Role of Leucine in Protein Metabolism

During Exercise and Recovery

Donald K. Layman

Cataiog Data
Layman, D.K. (2002). Role of leucine in protein metabolism during exercise and recovery.
Can. J. Appi. Physioi. 27(6): 646-662. ©2002 Canadian Society for Exercise Pbysiology.

Keywords: branched-chain amino acids, insulin, protein synthesis, skeletal muscle

Mots-cles: acides amines a chaine ramifiee, insuline, synthise de proteines, muscle
squelettique

Abstract/Resume
Exercise produces changes in protein and amino acid metabolism. These changes include
degradation ofthe branched-chain amino acids, production of aianine and glutamine, and
changes in protein tumover. One ofthe amino acid most affected by exercise is the branched-
chain amino acid leucine. Recently, there has been an increased understanding ofthe role
of leucine in metabolic regulations and remarkable new findings about the role of leucine in
intracellular signaling, Leucine appears to exert a synergistic role with insulin as a regula-
tory factor in the insulin/ phosphatidylinositol-3 kinase (PI3-K) signal cascade. Insulin
serves to activate the signal pathway, while leucine is essential to enhance or amplify the
signal for protein synthesis at the level ofpeptide initiation. Studies feeding amino acids or
leucine soon afier exercise suggest that post-exercise consumption of amino acids stimulates
recovery of muscie protein synthesis via translation regulations. This review focuses on the
unique roles of leucine in amino acid metabolism in skeletal muscle during and afier exercise.

L 'exercice modifie le metaboUsme des proteines et des acides amines. Parmi tes changements,
notons la degradation des acides amines a chaine ramifiee, la production d'aianine et de
glutamine ainsi que ie taux de renouveliement des proteines. La leucine, un acide amine a
chaine ramifiee, est un des acides amines les plus touches par I'exercice, Au cours des
demiires annees, de nombreuses etudes ont contribue a I 'enrichissement des connaissances
sur le role de la leucine dans la regulation du metaboUsme; les etudes ont egalement devoile

The author is with the Department of Rood Science and Human Nutridon, University
of Illinois, Urbana, IL.

646
 Leucine in Protein Metabolism • 647

les importantesfonctions de cet acide dans la signalisation intracellulaire. La leucine semble

jouer un role synergique avec I'insuline en tant que facteur de regulation dans la cascade
des signaux de l'msuline/phosphatidylinositol-3 kinase. L'insuline enclenche le processus
et la leucine est indispensable pour ameliorer ou amplifier le signal declenchant la synthise
des proteines au niveau des peptides. Les etudes sur la consommation d'acides amines ou
de leucine peu apres la fin de l'exercice mettent de Vavant que la consommation d'acides
amines apris la fin de l'exercice stimuie la recuperation des proteines musculaires par des
regulations de translation. Cet article met I'accent sur les roles particuliers de la leucine
dans le metabolisme intramusculaire des acides amines au cours et apres un exercice.

Introduction
Dietary protein has been assumed to be important for physical performance since
ancient times when consumption of meat was thought to enhance strength and
endurance. However, lipids and carbohydrates are clearly established as the major
fuels for muscle contraction (Wolfe, 1998) and relatively little additional protein is
believed necessary for muscle hypertrophy (RDA, 1989). Still most current day
athletes consume copious quantities of protein under the belief that protein intake
is a key to muscle development (Hickson and Wolinsky, 1996). For reviews of
exercise and protein metabolism see Paul et al. (1997), Wagenmakers (1998a), and
Rennie and Tipton (2000).
Evaluating the roles of protein and amino acids in maintaining the structure
and function of skeletal muscle is complicated by the diversity of the individual
amino acids. For carbohydrates and lipids, the basic molecules are glucose and
palmitate and the functions are largely limited to generation of energy via ATP or
storage as glycogen or fat. On the other hand, there are twenty different amino
acids required for protein structures and many of these amino acids also partici-
pate in numerous other metabolic reactions. In skeletal muscle, the amino acid
with the most complex of these metabolic roles may be the branched-chain amino
acid leucine.
Anabolic effects of leucine on muscle protein have been reported for over
twenty years (Buse and Reid, 1975; Li and Jefferson, 1978; Tischler et al, 1982),
however, only recently have researchers begun to understand and integrate the
overall metabolic roles of this amino acid (Hutson and Hards, 2001). Leucine
participates in metabolism in diverse ways including 1) as a substrate for protein
synthesis, 2) as a fuel, and 3) as a metabolic signal. The most obvious role for any
atnino acid is as the fundamental unit for building protein. As a substrate for muscle
protein synthesis, leucine's role is similar to the other twenty amino acids; how-
ever, in skeletal muscle leucine is disproportionately incorporated into proteins
accounting for approximately 9% of muscle atnino acids (RDA, 1989). Leucine
also functions as a metabolic fuel in muscle. Skeletal muscle is the principle site
for degradation of the three branched-chain amino acids, leucine, isoleucine and
valine. Degradation of these three amino acids in muscle provides carbon for use
as a direct energy source and also provides the stimulus for synthesis of alanine
and glutamine. Finally, the most recent and perhaps most intriguing role for leu-
cine is its participation in intracellular signal transduction as part of the insulin
signal cascade. Recent discovery of leucine's participation in signaling begins to
suggest links among the diverse metabolic roles of this essential atnino acid.
648 • Layman

Exercise Stimulates BCAA Metabolism

Collectively, the three branched-chain amino acids (BCAA) make up over 20% of
the amino acids in the food supply and account for three of the nine essential
amino acids in the diet. Further, the BCAA have the distinction of being the only
amino acids that are largely degraded in skeletal muscle. Associated with these
properties, the BCAA, exhibit rapid changes in blood and tissue concentrations
associated with dietary intake, metabolic stress, and exercise (Ahlborg et al., 1974;
Harper et al., 1984).
In 1974, Ahlborg et al. reported that exercise stimulated movement of amino
acids through the blood. They reported that interorgan movement of amino acids
was dominated by the three branched-chain amino acids (BCAA) and alanine. The
BCAA pass into the blood from the liver and gut and circulate to skeletal muscle
for use in synthesis of proteins or for degradation of the amino acids as a source of
energy. Degradation of the BCAA to energy results in release of the amino-nitro-
gen that is transferred from the BCAA on to either pyruvate or a-ketoglutarate to
form the non-essential amino acids alanine and glutamine (Figure 1). Alanine and
glutamine are released from muscle into the blood and circulate back to visceral

Liver Skeletal Muscle

BCAA
leucine
Isoleucine
valine

alanine

glutamine
Intestine

Figure 1. Picture represents movement of the BCAA from viseral tissues (liver and gut)
through blood circulation to uptake by skeletal muscle. In muscle tissue, BCAA are used
for protein synthesis, production of energy, and synthesis of alanine (Ala) and glutamine
(Gin). Alanine and glutamine are released from muscle and return to the liver and gut as
substrates for endogenous production of glucose. Abbreviations: BCAA branched-chain
amino acids, a-kg alpha-ketoglutarate.
 Leucine in Protein Metabolism • 649
tissues where tbe liver removes alanine and tbe gut, kidney and liver remove
glutamine. Tbis interorgan movement of amino acids serves multiple purposes
including delivering BCAA to muscle as a source of energy, supplying potential
intermediates for tbe TCA cycle, and providing a constant supply of alanine and
glutamine for endogenous production of glucose (Harper et al., 1984; Wagenmaker,
1998a; Young and Ajami, 2001).
Degradation of tbe tbree BCAA in skeletal muscle is largely dependent on
increases in tbe concentration of tbe amino acids (Harper et al., 1984). Tbe first
step in degradation is an aminotransferase tbat is sbared by tbe tbree BCAA and
driven by concentration. Tbe BCAA aminotransferase is predominately found in
muscle tissue and essentially absent in liver. Absence of tbe aminotransferase in
liver results in constant release of BCAA from visceral tissues, movement tbrougb
tbe blood and extraction by skeletal muscle. Witbin muscle, tbe aminotransferase
removes tbe a-amino group from tbe BCAA and transfers it either to pyruvate
forming alanine or to a-ketoglutarate forming glutamate. The second step in deg-
radation ofthe BCAA is also a shared enzyme known as the branched-chain ketoacid
debydrogenase (BCKAD) (Harris et al., 2001). BCKAD removes tbe alpha-car-
bon and comtnits the amino acid to complete degradation to energy.
Extraction of BCAA from plasma circulation increases during exercise in
proportion to intensity and duration (Ahlborg et al., 1974; Paul et al., 1996; Van
Hall et al., 1996). Movement of the BCAA into skeletal muscle stimulates their
oxidation (Wolfe et al., 1982). Early in exercise, changes in plasma amino acids
reflect an increased visceral release of BCAA and increased muscle production of
alanine. As the level of work progresses, production of alanine gives way to pro-
duction of glutatnine (Van Hall et al., 1995). These changes in tbe non-essential
amino acid products appear to be associated with the availability of pyruvate de-
rived from either blood glucose or muscle glycogen. At tbe beginning of exercise,
pyruvate appears to be readily available resulting in alanine production. As the
activity progresses, alanine production is replaced by production of glutamine from
a-ketoglutarate and glutamate. These reactions suggest a link between BCAA
metabolism and the balance of fuels during exercise (Wagenmaker, 1998a).
Supplementation of BCAA as ergogenic aids for muscle activity has been
tested with apparently minimal or no effects on performance (Blomstrand et al.,
1991; lackson et al., 1997; Madsen et al., 1996). However, the rate of BCAA deg-
radation is not insignificant with leucine oxidation ranging from 30 to 70 |xmol/
kg-hr for cycling activities at intensities of 30 - 55% VO^^^ (Evans et al., 1983;
Wolfe et al., 198^2). Depending on the size of the athlete and the intensity of tbe
exercise, leucine oxidation may reach nearly 1.0 g/hr. During intense exercise, we
observed a decline in plasma levels of BCAA after 90 minutes of intense cycling
and tbis decline continued into recovery (Paul et al, 1996).

Impact of Exercise on Muscle Protein Synthesis

Athletes have intense interest in muscle mass because ofthe relationship of muscle
mass to strength. Muscle mass is infiuenced by numerous factors but ultimately
the amount of protein is determined from the balance between the rates of protein
synthesis and protein breakdown. The combination of synthesis and breakdown is
called protein turnover. During muscle hypertrophy, the net balance of synthesis
650 • Layman
atid breakdown is positive producing muscle growth. To maximize muscle hyper-
trophy, athletes ofteti consume four to five times the RDA for protein (Hicksoti
and Wolitisky, 1996). However, maximum rates of growth or muscle hypertrophy
are less thati 0.5 kg per week. Assuming that protein accounts for 20% of this
weight change, maximum protein accretion is less than 15 grams per day for the
entire body, implying that the additional protein needs for muscle hypertrophy are
minimal. On the other hand, while the net balance is small, whole body protein
tumover accounts for over 300 grams of protein being synthesized and degraded
each day (Munro, 1982; Pacy et al., 1994) maximizing the potential for the body to
repair and remodel its structure. The discrepancy between the relatively small level
of daily protein deposition and the large rate of protein tumover makes evaluation
of protein needs challenging.
The individual processes of synthesis and breakdown are regulated by dif-
ferent mechanisms but appear to increase or decrease in a coordinate manner
(Millward et al., 1976). The relative importance of regulation ofsynthesis versus
breakdown in determining muscle mass is unknown. Under most conditions, the
anabolic response of protein tumover appears to be led by changes in protein syn-
thesis (Phillips et al., 1999). This review focuses on some remarkable new find-
ings about the molecular regulation of protein synthesis and the direct links to
insulin signaling and leucine levels.
Exercise is an anabolic stimulus resulting in increased muscle mass and
strength. Studies of isolated muscles demonstrate that constant stretch or electri-
cally induced contraction stimulate anabolic changes in muscle protein synthesis
at levels of both transcription and translation and produce muscle hypertrophy
(Goldspink, 1978; Laurant et al, 1978). While exercise has an anabolic effect on
muscle development, changes in protein tumover during exercise and key regula-
tory steps to achieve maximum muscle development have not been fully eluci-
dated.
In the early 198O's, a number of laboratories reported that immediately after
a bout of exercise muscle protein synthesis was reduced (Dohm et al., 1980; Rennie
et al., 1981; Wolfe et al.,1982). Research with rats indicated that the magnitude of
the inhibition was proportional to the intensity and duration of the activity such
that exhaustive, endurance running could depress muscle protein synthesis by as
much as 70% (Dohm et al., 1980). More recent work with experimental animals
appears to support these earlier findings (Balon et al., 1990; Davis and Karl, 1986;
Gautsch et al., 1998). The time-course for recovery of protein synthesis after exer-
cise is unknown, however, Tipton and Rennie (2000) suggest that protein metabo-
lism may remain negative in the absence of adequate food intake. Recently, we
found that protein synthesis remains depressed for at least eight hours after ex-
haustive exercise if an animal consumes nothing but fluids (Figure 2, unpublished
data). In total, these studies suggest that exercise of high intensity and long dura-
tion is a metabolic stress that inhibits that rate of protein synthesis. If this is true,
then the anabolic effects of exercise must be achieved during post-exercise recovery.
While intense exercise appears to depress protein synthesis in rats, the find-
ings from studies with humans are less clear. Investigators have reported decreases
(Rennie et al., 1981, Wolfe et al., 1982), no change (Carraro et al., 1990;
Wagenmakers , 1998b), or increases (Tipton et al., 1999) in protein synthesis after
exercise. The diversity of these findings has been attributed to differences in the
 Leucine in Protein Metabolism 651
14 •

* — Control
12 -
•*• Exercised

Protein ^
Synthesis
(%/day) 8 "

6 -

4 -
-2-1 0 1 2 3

Time (hours)

Figure 2. Time course changes in muscle protein synthesis after exhaustive exercise.
Rats were exercised for 2 hrs on a motor-driven treadmill at 36 m/min (~70% V02max) af-
ter a 12-hour fast. Rates of protein synthesis were determined by ineorporation of H-iso-
leucine into muscle protein using short-term bolus methods. Each data point represents 4
animals.

intensity or duration of the exercise protocol, to methodology differences among

isotopic tracers, and to differences between human and rats in their ability to sus-
tain intense exercise (Rennie and Tipton, 2000; Wagenmaker, 1998b).
Currently, it is not possible to fully reconcile these differences. An important
consideration in comparing animal and human exercise studies is to evaluate the
type of exercise. Most animal studies utilize endurance exercise with high inten-
sity (>70% VO^max), while most human studies use low intensity (40% VO^max)
or resistance exercise. Further, a problem inherent to human metabolic studies is
the need to make indirect measurements. Use of stable isotopes has been a tremen-
dous advancement in human metabolic research. However, use of stable isotopes
requires prolonged steady-state infusion in sufficient quantities to reach measur-
able enrichment ofthe amino acid pool. For these measurements to be interpreted,
methods require achieving and maintaining isotopic steady state. For exercise ex-
periments, this requirement is virtually impossible. Beginning with the metabolic
conditions at rest, exercise progresses from early metabolic adjustments toward
the physiology of exhaustion. Many investigators have attempted to minimize these
problems by using prolonged exercise. Unfortunately, prolonged exercise usually
requires exercise of lower intensity. Still other methods such as nitrogen balance
measurements appear too crude to reach definitive conclusions about short-term
regulations of protein synthesis. In the absence of full understanding of protein
metabolism during exercise, the molecular mechanisms and regulations obtained
from animals studies remain important.

Leucine Stimulation of Muscle Protein Synthesis

Regulation of protein synthesis occurs in numerous ways. The amount of protein
is controlled at the level of transcription of DNA into messenger and ribosomal
652 • Layman

Initiation Termination

Elongation

\
elF4
5"-mRNA —
AUG

ribosome peptide

Figure 3. Illustration represents translational regulation of protein synthesis emphasiz-

ing role of the initiation factors eIF2, eIF3 and eIF4. Initiation factor eIF2 participates in
formation of the ternary complex (43S pre-initiation complex) including the 40S riboso-
mal subunit and methionine-tRNA. The eIF4 is required for recognition and unfolding of
secondary structure at the 5'-end of the mRNA. The eIF3 is required to bind to the 40S
subunit during termination to maintain the subunit in a form active for protein synthesis.

RNA and at the level of translation of individual mRNA into peptides. In general,
transcription adjusts the capacity for synthesis of a protein. Short-term, minute-
by-minute controls of protein synthesis occur by regulation of the translation of
mRNA into individual proteins (Pain, 1996; Figure 3). Short-term controls are
modified by factors including hormones and the availability of energy or aniino
acids. These controls are exerted through at least twelve regulatory proteins called
eukaryote initiation factors (elF). Of these initiation factors, at least two of these
factors, eIF2 and eIF4 are subject to physiological regulations (Campbell et al.,
1999; Pain, 1996). Within skeletal muscle, the initiation factor eIF4 exhibits unique
regulation by the BCAA leucine (Anthony et al., 2001).
The first evidence that leucine could stimulate muscle protein synthesis ap-
peared in the 197O's. Using isolated diaphragm muscle and perfused himblimb
preparations, researchers demonstrated that supplementing the plasma or media
with a complete mixture of essential amino acids stimulated protein synthesis (Fulks
et al., 1975; Li and Jefferson, 1978). Further evaluation ofthe impact of individual
amino acids revealed that the stimulatory effect of the complete mixture could be
reproduced by the single amino acid leucine (Buse and Reid, 1975; Hong and
Layman, 1984; Tischler et al., 1978). Initially, these findings were interpreted to
suggest that leucine might have potential to stimulate muscle growth or reduce
muscle wasting in the critically ill. However, subsequent studies failed to show
anabolic effects of leucine during extended catabolic conditions (McNurlan et al.,
1982). Now it is recognized that leucine exerts its effect on short-term transla-
 Leucine in Protein Metabolism • 653
tional controls of protein synthesis and this translational effect is synergistic with
insulin (Anthony et al., 2001; Kimball et al., 1999).
Insulin is an anabolic hormone with a critical role in maintenance of muscle
protein synthesis (Kimball and Jefferson, 1994). The importance of insulin is clearly
demonstrated through use of chemical-induced diabetes (Flaim et al., 1980) or
anti-insulin antibodies (Preedy and Garlick, 1986) resulting in suppression of protein
synthesis. While insulin is essential for normal development, it is not sufficient
alone to stimulate muscle protein synthesis in postabsorptive conditions (Anthony
et al., 2000; Gautsch et al., 1998; Yoshizawa et al., 1998) unless supraphysiological
levels of insulin (>700 pmol/L) are provided (Garlick et al., 1983). Use of food
deprived rats demonstrates that feeding carbohydrates alone significantly increases
levels of plasma glucose and insulin but produces no effect on muscle protein
synthesis while feeding protein or amino acids can fully restore rates of protein
synthesis (Anthony et al, 2000; Gautsch et al., 1998). The anabolic effect of a
complete mixture of amino acids can be reproduced with the BCAA leucine alone
(Anthony et al., 2000). These findings suggest that the role of insulin in muscle
protein synthesis is more to potentate the translational system than for direct regu-
lation (Gautsch et al., 1998). The individual roles of insulin and leucine in intrac-
ellular signaling have begun to be elucidated (Anthony et al., 2001).

Leucine EfFects on Insulin Signaling

Intracellular hormone signaling is under intense investigation by researchers seek-
ing to define the mechanisms of action for growth factors including insulin, insu-
lin-like growth factor (IGF-1) and cytokines (Taha and Klip, 1999). Insulin and
other growth factors mediate a wide spectrum of biological responses including
cell division, regulation of gene expression, glucose transport, glycogen synthesis,
protein synthesis, and antilipolysis. These responses are initiated by the hormone
binding to a membrane bound receptor containing tyrosine kinase and triggering a
signal cascade beginning with phosphorylation of insulin receptor substrate-1
(IRS-1) (Figure 4). IRS-1 generates metabolic signals by binding to
phosphatidylinositol-3 kinase (PI3-K) and production of phosphatidyhnositol-3,4,5
phosphate (Tsakiridis et al., 1995). Through these phosphorylated inositol mol-
ecules, PI3-K plays a major role in regulation of the metabolic actions of insulin.
These actions include stimulation of glucose transport via stimulation ofthe GLUT4
transporter, synthesis of glycogen via glycogen synthase kinase-3, and stimulation
of protein synthesis via activation of downstream initiation factors eIF-4E and
p70^*K. Each of these physiological outcomes involves regulation by phosphory-
lation of key rate-limiting molecules.
While insulin is a primary stimulator of this pathway, chronic exposure of
cells to excess insulin produces down-regulation of the initial IRS-1 and PI3-K
steps (Cengel and Freund, 1999). These findings may represent important feed-
back regulations of the signal cascade and may be exerted by a downstream com-
ponent of the pathway named mTOR (Taha and Klip, 1999; Takano et al., 2001).
Further, the insulin signal pathway appears to be modulated by leucine which can
directly stimulate downstream components of this signal pathway independent of
changes in insulin concentration (Anthony et al., 2000a, 2000b; Gautsch et al.,
1998).
654 • Layman

Insulin
Glucose

Protein Synthesis

Figure 4. The intracellular signaling pathway illustrates the interaction of the hormone
insulin with the amino acid leucine. Leucine appears to amplify transmission of down-
stream signals via interaction at mTOR and has proposed upstream effects represented by
the dashed lines. Abbrevations: IRS-1 insulin receptor substrate-1, PI3-K
phosphatidylinositol-3 kinase, PKB protein kinase B, mTOR mammalian target of
rapamycin, eIF-4E is the 4E subunit ofthe eIF4 initiation complex, 4E-BP1 is an inhibi-
tory binding protein for eIF-4E.

The molecular mechanisms linking leucine and the signal pathway to con-
trol of muscle protein synthesis have begun to be elucidated (Hara et al., 1998;
Patti et al., 1998; Xu et al., 1999). The first critical finding was that leucine modi-
fies translational activity by stimulating a downstream protein kinase recognized
as mTOR (mammalian target of rapamycin). Through stimulation of mTOR, leu-
cine has the abihty to activate the initiation factor eIF4 and the 70-kDa ribosomal
protein S6 kinase (p70^*K) (Anthony et al., 2000; Hara et al., 1998; Xu et al.,
1999). The initiation factor eIF4 (also known as eIF4F) is a complex of three sub-
units: eIF4E, eIF4G and eIF4B, required for the initial recognition and unfolding
of secondary structure of the 5'-end of mRNA. To activate the eIF4 complex, first
requires release ofthe eIF4E subunit from an inhibitory binding protein (4E-BP1)
through phosphorylation of the binding protein by mTOR. Leucine serves to acti-
vate mTOR stimulating phosphorylation of 4E-BP1 causing release ofthe eIF4E
subunit. Once eIF4E is released from 4E-BP1, it is free to bind with the remaining
subunits (eIF4G and eIF4B) to form the active eIF4 initiation complex. Likewise,
mTOR also stimulates phosphorylation of p70^*K, which in tum activates the S6
ribosomal protein. Together, activation of eIF4 and p70^*K serve to initiate trans-
 Leucine in Protein Metabolism • 655
lation of significant components of muscle mRNA (Kimball and Jefferson, 2000).
These findings serve to link the anabolic response of dietary protein to muscle
protein synthesis through the ability to mTOR to detect changes in cellular leucine
concentrations.

Leucine Stimulates Protein Synthesis After Exercise

While changes in muscle protein tumover associated with exercise remain contro-
versial, studies evaluating changes in protein synthesis after exercise consistently
report post-exercise, anabolic responses enhanced by food intake (Farrell et al.,
1999; Gautsch et al., 1998; Tipton et al., 1999). Further, the anabolic responses to
post-exercise supplements appear to be derived from intake of relatively small
amounts of essential amino acids (Anthony et al., 2000; Rasmussen et al., 2000).
Recent work suggests that the anabolic response is associated with the signaling
role of leucine (Anthony et al., 2000; Gautsch et al., 1998).
As reviewed above, prolonged exercise with animals depresses muscle pro-
tein synthesis (Figure 2). Using this animal model, we tested the potential for a
pre-exercise meal to prevent the catabolic effects of exercise on protein synthesis
(Table 1). Our pre-exercise meal consisted of a complete nutritional drink (Ensure**,
Abbott Labs) and provided about 15% of the animal's daily energy needs. Use of
pre-exercise nutritional supplement had no impact on the exercise-induced de-
pression of muscle protein synthesis (Table 1).
Since exercise does not cause loss of muscle mass, the catabolic period dur-
ing exercise must be followed by an anabolic recovery period (Tipton and Rennie,
1999). We hypothesized that recovery of muscle protein synthesis after exercise
would require provision of protein or amino acids (Anthony et al., 1997).
Consumption of a complete nutrition supplement within the first 10 minutes after

Table 1 The Relationship of Endurance Exercise and a Pre-exercise Meal

on Muscle Protein Synthesis

Protein Synthesis'

Control (no exercise) 100%

Post-exercise^ 72%
Post-exercise with pre-exercise meaP
12 hrs 72%
5 hrs 74%
1.5 hrs 74%

'Protein synthesis determined by incorporation of ^H-isoleucine in to muscle protein and

expressed as a percentage of the control. ^Rats were exercised for 2 hrs on a motor-driven
treadmill at 36 m/min (~70% VO^^^^) after a 12-hour fast. ^The meal consisted of 5 ml of
Ensure" administered by oral gavage at the times indicated. (Unpublished data, Anthony,
Gautseh and Layman.)
656 • Layman

Tahle 2 Effects of Nutrient Supplements on Muscle Protein Synthesis

During Recovery After Exercise'

Protein Synthesis^

Control (no exercise) 100%

Post-exercise 71%
Post-exercise with recovery meal
Complete meaP 98%
Carbohydrate supplement" 70%
Protein supplement' (unpublished) 92%
Leucine supplement^ 99%
Leucine -i- carbohydrate' 108%

'Rats were exercised for 2 hrs on a motor-driven treadmill at 36 m/min (~70% VO^^^) after
a 12-hour fast. •^Protein synthesis determined by incorporation of ^H-isoleucine into muscle
protein and expressed as a percentage of the control. Measurements were made 1 hr after
completion of exercise. ^Complete meal provided 43.9 kJ and consisted of 5 ml of Ensure"*
administered by oral gavage immediately after exercise. "Carbohydrate supplement provided
43.9 kJ and consisted of 5 ml of a mixture of 50% glucose and 50% sucrose in water (525g/L)
administered by oral gavage immediately after exercise. 'Protein supplement provided 0.5 g
protein in 5 ml water. 'Leucine supplement provided 0.27 g leu in 5 rril water. 'Mixture of leu
and carhhydrate provided 43.9 kJ and consisted of 0.27 g leu in the Carbohydrate supplement.
(Summarized from Gautsch et al., 1998 and Anthony et al., 1999.)

exercise produced complete recovery of muscle protein synthesis within an hour

(Gautsch et al., 1998, data summarized in Table 2). On the other hand, use of a
carbohydrate drink with the same energy content produced dramatic increases in
blood glucose and insulin but failed to stimulate muscle protein synthesis (An-
thony et al., 1999; Gautsch et al., 1998). These data suggested that recovery of
muscle's ability to build protein after exercise requires dietary intake of protein
and that carbohydrate supplements alone are not sufficient to stimulate recovery
of muscle protein synthesis.
Similar post-exercise effects of amino acid supplements on muscle protein
synthesis have been reported for resistance exercise (Biolo et al., 1997; Rasmussen
et al, 2000) and dynamic exercise (Levenhagen et al., 2001) with humans. Biolo et
al. (1997) utiUzed normal volunteers who were not highly trained and examined
the effects of intravenous infusion of amino acids after 60 minutes of an intense
leg resistance exercise regimen. They reported that post-exercise infusion of amino
acids produced elevation of blood amino acids and a dramatic increase in muscle
protein synthesis (Biolo et al., 1997). They concluded that post-exercise intake of
amino acids may be important to anaboUc recovery after exercise. Subsequently,
they showed that oral administration of amino acids produced a comparable in-
crease in protein synthesis (Tipton et al., 1999). In this experiment, subjects drank
a supplement containing either 40 grams of a complete mixture of essential and
 Leucine in Protein Metabolism • 657
non-essential amino acids, or 40 grams of a mixture containing just the essential
amino acids immediately after the exercise. They found that the post-exercise
supplement of just the nine essential amino acids was sufficient to produce ana-
bolic recovery of muscle protein synthesis. Recently, Rasmussen et al. (2000) re-
ported that post-exercise stimulation of muscle protein synthesis can be achieved
with a supplement containing only 6 grams of a mixture of the essential amino
acids plus 35 grams of sucrose. In total, the post-exercise effects on muscle protein
synthesis appear similar for humans and animals. In each case, the research sug-
gests that intake of amino acids soon after exercise stimulates muscle protein syn-
thesis.
To further evaluate these findings, we examined the impact of protein and
leucine on recovery (Anthony et al., 1999). Using the same experimental design,
we found that animals fed only the protein fraction of the supplement or only
leucine could fully recover normal rates of protein synthesis within an hour after
exhaustive exercise (Table 2). These data are in agreement with reports suggesting
a unique effect of leucine on short-term recovery of muscle protein synthesis. A
second important finding from this study was that the leucine stimulated protein
synthesis in the absence of elevated plasma insulin. Subsequent research suggests
that insulin may be increased early after the amino acid supplement and that this
transient increase in insulin is sufficient to potentiate the signaling pathway (An-
thony etal., 2001).
Examining the molecular mechanism, we found that the response of muscle
protein synthesis was closely tied to changes in the initiation factor eIF4. At the
end of exercise, activation level of eIF4 was reduced by 70%. Feeding the carbo-
hydrate supplement had no effect on the activity state of eIF4, while consumption
of" the complete meal produced full recovery of eIF4 activity (Gautsch et al., 1998).
This was the first report identifying an initiation factor, eIF4, as a critical regulator
of muscle protein synthesis during and after exercise. It is now clear that the stimu-
latory effect of the meal on protein synthesis is produced by increased muscle
levels of leucine (Anthony et al., 1999) resulting in stimulation of mTOR and
activation of eIF4 and p70^'K (Anthony et al., 2001). These findings point toward
a mechanism that may allow the body to integrate metabolic signaJs of insulin
with nutrition to optimize muscle development.

Summary
Exercise produces changes in protein and amino acid metabolism, however the
impact of these changes on amino acid requirements remains unclear. Current evi-
dence indicates that supplementation of amino acids during exercise has little or
no beneficial effects on performance. On the other hand, emerging data suggests
that supplementation of amino acids soon after exercise may enhance the anabolic
nature of the post-exercise period.
During exercise, there is increased oxidation of the three branched-chain
amino acids in skeletal muscle. While oxidation ofthe BCAA contributes to muscle
energy needs the contribution appears minimal. A more likely role for the BCAA
in energy balance appears either through maintenance of TCA cycle intermediates
or through contributions to production of alanine and glutamine. Among the three
BCAA, oxidation of leucine appears to be the most dramatic reaching levels of
658 • Layman
perhaps 1 g of leucine per hour of exercise. Still, supplementation of the BCAA
during endurance exercise does not appear to enhance performance.
Changes in protein tumover associated with a bout of exercise remain con-
troversial. The majority of studies report that a single bout of intense or exhaustive
exercise produces a catabolic period for whole body protein tumover. This cata-
bolic period may be caused by depression in the rate of protein synthesis, increases
in protein breakdown, or both. \Vhile understanding changes in protein synthesis
and breakdown are of equal importance, current methods limit our evaluation of
molecular mechanism to studies of protein synthesis. Recently, there has been an
increased understanding of the role of leucine in metabolic regulations and re-
markable new findings about the role of leucine in intracellular signaling. It now
appears that leucine has a synergistic role with insulin as regulatory factors in the
PI3-K signal cascade. Insulin serves to activate the signal pathway, while leucine
is essential to enhance or amplify the signal for protein synthesis at the level of
peptide initiation factors. Studies feeding amino acids soon after exercise suggest
that post-exercise consumption of protein or amino acids stimulates recovery of
muscle protein synthesis via translation regulations. Presently the significance of
these molecular findings with experimental animal models to human perfonnance
or muscle development is unknown. It will be important to test these new findings
as components of defined training programs.

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