B. Drew, S. Phaneuf, A. Dirks, C. Selman, R. Gredilla, A. Lezza, G. Barja and C.
Leeuwenburgh
Am J Physiol Regulatory Integrative Comp Physiol 284:474-480, 2003. First published Oct 3, 2002;
doi:10.1152/ajpregu.00455.2002
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Am J Physiol Regul Integr Comp Physiol 284: R474–R480, 2003.
First published October 3, 2002; 10.1152/ajpregu.00455.2002.
Effects of aging and caloric restriction on mitochondrial
energy production in gastrocnemius muscle and heart
B. DREW,1 S. PHANEUF,1 A. DIRKS,1 C. SELMAN,1 R. GREDILLA,2
A. LEZZA,3 G. BARJA,2 AND C. LEEUWENBURGH1
1
University of Florida, Biochemistry of Aging Laboratory, College of Health and Human
Performance, Center for Exercise Science, Gainesville, Florida 32611; 2Complutense
University, Department of Animal Biology-II, Madrid 28040, Spain; and 3Department of
Biochemistry and Molecular Biology, University of Bari, Center of Study on Mitochondria
and Energetic Metabolism, National Council of Research, 70125 Bari, Italy
Submitted 25 July 2002; accepted in final form 30 September 2002
reactive oxygen species; 8-oxo-7,8-dihydro-2⬘-deoxyguanosine;
hydrogen peroxide; oxidative stress
MITOCHONDRIA produce ⬃90% of the required ATP necessary for cellular function during oxidative phosphor-
Address for reprint requests and other correspondence: C. Leeuwenburgh, Univ. of Florida, Biochemistry of Aging Laboratory, P.O.
Box 118206, Gainesville, FL 32611 (E-mail: [email protected]).
R474
ylation. A decline in mitochondrial oxidative function
and an increase in the incidence of mitochondrial DNA
(mtDNA) oxidative damage have been shown to occur
in various tissues with age, and there is strong support
that reactive oxygen species (ROS), such as superoxide
anion (O2⫺䡠) and H2O2, play a prominent role in these
processes (7, 12, 15, 17, 26, 43, 49, 52). Moreover,
mtDNA is more susceptible to damage by ROS than
nuclear DNA due to the lack of protective histones,
relative deficiency in repair mechanisms, and the proximity of mtDNA to the source of ROS, the inner mitochondrial membrane (15, 17, 43, 47, 49). The accumulation of mtDNA modification and mutations with age
can interfere with the synthesis of proteins and enzymatic pathways responsible for the transfer of electrons along the respiratory chain, as well as with the
production of ATP (38). These processes, particularly a
decreased energy production (43, 48, 49), have been
implicated in the reduction of cell viability as well as
an increase in cell necrosis and/or apoptosis (13, 34, 35,
39) with age, resulting in tissue dysfunction and ultimately disease pathologies (27, 49). It has been suggested that mitochondrial dysfunction, resulting from
an increase in oxidative stress, may be involved in both
sarcopenia (1, 13), i.e., the age-related decrease in
muscle mass and function, and the decrease in cardiomyocytes with age (32). In contrast, caloric restriction,
which significantly increases the maximum life span in
several mammalian species, attenuates the age-associated increase in ROS production and ROS-associated
damage to proteins, lipids, and DNA (16, 20, 24, 42, 43,
52). Caloric restriction has also been shown to decrease
proton leak in skeletal muscle and maintain mitochondrial and microsomal membrane order and fluidity
during aging in several tissues, including skeletal muscle and heart (20, 22, 52).
In the present study, we determined the effects of
aging and lifelong caloric restriction on ATP content
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Drew, B., S. Phaneuf, A. Dirks, C. Selman, R. Gredilla, A. Lezza, G. Barja, and C. Leeuwenburgh. Effects
of aging and caloric restriction on mitochondrial energy production in gastrocnemius muscle and heart. Am J Physiol
Regul Integr Comp Physiol 284: R474–R480, 2003. First
published October 3, 2002; 10.1152/ajpregu.00455.2002.—
Mitochondria are chronically exposed to reactive oxygen intermediates. As a result, various tissues, including skeletal muscle
and heart, are characterized by an age-associated increase in
reactive oxidant-induced mitochondrial DNA (mtDNA) damage. It has been postulated that these alterations may result in
a decline in the content and rate of production of ATP, which
may affect tissue function, contribute to the aging process, and
lead to several disease states. We show that with age, ATP
content and production decreased by ⬃50% in isolated rat
mitochondria from the gastrocnemius muscle; however, no decline was observed in heart mitochondria. The decline observed
in skeletal muscle may be a factor in the process of sarcopenia,
which increases in incidence with advancing age. Lifelong caloric restriction, which prolongs maximum life span in animals,
did not attenuate the age-related decline in ATP content or
rate of production in skeletal muscle and had no effect on the
heart. 8-Oxo-7,8-dihydro-2⬘-deoxyguanosine in skeletal muscle
mtDNA was unaffected by aging but decreased 30% with caloric
restriction, suggesting that the mechanisms that decrease oxidative stress in these tissues with caloric restriction are independent from ATP availability. The generation of reactive oxygen species, as indicated by H2O2 production in isolated
mitochondria, did not change significantly with age in skeletal
muscle or in the heart. Caloric restriction tended to reduce the
levels of H2O2 production in the muscle but not in the heart.
These data are the first to show that an age-associated decline
in ATP content and rate of ATP production is tissue specific, in
that it occurs in skeletal muscle but not heart, and that mitochondrial ATP production was unaltered by caloric restriction
in both tissues.
ATP PRODUCTION WITH AGE AND CALORIC RESTRICTION
and the rate of ATP production in rat skeletal muscle
and heart mitochondria and relate these effects to the
production of reactive oxidants (4). We chose skeletal
muscle and heart because they are postmitotic tissues
and exhibit tissue-specific aging effects (13, 34, 35, 39),
including differences in the accumulation of mutant
mtDNA (15). Additionally, the heart has a chronically
high aerobic requirement throughout life (3, 40),
whereas skeletal muscle is active only intermittently
throughout life. We compared these findings to those
observed after long-term caloric restriction, which is
known to attenuate the effects of oxidative stress (16,
20, 24, 42, 52) and reduce the incidence of mtDNA
deletions (2, 43). We hypothesized that an age-associated decline in mitochondrial ATP content and ATP
production would be observed in both tissues resulting
from a perturbation in mitochondrial function and that
these effects would be attenuated by long-term caloric
restriction.
Animals and experimental design. Ad libitum-fed and caloric-restricted male Fischer 344 rats were obtained from the
National Institute of Aging colony (Harlan Sprague Dawley,
Indianapolis, IN) several weeks before being killed. We used
12-mo-old ad libitum-fed (12AD, n ⫽ 14), 26-mo-old ad libitum-fed (26AD, n ⫽ 9), and 26-mo-old caloric-restricted animals (26CR, n ⫽ 12). Caloric restriction was started at 3.5 mo
of age (10% restriction), increased to 25% restriction at 3.75
mo, and maintained at 40% restriction from 4 mo throughout
the individual animal’s life (see Table 1 for additional information). Animals were anesthetized with an intraperitoneal
injection of pentobarbital sodium (5 mg/100 g body wt). Mitochondria were subsequently isolated from the right and left
ventricle of the heart and the gastrocnemius muscle as described previously (13, 17, 34). The gastrocnemius is a mix of
type II and type I fibers although it is predominately type II
and contains a mix of type II subtypes (i.e., types IIa, IIx, and
IIb). The gastocnemius was chosen because it shows significant atrophy with age, and one complete muscle yields sufficient mitochondria for our experiments. All treatment of
animals throughout this study conformed fully with the
“Guiding Principles for Research Involving Animals and Human Beings” of the American Physiological Society and in
addition received local institutional animal care and use
committee approval.
ATP content and production. Mitochondria were isolated
from gastrocnemius muscle and heart ventricles as previously described (13, 17, 34) and used immediately to determine mitochondrial ATP content and rate of ATP production.
During the isolation procedure, no protease was used; therefore the mitochondria obtained were primarily subsarcolemmal rather than interfibrillar. ATP production was determined using a luminometer (model TD-20/20, Turner
Designs, Sunnyvale, CA), employing an assay that utilizes
firefly luciferase, which fluoresces in proportion to the presence of ATP and d-luciferin. In brief, to determine ATP
content, freshly isolated mitochondria were added to a cuvette containing 1 mM pyruvate, 1 mM malate, and a luciferin-luciferase ATP monitoring reagent (ATP Determination
Kit A-22066, Molecular Probes, Eugene, OR). This was immediately followed by the addition of 2.5 mM ADP to determine ATP production. A blank cuvette containing no sample
was assayed to account for nonspecific ATP production, and
known concentrations of ATP were used to establish a standard curve. All mitochondrial samples were performed in
triplicate, and an average of these results was used in quantifying ATP content and rate of production. All mean values
were normalized to the 12AD rats.
Oxidant production. H2O2 production was measured at
37°C using a method developed by Barja and colleagues (4,
17). Fluorescence was determined using a fluorescent microplate reader (GeminiXS, Molecular Devices, Sunnyvale, CA),
and a standard curve was generated for each analysis using
glucose-glucose oxidase.
Measurement of oxidized bases. 8-Oxo-7,8-dihydro-2⬘-deoxyguanosine (8-oxodG) and deoxyguanosine (dG) concentrations were measured by HPLC with online electrochemical
and ultraviolet detection, respectively, using the method described in Lopez-Torres et al. (26). For quantification, peak
areas of dG standards and of three-level calibration of pure
8-oxodG standards (Sigma, St. Louis, MO) were analyzed
during each HPLC run. Comparison of areas of 8-oxodG
standards injected with and without simultaneous injection
of dG standards ensured that no oxidation of dG occurred
during HPLC analyses.
Statistical analysis. Unpaired Student’s t-tests were used
for comparisons between groups using a statistical package
from Prism (GraphPad, San Diego, CA). All data are expressed as means ⫾ SE. A P value of ⬍0.05 was considered
statistically significant.
RESULTS AND DISCUSSION
Table 1. Body mass and gastrocnemius and heart
muscle weight in 12AD, 26AD, and 26CR male
Fischer 344 rats
Body mass
Gastrocnemius
Heart
12AD
26AD
26CR
411 ⫾ 11.9
1.36 ⫾ 0.03
1.08 ⫾ 0.03
384 ⫾ 16.2
1.03 ⫾ 0.05†
1.12 ⫾ 0.04
266 ⫾ 6.3*
0.93 ⫾ 0.05
0.88 ⫾ 0.02‡
Values are means ⫾ SE. Groups: 12-mo-old ad libitum-fed (12AD),
26-mo-old ad libitum-fed (26AD), and 26-mo-old caloric-restricted
rats (26CR). Body mass did not change significantly with age (12AD
vs. 26AD), although caloric restriction (26CR) resulted in a significant reduction in body mass compared with 26AD (* P ⬍ 0.0001).
Furthermore, the mass of the gastrocnemius muscle declined significantly with age († P ⬍ 0.0001; 12AD vs. 26AD) but not with caloric
restriction. The heart mass was unaffected with age (12AD vs.
26AD), while caloric restriction decreased heart muscle mass significantly (‡ P ⬍ 0.001; 26AD vs. 26CR).
AJP-Regul Integr Comp Physiol • VOL
ATP content and rate of ATP production declines in
skeletal muscle, but not in heart, with age. The rate of
ATP production depends on the synthesis of ATP by
intact mitochondria as protons reenter the mitochondrial matrix through the rotary motor channels in the
ATP synthase enzyme complex (F1F0-ATPase) (46). In
the gastrocnemius muscle, ATP content (Fig. 1A) and
rate of ATP production (Fig. 1C) declined by ⬃50%
with age. Our findings correspond to those of an in vivo
study, employing NMR, which estimated that the
mean ATP production in the quadriceps muscle of old
human subjects (mean age 69 yr) was ⬃50% that of
younger subjects (mean age 39 yr) (9). The ATP results
for the heart were at variance with our initial expectations, as we found no significant decline with age in
ATP content (Fig. 1B) or rate of ATP production (Fig.
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METHODS
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ATP PRODUCTION WITH AGE AND CALORIC RESTRICTION
1D). Aged cardiac muscle exhibits smaller increases in
mtDNA deletions compared with skeletal muscle (15,
40); therefore, it is feasible that ROS-induced damage
is relatively lower in heart compared with skeletal
muscle and may explain why there was no significant
decline in ATP content or production with age.
Our data are in agreement with current literature on
mtDNA deletions, as it appears that mtDNA deletions
may interfere with respiratory chain ATP production
(8, 36) and are tissue specific (3, 15, 40). Barazzoni et
al. (3) found that a reduction in mtDNA copy number,
transcription of mitochondrially encoded cytochrome c
oxidase subunits, and oxidative capacity were significantly higher in aging skeletal muscle compared with
aging heart. They reported that both the mtDNA copy
number and mtDNA transcript levels of cytochrome c
oxidase I and III were significantly reduced during
aging by 23–25% and 17–22%, respectively, in the
gastrocnemius muscle of male Fischer 344 rats but
that no differences in these two parameters were observed in the heart (3) in parallel with the reduction in
oxidative capacity for the gastrocnemius muscle with
age relative to the heart. This disparity was attributed
to the differences in adaptive mechanisms that may
have evolved in response to the chronically high aerobic requirement of the heart to prevent and/or repair
oxidative damage (3, 40). Additionally, Liu et al. (25)
examined the relationship between the accumulation
of mtDNA4977, the most common mtDNA deletion accounting for 30–50% of all deletions (49), in skeletal
muscle and heart from humans ranging in age from 1 h
to 90 yr old. This study (25) found a strong positive
correlation between age and mtDNA4977 accumulation.
Moreover, the level of mtDNA4977 deletions was significantly higher in skeletal muscle (0.015–0.3% of
mtDNA) compared with heart (0.0003–0.004%) (25).
AJP-Regul Integr Comp Physiol • VOL
The mtDNA4977 deletion contains a portion of the mitochondrial genome that encodes for 7 of the 13 proteins that become subunits in the respiratory chain
complexes, including the two subunits for the ATP
synthase enzyme (8) responsible for ATP production
(49). Therefore, the relatively low accumulation of
mtDNA deletions in the heart, compared with the
skeletal muscle, may help explain the absence of an
age-associated decline in heart ATP content or production compared with the gastrocnemius muscle. In addition, regional differences in the subcellular location
of mitochondria, in both skeletal muscle and heart
tissue, exist (14, 18, 29, 33, 34). The subsarcolemmal
mitochondria population is located below the plasma
membrane and is metabolically distinct from the intermyofibrillar mitochondria, which are located between
the myofibers. In our study, only the subsarcolemmal
fraction was isolated, and it is not well established as
to what extent the rate of ATP production in each
subcellular mitochondrial population is affected by increasing age. However, recent evidence suggests that
there is a selective decrease in the rate of oxidative
phosphorylation in the intermyofibrillar population of
cardiac mitochondria but not in the subsarcolemmal
mitochondria (19, 28). These studies also used Fischer
344 rats as a model for studying the effects of aging on
the heart and found that intermyofibrillar mitochondria exhibited a decrease in oxidative phosphorylation
through complex III and IV in old compared with adult
controls, but no difference was observed in the subsarcolemmal population (19, 28).
Lifelong caloric restriction has no effect on ATP content or ATP production. Caloric restriction was unable
to attenuate the decline in oxidative metabolism observed in skeletal muscle and had no effect whatsoever
in the heart. Caloric restriction has been shown to
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Fig. 1. Skeletal and heart muscle ATP
content and ATP production rate in 12mo-old (12AD) and 26-mo-old (26AD)
ad libitum-fed rats and in 26-mo-old
caloric-restricted rats (26CR). A: ATP
content in gastrocnemius muscle decreased by 38% with age (12AD vs.
26AD, P ⫽ 0.02), but caloric restriction
had no effect on ATP content. B: neither aging nor caloric restriction had
any significant effect on ATP content
in the heart. C: ATP production in gastrocnemius muscle decreased significantly with age (59%, 12AD vs. 26AD,
P ⬍ 0.0001); however, caloric restriction did not enhance or attenuate this
decline. D: ATP production in the heart
was unaffected by aging and caloric
restriction. All mean values were normalized to the 12AD animals. Actual
rates of ATP production are shown in
Table 2. Values are means ⫾ SE. * P ⱕ
0.05, *** P ⱕ 0.001.
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ATP PRODUCTION WITH AGE AND CALORIC RESTRICTION
Table 2. ATP content and rate of production in gastrocnemius and cardiac muscle
Skeletal Muscle
ATP content
ATP production
Cardiac Muscle
12AD
26AD
26CR
12AD
26AD
26CR
1.21 ⫾ 0.18
7.29 ⫾ 1.01
0.78 ⫾ 0.18
3.17 ⫾ 0.99
1.10 ⫾ 0.21
2.77 ⫾ 0.53
0.23 ⫾ 0.04
18.31 ⫾ 1.46
0.26 ⫾ 0.05
17.92 ⫾ 2.02
0.22 ⫾ 0.03
18.86 ⫾ 1.84
Values are means ⫾ SE. ATP content is expressed as nmol ATP/mg protein, and ATP production is expressed as nmol ATP 䡠 mg
protein⫺1 䡠 min⫺1. These data represent the average of 2 separate batches of Fischer 344 rats analyzed at different times. The mean from each
batch was normalized to the 12 AD group, and results are shown in Fig. 1.
No effect of age on body mass was observed, but
caloric restriction reduced body weight by 31% (Table
1). Moreover, we observed a significant decline (24%) in
gastrocnemius muscle mass with age, similar to other
studies (1, 13). The caloric-restricted rats had a similar
decline in muscle mass compared with 12AD rats, and
therefore, the leaner CR animals had a greater muscle
mass relative to body weight compared with the ad
libitum-fed animals. With age there was no change in
heart weight, but caloric restriction reduced heart
weight significantly, as found by others (50). Musclecomposition changes with age, such as increases in
inner connective tissues, could have profound effects
on function, but it is unknown if changes in organ
weight could have an effect on the parameters determined in this study, such as ATP production.
Of interest are the differences between the ATP
content and rate of production between the gastrocnemius and heart muscle (Table 2). Skeletal muscle is
active only intermittently throughout an organism’s
lifetime. Hence because of the immediate force placed
on the gastrocnemius when the muscle is activated, it
is possible that it may require a higher reserve of free
ATP as opposed to the heart, which is constantly active. In contrast, the heart is capable of producing
⬃2.5–7 times more ATP than the gastrocnemius due to
the persistent high-energy demand of the active heart
(Table 2).
Oxidant production and oxidative damage. Age had
no effect on H2O2 production in mitochondria isolated
from skeletal muscle (Fig. 2A) or heart (Fig. 2B),
whereas caloric restriction tended to reduce oxidant
Fig. 2. Aging and caloric restriction influences on oxidant production in skeletal and heart muscle. To determine
the effects of long-term caloric restriction on oxidant production, we measured H2O2 production (dismutation
product derived from superoxide anion) in isolated gastrocnemius muscle (A) and heart mitochondria (B; Refs. 16,
17). A: age had no significant effect on H2O2 production in the gastrocnemius muscle. Caloric restriction tended to
reduce the levels of H2O2 (26AD vs. 26CR, P ⫽ 0.0661). B: additionally, H2O2 production in the heart was
unaffected by age and caloric restriction. Values are means ⫾ SE.
AJP-Regul Integr Comp Physiol • VOL
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decrease the production of reactive oxygen intermediates and oxidative stress with increasing age in heart
and skeletal muscle (17, 20, 42, 43). We initially hypothesized that lifelong caloric restriction would attenuate any age-associated decline in ATP concentration
and ATP production, assuming that ROS-induced damage to mitochondria leads to mitochondrial energy dysfunction. However, no differences were observed in
ATP content or rate of production in either gastrocnemius muscle or heart in 26CR animals compared with
the 26AD group due to caloric restriction (Fig. 1). Very
recently, Sreekumar et al. (45) also showed that mitochondrial ATP production and citrate synthase activity
in the gastrocnemius muscle were unaltered by longterm caloric restriction, supporting our findings in
skeletal muscle. It is perhaps surprising that caloric
restriction did not counteract the decrease in either
ATP content or ATP synthesis in skeletal muscle. Caloric restriction initiated during late middle age has
previously been shown to retard the age-associated
fiber loss and fiber type changes, decrease the number
of skeletal muscle fibers showing mitochondrial enzyme abnormalities, and lead to a decline in the accumulation of mtDNA deletions (2). Apparently, the requirement and demand for ATP were in balance with
caloric restriction. In addition, the amount of free ATP
available appears minimal, and therefore the rate of
ATP production probably better reflects the in vivo
situation. Additional studies are required to investigate variations in ATP production between the different mitochondria populations within different tissues
and fiber types.
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ATP PRODUCTION WITH AGE AND CALORIC RESTRICTION
production. There is still a significant amount of controversy as to whether oxidant production increases
with age. Recently, Barja and colleagues (5, 17) have
carefully reviewed this topic and concluded that there
is little evidence of an increase in H2O2 production in
isolated mitochondria with age. Bejma et al. (6) found
increases in mean cellular oxidative stress with age in
skeletal muscle using the 2⬘,7⬘-dichlorodihydrofluorescein method (4, 21), which indicates intracellular oxidant levels. Conversely, our method measured the
steady-state extracellular H2O2 production, which appears less prone to scavenging by matrix antioxidants.
Superoxide dismutase has been found in the inner
membrane space (31); however, it is unknown if glutathione peroxidase is located in the inner membrane
space of the mitochondria (I. Fridovich, personal communication). Consequently, it is unclear whether agerelated changes in antioxidant defenses in these two
tissues with age may explain our findings. We recently
showed that isolated rat heart mitochondria upregulate superoxide dismutase (⫹50%) and glutathione
peroxidase (⫹25%) significantly with age (34), suggesting a possible mechanism that leads to the increased
removal of H2O2 with age. Studies examining changes
in antioxidant enzyme activities in skeletal muscle also
tend to support an increase in enzymatic activities
with age (23, 30). This indicates that upregulation of
antioxidant enzymes may be due to chronic lifelong
exposure to oxidants. In any case, a lack of increase in
H2O2 production with age in vivo would be consistent
with the fact that aging is a progressive phenomenon,
and thus aging rate is roughly similar at different
adult ages (4, 5).
Previously, Gredilla et al. (17) found that 8-oxodG
levels in heart mtDNA did not increase with age, suggesting that mechanisms to remove oxidized bases are
very effective in the heart, consistent with the observation that 8-oxodG endonuclease activity in rat heart
AJP-Regul Integr Comp Physiol • VOL
Perspectives
This study indicates that age-related alterations in
energy metabolism are tissue specific and that caloric
restriction is unable to attenuate these effects in subsarcolemmal mitochondria of the heart and the gastrocnemius muscle, a predominately fast fiber type. An
overview of the changes in ATP content, rate of ATP
production, and H2O2 production in isolated mitochondria of gastrocnemius and heart muscle with aging and
caloric restriction is shown in Table 3. We suggest that
the decline in ATP content and ATP production in
skeletal muscle may result from an accumulation of
mtDNA damage with age, which interferes with respiratory chain ATP production. Consequently, this accrual of damage may lead to a fiber-specific decline in
skeletal muscle activity with age and may be a factor
involved in the increased incidence of sarcopenia in
skeletal muscle (1, 13, 50). Mitochondrial functional
measurements on different fiber types and muscle contractile property analysis need to be performed simultaneously to get a more complete picture of the effects
Table 3. Overview of changes in ATP content, rate of
ATP production, and H2O2 production in isolated
mitochondria of gastrocnemius and heart muscle
with aging and caloric restriction
Gastrocnemius muscle
ATP content
ATP production
H2O2 production
Gastrocnemius mass
Heart
ATP content
ATP production
H2O2 production
Heart mass
Aging
Caloric
Restriction
2
2
3
2
3
3
2
3
3
3
3
3
3
3
3
2
1, Increase; 2, decrease; 3, no change. 12AD, 26AD, and 26CR
male Fischer 344 rats were obtained from the National Institute of
Aging colony (Harlan Sprague Dawley, Indianapolis, IN).
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Fig. 3. 8-Oxo-7,8-dihydro-2⬘-deoxyguanosine (8-oxodG) levels in gastrocnemius muscle mitochondrial DNA (mtDNA). Aging had no effect on 8-oxodG levels in skeletal muscle mtDNA (4.41 ⫾ 0.40 vs.
4.35 ⫾ 0.43 8-oxodG/105 dG in mtDNA, P ⬎ 0.05), while caloric
restriction was able to reduce the levels by 30% (4.35 ⫾ 0.43 vs.
3.06 ⫾ 0.15 8-oxodG/105 dG in mtDNA, P ⫽ 0.006). Values are
means ⫾ SE. dG, deoxyguanosine. ** P ⱕ 0.01.
mitochondria increased with age (44). Due to the reduction in ATP production with age in skeletal muscle,
we also measured 8-oxodG levels in mtDNA isolated
from the gastrocnemius muscle and found no change
with aging (Fig. 3), consistent with the results observed for H2O2 production. Levels of 8-oxodG in skeletal muscle mtDNA (Fig. 3) decreased significantly by
30% (26AD vs. 26CR, P ⫽ 0.006), indicating that lifelong caloric restriction may lead to mechanisms that
inhibit the oxidation of DNA bases that lead to mtDNA
damage. While the decline in H2O2 production was not
statistically significant, it did show a strong trend
toward a reduction (⫺28%; P ⫽ 0.0661) in oxidant
production with caloric restriction, consistent with the
decline observed in 8-oxodG. These results support the
idea that caloric restriction may reduce the aging rate
at least in part by decreasing the rate of mitochondrial
ROS production and thus inhibiting reactive oxidantinduced mtDNA damage.
ATP PRODUCTION WITH AGE AND CALORIC RESTRICTION
We thank Drs. A. de Grey and J. O. Holloszy for input and for
critical reading of the manuscript.
This research was supported by National Institute on Aging Grant
AG-17994–01. R. Gredilla received a predoctoral fellowship from the
Culture Council of the Madrid Community.
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