IMF) and subsarcolemmal (SSM) mitochondria. Regardless of the degree of restriction, skeletal muscle mass was not affected by 3 days of FR. Mitochondrial basal proton conductance was significantly decreased in 50% restricted rats in both mitochondrial subpopulations (46 and 40% for IMF and SSM, respectively) but was unaffected in other groups compared with controls. State 3 and uncoupled state 3 respiration rates were decreased in SSM mitochondria only for 50% restricted rats when pyruvate + malate was used as substrate (–34.5 and –38.9% compared with controls, P < 0.05). IMF mitochondria respiratory rates remained unchanged. Three days of FR, particularly at 50% FR, were sufficient to lower mitochondria energetic metabolism in both mitochondrial populations. Our study highlights an early step in mitochondrial adaptation to FR and the influence of the severity of restriction on this adaptation. This step may be involved in an aging-retardation process.

proton leak; oxidative phosphorylation; mitochondria

AGING AND A NUMBER OF AGE-ASSOCIATED PATHOLOGIES such as Parkinson's disease and type 2 diabetes involve mitochondrial abnormalities (42). Although the precise mechanisms are unclear, mitochondria are thought to play an important role (38, 40). Given that ~90% of cellular oxygen consumption and the majority of reactive oxygen species (ROS) production in the cell takes place in the mitochondria (1), these organelles represent a highly exposed oxidative stress site. According to the oxidative stress theory of aging, the accumulation of free radical-induced macromolecule injuries in mitochondria and cells may lead to dysfunction and aging (17).

As far as Harman's free radicals theory of aging and diseases retardation is concerned, calorie restriction (CR) as a means of delaying aging has been widely studied. CR interventions are known for extending mean and maximum life span of a wide range of species (19). Long-term CR (lasting months to years) has been used in animal models to investigate different aspects of aging (28). Hypotheses proposed to account for the life-prolonging effect of CR include a decrease in ROS production and limited oxidative stress in macromolecules (41, 3, 4, 25).

The mechanisms that reduce ROS production during CR are still debated. However, Ramsey et al. (31) have proposed that a decrease in mitochondrial proton leak or a lowered membrane potential that reduces resting oxygen consumption is a likely explanation. Indeed, proton leak increases with age in both intact hepatocytes (18) and skeletal muscle mitochondria (24), as does ROS production (40, 44). Furthermore, in skeletal muscle, a postmitotic tissue that is highly susceptible to oxidative damage (37), maximum leak-dependent respiration (state 4) is decreased by 40% CR in the short and medium term (2 wk, 2 mo, and 6 mo) (3) as well as in the long term (12 mo) (4) and by 33% CR at 23 mo (24). No study has evaluated the impact of CR on mitochondria metabolism over a duration shorter than 5 days. Similarly, most trials have been performed with ~40% food restriction (FR), and it is not known whether similar conclusions can be made at restrictions that are higher or lower than this.

In skeletal muscle, mitochondria form a reticulum, with compartmentalization into subsarcolemmal (SSM) and intermyofibrillar (IMF) populations. SSM mitochondria provide energy for membrane-related processes including signal transduction, ion exchanges, substrate transport, and activation. IMF mitochondria are more involved in muscle contraction. Both populations appear to be regulated differently (6, 11, 20, 21, 36).

The aim of the present study was to investigate the early effect of FR on skeletal muscle mitochondrial proton leak kinetics and mitochondrial functions. To identify the mitochondrial subpopulation responsible for the adaptive mechanism observed, we focused on both IMF and SSM mitochondria populations. Furthermore, we chose to submit animals to various levels of FR over the same period of time (3 days).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals. Chemicals and reagents were purchased from Sigma Aldrich (Saint Quentin Fallavier, France) except for Tris and Tris-HCl, which were purchased from Eurobio (Les Ulis, France), and nagarse and KCN, which were purchased from Fluka (Saint Quentin Fallavier, France). Triphenylmethylphosphonium (TPMP) was purchased from Merck (VWR International, Strasbourg, France); BSA, NADH, and Triton X-100 were purchased from Roche-Boehringer (Meylan, France), and the bicinchoninic acid assay kit was purchased from Interchim (Montluçon, France).

Animals. Thirty male Sprague-Dawley rats, born and bred in our animal facility, were housed in individual cages at 12 wk of age (350–400 g) and maintained in a temperature-controlled room (22°C) with a 12:12-h dark-light cycle. Animals were provided with standard rat chow A04 (Scientific Animal Food and Engineering, Epinay-sur-Orge, France) consisting of 16% fish protein, 3% fat (0.4% fish and 99.6% vegetal), 60% carbohydrate (corn starch), 3.9% cellulose, 12% water, and 5.1% vitamins and minerals, and water was available ad libitum. The metabolizable energy content was 2.9 kcal/g. Rats were randomly separated into five groups of six rats as follows: control, 25% food restricted (FR), 50% FR, 75% FR, and 100% FR (fasted) groups. Control rats were fed ad libitum before and during the FR regimen. Baseline food intake [31 ± 4 g (mean ± SD)] was then measured to determine the amount of food required for restricted animals during the FR intervention phase. Rats were food restricted, receiving 75% (25% FR group), 50% (50% FR group), or 25% (75% FR group) of the total amount of food eaten by the control group. The fasted rats were totally deprived of feeding. The food restriction design was conducted over 3 full days. Rats were fed each day between 5:00 and 6:00 PM and killed on the morning of the fourth day. The present investigations were conducted in accordance with the guiding principles of the French Department of Animal and Environmental Protection for the care and use of laboratory animals and in accordance with the American Physiological Society "Guiding Principles for Research Involving Animals and Human Beings."

Rat skeletal muscle mitochondria. IMF and SSM mitochondria were isolated from the quadriceps and gastrocnemius muscles as previously described (36). On the fourth day, the animals were killed by decapitation. For each rat, quadriceps and gastrocnemius muscle of each hindlimb was quickly removed, weighed, and immediately placed in an ice-cold isolation medium consisting of 100 mM sucrose, 50 mM KCl, 50 mM Tris, and 5 mM EGTA, pH 7.4. Muscles were minced with scissors and homogenized using a Potter-Elvehjem homogenizer (5 passages). The homogenate was centrifuged at 600 g for 10 min. The supernatant containing the SSM was centrifuged at 1,000 g for 10 min. The pellet containing most of the IMF was resuspended in 40 ml of isolation medium and treated with nagarse (1 mg/g muscle wet weight) for 5 min in an ice bath. The mixture was diluted 1:2, homogenized, and then centrifuged at 1,000 g for 10 min. The resulting supernatants were filtered through cheesecloth and centrifuged at 10,000 g for 10 min to obtain the IMF and SSM pellets. The pellets were washed by resuspension in the isolation medium and centrifuged at 10,000 g for 10 min. Finally, IMF and SSM pellets were stored in ice-cold isolation medium. Protein concentration was determined using the bicinchoninic acid assay kit with bovine serum albumin used as a standard.

Mitochondrial respiration. Oxygen was measured using a Clark oxygen electrode (Oxytherm respirometer; Hansatech), in a 1.5-ml-volume glass cell, thermostatically controlled at 30°C, with constant stirring. Mitochondria (0.5 mg protein/ml) were incubated in a respiratory reaction medium consisting of 120 mM KCl, 5 mM KH2PO4, 1 mM EGTA, 2 mM MgCl2, and 3 mM HEPES, pH 7.4, supplemented with 0.3% (wt/vol) bovine serum albumin and saturated with room air. Substrate concentrations were 5 mM pyruvate + 2.5 mM malate and 40 µM palmitoyl-L-carnitine + 2.5 mM malate. The active state of respiration (state 3) was initiated by the addition of 500 µM ADP. The basal nonphosphorylating respiration rate (state 4 oligomycin) was obtained by adding 3 µg/ml oligomycin (6 µg/mg mitochondrial protein). The uncoupled state of respiration was initiated by the addition of 2 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP).

Mitochondrial ATP synthesis. ADP-stimulated mitochondria ATP production rate was measured by exploiting a coupled enzyme system linked to NADPH production as previously described by Cairns et al. (10). The coupled enzyme detection system (1 mM glucose, 0.5 mM NADP, 1 IU/ml glucose-6-phosphate dehydrogenase, and 0.9 IU/ml hexokinase) was then combined with 5 mM pyruvate + 2.5 mM malate, 40 µM palmitoyl-L-carnitine + 2.5 mM malate, and respiratory reaction medium, consisting of 120 mM KCl, 5 mM KH2PO4, 1 mM EGTA, 2 mM MgCl2, 3 mM HEPES, pH 7.4, and 0.3% (wt/vol) bovine serum albumin. After the addition of 0.2 mg/ml isolated mitochondria, the rate of NADPH formation in response to 0.3 mM ADP was measured at 37°C at 340 nm with a spectrophotometer (Beckman DU-640B). Parallel measurements were made with 3 µg/ml oligomycin. The rate of ATP synthesis specifically from oxidative phosphorylation was then determined by subtracting the oligomycin-insensitive rate from the total rate. P/O represents the ratio of ATP synthesis after the addition of ADP to the oxygen consumption rate under the same conditions.

Mitochondrial enzyme activities. Activities of citrate synthase and the electron transport chain complexes (complexes I–IV) were measured spectrophotometrically (Beckman DU-640B) at 37°C in the muscle mitochondria suspensions through modified procedures of Malgat et al. (27) in agreement with the Mitochondrial Diseases group of the Association Française de Myopathie.

The activity of citrate synthase was measured in reaction medium consisting of 100 mM Tris·HCl, 40 µg/ml 5,5'-dithio-bis(2-nitrobenzoic acid), 1 mM oxaloacetate, 0.3 mM acetyl-CoA, and 4% Triton X-100, pH 8.1. After 3 min of incubation, the reaction was initiated by adding the biological material (10 µg of proteins for IMF and SSM suspensions), and the change in optical density at 412 nm was recorded over 1.5 min.

Complex I activity was measured in reaction medium containing 50 mM phosphate buffer, 3.75 mg/ml fatty acid-free bovine serum albumin, and 0.1 mM decylubiquinone with or without 0.01 mM rotenone and biological material (20 µg of proteins for IMF and SSM suspensions). After 3 min of incubation at 37°C, the reaction was initiated by adding 0.1 mM NADH. The activity was measured at 340 nm by monitoring the oxidation of NADH.

The activity of succinate dehydrogenase (complex II) was measured after the reduction of 2,6-dichlorophenolindophenol (DCPIP) in the presence of phenazine methosulfate (PMS) at 600 nm. Isolated mitochondria (3 µg of proteins for IMF and SSM suspensions) were preincubated in a buffer containing 50 mM KH2PO4, 16 mM succinate, 1.5 mM KCN, and 100 µM PMS, pH 7.5 for 5 min. The reaction was initiated by the addition of 103 µM DCPIP, and the optical density was recorded for 1.5 min.

The activity of ubiquinone-cytochrome c reductase (complex III) was determined by monitoring the reduction of cytochrome c at 550 nm. Biological materials (10 µg of proteins for IMF and SSM suspensions) were incubated for 30 s in a reaction medium consisting of 35 mM KH2PO4, 5 mM MgCl2, 2.5 mg/ml bovine serum albumin, 1.8 mM KCN, 125 µM oxidized cytochrome c, 12.5 µM rotenone, and 62.5 mM EDTA, pH 7.5. The reaction was initiated by adding 80 µM decylubiquinol, and the optical density was measured over 3 min. The nonenzymatic reduction of cytochrome c was measured under the same conditions after the addition of 10 µg/ml antimycin A. The specific activity of complex III was calculated by subtracting the activity of the nonenzymatic reaction from that of the total activity of complex III.

The activity of cytochrome-c oxidase (complex IV) was measured by monitoring the oxidation of reduced cytochrome c at 550 nm. A 50 µM solution of reduced cytochrome c (92–97% reduced using dithionite) in 10 mM KH2PO4, pH 7.0, was preincubated over 5 min. The reaction was initiated by adding the isolated mitochondria (2 µg of proteins for IMF and SSM suspensions), and the change in optical density was measured over 1.5 min.

Kinetic response of proton leak. Respiration rate and membrane potential were measured simultaneously by using electrodes sensitive to oxygen and to the potential-dependent probe TPMP+. IMF and SSM (0.5 mg protein/ml) were suspended in assay medium containing 120 mM KCl, 5 mM KH2PO4, 1 mM EGTA, 2 mM MgCl2, 0.3% bovine serum albumin (wt/vol), and 3 mM HEPES, pH 7.4, and supplemented with 5 µM rotenone, 1 µg/ml oligomycin, and 80 ng/ml nigericin. The TPMP electrode was calibrated by sequential 0.5 µM additions up to 2 µM TPMP+, and 4 mM succinate was added to start the reaction. Respiration and membrane potential were progressively inhibited through successive steady states induced by the addition of malonate. After each run, 2 µM FCCP was added to dissipate the membrane potential and release all TPMP back into the medium for baseline correction. Membrane potentials were calculated as previously described by Brand et al. (7), assuming a TPMP binding correction of 0.35 (µl/mg protein)–1 for skeletal muscle mitochondria (35). For the interpretation of these results, we will assume that no redox slip occurred in the mitochondrial electron transport chain under any of the experimental conditions that we examined, keeping in mind that if a slip in the proton pumps did occur, then although our results would remain valid, their underlying mechanism could be due in part to slip.

Statistical analysis. Results are expressed as means ± SE. The relation between the intensity of FR and weight loss (total body and organ weights) was compared using linear regression. For the other parameters measured, means were compared using a one-way ANOVA and the Dunnett post hoc test. A value of P ≤ 0.05 was considered significant in all cases. Analyses were performed using SPSS for Windows (version 13.0).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Body weight and tissue masses. The body weight of the rats did not differ between experimental groups at the beginning of treatment (Table 1). Whereas control rats gained weight (+4.7%), all restricted rats progressively lost weight as the intensity of FR increased. Indeed, weight changes were negatively correlated with the intensity of FR (slope = –0.19%, R2 = 0.961). At any given percentage of FR, the gastrocnemius and quadriceps muscle weights and heart and kidney masses remained unchanged (Table 1). Liver mass and the liver-to-body weight ratio decreased linearly with the intensity of FR (slope = –0.073%, R2 = 0.68 and slope = –0.014%, R2 = 0.86 respectively), with a final loss of 49% (liver mass) and 36% (liver to weight ratio) in 3-day-fasted rats. The wet weight of spleen decreased in line with the intensity of FR (slope = –0.002%, R2 = 0.19), reaching –24% in 3-day-fasted rats but remaining unaffected relative to body weight (g/100 g body wt).


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  		Table 1. Effect of 3 days of gradual food restriction on total body, muscle (gastrocnemius and quadriceps), and organ (heart, kidney, liver, spleen) weight

 
Mitochondrial enzyme activities. The activity of cytochrome-c oxidase was significantly increased in SSM at 50–100% FR compared with control rats and reached a maximum in 50% FR rats (Table 2). All the other enzyme activities measured were not affected by FR in SSM or in any of the mitochondrial enzyme activities in IMF (Table 2).


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  		Table 2. Effect of 3 days of gradual food restriction on IMF and SSM mitochondrial enzymatic activities

 
Mitochondrial oxidative and phosphorylation rates. Table 3 reports the respiratory parameters of IMF mitochondria isolated from skeletal muscles of control, FR, and fasted rats. Three days of FR (regardless of the intensity) did not affect the rates of oxygen consumption in IMF mitochondria respiring on either pyruvate + malate or palmitoyl-carnitine + malate. Compared with ad libitum feeding, 3 days of fasting increased the rate of ATP synthesis in IMF mitochondria respiring on either pyruvate + malate or palmitoyl-carnitine + malate (Table 4). In contrast, a lower intensity of FR did not modify the rate of ATP synthesis in this mitochondrial population regardless of the respiratory substrate used.


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  		Table 3. Effect of 3 days of gradual food restriction on IMF mitochondria respiration rates with pyruvate/malate or palmitoyl-carnitine/malate as substrate

 

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  		Table 4. Effect of 3 days of gradual food restriction on IMF mitochondria ATP synthesis with pyruvate/malate or palmitoyl-carnitine/malate as substrate

 
Three days of FR elicited significant changes in respiratory parameters of SSM mitochondria respiring on pyruvate + malate (Table 5). Both state 3 and uncoupled state 3 were significantly decreased compared with control values after 50% FR (–34.5 and –38.9%, respectively; P < 0.05). Furthermore, it appears that the phenomenon was present only at 50% FR. Neither state 4 nor the respiratory control ratio was affected by FR. SSM mitochondria oxygen consumption, with palmitoyl-carnitine + malate used as substrate, was not affected by 3 days of FR regardless of the intensity of the restriction applied.


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  		Table 5. Effect of 3 days of gradual food restriction on SSM mitochondria respiration rates with pyruvate/malate or palmitoyl-carnitine/malate used as substrate

 
Figure 1 shows the oxygen consumed by phosphorylative mitochondria to drive ATP synthesis or proton leakage across the mitochondrial membrane with succinate as respiratory substrate. Total rates of oxygen consumption under state 3 in the experimental groups were not significantly different compared with controls in both IMF and SSM. Furthermore, there was no significant effect of FR on the oxygen consumed to drive ATP synthesis (~83 ± 2 and 79 ± 2% of total state 3 respiration rate in IMF and SSM, respectively) or proton leakage (~17 ± 2 and 21 ± 2% of total state 3 respiration rate in IMF and SSM, respectively) in both IMF and SSM. Consequently, mitochondrial oxidative phosphorylation yield (P/O), which represents the balance between the rates of ATP synthesis and proton leak, was not affected by FR irrespective of the mitochondrial population concerned (when data were combined, P/O was ~1.24 ± 0.02 in IMF and 1.19 ± 0.03 in SSM).


Figure 1
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  		Fig. 1. Effect of 3 days of gradual food restriction on effective ratio of ATP synthesis after the addition of ADP to the oxygen consumption rate under the same conditions (P/O) and on the contributions of ATP synthesis and proton leak to mitochondrial state 3 respiration in intermyofibrillar (IMF; A) and subsarcolemmal (SSM; B) mitochondria respiring on succinate. IMF and SSM mitochondria were isolated from skeletal muscles of control, food restricted, and fasted rats. Succinate (4 mM), in the presence of rotenone (5 µM), was used as substrate (see MATERIALS AND METHODS for details). Jh, respiration driving proton leak; Jp, respiration driving ATP synthesis.

 
Proton leak kinetics. Figure 2 shows the kinetics of proton leak in skeletal muscle mitochondrial subpopulations (IMF and SSM) respiring on succinate and isolated from control, FR, and fasted rats. At any membrane potential value, IMF from 50% FR rats displayed a lower rate of proton leak than control, 25% FR, 75% FR, and fasted groups. Basal proton conductance, calculated at 162 mV, the highest common membrane potential, was significantly decreased by 46% in IMF of 50% FR rats compared with corresponding control mitochondria (Fig. 2A). In contrast, basal proton conductance did not differ among the other three experimental groups (25% FR, 75% FR, and fasted groups) and the control group. Maximum leak-dependent respirations (state 4) of IMF mitochondria respiring on succinate were not significantly different when experimental groups were compared with controls.


Figure 2
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  		Fig. 2. Effect of 3 days of gradual food restriction on kinetics of proton leak in IMF (A) and SSM mitochondria (B). The kinetics of proton leak were measured in IMF and SSM mitochondria respiring on 4 mM succinate from skeletal muscles of control, food restricted, and fasted rats. Proton leak kinetics were measured with succinate and oligomycin and titrated with malonate. The conditions used for these measurements are described in MATERIALS AND METHODS.

 
At any membrane potential value, SSM from 50% FR rats displayed a lower rate of proton leak than control, 25% FR, 75% FR, and fasted groups. The basal proton conductance calculated at 162 mV was significantly decreased by 40% in 50% FR SSM mitochondria compared with controls (Fig. 2B). Basal proton conductance did not differ among the three experimental groups (25% FR, 75% FR, and fasted groups) and the control group. Maximum leak-dependent respirations (state 4) of SSM mitochondria respiring on succinate were not significantly different when experimental groups were compared with controls.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We demonstrated that very short-term FR was effective to trigger mitochondrial adaptive mechanisms in skeletal muscle. Three days of FR was sufficient to 1) significantly decrease the proton leak rate in both skeletal muscle mitochondrial populations, 2) decrease the oxidative phosphorylation activity in SSM but not in IMF, and 3) specifically increase the oxidative capacity of SSM as measured by the activity of cytochrome-c oxidase. Interestingly, we also observed that the degree of FR was a determining factor, because different adaptations were observed at different FR intensities, with most alterations occurring at 50% FR. Moreover, the two mitochondrial subpopulations were involved differently in this adaptive mechanism.

Our results show that 0% FR over 3 days elicited a 4.7% total weight gain in control animals compared with their initial weight, whereas 25–100% FR caused a proportional weight loss in experimental animals, reaching 15% in the fasted group (Table 1). Organs contributing to a total body weight decrease are known to be sequentially affected by FR and CR, with the liver being affected first, followed by the kidney and the heart. As shown in the present data, after 3 days of FR, liver and spleen weight had already decreased, whereas heart and kidney weights remained unaffected (14) (Table 1). In contrast, gastrocnemius and quadriceps muscle weights were unchanged whatever the FR degree applied, as shown in previous short- and long-term studies (2, 13). Rat skeletal muscle adaptive mechanisms induced by 3 days of FR appear to occur without any change in the mass of major hindlimb muscles and may vary later as described by Bevilacqua et al. (3), and in this way may not be due to a muscular remodeling.

The main objective of the present study was to determine the effect of short-term FR on mitochondrial proton leak. We focused on mitochondrial proton leak, since it is an important contributor to resting cellular energy expenditure (34). Because a muscle contains two types of mitochondria, which exhibit different biochemical properties and physiological functions, the study was performed on both SSM and IFM mitochondria.

The findings presented demonstrate that proton leak rate and basal proton conductance of 50% FR rats in both mitochondrial subpopulations were significantly decreased compared with all other groups (Fig. 2). Our results from skeletal muscle mitochondria provide evidence for the rapidity in which FR operates and, furthermore, they extend previous reports that show decreased skeletal proton leak rate following long-term CR. This is organ specific, given that liver mitochondrial proton leak has remained unaltered after 3 days of FR (14) and after 1 and 6 mo of CR (30), whereas it has been decreased by 18 mo of CR (15). This may reflect the tissue specificity of mitochondrial metabolism and the differences between mitotic (liver) and postmitotic (muscle) tissues. The result is also quite different from a decrease in state 4 associated with an unexpected decrease in protonmotive force as observed by Bevilacqua et al. (3), all occurring after 2 wk of CR at the same intensity. This highlights the impact of the duration of the CR and the need to better analyze the consequences of short-term CR.

The precise mechanism underlying the decrease in proton leak is uncertain; however, it likely involves a reduced permeability of the mitochondrial inner membrane to protons. Modifications of the lipids composition of the mitochondrial inner membrane may provide one explanation. Effectively, energy restriction was shown to decrease the concentration of long-chain polyunsaturated fatty acids and to increase the concentration of linoleic acid (22, 23), both modifications that lead to reduced membrane permeability. Another possibility may be through the reduction in uncoupling proteins. However, several reports have demonstrated that long- or short-term CR induces an increase in uncoupling protein-3 (UCP-3) level without any corresponding increase in proton leak (5, 9). Recently, Brand et al. (8) presented evidence to show that one-half to two-thirds of basal proton conductance in mitochondria depends on adenine nucleotide translocase (ANT) content. However, no information is available concerning FR and ANT.

Whatever the exact mechanism involved, according to Ramsey's hypothesis (31), a decrease in the rate of mitochondrial proton leak would lead to a reduced resting oxygen consumption, and hence a lower rate of reactive oxygen species (ROS) generation, by limiting the number of interactions between mitochondria and oxygen molecules. This mechanism could explain why proton leak is positively correlated to ROS synthesis (3, 4).

Mitochondrial function is usually assessed by state 3 and 4 oxygen respiration rates. Most studies report that a mixed mitochondrial population exhibits a decrease in state 3 or state 4 during short- or long-term CR (Table 6). In our study, mitochondrial oxidative capacities of both SSM and IFM mitochondria were investigated in the presence of substrates that were either nonlipidic (pyruvate, succinate) or lipidic (palmitoyl-carnitine). In both subpopulations of mitochondria, respiratory state 4 was found unchanged irrespective of the substrate oxidized. Decreasing state 3 respiratory rate was only observed in SSM mitochondria, with pyruvate + malate used as substrates. This is unlikely to be due to a change in mitochondrial mass, because citrate synthase activity was found to be unchanged (Table 2). In addition, activity of complex IV also was only increased in SSM. This could contribute to the maintenance of a high degree of oxidation of electron transporters by preventing the accumulation of electrons along the respiratory chain during transfer. Associated with the reduction in proton leak, these modifications that were only observed in the mitochondria of the 50% FR group could contribute in the control of SSM oxidative stress. Given the potential importance of SSM mitochondria for signal transduction, substrate transport (32) and the rapid adaptation of subsarcolemmal mitochondria (the decrease in both state 3 and proton leak) may contribute to the benefit of short-term FR.


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  		Table 6. Summary of studies showing an effect of calorie or food restriction on skeletal muscle mitochondrial respiration

 
It was already thought that IMF and SSM mitochondria might have different adaptive capacities toward various physiological situations such as endurance training (36, 6), immobilization (21), or a high-fat diet (20). The distinct response to 3 days of FR may be explained by regional metabolic differences between the two mitochondrial subpopulations. Given that SSM mitochondria occupy a smaller volume within skeletal muscle fibers compared with the IMF population, it is unlikely that the decreasing state 3 respiration rates reported exclusively in SSM from food-restricted rats would affect the whole skeletal oxygen consumption. Nevertheless, this adaptation may be important for local energy production. Indeed, SSM mitochondria, located near the sarcolemma, provide energy for membrane-related processes, including signal transduction, ion exchange, substrate transport, and activation, whereas the IFM mitochondria are more involved in muscle contraction. Thus mitochondria subpopulations react to FR, but not exactly in the same way, with SSM mitochondria being more reactive. SSM located under the cell membrane may be more sensitive to external variations in substrate availability. It also is possible that SSM exhibit differential hormonal regulation in mitochondrial energy production (33). In line with this view, it is interesting to note that a deficiency of SSM mitochondria was reported in muscle insulin resistance (32).

In conclusion, very short-term FR has been shown to differentially affect the two mitochondrial subpopulations. In IMF mitochondria, a decrease in proton leak was reported without modification of ATP synthesis rate. As proposed by Drew et al. (13) and Sreekumar et al. (39), the capacity of skeletal muscle mitochondria to generate ATP was preserved despite the FR, probably because ATP requirements were in balance with ATP synthesis. SSM mitochondria display a more complex regulatory mechanism, which may control oxidative stress in this population. Importantly, mitochondrial metabolism adjustments were principally induced by 50% FR and were shown not to occur in a linear way between fed and fasted states. This suggests the possibility of a specific response at this FR level. The precise regulation producing such effects remains largely unknown, but it does imply a role of insulin signaling. Indeed, insulin and insulin-like signaling have been proposed to play a role in the aging process, and they are decreased by CR.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Ritz, 4 rue Larrey, Medecine B, CHU, F-49033 Angers Cedex 01, France (e-mail: paritz{at}chu-angers.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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