Alterations of protein turnover underlying disuse atrophy in human

J Appl Physiol 107: 645–654, 2009.
First published July 16, 2009; doi:10.1152/japplphysiol.00452.2009.
Alterations of protein turnover underlying disuse atrophy in human skeletal
S. M. Phillips,1 E. I. Glover,1 and M. J. Rennie2
Exercise Metabolism Research Group, Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada;
and 2School of Graduate Entry Medicine and Health, University of Nottingham, Derby, United Kingdom
Submitted 28 April 2009; accepted in final form 10 July 2009
protein synthesis; proteolysis; proteasome; immobilization
are in a constant state of turnover, with
simultaneous synthesis and degradation. In adult human beings, in whom growth has ceased, the processes of synthesis
and breakdown are equal and opposite on a daily basis. Alterations in protein balance occur throughout the day depending
on environmental influences, such as the amount and composition of food and physical activity, which mainly affect
protein synthesis. Protein synthesis is, by far, the most dynamic
variable in the protein balance equation and changes severalfold throughout the day (Fig. 1A). Imbalances between protein
synthesis and degradation result in increases or reductions in
protein content, by a wide variety of possible changes in each
arm of protein turnover, separately or together. Whenever
protein breakdown chronically exceeds synthesis, the muscle
protein mass declines, but it has not been a trivial matter to
discover what actually happens to the two processes. It is now
generally agreed that in humans, protein synthesis is downregulated as a result of uncomplicated (i.e., nonpathological)
Address for reprint requests and other correspondence: S. M. Phillips,
McMaster Univ., Dept. of Kinesiology, Exercise Metabolism Research Group,
1290 Main St. West, Hamilton, ON, Canada L8S 4K1 (e-mail: phillis
disuse (38, 46 – 48, 90), but the relative importance of this
compared with protein breakdown and the actual changes in
direction and extent of breakdown in human muscle remain
somewhat unclear. Nevertheless, it seems that in an increasing
number of studies in situations in which there is a long-term
loss of muscle mass, alterations in protein synthesis are facilitative and responsible for the majority of the observed changes
in protein mass. Contrastingly, protein breakdown is often
adaptive to the fall in protein synthesis and actually falls or
remains unchanged but does not rise; this is a major conclusion
of our review.
Naturally the problem of clinical muscle wasting is one that
affects human beings rather than animals, but paradoxically,
most of the literature dealing with disuse atrophy concerns
animal models, which we suggest is inappropriate for reasons
that we lay out in detail. In particular, we highlight suggestions
for more definitive work on the mechanisms of human muscle
atrophy using robust methods in vivo. In this review, we focus
on results concerning human muscle in which, where possible,
the dynamic processes of muscle protein synthesis (MPS) and
muscle protein breakdown (MPB) have actually been measured
in vivo. These approaches are in contrast to inferences from
static measures of mRNA or protein expression or from mea-
8750-7587/09 $8.00 Copyright © 2009 the American Physiological Society
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Phillips SM, Glover EI, Rennie MJ. Alterations of protein turnover underlying
disuse atrophy in human skeletal muscle. J Appl Physiol 107: 645–654, 2009. First
published July 16, 2009; doi:10.1152/japplphysiol.00452.2009.—Unloading-induced atrophy is a relatively uncomplicated form of muscle loss, dependent almost
solely on the loss of mechanical input, whereas in disease states associated with
inflammation (cancer cachexia, AIDS, burns, sepsis, and uremia), there is a
procatabolic hormonal and cytokine environment. It is therefore predictable that
muscle loss mainly due to disuse alone would be governed by mechanisms
somewhat differently from those in inflammatory states. We suggest that in vivo
measurements made in human subjects using arterial-venous balance, tracer dilution, and tracer incorporation are dynamic and thus robust by comparison with
static measurements of mRNA abundance and protein expression and/or phosphorylation in human muscle. In addition, measurements made with cultured cells or in
animal models, all of which have often been used to infer alterations of protein
turnover, appear to be different from results obtained in immobilized human muscle
in vivo. In vivo measurements of human muscle protein turnover in disuse show
that the primary variable that changes facilitating the loss of muscle mass is protein
synthesis, which is reduced in both the postabsorptive and postprandial states;
muscle proteolysis itself appears not to be elevated. The depressed postprandial
protein synthetic response (a phenomenon we term “anabolic resistance”) may even
be accompanied by a diminished suppression of proteolysis. We therefore propose
that most of the loss of muscle mass during disuse atrophy can be accounted for by
a depression in the rate of protein synthesis. Thus the normal diurnal fasted-to-fed
cycle of protein balance is disrupted and, by default, proteolysis becomes dominant
but is not enhanced.
changes in muscle mass and thus are mechanistically equivalent. Thus we view disuse as the general broad descriptor that
encompasses these ground-based models, which are characterized by a mechanical unloading only. What these models do
not have as a feature is a marked increase in cortisol secretion
or cytokines (tumor necrosis factor-␣, interleukin-6, interleukin-10) seen in sepsis, uremia, burns, rapid cancer cachexia,
and acquired immunodeficiency syndrome (AIDS), which we
contend have different effects on the pattern of change in protein
turnover from those due to disuse alone (1, 8, 68, 73, 86).
Effects of Disuse on Muscle Mass
sures of physiological processes in muscle cells in vitro. For
descriptions of the ground-based (i.e., nonspaceflight) models
of disuse, we refer the reader to previous comprehensive
reviews (3, 34). In our view, commonly used techniques to
induce disuse atrophy in human muscle, such as chronic bed
rest, casting, limb suspension, and immobilization, are mechanically comparable and induce similar outcomes in terms of
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Fig. 1. Schematic representations of the processes of muscle protein synthesis
(MPS) and muscle protein breakdown (MPB) in the normal state (A). In this
situation, protein synthesis fluctuates with feeding (consumption of protein)
and fasting across the diurnal cycle, and changes in the gains in muscle protein
mass are balanced by losses. The horizontal line in the middle of each graph
is shown for reference and to make comparisons between graphs. In contrast,
the graph in B shows how atrophy would occur in disuse if breakdown were
chronically elevated. In this scenario, the smaller fed-state gain in muscle
protein mass and a greater fasted-state loss occur due to persistently elevated
proteolysis. Instead, we propose (C) that the primary reason for loss of muscle
protein, at least in humans with uncomplicated disuse atrophy, is due not to
elevated proteolysis but to a suppression of the rise in MPS with protein
feeding. Thus the decline in protein mass occurs due to a diminished accumulation of muscle protein mass with feeding and, as opposed to the situation
shown in B, there may be an adaptive reduction in proteolysis.
Muscle unloading results in a loss of muscle mass (i.e.,
atrophy) whenever muscle is not fulfilling its supportive and
locomotory functions. The extent of the wasting is variable
according to the different experimental regimes applied; for a
review of this topic and the actual degree of disuse atrophy
induced in ground-based models, see Adams et al. (3). For the
leg muscles and particularly the quadriceps head-down bed rest
induces the greatest rate of loss of muscle cross-sectional area
(CSA) (5, 6, 12, 18, 29, 39, 90, 92, 128); casting induces
slightly less atrophy (46, 47, 56, 58, 66), whereas unilateral leg
suspension (22, 28, 129) and knee immobilization induce
atrophy with rates of muscle loss somewhat less than bed rest
and casting (33, 48, 141). Nonetheless, based on available
evidence that has accumulated to investigate why there is
atrophy in all of these models, they share a common mechanism, which is a reduction in resting protein synthesis and no
measurable change in proteolysis (12, 38, 48, 90).
Rates of muscle loss in all models of disuse are marginally
faster within the first 30 days with a mean loss in muscle CSA
of ⬃0.6%/day (28, 29, 39, 46 – 48, 126, 141). After the first 30
days, rates of loss slow and reach a plateau in both the
quadriceps femoris and the soleus (3). Muscle fiber CSA loss
is apparently greater than that of muscle CSA with mean rates
of ⬃1.0%/day (7, 56, 126, 127, 141). It is unclear why there is
a discrepancy between changes in muscle fiber area and muscle
CSA; however, one may speculate that it is simply a methodological artifact of the histochemical method for determination
of muscle fiber CSA (3). For example, Trappe et al. (127)
reported changes in muscle CSA similar to that seen in single
fibers. Nonetheless, rates of reduction in muscle fiber CSA,
although apparently linear over 15–30 days (7, 56, 126, 127,
141), also show a plateau at longer periods of immobilization
(3). A perhaps underappreciated issue with immobilization is
that a muscle in an unloaded situation also shortens (13, 28).
When these findings are considered in concert with the wellknown changes in muscle CSA, it becomes apparent the
decrements in muscle volume as a product of CSA and length
changes would be more pronounced than either alone. For
example, if muscle CSA declines 5% in 14 days (28, 141) and
muscle length also declines by 5%, then at a first approximation, muscle volume should actually decline by ⬃10%. This
would be true of muscles that are unloaded in a shortened
position more so than those where muscle is held at a resting
One conspicuous difference between the results from human
and animal studies is that human muscle shows much smaller
differences, at least in the short-term, between fiber types in
their degree of disuse atrophy (7, 56, 126, 127, 141). Rodents
Muscle Protein Turnover: Normally and in Disuse
Feeding is a robust stimulator of muscle protein synthesis. In
the normal rested state, we know that feeding at a moderate
rate doubles muscle protein synthesis, a rise mainly due to the
effects of amino acids, particularly essential amino acids. The
effect appears to be dose related, with a hyperbolic relationship
between MPS and availability of essential amino acids (14) or
protein (88), with maximal effects at ⬃20 –25 g of protein or
8 –10 g of essential amino acids. It is likely that this effect is
not dependent on insulin (112), since it can be achieved when
insulin concentration is clamped by the use of somatostatin and
analogs (54, 113). Amino acids have a small inhibitory effect
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on MPB in their own right (114), but most of the effect of
dietary amino acids probably results from the release of insulin, which is a potent inhibitor of MPB (26, 43, 54, 80). With
normal feeding, the negative muscle net balance (i.e.,
MPB⬎MPS) observed in the postabsorptive state is rapidly
reversed due for the most part to a doubling of MPS with a
somewhat smaller (possibly 20 –30%) diminution of MPB
(Fig. 1A). However, these stereotypic responses have been
shown to be perturbed when muscle is in states of disuse.
We have known for 20 years that immobilization (i.e.,
unloading) of a human limb results in a reduced basal-fasted
rate of MPS (46). Gibson et al. (46) did not make direct
measures of MPB but calculated it from the observed loss of
muscle mass and the measured decline in MPS. By this means
they proposed that MPB could not have increased substantially
with immobilization, as suggested by results from studies of
mostly static markers in hindlimb unweighting in rodents (55,
70, 76, 81, 117, 118, 132), but actually adaptively decreased.
Thus, although the rates of MPS were measured only in the
fasted state, rather than across the daily diurnal cycle (Fig. 1A),
which would include the effects of feeding, it is highly likely
that daily rates of total muscle protein synthesis, including
those in the fed state, were markedly reduced, which is in fact
what we recently observed (48). Recently, de Boer et al. (28)
observed a decline in muscle CSA of ⬃10% with 21 days of
limb suspension. This reduction in muscle CSA of ⬃0.5%/day
was accompanied by reductions in the fasted state of MPS of
⬎50%, confirming the outcome of the earlier work (see also
the illustrative calculation below) (46). In immobilized human
quadriceps muscle, there is also “resistance” to the anabolic
effects of amino acids (48), strongly suggesting that the depressive effects of immobilization are seen across the daily
diurnal span of feeding and fasting (Fig. 1C). Even if the effect
on MPS were confined to the fasting state, which is not the case
according to our recent data (48), the decrease in rested MPS
could account for almost one-half of the observed wasting (46,
48). Thus a substantial net increase in protein breakdown is not
required to account for the observed atrophy of human muscle
over an extended period of immobilization. As a central hypothesis, we propose that a large part of the loss of muscle with
unloading is due not only to a reduction in fasted-state synthesis but also to an induction of anabolic resistance, that is, a
suppression of the normal amino acid-induced (i.e., feeding
induced) increase in protein synthesis (Fig. 1C). Because we
spend ⬃40% of our day under the influence of our last meal, it
makes sense that we should expect a depression of fed-state
MPS (48). The result is a disturbance of the normal rise and fall
of protein net balance, mediated for the most part by reductions
in MPS and not by increases in MPB. The consequences of this
change are such that the fed-state gains in protein mass are less
and that fasted losses remain the same (or possibly are increased early in immobilization; see Fig. 1, B and C). The
implications lead us to suggest a model for what actually
happens (Fig. 1C). First, assume, in steady state, that diurnal
rates of human MPS and MPB are equal at ⬃0.05%/h or
⬃1.2%/day. In disuse we have shown that the fasting rate of
MPS is depressed by ⬃60% and that the fed rate of MPS is
depressed by ⬃50%. If the relative durations per day of
postabsorptive and postprandial periods are ⬃9 and ⬃15 h,
respectively, then the total diurnal average is down by 58%
(i.e., to 0.03%/h or 0.72%/day). Therefore, if breakdown re-
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tend to lose greater quantities of their total muscle mass and
also show muscle-specific differences (76, 124). In rodents,
predominantly fast fiber-dominated muscles (extensor digitorum longus and tibialis anterior) losses are ⬃3 mg/day or
⬃1.6%/day, whereas they are ⬃6.2 mg/day or ⬃2.7%/day in
slow fiber-dominated muscles (usually soleus) (15, 36, 55, 61,
70, 93, 104, 133). Of course, muscles differing with respect to
their function (i.e., an extensor vs. a flexor) will also affect the
degree of atrophy observed. For example, flexor muscles (e.g.,
soleus and gastrocnemius) atrophy far more rapidly than extensor muscles (e.g., tibialis anterior, extensor digitorum longus) (103, 132), and the same is true in humans (29). The
greater atrophy seen in type I fibers in rodent muscles may
be partly a result of their being larger in CSA than those of type
II fibers in slow muscles like the soleus. Such dichotomies are
not seen in human muscle disuse atrophy, which show far more
homogeneous rates of decline in fiber CSA for type I and II
fibers within the same muscle (58, 141). One possible explanation for the apparent lack of fiber-specific differences in
human muscle during atrophy is that muscle biopsy samples
are too small to adequately detect differences (57, 82, 83).
However, when sufficient numbers of fibers are counted to
obtain reliable estimates of fiber sizes, no difference are seen in
the degree of fiber atrophy among type I, IIa, and IIx fibers
after 14 (141) or 21 days (58) of immobilization. In fact, even
with spinal cord injury, average changes in muscle fiber size
were 27, 37, and 28% for type I, IIa, and IIx fibers, respectively, between 6 and 24 wk after injury (24). The lack of
differential fiber-specific atrophy in human muscle is also
likely a consequence of the fact that the rates of protein
turnover are much more homogeneous between fiber types than
they are between fiber types in rodents. For example, Mittendorfer et al. (87) reported that biopsies taken from the soleus,
vastus, and triceps muscles of men exhibited virtually identical
rates of MPS at rest and in response to feeding, a finding also
reported for vastus and soleus by Carroll et al. (23). These
authors observed these rates despite observing differing concentrations of phosphorylated and total eukaryotic initiation
factor 4E binding protein-1 (4EBP-1) between muscles (23).
These findings are in marked contrast to data from rats (49 –51,
67, 78, 81), fowl (74, 75), and lagomorphs (53, 65, 71), all of
which display variations as high as twofold in rates of protein
turnover between muscles composed of slow and fast fiber
types. Of course, a study that examined fiber-specific changes
in rates of muscle protein turnover in humans would be
definitive in delineating the exact changes that occur with
atrophy; this has not been done to date.
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is a sharp decline in focal adhesion kinase (FAK) phosphorylation in the early stages of immobilization. FAK has been
shown in rodent and cell models to be responsive to loading
and unloading (40, 41, 52) and to cyclic mechanical stretch
(142), and we recently reported increases in phosphorylation of
FAK in response to bouts of resistance and endurance exercise
(139). As such, the acute regulation (i.e., phosphorylation) of
FAK after intense resistance exercise is a potential link of the
loading stimulus to metabolic effectors that induce an increase
in MPS. Such a possibility is predicted by the cellular tensegrity model, in which focal adhesion complexes anchor cytoskeletal proteins to the extracellular matrix of the cell to
transmit forces and activate relevant signaling cascades, reviewed in Ref. 60. The significance of the decrease in FAK
phosphorylation remains to be elucidated completely.
To summarize, in human muscle the available evidence
suggests that immobilization results in a reduced resting-fasted
rate of MPS (28, 46 – 48) and that, at least after 14 days of
immobilization, there is a reduced rate of MPS in response to
amino acid provision that cannot be overcome by high doses of
amino acids (AA) previously shown to maximally stimulate
MPS (14). The overall result is a reduction in the basal and
fed-state accretion of muscle protein, and as a result, the mass
of protein declines.
Muscle Protein Breakdown With Disuse
Far less is known about the regulation of MPB than MPS
(especially in human muscle), and our knowledge is mainly
confined to measurement of changes in gene expression
(mRNA) and, in some instances, protein content. Most work
has focused on changes in components of the pathways thought
to degrade protein. Mammalian muscle contains the three
major proteolytic systems. Lysosomal cathepsin expression,
although relatively low, occurs in adult muscle, cell lines, and
adult human muscle satellite cells (11). Cathepsins cleave a
variety of purified myofibrillar substrates in vitro (11). Where
cathepsins have been identified in normal human skeletal
muscle, it has been suggested that they are associated with
clearance of damaged protein structures and regeneration
rather than wholesale bulk degradation (138). To date, however, we have no knowledge of how this proteolytic pathway
responds in human muscle to disuse in nonpathological situations.
Skeletal muscle expresses the ubiquitous calpain-1 (␮-calpain) and calpain-2 (micro-calpain), as well as calpain-3 (p94)
(10), a muscle-specific form that binds to the giant sarcomeric
protein connectin/titin (108) and, when defectively expressed,
results in limb girdle muscle dystrophy type IIA (99). Jones
et al. (66) reported increases in mRNA coding for calpains-1
and -2 and calpastatin that were pronounced early in immobilization (24 h) but then declined over time. Calpain p94 mRNA
content was, in contrast, reduced with immobilization. Regrettably, neither protein contents nor enzyme activities were
It is well accepted that in skeletal muscle the ATP-dependent
ubiquitin proteasomal pathway (UPP) functions as the primary
degradative system for most muscle protein (62, 77, 117, 121).
Targeting of proteins to the proteasome begins with conjugation of an ubiquitin moiety by an ubiquitin carrier (E2) enzyme, which has been previously charged with ubiquitin by the
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mains at the predisuse rate, then the rate of loss of protein ⫽
kbreakdown ⫺ ksynthesis ⫽ 1.2 ⫺ 0.72, or ⬃0.5%/day, as observed
(12, 28, 38, 90, 92). Thus there is no reason for a substantive
increase in protein breakdown to see muscle mass decline.
Assuming that our estimated changes in protein synthesis are
correct, if proteolysis were also elevated by even 20%, then
losses of muscle CSA would be closer to ⬃0.7%/day and the
calculated loss of muscle CSA at 23 days of immobilization
would be 16.5%, not the 10% observed (28). In any case, there
are no reports in humans of MPB rates, measured by dynamic
methods (12, 38, 90, 116), being elevated in conditions of
decreased physical mobility; if anything MPB falls or remains
unchanged (38, 116). Anabolic resistance has been observed in
aging (27, 44, 101, 134) and immobilization (48) and also may
be pertinent in situations of transplant recipients (98) and
patients with cancer (97, 98). There are those, however, who
demur at our view and suggest that older persons can mount a
response to protein ingestion similar to that of younger persons
(69, 115) or, in a similar vein, that immobilization can be
rescued with daily doses of crystalline essential amino acids,
and thus immobilized muscle is not resistant to amino acidinduced anabolism (91, 92). In direct contrast to these reports
(91, 92), however, are reports that muscle atrophy during
short-term (28 days) (126 –128) and long-term (60 days) (126 –
128) bed rest failed to be impacted by daily amino acid
supplementation or by a daily leucine-enriched whey protein
supplement, respectively. Clearly, this is an area with some
important and clinically relevant discrepancies that requires
further investigation.
Recent data have suggested that there is an early increase in
MPB with disuse in humans, at least raising the possibility of
a biphasic rise and then fall in proteolysis (122); it also is
possible that such a biphasic response exists for MPS, something that has not been tested to date. However, we have
criticized the approach and validity of this work (122). One
possibility is that the normal feeding-induced suppression of
MPB (mediated for the most part by insulin) (26, 54) is
attenuated with immobilization; support for such a thesis can
be seen in human models (100). What very clearly needs to be
measured in a direct and detailed fashion is how MPB and
MPS change with short- and longer term durations of disuse.
Given that there is evidence of reduced synthesis but no
changes in proteolysis with disuse, at least to date (12, 38, 90,
109, 116), the possibility of a marked disuse-mediated atrophic
response underpinned by elevated MPB seems unlikely. Interestingly, two recent reports serve as an insightful look at what
happens in human skeletal muscle with disuse and also with a
countermeasure (20, 116). In one study, Symons et al. (116)
reported that, similar to previous studies in humans (28, 38, 46,
48, 90), MPS falls with 21 days of bed rest and provided a
direct dynamic estimate of MPB showing that it is not elevated
but remains unchanged. In a parallel study, Caiozzo et al. (20)
reported, from the same subjects, declines in mRNA abundance of all myosin heavy chains (MHC) but stereotypical
increases in type IIx MHC mRNA. Confirming previous results
(25, 28), these authors also reported minor (statistically nonsignificant) elevations in atrogin or myostatin mRNA abundance (20), and yet this occurred in the face of no increase in
MPB (116).
A potentially important and very interesting observation
made by de Boer et al. (28), which we recently confirmed (48),
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ing that, even in the face of elevations in transcript abundance
for elements of the UPP, there is no measurable change in
proteolysis or muscle mass in disease (16, 17, 63, 102) and
disuse (20, 116). It is difficult to reconcile such large increases
in the expression of genes coding for UPP proteins with a lack
of apparent increments in proteolysis; however, it may be that
the UPP is selectively degrading only a specific population
such as peripheral cellular structures, as suggested recently by
the data of Urso et al. (130, 131). Alternatively, the large
changes in expression of genes coding for UPP proteins may
not be resulting in proportional changes in protein content of
UPP proteins. Another final possibility is that the kinetics of
the UPP, which are still largely unknown, are altered and that
even changes in protein abundance do not translate into
changes in proteolytic activity due to unrecognized modes of
regulation. Thus there are a number of situations in which UPP
mRNA changes are discordant with actual measured rates of
proteolysis. In our view, static measurements of mRNA-encoding subunits of various proteolytic proteins (20), although
informative in their own right, are far removed from the actual
measured proteolytic rate (116), and thus these measures are
difficult to interpret in terms of their significance.
Thus a lacuna in our understanding of how disuse affects
muscle protein turnover in humans is what happens to protein
breakdown. We argue that, based on estimated rates (see
above), it appears unlikely that increased breakdown is playing
a role in the decline in muscle tissue mass, and there also is
tracer-based kinetic evidence to support this contention, at least
between 10 and 42 days (38, 90). The indirect evidence of
enhanced proteolysis, at least insofar as increases in MAFbx
and MuRF-1 mRNA are concerned, observed by de Boer et al.
(28) shows that early (between 0 and 10 days) after immobilization, proteolysis may be upregulated (122), and with longer
periods (at least ⬎30 days), there is a decline. If the anabolic
resistance we have seen occurs early in immobilization and
proteolysis is also elevated (Fig. 1, B and C), then this may
explain why loss of muscle mass is more rapid early (1–30
days; see above) and then subsequently reaches a plateau with
longer periods of disuse (i.e., 90 –120 days) (3). However, even
in patients with spinal cord injuries that completely or partially
block neural impulses to muscle there is preservation of muscle
mass, which is dependent on the level and severity of the
injury. Mild electrical stimulation (47) as well as high-intensity
and -load programs of resistance exercise (5, 22, 129) have
been shown to be effective in limiting human muscle wasting
with disuse.
Inherent Species Differences: A Tale of Rodents and Men
When considering the relevance of animal models to human
muscle protein turnover, species differences in protein metabolism should be taken into account. Total protein turnover in
adult rats is 3- to 4-fold higher than in adult humans, whereas
adult rat muscle protein synthetic rates are ⬃2.5-fold greater
(136). Therefore, it is not inconceivable that the response of
rodent muscle to reduced loading may differ in terms of scale,
pattern, and the relative contribution of each arm to turnover
from that of humans. This is supported by the more rapid and
severe atrophy observed in rodent models, as discussed above.
Indeed, Thomason et al. (123) reported a depression in soleus
muscle protein synthesis as early as 5 h after the onset of
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E1 ubiquitin-activating enzyme. For efficient targeting to the
proteasome, at least four ubiquitin moieties must be attached,
and this is accomplished by E2 in partnership with the E3
ligases, which confer specificity to the system because they
recognize a limited number of substrates (62, 77, 119). The
UPP cannot, however, degrade intact myofibrils (62, 77, 107,
121), and so enzymatic cleavage of “susceptible” sites in the
myofibrillar lattice is thought to occur via either activated
calpain (37, 137) or caspase-3 (35, 42, 135). In turn, these
site-specific cleavages then result in myofibrillar proteins being
exposed, partially degraded, and thus accessible for ubiquitination and thereafter degradable by the proteasome. To date,
however, despite numerous published reports of changes not
only in gene but also protein abundance for components of the
UPP (95), we lack the corresponding kinetic measures of MPB,
in any situation of disuse atrophy, in which its expression has
been up- or downregulated.
Our knowledge is relatively insecure concerning the scope
and regulation of proteolytic systems in human muscle. In fact,
what we know is, more often than not, extrapolated almost
exclusively from ex vivo studies of rodent muscle using inhibitors of one pathway or another (45, 85, 117, 121, 125).
Chloroquine, an inhibitor of acidification of the lysosome, has
been reported to suppress postabsorptive whole body protein
turnover in humans (30) but not that of forearm muscle
turnover (9), suggesting that lysosomal proteolysis does not
play a quantitatively significant role in human skeletal muscle
(64, 94). In addition, ubiquitinated proteins were increased in a
20-day bed rest study (89) in a model similar to that used by
others in which MPB was not found to increase (38, 90). It also
is important to realize that an increase in the concentration of
ubiquitinated proteins can come about due to either increased
rates of conjugation or decreased rates of enzymatic deconjugation. Moreover, inhibition (and not stimulation) of the proteasome can cause accumulation of ubiquitin-protein conjugates (121), which would indicate reduced but not increased
proteolysis. Increases in expression of transcripts for the ubiquitin ligases atrogin-1 and cbl-b also have been reported in
humans during bed rest (89), but the authors proposed that their
role in disuse atrophy may not be through the bulk degradation
of myofibrillar proteins but, rather, via suppression of protein
synthetic capacity by enhanced degradation of growth-promoting proteins. Evidence supporting such a hypothesis has been
presented in the form of modulation of eukaryotic initiation
factor-3 subunit f (eIF3-f) by MAFbx under proatrophic conditions (72). These data provide a link between the increased
ubiquitin ligase expression seen in models of atrophy and
downregulation of MPS (72). It also is possible that muscle
protein degradation in disuse atrophy is linked to degradation
of factors involved in myogenesis and cell cycle regulation;
anti-atrophy interventions have consistently been shown to be
associated with increased myogenic regulatory factor expression (2, 4, 56).
Interestingly, in a number of situations decreases in MPB
have been demonstrated to be dissociated from proteasome
subunit mRNA expression in subjects on a low-protein diet
(17). Furthermore, a mismatch has been observed in the insulin-induced suppression of protein breakdown and changes in
ubiquitin ligases and proteasome subunit C2 protein levels
(54). There also are a number of published reports in humans
in various disease- and pharmacologically based states show-
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unsatisfactory. This is mainly because of the difficulty of
getting the isolated muscle preparations to behave physiologically, in vitro, in terms of MPS and MPB. For example, none
of the ex vivo preparations are even able to sustain net positive
protein balance even in nonpathological conditions. In fact,
muscle preparations from normal young rats display much
lower synthetic rates and higher breakdown rates than those
observed in vivo (136). In addition, rodents are inherently
metabolically unstable because they spend a far greater proportion of their life span growing. During growth in rodents,
skeletal muscle MPS is still insulin sensitive (79) as it is in
humans during growth, but this stimulation is lost in adulthood
(54). On the basis of these criticisms, it would appear that
isolated rodent muscle preparations are inevitably predisposed
to show an increase in MPB in many situations. In fact, even
a decline in protein synthesis would be registered as an increase in breakdown in an ex vivo preparation in which
tyrosine release into the perfusion bath is the method of
measuring proteolysis, since synthesis cannot recapture amino
acids arising from proteolysis (84, 85, 117, 140). Almost 20
years ago, Thomason and Booth (124) stated in their review
that it is extremely difficult to obtain valid estimates of the true
rates of protein degradation in the unweighted soleus muscle.
They proposed estimating breakdown from a first-order model
of the decline in synthetic rates with hindlimb unloading in
adult rats (123) as an alternative to in vitro amino acid-release
rates of muscle preparations and in vivo estimates obtained by
subtracting growth rates from in vivo protein synthesis rates,
which were performed on growing rats (124). With this approach degradation is estimated to gradually increase, reaching
a peak by 15 days, but thereafter declines to below-baseline
rates. When measuring MPS in vivo, combined with estimates
of MPB by difference from rates of muscle loss, hindlimb
suspension can be seen to increase MPB. Thus there appears to
be marked difference in the importance of changes in MPS and
MPB in rat and human muscle. Similar criticisms also have
been made when comparing rats and humans in the field of
aging (31, 32).
By contrast to rats, in vivo human studies have a number of
advantages. For example, the muscle is, at least during a stable
isotope infusion, being perfused by an intact circulatory system. The muscle in this model can sustain a net positive protein
balance and behaves normally in the face of hyperaminoacidemia. In this model there is the ability to combine direct
measurements of protein turnover with simultaneous measures
of protein and gene expression from the same piece of muscle
that gave rise to the phenotypic response of MPS and MPB.
Thus all of these conditions would, we propose, yield a more
optimal experimental protocol from which to obtain a comprehensive description of how disuse affects muscle turnover
leading to atrophy.
In our view, the discrepancy between much of the accepted
descriptions, derived from animal models, of how disuse atrophy is mediated may be due to a combination of both methodological and inherent species-specific differences in how
muscle proteins turn over. Clearly, to resolve some of the
reported inconsistencies we highlight above, more human studies are required with simultaneous measurements of MPS,
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hindlimb unweighting, a phenomenon unlikely to be paralleled
in the human situation, where we spend ⬃8 h of each day
unloaded while sleeping and resting fasted and fed rates of
MPS are unchanged.
Interestingly, in a recent publication (25) it was observed
that aside from increases in MAFbx/atrogin-1, there were very
few changes in proteolytic gene expression in short-term immobilized human skeletal muscle. A number of other reports,
all using rodent models, have confirmed that hindlimb unloading, casting, and denervation result in elevations of mRNA
transcripts for components of the UPP (70, 84, 85, 117, 133).
These findings have led to the conclusion that muscle disuse
leads to elevations in MPB. We (96) recently commented that
the use of static measurements of proteolytic pathway components to provide “proof” that proteolysis is the main mechanism for muscle loss is potentially flawed. Specifically, we
pointed out that in animal studies static measurements of
concentrations of molecules thought to be markers of muscle
proteolysis have been taken in conditions, both physiological
and pathophysiological, in which there is disuse atrophy resulting from immobilization. However, few have ever been
shown to be coincident with the appropriate magnitude of
change in dynamically measured MPB. Thus our conclusion is
that static markers (mRNA and/or protein abundance) are not
often related to dynamic changes in protein breakdown and so
lack meaning when measured in isolation, and so such an
approach is flawed. Instead, we propose that changes in proteolytic components reflect “trimming” or modification of
components of the connective tissue matrix or the proteome
that have specific metabolic effects unrelated to the changes in
bulk protein turnover leading to net protein loss.
Some have argued that spinal isolation and denervation
models used in rodents (84, 104, 105, 140) may well induce
more “severe” atrophic changes than those seen with hindlimb
suspension (4, 21, 55, 59, 93, 117, 120, 132). However, a
comparison of the general programs of gene transcription
between these two models reveals they are similar in many
respects (104, 110). Notably, Stevenson et al. (110), who used
a model of hindlimb suspension, reported a robust activation of
proteasomal components, as well as MAFbx/atrogin-1, MuRF-1, and
lysosomal proteins. Sacheck et al. (104), using models of
spinal isolation and denervation, made similar observations
citing the rise in proteolytic components as the reason for
atrophy in these models in a manner similar to that seen in
uremia and sepsis. Simply put, do elevations in genes for these
pathways indicate that proteolysis is the dominant process?
Seemingly convincing evidence would appear to come from
studies of cells or mice (106, 111); however, at no point in any
of these studies have the processes of protein synthesis and
breakdown ever been measured simultaneously with static
measurements of genes or proteins.
Contributing to the confusion is the fact the measures of
UPP components, almost exclusively mRNA abundances for
proteins involved in proteolysis, appear to align with ex vivo
measurements of MPS and MPB with the use of isolated
muscle strips from rodents (84, 85, 117, 140). There are several
problems with measurements of muscle protein kinetics with
isolated muscles or muscle strips, however, particularly from
young growing rodents. For example, the results of studies
using isolated rodent muscles taken from animals that have
experienced some form of disuse atrophy are, we propose,
MPB, and cotemporal measurement of static markers of breakdown. It is imperative that these studies include examination of
dynamic measures of muscle protein turnover and putative
metabolic controllers early after immobilization (before 10
days) in the fed and fasted states. Factors such as sex, age, and
comorbidities will obviously affect the responses, but unless
we have a clear idea of the basic responses to immobilization
per se, the effects of such factors will not be easily teased out
and therapeutic goals will remain largely unattainable.
We acknowledge the following funding sources: The Collaborative Health
Research Program Award with grants from the Canadian Institutes of Health
Research (CIHR) and the National Science and Engineering Research
Council of Canada (to S. M. Phillips) and from the United Kingdom
Biotechnology and Biological Sciences Research Council and the European
Community EXEGENESIS program (to M. J. Rennie). E. I. Glover was
supported by a CIHR Doctoral Award.
1. Acharyya S, Guttridge DC. Cancer cachexia signaling pathways continue to emerge yet much still points to the proteasome. Clin Cancer Res
13: 1356 –1361, 2007.
2. Adams GR. Satellite cell proliferation and skeletal muscle hypertrophy.
Appl Physiol Nutr Metab 31: 782–790, 2006.
3. Adams GR, Caiozzo VJ, Baldwin KM. Skeletal muscle unweighting:
spaceflight and ground-based models. J Appl Physiol 95: 2185–2201,
4. Adams GR, Haddad F, Bodell PW, Tran PD, Baldwin KM. Combined isometric, concentric, and eccentric resistance exercise prevents
unloading-induced muscle atrophy in rats. J Appl Physiol 103: 1644 –
1654, 2007.
5. Alkner BA, Tesch PA. Efficacy of a gravity-independent resistance
exercise device as a countermeasure to muscle atrophy during 29-day bed
rest. Acta Physiol Scand 181: 345–357, 2004.
6. Andersen JL, Gruschy-Knudsen T, Sandri C, Larsson L, Schiaffino
S. Bed rest increases the amount of mismatched fibers in human skeletal
muscle. J Appl Physiol 86: 455– 460, 1999.
7. Bamman MM, Clarke MS, Feeback DL, Talmadge RJ, Stevens BR,
Lieberman SA, Greenisen MC. Impact of resistance exercise during
bed rest on skeletal muscle sarcopenia and myosin isoform distribution.
J Appl Physiol 84: 157–163, 1998.
8. Baracos VE, DeVivo C, Hoyle DH, Goldberg AL. Activation of the
ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats
bearing a hepatoma. Am J Physiol Endocrinol Metab 268: E996 –E1006,
9. Barrett EJ, Jahn LA, Oliveras DM, Fryburg DA. Chloroquine does
not exert insulin-like actions on human forearm muscle metabolism.
Am J Physiol Endocrinol Metab 268: E820 –E824, 1995.
10. Bartoli M, Richard I. Calpains in muscle wasting. Int J Biochem Cell
Biol 37: 2115–2133, 2005.
11. Bechet D, Tassa A, Taillandier D, Combaret L, Attaix D. Lysosomal
proteolysis in skeletal muscle. Int J Biochem Cell Biol 37: 2098 –2114,
12. Biolo G, Ciocchi B, Lebenstedt M, Barazzoni R, Zanetti M, Platen P,
Heer M, Guarnieri G. Short-term bed rest impairs amino acid-induced
protein anabolism in humans. J Physiol 558: 381–388, 2004.
13. Bleakney R, Maffulli N. Ultrasound changes to intramuscular architecture of the quadriceps following intramedullary nailing. J Sports Med
Phys Fitness 42: 120 –125, 2002.
14. Bohe J, Low A, Wolfe RR, Rennie MJ. Human muscle protein
synthesis is modulated by extracellular, not intramuscular amino acid
availability: a dose-response study. J Physiol 552: 315–324, 2003.
15. Boonyarom O, Inui K. Atrophy and hypertrophy of skeletal muscles:
structural and functional aspects. Acta Physiol (Oxf) 188: 77– 89, 2006.
16. Bossola M, Muscaritoli M, Costelli P, Nanni G, Tazza L, Panocchia
N, Busquets S, Argiles J, Lopez-Soriano FJ, Grieco G, Baccino FM,
Rossi FF, Castagneto M, Luciani G. Muscle ubiquitin m-rNA levels in
patients with end-stage renal disease on maintenance hemodialysis. J
Nephrol 15: 552–557, 2002.
J Appl Physiol • VOL
17. Brodsky IG, Suzara D, Furman M, Goldspink P, Ford GC, Nair KS,
Kukowski J, Bedno S. Proteasome production in human muscle during
nutritional inhibition of myofibrillar protein degradation. Metabolism 53:
340 –347, 2004.
18. Brooks N, Cloutier GJ, Cadena SM, Layne JE, Nelsen CA, Freed
AM, Roubenoff R, Castaneda-Sceppa C. Resistance training and timed
essential amino acids protect against the loss of muscle mass and strength
during 28 days of bed rest and energy deficit. J Appl Physiol 105:
241–248, 2008.
20. Caiozzo VJ, Haddad F, Lee SM, Baker M, Paloski WH, Baldwin
KM. Artificial gravity as a countermeasure to microgravity: a pilot study
examining the effects on knee extensor and plantar flexor muscle groups.
J Appl Physiol 107: 39 – 46, 2009.
21. Carlson CJ, Booth FW, Gordon SE. Skeletal muscle myostatin mRNA
expression is fiber-type specific and increases during hindlimb unloading.
Am J Physiol Regul Integr Comp Physiol 277: R601–R606, 1999.
22. Carrithers JA, Tesch PA, Trieschmann J, Ekberg A, Trappe TA.
Skeletal muscle protein composition following 5 weeks of ULLS and
resistance exercise countermeasures. J Gravit Physiol 9: 155–156, 2002.
23. Carroll CC, Fluckey JD, Williams RH, Sullivan DH, Trappe TA.
Human soleus and vastus lateralis muscle protein metabolism with an
amino acid infusion. Am J Physiol Endocrinol Metab 288: E479 –E485,
24. Castro MJ, Apple DF Jr, Rogers S, Dudley GA. Influence of complete
spinal cord injury on skeletal muscle mechanics within the first 6 months
of injury. Eur J Appl Physiol 81: 128 –131, 2000.
25. Chen YW, Gregory CM, Scarborough MT, Shi R, Walter GA,
Vandenborne K. Transcriptional pathways associated with skeletal
muscle disuse atrophy in humans. Physiol Genomics 31: 510 –520, 2007.
26. Chow LS, Albright RC, Bigelow ML, Toffolo G, Cobelli C, Nair KS.
Mechanism of insulin’s anabolic effect on muscle: measurements of
muscle protein synthesis and breakdown using aminoacyl-tRNA and
other surrogate measures. Am J Physiol Endocrinol Metab 291: E729 –
E736, 2006.
27. Cuthbertson D, Smith K, Babraj J, Leese G, Waddell T, Atherton P,
Wackerhage H, Taylor PM, Rennie MJ. Anabolic signaling deficits
underlie amino acid resistance of wasting, aging muscle. FASEB J 19:
422– 424, 2005.
28. de Boer MD, Selby A, Atherton P, Smith K, Seynnes OR, Maganaris
CN, Maffulli N, Movin T, Narici MV, Rennie MJ. The temporal
responses of protein synthesis, gene expression and cell signalling in
human quadriceps muscle and patellar tendon to disuse. J Physiol 585:
241–251, 2007.
29. de Boer MD, Seynnes OR, di Prampero PE, Pisot R, Mekjavic IB,
Biolo G, Narici MV. Effect of 5 weeks horizontal bed rest on human
muscle thickness and architecture of weight bearing and non-weight
bearing muscles. Eur J Appl Physiol 104: 401– 407, 2008.
30. De Feo P, Volpi E, Lucidi P, Cruciani G, Santeusanio F, Bolli GB,
Brunetti P. Chloroquine reduces whole body proteolysis in humans.
Am J Physiol Endocrinol Metab 267: E183–E186, 1994.
31. Demetrius L. Of mice and men. When it comes to studying ageing and
the means to slow it down, mice are not just small humans. EMBO Rep
6 Spec No: S39 –S44, 2005.
32. Demetrius L. Aging in mouse and human systems: a comparative study.
Ann NY Acad Sci 1067: 66 – 82, 2006.
33. Deschenes MR, Giles JA, McCoy RW, Volek JS, Gomez AL, Kraemer WJ. Neural factors account for strength decrements observed after
short-term muscle unloading. Am J Physiol Regul Integr Comp Physiol
282: R578 –R583, 2002.
34. Droppert PM. A review of muscle atrophy in microgravity and during
prolonged bed rest. J Br Interplanet Soc 46: 83– 86, 1993.
35. Du J, Wang X, Miereles C, Bailey JL, Debigare R, Zheng B, Price
SR, Mitch WE. Activation of caspase-3 is an initial step triggering
accelerated muscle proteolysis in catabolic conditions. J Clin Invest 113:
115–123, 2004.
36. Dupont Salter AC, Richmond FJ, Loeb GE. Prevention of muscle
disuse atrophy by low-frequency electrical stimulation in rats. IEEE
Trans Neural Syst Rehabil Eng 11: 218 –226, 2003.
37. Fareed MU, Evenson AR, Wei W, Menconi M, Poylin V, Petkova V,
Pignol B, Hasselgren PO. Treatment of rats with calpain inhibitors
prevents sepsis-induced muscle proteolysis independent of atrogin-1/
MAFbx and MuRF1 expression. Am J Physiol Regul Integr Comp
Physiol 290: R1589 –R1597, 2006.
107 • SEPTEMBER 2009 •
Downloaded from by on June 18, 2017
J Appl Physiol • VOL
59. Hunter RB, Stevenson E, Koncarevic A, Mitchell-Felton H, Essig
DA, Kandarian SC. Activation of an alternative NF-kappaB pathway in
skeletal muscle during disuse atrophy. FASEB J 16: 529 –538, 2002.
60. Ingber DE. Cellular mechanotransduction: putting all the pieces together
again. FASEB J 20: 811– 827, 2006.
61. Itai Y, Kariya Y, Hoshino Y. Morphological changes in rat hindlimb
muscle fibres during recovery from disuse atrophy. Acta Physiol Scand
181: 217–224, 2004.
62. Jagoe RT, Goldberg AL. What do we really know about the ubiquitinproteasome pathway in muscle atrophy? Curr Opin Clin Nutr Metab
Care 4: 183–190, 2001.
63. Jagoe RT, Redfern CP, Roberts RG, Gibson GJ, Goodship TH.
Skeletal muscle mRNA levels for cathepsin B, but not components of the
ubiquitin-proteasome pathway, are increased in patients with lung cancer
referred for thoracotomy. Clin Sci (Lond) 102: 353–361, 2002.
64. Jefferson LS, Li JB, Rannels SR. Regulation by insulin of amino acid
release and protein turnover in the perfused rat hemicorpus. J Biol Chem
252: 1476 –1483, 1977.
65. Jokl P, Konstadt S. The effect of limb immobilization on muscle
function and protein composition. Clin Orthop Relat Res 222–229, 1983.
66. Jones SW, Hill RJ, Krasney PA, O’Conner B, Peirce N, Greenhaff
PL. Disuse atrophy and exercise rehabilitation in humans profoundly
affects the expression of genes associated with the regulation of skeletal
muscle mass. FASEB J 18: 1025–1027, 2004.
67. Kelly FJ, Lewis SE, Anderson P, Goldspink DF. Pre- and postnatal
growth and protein turnover in four muscles of the rat. Muscle Nerve 7:
235–242, 1984.
68. Klaude M, Fredriksson K, Tjader I, Hammarqvist F, Ahlman B,
Rooyackers O, Wernerman J. Proteasome proteolytic activity in skeletal muscle is increased in patients with sepsis. Clin Sci (Lond) 112:
499 –506, 2007.
69. Koopman R, Verdijk L, Manders RJ, Gijsen AP, Gorselink M,
Pijpers E, Wagenmakers AJ, van Loon LJ. Co-ingestion of protein
and leucine stimulates muscle protein synthesis rates to the same extent
in young and elderly lean men. Am J Clin Nutr 84: 623– 632, 2006.
70. Krawiec BJ, Frost RA, Vary TC, Jefferson LS, Lang CH. Hindlimb
casting decreases muscle mass in part by proteasome-dependent proteolysis but independent of protein synthesis. Am J Physiol Endocrinol
Metab 289: E969 –E980, 2005.
71. Kurakami K. Studies on changes of rabbit skeletal muscle components
induced by immobilization with plaster cast. Nagoya Med J 12: 165–184,
72. Lagirand-Cantaloube J, Offner N, Csibi A, Leibovitch MP, Batonnet-Pichon S, Tintignac LA, Segura CT, Leibovitch SA. The initiation
factor eIF3-f is a major target for atrogin1/MAFbx function in skeletal
muscle atrophy. EMBO J 27: 1266 –1276, 2008.
73. Lang CH, Krawiec BJ, Huber D, McCoy JM, Frost RA. Sepsis and
inflammatory insults downregulate IGFBP-5, but not IGFBP-4, in skeletal muscle via a TNF-dependent mechanism. Am J Physiol Regul Integr
Comp Physiol 290: R963–R972, 2006.
74. Laurent GJ, Sparrow MP, Bates PC, Millward DJ. Turnover of
muscle protein in the fowl (Gallus domesticus). Rates of protein synthesis in fast and slow skeletal, cardiac and smooth muscle of the adult fowl.
Biochem J 176: 393– 401, 1978.
75. Laurent GJ, Sparrow MP, Millward DJ. Turnover of muscle protein in
the fowl. Changes in rates of protein synthesis and breakdown during
hypertrophy of the anterior and posterior latissimus dorsi muscles.
Biochem J 176: 407– 417, 1978.
76. Lawler JM, Song W, Demaree SR. Hindlimb unloading increases
oxidative stress and disrupts antioxidant capacity in skeletal muscle. Free
Radic Biol Med 35: 9 –16, 2003.
77. Lecker SH, Goldberg AL, Mitch WE. Protein degradation by the
ubiquitin-proteasome pathway in normal and disease states. J Am Soc
Nephrol 17: 1807–1819, 2006.
78. Lewis SE, Kelly FJ, Goldspink DF. Pre- and post-natal growth and
protein turnover in smooth muscle, heart and slow- and fast-twitch
skeletal muscles of the rat. Biochem J 217: 517–526, 1984.
79. Lobley GE. Species comparisons of tissue protein metabolism: effects of
age and hormonal action. J Nutr 123: 337–343, 1993.
80. Louard RJ, Fryburg DA, Gelfand RA, Barrett EJ. Insulin sensitivity
of protein and glucose metabolism in human forearm skeletal muscle.
J Clin Invest 90: 2348 –2354, 1992.
107 • SEPTEMBER 2009 •
Downloaded from by on June 18, 2017
38. Ferrando AA, Lane HW, Stuart CA, vis-Street J, Wolfe RR. Prolonged bed rest decreases skeletal muscle and whole body protein
synthesis. Am J Physiol Endocrinol Metab 270: E627–E633, 1996.
39. Ferrando AA, Tipton KD, Bamman MM, Wolfe RR. Resistance
exercise maintains skeletal muscle protein synthesis during bed rest.
J Appl Physiol 82: 807– 810, 1997.
40. Fluck M, Carson JA, Gordon SE, Ziemiecki A, Booth FW. Focal
adhesion proteins FAK and paxillin increase in hypertrophied skeletal
muscle. Am J Physiol Cell Physiol 277: C152–C162, 1999.
41. Fluck M, Ziemiecki A, Billeter R, Muntener M. Fibre-type specific
concentration of focal adhesion kinase at the sarcolemma: influence of
fibre innervation and regeneration. J Exp Biol 205: 2337–2348, 2002.
42. Franch HA, Price SR. Molecular signaling pathways regulating muscle
proteolysis during atrophy. Curr Opin Clin Nutr Metab Care 8: 271–275,
43. Fryburg DA, Jahn LA, Hill SA, Oliveras DM, Barrett EJ. Insulin and
insulin-like growth factor-I enhance human skeletal muscle protein
anabolism during hyperaminoacidemia by different mechanisms. J Clin
Invest 96: 1722–1729, 1995.
44. Fujita S, Rasmussen BB, Cadenas JG, Drummond MJ, Glynn EL,
Sattler FR, Volpi E. Aerobic exercise overcomes the age-related insulin
resistance of muscle protein metabolism by improving endothelial function and Akt/mammalian target of rapamycin signaling. Diabetes 56:
1615–1622, 2007.
45. Furuno K, Goodman MN, Goldberg AL. Role of different proteolytic
systems in the degradation of muscle proteins during denervation atrophy. J Biol Chem 265: 8550 – 8557, 1990.
46. Gibson JN, Halliday D, Morrison WL, Stoward PJ, Hornsby GA,
Watt PW, Murdoch G, Rennie MJ. Decrease in human quadriceps
muscle protein turnover consequent upon leg immobilization. Clin Sci
(Lond) 72: 503–509, 1987.
47. Gibson JN, Smith K, Rennie MJ. Prevention of disuse muscle atrophy
by means of electrical stimulation: maintenance of protein synthesis.
Lancet 2: 767–770, 1988.
48. Glover EI, Phillips SM, Oates BR, Tang JE, Tarnopolsky MA, Selby
A, Smith K, Rennie MJ. Immobilization induces anabolic resistance in
human myofibrillar protein synthesis with low and high dose amino acid
infusion. J Physiol 586: 6049 – 6061, 2009.
49. Goldspink DF. A comparative study of the effects of denervation,
immobilization, and denervation with immobilization on the protein
turnover of the rat soleus muscle. J Physiol 280: 64P– 65P, 1978.
50. Goldspink DF, Garlick PJ, McNurlan MA. Protein turnover measured
in vivo and in vitro in muscles undergoing compensatory growth and
subsequent denervation atrophy. Biochem J 210: 89 –98, 1983.
51. Goldspink DF, Morton AJ, Loughna P, Goldspink G. The effect of
hypokinesia and hypodynamia on protein turnover and the growth of four
skeletal muscles of the rat. Pflügers Arch 407: 333–340, 1986.
52. Gordon SE, Fluck M, Booth FW. Selected Contribution: Skeletal
muscle focal adhesion kinase, paxillin, and serum response factor are
loading dependent. J Appl Physiol 90: 1174 –1183, 2001.
53. Gossman MR, Rose SJ, Sahrmann SA, Katholi CR. Length and
circumference measurements in one-joint and multijoint muscles in
rabbits after immobilization. Phys Ther 66: 516 –520, 1986.
54. Greenhaff PL, Karagounis L, Peirce N, Simpson EJ, Hazell M,
Layfield R, Wackerhage H, Smith K, Atherton P, Selby A, Rennie
MJ. Disassociation between the effects of amino acids and insulin on
signalling, ubiquitin-ligases and protein turnover in human muscle. Am J
Physiol Endocrinol Metab 295: E595–E604, 2008.
55. Han B, Zhu MJ, Ma C, Du M. Rat hindlimb unloading down-regulates
insulin like growth factor-1 signaling and AMP-activated protein kinase,
and leads to severe atrophy of the soleus muscle. Appl Physiol Nutr
Metab 32: 1115–1123, 2007.
56. Hespel P, Op’t EB, Van LM, Urso B, Greenhaff PL, Labarque V,
Dymarkowski S, Van HP, Richter EA. Oral creatine supplementation
facilitates the rehabilitation of disuse atrophy and alters the expression of
muscle myogenic factors in humans. J Physiol 536: 625– 633, 2001.
57. Hikida RS, Staron RS, Hagerman FC, Walsh S, Kaiser E, Shell S,
Hervey S. Effects of high-intensity resistance training on untrained older
men. II. Muscle fiber characteristics and nucleo-cytoplasmic relationships. J Gerontol A Biol Sci Med Sci 55: B347–B354, 2000.
58. Hortobagyi T, Dempsey L, Fraser D, Zheng D, Hamilton G, Lambert
J, Dohm L. Changes in muscle strength, muscle fibre size and myofibrillar gene expression after immobilization and retraining in humans.
J Physiol 524: 293–304, 2000.
J Appl Physiol • VOL
103. Roy RR, Zhong H, Siengthai B, Edgerton VR. Activity-dependent
influences are greater for fibers in rat medial gastrocnemius than tibialis
anterior muscle. Muscle Nerve 32: 473– 482, 2005.
104. Sacheck JM, Hyatt JP, Raffaello A, Jagoe RT, Roy RR, Edgerton
VR, Lecker SH, Goldberg AL. Rapid disuse and denervation atrophy
involve transcriptional changes similar to those of muscle wasting during
systemic diseases. FASEB J 21: 140 –155, 2007.
105. Sandri M, Lin J, Handschin C, Yang W, Arany ZP, Lecker SH,
Goldberg AL, Spiegelman BM. PGC-1alpha protects skeletal muscle
from atrophy by suppressing FoxO3 action and atrophy-specific gene
transcription. Proc Natl Acad Sci USA 103: 16260 –16265, 2006.
106. Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A,
Walsh K, Schiaffino S, Lecker SH, Goldberg AL. Foxo transcription
factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause
skeletal muscle atrophy. Cell 117: 399 – 412, 2004.
107. Solomon V, Goldberg AL. Importance of the ATP-ubiquitin-proteasome pathway in the degradation of soluble and myofibrillar proteins in
rabbit muscle extracts. J Biol Chem 271: 26690 –26697, 1996.
108. Sorimachi H, Kinbara K, Kimura S, Takahashi M, Ishiura S,
Sasagawa N, Sorimachi N, Shimada H, Tagawa K, Maruyama K.
Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94-specific
sequence. J Biol Chem 270: 31158 –31162, 1995.
109. Stein TP, Schluter MD. Human skeletal muscle protein breakdown
during spaceflight. Am J Physiol Endocrinol Metab 272: E688 –E695,
110. Stevenson EJ, Giresi PG, Koncarevic A, Kandarian SC. Global
analysis of gene expression patterns during disuse atrophy in rat skeletal
muscle. J Physiol 551: 33– 48, 2003.
111. Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO,
Gonzalez M, Yancopoulos GD, Glass DJ. The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by
inhibiting FOXO transcription factors. Mol Cell 14: 395– 403, 2004.
112. Svanberg E. Amino acids may be intrinsic regulators of protein synthesis in
response to feeding. Clin Nutr 17: 77–79, 1998.
113. Svanberg E, Jefferson LS, Lundholm K, Kimball SR. Postprandial
stimulation of muscle protein synthesis is independent of changes in
insulin. Am J Physiol Endocrinol Metab 272: E841–E847, 1997.
114. Svanberg E, Moller-Loswick AC, Matthews DE, Korner U, Andersson M, Lundholm K. Effects of amino acids on synthesis and degradation of skeletal muscle proteins in humans. Am J Physiol Endocrinol
Metab 271: E718 –E724, 1996.
115. Symons TB, Schutzler SE, Cocke TL, Chinkes DL, Wolfe RR,
Paddon-Jones D. Aging does not impair the anabolic response to a
protein-rich meal. Am J Clin Nutr 86: 451– 456, 2007.
116. Symons TB, Sheffield-Moore M, Chinkes DL, Ferrando AA, PaddonJones D. Artificial gravity maintains skeletal muscle protein synthesis
during 21 days of simulated microgravity. J Appl Physiol 107: 34 –38,
117. Taillandier D, Aurousseau E, Meynial-Denis D, Bechet D, Ferrara
M, Cottin P, Ducastaing A, Bigard X, Guezennec CY, Schmid HP.
Coordinate activation of lysosomal, Ca2⫹-activated and ATP-ubiquitindependent proteinases in the unweighted rat soleus muscle. Biochem J
316: 65–72, 1996.
118. Taillandier D, Bigard X, Desplanches D, Attaix D, Guezennec CY,
Arnal M. Role of protein intake on protein synthesis and fiber distribution in the unweighted soleus muscle. J Appl Physiol 75: 1226 –1232,
119. Taillandier D, Combaret L, Pouch MN, Samuels SE, Bechet D,
Attaix D. The role of ubiquitin-proteasome-dependent proteolysis in the
remodelling of skeletal muscle. Proc Nutr Soc 63: 357–361, 2004.
120. Taillandier D, Guezennec CY, Patureau-Mirand P, Bigard X, Arnal
M, Attaix D. A high protein diet does not improve protein synthesis in
the non-weight-bearing rat tibialis anterior muscle. J Nutr 126: 266 –272,
121. Tawa NE Jr, Odessey R, Goldberg AL. Inhibitors of the proteasome
reduce the accelerated proteolysis in atrophying rat skeletal muscles.
J Clin Invest 100: 197–203, 1997.
122. Tesch PA, von WF, Gustafsson T, Linnehan RM, Trappe TA.
Skeletal muscle proteolysis in response to short-term unloading in humans. J Appl Physiol 105: 902–906, 2008.
123. Thomason DB, Biggs RB, Booth FW. Protein metabolism and betamyosin heavy-chain mRNA in unweighted soleus muscle. Am J Physiol
Regul Integr Comp Physiol 257: R300 –R305, 1989.
107 • SEPTEMBER 2009 •
Downloaded from by on June 18, 2017
81. Loughna P, Goldspink G, Goldspink DF. Effect of inactivity and
passive stretch on protein turnover in phasic and postural rat muscles.
J Appl Physiol 61: 173–179, 1986.
82. McCall GE, Byrnes WC, Dickinson AL, Fleck SJ. Sample size
required for the accurate determination of fiber area and capillarity of
human skeletal muscle. Can J Appl Physiol 23: 594 –599, 1998.
83. McGuigan MR, Kraemer WJ, Deschenes MR, Gordon SE, Kitaura
T, Scheett TP, Sharman MJ, Staron RS. Statistical analysis of fiber
area in human skeletal muscle. Can J Appl Physiol 27: 415– 422, 2002.
84. Medina R, Wing SS, Goldberg AL. Increase in levels of polyubiquitin
and proteasome mRNA in skeletal muscle during starvation and denervation atrophy. Biochem J 307: 631– 637, 1995.
85. Medina R, Wing SS, Haas A, Goldberg AL. Activation of the ubiquitin-ATP-dependent proteolytic system in skeletal muscle during fasting
and denervation atrophy. Biomed Biochim Acta 50: 347–356, 1991.
86. Mitch WE. Proteolytic mechanisms, not malnutrition, cause loss of
muscle mass in kidney failure. J Ren Nutr 16: 208 –211, 2006.
87. Mittendorfer B, Andersen JL, Plomgaard P, Saltin B, Babraj JA,
Smith K, Rennie MJ. Protein synthesis rates in human muscles: neither
anatomical location nor fibre-type composition are major determinants.
J Physiol 563: 203–211, 2005.
88. Moore DR, Tang JE, Burd NA, Rerecich T, Tarnopolsky MA,
Phillips SM. Differential stimulation of myofibrillar and sarcoplasmic
protein synthesis with protein ingestion at rest and after resistance
exercise. J Physiol 587: 897–904, 2009.
89. Ogawa T, Furochi H, Mameoka M, Hirasaka K, Onishi Y, Suzue N,
Oarada M, Akamatsu M, Akima H, Fukunaga T, Kishi K, Yasui N,
Ishidoh K, Fukuoka H, Nikawa T. Ubiquitin ligase gene expression in
healthy volunteers with 20-day bedrest. Muscle Nerve 34: 463– 469,
90. Paddon-Jones D, Sheffield-Moore M, Cree MG, Hewlings SJ, Aarsland A, Wolfe RR, Ferrando AA. Atrophy and impaired muscle protein
synthesis during prolonged inactivity and stress. J Clin Endocrinol Metab
91: 4836 – 4841, 2006.
91. Paddon-Jones D, Sheffield-Moore M, Urban RJ, Aarsland A, Wolfe
RR, Ferrando AA. The catabolic effects of prolonged inactivity and
acute hypercortisolemia are offset by dietary supplementation. J Clin
Endocrinol Metab 90: 1453–1459, 2005.
92. Paddon-Jones D, Sheffield-Moore M, Urban RJ, Sanford AP, Aarsland A, Wolfe RR, Ferrando AA. Essential amino acid and carbohydrate supplementation ameliorates muscle protein loss in humans during
28 days bedrest. J Clin Endocrinol Metab 89: 4351– 4358, 2004.
93. Pesce V, Cormio A, Fracasso F, Lezza AM, Cantatore P, Gadaleta
MN. Rat hindlimb unloading: soleus and extensor digitorum longus
histochemistry, mitochondrial DNA content and mitochondrial DNA
deletions. Biosci Rep 22: 115–125, 2002.
94. Rannels DE, Kao R, Morgan HE. Effect of insulin on protein turnover
in heart muscle. J Biol Chem 250: 1694 –1701, 1975.
95. Reid MB. Response of the ubiquitin-proteasome pathway to changes in
muscle activity. Am J Physiol Regul Integr Comp Physiol 288: R1423–
R1431, 2005.
96. Rennie MJ, Atherton P, Selby A, Smith K, Narici M, de BM, Phillips
S, Glover E. Letter to the editor on the Journal Club article by Barker and
Traber. J Physiol 586: 307–308, 2008.
97. Rennie MJ, Wackerhage H, Spangenburg EE, Booth FW. Control of
the size of the human muscle mass. Annu Rev Physiol 66: 799 – 828,
98. Rennie MJ, Wilkes EA. Maintenance of the musculoskeletal mass by
control of protein turnover: the concept of anabolic resistance and its
relevance to the transplant recipient. Ann Transplant 10: 31–34, 2005.
99. Richard I, Broux O, Allamand V, Fougerousse F, Chiannilkulchai N,
Bourg N, Brenguier L, Devaud C, Pasturaud P, Roudaut C. Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular
dystrophy type 2A. Cell 81: 27– 40, 1995.
100. Richter EA, Kiens B, Mizuno M, Strange S. Insulin action in human
thighs after one-legged immobilization. J Appl Physiol 67: 19 –23, 1989.
101. Rieu I, Balage M, Sornet C, Giraudet C, Pujos E, Grizard J, Mosoni
L, Dardevet D. Leucine supplementation improves muscle protein synthesis in elderly men independently of hyperaminoacidaemia. J Physiol
575: 305–315, 2006.
102. Roberts RG, Redfern CP, Graham KA, Bartlett K, Wilkinson R,
Goodship TH. Sodium bicarbonate treatment and ubiquitin gene expression in acidotic human subjects with chronic renal failure. Eur J Clin
Invest 32: 488 – 492, 2002.
J Appl Physiol • VOL
133. Vermaelen M, Marini JF, Chopard A, Benyamin Y, Mercier J,
Astier C. Ubiquitin targeting of rat muscle proteins during short periods
of unloading. Acta Physiol Scand 185: 33– 40, 2005.
134. Volpi E, Mittendorfer B, Rasmussen BB, Wolfe RR. The response of
muscle protein anabolism to combined hyperaminoacidemia and glucose-induced hyperinsulinemia is impaired in the elderly. J Clin Endocrinol Metab 85: 4481– 4490, 2000.
135. Wang X, Hu Z, Hu J, Du J, Mitch WE. Insulin resistance accelerates
muscle protein degradation: activation of the ubiquitin-proteasome pathway
by defects in muscle cell signaling. Endocrinology 147: 4160 – 4168, 2006.
136. Waterlow JC, Garlick PJ, Millward DJ. Protein Turnover in Mammalian Tissues and in the Whole Body. Amsterdam: North Holland,
137. Wei W, Fareed MU, Evenson A, Menconi MJ, Yang H, Petkova V,
Hasselgren PO. Sepsis stimulates calpain activity in skeletal muscle by
decreasing calpastatin activity but does not activate caspase-3. Am J
Physiol Regul Integr Comp Physiol 288: R580 –R590, 2005.
138. Whitaker JN, Bertorini TE, Mendell JR. Immunocytochemical studies
of cathepsin D in human skeletal muscle. Ann Neurol 13: 133–142, 1983.
139. Wilkinson SB, Phillips SM, Atherton PJ, Patel R, Yarasheski KE,
Tarnopolsky MA, Rennie MJ. Differential effects of resistance and
endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle. J Physiol 586: 3701–3717,
140. Wing SS, Haas AL, Goldberg AL. Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle
during starvation and atrophy denervation. Biochem J 307: 639 – 645,
141. Yasuda N, Glover EI, Phillips SM, Isfort RJ, Tarnopolsky MA.
Sex-based differences in skeletal muscle function and morphology with
short-term limb immobilization. J Appl Physiol 99: 1085–1092, 2005.
142. Zhang SJ, Truskey G, Kraus WE. Effect of cyclic stretch on ␤1Dintegrin expression and activation of FAK and RhoA. Am J Physiol Cell
Physiol 292: C2057–C2069, 2007.
107 • SEPTEMBER 2009 •
Downloaded from by on June 18, 2017
124. Thomason DB, Booth FW. Atrophy of the soleus muscle by hindlimb
unweighting. J Appl Physiol 68: 1–12, 1990.
125. Tischler ME, Rosenberg S, Satarug S, Henriksen EJ, Kirby CR,
Tome M, Chase P. Different mechanisms of increased proteolysis in
atrophy induced by denervation or unweighting of rat soleus muscle.
Metabolism 39: 756 –763, 1990.
126. Trappe S, Creer A, Minchev K, Slivka D, Louis E, Luden N, Trappe
T. Human soleus single muscle fiber function with exercise or nutrition
countermeasures during 60 days of bed rest. Am J Physiol Regul Integr
Comp Physiol 294: R939 –R947, 2008.
127. Trappe S, Creer A, Slivka D, Minchev K, Trappe T. Single muscle
fiber function with concurrent exercise or nutrition countermeasures
during 60 days of bed rest in women. J Appl Physiol 103: 1242–1250,
128. Trappe TA, Burd NA, Louis ES, Lee GA, Trappe SW. Influence of
concurrent exercise or nutrition countermeasures on thigh and calf
muscle size and function during 60 days of bed rest in women. Acta
Physiol (Oxf) 191: 147–159, 2007.
129. Trappe TA, Carrithers JA, Ekberg A, Trieschmann J, Tesch PA. The
influence of 5 weeks of ULLS and resistance exercise on vastus lateralis
and soleus myosin heavy chain distribution. J Gravit Physiol 9: 127–128,
130. Urso ML, Chen YW, Scrimgeour AG, Lee PC, Lee KF, Clarkson
PM. Alterations in mRNA expression and protein products following
spinal cord injury in humans. J Physiol 579: 877– 892, 2007.
131. Urso ML, Scrimgeour AG, Chen YW, Thompson PD, Clarkson PM.
Analysis of human skeletal muscle after 48-h immobilization reveals
alterations in mRNA and protein for extracellular matrix components.
J Appl Physiol 101: 1136 –1148, 2006.
132. Vazeille E, Codran A, Claustre A, Averous J, Listrat A, Bechet D,
Taillandier D, Dardevet D, Attaix D, Combaret L. The ubiquitinproteasome and the mitochondria-associated apoptotic pathways are
sequentially downregulated during recovery after immobilization-induced muscle atrophy. Am J Physiol Endocrinol Metab 295: E1181–
E1190, 2008.