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Human Nutrition Research Center of Clermont-Ferrand and Institut National de la Recherche Agronomique, Unité d’Etude du Métabolisme Azoté, 63122 Ceyrat, France
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • rats • protein synthesis • leucine • age • muscle
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and rodents (Holloszy et al. 1991
, Klitgaard et al. 1989
). This loss of
protein results from an imbalance between protein synthesis and
degradation rates. The imbalance is not clearly apparent when basal
rates of protein turnover are measured (Dardevet et al. 1994
, Goldspink et al. 1987
, Mosoni et al. 1993
). A reduction in the stimulation of muscle protein
synthesis was detected in rats during the postprandial period
(Mosoni et al. 1995
), suggesting that the "meal
signal" was altered with age. The origin of this alteration remains
obscure because muscle protein synthesis responds normally if amino
acids are perfused continuously into old rats (Mosoni et al. 1993
). Similarly, Volpi et al. (1999)
showed
that muscle protein synthesis was still able to respond positively to
an increase of amino acid availability in elderly humans after oral
amino acid administration. Moreover, a recent study of Arnal et al. (1999)
demonstrated that the response of protein turnover
was restored in elderly humans if a "protein-pulse feeding" pattern
(80% of dairy proteins in one meal) was used instead of
"spread-protein feeding" (dairy proteins equally distributed).
Taken together, these results suggest that aged muscle is less
sensitive to the stimulatory effect of amino acids at physiologic
concentrations but is still able to respond if the increase in amino
acidemia is sufficiently large.
Amino acids play an important role in regulating muscle protein
synthesis [see May and Buse (1989)
for a review]. Many
mechanisms have been proposed to explain this effect, including
regulation of cell volume (Lobley et al. 1998
) and
regulation of gene expression (Bruhat et al. 1997
,
Jousse et al. 1998
). Recently, several studies conducted
in vitro showed that in addition to being the precursors of protein
synthesis, amino acids also mimic the effect of insulin and modulate
translation initiation. Incubation of cells [Fao and H4IIE hepatocytes
(Patti et al. 1998
)], CHO (Wang et al. 1998
) and adipocytes (Fox et al. 1998
) without
amino acids caused a very rapid decrease in p70 S6 kinase
(p70S6K)3
activity, dephosphorylation of eukariotic initiation factor 4E (eIF-4E)
binding protein (4E-BP1) and an increased binding of initiation factor
eIF-4E to the inhibitory regulator protein 4E-BP1. These effects were
rapidly reversed by supplying a mixture of amino acids, but blocked by
inhibitors of phosphatidylinositol 3' kinase (PI3 kinase) and the
mammalian target of rapamycin (mTOR). This suggested a role for these
two kinases in the signaling pathway linking amino acids with the
control of p70S6K and these translation factors.
However, no measurement of protein synthesis was performed
simultaneously in the presence of rapamycin, which blunts the
regulation of the eIF-4E /4E-BP1; thus, there is no evidence
that these initiation factors represent the limiting step of the
stimulation of protein synthesis by amino acids. In skeletal muscle,
few data are available concerning the role of amino acids in modulating
intracellular factors and kinases. Only the works of Svanberg et al. (1997)
and Yoshizawa et al. (1998)
have
shown that amino acids are also potent regulators of the 4E-BP1/eIF-4E
complexes in vivo.
In this study, we hypothesized that the defect in the postprandial stimulation of muscle protein synthesis recorded in old rats might be the consequence of an alteration in the signaling cascade responsible for the amino acid–induced stimulation of protein synthesis in skeletal muscle. To assess and further understand the physiologic role of amino acids and the signaling pathway(s) involved in the regulation of protein synthesis, we performed this study on rat epitrochlearis muscle in vitro to demonstrate the direct effects of amino acids and also to eliminate the interactions with other in vivo potent regulators of protein synthesis such as insulin. To address this question, we measured the effect of inhibitors of selected intracellular kinases (PD98059, LY294002, rapamycin) on amino acid–stimulated protein synthesis in young rat muscle and the effect of amino acids or a single amino acid on p70 S6 kinase activity in young, adult and old rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experiments were conducted in accordance with NIH guidelines (NRC 1985
). Young (4–5 wk old; 120–150 g), adult (6–8 mo old;
550–650 g) and old (20 mo old; 600–650 g) male Wistar rats were
housed under controlled environmental conditions (temperature, 22°C;
12-h dark period starting at 1800 h). Rats had free access to a
commercial laboratory diet (22% protein, 4.3% lipid, 53%
carbohydrate; UAR, Villemoisson-sur-orge, France) and water. They were
deprived of food overnight before the incubation experiment. Rats were
anesthetized with sodium pentobarbital (6.0 mg/100 g body) and
epitrochlearis muscles were dissected intact for incubation (see
below). The weights of epitrochlearis muscles were 24.8 ± 1.16
(n = 26), 166.3 ± 4.9 (n
= 15), 144.2 ± 5.3 (n = 16) in young,
adult and old rats, respectively (P < 0.05 adult
and old vs. young; P < 0.05 old vs. adult).
Effect of amino acids on muscle protein synthesis in the presence of PD98059, LY294002 and rapamycin.
Muscles were preincubated for 30 min in Krebs-Henseleit
buffer (KHB) (NaCl, 120 mmol/L; KCl, 4.8 mmol/L; NaHCO3, 25
mmol/L CaCl2, 2.5mmol/L; KH2PO4,
1.2 mmol/L; and MgSO4, 1.2 mmol/L; pH 7.4) supplemented
with 5 mmol/L HEPES, 5 mmol/L glucose, 1 g/L bovine serum albumin (BSA;
99% fatty acid free) saturated with a 95% O2/5%
CO2 gas mixture. They were then transferred into fresh
medium of the same composition for 2 h in the presence or absence
of an amino acid mixture that was representative of the arterial
postabsorptive (PA) or postprandial (PP) status found in control rats
(Table 1
). Muscles were then incubated for an additional 75 min in fresh medium
containing 0.5 mmol/L
L-[14C]phenylalanine (8.5 MBq/L).
In an additional experiment, the effects of leucine, arginine and
histidine alone were compared with that of the total amino acid mixture
(PP) or in terms of the dose-response curves at the concentrations
indicated. Viability of such muscle preparations has been assessed
previously. ATP, phosphocreatine and lactate levels were similar in
muscles of young, adult and old rats and remained constant throughout
the incubation period (Dardevet et al. 1994
). At the end
of the incubation, muscles were blotted and homogenized in 0.61 mol/L
trichloroacetic acid (TCA). Samples were centrifuged at 10,000 x g for 10 min at 4°C and TCA-insoluble material was
washed 3 times with 0.61 mol/L TCA. The resultant pellet was
solubilized in 1 mol/L NaOH at 37°C for determination of protein and
radioactivity incorporated into muscle proteins. Tissue protein mass
was determined using the bicinchoninic acid procedure (Pierce Chemical,
Rockford, IL) and protein-bound radioactivity was measured using
scintillation counting. Protein synthesis was calculated by dividing
the protein-bound radioactivity by the specific activity of free
phenylalanine in the incubation medium and expressed as nanomoles of
phenylalanine incorporated per milligram protein per 75 min.
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Effect of amino acids and leucine on p70S6K activation.
Epitrochlearis muscles were preincubated for 45 min in KHB supplemented with 5 mmol/L HEPES, 5 mmol/L glucose, 1 g/L BSA and transferred into fresh medium of the same composition in the presence or absence of amino acid mixture (PP) or leucine (200 µmol/L) for 60 min. Pilot experiments established that maximal phosphorylation of p70S6K was observed 40 min after the addition of either PP or leucine (data not shown). Muscles were homogenized in ice-cold extraction buffer [50 mmol/L Tris-acetate, 50 mmol/L NaF, 2.5 mmol/L EDTA, 1 mmol/L EGTA, 5 mmol/L sodium pyrophosphate, 5 mmol/L ß-glycerophosphate, 1 mmol/L Na3VO4, 2 mmol/L dithiothreitol (DTT), 1 mmol/L benzamidine, 4 µg/L leupeptin and 0.5% Triton X-100; pH 7.2] and centrifuged at 10,000 x g for 10 min at 4°C. The activity of p70S6K was assessed by an immune complex kinase assay. Normalized amounts of muscle proteins (100 and 200 µg for young and adult-old rats, respectively) were incubated for 4 h at 4°C with 3 µL of p70S6K antibodies preabsorbed to protein A-agarose beads. The immune complexes were washed three times with S6 kinase assay buffer [25 mmol/L 2-(N-morpholino)propanesulfonic acid, 15 mmol/L MgCl2, 1 mmol/L DTT, 0.1% BSA; pH 7.2]. The beads were then resuspended in 50 µL of S6 kinase assay buffer containing 0.1 mmol/L of the S6 peptide RRRLSSLRA, 2 µmol/L peptide inhibitor of cAMP-dependent protein kinase and 100 µmol/L ATP (specific activity 3000 dpm/pmol). After a 60-min incubation at 30°C, the phosphorylation reaction was stopped with 10 µL of 50 mmol/L unlabeled ATP. The reaction mixture was spotted onto Whatman P-81 phosphocellulose filter paper squares (Whatmay, Kenk UK); after three washes in 75 mmol/L phosphoric acid, the squares were counted in a ß-scintillation counter. Pilots experiments showed that 32P incorporation into S6 substrate was linear for at least 60 min and proportional to the amount of protein used (100–200 µg).
Statistical analysis.
Data are expressed as means ± SD. Statistical evaluation of the data was performed using Student’s t test or by ANOVA when appropriate. When differences were detected, a Student-Newman-Keuls post-hoc test was performed to determine pairwise differences. Differences among means were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Addition of amino acids to the incubation medium significantly
increased the rate of muscle protein synthesis in epitrochlearis muscle
from young rats [0.224 ± 0.009, 0.267 ± 0.012, 0.302
± 0.007 nmol phenylalanine/(mg protein · 75min) for basal, PA
and PP, respectively, P < 0.05, PA and PP vs. basal;
P < 0.05, PP vs. PA]. The maximal stimulation of
protein synthesis was systematically recorded with the PP mixture,
whereas the PA mixture gave variable stimulations between basal and PP
values. Among the amino acids, branched-chain amino acids, and
leucine in particular, were the most efficient in this stimulation
(May and Buse 1989
). In our muscle preparations, leucine
alone, at the same concentration as that present in PP, was as potent
as all amino acids combined in stimulating muscle protein synthesis
(Fig. 1
). The other amino acids tested (arginine and histidine) did not
significantly stimulate muscle protein synthesis when added at the
postprandial concentration. Increasing leucine concentration
progressively increased muscle protein synthesis, and the maximal
effect was recorded at 200 µmol/L, which is similar to the
arterial postprandial leucinemia (Fig. 2
). The half-maximum effect (IC50) was recorded
with a leucine concentration close to the normal postabsorptive
concentration.
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). As observed in young rats, maximal stimulation was recorded at a
leucine concentration close to 200 µmol/L and the
IC50 occurred at 110–120 µmol/L
leucine. However, when the effect of leucine was considered in term of
incremental increase, its effect was lower in adult than in young rats
[0.025 vs. 0.091 nmol phenylalanine/(mg protein · 75min),
respectively]. In muscles of old rats, leucine also stimulated
protein synthesis to the same extent as in adult rats, but the maximal
response was recorded at a leucine concentration of 
400
µmol/L, with an IC50 at 200–250
µmol/L leucine (Fig. 3)
.
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The addition of PD98059 (an inhibitor of MAP-kinase pathways) into
the incubation medium did not modify the rate of basal muscle protein
synthesis [0.281 ± 0.013 vs. 0.297 ± 0.015 nmol
phenylalanine/(mg protein · 75 min) without or with the inhibitor,
respectively, not shown] nor the stimulatory effect of amino acids
[0.373 ± 0.025 vs. 0.387 ± 0.017 nmol phenylalanine/(mg
protein · 75 min) without or with the inhibitor, respectively]
(Fig. 4
). Therefore the MAP-kinase pathways are not involved in the acute
regulation of muscle protein synthesis by amino acids.
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The addition of rapamycin to the incubation medium did not affect basal
protein synthesis [0.279 ± 0.012 vs. 0.280 ± 0.010 nmol
phenylalanine/(mg protein · 75 min) min without or with the
inhibitor, respectively] but completely abolished the stimulatory
effect of amino acids on muscle protein synthesis (Fig. 4)
. Similarly,
rapamycin also completely inhibited stimulation of muscle protein
synthesis when leucine alone was added to the incubation medium (data
not shown).
Effects of amino acids and leucine on p70S6K activity.
The addition of amino acids to the incubation medium stimulated
p70S6K activity. When leucine alone was added,
p70S6K was stimulated but to a lesser extent than
with the total amino acid mixture (Fig. 5
). Thus, p70S6K, which is part of the
rapamycin-dependent pathways, might be responsible in part for the
effects of amino acids on muscle protein synthesis. However, because
leucine alone was less effective than total amino acids, this suggests
that other amino acids have minor effects on this kinase activity. In
adults rats, leucine also stimulated the activity of
p70S6K with a maximal effect at 200
µmol/L (Fig. 6
). The basal activity of p70S6K was not different
in muscles of old and adult rats, but the maximal stimulation in muscle
of old rats was at a higher leucine concentration (400
µmol/L). For protein synthesis,
p70S6K activation in muscle of old rats was less
sensitive to leucine compared with muscle of adults (Fig. 6)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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showed that leucine was the more potent amino acid in
stimulating 4E-BP1 phosphorylation in adipocytes.
It seems likely that leucine is involved in the regulation of
protein synthesis in vivo. A recent study of Anthony et al. (2000)
showed that orally administered leucine stimulated
muscle protein synthesis independently of insulin in vivo. We found
that a modest increase in plasma leucine above postabsorptive levels
had no significant effect, whereas a twofold increase stimulated
whole-body protein synthesis in healthy humans
(Bergström et al. 1990
, Elia et al. 1989
, Giordano et al. 1996
). A similar
conclusion could be drawn from experiments using intravenous amino acid
infusion in rats. Only a sustained large hyperaminoacidemia (leucine
increased two- to threefold) stimulated muscle protein
synthesis (Garlick and Grant 1988
,
McNulty et al. 1993
, Mosoni et al. 1993
,
Sinaud et al. 1999
). Moreover, the postprandial
stimulation of translation of muscle protein synthesis in rats
originated mainly from absorbed amino acids because this stimulation
was observed after feeding a high protein meal but not after an
isoenergetic protein-free meal (Yoshizawa et al. 1998
). It is important to emphasize that amino acids or feeding
affects plasma insulin, which also participates in the stimulation of
muscle protein synthesis, especially in young growing animals
(Garlick and Grant 1988
). Dose-response curves of
insulin action showed a very high sensitivity in vivo. Indeed, the
effect of exogenous insulin was visible only in the postabsorptive
state and not during the postprandial state when insulin levels are
already elevated (Garlick and Grant 1988
). The acute
decrease of postprandial insulinemia below the postabsorptive levels
due to diazoxide treatment greatly impaired muscle protein synthesis
(Sinaud et al. 1999
). The present experiment was
therefore performed in vitro in the absence of insulin to analyze
specifically the effect of total amino acids and leucine.
Because leucine alone reproduced the effect of total amino acids on
muscle protein synthesis in our experiment, it might be hypothesized
that this effect was not dependent on the amino acid concentration
itself but on a specific signal initiated by leucine. However, to our
knowledge, the effect of inhibitors of specific kinases has not been
investigated on amino acid–stimulated protein synthesis itself in
isolated muscles. We demonstrated, for the first time, that inhibition
of PI3 kinase by LY294002 completely abolished amino acid–stimulated
protein synthesis in rat skeletal muscle. Moreover, the MAP kinase
pathway was not involved in this stimulation because PD98059, an
inhibitor of the upstream activator of MAP kinases, MEK, had no
effect. We found that amino acids stimulated muscle protein synthesis
through intracellular kinases, which are PI3 kinase dependent. The
downstream events linked to PI3 kinase remain poorly defined; in
several cell lines, however, (Cheatham et al. 1994
,
Cross et al. 1994
, Welsh et al. 1994
) as well as in rat skeletal muscle (Dardevet et al. 1996
), PI3 kinase activation was required for stimulation of
p70S6K. This kinase has been shown to be
partially involved in the regulation of protein synthesis in rat
epitrochlearis muscle and inhibited by rapamycin (Dardevet et al. 1996
). Although rapamycin caused a deactivation of
p70S6K, the direct target of this inhibitor is in
fact the protein known as mTOR (mammalian target of rapamycin). The
role of mTOR as the rapamycin target responsible for inhibition of
p70S6K was demonstrated by Brown et al. (1995)
who showed that mutant TOR, which lack the ability to
bind rapamycin, prevent the inhibition of p70S6K
by the drug. Our results clearly showed that addition of rapamycin
completely inhibited the stimulation of muscle protein synthesis by
amino acids or leucine alone and that mTOR represented the downstream
target of amino acid–stimulated PI3 kinase in skeletal muscle. We also
demonstrated that amino acids or leucine alone at physiologic
concentrations stimulated p70S6K activity.
However, leucine alone seemed to be less effective than total amino
acids in increasing p70S6K activity, suggesting
than other amino acids might have minor stimulating effects on this
kinase. Very recently, p70S6K activation by amino
acids was investigated in culture cells and it was shown that in Fao
hepatocytes (Patti et al. 1998
), CHO cells (Hara et al. 1998
, Wang et al. 1998
) and L6 myoblasts
(Kimball et al. 1999
), amino acids are also potent
stimulators of this kinase. As we found, leucine is the major amino
acid responsible for this effect. Activation of
p70S6K by leucine has been reported to be very
specific; the structural requirement of this amino acid to induce
p70S6K activation is very strict and precise
(Shigemitsu et al. 1999
). However, to our knowledge, the
target of leucine that would initiate the activation of the PI3
kinase/mTOR pathways is not known.
It was shown previously that stimulation of muscle protein synthesis
was lower in old rats than in adults (Mosoni et al. 1995
). Therefore, we investigated whether a decrease in the
sensitivity of muscle protein synthesis to amino acids with age may
explain the defect in the postprandial anabolism. We found that muscle
protein synthesis still responded to the leucine signal in old rats,
but the IC50 was observed with amino acid levels
two to three times greater than in young or adult rats. This indicated
that at postprandial amino acid concentrations, muscle protein
synthesis was maximally stimulated in adult rats but poorly stimulated
in old rats. Indeed, we measured the plasma amino acid concentrations
in old and adult rats during the postaborptive and postprandial states
and no difference of aminoacidemia was found (data not shown).
Similarly, Volpi et al. (1999)
showed that amino acid
concentrations were not different in adult and elderly humans in both
basal and fed states. However, it is important to note that muscle
protein synthesis was stimulated to the same extent in old and adult
rats when leucine levels reached supraphysiologic concentrations,
demonstrating that muscle from old rats did not lose the ability to
respond to the amino acid signal. Moreover, these results suggest that
a difference in postprandial stimulation of muscle protein synthesis
with age will be detectable only if the plasma leucine concentration is
moderately increased (1.5–2 times) as observed after a standard oral
feeding (Bergström et al. 1990
). This observation
might explain why amino acid perfusion, which induced a threefold
increase in plasma leucine, stimulated muscle protein synthesis to the
same extent in adult and old rats (Mosoni et al. 1993
),
whereas a defect in this stimulation was observed after "normal"
feeding. A similar conclusion could be reached from the study of
Volpi et al. (1999)
who observed a similar effect of
oral amino acid administration on muscle protein synthesis in adult and
elderly humans. Indeed, arterial leucine concentrations were increased
threefold in both age groups. Our hypothesis is also in agreement with
the work of Arnal et al. (1999)
who showed that
protein-pulse feeding improved protein retention in elderly women,
whereas protein-spread feeding did not. In these experiments,
plasma amino acid concentrations were not determined; we assume,
however, that amino acid availability (i.e., leucine) to peripheral
tissues was higher with protein-pulse feeding than with
protein-spread feeding, and reached the level that would
significantly stimulate muscle protein synthesis in elderly humans.
The decreased sensitivity of muscle protein synthesis to leucine in aged rats suggested that the signaling pathway that carries the leucine signal to the protein translation machinery was less responsive to the amino acid. Our study demonstrated that p70S6K activity was stimulated by leucine in both adult and old rats, but as observed with protein synthesis, this activation occurred with higher, supraphysiologic levels of leucine (IC50: 110 vs. 260 µmol/L, respectively). Thus, for full activation, postprandial leucine concentrations are sufficient in adult rats, whereas up to 400 µmol/L is required in old rats. Our results showed a similar alteration of the sensitivity of muscle protein synthesis and sensitivity of p70S6K activation to leucine. This confirms that the signaling pathway PI3 kinase/mTOR/p70S6K is involved in the stimulation of muscle protein synthesis by leucine.
In conclusion, we showed that amino acids directly stimulated muscle protein synthesis in vitro, independently of insulin. Furthermore, among the amino acids, leucine alone reproduced this effect, suggesting that this branched-chain amino acid acted as a mediator of muscle protein synthesis stimulation. To date, no studies have been performed on skeletal muscle in vitro to identify the signaling pathways involved. We demonstrated that the amino acid signal occurred through the stimulation of PI3 kinase and its downstream substrate, mTOR. We also demonstrated that amino acids were potent stimulators of p70S6K activity.
Moreover, our results suggested that the defect of postprandial muscle
protein anabolism during aging may result from a decrease of protein
synthesis sensitivity to amino acids, particularly leucine. This defect
was associated with the inability of leucine to stimulate
p70S6K. Because old rats remain able to
respond normally to higher leucine concentrations, it is possible that
increasing plasma leucine may be beneficial in maintaining the
postprandial stimulation of muscle protein metabolism. However, it has
been demonstrated by Boirie et al. (1997)
that the
first-pass splanchnic uptake of leucine increases with age and may
limit the availability of amino acids to peripheral tissues. Recently,
Volpi et al. (1999)
confirmed this observation but
showed that despite this phenomenon, the delivery of amino acids to the
leg increased to the same extent in both adult and elderly humans.
Thus, an oral leucine supplementation may increase leucine availability
to peripheral tissues in aged animals and may restore normal
postprandial anabolism.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 BSA, bovine serum albumin; 4E-BP1, eukariotic factor 4E binding protein; eIF-4E, eukariotic factor 4E; IC50, half-maximum effect; KHB, Krebs-Henseleit buffer; mTOR, mammalian target of rapamycin; MAP, mitogen-activated protein; p70S6K, p70 S6 kinase; PA, postabsorptive amino acid concentration; PI3 kinase, phosphatidylinositol 3' kinase; PP, postprandial amino acid concentration; TCA, trichloroacetic acid.
Manuscript received May 9, 2000. Initial review completed May 29, 2000. Revision accepted July 18, 2000.
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