![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033
2To whom correspondence should be addressed.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
KEY WORDS: • leucine • protein synthesis • translation initiation • skeletal muscle • rats
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, Yoshizawa et al. 1995
and 1998
). This anabolic effect may be attributed in part to an
increase in amino acid supply to the muscle, thereby augmenting
substrate availability for peptide synthesis. Additionally, individual
amino acids may function as nutritional signaling molecules that
regulate mRNA translation. Indeed, we recently demonstrated that oral
administration of leucine independently stimulates protein synthesis in
muscle in association with enhanced rates of translation initiation
(Anthony et al. 1999
and 2000
). The remaining
branched-chain amino acids
(BCAA),3
isoleucine and valine, are similar in structure to leucine, and like
leucine, are degraded extensively in skeletal muscle. Therefore,
dietary isoleucine or valine may also signal independently for enhanced
rates of translation initiation in muscle. Whether these BCAA exhibit
anabolic potential similar to that of leucine remains to be determined.
A principal site in the regulation of translation initiation involves
the binding of mRNA to the 40 S ribosome [reviewed by Pain (1996)
and Voorma et al. (1994)
]. Oral
administration of leucine facilitates this process by increasing the
availability of eukaryotic initiation factor (eIF) 4E, a protein that
binds the m7GTP cap present at the 5'-end of the
mRNA, for binding eIF4G, a large, 220-kDa polypeptide that functions as
a scaffold for eIF4E, the mRNA (via association with eIF4E) and the
ribosome (via association with eIF3) (Anthony et al. 2000
). The increase in eIF4E availability is due in part to the
leucine-dependent hyperphosphorylation of the translational
repressor, eIF4E-binding protein 1 (4E-BP1). Increased phosphorylation
of 4E-BP1 decreases its affinity for eIF4E, thereby facilitating the
association of eIF4E with eIF4G.
Increased activity of the 70-kDa ribosomal protein S6 kinase (S6K1) has
been implicated in stimulating protein synthesis under conditions that
promote 4E-BP1 phosphorylation (Sonenberg 1996
). We
demonstrated previously that oral administration of leucine enhances
the phosphorylation state of S6K1 (Anthony et al. 2000
).
Because phosphorylation of the kinase is associated with its activation
(Cheatham et al. 1994
, Chung et al. 1994
), our previous observations suggest the involvement of
S6K1 in stimulating protein synthesis after oral administration of
leucine. The ability of leucine to promote the hyperphosphorylation of
both 4E-BP1 and S6K1 suggests a common signaling pathway through which
the amino acid upregulates translational efficiency.
Recent studies using cells in culture indicate that the
hyperphosphorylation of 4E-BP1 and S6K1 by amino acids, and leucine, in
particular, involves a signaling pathway that includes the protein
kinase mTOR that is inhibited by the immunosuppressant drug rapamycin
(Kimball et al. 1999
, Patti et al. 1998
,
Xu et al. 1998
). Therefore, leucine may stimulate
translation initiation by modulating the activity of mTOR in vivo. The
involvement of mTOR in stimulating protein synthesis in skeletal muscle
after oral administration of leucine remains to be determined.
The objectives of the present study were twofold: 1) to determine whether leucine is unique among the BCAA in its ability to stimulate protein synthesis in skeletal muscle of food-deprived rats; and 2) to investigate whether changes in muscle protein synthesis after leucine administration involve a signaling pathway that includes mTOR. To investigate the role of mTOR signaling in the stimulation of protein synthesis and translation initiation in vivo, food-deprived rats were injected intravenously with the immunosuppressant drug rapamycin, a specific inhibitor of mTOR, before leucine administration.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The animal facilities and protocol were reviewed and approved by the
Institutional Animal Care and Use Committee of the Pennsylvania State
University, College of Medicine. Male Sprague-Dawley rats (
200
g) were maintained on a 12-h light:dark cycle with food (Harlan-Teklad
Rodent Chow, Madison, WI) and water provided freely. The food contained
24% protein and 4% fat.
Study 1.
Food-deprived (18 h) rats were assigned randomly to one of the
following four dietary treatments: control (Con), or administered 1.35
g/kg body weight L-valine (Val), L-isoleucine
(Ile) or L-leucine (Leu) by oral gavage. The dose for each
amino acid was 2.5 mL/100 g body weight (prepared as 54.0 g/L in
distilled water). Control rats were fed 2.5 mL saline/100 g body weight
(0.155 mol/L). After amino acid administration, rats were returned to
their cages where they were permitted free access to water only. The
amount of each amino acid administered was equivalent to the amount of
leucine consumed by rats of this age and strain during 24 h
(Gautsch et al. 1998
) of free access to an
AIN-93 powdered diet (Harlan-Teklad, Madison, WI).
Study 2.
Rats were food-deprived for 16 h and then randomly administered 0.75 mg rapamycin (Rap)/kg body weight (Calbiochem-Novabiochem, La Jolla, CA) or an equal volume of excipient (Con; 0.155 mol/L NaCl, 2% v/v ethanol) via the tail vein. Two hours later, one half of the rats in the Rap and Con groups were orally administered 1.35 g L-leucine/kg body weight as described in Study 1 (RapLeu and ConLeu, respectively). Rats not receiving leucine were gavaged with 2.5 mL saline/100 g body weight (0.155 mol/L).
Administration of metabolic tracer and sample collection.
A flooding dose (1.0 mL/100 g body weight) of
L-[2,3,4,5,6-3H] phenylalanine (150 mmol/L
containing 3.70 GBq/L) was injected via the tail vein 50 min after oral
administration of amino acids for the measurement of synthesis of total
mixed proteins in skeletal muscle (Garlick et al. 1980
).
Exactly 1 h after oral administration of amino acids, rats were
killed by decapitation. Trunk blood was collected and centrifuged at
1800 x g for 10 min at 4°C to obtain serum. The
gastrocnemius and plantaris muscles were excised as a unit, weighed and
homogenized in 7 volumes of buffer consisting of (in mmol/L) 20
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (pH 7.4), 100 KCl, 0.2 EDTA, 2 ethylene glycol-bis
(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 1
dithiothreitol, 50 sodium fluoride, 50 ß-glycerophosphate, 0.1
phenylmethylsulfonyl fluoride, 1 benzamidine and 0.5 sodium vanadate.
An aliquot (0.5 mL) was used for the measurement of skeletal muscle
protein synthesis as described below. The remainder of the homogenate
was immediately centrifuged at 10,000 x g for 10
min at 4°C. The supernatant was used for measurement of eIF
distribution and phosphorylation as described below.
Serum measurements.
Serum insulin concentrations were analyzed using a commercial RIA kit
for rat insulin (Linco Research, St. Charles, MO). Serum was analyzed
for amino acids by derivatizing with phenylisothiocyanate and HPLC
analysis as described previously (MacLean et al. 1991
).
Measurement of skeletal muscle protein synthesis.
Fractional rates of skeletal muscle protein synthesis were estimated
from the rate of incorporation of radioactive phenylalanine into muscle
protein using the specific radioactivity of serum phenylalanine as
representative of the precursor pool (Kimball et al. 1992
). The elapsed time from injection of the metabolic tracer
until homogenization of muscle was recorded as the actual time for
incorporation of labeled amino acid into protein (13 min).
Quantitation of 4E-BP1 · eIF4E and eIF4G · eIF4E complexes.
eIF4E was immunoprecipitated from 10,000 x g
supernatants of muscle homogenate using a monoclonal antibody to eIF4E
(Kimball et al. 1997
). Next, samples were subjected to
immunoblot analysis using polyclonal antibodies to either 4E-BP1 or
eIF4G to determine the association of 4E-BP1 and eIF4G with eIF4E,
respectively (Kimball et al. 1997
). Results were
normalized to the amount of eIF4E in the immunoprecipitates.
Quantitation of phosphorylated and unphosphorylated eIF4E.
The phosphorylated and unphosphorylated forms of eIF4E were separated
by isoelectric focusing of 10,000 x g supernatants
on a slab gel and quantitated by protein immunoblot analysis as
described previously (Kimball et al. 1997
).
Examination of 4E-BP1 phosphorylation state.
4E-BP1 was immunoprecipitated from 10,000 x g
supernatants of skeletal muscle with an anti-4E-BP1 monoclonal antibody
and then was subjected to protein immunoblot analysis as described
previously (Kimball et al. 1997
).
Phosphorylation of S6K1.
Phosphorylation of S6K1 was determined in 10,000 x g supernatants by protein immunoblot analysis as
previously described (Gautsch et al. 1998
).
Phosphorylation of S6K1 at Thr389.
Phosphorylation of S6K1 at Thr389 was determined in 10,000
x g supernatants by protein immunoblot analysis as
described previously (Kimball et al. 1997
). Membranes
were incubated with a rabbit polyclonal antibody, which specifically
recognizes phosphorylation of S6K1 at Thr389 (New England
Biolabs, Beverly, MA).
Statistical analysis.
All data were analyzed by the STATISTICA statistical software package for the Macintosh, volume II (StatSoft, Tulsa, OK). All data are expressed as means ± SEM. If the variance was heterogeneous, an appropriate transformation of the data was performed. In Study 1, data were analyzed using a one-way ANOVA with treatment group as the independent variable. When a significant overall effect was detected, differences among individual means were assessed with Duncan’s Multiple Range post-hoc test. In Study 2, a two-way ANOVA was performed to assess main vs. interaction effects with leucine administration and drug treatment as independent variables. If no significant interaction was detected, a one-way ANOVA was performed with treatment group (leucine + drug) as the independent variable. When a significant interaction or overall effect was detected, differences among individual means were assessed with Duncan’s Multiple Range post-hoc test. The level of significance was set at P < 0.05 for all statistical tests.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In contrast, provision of valine, isoleucine or leucine elevated
serum concentrations of the administered amino acid (Table 1)
.
Additionally, leucine administration reduced circulating concentrations
of isoleucine and valine compared with control rats. This reduction of
serum isoleucine and valine after leucine administration may reflect an
increase in uptake of those amino acids to support enhanced rates of
protein synthesis (Table 1)
. Leucine was unique among the BCAA in its
ability to stimulate skeletal muscle protein synthesis in vivo.
Provision of leucine stimulated protein synthesis 65% compared with
control rats. In contrast, neither valine nor isoleucine administration
affected rates of protein synthesis.
|
). Leucine administration reduced the amount of the inactive
4E-BP1 · eIF4E complex to 17% of values observed in controls (Fig. 1A
). Concomitantly, the association of eIF4G with eIF4E was
fourfold greater in rats fed leucine compared with food-deprived
rats (Fig. 1C
). This increase in the availability of eIF4E
for binding eIF4G resulted from hyperphosphorylation of 4E-BP1. 4E-BP1
phosphorylation was fivefold greater in rats fed leucine compared with
controls (Fig. 1B
).
|
), and the
amount of 4E-BP1 in the eIF4E immunoprecipitate was reduced to 65% of
control values (Fig. 1A
). Furthermore, the association of
eIF4E with eIF4G doubled and was statistically intermediate between
control rats and rats administered leucine (Fig. 1C
). In
contrast, valine administration did not alter either 4E-BP1
phosphorylation or the association of eIF4E with either 4E-BP1 or
eIF4G. These results indicate that although oral administration of
isoleucine promotes increased availability of eIF4E, these alterations
in translation initiation do not lead to a stimulation of protein
synthesis.
The effect of BCAA administration on eIF4E phosphorylation was also
examined. Phosphorylation of eIF4E in cells in culture has been shown
to be increased under a variety of conditions in which rates of
translation initiation are accelerated (Sonenberg 1996
).
In Study 1, we did not observe any significant differences among
treatment groups in the percentage of eIF4E in the phosphorylated form
(Fig. 1D
). However, the absolute values obtained for eIF4E
phosphorylation in rats fed leucine are less than those observed in the
other treatment groups. Additionally, leucine administration resulted
in an inhibition of eIF4E phosphorylation in Study 2. Further,
we demonstrated previously that leucine reduces the amount of eIF4E in
the phosphorylated form (Anthony et al. 2000
).
Collectively, these observations indicate that leucine administration
reduces the amount of eIF4E in the phosphorylated form.
To further evaluate the effects of BCAA administration on translation
initiation, we examined the relative abilities of leucine, isoleucine
and valine to enhance phosphorylation of S6K1. During SDS-PAGE,
S6K1 resolves into multiple electrophoretic forms, with increased
phosphorylation corresponding to decreased electrophoretic mobility.
The slowest migrating electrophoretic forms represent S6K1 phosphoryled
on multiple residues including Thr389, a residue
whose phosphorylation is associated with increased activation of the
protein (Burnett et al. 1998
). After food deprivation,
the kinase became hypophosphorylated, and only the fastest migrating
electrophoretic forms were observed (Fig. 2
). Leucine was most effective among the BCAA in its ability to stimulate
phosphorylation of S6K1 (Fig. 2A
), particularly on
Thr389 (Fig. 2B
). Moreover, isoleucine
administration also promoted phosphorylation of the kinase on
Thr389 although to a lesser extent than leucine.
Finally, administration of valine did not alter S6K1 phosphorylation
compared with control rats.
|
). In contrast, both leucine administration and drug treatment raised
serum leucine concentrations (Table 2)
. Moreover, rapamycin
independently elevated serum 3-methylhistidine concentrations (Table 2)
. Circulating 3-methylhistidine concentrations have been shown to
correlate with rates of myofibrillar protein breakdown (Nagasawa et al. 1996
). Therefore, the increase in serum leucine values
in rats fed rapamycin may be due in part to enhanced degradation of
myofibrillar proteins. Further studies are required to determine the
involvement of mTOR in the regulation of muscle protein breakdown.
|
. Protein synthesis rates in
rats administered leucine alone were 42% greater than in
food-deprived controls. Similarly, leucine also stimulated protein
synthesis 35% in rats injected with rapamycin (Rap vs. RapLeu). On the other hand, rapamycin treatment reduced rates of protein synthesis independently of leucine administration. Protein synthesis rates in rats treated with rapamycin and then administered leucine were only 72% of those in rats fed leucine alone and were equal to those of food-deprived controls administered saline. Additionally, rapamycin also tended to inhibit protein synthesis in food-deprived rats (Con vs. Rap; P = 0.058). These results suggest that the leucine-dependent stimulation of muscle protein synthesis is rapamycin sensitive in part and involves mTOR.
Administration of leucine increased the availability of eIF4E for
binding eIF4G (Fig. 3
). Leucine reduced the association of 4E-BP1 with eIF4E to 50% of that
in food-deprived rats (Fig. 3A
). The
leucine-dependent inhibition of eIF4E binding 4E-BP1 was associated
with 4E-BP1 phosphorylation values that were fourfold greater than
those of food-deprived controls (Fig. 3B
). Consistent
with the above data, eIF4G·eIF4E complex formation was more than
doubled in rats fed leucine (Fig. 3C
). Finally, leucine
reduced the amount of eIF4E in the phosphorylated form compared with
food-deprived rats.
|
). Rapamycin administration was also associated with
reduced 4E-BP1 phosphorylation (Fig. 3B
). 4E-BP1
phosphorylation in drug-treated rats (Rap and RapLeu) was < 10% of that in rats fed leucine alone and significantly lower than
that in food-deprived controls. Furthermore, rapamycin treatment
reversed the effects of leucine on formation of the active
eIF4G · eIF4E complex, resulting in values that were 50% less than
those of ConLeu and not different than those of food-deprived
controls (Fig. 3C
). Additionally, rapamycin also inhibited
the association of eIF4E with eIF4G in rats not receiving leucine (Con
vs. Rap). Finally, rapamycin reversed the leucine-dependent
decrease in eIF4E phosphorylation (Fig. 3D
), resulting in
values that were not different than those of food-deprived rats
(Con and Rap). Collectively, these results suggest that the
leucine-dependent increase in eIF4E availability for formation of
the active eIF4G · eIF4E complex occurs via a rapamycin-sensitive
signaling pathway.
Rapamycin also inhibited the leucine-dependent hyperphosphorylation
of S6K1 (Fig. 4
). Leucine promoted hyperphosphorylation of the kinase and resulted in
bands with the slowest electrophoretic mobility (Fig. 4A
).
The ability of leucine to stimulate phosphorylation of S6K1 was ablated
in the presence of rapamycin. This was particularly evident when
examining the phosphorylation of Thr389 (Fig. 4B
). Phosphorylation of Thr389 was
observed only in rats administered leucine alone. Little or no
phosphorylation of this residue was observed in any other treatment
group. These observations indicate that rapamycin reverses the
leucine-dependent phosphorylation of S6K1 in skeletal muscle.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, Patti et al. 1998
). Thus, amino acids function independently as nutritional
signaling molecules that regulate protein synthesis. Numerous reports
have established that in skeletal muscle, the indispensable BCAA,
leucine, is unique in this regard (Anthony et al. 2000
,
Buse et al. 1979
, Buse and Reid 1975
,
Li and Jefferson 1978
).
The data presented here demonstrate that leucine is the only BCAA that
stimulates skeletal muscle protein synthesis in food-deprived rats
and is most effective at promoting hyperphosphorylation of both 4E-BP1
and S6K1. Leucine-dependent hyperphosphorylation of 4E-BP1 resulted
in increased availability of eIF4E to form the active eIF4G · eIF4E
complex. To a lesser extent, isoleucine also promoted eIF4E
availability and facilitated hyperphosphorylation of S6K1. These
changes did not result in increased rates of protein synthesis at
1 h. However, the possibility that isoleucine may have altered
protein synthesis at a different sampling time or if provided in
greater amounts cannot be eliminated. Alternatively, the reduced
ability of isoleucine to enhance the phosphorylation state of 4E-BP1
and S6K1 may indicate specific structural requirements of leucine for
regulating signaling pathways that modulate translation initiation
(Lynch et al. 2000
, Shigemitsu et al. 1999
).
Several investigators have reported that in cells in culture, amino
acids, and in particular, leucine, enhance the phosphorylation of
4E-BP1 and S6K1 through a signaling pathway that includes the protein
kinase mTOR (Kimball et al. 1999
, Patti et al. 1998
, Xu et al. 1998
). Studies using L6 cells in
culture show that treatment with rapamycin blocks the activation of
S6K1 as well as the phosphorylation of 4E-BP1 caused by leucine
(Kimball et al. 1999
). Similarly, rapamycin inhibits the
leucine-induced activation of S6K1 in H4IIE cells. In this study,
we demonstrated for the first time that rapamycin inhibits
leucine-dependent hyperphosphorylation of 4E-BP1 and S6K1 in
skeletal muscle in vivo. These results indicate that oral
administration of leucine increases the availability of eIF4E for
active eIF4G · eIF4E complex formation as well as the activity of
S6K1 through mTOR signaling in skeletal muscle.
Although mTOR signaling appears requisite for increasing the
availability of eIF4E and the activity of S6K1, the data indicate that
the leucine-dependent increase in skeletal muscle protein synthesis
involves additional intracellular signaling pathways. For example,
isoleucine administration was able to increase 4E-BP1 and S6K1
phosphorylation without altering rates of protein synthesis.
Additionally, leucine was able to stimulate protein synthesis in rats
treated with rapamycin even though the absolute increase in protein
synthesis was not as great as that seen in rats administered excipient.
Finally, there was no statistical interaction between leucine
administration and drug treatment on rates of protein synthesis (Table 2)
. Collectively, the results presented here suggest that signaling
through mTOR alone is not sufficient to explain the
leucine-dependent stimulation of muscle protein synthesis in
postabsorptive rats. Therefore, leucine administration may upregulate
additional steps in translation initiation.
The physiologic consequences of modifying the phosphorylation state of
eIF4E remain to be determined. Studies using cells in culture suggest
that an increase in eIF4E phosphorylation enhances mRNA cap-binding
affinity and/or association with eIF4G. These changes augment rates of
protein synthesis and cell growth (Bu et al. 1993
,
Minich et al. 1994
). In contrast, experiments in vivo
demonstrate that eIF4E phosphorylation either does not change, or
increases and then decreases after food deprivation and refeeding,
respectively (Yoshizawa et al. 1997
and 1998
).
Furthermore, we demonstrated previously that oral administration of
leucine results in a net dephosphorylation of eIF4E compared with
food-deprived rats (Anthony et al. 2000
). The
results of the present study support these findings because the
proportion of eIF4E in the phosphorylated form was reduced in rats
administered leucine. The basis for the leucine-dependent decrease
in eIF4E phosphorylation is unknown; however, the data presented here
suggest that this inhibition is rapamycin sensitive, implicating mTOR
in the regulation of eIF4E phosphorylation.
In conclusion, these data suggest that leucine is unique among the BCAA in its ability to stimulate protein synthesis in muscle of food-deprived rats. Further, leucine was also most effective in enhancing translation initiation by increasing the availability of eIF4E for formation of the active eIF4G · eIF4E active complex and through hyperphosphorylation of S6K1. Administration of rapamycin inhibited the stimulatory effects of leucine on both protein synthesis and translation initiation. These results demonstrate that leucine-dependent stimulation of translation initiation in food-deprived rats occurs via a rapamycin-sensitive pathway and likely involves mTOR. However, the ability of isoleucine to hyperphosphorylate 4E-BP1 and S6K1 in the absence of increased rates of protein synthesis as well as the ability of leucine to stimulate protein synthesis in drug-treated rats would indicate that mTOR signaling alone does not account for the stimulatory effect of leucine on muscle protein synthesis.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: BCAA, branched-chain amino acids; Con, food-deprived rats; ConLeu, food-deprived rats injected with excipient and orally administered 1.35 g/kg leucine; 4E-BP1, eIF4E-binding protein 1; eIF, eukaryotic initiation factor; Ile, food-deprived rats, orally administered 1.35 g/kg isoleucine; Leu, food-deprived orally administered 1.35 g/kg leucine; mTOR, mammalian target of rapamyein kinase; Rap, food-deprived, injected with 0.75 mg/kg rapamycin; RapLeu, food-deprived rats, injected with 0.75 mg/kg rapamycin and orally administered 1.35 g/kg leucine; S6K1, 70-kDa ribosomal protein S6 kinase; Val, food-deprived, administered 1.35 g/kg valine.
Manuscript received February 4, 2000. Initial review completed March 11, 2000. Revision accepted May 23, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1.
Anthony J. C., Gautsch Anthony T., Kimball S. R., Vary T. C., Jefferson L. S. Orally administered leucine stimulates protein synthesis in skeletal muscle of postabsorptive rats in association with increased eIF4F formation. J. Nutr. 2000;130:139-145
2.
Anthony J. C., Gautsch Anthony T., Layman D. K. Leucine supplementation enhances skeletal muscle recovery in rats following exercise. J. Nutr. 1999;129:1102-1106
3.
Bu X., Haas D. W., Hagedorn C. H. Novel phosphorylation sites of eukaryotic initiation factor-4F and evidence that phosphorylation stabilizes the interactions of the p25 and p220 subunits. J. Biol. Chem. 1993;268:4975-4978
4.
Burnett P. E., Barrow R. K., Cohen N. A., Snyder S. H., Sabatini D. M. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc. Natl. Acad. Sci. U.S.A. 1998;95:1432-1437
5. Buse M. G., Atwell R., Mancusi V. In vitro effect of branched chain amino acids on the ribosomal cycle in muscles of fasted rats. Horm. Metab. Res. 1979;11:289-292[Medline]
6. Buse M. G., Reid S. S. Leucine: a possible regulator of protein turnover in muscle. J. Clin. Investig. 1975;56:1250-1261
7.
Cheatham B., Vlahos C. J., Cheatham L., Wang L., Blenis J., Kahn C. R. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70S6 kinase, DNA synthesis, and glucose transporter translocation. Mol. Cell. Biol. 1994;14:4902-4911
8. Chung J., Grammer T. C., Lemon K. P., Kazlauskas A., Blenis J. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature (Lond.) 1994;370:71-75[Medline]
9. Garlick P. J., McNurlan M. A., Preedy V. R. A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H] phenylalanine. Biochem. J. 1980;192:719-723[Medline]
10. Gautsch T. A., Anthony J. C., Kimball S. R., Paul G. L., Layman D. K., Jefferson L. S. Eukaryotic initiation factor 4E availability regulates skeletal muscle protein synthesis during recovery from exercise. Am. J. Physiol. 1998;274:C406-C414
11.
Hara K., Yonezawa K., Weng Q.-P., Kozlowski M. T., Belham C., Avruch J. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 trough a common effector mechanism. J. Biol. Chem. 1998;273:14484-14494
12.
Kimball S. R., Jurasinski C. V., Lawrence J. C., Jefferson L. S. Insulin stimulates protein synthesis in skeletal muscle by enhancing the association of eIF4E and eIF4G. Am. J. Physiol. 1997;272:C754-C759
13.
Kimball S. R., Shantz L. M., Horetsky R. L., Jefferson L. S. 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. 1999;274:11647-11652
14. Kimball S. R., Vary T. C., Jefferson L. S. Age-dependent decrease in the amount of eukaryotic initiation factor 2 in various tissues. Biochem. J. 1992;286:263-268
15. Li J. B., Jefferson L. S. Influence of amino acid availability on protein turnover in perfused skeletal muscle. Biochem. Biophys. Acta 1978;544:351-359[Medline]
16. Lynch C. J., Fox H. L., Vary T. C., Jefferson L. S., Kimball S. R. Regulation of amino acid-sensitive TOR signaling by leucine analogs in adipocytes. J. Cell. Biochem. 2000;77:234-251[Medline]
17.
MacLean D. A., Spriet L. L., Hultman E., Graham T. E. Plasma and muscle amino acid and ammonia responses during prolonged exercise in humans. J. Appl. Physiol. 1991;70:2095-2103
18.
Minich W. B., Balasta L., Goss D. J., Rhoads R. E. Chromatographic resolution of in vivo phosphorylated and non-phosphorylated eukaryotic translation initiation factor eIF4E: increased cap affinity of the phosphorylated form. Proc. Natl. Acad. Sci. U.S.A. 1994;91:7668-7672
19.
Nagasawa T., Yoshizawa F., Nishizawa N. Plasma N
-methylhistidine concentration is a sensitive index of myofibrillar protein degradation during starvation in rats. Biosci. Biotechnol. Biochem. 1996;60:501-502[Medline]
20. Pain V. M. Initiation of protein synthesis in eukaryotic cells. Eur. J. Biochem. 1996;236:747-771[Medline]
21. Patti M.-E., Brambilla E., Luzi L., Landaker E. J., Kahn C. R. Bidirectional modulation of insulin action by amino acids. J. Clin. Investig. 1998;101:1519-1529[Medline]
22. Shigemitsu K., Tsujishita Y., Miyake H., Hidayat S., Tanaka N., Hara K., Yonezawa K. Structural requirement of leucine for activation of p70 S6 kinase. FEBS Lett 1999;447:303-306[Medline]
23. Sonenberg N. mRNA 5' cap-binding protein eIF4E and control of cell growth. Hershey J.W.B. Mathews M. B. Sonenberg N. eds. Translational Control 1996:245-289 Cold Spring Harbor Laboratory Press Plainview, NY.
24. Voorma H. O., Thomas A.A.M., Van Heugten H.A.A. Initiation of protein synthesis in eukaryotes. Mol. Biol. Rep. 1994;19:139-145[Medline]
25.
Xu G., Kwon G., Marshall C. A., Lin T.-A., Lawrence J. C., Jr, McDaniel M. L. Branched-chain amino acids are essential in the regulation of PHAS-I and p70 S6 kinase by pancreatic ß-cells. J. Biol. Chem. 1998;273:28178-28184
26. Yoshizawa F., Endo M., Ide H., Yagasaki K., Funabiki R. Translational regulation of protein synthesis in the liver and skeletal muscle of mice in response to refeeding. J. Nutr. Biochem. 1995;6:130-136
27. Yoshizawa F., Kimball S. R., Jefferson L. S. Modulation of translation initiation in rat skeletal muscle and liver in response to food-intake. Biochem. Biophys. Res. Commun. 1997;240:825-831[Medline]
28.
Yoshizawa F., Kimball S. R., Vary T. C., Jefferson L. S. Effect of dietary protein on translation initiation in rat skeletal muscle and liver. Am. J. Physiol. 1998;275:E814-E820
This article has been cited by other articles:
|
|
 |
|
  D. M. Thomson, C. A. Fick, and S. E. Gordon AMPK activation attenuates S6K1, 4E-BP1, and eEF2 signaling responses to high-frequency electrically stimulated skeletal muscle contractions J Appl Physiol, March 1, 2008; 104(3): 625 - 632. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. C. Dreyer, M. J. Drummond, B. Pennings, S. Fujita, E. L. Glynn, D. L. Chinkes, S. Dhanani, E. Volpi, and B. B. Rasmussen Leucine-enriched essential amino acid and carbohydrate ingestion following resistance exercise enhances mTOR signaling and protein synthesis in human muscle Am J Physiol Endocrinol Metab, February 1, 2008; 294(2): E392 - E400. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Escobar, J. W. Frank, A. Suryawan, H. V. Nguyen, and T. A. Davis Amino acid availability and age affect the leucine stimulation of protein synthesis and eIF4F formation in muscle Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1615 - E1621. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. L. Eley, S. T. Russell, J. H. Baxter, P. Mukerji, and M. J. Tisdale Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli Am J Physiol Endocrinol Metab, October 1, 2007; 293(4): E923 - E931. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sharma, P. H. Guthrie, S. S. Chan, S. Haq, and H. Taegtmeyer Glucose phosphorylation is required for insulin-dependent mTOR signalling in the heart Cardiovasc Res, October 1, 2007; 76(1): 71 - 80. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. C. Vary 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., September 1, 2007; 137(9): 2074 - 2079. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T B. Symons, S. E Schutzler, T. L Cocke, D. L Chinkes, R. R Wolfe, and D. Paddon-Jones Aging does not impair the anabolic response to a protein-rich meal Am. J. Clinical Nutrition, August 1, 2007; 86(2): 451 - 456. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. C. Vary and C. J. Lynch Nutrient Signaling Components Controlling Protein Synthesis in Striated Muscle J. Nutr., August 1, 2007; 137(8): 1835 - 1843. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. C. Vary, G. Deiter, and C. J. Lynch Rapamycin Limits Formation of Active Eukaryotic Initiation Factor 4F Complex Following Meal Feeding in Rat Hearts J. Nutr., August 1, 2007; 137(8): 1857 - 1862. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. C. Vary, J. C. Anthony, L. S. Jefferson, S. R. Kimball, and C. J. Lynch Rapamycin blunts nutrient stimulation of eIF4G, but not PKC{varepsilon} phosphorylation, in skeletal muscle Am J Physiol Endocrinol Metab, July 1, 2007; 293(1): E188 - E196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Pospisilova, J. Cmejlova, J. Hak, T. Adam, and R. Cmejla Successful treatment of a Diamond-Blackfan anemia patient with amino acid leucine Haematologica, May 1, 2007; 92(5): e66 - e67. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Murata and M. Moriyama Isoleucine, an Essential Amino Acid, Prevents Liver Metastases of Colon Cancer by Antiangiogenesis Cancer Res., April 1, 2007; 67(7): 3263 - 3268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. G. Anthony, B. J. McDaniel, P. Knoll, P. Bunpo, G. L. Paul, and M. A. McNurlan Feeding Meals Containing Soy or Whey Protein after Exercise Stimulates Protein Synthesis and Translation Initiation in the Skeletal Muscle of Male Rats J. Nutr., February 1, 2007; 137(2): 357 - 362. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Wang, N. Kubica, L. W. Ellisen, L. S. Jefferson, and S. R. Kimball Dexamethasone Represses Signaling through the Mammalian Target of Rapamycin in Muscle Cells by Enhancing Expression of REDD1 J. Biol. Chem., December 22, 2006; 281(51): 39128 - 39134. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Holecek, T. Muthny, M. Kovarik, and L. Sispera Simultaneous Infusion of Glutamine and Branched-Chain Amino Acids (BCAA) to Septic Rats Does Not Have More Favorable Effect on Protein Synthesis in Muscle, Liver, and Small Intestine Than Separate Infusions JPEN J Parenter Enteral Nutr, November 1, 2006; 30(6): 467 - 473. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. B. Reinert, L. M. Oberle, S. A. Wek, P. Bunpo, X. P. Wang, I. Mileva, L. O. Goodwin, C. J. Aldrich, D. L. Durden, M. A. McNurlan, et al. Role of Glutamine Depletion in Directing Tissue-specific Nutrient Stress Responses to L-Asparaginase J. Biol. Chem., October 20, 2006; 281(42): 31222 - 31233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Fujita, B. B. Rasmussen, J. G. Cadenas, J. J. Grady, and E. Volpi Effect of insulin on human skeletal muscle protein synthesis is modulated by insulin-induced changes in muscle blood flow and amino acid availability Am J Physiol Endocrinol Metab, October 1, 2006; 291(4): E745 - E754. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Lynch, B. Gern, C. Lloyd, S. M. Hutson, R. Eicher, and T. C. Vary Leucine in food mediates some of the postprandial rise in plasma leptin concentrations Am J Physiol Endocrinol Metab, September 1, 2006; 291(3): E621 - E630. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Rieu, M. Balage, C. Sornet, C. Giraudet, E. Pujos, J. Grizard, L. Mosoni, and D. Dardevet Leucine supplementation improves muscle protein synthesis in elderly men independently of hyperaminoacidaemia J. Physiol., August 15, 2006; 575(1): 305 - 315. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Crozier, X. Zhang, J. Wang, J. Cheung, S. R. Kimball, and L. S. Jefferson Activation of signaling pathways and regulatory mechanisms of mRNA translation following myocardial ischemia-reperfusion J Appl Physiol, August 1, 2006; 101(2): 576 - 582. [Abstract] [Full Text] [PDF]
|
|
- and ß-forms of 4E-BP1 noted to the right. (B)
Amount of 4E-BP1 in the 
-phosphorylated form as a percentage of the
total 4E-BP1. Inset shows a representative immunoblot with positions of
