Special Article

EV McCollum Award Lecture, 2004

Aging muscle15
K Sreekumaran Nair

KEY WORDS
sin, hypothalamus

Aging, muscle, proteins, mitochondria, myo-

INTRODUCTION

All species share the common destinies of birth, growth, aging,

and death. We are born with a set of genes that largely determine
our lives on the planet. The expression of genes is influenced by
environmental factors, which can alter the rate of absolute
growth, aging, and death. Environmental factors affect not only
gene transcription but also gene translation (synthesis of proteins) and the posttranslational modification of proteins. The
major determinants of alterations in body functions are proteins.
Cellular functions are dependent on the ability to synthesize

proteins with specific functions, interactions of proteins to proteins, proteins and genes, and proteins and metabolites. There are
gradual and progressive alterations in body functions from birth
to death. The changes are rapid during the growth phase, but the
changes are rather slow during a rather prolonged period of
agingfrom 30 y to death. The aging process or changes
related to secondary events or diseases cause rapid deterioration
of body functions in most species in the last phase of life. The
topic covered in this review is the aging of skeletal muscle;
hypotheses that will hopefully stimulate new research in this area
are discussed. Most of the presentation is based on results from
our laboratory.

AGING IN HUMANS

Aging affects all species and substantially influences the scope

of our activities and quality of life but what aging is exactly
remains to be fully understood. The definition of aging is complicated by the occurrence of various diseases that modify body
functions and tissue structures. The structural and functional
changes related to diseases that are common in older persons are
often hard to delineate from the aging process per se. Many
disease processes and environmental factors profoundly influence the rate of aging. Moreover, the aging process occurs at
different rates among different tissues, and the functional manifestations also vary. Aging-related changes in one organ might
affect the functions of other organs. The aging process also differs substantially in different species. Aging in humans is vastly
different from that in most other species because of the relatively
long duration of life in humans after the genetic potential for
growth is complete. All of these reasons make it difficult to study
aging, and studies done in many other species cannot always be
directly correlated to human aging.
1
From the Mayo Clinic College of Medicine, Division of Endocrinology
and Endocrine Research, Rochester, MN.
2
Presented at the American Society for Clinical Nutrition 44th Annual
Meeting, April 18, 2004, Washington, DC.
3
Presentation of the EV McCollum Award supported by Wyeth Nutrition.
4
Supported by NIH grants R01 AG09531, R01 DK41973, and M01
RR00585 (General Clinical Research Center). KSN was supported by the
David Murdock Dole Professorship and Mayo Foundation.
5
Address reprint requests to KS Nair, Mayo Clinic College of Medicine,
Division of Endocrinology and Endocrine Research, 200 First Street SW,
5-194 Joseph, Rochester, MN 55905. E-mail: nair.sree@mayo.edu.
Received July 22, 2004.
Accepted for publication January 19, 2005.

Am J Clin Nutr 2005;81:953 63. Printed in USA. 2005 American Society for Clinical Nutrition

953

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ABSTRACT
Age causes structural and functional changes in skeletal muscle in a
wide range of species, including humans. Muscle changes in humans
start in the fourth decade of life and cause frailty and disabilities.
Associated changes in body composition form the basis of many
metabolic disorders, such as insulin resistance, type 2 diabetes, hypertension, and hyperlipidemia, which result in an increased incidence of cardiovascular death. Decreases in the synthesis rates of
many muscle proteins, specifically of myosin heavy chain and mitochondrial proteins, occur with age. The underlying causes of the
reduction in mitochondrial biogenesis and ATP production seem to
be decreases in mitochondrial DNA and messenger RNA. Reduced
ATP production could be the basis of reduced muscle protein turnover, which requires energy. Both aerobic exercise and resistance
exercise enhance muscle protein synthesis and mitochondrial biogenesis. Insulin and amino acids have also been shown to enhance
muscle mitochondrial biogenesis and mitochondrial protein synthesis. However, the insulin-induced increase in muscle mitochondrial
ATP production is defective in type 2 diabetic patients with insulin
resistance. Moreover, a dissociation between increases in muscle
mitochondrial biogenesis and insulin sensitivity after exercise has
been noted in older persons. It remains to be determined whether
muscle mitochondrial dysfunction causes or results from insulin
resistance. Exercise seems to enhance the efficiency of muscle mitochondrial DNA in rodents. Reduced physical activity as a contributor of age-related mitochondrial dysfunction remains to be determined. It is proposed that a reduction in tissue mitochondrial ATP
production signals the hypothalamic centers to reduce spontaneous
physical activities. Voluntary physical activity is regulated by cognitive centers and could attenuate the progressive decline in mitochondrial functions that occurs with age.
Am J Clin Nutr 2005;
81:953 63.

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EFFECTS OF AGE ON MUSCLE

FIGURE 1. Age-related decreases in thigh muscle area (based on one-cut computed tomography scan), knee extensor strength (180/s), and aerobic capacity
[measured as maximal oxygen volume (VO2max) adjusted for fat-free mass] in 78 healthy persons who did not regularly exercise (2). The significant decline
in muscle strength persisted even after normalization for leg fat-free mass (data not shown). Adapted from references 2 and 5.

MUSCLE CHANGES IN AGING

Of interest, sarcopenia of aging is noted in a wide range of

species, from nematodes, flies, rodents, and nonhuman primates
to humans. A recent study in Caenorhaleditis elegans showed
gradual, progressive deterioration of muscle in this short-lived
nematode that resembled human sarcopenia from midlife (8).
The observed muscle deterioration occurred not only in body
wall muscle but also in myofibrils of pharyngeal muscles. The
sarcomeres of older C. elegans progressively became disorganized and contained fewer myosin thick filaments. Thus, it appears that C. elegans, like humans, undergo progressive loss of
muscle mass and function. This nematode, which has a short life
span of 20 25 d and whose genome is completely sequenced, is
extensively studied to define the aging process. A remarkable
finding of Herndon et al (8) was that these nematodes have a
preserved central nervous system, even at an advanced age, and
that muscle changes, which appear to occur at the beginning of
midlife, indicate a dissociation between neuronal and muscle

FIGURE 2. Aging-related functional changes in skeletal muscle.

Changes with age are a major contributor to obesity, insulin resistance, and
the components of metabolic syndrome leading to cardiovascular death
(CVD). Reduced glucose disposal also may occur because of reduced metabolically active muscle mass. T2DM, type 2 diabetes mellitus.

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The current review focuses on aging in muscle. For the purposes of this discussion, I assume that the aging process in humans starts from the fourth decade of life (at 30 y of age). This
concept is supported by studies performed in human skeletal
muscle (13), although it is not well established in all tissues. In
healthy persons who died in motor vehicle accidents, it was
observed that both muscle area and fiber numbers decreased from
as early as the fourth decade of life (1, 4). We recently measured
the cross-sectional area of muscle in the midthigh using computed tomography imaging in healthy human subjects ranging in
age from 18 to 88 y. We observed an age-related decrease in
muscle area starting from the fourth decade of life (2, 5) (Figure
1). In addition, a parallel decrease in muscle strength (knee extension) occurred with the decrease in muscle mass. We also
noted that muscle strength normalized for fat-free mass in the leg
also decreases with age, indicating that not only muscle mass
declines with age but the efficiency of muscle mass (quality) does
as well. Another key performance measurement that declines
with age is maximal oxygen uptake (2), as noted in several
previous studies (6, 7). It is a common belief that the muscle of
older persons also fatigues faster than does that of young persons,
although this has not been well documented by objective measurements. However, age-related changes vary substantially
among persons, depending on their activity levels and other environmental factors.
It is likely that the decrease in muscle mass and muscle
strength, in combination with reduced endurance, causes reduced physical activity (Figure 2). A reduction in muscle mass
(responsible for 30% of resting energy expenditure and protein
turnover as well as 70% body cell mass) and physical activity
levels (contributes variable components to daily energy expenditure: 10 60%) decrease total energy expenditure in older persons. This reduction in overall energy expenditure in elderly
persons results in an increased prevalence of obesity, especially
abdominal fat accumulation. These alterations in body composition cause insulin resistance, which contributes to the development of a high prevalence of type 2 diabetes, hyperlipidemia, and
hypertension in a genetically susceptible population (Figure 2).
The combined effect of these metabolic abnormalities is increased cardiovascular death and other morbidities.

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changes. It is also fascinating that locomotor activity was found

to be a predictor of life expectancy; those nematodes that showed
earlier locomotor dysfunction had lower life expectancies. They
found that mutation of age-1 (hx 546), which enhances locomotor activity in the aging population, delayed the age-related muscle nuclear changes. This finding suggests that the age-1
phosphatidylinositol-3-OH kinase might exert its effects on longevity in part by prolonging muscle integrity (8). Of note, an
age-related decline in the synthesis rate of skeletal muscle myosin
heavy chain (MHC) (Figure 3) has been reported in humans (1,
9-11) and may explain the decline in myosin content reported in C.
elegans. Because MHC is a key contractile protein, its reduction is
likely to contribute to a decline in locomotor function.
The underlying mechanism that causes a decrease in muscle
fibers, muscle mass, and muscle functionwhich are reported in
human aging has been extensively investigated. Many studies
have reported an overall decrease in the skeletal muscle protein
synthesis rate (2, 10, 12-14). Studies that did not normalize the
activity levels and diets of the participants before measuring the

fractional muscle protein synthesis rate failed to show any differences between young and older persons (15). There is substantial evidence that the muscle protein synthesis rate is responsive to exercise. In studies performed in young and old persons,
resistance exercise stimulates the synthesis rate of mixed muscle
proteins after 2 wk as well as it does after 3 mo, although the
increment attenuates from 2 wk to 3 mo (16, 17). Resistance
training for 3 mo also increased specific muscle proteins, such as
MHC proteins, in older persons (10) (Figure 3). Studies have also
shown that synthesis rates of mixed muscle proteins increase 3 4
h after resistance exercise (18). Mixed muscle protein synthesis
has been shown to increase after 4 mo of aerobic exercise (2).
Together, these results indicate that mixed muscle protein synthesis in both young and older persons responds to aerobic and
resistance exercise programs. It is, however, important to appreciate that the effects of aerobic exercise and resistance exercise
on muscle metabolism and muscle size are different. Aerobic
exercise improves many metabolic functions, including insulininduced glucose disposal and mitochondrial functions. It is likely

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FIGURE 3. Changes in the fractional synthesis rate (FSR) of myosin heavy chain (MHC) and in ribosomal messenger RNA (mRNA) MHC isoforms by
age [young (20-30 y), middle-aged (45-55 y), and old (65 y) subjects] and in response to a 3-mo resistance exercise intervention. MHC FSRs decreased
beginning in middle age and continued with advancing age (10). This decrease was related to a reduction in mRNA abundance of MHCIIa and x isoforms (9)
and a relative increase in type 1 fibers (1, 11). *Significantly different from young subjects, P 0.05. #Significantly different from middle-aged subjects, P
0.05. **Significantly different from baseline, P 0.05. AU, arbitrary units.

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EFFECTS OF AGE ON MUSCLE

DOES AGE REDUCE ACTIVITY LEVELS?

There is substantial evidence from various species that activity

levels decline with age (8, 23). Age-related declines in locomotor
activity levels have been documented in C. elegans (8, 23), flies
(24), and animals (25, 26). In humans, indirect measurements
suggest an age-related decline in activity levels (27), and it is a
common dogma that humans slow down with age. This general
impression remains to be confirmed by reliable and objective
measurements.
MITOCHONDRIAL DYSFUNCTION AS A
DETERMINANT OF PHYSICAL ACTIVITY LEVELS

There is increasing evidence in rodents (28) and in humans

(14) (Figure 4) that muscle mitochondrial dysfunction occurs
with age. Changes in muscle mitochondrial function include
decreases in mitochondrial DNA copy numbers, decreased
mRNA concentrations in genes encoding muscle mitochondrial
proteins (5, 28), reduced muscle mitochondrial oxidative enzyme activities, and reduced mitochondrial protein synthesis

FIGURE 4. Fractional synthesis rates (FSRs) of muscle mitochondrial

proteins and activity of oxidative enzymes. Data indicate mitochondrial
dysfunction with aging. **Significantly different from young subjects, P
0.05 (14).

rates (5, 14) (Figure 4 and Figure 5). There are conflicting results
on whether the actual muscle mitochondrial ATP production
decreases with age (Table 1) (29-39). Our own preliminary
results strongly indicate that there is an age-related decrease in
muscle mitochondrial ATP production (40). A plausible hypothesis is that muscle mitochondrial ATP production is a determinant of physical activity levels (Figure 6). The rationale for the
above hypothesis is that humans and other species need ATP for
muscle contractile activities. The availability of ATP may signal,
via an activity center, an alteration in activity levels. It is likely
that the effects of mitochondrial ATP concentrations are spontaneous activities under the control of the hypothalamus. Evidence from animal studies supports the fact that activity levels
are regulated by the hypothalamus, possibly in the paraventricular nucleus. Orexin A (hypocretin 1), when injected into the
hypothalamic paraventricular nucleus, increased spontaneous
activity levels (41) and food intake (42) in rodents. An intriguing
possibility is that a neuropeptide that stimulates physical activity
levels (needed for gathering food) also increases food intake. A
recent review (43) outlines various catabolic neuronal pathways
that reduce food intake and increase energy expenditure (eg,
melanocortin neurons in hypothalamic arcuate nucleus) and are
stimulated by leptin and insulin. In contrast, those anabolic pathways (eg, neurons containing neuropeptide Y) that enhance food
intake and decrease energy expenditure appear to be inhibited by

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that the muscle functional changes induced by aerobic exercise

are mediated by changes in specific proteins, especially the enzymes. Many of these proteins are likely to be of low concentration in muscle tissue and may not contribute much to the muscle
mass. In contrast, resistance exercise mainly affects muscle
strength and muscle mass, which may include many structural
proteins. It is therefore likely that the proteins involved are different in response to resistance exercise from the aerobic exercise. Measurement of the fractional synthesis rate (FSR) of
mixed muscle proteins represents an average of many proteins
(low- and high-abundant proteins with low and high FSRs) and
the changes in the FSR of mixed muscle protein synthesis rates
may not translate into changes in muscle mass. Moreover,
changes in muscle protein breakdown in response to different
types of exercise also may occur at different rates for different
muscle proteins. These issues are difficult to resolve but offer
challenging opportunities for future research. Changes in the
composition of meals or in substrate milieu have also been shown
to affect muscle protein synthesis rates and protein turnover
(19-21). The previous days meals can affect not only protein
turnover but also insulin sensitivity (22), which in turn may affect
muscle protein turnover. It is therefore critical to standardize the
diet and exercise status in young and older persons before protein
turnover studies to determine whether age per se has any effect on
muscle protein turnover. Additional studies are needed to assess
whether activity levels and diet have differential effects in young
and older persons.
The important question then is: What causes the reduced baseline muscle protein synthesis rate? It is possible that younger
persons are physically more active than are older persons, which
by itself may affect muscle protein turnover. In our studies, we
selected subjects who did not engage in any sport activities and
did not deliberately exercise regularly. On the basis of a leisuretime activity questionnaire, the young and older subjects had
similar activity levels. It remains to be determined whether
younger persons are generally more physically active than are
older persons on the basis of more objective measures and
whether low activity levels in older persons caused the reduced
muscle protein synthesis.

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FIGURE 6. Hypothesis for the regulation of physical activity. It is proposed that spontaneous activity levels are regulated by hypothalamic centers,
possibly the paraventricular nucleus. Peripheral (mostly skeletal muscle)
mitochondrial ATP send signals to the hypothalamus (afferent sympathetic
nerve or chemical signal), which, via efferent sympathetic or chemical signals, up- or down-regulate activity levels. Voluntary activity levels are
largely under cognitive control, although signals from hypothalamic centers
may provide signals.

insulin and leptin. It remains to be determined how these catabolic and anabolic regulatory centers are involved in the regulation of activity levels. Triiodothyronine administration increases
activity levels in association with metabolic rate (44). It also

TABLE 1
Effect of age on muscle mitochondrial ATP production1
Reference
In vivo NMR studies
Petersen et al, 2003 (29)
Conley et al, 2000 (30)
Chilibeck et al, 1998 (31)
Kent-Braun and Ng, 2000 (32)
Taylor et al, 1997 (33)
In vitro respiration studies
Tonkonogi et al, 2003 (34)
Trounce et al, 1989 (35)
Rasmussen et al, 2003 (36)
Chretien et al, 1998 (37)
Barrientos et al, 1996 (38)
Brierley et al, 1996 (39)

Age effect
2
2
2
7
7
2
2
7
7
7
7

1
2, lower ATP production in older persons; 7, no differences between
young and old persons. NMR, nuclear magnetic resonance.

EFFECT OF REDUCED PHYSICAL ACTIVITY LEVEL

ON MUSCLE CHANGES

As discussed earlier in this review, synthesis rates of several

muscle proteins are responsive to both aerobic and resistance
exercise program (Figures 7 and 8) (2, 9). Substantial evidence
indicates that mitochondrial biogenesis is stimulated by aerobic
exercise (45). Muscle mitochondrial oxidative enzyme concentrations, such as cytochrome-c oxidase and citrate synthase, and
mRNA concentrations of several genes encoding mitochondrial

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FIGURE 5. Progressive decrease in muscle mitochondrial messenger

RNA concentrations of cytochrome-c oxidase subunit 4 (Cox 4) and NADH
subunit 4 (ND4) in men and women from 20 to 80 y of age (5). AU, arbitrary
units.

remains to be determined whether neuronal or chemical mediators are involved in signaling hypothalamus from periphery to
stimulate the center regulating activity levels. The different signals to the periphery from the activity regulating centers also
remain to be determined. It is proposed that afferent sympathetic
nerves signal the hypothalamic center (likely the paraventricular
nucleus) about the status of muscle ATP production. The signal
for changing activity level (spontaneous and possibly voluntary)
is proposed to occur via efferent sympathetic nerves (Figure 6).
It is also possible that the signals are mediated by chemicals.
Physical activity levels are likely regulated in 2 ways. Spontaneous physical activities are likely regulated by hypothalamic
centers, and voluntary physical activities are regulated largely
under cognitive control. It is proposed that spontaneous physical
activity is regulated by hypothalamic centers in response to signals from the peripheral tissues, especially from skeletal muscle,
and that spontaneous physical activity is reduced in older persons
because of reduced muscle mitochondrial functions. The downregulation of spontaneous activity may also influence the initiation of voluntary activities. Although voluntary activities are
mostly under cognitive control, it is proposed that reduced spontaneous activity and other unknown regulatory factors interact
with muscle mitochondrial functions to reduce the motivation
of older persons to engage in voluntary physical activity. As a
result, both spontaneous and voluntary physical activities decrease with age. Although it is a common dogma that humans
slow down with age and are physically less active than are
younger persons, hard supporting evidence is still not available.

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EFFECTS OF AGE ON MUSCLE

proteins and genes regulating mitochondrial transcription [(peroxisome proliferatorsactivated receptor co-activator 1 (PGC1), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (TFAM)] are enhanced by 4 mo of aerobic
exercise (2). It was previously shown that the master regulator of
mitochondrial biogenesis is PGC-1, a protein localized in nucleus (46, 47). PGC-1, a cold-inducible co-activator of nuclear
receptors, stimulates mitochondrial biogenesis and respiration in
muscle cells and through regulation of NRF-1 and NRF-2. In
addition, PGC-1 also binds to and coactivates the transcriptional
function of NRF-1 on the promoter for TFAM. TFAM is a direct
regulator of mitochondrial DNA replication and transcription
(48). Overexpression of PGC-1 has been shown to enhance
slow twitch muscle fibers in a mouse model (49), thus favoring
the oxidative phenotype. We previously showed that aerobic
exercise enhances the mRNA expression of PGC-1 in human
skeletal muscle (2), thus favoring the oxidative phenotype.
Moreover, as discussed earlier, in human skeletal muscle mRNA
concentrations of NRF-1 and TFAM along with those of NADH
subunit 4 (NADH 4) and cytochrome-c oxidase subunit 4 mRNA
concentrations are enhanced by exercise programs (5). Longterm aerobic exercise seems to favor the phenotype of type I
fibers (50) in skeletal muscle. It is clear that there is a common
pathway for regulating the phenotype of oxidative muscle fiber
type.
Mitochondrial biogenesis and mitochondrial DNA replication
are stimulated by overexpression of a constitutively active form
of calcium/calmodulin-dependent protein kinase IV (CaMK IV)
(51). This effect of CaMK is mediated through induction of
PGC-1 expression (51). Chronic electrical stimulation of skeletal muscle causes a sustained elevation of intracellular calcium
and activates calcium-regulated enzymes such as calcineurin and

FIGURE 8. Fractional synthesis rates (FSRs) of mixed muscle protein by

sex and age and in response to 4 mo of aerobic exercise and changes in FSRs
by sex and age. *Significantly different from baseline, P 0.05 (2).

CaMK, both of which have been shown to synergistically activate slow and oxidative fiber-specific gene expression in myocytes (52). Muscle contractile activity through CaMK, AMPK,
and calcineurin (51, 5355) enhances mitochondrial biogenesis
and oxidative phenotype of skeletal muscle. It appears that exercise favors oxidative muscle fiber type by well-defined regulatory pathways; CAMK and AMP kinase are key proteins in the
cytoplasm of muscle cells that signal nuclear regulators involved
in the biogenesis of mitochondria and expression of isoforms of
MHC that determine the phenotype of muscle fibers. It remains
to be determined whether this pathway of determining muscle
phenotype is intact in aging. Future research may focus on the
effect exercise on this regulatory pathway to determine the potential areas affected by aging.
Studies performed in rodents showed that mitochondrial DNA
copy numbers are lower in skeletal muscle and liver (28) in older
rats. The magnitude of the differences in mitochondrial copy
numbers between young and older rats is related to the oxidative

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FIGURE 7. Fractional synthesis rates (FSRs) of both mixed muscle

protein (MMP) and myosin heavy chain (MHC) after 3 mo of a resistance
exercise intervention. *Significantly different from baseline, P 0.05 (9).

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FIGURE 10. Fractional synthesis rates (FSRs) of muscle mitochondrial

protein after infusion of high and low amounts of physiologic insulin and
saline while maintaining glucose and amino acid concentrations. *,Significantly different from saline: *P 0.05, P 0.005 (59).

capacity of the tissues. For example, the lowering of mitochondrial copy numbers from young to older rats is greater in soleus
(a highly oxidative tissue) than in gastrocnemius (Figure 9).
However, DNA copy numbers in heart muscle are not different
between young and older rats, despite the heart being a tissue
with a high oxidative capacity. The potential explanation for this
discrepancy is that heart muscle is in constant contractile activity
and, as discussed in the previous paragraph, contractile activity
may stimulate mitochondrial DNA replication and biogenesis
through a CaMK-mediated pathway. Of note, in rodents, mRNA
concentrations per DNA copy numbers are higher in the soleus of
older rats than of younger rats (Figure 9). This intriguing observation suggests either an increased transcriptional efficiency
(transcripts/DNA) or enhanced mRNA stability in older rats. If
there is a higher transcription rate per DNA in older persons, a
potential problem is the limit of the transcription rate, which may
set the threshold for the ATP-producing capacity of DNA in a
tissue. This is an intriguing possibility that requires further experimental evidence and may determine whether mitochondrial
DNA copy numbers are a key determinant of the ATP-producing
capacity of a tissue.
POTENTIAL MECHANISM OF AGING MUSCLE

The underlying mechanism of functional and structural

changes in aging muscle remains to be fully understood. There is
a generalized decline in muscle protein synthesis in humans with

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FIGURE 9. Effect of age on mitochondrial (mt) DNA copy number in

young and old rats and the ratio of messenger RNA (mRNA) to mtDNA
in rats. *Significantly different from young animals, P 0.05. Reductions in
mtDNA were greater in the more oxidative tissues, such as the soleus and the
liver (but not in the heart, which is continuously engaged in contractile
activity), than in gastrocnemius medial (gastroc. med.) and gastrocnemius
lateral (gastroc. lat.) tissues. mRNA expression per DNA copy number was
greater in older animals in tissues such as the soleus muscle and liver. The
values represent the percentage of the average value in young rats. Cox 1 and
Cox 3, cytochrome-c oxidase subunits 1 and 3.

aging (2, 9, 10, 12, 14, 56). However, studies have shown that
whereas the FSR of muscle mitochondrial protein and MHC (10)
are lower in older persons, the synthesis rate of sarcoplasmic
proteins is relatively higher in older persons, which suggests that
the age-related inhibition of muscle protein is not a global effect
on all proteins but is selective for certain proteins. Almost all of
the previous studies were performed during the postabsorptive
state, whereas muscle protein balance becomes positive only
after a meal (19, 20). Studies to understand the regulation of
postprandial protein dynamics are difficult to perform and are
fraught with theoretical problems when interpreting the results.
One potential approach is to label the proteins in a meal with
stable isotopes and measure their incorporation into multiple
muscle proteins. Advances in protein purification techniques and
mass spectrometry will soon enable us to perform such studies.
Studies have shown that milk proteins can be labeled with stable
isotope of amino acids by infusing the isotopes in lactating cows
(57). However, many studies using the intravenous infusion of
substrates and insulin have been conducted. Amino acids alone
(20) and insulin with glucose (19) achieve a positive protein
balance across muscle bed. The main anticatabolic effect of insulin in the postabsorptive state is due to inhibition of muscle
protein breakdown (19, 58), whereas the anticatabolic effect of
amino acids is due to stimulation of protein synthesis and inhibition of protein breakdown (20). Both insulin and amino acids
are largely responsible for protein accretion in skeletal muscle
after a meal. Acute studies indicate that the main effect of insulin
and amino acids is on the stimulation of muscle mitochondrial
protein synthesis (59, 60) (Figure 10). Insulin and amino acids
also enhance mRNA abundance in genes encoding mitochondrial proteins (Figure 11). It remains to be fully elucidated
whether the transcriptional regulation of insulin and amino acids
is selective for specific genes involved in specific functions.
Insulin and amino acids have been shown to act via the signaling
pathways (mainly by PI3 kinase and mTOR) to facilitate the
initiation of the translation of messages in transcripts, thus promoting protein synthesis. The enzyme activities of selective mitochondrial enzymes and ATP production are also enhanced by
insulin and amino acids (59) (Figure 11). It is possible that the
effect of insulin may be secondary to other events, such as substrate oxidation or changes in substrate availability. The role of
substrate oxidation, especially the role of free fatty acids and
glucose on mitochondrial function, remains to be determined. In
our studies, we replaced glucose during an insulin infusion

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FIGURE 11. Effect of an infusion of insulin, while maintaining glucose

and amino acid (AA) concentrations, on muscle mitochondrial ATP production (with the use of the substrates glutamate malate and palmitoyl-Lcarnitine malate) and on ribosomal messenger RNA concentrations of
genes encoding mitochondrial proteins [in the subunits NADH4 and
cytochrome-c oxidase 3 and 4 (Cox 3 and Cox 4)]. *Significantly different
from saline, P 0.05. AU, arbitrary units.

(achieving similar insulin concentrations) to prevent hypoglycemia and to maintain similar glucose concentrations in both diabetic and nondiabetic persons. The amount of glucose that was
required to maintain similar blood glucose concentrations was
lower in persons with type 2 diabetes. In type 2 diabetic patients,
muscle mitochondrial ATP production was lower than in nondiabetic control subjects and insulin concentrations increased (59),
although insulin concentrations were identical in both groups at
low and high levels. The mechanism for the lack of stimulation
of muscle mitochondrial ATP production in type 2 diabetic patients by insulin or amino acids remains to be determined. Reduced phosphorylation of signaling proteins and lower synthesis
of mitochondrial proteins in type 2 diabetic patients may cause
the lack of stimulation of muscle mitochondrial ATP production.
Alternatively, a reduced delivery rate of glucose (and thus a
reduced flux of glucose oxidation in muscle tissue) may affect
muscle oxidative phosphorylation by an unknown mechanism. If
insulin-induced signaling is important for muscle mitochondrial
ATP production, insulin resistance in aging and type 2 diabetes
could reduce muscle mitochondrial ATP production. Because
insulin resistance occurs with aging, what remains to be determined is whether reduced insulin resistance causes a blunted
increase in muscle mitochondrial ATP production after a meal or
after an infusion of insulin with amino acids and glucose.
The reasons for the selective decrease in synthesis rates of
certain muscle proteins in older persons also remain to be determined. The previous studies were based mainly on either the
measurement of FSR of mixed muscle proteins (2, 13, 61) or
selective fractions of muscle proteins, such as myofibrillar (56)
or mitochondrial (14, 62) proteins. The FSRs of these mixed
proteins represent the average of several proteins with a wide
range of FSRs. Many of these proteins have different or even
opposing functions. It is likely that, whereas aging inhibits the
synthesis rate of some of these proteins, other proteins may not be
affected by aging. In the case of mitochondrial proteins, the
measurement of FSR of mixed proteins has definite functional
importance because the main function of mitochondrial proteins
is oxidative phosphorylation. However, 85% of these proteins
(5 protein complexes with many subunits) are encoded by the
nucleus, whereas others are encoded by mitochondrial genes. It
is possible that if protein complexes involved at certain levels of
electron transport are not available in sufficient quantities, ATP
production may not occur. It is therefore important to study the
abundances and FSRs of different mitochondrial proteins.
The interpretation of studies that show changes in muscle
protein synthesis in response to interventions or in the basal
steady state should be done with caution for a variety of reasons.
One cannot assume that changes in the synthesis rate of a protein
mixture can be translated to changes in the concentration of that
protein mixture in the cell. First, a balance between protein synthesis and breakdown occurring in both the postabsorptive and
postprandial states determines the change in protein content in a
tissue. Second, the average synthesis rate of mixed muscle proteins measured in a tissue represents different proteins of a wide
range of concentrations and turnover rates. For example, proteins
that have a high turnover rate, such as enzymes, may make a
larger contribution to the FSR but they make up a very small
fraction of the concentration of mixed proteins. Proteins such as
myosin, although constituting a major component of mixed muscle proteins, have a slow turnover rate and thus contribute only a
small fraction to the synthesis rate of mixed muscle protein.

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SUMMARY AND CONCLUSIONS

Structural and functional changes in muscle during aging occur in a wide range of species, ranging from C. elegans to humans. The structural changes include a reduction in muscle mass
and muscle fibers, and a shift of muscle fibers toward type 1
fibers. These structural changes are associated with muscle
weakness, reduced endurance capacity, and insulin resistance.
Muscle weakness is largely related to reduced mass but the muscle strength per unit mass of muscle also declines. A reduction in
the synthesis rate of MHC, the key protein in the contractile
apparatus, is likely to contribute to the muscle weakness. Myosin
is deficient in the muscle of C. elegans, which reduces its locomotion. It remains to be determined whether the concentration of
myosin and other key proteins involved in muscle contraction are
reduced with aging in humans. In long-distance runners and in
animal studies, type 1 fibers are rich in mitochondria and are
relatively fatigue resistant. In contrast, the relative increase in
type 1 fibers does not make older muscle fatigue resistant, perhaps because of a reduction in mitochondrial content with age. A

FIGURE 12. Proposed mechanism for factors contributing to age-related

reductions in maximal oxygen uptake (VO2max), endurance, and impaired
glucose tolerance (IGT). mRNA, messenger RNA.

reduction in mitochondrial ATP production could contribute to

reduced endurance and muscle weakness. Increased mitochondrial DNA oxidative damage with aging and cumulative DNA
damage could explain an overall reduction in mitochondrial
DNA copy numbers in oxidative tissue, such as skeletal muscle
(Figure 12) (68). A reduced mitochondrial DNA copy number
may contribute to reduced mRNA abundance, which results in
reduced mitochondrial protein synthesis and enzyme activity.
The overall effect is a reduced capacity for oxidative phosphorylation. The reduced availability of ATP may contribute to an
overall reduction in the remodeling process that involves the
synthesis and breakdown of proteins, both of which are energyconsuming reactions in muscle.
Contractile muscle activity enhances muscle mitochondrial
biogenesis through CaMK, AMP-activated kinase, and PGC-1.
Physical activity in the form of resistance and aerobic exercise
stimulates muscle protein synthesis in both young and older
persons. It remains to be determined whether different exercise
programs have variable effects on different muscle proteins.
Resistance exercise programs increase muscle mass; therefore, it
is likely that the synthesis of structural proteins is enhanced by
resistance exercise. In contrast, many metabolic changes occur
with aerobic exercise with no increase in muscle mass. It is
therefore likely that aerobic exercise stimulates the synthesis of
many muscle proteins involved in metabolic processes. There is
evidence in various species, from worms to rodents, that physical
activity levels decrease with age. Although it is a common belief,
supported by some experimental evidence, that physical activity
levels decrease with age, further direct evidence is needed to
verify this in humans. It is proposed that spontaneous activity
levels in humans are regulated via the hypothalamus (possibly
the paraventricular nucleus), and peripheral tissue mitochondrial
ATP production is a determinant of hypothalamic control. In
contrast, voluntary activities are likely to be mainly regulated by
cognitive centers. Spontaneous activities decline with age in
response to declining peripheral tissue mitochondrial function
(Figure 13). This, together with a reduction in voluntary activities, further reduces mitochondrial biogenesis and functions as
well as the synthesis rates of contractile proteins. Maintaining
voluntary physical activities will partly prevent the age-related
decline in muscle mitochondrial and contractile functions. Moreover, physical activities and related changes are also likely to
delay or prevent insulin resistance. It remains to be determined
whether age-related insulin resistance is due to or the cause of
muscle mitochondrial dysfunction. It is likely that increases in

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Moreover, the concentration and synthesis rate of proteins such

as myosin are unlikely to change during a short intervention
periodat least most techniques are not sufficiently precise to
detect the change. In contrast, many proteins with a fast turnover
but in low concentrations may change during short interventions.
Because proteins in relatively high concentrations (but low turnover rates), such as myosin, contribute more to muscle protein
mass than do proteins in low concentrations (with fast turnover),
the changes in synthesis rates of mixed proteins may not necessarily equate with changes in total protein mass. Arteriovenous
balance studies are extremely useful for determining changes in
the balance of total proteins in the muscle bed. However, these
measurements are not precise because the measurements of
many factors, such as blood flow, are known to be widely variable. Moreover, because these measurements have to be normalized for lean tissue mass or area in the leg or forearm, crosssectional comparisons are fraught with many problems. Older
persons have a greater proportion of fibrous tissues in muscle
mass and less metabolically active tissue with a higher water
content than do younger persons (63). As a result, the normalization of flux values per unit mass introduces further inaccuracy
in cross-sectional comparisons.
The measurement of the synthesis rate of MHC (9, 10, 64, 65)
is an advance over previous techniques that measure the synthesis rate of mixed proteins. However, even MHC exists in at least
3 isoforms in human muscle, and the relative compositions of
these isoforms have a substantial effect on muscle phenotypes.
When the MHC isoform 1 is predominant, it favors the phenotype
of oxidative fatigue-resistant slow twitch type 1 fibers; MHCIIa
and IIx favor type IIa and type IIb fibers, respectively, which are
fast twitch and glycolytic. Currently, no techniques are available
to measure the synthesis rates of these isoforms or other key
proteins in skeletal muscle. There are many promising approaches for purifying and measuring synthesis rates and concentrations of multiple muscle proteins in humans (66, 67). Application of these novel approaches may, in combination with
mRNA measurements, DNA studies, and studies to determine
regulation of both transcription and translation, are likely to
provide a new understanding of the underlying mechanism of
aging muscle.

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EFFECTS OF AGE ON MUSCLE

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muscle mitochondrial function as a result of physical activity,

and related changes, can prevent insulin resistance and related
metabolic disorders that cause an increased incidence of cardiovascular deaths in elderly persons.

16.

I acknowledge with profound gratitude the hard work and intellectual

contributions of several investigators trained in my laboratory, specifically
Kevin Short, P Balagopal, Rocco Barazzoni, Olav Rooyackers, Craig Stump,
Yves Boirie, Sreekumar Raghavakaimal, David Proctor, Laura Greenlund,
Olle Ljungqvist, Bo Ahlman, Panos Halvatsiotis, Jonas Nygren, Niels
Moller, Aizhong Fu, Abdul Jaleel, and Sean Meek. I acknowledge the outstanding and skillful technical support of Jane Kahl, Jill Coenen-Schimke,
Dawn Morse, Kate Klaus, Paul Rys, Charles Ford, and Mai Persson and the
continuing support and valuable collaboration of my coinvestigators, especially Robert Rizza, James Levine, Michael Joyner, Michael Jensen, Cheryl
Conover, Janet Vittone, and Michael Charlton. My research career was
initiated by an outstanding mentor, John Garrow (Medical Research Council,
United Kingdom), and I thank David Halliday (Medical Research Council,
United Kingdom) for teaching me the methods for the stable-isotope studies.
I also thank my devoted nursing staff, especially Maureen Bigelowmy
study coordinatorand the metabolic research nurses. I also acknowledge
the valuable secretarial support of Melissa Aakre.
There was no conflict of interest.

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