Skeletal Muscle Protein Synthesis and mTORC1 Nutritional and Contractile Regulation of Human

doi:10.1152/japplphysiol.91397.
2008
106:1374-1384, 2009. First published 15 January 2009; J Appl Physiol
Blake B. Rasmussen
Micah J. Drummond, Hans C. Dreyer, Christopher S. Fry, Erin L. Glynn and
signaling
skeletal muscle protein synthesis and mTORC1
Nutritional and contractile regulation of human
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Review
HIGHLIGHTED TOPIC Regulation of Protein Metabolism in Exercise and Recovery
Nutritional and contractile regulation of human skeletal muscle protein
synthesis and mTORC1 signaling
Micah J. Drummond,
1,2,3
Hans C. Dreyer,
1,2,3
Christopher S. Fry,
2
Erin L. Glynn,
2
and Blake B. Rasmussen
1,2,3
1
Department of Physical Therapy,
2
Division of Rehabilitation Sciences, and
3
Sealy Center on Aging, University of Texas
Medical Branch, Galveston, Texas
Submitted 21 October 2008; accepted in nal form 13 January 2009
Drummond MJ, Dreyer HC, Fry CS, Glynn EL, Rasmussen BB. Nutritional
and contractile regulation of human skeletal muscle protein synthesis and mTORC1
signaling. J Appl Physiol 106: 13741384, 2009. First published Janaury 15, 2009;
doi:10.1152/japplphysiol.91397.2008.In this review we discuss current ndings
in the human skeletal muscle literature describing the acute inuence of nutrients
(leucine-enriched essential amino acids in particular) and resistance exercise on
muscle protein synthesis and mammalian target of rapamycin complex 1
(mTORC1) signaling. We show that essential amino acids and an acute bout of
resistance exercise independently stimulate human skeletal muscle protein synthe-
sis. It also appears that ingestion of essential amino acids following resistance
exercise leads to an even larger increase in the rate of muscle protein synthesis
compared with the independent effects of nutrients or muscle contraction. Until
recently the cellular mechanisms responsible for controlling the rate of muscle
protein synthesis in humans were unknown. In this review, we highlight new
studies in humans that have clearly shown the mTORC1 signaling pathway is
playing an important regulatory role in controlling muscle protein synthesis in
response to nutrients and/or muscle contraction. We propose that essential amino
acid ingestion shortly following a bout of resistance exercise is benecial in
promoting skeletal muscle growth and may be useful in counteracting muscle
wasting in a variety of conditions such as aging, cancer cachexia, physical
inactivity, and perhaps during rehabilitation following trauma or surgery.
essential amino acids; contraction; translation initiation; leucine
HUMAN SKELETAL MUSCLE PROTEIN metabolism has received sig-
nicant attention over the past few decades because of its
relevance to aging, disease processes, and physical inactivity.
The importance of skeletal muscle is obvious since it comprises
nearly 40%of body weight, constitutes between 50 and 75%of all
proteins (73), and is imperative for locomotion. However, it is also
important as an amino acid reservoir, for energy consumption and
for fuels for other tissues (e.g., brain, immune cells).
Skeletal muscle proteins turnover regularly such that 12%
of proteins are synthesized and broken down daily (111). The
turnover of proteins involves the ongoing processes of protein
synthesis and breakdown. A positive net protein balance occurs
when proteins accumulate in excess of their removal (e.g.,
following nutrient ingestion), whereas a negative net protein
balance occurs when the breakdown of proteins exceeds that of
their synthesis (e.g., fasting). The initial work of Professors
Michael J. Rennie and Robert R. Wolfe contributed signi-
cantly to the understanding of the muscle protein metabolism
eld in humans by incorporating stable isotope techniques
concurrently with skeletal muscle biopsies in the 1980s and
1990s. Because of these breakthroughs, researchers can now
study the dynamics of skeletal muscle protein turnover under
various interventions. Although the turnover of skeletal muscle
protein is relatively slow compared with other proteins, such as
blood (albumin) and gastric intestinal tract proteins, changes in
protein turnover can be identied within hours following acute
anabolic interventions. Indeed, essential amino acids (particu-
larly leucine) and resistance exercise are powerful stimulators
of skeletal muscle protein synthesis in animal and human
models (1, 30, 42). Less understood are the cellular and
molecular mechanisms regulating protein turnover following
nutrition and resistance exercise in humans, although great
strides have been made in the last decade. Thus this review
highlights the most recent peer-reviewed articles capturing the
inuence of nutrition and resistance exercise on protein syn-
thesis and cellular signaling in human skeletal muscle.
TRANSLATIONAL CONTROL OF MUSCLE
PROTEIN SYNTHESIS
The regulation of skeletal muscle protein turnover is com-
plex but, in general, involves interactions of gene transcription,
Address for reprint requests and other correspondence: B. B. Rasmussen,
Univ. of Texas Medical Branch, Dept. of Physical Therapy, Div. of Rehabil-
itation Sciences, 301 Univ. Blvd. Galveston, TX 77555-1144 (e-mail: blrasmus
@utmb.edu).
J Appl Physiol 106: 13741384, 2009.
First published Janaury 15, 2009; doi:10.1152/japplphysiol.91397.2008.
8750-7587/09 $8.00 Copyright 2009 the American Physiological Society http://www. jap.org 1374
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translation and protein breakdown (as well as various pre- and
posttranscriptional modications). This section emphasizes
translational control of protein synthesis [particularly transla-
tion initiation through the mammalian target of rapamycin
(mTOR) pathway].
Translation initiation involves a series of events necessary
for ribosomal complex assembly and binding of the target
mRNA. A plethora of initiation factors and signaling mole-
cules (through phosphorylation and association steps) regulate
the formation of the translational apparatus, thus implicating
alternative, but tight, levels of control of the synthesis of new
proteins. Interestingly, these regulatory mechanisms can occur
within minutes of a particular stimulus (12).
mTOR is a key regulator of translational control. Nutrient,
hormonal, and contractile stimuli often (but not always) con-
verge at this protein, suggesting that mTOR is an important
modulator of protein synthesis. For example, an initial study
conducted by Baar and Esser (3) showed that the mTOR
pathway may be associated with long-term increases in muscle
mass due to the positive correlation between the acute phos-
phorylation of the downstream mTOR effector, p70 ribosomal
S6 kinase 1 (S6K1), and the increase in muscle mass over 6 wk
of electrical stimulation in rodent hindlimb muscles. Interest-
ingly, the association of S6K1 phosphorylation and muscle
mass was also demonstrated in human subjects after 12 wk of
resistance exercise training (98). To more fully elucidate the
role of mTOR, rapamycin, a specic inhibitor of mTOR
function, is commonly used to study muscle growth in cell and
animal models (2, 61). This was well demonstrated by Bodine
and colleagues (11) in which rapamycin was used to block
muscle hypertrophy following functional overload in rodents
further suggesting an important role for the mTOR pathway in
controlling skeletal muscle growth.
The mTOR protein is a very large molecule (289 kDa) with
many regulatory domains and is found within two protein com-
plexes: mTORC1 and mTORC2. In addition to mTOR, two
known proteins make up the mTORC1 complex [G protein
-subunit-like protein (GL) and regulatory associated protein of
mTOR (raptor)], while the mTORC2 complex is composed of
GL, mSin, and rapamycin-insensitive companion of mTOR
(rictor). The mTORC1 complex is sensitive to rapamycin and
plays an important regulatory role during the hypertrophy process
of skeletal muscle cells (11). Less is known about the regulation
of mTORC2 although it is not thought to be involved in the
regulation of translation initiation and elongation (94).
Two well-studied downstream targets of mTORC1 inuence
translation initiation and elongation: S6K1 and eukaryotic initia-
tion factor 4E binding protein (4E-BP1) (Fig. 1). Additionally,
mTORC1 has also been implicated in enhancing ribosomal bio-
genesis through cMyc (45, 97). S6K1, when activated, can phos-
phorylate at least nine different targets (92), including ribosomal
protein S6 (rpS6) (89) and eukaryotic elongation factor 2 (eEF2)
kinase (108). Phosphorylation of rpS6 has been reported to en-
hance cell size and proliferation (91), although its role has been
questioned (91). Reduced phosphorylation of eEF2 due to
eEF2 kinase inhibition results in mediation of the ribosome
to the mRNA following each amino acid addition, thereby
contributing to a growing peptide chain (16). mTORC1
phosphorylation of 4E-BP1 inhibits the binding of eukary-
otic initiation factor (eIF) 4E to 4E-BP1, thereby promoting
Fig. 1. A simplied schematic representation
of the mammalian target of rapamycin complex
1 (mTORC1) signaling pathway driven by
muscle contraction, insulin, essential amino ac-
ids (leucine), and/or energy. Proteins have
been labeled to designate them as a positive
(green) or negative (red) regulators of
mTORC1 and muscle protein synthesis.
AMPK, AMP-activated protein kinase; Akt,
protein kinase B; TSC1, tuberous sclerosis
complex 1; TSC2, tuberous sclerosis complex
2; REDD1/2, regulated in development and
DNA damage responses; Rheb, Ras-homo-
logue enriched in brain; TCTP, translation-
ally controlled tumor protein; PAM, protein
associated with Myc; Raptor, regulatory as-
sociated protein of mTOR; GL, G protein
-subunit-like protein; MAP4K3, mitogen
activated protein kinase kinase kinase ki-
nase-3; hVps34, human vacuolar protein
sorting-34; S6K1, p70 ribosomal S6 kinase 1;
4E-BP1, 4E binding protein 1; eEF2k, eu-
karyotic elongation factor 2 kinase; eEF2,
eukaryotic elongation factor 2; rpS6, ribo-
somal protein S6; PRAS40, proline-rich Akt
substrate-40.
Review
1375 MUSCLE PROTEIN SYNTHESIS AND MTORC1 SIGNALING
J Appl Physiol VOL 106 APRIL 2009 www.jap.org
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eIF4E to complex with eIF4G, further enhancing formation
of the translation initiation complex (109).
There are several proteins that respond to nutrients, hormones,
and muscle contraction, which also regulate the function of
mTORC1. Rheb (Ras-homologue enriched in brain), the imme-
diate upstream guanosine triphosphate (GTPase), is often targeted
in the regulation of mTORC1. For instance, translationally con-
trolled tumor protein (TCTP) has been proposed to catalyze the
GDP 3GTP reaction, thereby enhancing mTOR activity and cell
growth (53). Added to the rapidly growing eld of mTORC1
regulation is the E3 ubiquitin ligase, protein associated with Myc
(PAM), which has also been postulated to regulate GTP/GDP
exchange of Rheb directly thereby activating mTOR (70). Akt
(protein kinase B) can directly activate mTORC1 through phos-
phorylation (76) or indirectly by phosphorylating (and inhibiting)
tuberous sclerosis complex 2 (TSC2) (54, 71). mTORC1 is also
positively regulated by amino acids, particularly leucine (1, 2). To
date, two proteins have been implicated in mediating signals from
amino acids to mTORC1: human vacuolar protein sorting-34
(hVps34) and mitogen activated protein kinase kinase kinase
kinase-3 (MAP4K3) (17, 38). Interestingly, mTORC1 can also be
activated by contraction independent of amino acids, Akt, and
growth factors (50, 51). This has been described to occur through
phospholipase D1 and 2 using phosphatidic acid (PA) as a second
messenger (52). Unfortunately, few experiments have explored
the role of these positive mTORC1 regulators in animal and
human skeletal muscle.
On the other hand, REDD (regulated in development and
DNA damage responses) 1 and 2 have been identied as
negative regulators of mTORC1 (23), possibly through their
interaction with TSC2 (29). Another novel negative regulator
of mTORC1 is PRAS40 (proline-rich Akt substrate-40). While
its function remains elusive, PRAS40 has been shown to bind
mTOR via raptor to repress mTORC1 signaling (106, 107).
The inhibitory function of PRAS40 is reduced when phosphor-
ylated by Akt or mTORC1 (107). Finally, because synthesizing
proteins is an energetically expensive cellular process, it is not
surprising to nd a specic regulator of mTORC1 function
when energy is not sufcient. Indeed, mTORC1 has been
shown to be inhibited by AMP-activated protein kinase
(AMPK) through enhanced TSC2 activity (55) and, recently,
by phosphorylating raptor (47).
Another important signaling pathway involved in regulating
translation initiation and elongation is the extracellular signal-
regulated kinase 1/2 (ERK1/2) pathway (Fig. 2). Cross talk of
the ERK1/2 and mTORC1 signaling pathways has been iden-
tied to occur through the ERK1/2 proteins, most likely
through direct interaction with TSC2 (67, 88) or indirectly by
phosphorylation of p90 ribosomal protein S6 kinase polypep-
tide 1 (RSK1) (89). ERK1/2 can also enhance protein synthesis
independent of the mTORC1 pathway (40) through MAP
kinase-interacting kinase 1 (MNK1) signaling to eIF4E (110).
Much of these data are generated from rodents, Drosophila,
and cell lines. Few data, if any, are available on mTORC1
signaling in human skeletal muscle in response to an ana-
bolic stimulus. The following describes current data from
human skeletal muscle in which muscle protein synthesis
and mTORC1 signaling were assessed following nutrition,
resistance exercise, or the two in combination.
NUTRITIONAL AND CONTRACTILE REGULATION OF HUMAN
MUSCLE GENE EXPRESSION
Traditionally (although now heavily debated), the mTORC1
pathway has been postulated to regulate the transcription
of specic genes particularly the 5 terminal oligopyrimidine
(5 TOP) mRNAs possibly through rpS6. Recent evidence in
drosophila has indicated that TOR can increase expression of
genes associated with ribosomal biogenesis such as cMyc (45,
97), although the mechanism is not well understood. However,
few studies have evaluated the impact of resistance exercise
and/or essential amino acids on gene expression responses
associated with proteins of the mTORC1 signaling pathway in
skeletal muscle. Although the mRNA species is a precursor to
the proteins that inuence translation initiation and elongation,
repeated transient changes in genes may inuence specic
protein accumulation or removal. For instance, our research
group identied a decrease in basal Akt and S6K1 mRNA
expression after 10 wk of paraplegia in rodents compared with
healthy controls (35). This nding paralleled the decreased
protein expression of Akt and S6K1 and a concomitant de-
crease in protein synthesis (31). In another study, our labora-
tory evaluated the expression pattern of mRNAs associated
with the mTORC1 pathway following a single bout of low-
intensity resistance exercise in human skeletal muscle. Al-
though mTOR and S6K1 mRNA expression were unchanged
3 h postexercise, REDD1 mRNA expression, a negative regu-
lator of mTORC1, was signicantly decreased (34). The rapid
change in REDD1 mRNA expression was not entirely surpris-
ing because REDD1 protein has a fast turnover rate (5 min)
(62), but instead suggests that acute changes in the mRNAs
associated with the regulation of mTORC1 may indirectly
impact the function of this integral protein. In another recent
publication, our laboratory found that Rheb mRNA expression,
the primary positive regulator of mTORC1, was increased,
whereas TSC1 and TSC2 mRNA was decreased in human
skeletal muscle within hours of resistance exercise and essen-
tial amino acid ingestion (36). Thus it is tempting to speculate
that with repeated bouts of exercise and/or supplementation
with essential amino acids, transient changes in expression of
mTORC1 pathway-related genes may change expression of
specic proteins over time.
CONTRACTILE REGULATION OF HUMAN MUSCLE
PROTEIN SYNTHESIS
Early work demonstrated that exercise produces signicant
changes in whole body protein metabolism and hinted at the
possibility that muscle protein turnover was depressed (84, 85).
Our laboratory conrmed that muscle protein synthesis was
indeed depressed during an acute bout of resistance exercise
(30). However, the inhibition of muscle protein synthesis that
occurs during muscle contraction is rapidly reversed during
postexercise recovery. For example, several human studies in
the early 1990s demonstrated that an acute bout of resistance
exercise stimulated muscle protein synthesis during the rst
few hours of recovery (8, 21, 68, 113). From these and later
investigations it is now well established that a single bout of
resistance exercise, even when performed in the fasted state,
increases human skeletal muscle protein synthesis.
Muscle protein breakdown is also increased after resistance
exercise (80, 81). A positive correlation has been identied
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between the rate of muscle protein synthesis and the rate of
muscle protein breakdown 3 h following resistance exercise,
suggesting a link between the two processes during the early
recovery hours (80). A potential mechanism for this associa-
tion may be that high-resistance muscle contractions increased
the activity of the nutrient sensor hVps34 (69), a protein
identied as being an amino acid sensor that activates
mTORC1 (17) and an activator of autophagy through its
binding partner hVps15 and becklin-1 (4). It has been sug-
gested that an increased S6K1 activity (which correlates with
increased hVps34 activity) may concurrently stimulate muscle
protein synthesis and protein breakdown in rodents (69). None-
theless, muscle protein synthesis is stimulated to a greater
extent than breakdown, leading others to propose that the
higher rate of protein synthesis is a key factor in promoting
skeletal muscle hypertrophy due to resistance exercise train-
ing (80).
The mechanisms for the increase in human muscle protein
synthesis following resistance exercise were relatively un-
known a few years ago. Recent work has begun to combine
traditional stable isotope infusion studies with analysis of key
cell signaling pathways known to be involved in the regulation
of translation initiation and protein synthesis following human
muscle contraction (30, 37, 44). From these initial studies, it
appears that activation of the mTORC1 signaling pathway is
playing an important role in stimulating muscle protein syn-
thesis following muscle contraction. For example, a key down-
stream effector of mTORC1, S6K1, is readily phosphorylated
in the rst 16 h following a bout of resistance exercise
concurrently with an increase in muscle protein synthesis (30,
44). Another downstream effector of mTORC1 is 4E-BP1, and
our laboratory has consistently shown that 4E-BP1 phosphor-
ylation is reduced during resistance exercise and is un-
changed during postexercise recovery (30, 32). Thus, in our
Fig. 2. A simplied schematic representation of the inter-
action between the mTORC1 and ERK1/2 signaling path-
ways following muscle contraction and/or insulin stimu-
lation. mTORC1 pathway is indicated in blue, while the
ERK1/2 pathway is in purple. MEK1/2, mitogen-activated
protein kinase kinase-1/2; ERK1/2, extracellular signal-
regulated kinase 1/2; MNK1, MAP kinase-interacting ki-
nase 1; PI3K, phosphatidylinositol 3-kinase; RSK1, p90
ribosomal protein S6 kinase.
Review
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laboratorys hands, it appears that the contraction induced
increase in muscle protein synthesis is independent of changes
in 4E-BP1 phosphorylation which is in stark contrast to the
large increase in 4E-BP1 phosphorylation detected when es-
sential amino acids are ingested (32, 33).
Time course studies for the signaling events discussed above
have also been carried out because previous work has shown
that the rate of human skeletal muscle protein synthesis re-
mains elevated for up to 24 h in trained individuals (68) and for
48 h (80) and 72 h (74) in untrained individuals. Deldicque and
colleagues (27) showed that S6K1 phosphorylation remained
elevated at 24 and 72 h after completing 10 sets of 10
repetitions of leg exercise performed at 80% of a one repetition
maximum (27). This suggests that sustained phosphorylation
of S6K1 may play a role in maintaining synthesis rates of
muscle proteins above baseline for similar durations in un-
trained individuals. At present, no concurrent measurements of
human muscle protein synthesis and mTORC1 signaling data
exist for time points greater than 6 h postresistance exercise.
However, it should be noted that other studies have shown
increases in muscle protein synthesis using different modes of
contraction such as endurance exercise (19) and dynamic
shortening and lengthening exercise (26). It is likely that
different modes of contraction may result in a somewhat
different cell signaling response.
Resistance exercise in humans has also been shown to alter
other components of the mTORC1 signaling pathway includ-
ing Akt and eukaryotic elongation factor 2 (eEF2). Akt is an
upstream regulator of mTORC1 (Fig. 1) which is responsive to
both insulin and muscle contraction. Akt phosphorylation is
increased at 10 min (24) and at 1 and 3 h postresistance
exercise (30, 44). However, changes in Akt phosphorylation
have not been shown in all studies (33, 37, 72, 96). Possible
explanations for the varying results may include glycogen
status, differences in exercise intensity, antibody specicity,
and isoform expression.
Our laboratory has recently shown that translation elonga-
tion also appears to be sensitive to muscle contraction since
eEF2 phosphorylation is decreased (indicator of enhanced
elongation) following resistance exercise (30, 33). Contractile
regulation of eEF2 may be mediated by S6K1 phosphorylation
(and inactivation) of eEF2 kinase (108). It has also been shown
that resistance exercise decreases the phosphorylation of
eIF2B, which likely plays a key regulatory role in stimulating
muscle protein synthesis during recovery (44). Although these
studies suggest that the mTORC1 pathway is involved in
regulating muscle protein synthesis postexercise, it should be
acknowledged that other important signaling pathways which
are not necessarily a component of the mTORC1 network may
also be involved in regulating muscle protein synthesis such as
the ERK1/2 pathway (Fig. 2). Our laboratory and others have
shown that ERK1/2 and MNK1 are readily phosphorylated
during postexercise recovery (33, 112). Our laboratory has
recently proposed that maximal stimulation of muscle protein
synthesis during postexercise recovery likely requires dual
activation of both the mTORC1 and the ERK1/2 signaling
pathways; however, much more work needs to be done in this
area (33). It should be noted that some studies fail to demon-
strate signicant increases in the phosphorylation of some
components of these signaling pathways, perhaps due to dif-
ferences in exercise intensity (57), mode of contraction (37),
and/or glycogen status (24). In any event, evidence from the
data highlighted in this section clearly shows that the increase
in muscle protein synthesis following a bout of resistance
exercise is associated with an activation of the mTORC1
signaling pathway in humans.
NUTRITIONAL CONTROL OF HUMAN MUSCLE
PROTEIN SYNTHESIS
The effect of nutrients on muscle protein turnover was
originally measured in humans using stable-isotopic tech-
niques in vivo by Rennie et al. in 1982 (86). Stable isotopic
methodologies developed over the past 25 yr as well as
advances in measuring intracellular signaling events and gene
expression have greatly advanced our knowledge of nutritional
regulation of muscle protein turnover. Although earlier studies
showed increases in muscle protein synthesis of 30100%
following nutrient ingestion or infusions of mixed amino acids
(6, 86), we now know that this can primarily be attributed to
essential amino acids alone (104).
Ingestion of essential amino acids with or without carbohy-
drate has been shown to increase muscle protein synthesis (42,
104). In humans, increases in protein synthesis caused by the
ingestion of essential amino acids in combination with carbo-
hydrate are associated with increased mTORC1 signaling as
well (25, 42). In particular, our laboratory previously reported
that phosphorylation of Akt, mTOR, 4E-BP1 and S6K1 were
increased and eEF2 was decreased in association with an
100% increase in muscle protein synthesis following inges-
tion of a leucine-enriched essential amino acid and carbohy-
drate nutrient solution (42). The branched-chain amino acid
leucine has received much attention for its ability to increase
muscle protein synthesis and activate mTORC1 without the
addition of other essential amino acids (2). The specic cellular
mechanism for the unique ability of leucine to independently
stimulate muscle protein synthesis remains to be elucidated,
although evidence in animal models indicates (see below) it is
most likely through enhanced translation initiation (1, 2, 13).
The role of carbohydrate on protein turnover has been less
clearly dened. The major consensus is that essential amino
acids stimulate protein synthesis but do not affect breakdown,
while insulin reduces protein breakdown with little to no
signicant effect on protein synthesis if blood amino acid
concentrations are not maintained (7, 22, 25, 41). A recent
study by Chow et al. (22) comparing several surrogate mea-
sures of muscle protein synthesis and breakdown with mea-
sures of amino-acyl tRNA (a precursor for synthesis) and mass
balance of trace amino acid (for breakdown) conrmed these
hypotheses (22). Other studies examining the effect of carbo-
hydrate following resistance exercise have also shown a de-
crease in protein breakdown with either local hyperinsulinemia
(10) or ingestion of carbohydrate (15) with no additional effect
on protein synthesis. Carbohydrates may also affect AMPK,
decreasing its activation and thereby reducing the AMPK
inhibition of the mTOR pathway (for a review, see Ref. 49).
New evidence has revealed that essential amino acids may
not stimulate the mTORC1 pathway by activating the p85/p110
phosphatidylinositol 3-kinases (PI3K) and downstream targets
PDK1 and Akt, traditionally seen in insulin and growth factor
signaling (77). Instead, new data from cell culture and Dro-
sophila experiments demonstrate that essential amino acids can
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directly stimulate mTOR via a recently characterized class 3
phosphatidylinositol kinase, hVps34 (17) as well as through
the Sterile-20 (Ste20) protein kinase, MAP4K3 (38). A family
of small GTPases known as Rag proteins may also be impor-
tant in promoting the intracellular localization of mTORC1
toward Rheb during amino acid stimulation, although this has
only been observed in human embryonic kidney cell cultures to
date (93). Currently, mTORC1 is thought to be regulated by at
least three known inputs: glucose via insulin signaling stimu-
lation and decreased AMPK activation (20, 55); amino acids
via hVps34 (17), MAP4K3 (38) and the Rag GTPases (93);
and growth factors through Akt and the TSC1/2 complex (66).
Nutrients can directly or indirectly affect each of these
inputs, conrming the importance of nutrition in regulating
the mTORC1 pathway.
CONTRACTILE AND NUTRITIONAL REGULATION OF HUMAN
MUSCLE PROTEIN SYNTHESIS
Resistance exercise is clearly a strong stimulus for promot-
ing skeletal muscle hypertrophy. A single bout of resistance
exercise increases both the rate of muscle protein synthesis and
the rate of muscle protein breakdown, and thus the net balance
of muscle proteins remains negative in the absence of nutri-
tional intake. Muscle protein accretion can only occur with a
positive net balance of proteins. Protein, amino acids, and
carbohydrate ingestion following resistance exercise improves
net muscle protein balance by stimulating muscle protein
synthesis and possibly by also inhibiting muscle protein break-
down.
Increasing the availability of amino acids provides substrate
for de novo protein synthesis and also leads to an increase in
the circulating plasma insulin concentration (39). For example,
an intravenous administration of amino acids following a bout
of resistance exercise stimulates muscle protein synthesis (9).
The oral administration of amino acids has also been studied,
as a more practical delivery method for increasing amino acid
availability to the muscle, and large amounts of amino acids
ingested after a bout of exercise also stimulates muscle protein
synthesis (15, 75, 99101). When smaller amounts of amino
acids are ingested, both with and without carbohydrate, similar
increases in postresistance exercise muscle protein synthesis
are observed (14, 83). The increase in muscle protein synthesis
observed with the ingestion of amino acids following exercise
can likely be explained by the increase in intracellular avail-
ability of amino acids activating mTORC1 (42). Another
explanation is that increased muscle protein synthesis is due to
a particular amino acid or group of amino acids, such as the
branched-chain amino acids. A recent paper suggests that the
addition of leucine to protein hydrolysate and carbohydrate led
to a greater increase in protein synthesis than protein hydroly-
sate and carbohydrate without leucine alone following resis-
tance exercise (63). The added leucine may exert its effects
through an increase in plasma insulin concentrations and/or
through a more pronounced activation of mTORC1 signaling
pathway (63).
Following a bout of resistance exercise, the ingestion of
carbohydrate in the form of glucose resulted in a decrease in
urea excretion, typical of a reduction in muscle protein break-
down (90). The ingestion of carbohydrate following exercise
does not stimulate muscle protein synthesis, so net protein
balance remains negative (15). The changes in muscle
protein metabolism can be attributed to the increase in plasma
insulin concentrations, although insulin has only a modest
effect on protein synthesis in the absence of amino acids (7).
Our laboratory and others have studied the effects of a
combination of amino acids and carbohydrate on postresistance
exercise muscle recovery. Not only has it been shown that
muscle protein synthesis is stimulated to a larger extent than
with nutrients or exercise alone but also that the addition of
carbohydrate to amino acids does not have an additive effect
when ample amounts of amino acids are ingested (15, 42). Our
laboratory recently reported an enhanced activation of muscle
protein synthesis and a substantial increase in phosphorylation
of Akt, mTOR, S6K1, and 4E-BP1 with ingestion of nutrients
(leucine-enriched essential amino acids carbohydrate) 1 h
following a bout of resistance exercise, indicating improved
translation initiation (32). Translation elongation also appeared
to be enhanced, as seen by a decrease in the phosphorylation of
eEF2 (32). One previous study found that ingestion of a small
amount of essential amino acids and carbohydrate immediately
before a bout of resistance exercise resulted in a higher rate of
estimated muscle protein synthesis than when the nutrients
were ingested immediately postexercise (100). However, when
using direct measures of muscle protein synthesis (i.e., frac-
tional synthetic rate), the ingestion of nutrients following
exercise has been shown to yield the greatest increase in
muscle protein synthesis (32). This is similar to resistance
exercise (30) and nutrient ingestion (42) individually or when
nutrients are provided before resistance exercise (43) (Fig. 3).
This potent anabolic muscle protein synthesis response is
supported with enhanced mTORC1 signaling, including a ro-
bust phosphorylation of S6K1 following the intervention (32).
The addition of carbohydrate to an amino acid nutritional
supplement following resistance exercise may improve net
Fig. 3. Effect of resistance exercise and the timing of nutrient (leucine-
enriched essential amino acids carbohydrates) ingestion on muscle protein
synthesis in young human subjects. Data are presented as percent change in
fractional synthetic rate (FSR) from baseline. Resistance Exercise, percent
increase in FSR for 2-h period of postexercise recovery (n 11; 7 men and 4
women) (Ref. 30); Nutrient Ingestion before Resistance Exercise, percent
increase in FSR for 2 h period of postexercise recovery (nutrients ingested 1 h
before exercise) [n 11, 6 men (Ref. 43) and new data from 5 female
subjects]; Nutrient Ingestion, percent increase in FSR 1 h following nutrient
ingestion (n 11; 6 men and 5 women) (Ref. 42); Nutrient Ingestion after
Resistance Exercise, percent increase in FSR 1 h following nutrient ingestion
when nutrients were ingested 1 h after resistance exercise (n 6 men) (Ref.
30). *Signicantly greater than baseline, P 0.05.
#
Signicantly greater than
nutrient ingestion before resistance exercise, P 0.05.
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protein balance through a decrease in muscle protein break-
down and/or by increasing insulin levels to support muscle
protein synthesis (15).
Our laboratory published a study identifying the effects of
ingesting an essential amino acid and carbohydrate solution
before a bout of heavy resistance exercise on muscle protein
synthesis (43). Ingestion of nutrients before exercise did not
improve the anabolic muscle protein synthesis response com-
pared with resistance exercise alone (30, 43). The ingestion of
amino acids before resistance exercise did prevent the exercise-
induced decrease in muscle protein synthesis, as identied
previously when resistance exercise is performed in the fasted-
state (30), but did not improve the muscle protein synthesis
response following the bout of exercise compared with resis-
tance exercise in the fasted state. However, a recent paper has
shown that when protein is ingested during a 2-h workout that
includes resistance exercise, muscle protein synthesis was
increased (5), although the increase was much less than seen
when essential amino acids are ingested postexercise (32).
These recent ndings suggest that the increased availability of
amino acids and elevated plasma insulin levels during postex-
ercise recovery enhance muscle protein synthesis likely via
activation of the Akt/mTORC1 signaling pathway.
AGING, NUTRITION, AND RESISTANCE EXERCISE
Numerous studies have examined the cellular and molecular
mechanisms associated with the decline in muscle mass that
accompanies aging. Because basal rates of muscle protein
synthesis are similar between age groups (103), older humans
may be impaired in their ability to properly respond to an
anabolic stimuli (e.g., resistance exercise and/or essential
amino acids) compared with the young. Volpi and colleagues
(102) identied that a 3-h amino acid infusion stimulated
muscle protein synthesis to a similar extent in young and older
subjects. However, in response to ingestion of essential amino
acids, older subjects are less sensitive to smaller doses of
essential amino acids (7 vs. 15 g) compared with the young
(58, 78). Cuthbertson et al. (25) and Guillet et al. (46) have
shown that the reduced anabolic response to nutrients in older
adults may be associated with dysregulated mTORC1 signal-
ing. However, it appears that adding additional leucine to a
nutritional supplement in older adults may be necessary to
optimally stimulate muscle protein synthesis to a similar rate as
in the young (59). Interestingly, Rieu et al. (87) supplemented
meals with leucine, which restored the anabolic effects of
feeding on muscle protein synthesis in older adults. Therefore,
it may be important for older individuals to eat a sufcient
amount of protein with each meal to ensure sufcient leucine
availability for maximizing muscle protein synthesis during
feeding (79). Although it should be kept in mind that a
high-protein diet in older adults may present some physiolog-
ical concerns. For example, in one study (105), older subjects
demonstrated a tendency for a reduced glomular ltration rate
compared with healthy young subjects, who signicantly in-
creased glomular ltration rate following the high protein diet
(3 g protein/kg fat-free mass). This may result in a detrimental
impact on renal function if continued over time. Together,
ingesting a sufcient amount of essential amino acids can
stimulate muscle protein synthesis in older adults to a similar
extent as in the young (102).
As mentioned previously, resistance exercise is an excellent
way to stimulate muscle protein synthesis. However, earlier
work indicated that muscle protein synthesis was unchanged in
older adults 3 h following a single bout of heavy resistance
exercise compared with the increase seen in the younger
subjects (95). Additionally, a study by Kumar et al. (65)
showed that older adults have a blunted muscle protein syn-
thesis response compared with young adults following resis-
tance exercise (6090% 1 repetition maximum). These nd-
ings are novel and support the idea of an impaired ability in
older adults to respond to a protein anabolic stimulus, with the
end result being an acute dysregulation of muscle protein
synthesis. The signaling pathways associated with acute dys-
regulation in muscle protein synthesis following resistance
exercise in older adults are not yet clear. Several articles have
indicated an altered anabolic response at the gene and protein
level during the early recovery hours following resistance
exercise in older human skeletal muscle. The impairment
covers a vast array of molecules from anabolic and cell cycle
to remodeling and inammatory genes and proteins (18, 28, 33,
36, 56, 60, 112). In previous work, our laboratory suggested
that older men were not able to generate the same level of
muscular tension as the young during a bout of heavy resis-
tance exercise (33). In addition to the suggested age-related
differences in ber type, muscle mass and postresistance ex-
ercise lactate responses, we attributed this claim to a dysregu-
lation in translation initiation-associated cellular signaling.
Following resistance exercise, older subjects had a tendency
for lower S6K1 phosphorylation but, more interesting, a
blunted ERK1/2 and MNK1 signaling compared with the
young when exercising at the same relative exercise intensity
(33). Although mTORC1 cellular signaling responses were
somewhat similar between age groups in our laboratorys study
(33), we propose, as mentioned previously, that ERK1/2 and
mTORC1 pathways must be dually stimulated to achieve
maximal activation of muscle protein synthesis during the early
recovery hours following resistance exercise. In support of this
hypothesis, IGF-I induced muscle hypertrophy was blunted
(reduced S6K1 phosphorylation) following inhibition of the
ERK1/2 pathway in rodent skeletal muscle (48). Thus it is
tempting to speculate that a specic site on S6K1 is targeted
(directly or indirectly) by ERK1/2 and that a lack of ERK1/2
activity would prevent the activation of all S6K1 phosphory-
lation sites that are required for full activity of this enzyme
(82). However, this may not be the case when essential amino
acids are ingested. According to Karlsson et al. (57), the
branch-chained amino acids activate translation initiation
that is dependent on mTORC1, but not ERK1/2 signaling in
humans. Together, these data suggest that an impaired
ERK1/2 signaling pathway may be associated with the
blunted muscle protein synthesis response in older subjects
following an acute bout of resistance exercise. Further work
is needed to evaluate whether additional cellular pathways
contribute to the acute impaired muscle protein synthesis
response in aging human skeletal muscle.
Over the last couple of years, the ingestion of essential
amino acids or protein following resistance exercise has been
evaluated in older humans. In these studies, muscle protein
synthesis was similar in young and older men 6 h following
resistance exercise (33, 64). Interestingly, muscle protein syn-
thesis was delayed at 3 h postexercise but was restored a few
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hours later (33). Although the muscle protein synthesis re-
sponse was similar over the 6 h postresistance exercise period
in both age groups, we are unsure whether this muscle protein
synthesis response remains the same or diverges over a longer
time course (e.g., 24 h postresistance exercise). Further re-
search is required to evaluate the effectiveness of essential
amino acid supplementation following repeated bouts of resis-
tance exercise to determine whether the muscle protein syn-
thesis response continues to be similar between young and
older subjects.
SUMMARY AND CONCLUSIONS
In summary, human skeletal muscle protein synthesis is
increased following the ingestion of essential amino acids
carbohydrates and following an acute bout of resistance exer-
cise. However, muscle protein synthesis is increased to a
greater extent when the nutrients are ingested following resis-
tance exercise. Our data, as well as others, indicate that the
mTORC1 signaling pathway and alterations in the expression
of key genes associated with the regulation of protein turnover
may contribute to the acute increase in muscle protein synthe-
sis response following resistance exercise and nutrition. Fur-
thermore, muscle protein synthesis is impaired in the old
following a single bout of resistance exercise in conjunction
with altered anabolic gene and cell signaling responses. Pro-
viding nutrition following resistance exercise in older adults
appears to increase muscle protein synthesis to levels similar to
the young but in a delayed manner. We conclude that essential
amino acids and/or resistance exercise stimulate human muscle
protein synthesis in association with enhanced mTORC1 sig-
naling. By better understanding the cellular mechanisms reg-
ulating muscle protein synthesis, we are now able to scientif-
ically design exercise and nutritional strategies to include
ingestion of leucine-enriched essential amino acids following
resistance exercise to maximally promote muscle protein syn-
thesis and growth. These strategies may have important clinical
application in patients with muscle wasting conditions such as
aging, cancer, physical inactivity/bed rest, and they may be
useful in improving muscle rehabilitation following trauma
and/or surgery.
GRANTS
This work was supported by National Institute of Arthritis and Musculo-
skeletal and Skin Disease Grant R01 AR-049877. Additional support came
from Grant P30 AG-024832 [National Institutes of Health (NIH)/National
Institute on Aging] and M01 RR-00073 from the General Clinical Research
Branch, National Center for Research Resources (NIH).
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Skeletal Muscle Protein Synthesis and mTORC1 Nutritional and Contractile Regulation of Human

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