TRIENNIAL GROWTH SYMPOSIUM: Leucine acts as a nutrient signal to stimulate

protein synthesis in neonatal pigs

A. Suryawan, R. A. Orellana, M. L. Fiorotto and T. A. Davis

J ANIM SCI 2011, 89:2004-2016.

doi: 10.2527/jas.2010-3400 originally published online October 8, 2010

The online version of this article, along with updated information and services, is located on
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 TRIENNIAL GROWTH SYMPOSIUM: Leucine acts as a nutrient
signal to stimulate protein synthesis in neonatal pigs1,2
A. Suryawan, R. A. Orellana, M. L. Fiorotto, and T. A. Davis3
USDA/ARS, Children’s Nutrition Research Center, Baylor College of Medicine, Houston, TX 77030
ABSTRACT: The postprandial increases in AA
and insulin independently stimulate protein synthesis
in skeletal muscle of piglets. Leucine is an important
mediator of the response to AA. We have shown that
the postprandial increase in leucine, but not isoleucine
or valine, acutely stimulates muscle protein synthesis
in piglets. Leucine increases muscle protein synthesis
by modulating the activation of mammalian target of
rapamycin (mTOR) complex 1 and signaling compo-
nents of translation initiation. Leucine increases the
phosphorylation of mTOR, 70-kDa ribosomal protein
S6 kinase-1, eukaryotic initiation factor (eIF) 4E-
binding protein-1, and eIF4G; decreases eIF2α phos-
phorylation; and increases the association of eIF4E
with eIF4G. However, leucine does not affect the up-
stream activators of mTOR, that is, protein kinase B,
adenosine monophosphate-activated protein kinase,
Key words: amino acid, leucine, muscle, newborn, pig, translation initiation
©2011 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2011. 89:2004–2016
doi:10.2527/jas.2010-3400
INTRODUCTION
Leucine, a branched-chain AA (BCAA), is an es-
sential AA (EAA) that cannot be synthesized de novo
1
Based on a presentation at the Triennial Growth Symposium
titled “Dietary Regulation of Growth and Development,” a sympo-
sium preceding the Joint Annual Meeting, July 11, 2010, Denver,
Colorado, sponsored by the European Federation of Animal Science
(EAAP, Rome, Italy), Pfizer Animal Health (Kalamazoo, MI), and
Elanco Animal Health (Greenfield, IN) with publication sponsored,
in part, by the Journal of Animal Science and the American Society
of Animal Science.
2
This project was funded, in part, by the NIH R01 AR-44474,
USDA/ARS Cooperative Agreement No. 58-6250-6-001, and the Aji-
nomoto Amino Acid Research Program. This work is a publication
of the USDA/ARS Children’s Nutrition Research Center, Depart-
ment of Pediatrics, Baylor College of Medicine, Houston, TX. The
contents of this publication do not necessarily reflect the views or
policies of the USDA, nor does the mention of trade names, com-
mercial products, or organizations imply endorsement by the US
government.
3
Corresponding author: tdavis@bcm.tmc.edu
Received August 5, 2010.
Accepted October 2, 2010.
2004
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and tuberous sclerosis complex 1/2, or the activation
of translation elongation regulator, eukaryotic elonga-
tion factor 2. The action of leucine can be replicated
by α-ketoisocaproate but not by norleucine. Interfer-
ence by rapamycin with the raptor-mTOR interaction
blocks leucine-induced muscle protein synthesis. The
acute leucine-induced stimulation of muscle protein
synthesis is not maintained for prolonged periods, de-
spite continued activation of mTOR signaling, because
circulating AA fall as they are utilized for protein syn-
thesis. However, when circulating AA concentrations
are maintained, the leucine-induced stimulation of mus-
cle protein synthesis is maintained for prolonged peri-
ods. Thus, leucine acts as a nutrient signal to stimulate
translation initiation, but whether this translates into a
prolonged increase in protein synthesis depends on the
sustained availability of all AA.
by humans and other mammals (Hutson, 2006). It is
now well recognized that leucine not only has a role as
a substrate for protein synthesis, but also acts as a nu-
trient signal that regulates protein synthesis in various
tissues of the body, including skeletal muscle (Anthony
et al., 2001). Several studies have shown that leucine
administration increases skeletal muscle protein synthe-
sis in rodents (Crozier et al., 2005) and humans (Drum-
mond and Rasmussen, 2008). Although the ability of
leucine to stimulate protein synthesis is apparent, its
mode of action is less clear (Proud, 2007). It has been
established in studies originating with yeast and mam-
malian cell culture that leucine activates the mRNA
translation machinery through the mammalian target
of rapamycin (mTOR), a master kinase that is crucial
for cell growth (Kimball et al., 1999; Du et al., 2007).
One of the main goals of domestic animal production
is to improve animal growth by means of increasing
skeletal muscle mass. Thus, consideration of leucine in
the nutritional management of farm animals is particu-
larly important because of its role in the regulation
of muscle protein synthesis. In this review, we present
an overview of our studies on the effect of leucine ad-
 Leucine stimulates protein synthesis
ministration on protein synthesis conducted in vivo in
neonatal pigs.
REGULATION OF NEONATAL GROWTH
During the neonatal period, the growth rate is great-
er than at any other state of postnatal life, and this
is crucial for the long-term well-being of the organism
(Reeds et al., 2000). This period is characterized by
rapid protein deposition and greater nutritional ef-
ficiency, as well as marked differences in the growth
rates of individual tissues and a series of maturational
changes (Goldspink and Kelly, 1984; Denne and Kal-
han, 1987; Davis et al., 1989; Reeds et al., 2000). More
specifically, our early work and that of others show that
neonates are very efficient in utilizing dietary AA for
protein deposition, and this ability decreases markedly
with development (McCracken et al., 1980; Burrin et
al., 1991, 1992; Davis et al., 1991, 1993, 1996; Fiorotto
et al., 1991; Wray-Cahen et al., 1998). Furthermore, the
gain in protein mass is more rapid in skeletal muscle
than in other tissues in the body (Davis and Fiorotto,
2009).
Early postnatal morbidity and mortality have posed
significant challenges to the swine industry and in-
creased the cost of animal production (Odle et al.,
1996). Promotion of growth, muscling, and feed effi-
ciency are primary goals of swine production. Thus,
optimization of the diet to increase growth, sustain
health, and minimize the consequences of disease dur-
ing the early postnatal period is critically important for
animal agriculture.
ROLE OF AA IN THE REGULATION
OF PROTEIN SYNTHESIS
Protein synthesis is a large energy-consuming, com-
plex process that is governed by mediators, such as hor-
mones and nutrients (Ma and Blenis, 2009). More than
3 decades ago, investigators demonstrated that AA
alone can stimulate protein synthesis in skeletal muscle
in vitro (Buse and Reid, 1975; Fulks et al., 1975). Fulks
et al. (1975) used diaphragms isolated from young rats
to study the effect of insulin, glucose, and AA on pro-
tein turnover. In this study, the diaphragms from young
rats were desirable because the muscle is thin and per-
mits the rapid diffusion of nutrients or hormones. Fulks
et al. (1975) found that addition of AA mixtures to the
medium stimulated protein synthesis. Although the re-
sults of AA studies in vitro are important, Garlick and
Grant (1988) questioned the physiological significance
of those findings. One of the obvious concerns was that
the AA concentrations used were 5 to 10 times greater
than normal physiological concentrations (Lundholm
and Schersten, 1977; Li and Jefferson, 1978). Thus,
Preedy and Garlick (1986) conducted an in vivo study
in which a normal physiological concentration of an AA
mixture was infused into young rats and found that
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2005
AA did not stimulate protein synthesis unless glucose
was also infused. Preedy and Garlick (1986) concluded
that AA increased the sensitivity of muscle to insu-
lin, allowing it to stimulate protein synthesis at normal
physiological concentrations (Preedy and Garlick, 1986;
Garlick and Grant, 1988).
In our early studies, we developed the pancreatic-
substrate clamp technique to identify the individual
roles of AA and insulin in the regulation of protein syn-
thesis in neonatal pigs (Wray-Cahen et al., 1997; Davis
et al., 2002); we used an AA mixture containing EAA
and nonessential AA that was similar to the AA com-
position of body proteins (Davis et al., 2002). When we
infused the AA mixture to raise circulating AA to fed
amounts, we demonstrated that AA alone could stimu-
late muscle protein synthesis (Davis et al., 2002). This
response of protein synthesis to AA decreases with age
in parallel with the age-related reduction in the post-
prandial rate of muscle protein synthesis. Furthermore,
we determined that the stimulatory effect of AA occurs
in the presence of fasting concentrations of insulin, at
concentrations of insulin that are intermediate between
the fasting and the fully fed levels, and even at below
fasting insulin concentrations that are undetectable by
RIA (O’Connor et al., 2003).
Now that several studies in different species ranging
from rodents to humans (Preedy and Garlick, 1986; Gar-
lick and Grant, 1988; Zanetti et al., 1999) have shown
that AA can stimulate muscle protein synthesis, the
obvious question would be: is the ability of each AA to
induce protein synthesis equal? Surprisingly, this ques-
tion was addressed by 2 independent groups approxi-
mately 35 yr ago (Buse and Reid, 1975; Fulks et al.,
1975). In their studies, Fulks et al. (1975) demonstrated
that of the AA in their mixture, only the BCAA en-
hanced protein synthesis. Further experiments showed
that leucine alone or isoleucine and valine together
stimulated protein synthesis. Using a hemidiaphragm
preparation, Buse and Reid (1975) found that leucine
acts as regulator that can stimulate protein synthesis
and inhibit protein degradation in vitro. Since these re-
markable findings, investigators have been seeking the
answer as to whether leucine has similar effect in vivo
in several species, including humans (Matthews, 2005).
The uniqueness and the ability of leucine to regulate
protein synthesis are well-established (Garlick, 2005).
However, the notion that leucine acts as a signaling
molecule that activates cellular protein synthetic ma-
chineries is still unsettled (Avruch et al., 2009). Bio-
chemical and genetic approaches have been used to
dissect the molecular mechanisms by which leucine
stimulates protein synthesis (Proud, 2007). There is a
general consensus that in order for leucine to stimu-
late protein synthesis, it must first activate mTOR
complex 1 [mTORC1; Figure 1 (Avruch et al., 2009;
Kim, 2009)]. The master protein kinase, mTOR, is
the central component of 2 independently regulated
complexes: mTORC1 and mTORC2 (Sabatini, 2006;
Lian et al., 2008). The mTORC1 consists of mTOR,
2006 Suryawan et al.

Figure 1. A simple model of the molecular mechanisms by which insulin and AA/leucine regulate mRNA translation in the cytosol of eukary-
otic cells. IRS-1 = insulin receptor substrate 1; PI-3K = phosphoinositide 3-kinase; PDK = phosphoinositide-dependent kinase 1; PKB = protein
kinase B; MAP4K3 = mitogen-activated protein kinase kinase kinase kinase 3; Vps34 = vacuolar sorting protein 34; Rag A-D = Ras-related
GTP-binding protein A-D; eIF = eukaryotic initiation factor; mTOR = mammalian target of rapamycin; eEF2 = eukaryotic elongation factor
2; PRAS40 = proline-rich Akt substrate 40 kDa; Rheb = Ras homolog enriched in brain; TSC1/2 = tuberous sclerosis complex 1/2; S6K1 =
ribosomal protein S6 kinase 1; AMPK = adenosine monophosphate-activated protein kinase.

raptor, and the G-protein β-like protein, GβL/LST8,
whereas mTORC2 is composed of mTOR, rictor, GβL,
and mammalian stress-activated protein kinase-inter-
action protein 1. Whereas mTORC1 participates in
major metabolic processes, including protein synthesis,
mTORC2 has a relatively minor role (Huang and Man-
ning, 2009). The most notable function of mTORC2 is
to partly activate protein kinase B (PKB) by phos-
phorylation of the S473 site (Liao and Hung, 2010).
Whereas the AA signaling pathway is critical to the
cellular regulation of growth, the insulin signaling path-
way senses and coordinates general nutrient status (Hi-
etakangas and Cohen, 2009). Both the insulin and AA
signaling pathways converge at mTOR to regulate pro-
tein synthesis and, thus, growth. The insulin signaling
pathway is well-characterized (Bevan, 2001). Briefly,
upon insulin binding to its receptor, early steps in the
insulin signaling cascade (i.e., insulin receptor, insulin
receptor substrate 1, and phosphoinositide 3-kinase)
are activated, resulting in the activation of PKB (Fig-
ure 1). By several proposed mechanisms, the activated
PKB stimulates the activation of mTORC1. Activated
PKB can phosphorylate the tuberous sclerosis complex
(TSC)2, resulting in the disruption of the TSC1-TSC2
complex, a potent inhibitor of mTORC1 activation. In
contrast, adenosine monophosphate-activated protein
kinase (AMPK), an energy sensor that is activated by
energy deficiency in the cell, can activate TSC1-TSC2,
resulting in the inhibition of mTORC1. In other mecha-
nisms, PKB can also phosphorylate proline-rich Akt
substrate 40 kDa (PRAS40) and alleviate the sup-
pression effect of PRAS40 on mTORC1.
Unlike insulin, the mechanism by which leucine stim-
ulation leads to the activation of mTORC1 is not well
understood (Avruch et al., 2009). Several possible sig-
naling components have been identified that respond to
changes in intracellular leucine with subsequent activa-
tion of mTORC1 (Figure 1). These include Ras homo-
log enriched in brain, mitogen-activated protein kinase
kinase kinase kinase 3, vacuolar sorting protein 34, and
Ras-related GTP-binding protein A-D (Avruch et al.,
2006; Findlay et al., 2007; Backer, 2008; Shaw, 2008).
Reviews of postulated mechanisms by which leucine ac-
tivates these signaling components are presented else-
where (Avruch et al., 2006; Findlay et al., 2007; Backer,
2008; Shaw, 2008).

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 Leucine stimulates protein synthesis 2007

Figure 2. Fractional rates of protein synthesis in the LM of neonatal pigs treated with different doses of leucine (panel A) and in the LM
and masseter (Mass) muscles of neonatal pigs treated with branched-chain AA. Leu, Ile, or Val, compared with saline (Sal; panel B). Results are
presented as means ± SEM. *Differs from saline group (P < 0.05). Data are from Escobar et al. (2005).

A common consensus is that both insulin and leu-
cine utilize and activate similar signaling components
downstream of mTORC1, leading to the stimulation of
mRNA translation and translation elongation (Proud,
2002; Figure 1). The mTORC1 regulates mRNA trans-
lation by phosphorylating 2 of its effectors, ribosomal
protein S6 kinase 1 (S6K1) and eukaryotic initiation
factor (eIF) 4E-binding protein-1 (4EBP1; Kimball,
2001; Proud, 2002). Phosphorylated 4E-BP1 releases
eIF4E from the inactive eIF4E·4EBP1 complex, allow-
ing the formation of the active eIF4E·eIF4G complex
(eIF4F complex). The increased formation of eIF4F
preferentially induces translation of mRNA containing
long, highly-structured 5′-untranslated region (Kim-
ball, 2001). Through the mTORC1-dependent path-
way, leucine can also stimulate translation elongation
by inhibiting the phosphorylation of eukaryotic elon-
gation factor 2 (eEF2; Kimball, 2001; Proud, 2002).
Amino acids also can influence protein synthesis in an
mTORC1-independent fashion by preserving eIF2B ac-
tivity (Kimball, 2001; Figure 1). Leucine inhibits phos-
phorylation of the α subunit of eIF2, which permits the
activation of eIF2B, and promotes the translation of
all mRNA (i.e., global protein synthesis; see review by
Kimball, 2001).
ACUTE EFFECTS OF LEUCINE ON
PROTEIN SYNTHESIS
AND ACTIVATION OF SIGNALING
COMPONENTS THAT LEAD TO MRNA
TRANSLATION IN NEONATAL PIGS
Our early work demonstrated that AA were very ef-
fective in promoting the activation of components of
mRNA translation and protein synthesis in muscle
of neonatal pigs (Davis et al., 2002; O’Connor et al.,
2003). Because leucine is considered a unique regula-
tor of protein synthesis (Buse and Reid, 1975), we first
determined the acute effect of different physiological
amounts of leucine on protein synthesis in skeletal mus-
cle of neonatal pigs (Escobar et al., 2005). We found
that an acute (i.e., 1 h) infusion of leucine stimulated
muscle protein synthesis in a dose-dependent manner
(Figure 2A). To determine whether the ability to pro-
mote muscle protein synthesis is specific to leucine, we
compared the response to leucine with the response to
the other BCAA, isoleucine and valine, administered in
equimolar concentrations. The results clearly demon-
strated that leucine, but not isoleucine or valine, stimu-
lated muscle protein synthesis (Figure 2B). In addition,
leucine induced-stimulation occurred in the LM, which
contains primarily fast-switch glycolytic fibers, and the
masseter muscle, which contains more oxidative fibers
(Figure 2B).
Although these studies demonstrated that leucine
could stimulate muscle protein synthesis for up to 1 h,
the response was not maintained after 2 h of infusion
(Figure 3A and 3B). To further examine these respons-
es, we evaluated the signaling components downstream
of mTORC1. The increase in muscle protein synthesis
after 1 h of leucine infusion was associated with an in-
crease in the phosphorylation of S6K1 and 4EBP1 (Fig-
ure 3C and 3E; Escobar et al., 2005), and this increase
in phosphorylation was sustained even after 2 h (Figure
3D and 3F) despite the absence of a protein synthesis
response at 2 h. These data indicated that other factors
are responsible for the lack of any effect of leucine on
protein synthesis after 2 h. Importantly, we found that
the 2- to 3-fold increase in circulating leucine concen-
trations was associated with a 50% reduction in the cir-
culating concentrations of the other BCAA, valine and
isoleucine (Figure 4A and 4B). Likewise, the concentra-
tions of the other EAA had also decreased by the same
magnitude (Figure 4C and 4D; Escobar et al., 2005).
The results led us to speculate that the decline in the
other EAA, as they are used for protein synthesis, may
have limited the ability of leucine to continue to stimu-
late protein synthesis. Thus, the ability of leucine to

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2008 Suryawan et al.

Figure 3. Fractional rates of protein synthesis (panels A and B), 70-kDa ribosomal protein S6 kinase-1 (S6K1) phosphorylation (panels C
and D), and eukaryotic initiation factor 4E-binding protein-1 (4EBP1) phosphorylation (panels E and F) in the LM of neonatal pigs treated with
different doses of leucine for 60 or 120 min. Results are presented as means ± SEM. *Differs from saline group (P < 0.05). AU = arbitrary units.
Data are from Escobar et al. (2005).

stimulate protein synthesis may be dependent upon the
availability of substrate AA for protein synthesis.
To determine whether the leucine-induced stimula-
tion of protein synthesis in skeletal muscle of neonatal
pigs is dependent upon the availability of EAA, we in-
fused neonatal pigs for 2 h with saline, leucine alone,
or leucine in the presence of an AA clamp where a
balanced AA mixture (without leucine) was infused to
maintain circulating concentrations of all AA at fast-
ing levels during the increase of leucine (Escobar et al.,
2007). The amount of leucine achieved was about 2- to
3-fold greater than fasting levels and was similar to fed
leucine levels regardless of whether we infused leucine
alone or leucine in the presence of an AA clamp. This
AA clamp was successful in preventing the reduction
in the circulating concentration of other EAA during
the infusion of leucine (Figure 5). With this protocol,
the 2-h leucine infusion increased the phosphorylation
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of S6K1 and 4EBP1, as well as the formation of the
active complex of eIF4E bound to eIF4G, similar to
that which occurred at 1 h (Figure 6A). Importantly,
when hypoaminoacidemia was prevented and euami-
noacidemia was maintained with the AA clamp, the
leucine-induced stimulation of muscle protein synthesis
was sustained for 2 h (Figure 6B). The results indicate
that the stimulation of protein synthesis by parenteral
leucine infusion is dependent on the availability of AA.
LEUCINE-INDUCED STIMULATION
OF MUSCLE PROTEIN SYNTHESIS
IS MTORC1 DEPENDENT
The majority of information obtained on the mo-
lecular mechanism by which AA or leucine enhance
protein synthesis has been based on in vitro or cell
 Leucine stimulates protein synthesis 2009

Figure 4. Plasma concentrations of valine (panel A), isoleucine (panel B), lysine (panel C), and methionine (panel D) during leucine infusion
for 120 min. Results are presented as means ± SEM. After 2 h of leucine infusion, AA concentrations were reduced (P < 0.05). Data are from
Escobar et al. (2005).

culture studies (Du et al., 2007; Atherton et al., 2010).
Because little was known of the mechanism of action
of leucine in vivo, we examined the effect of rapamy-
cin, a drug that acutely inhibits only mTORC1 acti-
vation, on the leucine-induced stimulation of protein
synthesis and the activation of signaling components in
skeletal muscle of 7-d-old pigs (Suryawan et al., 2008).
We found that leucine and rapamycin had no effect
on the phosphorylation of AMPK, PKB, and TSC2,
but it severely reduced raptor-mTOR interaction. The
inhibitory effect of rapamycin was absent in both the
rictor-mTOR interaction and the GβL-mTOR inter-
action, verifying cell culture work showing that acute
treatment with rapamycin has no effect on mTORC2
activation (Toschi et al., 2009). Consistent with these
observations, rapamycin completely blocked leucine-
induced phosphorylation of mTOR (Figure 7A), S6K1,
and 4EBP1, as well as the leucine-induced formation of
an active eIF4E·eIF4G complex (Figure 7B), whereas
the formation of the inactive eIF4E·4EBP1 complex
was increased significantly (Figure 7C; Suryawan et al.,
2008). We did not detect any effect of rapamycin on
the phosphorylation of eEF2, although studies in cell
cultures indicate that eEF2 phosphorylation is mTOR-
dependent (Proud, 2004). Importantly, we found that
disruption of leucine-induced activation of mTORC1
has a detrimental effect on protein synthesis in vivo
because rapamycin completely blocked leucine-induced
muscle protein synthesis (Figure 7D; Suryawan et al.,
2008), consistent with findings in cell culture (Talvas et
al., 2006). In contrast, other studies from our laborato-
ry demonstrated that rapamycin only partially blocked
the feeding-induced stimulation of muscle protein syn-
thesis in neonatal pigs (Kimball et al., 2000). Together
the results indicate that, whereas leucine-stimulated
protein synthesis is mTOR-dependent, the portion of
the feeding-induced stimulation of muscle protein syn-
thesis that is mediated by insulin or nonleucine AA or
both is, at least in part, mTOR-independent.
LEUCINE OR KETOISOCAPROATE?
Recognition of leucine as a nutrient signal is chal-
lenged by the fact that leucine can be easily me-
tabolized by most tissues, including skeletal muscle
(Suryawan et al., 1998). Thus, at this point it is not

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2010 Suryawan et al.

Figure 5. Plasma concentrations of valine (panel A), isoleucine (panel B), lysine (panel C), and threonine (panel D) during leucine infusion
for 120 min, with or without an AA clamp, compared with saline infusion. Results are presented as means ± SEM. Without an AA clamp, 2 h of
leucine infusion reduced (P < 0.05) plasma AA concentrations. The AA clamp prevented the fall in AA concentrations. Data are from Escobar
et al. (2007).

clear whether the anabolic effects of leucine are due which is readily reversible, is the transamination of the
to leucine itself or one of its metabolites. There are BCAA, leucine, isoleucine, and valine, by branched-
2 primary steps of the BCAA catabolic pathways in chain aminotransferase (BCAT). There are 2 mam-
tissues (Hutson et al., 2005; Figure 8). The first step, malian BCAT: a mitochondrial (BCATm) and a cy-

Figure 6. The eIF4E·eIF4G complex abundance (panel A) and fractional rates of protein synthesis (panel B) in the LM of neonatal pigs
treated with leucine, with or without AA clamp for 120 min, compared with saline (Sal) treatment. Results are presented as means ± SEM. *Dif-
fers from saline group (P < 0.05). AU = arbitrary units; eIF4E·eIF4G complex = eukaryotic initiation factor (eIF) 4E·eIF4G complex. Data are
from Escobar et al. (2007).

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 Leucine stimulates protein synthesis 2011

Figure 7. Effect of rapamycin (Rap) on the phosphorylation of mammalian target of rapamycin (mTOR; panel A), the formation of an active
eukaryotic initiation factor (eIF) 4E·eIF4G complex (panel B), the inactive eIF4E·eIF4E-binding protein-1 (eIF4E·4EBP1) complex (panel C),
and fractional rates of protein synthesis (panel D) in the LM of neonatal pigs treated with leucine for 1 h. Results are presented as means ± SEM.
*Differs from saline (Sal) group (P < 0.05). AU = arbitrary units. Data are from Suryawan et al. (2008).

tosolic (BCATc) isozyme (Hutson et al., 2005). The
transamination products are α-ketoisocaproate (KIC),
α-keto-β-methylvalerate, and α-ketoisovalerate, respec-
tively. The second step, which is irreversible, is oxida-
tive decarboxylation catalyzed by the branched-chain
keto acid dehydrogenase complex. The products of this
process can enter the tricarboxylic acid cycle to pro-
duce energy or other metabolic components.
The notion of whether or not the molecular structure
of leucine is crucial for its ability to stimulate protein
synthesis is unclear. Several studies in cell culture (Ya-
gasaki et al., 2003) and rodent models (Lynch et al.,
2002a) have examined the effects of a metabolite of
leucine, KIC, and norleucine, an isomer of leucine, on
protein synthesis, with conflicting results. In our recent
study, we evaluated the effects of leucine, KIC, and nor-
leucine infusion for 60 min on muscle protein synthesis
and the activation of translation initiation factors in
overnight fasted neonatal pigs (Escobar et al., 2010).
After infusion, as expected, the concentration of each
infused individual compound was increased significant-
ly when compared with the control treatment, and the
infusion of KIC and leucine increased the concentra-
tions of each other. When we examined the activation
of translation initiation, we found that both leucine and
KIC, but not norleucine, enhanced the phosphorylation
of 4EBP1 and the formation of the eIF4E·eIF4G com-
plex (Figures 9A and C), and reduced the formation
of the inactive eIF4E·4EBP1 complex (Figure 9B). As
a consequence, only leucine and KIC increased muscle
protein synthesis in neonatal pigs (Figure 9D).
Our findings regarding the effect of KIC are consis-
tent with other studies performed in cell culture (Yaga-
saki et al., 2003) and in rodents (Lynch et al., 2002b).
Unfortunately, the true value of this observation is still
highly debatable. This is because KIC easily can be
converted into leucine by BCAT in most tissues (see
Figure 8). Thus, the use of BCATm (–/–) transgenic
mice (She et al., 2007) would be appropriate to study
the actual effect of KIC on muscle protein synthesis.
Unlike the KIC data, our data on norleucine are not
consistent with studies in rats (Lynch et al., 2002b).
Lynch et al. (2002b) found that, acutely, norleucine is
as effective as leucine in stimulating protein synthesis
and the phosphorylation of S6K1 and 4EBP1 in skeletal
muscle of mature rats. Furthermore, these investigators
showed that chronic norleucine and leucine supplemen-
tation (12 d) produced similar results (Lynch et al.,
2002a). The reason for the discrepancy between our
study and theirs is unknown; thus, further study of the
effect of norleucine in domestic animals is warranted.
PROLONGED EFFECTS OF LEUCINE
ON THE REGULATION
OF TRANSLATION INITIATION
Although there is ample evidence that leucine
acutely stimulates muscle protein synthesis in neona-

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2012 Suryawan et al.
Figure 8. A schematic of the branched-chain AA (BCAA) meta-
bolic pathway. The first step of BCAA metabolism is the reversible
transamination of Leu, Ile, or Val, which is catalyzed by the branched-
chain aminotransferase (BCAT) isozymes (BCATm and BCATc). In
the second step, the products, α-ketoisocaproate (KIC), α-keto-β-
methylvalerate (KMV), or α-ketoisovalerate (KIV), undergo oxidative
decarboxylation, which is catalyzed by the branched-chain keto acid
dehydrogenase (BCKD) enzyme complex. This irreversible process
produces branched-chain acetyl CoA, which can enter the tricarbox-
ylic acid (TCA) cycle.
tal pigs, studies of longer duration are needed to test
the potential efficacy of leucine in promoting protein
synthesis and muscle accretion. To determine whether
the stimulation of muscle protein synthesis by leucine
can be maintained for a longer duration and whether
other AA are required, we evaluated the response to
a 24-h leucine infusion alone or in combination with
an AA clamp to maintain AA at the baseline fasting
level in neonatal pigs (Wilson et al., 2010a). Circulating
concentrations of leucine increased about 2- to 3-fold
during the first 12 h of leucine infusion, but by 24 h
had increased about 4-fold. Leucine infusion alone re-
duced EAA and nonessential AA concentrations by ap-
proximately 50%; however, the decrease was prevented
when the AA clamp was employed. Circulating insu-
lin concentrations were maintained at fasting amounts
throughout the infusion.
The effects of chronic leucine infusion on the activa-
tion of signaling components leading to mRNA transla-
tion in skeletal muscle were determined. We found that
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chronic increases in leucine had no effect on the phos-
phorylation of PKB, indicating that leucine infusion
did not activate the insulin signaling pathway (Wil-
son et al., 2010a). The phosphorylation of AMPK was
not increased, indicating that there was no significant
change in energy status in the cell. Furthermore, TSC2
phosphorylation was unchanged. This is important be-
cause studies in cell culture had provided controversial
evidence that TSC2 has a role in AA signaling (Smith
et al., 2005), although our recent in vivo studies dem-
onstrated that insulin, but not AA, increase TSC2
phosphorylation and, thus, decrease the activation of
the mTORC1 inhibitor (Suryawan et al., 2007). The
interactions among the components of mTORC1 and
mTORC2 are necessary for their activation (Toschi et
al., 2009). Leucine, with or without an AA clamp, did
not affect the interaction between mTOR and raptor, in
mTORC1 (Wilson et al., 2010a), and had no effect on
the interaction of mTOR with GβL, in both mTORC1
and mTORC2, and the interaction of mTOR with ric-
tor, in mTORC2 in muscle. As predicted, prolonged
leucine infusion, with or without an AA clamp, induced
the phosphorylation of mTOR, but had no effect of
the phosphorylation of PRAS40 and raptor. In con-
trast, we found that prolonged leucine infusion, with or
without an AA clamp, enhanced the phosphorylation of
S6K1 and 4EBP1, increased the formation of the active
eIF4E·eIF4G complex (Figure 10A), and reduced the
formation of the inhibitory eIF4E·4EBP1 complex. The
elongation process, which is partly regulated by eEF2,
is essential for mRNA translation (Proud, 2004). Previ-
ous studies examining the effect of feeding on the regu-
lation of translation in neonatal muscle were unable to
detect any change in eEF2 phosphorylation, suggesting
that the elongation process is not a limiting step in this
condition (Proud, 2004). Thus, it is not surprising that
in this study, leucine did not alter the phosphorylation
of eEF2 (Figure 10B; Wilson et al., 2010a). We also
determined the phosphorylation of eIF2α, an inhibitor
of eIF2B (see Figure 1). The results show that long-
term treatment with leucine, with or without the AA
clamp, decreased the phosphorylation of eIF2α (Fig-
ure 10C), which would release the inhibitory effect of
this factor, and thus promote the binding of methionyl-
tRNA to the 40S ribosomal complex and allow mRNA
translation to proceed. Most importantly, we showed
that long-term leucine infusion increased muscle pro-
tein synthesis, but only in the presence of an AA clamp
when euaminoacidemia was maintained (Figure 10D).
These results stress the importance of AA availability
for supporting the leucine-induced stimulation of pro-
tein synthesis.
The aforementioned data presented were obtained
from the LM, which is composed of primarily fast-
twitch glycolytic fibers. Because of the potential for use
of leucine clinically and in animal production, it was
also important to examine the effect in muscles of dif-
ferent fiber types (Wilson et al., 2010b). Therefore, we
examined the gastrocnemius muscle, which has a more
 Leucine stimulates protein synthesis 2013

Figure 9. Effect of saline (Sal), Leu, α-ketoisocaproic acid (KIC), and norleucine (Nleu) infusion for 1 h on eukaryotic initiation factor (eIF)
4E·eIF4E-binding protein-1 (4EBP1) phosphorylation (panel A), the abundance of eIF4E·4EBP1 complex (panel B), the abundance of the
eIF4E·eIF4G complex (panel C), and fractional protein synthesis rates (panel D) in skeletal muscle of neonatal pigs. Results are presented as
means ± SEM. *Differs from saline group (P < 0.05). AU = arbitrary units. Data are from Escobar et al. (2010).

Figure 10. Effect of 24-h Leu infusion, with or without AA clamp, on the eukaryotic initiation factor (eIF) 4E·eIF4G (eIF4E·eIF4G) complex
abundance (panel A), the phosphorylation of eukaryotic elongation factor 2 (eEF2; panel B) and eIF2α (panel C), and fractional protein synthesis
rates (panel D) in skeletal muscle of neonatal pigs. Results are presented as means ± SEM. *Differs from saline (Sal) group (P < 0.05). AU =
arbitrary units. Data are from Wilson et al. (2010a).

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2014 Suryawan et al.
mixed fiber type, and the masseter muscle, which is
composed of primarily oxidative fibers. We also exam-
ined the right and left ventricles of the heart because
the left heart undergoes hypertrophy during the first
few days after birth (Escobar et al., 2006). We found
that leucine increased protein synthesis in the gastroc-
nemius and masseter muscles but this only occurred
in the presence of an AA clamp where hypoaminoaci-
demia was prevented (Wilson et al., 2010b). However,
there was no significant effect of leucine on protein syn-
thesis in the heart. Consistent with protein synthesis
data, we found that leucine, with or without an AA
clamp, stimulated the phosphorylation of S6K1 and
4EBP1 in the gastrocnemius and masseter muscles but
not in the heart.
In this study, we also determined protein synthesis
rates and the phosphorylation of S6K1 and 4EBP1 in
visceral tissues (Wilson et al., 2010b). We found that
leucine alone, and in the presence of an AA clamp,
increased the phosphorylation of S6K1 and 4EBP1 in
liver and pancreas, but not in the kidney and jejunum.
However, maintenance of euaminoacidemia, using an
AA clamp, was required for the stimulation of protein
synthesis in the liver and pancreas by leucine. Taken
together, these data indicate that prolonged parenter-
al infusion of leucine enhances the activation of the
mTOR signaling pathway in skeletal muscle, liver, and
pancreas of neonatal pigs. However, the leucine-induced
activation of translation initiation factors is not affect-
ed by changes in the circulating concentrations of other
AA. Most important, prolonged parenteral infusion of
leucine stimulates protein synthesis in most peripheral
and visceral tissues but this effect is dependent on the
availability of all other AA.
SUMMARY AND CONCLUSIONS
There is a growing body of evidence indicating that
leucine acts as an anabolic agent that stimulates pro-
tein synthesis in various tissues in the body. But un-
til now, the beneficial effect of physiological leucine
administration on protein synthesis in newborn pigs
had not been elucidated. Our data from several stud-
ies show that acute (i.e., 1 h) administration of leu-
cine promotes muscle protein synthesis by activating
translation initiation factors downstream of mTORC1.
Our finding that KIC, but not norleucine, can replace
leucine action is interesting, but more studies are nec-
essary to evaluate the chemical structures required for
the leucine-induced stimulation of protein synthesis.
Using rapamycin, we also demonstrated that mTORC1
plays a crucial role in mediating the action of leucine on
muscle protein synthesis in vivo. Although the activa-
tion of translation initiation factors was preserved after
a 2-h leucine infusion, muscle protein synthesis stimu-
lation was not, unless the leucine-induced reduction in
EAA concentrations was rescued by an AA clamp. This
novel and important finding also applied to chronic (24
h) leucine infusion studies. Moreover, our observations
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that chronic leucine administration promotes protein
synthesis in skeletal muscles of different fiber types and
several visceral tissues are encouraging. However, the
ultimate challenge relevant to animal production would
be the use of leucine supplementation in newborn feed-
ing practices to promote growth, the challenge that we
are currently pursuing.
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