1

Leucine Stimulates Protein Synthesis in Skeletal Muscle of Neonatal Pigs by Enhancing

mTORC1 Activation

Agus Suryawan, Asumthia S. Jeyapalan, Renan A. Orellana, Fiona A. Wilson, Hanh V. Nguyen,

and Teresa A. Davis

United States Department Agriculture/Agriculture Research Service, Children’s Nutrition

Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030

Running Title: Leucine-Induced mTOR Signaling in Neonatal Muscle

Correspondence: Teresa A. Davis, Ph.D., USDA/ARS Children’s Nutrition Research Center,

Baylor College of Medicine, 1100 Bates St., Houston, TX 77030 (phone: 713-798-7169, fax:

713-798-7171, email: tdavis@bcm.tmc.edu).

Articles in PresS. Am J Physiol Endocrinol Metab (August 5, 2008). doi:10.1152/ajpendo.90314.2008

Copyright © 2008 by the American Physiological Society.

2

ABSTRACT

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the

postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by

which leucine stimulates protein synthesis in neonatal muscle, overnight fasted 7-day-old piglets

were treated with rapamycin (an inhibitor of mammalian target of rapamycin complex

1/mTORC1) for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis

and activation of signaling components that lead to mRNA translation were determined in

skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis.

Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation.

Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6K1, and 4EBP1 and

formation of the eIF4E•eIF4G complex and increased eIF4E•4EBP1 complex abundance.

Rapamycin had no effect on the association of mTOR with rictor, a crucial component for

mTORC2 activation, or GβL, a component of mTORC1 and mTORC2 complexes. Neither

leucine nor rapamycin affected the phosphorylation of AMPK, PKB, or TCS2, signaling

components that reside upstream of mTOR. eEF2 phosphorylation was not affected by leucine

or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR-dependent.

Taken together, these in vivo data suggest that leucine stimulates muscle protein synthesis in

neonates by enhancing mTORC1 activation and its downstream effectors.

Keywords: mRNA translation; eukaryotic initiation factor 4G; AMP-activated protein kinase;

raptor; rictor

3

INTRODUCTION

The neonatal period is characterized by rapid growth that is supported by a high rate of

protein synthesis (3). However, most infants that are born of low birth weight are discharged

weighing less than the tenth percentile for age despite improvements in their nutritional

management (9). Some remain small to adulthood and exhibit adverse long-term developmental

outcomes including reduced work capacity (27). To improve strategies for the nutritional

management of low birth weight infants, we have used the neonatal pig as a model of the human

neonate so as to identify the mechanisms that regulate protein deposition in neonates. Our

studies have shown that the profound accretion of skeletal muscle protein is in part due to the

ability of neonatal muscle to markedly increase protein synthesis in response to feeding, a

response that declines rapidly with development (5). In skeletal muscle, the feeding-induced

stimulation of protein synthesis is independently modulated by the rise in insulin and amino acids

(6; 24). Recently, we demonstrated that the postprandial rise in leucine alone, but not isoleucine

or valine alone, stimulates skeletal muscle protein synthesis in neonatal pigs and this response

decreases with age (10-12).

Signaling through the mammalian target of rapamycin (mTOR) plays a significant role in

cell growth regulation including protein synthesis (4). Recent studies have revealed that mTOR

exists in two protein complexes (Fig. 1): mTOR complex 1 (mTORC1) and mTOR complex 2

(mTORC2) (4). mTORC1 is rapamycin-sensitive and consists of mTOR, raptor, and G-protein β

subunit-like protein (GβL). This complex is activated by amino acids (especially leucine),

hormones/growth factors, and energy signals (17). mTORC1 regulates mRNA translation by

phosphorylating two of its effectors, ribosomal protein S6 kinase 1 (S6K1) and eIF4E-binding

protein-1 (4E-BP1) (4). Phosphorylated 4E-BP1 releases eIF4E from the inactive eIF4E•4EBP1

4

complex, allowing the formation of the active eIF4G•eIF4E complex (29). mTORC1 has also

been implicated in the regulation of elongation factor 2 (eEF2) activation (38). The second

protein complex, mTORC2, is rapamycin insensitive and consists of mTOR, rictor, and GβL (4).

Recent biochemical studies suggest that one major function of mTORC2 is to activate the growth

factor-regulated protein kinase B (PKB) (28).

The molecular mechanisms by which amino acids or leucine modulate the activation of

mTORC1 in vivo is unknown. Recent studies suggest that amino acids or leucine stimulate

mTORC1 independent of PKB, AMPK and TSC1/TSC2 activation (4). Furthermore, studies

conducted in cell culture suggest that the activation of mTORC1 is partly regulated by the

protein-protein interaction among members of mTORC1, in particular, the interaction of mTOR

with raptor (16). However, we (31) and others (37) have found that feeding stimulates the

activation of mTOR in vivo without affecting the interaction of mTOR with raptor.

Until recently (1; 11; 35), information regarding the molecular mechanism by which

leucine regulates the activation of mTOR leading to the stimulation of muscle protein synthesis

were obtained from cell culture studies. We demonstrated that a physiological rise in plasma

leucine stimulates muscle protein synthesis in neonatal pigs by enhancing translation initiation

factor activation (11). However, detailed study of the effect of physiological levels of leucine on

the activation of mTORC1 leading to the stimulation of protein synthesis in skeletal muscle of

the neonate has not been conducted previously. Therefore, our objective was to determine the

molecular mechanism by which leucine modulates mTORC1 activation in vivo using rapamycin,

a potent inhibitor of mTORC1. Previously we demonstrated that the feeding-induced stimulation

of muscle protein synthesis in neonatal pigs was only partially inhibited by rapamycin (21).

5

MATERIALS AND METHODS

Animals and housing. Two crossbred (Landrace x Yorkshire x Duroc x Hampshire)

pregnant sows (Agriculture Headquarters, Texas Department of Criminal

Justice, Huntsville, TX)

were housed in lactation crates in individual, environmentally controlled rooms 2 wk before

farrowing. Sows were fed a commercial diet (no. 5084; PMI Feeds, Richmond,

IN) and provided

water ad libitum. After farrowing, piglets remained with the sow but were not allowed access to

the sow's diet. A total of 23 piglets from three litters, weighing 2 kg, were studied at 7 days of

age, respectively. Three days prior to the experiment, piglets were anesthetized for sterile

catheter insertion into a jugular vein and carotid artery. Piglets were then returned to the sow

and allowed to suckle freely until studied. The protocol was approved by the Animal Care and

Use Committee of Baylor College of Medicine and was conducted in accordance with the

National Research Council's Guide for the Care and Use of Laboratory Animals.

Treatments and infusion. Piglets were fasted for 12–14 h before infusion and placed in a

sling restraint system. The carotid catheter was used to infuse saline, leucine, rapamycin, and L-

[4-3H]phenylalanine,

whereas the jugular catheter was used for repeated blood sample

collection.

Pigs were randomly assigned to one of four treatment groups: 1) saline (control), 2) saline +

rapamycin, 3) leucine, and 4) leucine + rapamycin. Piglets assigned to the rapamycin groups

were injected with a rapamycin solution (0.75 mg/kg in 5% dimethyl sulfoxide) 1 hour prior to

the initiation of the leucine infusion; other pigs were injected with diluent. Leucine infusion was

initiated with a primed dose (148 µmol/kg) for 10 min, followed by a constant infusion of

leucine at 400 µmol·kg–1

·h–1

for 1 hour. Previous studies (11) have shown that a 2-3 fold

elevation in plasma leucine concentration, similar to that observed with feeding, is achieved by

6

this rate of leucine infusion. During the priming and constant infusion period, saline-infused

pigs received an equal volume of saline as those receiving leucine.

Tissue protein synthesis in vivo. Fractional rates of protein synthesis were measured

using a modification of the flooding dose method (14). At 30 min before the end of the infusion,

pigs were injected with 10 ml/kg body weight of a flooding dose of phenylalanine (Amersham

Biosciences, Piscataway, NJ) which provided 1.5 mM phenylalanine/kg body weight and 0.5

mCi of L-[4-3H]phenylalanine/kg body weight. Samples of whole blood were taken 5, 15, and

30 min after the injection for measurement of the specific radioactivity of the extracellular free

pool of phenylalanine. Pigs were killed at 60 min, and longissimus dorsi muscle samples were

collected and immediately frozen in liquid nitrogen and stored at –70°C until analyzed, as

previously described (8).

Protein synthesis (Ks expressed as % protein synthesized in a day) was calculated

as: Ks

(%/day) = [(Sb/Sa) x (1,440/t)] x 100, where Sb is the specific radioactivity of the protein-bound

phenylalanine; Sa is the specific radioactivity of the tissue free phenylalanine

for the labeling

period, determined from the value of the animal at the time of tissue collection, corrected by the

linear regression of the blood specific radioactivity of the animal against time;

and t is the time of

labeling in minutes. Previous studies have demonstrated that after a flooding dose of L-[4-

3H]phenylalanine is administrated, the specific radioactivity of tissue free phenylalanine is in

equilibrium with the aminoacyl tRNA specific radioactivity, and therefore the tissue free

phenylalanine is a valid measure of the tissue precursor pool specific radioactivity (7).

Tissue extraction and immunoblot analysis. Freshly collected longissimus dorsi muscle

tissue samples were homogenized, centrifuged at 10,000 g for 10 min

at 4°C. Supernatants were

diluted in sample buffer, frozen in liquid nitrogen, and stored at -70oC until analysis. Equal

7

amounts of protein samples were electrophoretically separated in polyacrylamide gels and

transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA), which

was incubated with appropriate primary antibodies, washed, and exposed to an appropriate

secondary antibody as previously described (32).

For normalization, immunoblotting performed with antiphospho-specific antibodies were

stripped in stripping buffer (Pierce Biotechnology, Rockford, IL) and reprobed with the

corresponding nonphospho-specific antibodies. Blots were developed

using an enhanced

chemiluminescence kit (Amersham, Piscataway, NJ), visualized,

and analyzed using a

ChemiDoc-It Imaging System (UVP, Upland, CA). Primary antibodies that were used in the

immunoblotting were: PKB (total and Ser 473, Cell Signaling Technology, Inc., Beverly, MA),

AMPKα (total and Thr 172, Cell Signaling), TSC2 (total and Thr 1462, Cell Signaling), mTOR

(total, Ser 2448 and Ser 2481, Cell Signaling), S6K1 (total and Thr 398, Cell Signaling), 4E-BP1

(total, Bethyl Laboratories, Montgomery, TX, and Thr 70, Cell Signaling), and eEF2 (total and

Thr 56, Cell Signaling).

Quantification of eIF4E·4EBP1 and eIF4E·eIF4G complexes. These complexes were

immunoprecipitated using an anti-eIF4E monoclonal antibody (gift of Dr. Leonard Jefferson,

Penn State University College of Medicine, Hershey, PA) from aliquots of fresh tissue

homogenates. Briefly, samples were homogenized in seven volumes of buffer (in mM: 20

HEPES, 2 EGTA, 50 NaF, 100 KCl, and 0.2 EDTA, pH 7.4) containing Sigma P3840 Protease

Inhibitor Cocktail (Sigma Chemical, St. Louis, MO) and centrifuged at 10,000 g for 10

min at

4°C. Supernatants were incubated overnight at 4°C with constant rocking with anti-eIF4E

antibody. Immunoprecipitates

were recovered with goat anti-rabbit IgG magnetic beads

(Polysciences,

Warrington, PA), washed and resuspended in sample buffer as

described

8

elsewhere (8), and immediately subjected to protein immunoblot analysis using rabbit anti-4E-

BP1 (Cell Signaling) antibody or rabbit anti-eIF4G (Bethyl Laboratories). Amounts of 4E-BP1

and eIF4G were corrected by the eIF4E recovered from the immunoprecipitate.

Analysis of mTORC1 and mTORC2 complexes. To determine the association between

mTOR and its partners (with raptor and GβL for the mTORC1 complex, or with rictor and GβL

for the mTORC2 complex), muscle samples were homogenized in CHAPS buffer according to

Williamson et al (39). The CHAPS buffer consisted of 40 mM HEPES, pH 7.5, 120 mM NaCl, 1

mM EDTA, 10 mM pyrophosphate, 10 mM β-glycerolphosphate, 40 mM NaF, 1.5 mM sodium

vanadate, 0.3% CHAPS, 0.1 mM PMSF, 1 mM benzamidine, and 1 mM DTT. The homogenate

was mixed on a platform rocker for 30 min at 4°C and then centrifuged at 1,000 g for 3 min

(4°C). The supernatant containing 500 µg of protein was combined with 2 µl of anti-mTOR

antibody (Cell Signaling) and mixed on a platform rocker overnight at 4°C. Following the

incubation, the immune complexes were isolated with a goat anti-mouse

BioMag IgG

(PerSeptive Diagnostics, Cambridge, MA) bead slurry. The magnetic bead complexes were

collected using a magnetic stand, washed twice with CHAPS buffer, and once in CHAPS buffer

containing 200 mM instead of 120 mM NaCl and 60 mM instead of 40 mM HEPES.

The

precipitates were rinsed with 100 µl of 1X SDS sample buffer and then boiled for 5 min and

centrifuged to collect the supernatant. The samples were subjected to SDS-PAGE followed by

immunoblotting with anti-raptor antibody (Cell Signaling), anti-rictor antibody (Cell Signaling)

and anti-GβL antibody (Cell Signaling). The mTOR-protein complexes were normalized by the

amount of total mTOR in the precipitates.

Statistics. All data were analyzed using one-way analysis of variance (ANOVA) with

saline-treated animals as the independent variable. When a significant overall effect was

9

observed, differences among individual means were assessed by the Tukey-Kramer comparisons

test. Probability values of P < 0.05 were considered statistically significant. Data are presented

as means ± SE. Data are presented as means ± SE.

RESULTS

Fractional protein synthesis rates. Several laboratories have demonstrated that leucine

administration increases in vivo skeletal muscle protein synthesis (1; 2). Recently, we also

provided evidence that leucine administration at physiological levels stimulates protein synthesis

in skeletal muscle of neonatal pigs (11). In this study, we examined whether treatment with

rapamycin could suppress the leucine-induced stimulation of protein synthesis in skeletal muscle

of neonatal pigs. As shown in Fig. 2, leucine infusion, compared to saline infusion, increased the

fractional rates of protein synthesis in skeletal muscle (P < 0.05). Administration of rapamycin

completely blocked the stimulatory effect of leucine on skeletal muscle protein synthesis.

Because it is important to take into account any possible effect of rapamycin on the basal fasting

rate of protein synthesis, rapamycin was also administrated to saline-infused pigs. We found that

rapamycin administration to saline-infused pigs did not reduce the fractional rate of skeletal

muscle protein synthesis below the basal fasting level.

mTORC1 and mTORC2 activation. We previously showed that administration of leucine

to neonatal pigs increases skeletal muscle protein synthesis through the activation of translation

initiation factors (11). However, the mechanism underlying the effect of leucine on mTORC1

was not investigated. Therefore, in this study we wished to determine whether physiological

changes in circulating leucine levels modulates the activation of signaling components upstream

of mTOR (AMPK, PKB, and TSC2) and the protein-protein interactions between members of

10

the mTORC1 and mTORC2 complexes and whether the response is affected by rapamycin

treatment. We found that neither leucine infusion nor rapamycin treatment affected the

phosphorylation of AMPK, PKB, or TSC2 (Fig. 3).

In cell culture (17), rapamycin’s potency for dissociating the interaction of raptor with

mTOR is increased under leucine-rich conditions and this is correlated with a decrease in

mTORC1 kinase activity. Therefore, we hypothesized that rapamycin’s ability to disturb the

raptor-mTOR interaction would be greater in skeletal muscle of leucine-infused piglets than in

saline-infused piglets. As shown in Fig. 4A, rapamycin severely destabilized the raptor-mTOR

complex. Although, there was no significant difference between the presence or absence of

leucine in the potency of rapamycin in perturbing the interaction of raptor with mTOR, there was

a tendency (P=0.20) for the rapamycin-induced dissociation of the raptor-mTOR complex to be

greater in the leucine-treated group compared to the control group. Furthermore, rapamycin did

not affect the GβL-mTOR complex (Fig. 4B), as well as the rictor-mTOR complex (mTORC2)

(Fig. 4C).

Activation of mTOR and its downstream signaling components. Next, we wished to

investigate the effect of rapamycin on the leucine-induced activation of mTOR and its

downstream components leading to the stimulation of skeletal muscle protein synthesis in

neonatal pigs. In this study we determined mTOR activation by measuring mTOR

phosphorylation at Ser 2448 and Ser 2481. Leucine enhanced mTOR phosphorylation at both

sites (P < 0.05) and this effect was abolished by rapamycin (Fig. 5).

mTOR regulates mRNA translation in response to nutrients/leucine and growth factors

via activation of its downstream substrates, S6K1 and 4E-BP1 (4). To determine the effects of

leucine and rapamycin on the activation of these important effectors of mTOR, the

11

phosphorylation state of S6K1 at Thr 398 and 4E-BP1 at Thr 70 were analyzed using

immunoblot analysis. Leucine significantly enhanced the phosphorylation of S6K1 and 4E-BP1

in skeletal muscle (P < 0.05) (Figs. 6A and 6B, respectively) and its effects on the

phosphorylation of these mTOR effectors were completely abolished by rapamycin.

One of the major factors that regulate the formation of an active eIF4E∙eIF4G complex is

4E-BP1 (29). In the hypo-phosphorylated state, this protein binds eIF4E to form an inactive

complex. Conversely, in the hyper-phosphorylated state, 4E-BP1 detaches from eIF4E, allowing

eIF4G to assemble an active eIF4E∙eIF4G complex and initiate mRNA translation. To determine

the effects of leucine and rapamycin on the assembly of an active eIF4E∙eIF4G complex and an

inactive eIF4E∙4EBP1 complex, immunoprecipitation and immunoblot analysis were performed.

As shown in Fig. 7A and 7B, leucine increased the formation of the active eIF4E∙eIF4G complex

(P < 0.05) and inhibited the formation of an inactive eIF4E∙4EBP1 complex (P < 0.05). The

leucine-induce formation of the active eIF4E∙eIF4G complex was completely prevented by

rapamycin (Fig. 7A). Likewise, the inhibitory effect of leucine on the formation of the inactive

eIF4E∙4EBP1 complex was eliminated by rapamycin (Fig. 7B).

Translation elongation in mammalian cells requires two eEFs (eukaryotic elongation

factors), eEF1 and eEF2. eEF2 mediates the translocation of the ribosome by three nucleotides

along the mRNA after the addition of each new amino acid. Phosphorylation of eEF2 on Thr 56

impairs its ability to bind ribosomes, thus inactivating this signaling protein and inhibiting

mRNA translation (18). In this study, we determined the effects of leucine on the

phosphorylation of eEF2 at Thr 56. As shown in Fig. 8, neither leucine nor rapamycin altered

the phosphorylation of eEF2 in skeletal muscle.

12

Effect of rapamycin on the protein abundance of the signaling components. Treatment

with rapamycin, which down regulates mTORC1 activation, has been associated with increased

autophagy, one of the intracellular protein degradation pathways (4). To determine whether

short-term exposure to rapamycin alters the degradation of several signaling proteins, we

measured their protein abundance. We found that the protein abundance of all signaling

components measured in the current study was not affected by short-term exposure to rapamycin

(data not shown)

DISCUSSION

Amino acids, especially the branched-chain amino acid, leucine, serve not only as

precursors for protein synthesis but also as signals that activate protein synthesis via activation of

intracellular signal transduction pathways that regulate mRNA translation (19). However,

despite tremendous efforts, the molecular mechanisms by which amino acids regulate mRNA

translation remain largely unknown. Studies using different cell culture systems have reported

conflicting results. Early data suggest that TSC1 and TSC2 are required for amino acid-induced

signaling through mTOR (34). Other studies indicate that rheb activation, but not TSC1/2

activation, is required for amino acid control of mTOR activation (30). A recent study suggested

that amino acid input into mTOR signaling was not mediated by the TSC1/2 complex or rheb but

by a class 3 phosphatidylinositol 3OH-kinase (PI3K) or human vacuolar protein sorting 34

(hVps34) (23). Our recent work demonstrated that a physiological rise in amino acids in vivo

had no effect on the phosphorylation of AMPK, PKB and TSC2 in skeletal muscle of neonatal

pigs, suggesting that amino acids transmit their signal downstream of the TSC1/2 complex (33).

13

Our in vivo studies have revealed that leucine is effective in stimulating protein synthesis

in skeletal muscle of the neonate although the response to leucine is less than that to a complete

amino acid mixture (11). The leucine-induced stimulation of skeletal muscle protein synthesis

involves the activation of signaling components downstream of mTOR leading to mRNA

translation. We further demonstrated that the acute (1 hr) leucine-induced stimulation of protein

synthesis is not maintained (2 hr), despite continued activation of the mTOR signaling pathway,

because circulating concentrations of essential amino acids fall to less than fasting levels as they

are utilized as substrates for protein synthesis (10). However, when circulating amino acid levels

are maintained at baseline levels for 2 hr, the leucine-induced stimulation of muscle protein

synthesis is maintained. In addition, we demonstrated that the stimulation of the mTOR

signaling pathway and protein synthesis in muscle by leucine, like that by feeding, insulin and

balanced amino acid mixtures, decreases with age (8; 10; 33). In this study, we wished to further

characterize the acute action of leucine by utilizing rapamycin, a potent inhibitor of mTOR.

Amino acids, especially leucine, regulate signaling through mTOR and thereby control a

number of components of the protein synthetic machinery, including initiation and elongation

factors (26). Rapamycin, a well-characterized mTOR inhibitor, has been widely used to study

mTOR functions including protein synthesis (21). Previously, we showed that rapamycin

completely blocked the feeding-induced stimulation of protein synthesis in liver, but only

attenuated 60% of the feeding-induced stimulation of skeletal muscle protein synthesis in

neonatal pigs (21). However, in the current study, infusion of leucine to raise circulating levels

of leucine to fed levels, similar to that achieved in previous feeding studies (5; 13), stimulated

muscle protein synthesis and this was completely blocked by rapamycin.

14

Whilst there is evidence of the involvement of the TSC1/2 complex on the amino acid-

induced activation of mTORC1 (34), in the current in vivo study we did not find any effect of

physiological levels of leucine on the phosphorylation of TSC2 and its kinases, PKB, and

AMPK. This is consistent with our previous finding that fed amino acid levels have no effect on

AMPK, PKB, or TSC2 phosphorylation, although raising insulin to fed levels increased PKB and

TSC2 phosphorylation but not AMPK (33). To the best of our knowledge, this is the first in vivo

study to examine the effect of leucine on the activation of TSC1/TSC2 complex.

Mechanistic studies using cell cultures showed that amino acids regulate the interaction

of raptor with mTOR, resulting in the activation of mTORC1 and its downstream effectors,

S6K1 and 4E-BP1 (4). A recent cell culture study indicates that under nutrient or leucine-rich

conditions, the mTOR-raptor association is destabilized resulting in the activation of mTOR (16).

However, our in vivo studies in skeletal muscle (31; 33) and other studies in heart (37) showed

that although both feeding and amino acid infusion increase S6K1 and 4E-BP1 activation, no

change in the interaction of mTOR with raptor is detected. In this study, leucine infusion also

did not have any effect on the interaction of raptor with mTOR. Thus, further study of the

association of mTOR and raptor is required.

Rapamycin, like nutrient deprivation, inhibits the activation of downstream effectors of

mTOR, but exactly how the drug perturbs mTOR function is unknown (4). A recent study

showed that one of the actions of rapamycin is to interfere with the ability of raptor to effectively

present substrates (such as S6K1 and 4E-BP1) to mTOR (4). In support to this model, we found

that rapamycin significantly reduced the interaction of raptor with mTOR in both control and

leucine-treated groups. In cell culture conditions, additional of leucine to nutrient-poor media

enhanced the destabilizing effect of rapamycin on the raptor-mTOR complex (17). Interestingly,

15

we found that there was a tendency (P=0.20) for the rapamycin-induced dissociation of the

raptor-mTOR complex to be greater in the leucine-treated group compared to the control group.

GβL is a crucial member of mTORC1 because its binding to the kinase domain of mTOR

stabilizes the interaction of raptor with mTOR (4). Furthermore, mTOR, GβL and rictor are

members of mTORC2, a rapamycin insensitive complex (4). While we were able to detect both

GβL and rictor in mTOR-immunoprecipitants, their interactions were not affected by rapamycin

treatment in vivo. To the best of our knowledge, this is the first in vivo study to examine the

effect of rapamycin on leucine regulation of the integrity of the mTORC1 and mTORC2

complexes.

Several lines of evidence suggest that leucine induces the activation of mTOR and its

downstream substrates (S6K1 and 4E-BP1) that control in part the step in translation initiation

involving the binding of mRNA to 40S ribosomal subunit (20). In this study, we found that in

fasting pigs, rapamycin did not reduce the phosphorylation of mTOR, S6K1, and 4E-BP1 below

the basal level. In addition, rapamycin completely blocked the leucine-induced phosphorylation

of mTOR, S6K1 and 4E-BP1 in skeletal muscle. Furthermore, rapamycin completely blunted

the eIF4G association with eIF4E and restored the association between 4E-BP1 and eIF4E. Most

of these observations are in agreement with a study from Vary et al (36).

Recent studies suggest that one of the key roles of mTOR is to regulate the activation of

elongation factors (25). Previously we found that neither amino acids nor leucine alter eEF2

phosphorylation (12; 33). Interestingly, rapamycin did not have any effect on the

phosphorylation of eEF2. Since eEF2 is thought to be one of the major substrates of mTOR

(25), the lack of effect of rapamycin on eEF2 phosphorylation is surprising but it could be due

to the physiological dose of leucine used in this study.

16

In summary, in the present study, we showed that the leucine-induced stimulation of

protein synthesis and the activation of signaling components leading to mRNA translation in

skeletal muscle of the neonate are sensitive to rapamycin. This contrasts with a study published

while we were preparing this manuscript which showed that the feeding-induced activation of 5’

mRNA translation is resistant to rapamycin in fetal liver (hepatocytes) but not adult liver (15).

In the current study, rapamycin blocked leucine-induced phosphorylation of mTOR, S6K1, and

4EBP1 and the formation of active eIF4E•eIF4G complex in neonatal muscle, in agreement with

data from cell culture studies (17; 22). However, study of the effect of leucine on the protein-

protein interaction of members of the mTORC1 complex is more challenging as we were unable

to detect an effect of leucine on raptor-mTOR association in vivo although we did show a

tendency toward leucine-induced loosening of the raptor-mTOR association, a condition that has

been documented in cell culture studies (17). Nevertheless, it is clear that our understanding of

the molecular mechanisms by which leucine regulates the signaling function of mTOC1 in vivo

is incomplete. Considering the potential significance of leucine’s action as a nutrient signal to

stimulate muscle protein synthesis in vivo, further studies to elucidate its mechanistic functions

are warranted.

17

ACKNOWLEDGMENTS

We thank D. E. Miller and J. C. Stubblefield for care of animals and L. F. Weiser for

secretarial assistance.

GRANTS

This work is publication of the United States Department of Agriculture/Agricultural

Research Service (USDA/ARS) Children’s Nutrition Research Center, Department of Pediatrics,

Baylor College of Medicine. This project has been funded in part by National Institutes of

Health (NIH) grant AR-44474 (T. A, Davis), and by the USDA/ARS under Cooperative

Agreement no. 6250510000-33 (T. A. Davis). The contents of this publication do not necessarily

reflect the views or policies of U.S. Department of Agriculture, nor does mention of trade names,

commercial products, or organizations imply endorsement by the U.S. Government.

18

REFERENCES

1. Anthony JC, Yoshizawa F, Anthony TG, Vary TC, Jefferson LS and Kimball SR.

Leucine stimulates translation initiation in skeletal muscle of postabsorptive rats via a

rapamycin-sensitive pathway. J Nutr 130: 2413-2419, 2000.

2. Bolster DR, Vary TC, Kimball SR and Jefferson LS. Leucine regulates translation

initiation in rat skeletal muscle via enhanced eIF4G phosphorylation. J Nutr 134: 1704-

1710, 2004.

3. Chien PF, Smith K, Watt PW, Scrimgeour CM, Taylor DJ and Rennie MJ. Protein

turnover in the human fetus studied at term using stable isotope tracer amino acids. Am J

Physiol 265: E31-E35, 1993.

4. Corradetti MN and Guan KL. Upstream of the mammalian target of rapamycin: do all

roads pass through mTOR? Oncogene 25: 6347-6360, 2006.

5. Davis TA, Burrin DG, Fiorotto ML and Nguyen HV. Protein synthesis in skeletal

muscle and jejunum is more responsive to feeding in 7-than in 26-day-old pigs. Am J

Physiol 270: E802-E809, 1996.

6. Davis TA, Fiorotto ML, Burrin DG, Reeds PJ, Nguyen HV, Beckett PR, Vann RC and

O'Connor PM. Stimulation of protein synthesis by both insulin and amino acids is unique

to skeletal muscle in neonatal pigs. Am J Physiol Endocrinol Metab 282: E880-E890, 2002.

19

7. Davis TA, Fiorotto ML, Nguyen HV and Burrin DG. Aminoacyl-tRNA and tissue free

amino acid pools are equilibrated after a flooding dose of phenylalanine. Am J Physiol 277:

E103-E109, 1999.

8. Davis TA, Nguyen HV, Suryawan A, Bush JA, Jefferson LS and Kimball SR.

Developmental changes in the feeding-induced stimulation of translation initiation in

muscle of neonatal pigs. Am J Physiol Endocrinol Metab 279: E1226-E1234, 2000.

9. Ehrenkranz RA. Early, aggressive nutritional management for very low birth weight

infants: what is the evidence? Semin Perinatol 31: 48-55, 2007.

10. Escobar J, Frank JW, Suryawan A, Nguyen HV and Davis TA. Amino acid

Availability and Age Affect the Leucine Stimulation of Protein Synthesis and eIF4F

Formation in Muscle. Am J Physiol Endocrinol Metab 2007.

11. Escobar J, Frank JW, Suryawan A, Nguyen HV, Kimball SR, Jefferson LS and Davis

TA. Physiological rise in plasma leucine stimulates muscle protein synthesis in neonatal

pigs by enhancing translation initiation factor activation. American Journal of Physiology-

Endocrinology and Metabolism 288: E914-E921, 2005.

12. Escobar J, Frank JW, Suryawan A, Nguyen HV, Kimball SR, Jefferson LS and Davis

TA. Regulation of cardiac and skeletal muscle protein synthesis by individual branched-

chain amino acids in neonatal pigs. American Journal of Physiology-Endocrinology and

Metabolism 290: E612-E621, 2006.

20

13. Frank JW, Escobar J, Suryawan A, Nguyen HV, Kimball SR, Jefferson LS and Davis

TA. Dietary protein and lactose increase translation initiation factor activation and tissue

protein synthesis in neonatal pigs. American Journal of Physiology-Endocrinology and

Metabolism 290: E225-E233, 2006.

14. Garlick PJ, McNurlan MA and Preedy VR. A rapid and convenient technique for

measuring the rate of protein synthesis in tissues by injection of [3H]phenylalanine.

Biochem J 192: 719-723, 1980.

15. Gruppuso PA, Tsai SW, Boylan JM and Sanders JA. Hepatic translation control in the

late gestation fetal rat. Am J Physiol Regul Integr Comp Physiol In Print, 2008.

16. Kim DH and Sabatini DM. Raptor and mTOR: subunits of a nutrient-sensitive complex.

Curr Top Microbiol Immunol 279: 259-270, 2004.

17. Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst

P and Sabatini DM. mTOR interacts with raptor to form a nutrient-sensitive complex that

signals to the cell growth machinery. Cell 110: 163-175, 2002.

18. Kimball SR. Regulation of translation initiation by amino acids in eukaryotic cells. Prog

Mol Subcell Biol 26: 155-184, 2001.

19. Kimball SR and Jefferson LS. Regulation of protein synthesis by branched-chain amino

acids. Current Opinion in Clinical Nutrition and Metabolic Care 4: 39-43, 2001.

21

20. Kimball SR and Jefferson LS. Role of amino acids in the translational control of protein

synthesis in mammals. Semin Cell Dev Biol 16: 21-27, 2005.

21. Kimball SR, Jefferson LS, Nguyen HV, Suryawan A, Bush JA and Davis TA. Feeding

stimulates protein synthesis in muscle and liver of neonatal pigs through an mTOR-

dependent process. Am J Physiol Endocrinol Metab 279: E1080-E1087, 2000.

22. Kimball SR, Shantz LM, Horetsky RL and Jefferson LS. Leucine regulates translation

of specific mRNAs in L6 myoblasts through mTOR-mediated changes in availability of

eIF4E and phosphorylation of ribosomal protein S6. J Biol Chem 274: 11647-11652, 1999.

23. Nobukuni T, Joaquin M, Roccio M, Dann SG, Kim SY, Gulati P, Byfield MP, Backer

JM, Natt F, Bos JL, Zwartkruis FJ and Thomas G. Amino acids mediate mTOR/raptor

signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proc Natl Acad

Sci U S A 102: 14238-14243, 2005.

24. O'Connor PM, Bush JA, Suryawan A, Nguyen HV and Davis TA. Insulin and amino

acids independently stimulate skeletal muscle protein synthesis in neonatal pigs. Am J

Physiol Endocrinol Metab 284: E110-E119, 2003.

25. Proud CG. Role of mTOR signalling in the control of translation initiation and elongation

by nutrients. Curr Top Microbiol Immunol 279: 215-244, 2004.

22

26. Proud CG. Amino acids and mTOR signalling in anabolic function. Biochem Soc Trans

35: 1187-1190, 2007.

27. Saigal S, Stoskopf BL, Streiner DL and Burrows E. Physical growth and current health

status of infants who were of extremely low birth weight and controls at adolescence.

Pediatrics 108: 407-415, 2001.

28. Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, Markhard AL and

Sabatini DM. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.

Mol Cell 22: 159-168, 2006.

29. Shah OJ, Anthony JC, Kimball SR and Jefferson LS. 4E-BP1 and S6K1: translational

integration sites for nutritional and hormonal information in muscle. Am J Physiol

Endocrinol Metab 279: E715-E729, 2000.

30. Smith EM, Finn SG, Tee AR, Browne GJ and Proud CG. The tuberous sclerosis protein

TSC2 is not required for the regulation of the mammalian target of rapamycin by amino

acids and certain cellular stresses. J Biol Chem 280: 18717-18727, 2005.

31. Suryawan A, Escobar J, Frank JW, Nguyen HV and Davis TA. Developmental

regulation of the activation of signaling components leading to translation initiation in

skeletal muscle of neonatal pigs. Am J Physiol Endocrinol Metab 291: E849-E859, 2006.

23

32. Suryawan A, Nguyen HV, Bush JA and Davis TA. Developmental changes in the

feeding-induced activation of the insulin-signaling pathway in neonatal pigs. Am J Physiol

Endocrinol Metab 281: E908-E915, 2001.

33. Suryawan A, Orellana RA, Nguyen HV, Jeyapalan AS, Fleming JR and Davis TA.

Activation by Insulin and Amino Acids of Signaling Components Leading to Translation

Initiation in Skeletal Muscle of Neonatal Pigs Is Developmentally Regulated. Am J Physiol

Endocrinol Metab 2007.

34. Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC and Blenis J.

Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian

target of rapamycin (mTOR)-mediated downstream signaling. Proc Natl Acad Sci U S A

99: 13571-13576, 2002.

35. Vary TC. Acute oral leucine administration stimulates protein synthesis during chronic

sepsis through enhanced association of eukaryotic initiation factor 4G with eukaryotic

initiation factor 4E in rats. J Nutr 137: 2074-2079, 2007.

36. Vary TC, Anthony JC, Jefferson LS, Kimball SR and Lynch CJ. Rapamycin blunts

nutrient stimulation of eIF4G, but not PKCepsilon phosphorylation, in skeletal muscle. Am

J Physiol Endocrinol Metab 293: E188-E196, 2007.

24

37. Vary TC, Deiter G and Lynch CJ. Rapamycin limits formation of active eukaryotic

initiation factor 4F complex following meal feeding in rat hearts. J Nutr 137: 1857-1862,

2007.

38. Wang X and Proud CG. Methods for studying signal-dependent regulation of translation

factor activity. Methods Enzymol 431: 113-142, 2007.

39. Williamson DL, Bolster DR, Kimball SR and Jefferson LS. Time course changes in

signaling pathways and protein synthesis in C2C12 myotubes following AMPK activation

by AICAR. Am J Physiol Endocrinol Metab 291: E80-E89, 2006.

25

FIGURE LEGENDS

Figure 1. Current concepts of the amino acid signaling pathway leading to protein

synthesis.

Figure 2. Fractional rates of protein synthesis in skeletal muscle of 7-day-old-pigs after

60 min of infusion of saline (Sal), saline with rapamycin (Sal+Rap), 400 µmol·kg–1

·h–1

of

leucine without rapamycin (Leu), and 400 µmol·kg–1

·h–1

of leucine with rapamycin (Leu+Rap).

Values are means ± pooled SEM; n = 5-7 per treatment. a,b

Values with different superscripts

differ significantly (P < 0.05).

Figure 3. Phosphorylation of protein kinase B (PKB) at Ser473 (A), AMP-activated

protein kinase (AMPK) at Thr172 (B), and tuberous sclerosis complex-2 (TSC2) at Thr1462 (C)

in skeletal muscle of 7-day-old-pigs after 60 min of infusion of saline (Sal), saline with

rapamycin (Sal+Rap), 400 µmol·kg–1

·h–1

of leucine without rapamycin (Leu), and 400 µmol·kg–

1·h

–1 of leucine with rapamycin (Leu+Rap). Phosphorylation of PKB, AMPK, and TSC2 were

corrected by total PKB, AMPK, and TSC2, respectively. Values are means ± pooled SEM; n =

5-7 per treatment.

Figure 4. Association between mTOR and raptor (A), mTOR and GβL (B), and mTOR

and rictor (C) in skeletal muscle of 7-day-old-pigs after 60 min of infusion saline (Sal), saline

with rapamycin (Sal+Rap), 400 µmol·kg–1

·h–1

of leucine without rapamycin (Leu), and 400

µmol·kg–1

·h–1

of leucine with rapamycin (Leu+Rap). Association between mTOR and its

partners was corrected by total mTOR in the immunoprecipitant. Values are means ± pooled

SEM; n = 5-7 per treatment. a,b

Values with different superscripts differ significantly (P < 0.05).

Figure 5. Phosphorylation of mammalian target of rapamycin (mTOR) at Ser2481 (A),

at Ser2448 (B) in skeletal muscle of 7-day-old-pigs after 60 min of infusion of saline (Sal), saline

26

with rapamycin (Sal+Rap), 400 µmol·kg–1

·h–1

of leucine without rapamycin (Leu), and 400

µmol·kg–1

·h–1

of leucine with rapamycin (Leu+Rap). Phosphorylation of mTOR were corrected

by total mTOR. Values are means ± pooled SEM; n = 5-7 per treatment. a,b

Values with different

superscripts differ significantly (P < 0.05)

Figure 6. Phosphorylation of the 70-kDa ribosomal protein S6 kinase (S6K1) at Thr389

(A), and eukaryotic initiation factor (eIF) binding protein-1 (4E-BP1) at Thr70 (B) in skeletal

muscle of 7-day-old-pigs after 60 min of infusion of saline (Sal), saline with rapamycin

(Sal+Rap), 400 µmol·kg–1

·h–1

of leucine without rapamycin (Leu), and 400 µmol·kg–1

·h–1

of

leucine with rapamycin (Leu+Rap). Phosphorylation of S6K1 and 4E-BP1 were corrected by

total S6K1 and 4E-BP1, respectively. Values are means ± pooled SEM; n = 5-7 per treatment.

a,bValues with different superscripts differ significantly (P < 0.05).

Figure 7. Association of eukaryotic initiation factor (eIF) 4G with eIF4E (A) and 4E-

BP1 with eIF4E (B) in skeletal muscle of 7-day-old-pigs after 60 min of infusion of saline (Sal),

saline with rapamycin (Sal+Rap), 400 µmol·kg–1

·h–1

of leucine without rapamycin (Leu), and 400

µmol·kg–1

·h–1

of leucine with rapamycin (Leu+Rap). The formation of eIF4G•eIF4E and 4E-

BP1•eIF4E complexes were corrected by total eIF4E in the immunoprecipitant. Values are

means ± pooled SEM; n = 5-7 per treatment. a,b

Values with different superscripts differ

significantly (P < 0.05).

Figure 8. Phosphorylation of eukaryotic elongation factor 2 (eEF2) at Thr56 in skeletal

muscle of 7-day-old-pigs after 60 min of infusion of saline (Sal), saline with rapamycin

(Sal+Rap), 400 µmol·kg–1

·h–1

of leucine without rapamycin (Leu), and 400 µmol·kg–1

·h–1

of

leucine with rapamycin (Leu+Rap). Phosphorylation of eEF2 was corrected by total eEF2.

Values are means ± pooled SEM; n = 5-7 per treatment.

Figure 1

Growth Factors(Insulin)

IRS 1/2P

PKBP

AMPK

Amino Acids(Leucine)

Rheb

TSC2TSC1P

mTORGβL Rictor mTORC2mTORC1 mTOR

GβL

Raptor

Rapamycin

P

S6K14EBP1

eIF4E eIF4G

Raptor

4EBP1eIF4E

P

P

P

P

eEF2K

eEF2 P

AMPK

S6eIF4E eIF4GeEF2

Initiation Elongation

Protein Synthesis

Figure 2n

Synt

hesi

s %

/day

)

10

15

20

a aa

b

Sal Sal+Rap Leu Leu+Rap

Prot

ein (%

0

5

A1.5

Figure 3

PKB

PKBSer 473

P-PK

B (

AU

)

0.5

1.0P

Sal Sal+Rap Leu Leu+Rap0.0

AMPKThr 172

K (A

U)

B

0.75

1.00

AMPK

AMPK

P-A

MPK

0.25

0.50

C

Sal Sal+Rap Leu Leu+Rap0.00

TSC2

TSC2Thr 1462

0.3

0.4

0.5

0.6

C2

(AU

)

0.0

0.1

0.2

P-TS

Sal Sal+Rap Leu Leu+Rap

plex

Figure 4

2.0A

mTOR

Raptor

or -

mTO

R c

omp

(AU

)

0 5

1.0

1.5 aa

Sal Sal+Rap Leu Leu+Rap

Rap

to

0.0

0.5b

b

GβL

ompl

ex

3

4

5B mTOR

β

GβL

-m

TOR

co

(AU

)

0

1

2

3

Sal Sal+Rap Leu Leu+Rap0

7.5C mTOR

Rictor

mTO

R c

ompl

ex

(AU

)

2 5

5.0

7.5

Ric

tor -

m

Sal Sal+Rap Leu Leu+Rap0.0

2.5

Figure 5

Ser 2448

A4

b

mTOR

mTORSer 2448

mTO

R (

AU

)

2

3

a aa

b

P-m

Sal Sal+Rap Leu Leu+Rap0

1

mTOR

mTORSer 2481

0.75

1.00

b

(AU

)

B

0.00

0.25

0.50

a aaP-m

TOR

Sal Sal+Rap Leu Leu+Rap

Figure 6

A1 5

S6K1

S6K1Thr 389

P-S6

K1

(AU

)

1.0

1.5

b

P

Sal Sal+Rap Leu Leu+Rap0.0

0.5

a aa

4EBP1

4EBP1Thr 70

B

P1 (A

U)

0.50

0.75

b

P-4E

BP

Sal Sal+Rap Leu Leu+Rap0.00

0.25a aa

Sal Sal Rap Leu Leu Rap

Figure 7

AeIF4E

eIF4G

F4E

com

plex

(A

U) 5.0

7.5

b

eIF4

G-e

IF

Sal Sal+Rap Leu Leu+Rap0.0

2.5

a aa

IF4E

4EBP1

ompl

ex

B

0.50

0.75a

aa

eIF4E

4EB

P1-e

IF4E

co

(AU

)

0 00

0.25

b0.00

Sal Sal+Rap Leu Leu+Rap

Figure 8

eEF2Thr 56

15

U)

eEF2

5

10

P-eE

F2 (A

U

0Sal Sal+Rap Leu Leu+Rap