Critical Review
Assessment of Skeletal Muscle Proteolysis
and the Regulatory Response to Nutrition
and Exercise
1
Military Nutrition Division, US Army Research Institute of Environmental
Medicine, Natick, MA, USA
2
School of Health Sciences, Eastern Michigan University, Ypsilanti, MI,
USA
Abstract
Skeletal muscle proteolysis is highly regulated, involving com-
plex intramuscular proteolytic systems that recognize and
degrade muscle proteins, and recycle free amino acid precur-
sors for protein synthesis and energy production. Autophagy-
lysosomal, calpain, and caspase systems are contributors to
muscle proteolysis, although the ubiquitin proteasome system
(UPS) is the primary mechanism by which actomyosin frag-
ments are degraded in healthy muscle. The UPS is sensitive to
mechanical force and nutritional deprivation, as recent reports
have demonstrated increased proteolytic gene expression and
activity of the UPS in response to resistance and endurance
exercise, and short-term negative energy balance. However,
consuming dietary protein alone (or free amino acids), or as a
Keywords: ubiquitin proteasome; lysosomes; protein breakdown;
exercise; amino acids
Introduction
Studies examining human skeletal muscle protein turnover
have focused predominantly on muscle protein synthesis (1,2).
This is not surprising, considering protein synthetic responses
to a variety of stressors in healthy muscle, including nutrition
and exercise, are generally more robust and sustained than
those related to protein degradation (3,4). As such, the role of
C 2014 International Union of Biochemistry and Molecular Biology
V
Volume 66, Number 7, July 2014, Pages 478–484
Address correspondence to: Stefan M. Pasiakos, 15 Kansas St. Bldg. 42,
Natick, MA 01760, USA. Tel.: 508-233-6474. Fax: 508-233-4869.
E-mail: stefan.pasiakos@us.army.mil
Received 23 May 2014; Accepted 1 July 2014
DOI 10.1002/iub.1291
Published online 23 July 2014 in Wiley Online Library
(wileyonlinelibrary.com)
478
Stefan M. Pasiakos1*
John W. Carbone2
primary component of a mixed meal, may attenuate intramus-
cular protein loss by down-regulating proteolytic gene expres-
sion and the catabolic activity of the UPS. Although these
studies provide novel insight regarding the intramuscular reg-
ulation of skeletal muscle mass, the role of proteolysis in the
regulation of skeletal muscle protein turnover in healthy
human muscle is not well described. This article provides a
contemporary review of the intramuscular regulation of skele-
tal muscle proteolysis in healthy muscle, methodological
approaches to assess proteolysis, and highlights the effects of
nutrition and exercise on skeletal muscle proteolysis.
C 2014 IUBMB Life, 66(7):478–484, 2014
V
proteolysis in the regulation of healthy human skeletal muscle
protein turnover is often overlooked (5). We contend that pro-
teolysis, a fundamental biological process and determinant of
net protein balance, should be better studied, as proteolysis
provides amino acid precursors for synthesis of vital organs
and tissues (6), the repair, remodeling, and synthesis of muscle
proteins (7), and hepatic gluconeogenesis (8).
Skeletal muscle proteolysis is regulated by intramuscular
systems sensitive to alterations in nutritional status, exercise,
and their associated hormonal milieu. This article provides a
concise review of the literature regarding the intramuscular
regulation of skeletal muscle proteolysis in healthy muscle.
Advantages, disadvantages, and the assumptions associated
with contemporary methodological approaches to measure
proteolysis are described to demonstrate the inherent difficul-
ties associated with human muscle proteolytic assessment. We
highlight the effects of nutrition (e.g., free amino acids, intact
protein, mixed-meal feeding, and energy deficit), exercise
IUBMB Life
(resistance and endurance), and when appropriate, their asso-
ciated hormonal milieu on skeletal muscle proteolysis and the
contribution of proteolysis to net protein balance, as nutrition
and exercise are potent independent regulators of healthy
muscle protein turnover.
Intramuscular Regulation of Skeletal
Muscle Proteolysis
Skeletal muscle protein breakdown is regulated primarily by
four proteolytic systems; autophagy-lysosomal, calcium-
dependent calpains, the cysteine protease caspase enzymes,
and the UPS. The contribution of each system to protein
breakdown in healthy muscle is dependent on endogenous and
exogenous stimuli, including nutrition (e.g., amino acids,
energy status, and insulin) and the application of mechanical
force during exercise (9–11).
Autophagy is a highly conserved proteolytic homeostatic
mechanism by which bulk cytoplasmic, long-lived proteins,
and organelles are degraded by lysosomal machinery (i.e.,
cathepsins) (12). Cathepsins are also capable of degrading
muscle proteins with specificity towards particular myofibrillar
substrates (e.g., troponin T, myosin heavy chain, and tropo-
myosin) (13). Slow-twitch muscle fibers exhibit higher cathep-
sin levels than fast-twitch muscle fibers, although expression
of these endopeptidases is generally low in skeletal muscle. In
cultured myotubes, autophagy-lysosomal proteolysis increases
in response to amino acid deprivation, particularly leucine
starvation (14). Autophagic gene expression in rats is also
increased following short-term fasting (15), although cathepsin
activity decreases with sustained energy deficit (16).
Calcium-dependent m-calpain, m-calpain, and p94 (cal-
pains 1, 2, and 3, respectively) are contributors to muscle pro-
teolysis (17), with p94 being the most highly expressed in skel-
etal muscle (18). Although calpains have limited capability to
fully degrade complex intramuscular substrates (19), calpain
enzymatic activity is upregulated in response to proteolytic
stressors, including endurance exercise, muscular dystrophy,
and disuse (17,20). The mechanism by which calpains contrib-
ute to muscle proteolysis is likely by cleaving myofibrillar pro-
teins into smaller actomyosin fragments (21). Intact myofibrils
are unable to be degraded by the UPS due to their relatively
large size. This initial calpain-mediated cleavage consequently
increases substrate accessibility to the proteasome (19).
The caspase enzymes also initiate skeletal muscle proteol-
ysis (22). Caspase-3, a cysteine protease activated after its pro-
enzyme is cleaved by caspase-9, initiates proteolysis by cleav-
ing myofibrillar proteins into smaller fragments, including a
characteristic 14 kDa actin fragment (22). We have recently
shown significant increases in caspase-3 activation in human
skeletal muscle following a modest, short-term energy deficit
(23). We have also demonstrated a reduction in post-exercise
caspase-3 activation following mixed protein-carbohydrate
consumption, relative to carbohydrate alone (24). In addition
Pasiakos and Carbone
to cleaving myofibrils, caspase-3 may also serve to stimulate
proteasome activity (25), thereby both providing substrate to
the UPS and increasing UPS-mediated proteolysis.
Prior to being degraded by the proteasome, cleaved myofibril
segments are ubiquitylated through energy-dependent reactions
by muscle-specific ubiquitin ligases, namely atrogin-1 (MAFbx)
and muscle RING finger-1 (MuRF-1). Poly-ubiquitylated proteins
are subsequently degraded by amino acid hydrolysis within the
26S proteasome of the UPS (26). Nutritional stressors, such as
short-term energy deficit, can induce intramuscular expression
of the proteasomal a-subunit PSMA2 (23). Muscle Akt phospho-
rylation is also reduced during short-term energy deficit, with
concomitant reductions in muscle protein synthesis (27), which
likely activates the FOXO family of transcription factors (28) and
upregulates atrogin-1 and MuRF-1 expression (29,30). Consum-
ing a protein-containing mixed meal, however, inhibits substrate
ubiquitylation and decreases the activities of the catalytic sites
within 26S proteasome (31).
It is likely that these four proteolytic systems function in
concert to regulate muscle protein degradation in response to
nutrition and exercise perturbations (32). For example, FOXO
transcription factors regulate UPS activity (29), and FOXO3 is
also involved in control of autophagy-related gene expression
(33). Similarly, ubiquitylated proteins are not only processed
by the 26S proteasome; some are also degraded by the lyso-
some (34).
Analysis of Skeletal Muscle Protein
Degradation
Several qualitative and quantitative methods can be used to
assess proteolysis. An applied approach to estimate skeletal
muscle proteolysis is the measurement of urinary 3-
methylhistidine, an amino acid generated by post-translational
methylation of histidine residues derived from actin and myo-
sin degradation (35). Urinary 3-methylhistidine is a valid
marker of muscle proteolysis because it cannot be further cat-
abolized, nor recycled for protein synthesis (36,37). Although
3-methylhistidine assessment is a noninvasive means of evalu-
ating skeletal muscle proteolysis in humans, it is important to
recognize that this amino acid is not solely representative of
skeletal muscle proteolysis, though its presence in non-muscle
tissue is relatively low (38). Cardiac and smooth muscle pro-
tein degradation also produce 3-methylhistidine as does the
metabolism of dietary protein from animal meat. Therefore,
relying on 3-methylhistidine as an indicator of muscle proteol-
ysis also requires significant dietary control (i.e., meat-free).
Nevertheless, 3-methylhsitidine can be used as a non-invasive
indicator of muscle proteolysis, which can readily be assessed
in urine and, perhaps more directly, by collecting muscle
interstitial fluid by microdialysis (39). It is also possible that
combining stable isotope labeled 3-methylhistidine with the
arteriovenous (AV, see below) balance approach can be used
as a consistent and valid measure of muscle proteolysis (37).
479

More complex measures of muscle proteolysis include
kinetic assessment of AV balance, and stable isotope tracee
release and flooding dose methodologies. These procedures
are invasive; requiring arterial and venous catheterizations,
stable isotope infusions, and percutaneous muscle biopsies,
procedures typically performed on the thigh (biopsies are gen-
erally obtained from the vastus lateralis) (40). The AV balance
method measures protein turnover of the muscle by collecting
arterial and venous blood samples from the vessels feeding
and draining the muscle (41). Protein degradation, as well as
protein synthesis, and net protein balance can be derived by
measuring blood flow across the muscle and the differences in
isotopic enrichments in the arterial and venous free amino
acid pools (40). It is important to note that AV balance does
not provide kinetic measures of muscle protein turnover per
se, rather AV balance measures primarily reflect amino acid
uptake or release from skeletal muscle. There is also the
potential for non-muscle sources to contribute to isotopic AV
balance differences (e.g., skin), confounding the validity of AV
balance for estimating muscle protein turnover (42). The con-
tribution of these non-muscle sources of isotopic enrichment
may become significant when evaluating degradation in indi-
viduals suffering from hyper-catabolic conditions (e.g., burns);
however, using the AV balance method to assess protein deg-
radation has been validated in healthy individuals (40).
Direct measures of muscle protein degradation are
attained using stable isotope-labeled amino acids infusions,
particularly phenylalanine, because skeletal muscle cannot
metabolize nor synthesize this amino acid (41). Isotopically
labeled phenylalanine (tracer) appearance in intact muscle
protein indicates protein synthesis, whereas isotopic disap-
pearance (i.e., dilution) in the intramuscular and plasma-free
amino acid pools indicates protein degradation (3). To consider
stable isotope-derived protein degradation rates valid requires
a number of assumptions to be made, including: 1) that the
tracer is metabolized in an identical fashion to the tracee
(unlabeled amino acid); 2) that the tracer does not alter tracee
metabolism; 3) that the lowest atomic weight of the tracer
atom represents its most abundant form; and 4) that the sub-
strate pool (blood) must be considered homogenous, with rapid
mixing to effectively circulate infused tracer (41).
The flooding dose method, which has been used effectively
to determine muscle protein synthesis (41,43), can also be
used to assess protein degradation. This approach first
requires labeling the intact muscle protein pool of amino acids,
and then monitoring isotopic dilution. A theoretically similar,
although less invasive, approach has recently been established,
involving the administration of a single oral dose of deuterium
oxide, which donates deuterium to endogenous amino acids
(e.g., alanine), which are subsequently incorporated into body
proteins (44). Protein degradation is estimated by determining
the rate of disappearance or isotopic dilution of deuterated
alanine of particular proteins. Although the flooding dose and
deuterium oxide methods are valid, turnover rates can vary
considerably and muscle proteins, in particular, typically turn
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over at a rate of only 2% per day (41), suggesting that meas-
urable changes would require several days of monitoring.
A time-efficient stable isotope approach to estimate muscle
proteolysis, which provides distinct advantages and estimates
of protein degradation over the methods previously reviewed,
is the tracee release method. This method models three pools
of amino acids; two precursors (plasma and intact muscle pro-
tein) and one product (intramuscular free amino acid pool)
(41). Primed, constant infusions of stable isotopically labeled
phenylalanine are infused to attain steady state conditions.
The infusion is then stopped and proteolysis is quantified from
the rate the tracee is released from intact muscle protein,
diluting intramuscular and plasma tracer enrichment (23,45).
The time course for these procedures is generally much
smaller than those required to detect measurable differences
in protein degradation using the flooding dose and deuterium
oxide approaches. When these tracee release methods have
been applied, muscle protein degradation rates have generally
been estimated using a primed, 2 H constant infusion followed
by a short dilution period (e.g., 1 H) (23,46).
The pulse tracer injection method is similar to the tracee
release method, such that arterial isotopic dilution is meas-
ured for a fixed period of time, but the pulse tracer injection
method does not require isotopic steady state (47). This allows
the duration of the infusion protocol (60 Min) and the total
number of biopsies (one biopsy) necessary to assess muscle
protein degradation, to both be reduced. However, the tracee
release and pulse tracer injection methods both assume physi-
ological steady state exists during the evaluation period,
thereby limiting the application of these methods when study-
ing the effects of acute nutrient (e.g., protein, amino acids, and
carbohydrate) provision and exercise. A modified calculation
has been introduced to the pulse tracer injection method to
account for non-isotopic steady state conditions, which
assumes that amino acid transport into the myocyte correlates
with plasma amino acid concentrations (48). This new method
illustrates a high degree of correlation with traditional pulse
tracer injection, although further work is needed to evaluate
the potential influence of mixed meal consumption relative to
the parenteral amino acid injections used in the initial study.
Analysis of Intramuscular Proteolysis
The effects of nutrition and exercise on muscle proteolysis can
also be assessed using qRT-PCR to analyze mRNA expression
patterns for known regulators of proteolysis. Although transla-
tion may serve as an important regulatory step in the expres-
sion of proteolytic factors (23), alterations in mRNA levels may
not always manifest into biologically relevant changes in the
expression and the activity of the protein. Therefore, Western
blotting and multiplex assays are used to obtain qualitative
measures of proteolytic protein expression and activity
through phosphorylation. However, mRNA and protein expres-
sion only provide a snapshot of proteolysis at one point in time,
and often fail to represent the cumulative changes in muscle
Protein Degradation in Healthy Muscle
protein degradation that occurs over time (49). We recently
demonstrated no change in atrogin-1 protein expression in
response to short-term energy deficit, although kinetic meas-
ures of muscle protein degradation were higher after energy
deficit compared to weight maintenance (23). Assessing nuclear
translocation of atrogenic transcription factors (50) and proteo-
lytic enzymatic activity (23,24,31) may provide additional
insight and clarification to sometimes discrepant kinetic meas-
ures of protein degradation and mRNA and protein expression.
Assuming genetic sequences, primary antibodies, and
fluorophore-linked substrates are available, these quantitative
methods can be applied to assess any enzymatic component of
the four primary proteolytic pathways in human muscle.
Exercise and Nutritional Modulation
of Skeletal Muscle Proteolysis
Nutrition and exercise are potent regulators of muscle protein
turnover in healthy muscle (2). As such, the influences of each
alone, and in combination, on the regulation of muscle protein
degradation are reviewed.
Kinetic measures of skeletal muscle proteolysis in response
to exercise are differentially influenced by training status and
the type of exercise performed (51). Using the tracee release
method, resistance exercise has been shown to result in a
transient but significant increase in protein degradation during
the first 24 H post-exercise, followed by a return to resting lev-
els by 48 H (3). Similar acute proteolytic responses to resist-
ance exercise have been reported by others using the AV bal-
ance approach (52,53).
The skeletal muscle proteolytic responses to resistance
exercise are generally less pronounced in trained versus
untrained individuals (53,54). Endurance exercise may also be
acutely catabolic, particularly in the postabsorptive state, as
proteolysis is likely increased during endurance exercise to
support the energy requirement of exercising skeletal muscle
(55). Whether endurance exercise elicits a similar post-
exercise proteolytic response in trained versus untrained indi-
viduals as resistance exercise is not well described. We dem-
onstrated no change in muscle protein degradation using the
tracee release method in response to moderate intensity
endurance exercise in physically active adults during a well-
controlled energy deficient dietary intervention (23). However,
we studied trained volunteers and suspect that the exercise
stimulus may have been inadequate to elicit measureable
changes in protein degradation in that population (56). In
addition, endurance training upregulates resting muscle pro-
tein turnover, which may have confounded our ability to detect
changes in post-exercise protein degradation as our study par-
ticipants were permitted to continue their exercise training
program throughout the intervention (46).
It is well established that consuming protein or free amino
acids alone, or in combination with carbohydrate, stimulates a
robust increase in muscle protein synthesis and anabolic
Pasiakos and Carbone
intramuscular signaling at rest and after exercise (2). Addi-
tionally, some research suggests that protein consumption may
attenuate muscle proteolysis, as physiologic hyperinsulinemia
and amino acid administration were observed to lower resting
and post-resistance exercise kinetic measures of muscle pro-
tein degradation and 3-methylhistidine excretion (57–60).
However, other studies have demonstrated opposite effects,
whereby amino acid administration at rest or during recovery
from resistance exercise upregulates protein degradation (61),
and still others have demonstrated minimal or no changes in
kinetic measures of protein degradation regardless when an
amino acid (with or without carbohydrate) supplement was
consumed at rest, before, or after resistance and endurance
exercise (62–66). Regardless of what effects on protein degra-
dation were observed, in all these studies changes in protein
synthesis and positive shifts in net protein balance in response
to feeding and exercise were observed. Although proteolysis is
undoubtedly a contributor to net protein turnover, these stud-
ies do suggest that changes in protein synthesis are likely to
exert a more pronounced regulatory effect on net protein bal-
ance than protein degradation in healthy muscle.
Several recent studies have assessed the autophagy-
lysosomal and UPS-mediated proteolytic response to
endurance-type exercise (67–70). Specifically, autophagy-
related mRNA gene expression was upregulated, including a
123% increase in the expression of the lysosomal cathepsin-L
when compared to resting values after an ultra-endurance
(200-km run) exercise event. These findings occurred with
concomitant increases in the expression of UPS components,
in particular atrogin-1, MuRF-1, and PSMA1, although the
activity of the 26S proteasome itself was interestingly
repressed after exercise (71). Laboratory animal studies have
also demonstrated a more pronounced upregulation in
autophagy-lysosomal gene expression and associated FOXO-
related activity when endurance exercise is performed in the
fasted state compared with the fed state (69), particularly in
slow-twitch muscle fibers (70), although these findings have
not been confirmed in human muscle. Results from our labora-
tory confirm the effects of endurance exercise on UPS-
mediated proteolysis, as atrogin-1 and MuRF-1 expression
were upregulated during recovery from moderate endurance
exercise in recreationally active individuals (72). Louis et al.
(73) also demonstrated upregulated MuRF-1, atrogin-1, and
FOXO3A expression during the first 4 H of recovery from
endurance exercise, a proteolytic response that was more
robust than those observed after resistance exercise.
Intramuscular proteolytic responses to resistance exercise
often vary and data from studies that rely solely on qRT-PCR
and Western blotting to elucidate the effects of exercise and
nutrition on proteolysis can be difficult to interpret. For exam-
ple, UPS-related mRNA expression is dependent on the type of
contraction performed. Eccentric exercise appears to upregu-
late proteasome subunit expression exclusively, whereas
FOXO1 and MuRF-1 mRNA expressions were increased only
after concentric muscle contractions (74). Atrogin-1 expression
481

was either unchanged or lower after eccentric and concentric
contractions compared to rest. The authors hypothesized a
minimal role for the ubiquitin ligases in actual proteolysis but
rather changes in ubiquitin ligase expression may be due to an
increased demand to remodel muscle after eccentric exercise
and higher demand for energy with concentric compared to
eccentric exercise. Their assertions are supported, in part by
others, where changes in UPS-related gene expression
appeared unrelated and did not concur with changes in 26S
proteasome activity or kinetic measures of muscle protein deg-
radation (49,69,75).
Energy balance likely influences intramuscular proteolysis
at rest and in response to exercise. We recently demonstrated
increased postabsorptive muscle protein degradation and UPS-
related gene expression at rest after both short-term and long-
term moderate energy deficit (23,31). However, consuming a
mixed-meal with 20 g of dietary protein attenuated intramuscu-
lar proteolysis, in particular 26S proteasome activity (31). We
and others have also observed similar downregulations in intra-
muscular proteolysis in response to consuming protein at rest
and after exercise (24,76). Most recently, Stefanetti et al. (77)
compared the effects of whey protein versus isocaloric carbohy-
drate supplementation on atrogin-1, MuRF-1, and FOXO1 and
FOXO3A expression after a single bout of eccentric and concen-
tric exercise and after 12 weeks of training, in previously
untrained individuals. UPS-related gene expression varied dra-
matically by contraction type and in response to training, and
although whey protein supplementation had no impact on
mRNA expression, it was associated with greater increases in
muscle cross sectional area (77), findings that highlight the high
degree of complexity involved in the regulation of human skele-
tal muscle proteolysis in response to exercise and nutrition.
Conclusions and Future Applications
As the regulation of muscle protein synthesis becomes better
understood, it is clear that increased attention needs to also be
paid to muscle proteolysis to gain a more holistic view of the
effects of endogenous and exogenous stimuli on skeletal mus-
cle protein turnover. Recent advances in stable isotope meth-
odologies, particularly the development of the pulse tracer
injection method and its subsequent non-steady-state modifica-
tion, increase the likelihood of being able to attain credible,
reproducible kinetic measures of muscle breakdown in
response to variations in nutritional status and exercise.
Future studies coupling kinetic data with measures of expres-
sion and activity of intramuscular regulators of proteolysis are
paramount to deciphering the complex pathways that mediate
muscle degradation and the conservation of skeletal muscle
mass in healthy individuals.
Acknowledgements
The authors thank Dr. Andrew Young and Dr. Scott Montain for
their critical review in support of the development of this
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IUBMB LIFE
manuscript. All authors have read and approved the final manu-
script. This work has been supported by the US Army Military
Research and Material Command and the Eastern Michigan Uni-
versity College of Health and Human Services. The opinions or
assertions contained herein are the private views of the authors
and are not to be construed as official or as reflecting the views
of the Army or the Department of Defense. Any citations of com-
mercial organizations and trade names in this report do not con-
stitute an official Department of the Army endorsement of
approval of the products or services of these organizations.
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Protein Degradation in Healthy Muscle