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* INRA, UMR1019, UNH, Clermont-Ferrand, France;
Université Clemont 1, UFR Médicine,
UMR1019, Clermont-Ferrand, France;
CRNH Auvergne, Clemont-Ferrand, France; and
|| CHU Clermont-Ferrand, Hôpital Gabriel Montpied, Clemont-Ferrand, France
1Correspondence: Unité de Nutrition Humaine, UMR Université d’Auvergne/INRA, Centre de Recherche en Nutrition Humaine, CHU de Clermont-Ferrand, Clermont-Ferrand, France. E-mail: boirie{at}sancy.clermont.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: age • skeletal muscle • mitochondria • caloric restriction • protein intake
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The prevalence of sarcopenia-related physical disability and metabolic disorders is a significant healthcare problem.
Although a number of mechanisms have been proposed as the underlying causes of sarcopenia, including loss of motor units, fiber atrophy, decrease in satellite cell recruitment, and alteration of anabolic response to hormones and nutrients (2
, 3)
, mitochondrial abnormalities have been suggested as the key factors in muscle alterations (4
5
6)
. Research on the mitochondrial electron transport chain (ETC) in skeletal muscle has clearly demonstrated deficient ETC activity in muscles exhibiting the greatest loss of muscle mass with age (7)
. The accumulation of oxidative damage due to ROS generated during normal metabolism appears to play a central role in mitochondrial dysfunction. Accordingly, mitochondrial DNA-damaged lesions due to ROS overproduction have been detected in most of the ETC abnormal fibers in sarcopenic animals (8
9
10)
. Yarovaya et al. reported decreased ETC activity in the soleus muscle in both middle-aged (18–20 months) and very old (28 months) rats compared with young animals (4 months) (11)
. This ETC dysfunction was accompanied by a reduction in the amount of full-length mitochondrial DNA and by an increase in the number of mitochondrial DNA deletions in both the middle-aged and old rats (11)
. In addition to DNA, oxidative modifications to mitochondrial proteins and membrane lipids have been suggested to play a role in ETC dysfunction during aging (12)
. Therefore, the mitochondrion, which is believed to be the most important source of potentially harmful ROS (13
, 14)
, is also the dominating target of oxidative damage in aged skeletal muscle.
Caloric restriction (CR) attenuates the age-associated increase in ROS production and ROS-associated damage to proteins, lipids, and DNA in skeletal muscle (15
16
17)
. However, although ROS-induced damage to mitochondria is thought to be closely linked to mitochondrial dysfunction, it is interesting to note that caloric restriction does not prevent the age-associated decline in skeletal muscle ATP production (18
, 19)
. Normal ATP production in skeletal muscle is of key importance not only in sustaining contractile activities, but also for the high energetic demand of particular metabolic pathways such as protein turnover. Previous studies have reported a reduced mitochondrial protein synthesis rate with aging (20)
. Of note, the alterations in mitochondrial protein turnover rate (20)
as well as the decline in mitochondrial activity (11)
occur from middle age in both rats and humans. This decrease in mitochondrial protein synthesis rate could be partly responsible for the loss of mitochondrial efficiency in aged skeletal muscle. However, although previous studies have evaluated the impact of CR on transcript levels of genes associated with mitochondrial ATP production (10
, 19
, 21)
, the effect of CR on the synthesis rates of mitochondrial and contractile proteins has never been studied together with the determination of mitochondrial functions.
We hypothesized that a limited ATP production during total CR (i.e., calorie and protein restrictions) is associated with a reduction in mitochondrial protein turnover (22
, 23)
. We also hypothesized that maintaining protein intake during CR stimulates mitochondrial protein turnover and subsequently improves mitochondrial protein oxidation and function. If there is a causal relationship between age-related mitochondrial abnormalities and muscle weakness, the improvement in mitochondrial activity should be accompanied by an increase in contractile muscle function. Therefore, we hypothesized that increased mitochondrial activity with a CR diet maintaining protein intake enhances the rate of myofibrillar protein synthesis and, consequently, improves skeletal muscle strength during aging. To address these hypotheses, we measured mitochondrial function, mitochondrial protein, myosin and actin synthesis rates, and grip force in 21-month-old rats under medium-term (5 months) CR with the usual protein intakes. We chose to begin the CR diet at middle age in order to monitor age-related alterations of mitochondrial activity in skeletal muscles from the outset (11
, 20)
. Accordingly, we first measured muscle mitochondrial activity, protein synthesis rates, and grip force in 4- and 21-month-old animals. Furthermore, we studied two different skeletal muscle types (soleus, a slow-twitch type and tibialis anterior, a fast-twitch type) because, despite their close metabolic activity, they exhibit fiber type-specific effects of aging (24
, 25)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Fig. 1
). This reduction only concerned protein and carbohydrate contents, with no modification in fat content, as in the vast majority of CR studies (26)
. Rats in the energy-restricted group (ER group) received 60% of the food intake of the control animals, but daily intake of proteins was maintained at the same level as for the control rats. To reach a protein intake in the ER group equal to the ad libitum group while maintaining caloric restriction, we replaced a part of carbohydrates by proteins (see Table 1
, Fig. 1
). Therefore, the ER group corresponded to the AL group with a 40% reduction in carbohydrates. The rats were randomly assigned to one of the three diet regimens (n=10 animals/group). Fat, vitamin, and mineral intakes were equivalent between CR and control rats. All animals had free access to water and were housed individually in plastic cages at 22 ± 1°C with lights on from 08:00 a.m. to 8.00 p.m.
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Two additional groups of 4- and 21-month-old male Wistar rats were purchased in order to study the effects of aging on mitochondrial function (n=7 for each group, see Table 2
). The study protocol was approved by the French Animal Care and Use Committee before the study began. All animals were cared for and handled in accordance with the National Institute of Health guidelines for the use of animals in experimental studies.
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Grip force
Two weeks before the end of the study, grip force was measured using a grip force meter (Bioseb, Chaville, France). This apparatus is widely used to measure the muscular performance of rodents (27)
. Previous studies have shown that grip force is closely related to fore- and hind-limb muscle contractions (27)
. During testing, each rat was held by its tail and gently moved in a rostral-caudal direction in order to apply force to the mesh grid. The gauge converts grip force into a digitized signal, which is displayed on-line. We repeated two successive measurements 3 days a week. Comparisons were performed between the amplitude of the forces exerted by control and experimental animals in N or N/g of body weight. Data were not standardized for nonmuscle weight changes induced by the different diets. The intersubject coefficient of variation for this method is 12%.
Tissue collection and preparation
At the end of the study period, the soleus (type I slow-twitch oxidative muscle) and tibialis anterior (TA, type II fast-twitch glycolytic muscle) muscles were rapidly removed and weighed. One portion of each muscle was kept on ice for mitochondrial studies. The rest of the muscles were immediately frozen in liquid nitrogen and stored at –80°C until analysis.
Preparation of skinned muscle fibers
Skinned muscle fibers were prepared according to the method described by Saks et al. (28)
. Muscle fiber bundles (3–4 mm long, 
1 mm in diameter) were separated under a magnifying glass using needles. The fibers were then permeabilized using saponin (60 µg/ml) and incubated for 20 min under gentle stirring. The permeabilized skinned fibers were then washed for 10 min three times in order to completely remove saponin and all metabolites, especially traces of ADP. The fiber bundles were kept on ice for immediate determination of respiratory activity.
Mitochondrial respiration rate
The oxygen consumption rate of skinned fibers was measured polarographically at 25°C using a Clark-type oxygen electrode (Oxygraph, Hansatech, Norfolk, UK). Skinned fibers were incubated in the micro-oxygen chamber with freshly prepared EDTA (1 mM) in order to inhibit ATPase activities. Substrates (5 mM glutamate, 2 mM malate, and 5 mM succinate) were added to initiate respiration. The addition of these respiratory substrates allowed measurement of state 2 respiration, and was followed by the addition of 1 mM ADP to obtain coupled state 3 respiration. The respiratory control ratio (RCR) was calculated as the state 3-to-state 2 ratio. All measures were performed in duplicate. Data are expressed as natom·min–1·mg–1 of muscle fibers.
Mitochondrial ATP production rate
ATP production was determined as described previously (29)
using a firefly luciferase-based ATP Bioluminescence Assay (HSII Kit, Boehringer Mannheim). In brief, ATP synthesis was initiated by addition of 1 mM ADP in the micro-oxygen chamber, then left to continue for 1 min. At 15 s intervals after ADP addition, 10 µl were withdrawn, quenched in 100 µl dimethyl sulfoxide, and diluted in 5 µl ice-cold distilled water. ATP was quantified using a luminometer. A solution with known concentrations of ATP was used to establish a standard curve. ATP production rate was used to calculate the respective molar quantities of ATP generated per mole of oxygen consumed (P:O ratio). All measures were performed in duplicate. Results are expressed as nmolATP·min–1·mg–1 of muscle fibers.
Mitochondrial enzyme activities
A piece of TA and soleus was used to isolate mitochondrial proteins as described previously (30)
. Muscle samples were homogenized in a 5% ice-cold buffer containing 0.25 M sucrose, 2 mM EDTA, and 10 mM Tris-HCl (pH 7.4) using a Potter-Elvehjem homogenizer. The homogenate was centrifuged at low speed (600 g). The supernatant was recovered and recentrifuged at 7000 g for 10 min. The mitochondria-containing pellet was washed twice using ice-cold buffer containing 100 mM KCl, 5 mM EGTA, 5 mM MgSO4, 50 mM Tris-HCl (pH 7.4), and 1 mM ATP and centrifuged at 7000 g. The final mitochondrial pellet was suspended in the sucrose/EDTA/Tris-HCl buffer for analysis.
Citrate synthase (CS) and the activities of complexes I to IV were spectrophotometrically assayed in the mitochondrial suspension as described previously (19)
. Only CS activity was measured in the soleus due to the limited size of this muscle. We chose this enzyme for its importance in flux-generating reactions in the Krebs cycle and because it displays modifications during aging and caloric restriction (19)
. All measures were performed in triplicate. Enzyme activities were expressed in mmol·min–1·mg–1 mitochondrial proteins or as activity ratios.
Mitochondrial superoxide anion production
The chemiluminescence triggered by superoxide radical production in the presence of lucigenin (31)
was measured in the TA muscle following mitochondrial isolation. Mitochondria were placed in microwell plates containing 25 µmol/L lucigenin in a final volume of 200 µl buffer (saccharose 250 mM, trisbase 10 mM, EGTA 0.1 mM, pH 7.4). Experiments were performed using 0.1 mg of proteins. All manipulations were conducted in the dark at 37°C. Microwells containing all components were counted 320 times for 80 min using a luminometer. Results are expressed as the lucigenin luminescence AUC and are carried over to CS activity to correct for the mitochondrial density in the vial.
Protein carbonyl contents
The reactive carbonyl contents in mitochondrial proteins and myosin were measured by the widely applied 2,4-dinitrophenylhydrazine (DNPH) procedure (32)
. A 300 mg piece of TA was used to isolate mitochondrial proteins and myosin, as we have previously described (30
, 33)
. Proteins (1–1.5 mg/ml) were separated into two 150 µl portions (i.e., a test sample and a blank sample). Five hundred milliliters of 12.5 mM DNPH in 2 M HCl was added to the test sample fraction while 500 µl of 2 M HCl alone was added to the blank sample fraction. Both fractions were then incubated in the dark and at room temperature for 15 min without antioxidants. The samples were precipitated with 500 µl of 30% trichloroacetic (TCA) and subsequently treated via a washing procedure consisting of washing with 10% TCA, followed by washing in ethanol:ethyl acetate (1:1, v/v, four times). The final pellets were dissolved in 1 µl of 6 M guanidine-HCl in the presence of 20 mM phosphate buffer:trifluoroacetic acid (pH 2.3) and left for 30 min at 50°C with vortexing. The reactive carbonyl content was calculated from its peak absorption at 380 nm using a molar absorption coefficient (
) of 22,000 M–1 cm–1. Reactive carbonyl content (µmol/L) was calculated using the Beer-Lambert equation: Abs380nm (test-blank) x 106/. The final carbonyl content in the proteins was expressed as µmol·g–1 protein.
Mitochondrial proteins, actin and myosin synthesis rates
Just before sacrifice, each rat (in postabsorptive state) received a subcutaneous injection of a large dose of L-[13C]-valine (99 atom%, 300 µmol/100 g, Cambridge Isotope Laboratories, Andover, MA, USA) to flood the protein synthesis precursor pool. The tracer incorporation time within the groups was different for each rat. In each group, a rat was killed at 20, 25, 30, 35, 40, 45, or 50 min after the tracer injection (n=1 per time point). Thus, the same kinetics of incorporation from 20 to 50 min was performed for each group of animals. The kinetics of tracer incorporation tracer in the mitochondrial proteins, myosin, and actin was used to calculate their synthesis rates (see equation below). A 200 mg piece of soleus or TA was used to isolate mitochondrial proteins, myosin, and actin as described previously (26
, 29)
. Measurement of L-[13C]-valine enrichment in hydrolyzed proteins was performed using a GC-C-IRMS (µGas System, Fisons Instruments, VG Isotech, Middlewich, UK). Amino acids in the tissue fluid were derivatized and valine enrichments were used as precursor pool enrichment to calculate fractional synthesis rates as described previously (34
, 35)
. Another set of four rats per regimen was used to determine basal isotopic abundance in proteins.
The fractional synthesis rate (FSR) of mitochondrial proteins, actin, and myosin was calculated using the following equation: FSR = (Eix100)/(Eprecxt), where Ei is the enrichment as atom percent excess of 13C derived from decarboxylation of valine from proteins at time t (minus basal enrichment), Eprec is the mean enrichment in the precursor pool (tissue fluid [13C] valine), and t is the incorporation time in hours. Data are expressed as %/h.
Statistical analysis
All data are presented as means ± SE. ANOVA (StatView program 4.02; Abacus Concepts, Berkley, CA, USA) was used to calculate differences between treatment groups. When the ANOVA was statistically significant, a Fisher’s test for a posteriori analysis was applied to identify pairwise differences between groups. An unpaired t test was used when appropriate. Values of P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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highlights an overall marked decline in aging-related skeletal muscle oxidative capacity and mitochondrial function in both the soleus and tibialis anterior muscles. The soleus and TA muscles showed aging-related reductions in RCR of 39% and 53%, respectively (P<0.0001). Moreover, ATP production decreased 56% (P<0.0001) in the TA muscle of the 21-month-old rats compared with the young animals. This same pattern was also found to a far greater extent in the soleus muscle (–73%, P<0.01). Aging seemed to have a stronger effect on COX than CS activity, especially in the soleus muscle, where a 75% reduction in COX activity was observed (P<0.0001). Although mitochondrial protein synthesis was not affected by age in the soleus muscle, there was a strong decrease in mitochondrial protein FSR in the TA muscle (–54%; P<0.0001, Table 2
).
Effect of CR in 21-month-old rats after 5 months of experimentation
Body mass, muscle mass, and muscle mass-to-body mass ratio
At the time of experimentation, average body weight was greater in the AL group than in both CR groups (P<0.0001, Table 3
). The mean weight gain of the AL group during the experiment was 12.2 ± 5.3%, while the PER and ER groups showed mean weight losses of 13.0 ± 5.6% and 16.2 ± 5.9%, respectively. There were no significant differences in weight loss between the PER and ER groups. Although CR, as expected, led to a significant decrease in body weight (P<0.05 vs. AL), the absolute muscle weights remained unaltered with the diet except for the tibialis anterior muscle in the PER group (P<0.05 vs. AL).
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Mitochondrial oxygen consumption
The effect of energy restriction, with or without modification of protein intake, on mitochondrial respiration was different according to the muscle type (Table 4
). State 2 respiration in the soleus muscle was down-regulated by 35% (P<0.01 vs. AL) and 27% (P<0.05 vs. AL) in the PER and ER groups, respectively. An important observation is that state 3 oxygen consumption in the soleus decreased by 25% (P<0.05 vs. AL) in the PER group but was maintained in rats receiving the ER diet. RCR was enhanced in the PER and ER groups compared with the control group (P<0.05). ER was associated with a lower level (–35%) of resting oxygen consumption (i.e., state 2) in the TA (P<0.05 vs. AL). Contrary to observations in the soleus muscle, CR had no effect on state 3 oxygen consumption in the tibialis anterior muscle. Moreover, in the TA muscle, the RCR was improved only in the ER rats (P<0.05 vs. AL and PER groups).
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Rate of ATP production and P:O ratio
The PER diet was not associated with modification of mitochondrial ATP production irrespective of muscle type (Fig. 2
). However, ATP generation increased in the ER-fed group by 30% (P=0.07 vs. PER) and by 27% (P<0.05 vs. PER) in the soleus and the TA muscles, respectively. Table 4
indicates that hypoenergetic feeding did not change the P:O ratio in the soleus in the ER or PER groups. In contrast, the TA displayed improved coupling in mitochondria from the ER group (+46%; P<0.05 et al. AL).
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Enzyme activities
CS activity in the TA muscle decreased by 25% and 20% in the PER and ER group, respectively (P<0.05 vs. AL, Table 5
). The same modification pattern was observed in the soleus of the ER-fed rats (P<0.05 vs. AL).
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Given that a balanced proportion among complex activities is required for normal ETC function (36)
, ratios between complex activities were calculated (Table 5)
. In the TA, the ratios of complex I to the other complex activities (II, III, and IV) were markedly reduced in the ER-fed rats compared with the PER-fed rats (P<0.05). This finding was related to a 33% reduction of complex I activity in the ER-fed animals (P<0.05 vs. AL and PER, data not shown). There was no apparent disarray in the stochiometric relationships between the other complexes.
Superoxide radical production
No remarkable change was noticed in the PER or ER group concerning muscle mitochondrial superoxide production rate (Fig. 3
).
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Oxidative damage to mitochondrial proteins and myosin
Protein carbonyl levels have been used as a marker of protein oxidation, and there is strong evidence that carbonyl content increases with aging in a range of tissues (5)
. As expected, mitochondrial protein carbonyl content decreased strongly (33%) in both CR groups, the PER and ER rats (P<0.005 vs. AL, Fig. 4
). However, there was no modification of carbonyl concentration in myosin in either group in response to the diets.
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FSR of mitochondrial proteins, myosin, and actin
Although the diets had no effect on mitochondrial and myofibrillar (i.e., myosin and actin) protein synthesis rates in the soleus muscle (Fig. 5
), results were significantly different in the TA. In the TA, PER-fed rats exhibited the lowest mitochondrial protein FSR of all animals (P<0.05 vs. AL). However, the PER-induced reduction in muscle mitochondrial protein synthesis was attenuated by preserving the level of daily protein intake during CR (P<0.05 ER vs. PER). The same diet (i.e., ER) led to a 30% and 29% improvement in actin and myosin FSR, respectively, in the TA of aged rats (P<0.05 ER diet vs. PER diet).
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Muscle force and force-to-body ratio
Absolute muscular force (Fig. 6
) significantly increased in the ER-fed rats compared with both the PER (+7%, P<0.01) and AL (+18%, P<0.0001) regimens. Furthermore, the muscular force-to-body ratio was significantly increased in the PER group (+30%, P<0.0001 vs. AL) but increased to a still greater extent in the ER rats (+49%, P<0.0001 vs. AL and+13%, P<0.01 vs. PER).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Our hypothesis was that a CR diet with maintained protein intake counteracts mitochondrial abnormalities and improves muscle protein synthesis rate, and as a result improves muscle strength. We chose to focus on mitochondria because of their increasingly important role in explaining aging-related loss of muscle mass and strength (12)
. Accordingly, we first confirmed in 4- and 21-month-old rats that age affects type 2 muscle mass, mitochondrial function, and mitochondrial protein synthesis rate. This is in accordance with previous data showing that modifications of muscle metabolism (in particular, mitochondrial functions) occur by middle age in humans (20)
as well as in rats (11)
. In a second step, we observed that the ER diet was able to increase ATP production, improve mitochondrial coupling, reduce oxidative damage to mitochondrial proteins, and enhance mitochondrial protein synthesis rates. Furthermore, ER-fed rats exhibited a significant increase in the synthesis rates of myosin and actin, the major contractile proteins, compared with total protein-caloric restricted animals. We also demonstrated a beneficial effect of CR without protein deprivation on muscle strength in aged rats, as assessed by the grip force test; however, it is not clear that the improvements in mitochondrial function and contractile apparatus directly affected skeletal muscle strength in our study. In addition, all these indices will have to be studied in young adult as well as old and very old rats.
In accordance with recent reports (18
, 19
, 37)
, dietary restriction of both protein and calorie intakes had no effect on skeletal muscle ATP production in the present study. Lifelong CR was unable to attenuate the age-related decline in ATP production and content in the gastrocnemius muscle (i.e., a predominately fast-twitch fiber type muscle) in 26-month-old rats (18)
. We confirmed this observation and revealed that total CR had no effect on ATP production in either fast-twitch or slow-twitch fiber type. However, it should be underlined that the capacity to generate ATP was maintained in the CR rats despite reduced food intakes, thus supporting the hypothesis of a potential increase in mitochondrial efficiency. Accordingly, an improvement in the mitochondrial RCR was observed in the soleus muscle under the PER diet. However, RCR was not modified by the PER diet in the TA muscle. Recent studies (38
, 39)
have highlighted the fact that muscle mitochondrial capacity is not only dependent on mitochondrial density, but also on the existence of qualitative differences in mitochondrial functioning according to muscle type. For example, complex I activity and ADP-stimulated respiration rate were higher in mitochondria from a pure slow-twitch pig muscle than a pure fast-twitch pig muscle (38)
. Therefore, muscle fiber type-dependent differences in mitochondrial activity, and its regulation by substrates may explain the differences in RCR ratios between the soleus and TA muscles under the PER diet. These observations imply that future studies should be carried out on pure fiber types in order to better understand the potential link between the contractile and metabolic functions of each fiber type and the modifications induced by aging and nutritional treatments.
In agreement with the data on ATP production, assessment of the mitochondrial complex activities showed no significant changes in complex I, II, III, or IV with total CR (i.e., in the PER rats). The reduced energy intake may have slowed electron flow through the ETC. This hypothesis is supported by the reduced CS activity following total CR. CS is the starting point of the Krebs cycle, and it defines the supply of electrons from reduced substrates to the mitochondrial respiratory chain. Therefore, the decrease in CS activity may be the rate-limiting step in energy production. Moreover, a PER diet reduces mitochondrial protein synthesis rates in the TA muscle of aged rats, probably due to the low protein intakes (35)
. In contrast, Sreekumar et al. (19)
demonstrated that total CR enhanced the muscular transcript levels of genes involved in ETC. There may be a lack of translation of transcripts to the corresponding proteins in total food restriction conditions.
One of the most interesting findings of this study was that energy restriction with maintained protein intake (i.e., by the ER diet) increased mitochondrial ATP production in skeletal muscle. This effect of the ER diet in both the soleus and the TA muscles was unexpected, since the two muscles possess differentcontractile properties. As mentioned above, the soleus muscle is a pure type I muscle whereas the TA muscle is a mix of type IIa, IIb, and IIx fibers, although it predominately expresses type IIa fibers (40)
. Despite their different contractile properties, recent data (38
, 39)
have shown that the previous metabolic distinction between fast and slow fibers is not sufficient to describe the differences in the regulation of mitochondrial activity between fiber types. Although classified in the fast-twitch fibers (according to their contractile properties), type IIa fibers (predominately expressed in the TA muscle) seem to be closer to mitochondrial functioning observed in type I fibers (soleus) than type IIx and IIb fibers (39)
. For example, mitochondria from type I and IIa fibers displayed a lower affinity for ADP (i.e., a lower stimulation of ATP production by ADP) and higher functional coupling than type IIb or type IIx fibers. In our study, we chose to use a pure type I muscle and a predominantly type IIa muscle (i.e., two fiber types with different contractile activities but similar mitochondrial functioning). This may explain the similar data obtained for CS activity and ATP production in the soleus and TA muscles in the ER-fed group. These data also raise the question of why, despite their close metabolism, type IIa and type I skeletal muscles display different fate during the aging process (i.e., loss of type IIa fibers and maintenance of type I). This observation would mean that specific muscle loss during aging would be associated with the velocity of the muscle rather than its metabolic specificity.
Increased ATP production indicates that the ER regimen increased the efficiency of the ETC and oxidative phosphorylation in the mitochondria. In recent years, analysis of ETC enzyme ratios has been reported to be a better tool for detecting subtle ETC deficiencies that enzyme activities per se due to the enormous variation in normal ETC enzyme activities (36)
. When this parameter was taken into account in the present study, a marked reduction of complex I-to-complex II, III, and IV ratios was revealed in the ER-fed group. These changes reflected ER diet-induced modifications of the balanced proportion among complex activities, particularly at complex I level. This new stochiometric relationship may reverse deficiencies and/or imbalances in ETC functioning with age, and therefore improve skeletal muscle ATP production (36
, 41)
. Of note, we observed that complex I was invariably involved in modifications of complex activity ratios. Obviously, these modifications were linked to the significant reduction in complex I activity in ER rats. Several recent reports have underlined the decisive role of this particular complex in the aging process, as deduced from CR studies (16
, 42
43
44)
. Accordingly, Gredilla et al. (42)
recently reported that the decrease in ROS production observed in animals subject to CR takes place exclusively at complex I level in skeletal muscle. Therefore, it makes sense that a beneficial impact of the ER diet on complex I activity occurred in our study. The efficiency of ETC activity in the ER group was also confirmed by changes in state 2 and state 3 respiration rates in both the soleus and TA muscles. The present study clearly demonstrated that the ER diet is able to decrease state 2 and to maintain state 3 respiration rates, with subsequent improvements in RCR ratio. State 2 is defined as oxygen consumption by mitochondria on particular substrates in the absence of ADP. Hence, oxygen consumption is exclusively due to leakage of protons across the mitochondrial inner membrane, a situation where ROS generation is at its highest. Thus, the decrease in maximum leak-dependent oxygen consumption would lead to reduced ROS production and less oxidative damage (15
, 37
, 45
46
47
48
49)
. Accordingly, we noted a reduction in mitochondrial carbonyl content. These results support the idea that restricting energy intake without decreasing protein intake may attenuate the aging process in skeletal muscle at least in part by decreasing the rate of oxidative protein damage. A recent study (50)
showed that restricting dietary protein intake without strongly decreasing caloric intake led to lower mitochondrial ROS production and less oxidative damage to mitochondrial DNA in the liver of young rats. It was postulated that the CR-induced decrease in aging rate was at least partly due to the reduced proteins intake (50)
. However, these data obtained in the liver remain to be confirmed in skeletal muscles. Although protein restriction seems to be beneficial in young rats, it remains to be clarified whether the same effect would be observed in middle-aged or aged animals.
State 3 respiration rate is demonstrated by adding ADP to the preparation in the presence of suitable substrates. In state 3, ATP synthesis increases while mitochondrial membrane potential decreases, and the ETC consumes oxygen in order to restore membrane potential. The maintenance of state 3 by the ER diet despite a decrease in food intake highlighted a better ETC coupling between oxygen consumption and ADP phosphorylation rates in the mitochondria. The increase in the ATP produced-to-oxygen consumed ratio obviously confirmed this observation.
The improvement in ETC activity together with the decrease in mitochondrial protein oxidation could also be related to the enhanced synthesis of mitochondrial proteins in the ER-fed animals. The ER diet may have triggered a metabolic shift toward an increase in protein turnover rate and a decrease in molecular damage in the mitochondria. These observations confirmed the data identified by high-density oligonucleotide arrays on muscle mitochondrial transcription patterns in response to CR (10
, 19
, 21)
. Taken together, these observations are consistent with the hypothesis that one life span-extending component of CR with maintained protein intakes could be the acceleration of protein turnover rates and the maintained pools of healthy proteins free of oxidative damage, particularly in mitochondria.
One of the most striking outcomes of this study was the positive effect of the ER diet on the contractile proteins. The ER regimen, albeit deprived in energy, stimulated the synthesis rate of myosin and actin in the TA muscle of 21-month-old rats. The factors affecting general fiber atrophy may stem in part from an age-associated reduction in the capacity of muscles to synthesize muscle protein—in particular, functional contractile proteins (i.e., actin and myosin). Both protein synthesis and degradation are energy-requiring processes that have a major impact on cellular energy expenditure (51)
. The protective effect of the ER diet on muscle protein synthesis rate could therefore be explained by an improvement in ATP availability. As oxidative stress has also been proposed as a potential mechanism for muscle fiber atrophy (8)
, we determined the carbonyl content of a specific myofibrillar protein (i.e., myosin). Our hypothesis was that a reduction in protein turnover could contribute significantly to an aging-related accumulation of oxidative damage to myofibrillar proteins. Surprisingly, despite increased myosin synthesis rates in the ER group. In addition, we observed no change in oxidative damage to myosin. Mitochondria are the primary cellular source of endogenous ROS. Therefore, due to their location, mitochondrial proteins may be the primary target of the protection conferred by the ER diet. No antioxidant was included in the method preparation. In these conditions, ex vivo oxidation may have contributed to modify myosin carbonyl content. Ramamurthy et al. have also demonstrated (52)
that nonenzymatic glycosylation of myosin is an important posttranslational modification underlying aging-related alterations in myosin structure and motility speed. Although myosin glycation was not investigated in the present study, it is tempting to hypothesize that the ER diet decreased protein glycation and improved actin-myosin cross-bridge stability, consequently affecting muscle contraction ability.
Another important finding of this study were the marked differences between type 1 (soleus) and type 2 (TA) muscles. Despite the positive impact of the ER regimen on soleus mitochondrial activity (i.e., ATP production), contractile protein synthesis rate remained unaffected. This result means that 1) there is no direct link between the increase in ATP production and contractile protein synthesis rate, and 2) increased ATP availability has a different metabolic fate according to the muscle type. Furthermore, it has been suggested (23)
that the response of protein synthesis rate to nutritional modifications in each muscle type may be related to the intrinsic characteristics of the fiber (i.e., its intrinsic protein synthesis rate). We confirmed the observation that type I fibers display a higher protein synthesis rate than type II fibers. Therefore, the higher intrinsic protein synthesis rate in the soleus muscle may explain its lower reactivity. This concept has been proposed in different models displaying strong modifications of muscle protein synthesis, e.g., glucocorticoid treatment (53
, 54)
. Finally, protein synthesis rate was not modified by aging in the soleus muscle (see Table 1
). This observation confirms that both the number and sectional area of type II fibers are affected by aging whereas type I fibers remain unchanged (55)
. Furthermore, the lack of effect of the ER diet in the soleus muscle of the 21-month-old rats may be partly explained by the stability of intrinsic protein synthesis rates in the soleus.
As the primary function of skeletal muscle is contraction, it seems logical that an improvement in energetic production and protein turnover rate would affect muscle strength. We demonstrated that energy restriction with maintenance of protein intakes leads to a significant increase in the grip force of 21-month-old rats. It is remarkable that despite the absence of nutritional treatment-related changes in muscle mass, both grip force and grip force-to-body size ratio increased in the ER group. This observation suggests that the decreased strength with aging is not due solely to decreased muscle mass, but may also be linked to alterations in muscle quality. Accordingly, comparison of grip force between young and old rats has showed no change associated with age despite a greater body weight and a lower TA weight in the older group. However, because the method of measuring grip force is not the best representation of muscle strength, more studies are needed to estimate the effect of CR on muscle strength in aged rats. In addition, the grip force measurement is a global determination of muscle force in rats. Therefore, we cannot definitively conclude that an increase in muscle force as assessed by grip force is directly linked to the improved muscle metabolism observed in this study in the soleus and TA muscles. The grip face technique has been validated in many studies assessing muscular function during drug treatments or pathologies (56
57
58
59)
. Studies have been performed (27)
to evaluate the anatomical specificity of this measurement by using injection of molecules known to activate or inhibit muscle contraction. These studies have demonstrated that grip force is strongly related to fore- and hind-limb muscle contractions. Several authors (60
, 61)
have reported that CR was able to attenuate the age-associated decrease in in vitro contractile properties of the fast-twitch type 2 extensor digiturum longus muscle in old rats. This effect was not observed in the soleus muscle (46)
, which is in accordance with our results on soleus protein synthesis rates. Mechanically speaking, ATP is needed for skeletal muscle contraction—in particular, to sustain myosin ATPase activity. Therefore, the stimulation of ATP production by the ER diet may also have contributed to the increased muscle contraction ability in the 21-month-old rats.
In conclusion, we clearly demonstrated a synergistic effect of a combination of caloric restriction with maintained protein intake on muscle functionality. Our data indicate that deprivation of energy intake initiated in late middle age improves primary mitochondrial function, while the maintenance of protein intake leads to enhanced protein turnover in the skeletal muscle of 21-month-old rats. The two phenomena are certainly interdependent, and it is tempting to establish a direct link between improved ATP availability in muscle cells, enhanced myosin and actin synthesis rates, and increased muscle strength. Furthermore, the present study supports the hypothesis that sarcopenia is nutritionally preventable by using strategies associating energy intake restriction but favoring a sufficient protein intake in the diet.
Received for publication June 28, 2006. Accepted for publication July 17, 2006.
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