The Journal of Nutrition

Nutrition and Disease

Acute Oral Leucine Administration Stimulates

Protein Synthesis during Chronic Sepsis
through Enhanced Association of Eukaryotic
Initiation Factor 4G with Eukaryotic Initiation
Factor 4E in Rats1,2
Thomas C. Vary*

Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey, PA 17033

Abstract
Sepsis induces the loss of muscle proteins by impairing skeletal muscle protein synthesis through an inhibition of messenger
RNA (mRNA) translation initiation. Amino acids and Leu (Leu) in particular stimulate mRNA translation initiation. The
experiments were designed to test the effects of Leu on potential signal transduction pathways that may be important in
accelerating mRNA translation initiation in skeletal muscle of rats with chronic (5–6 d) septic intra-abdominal abscess.
Gastrocnemius from male Sprague Dawley rats gavaged with Leu or water were sampled 5–6 d following development of an
intra-abdominal sterile or septic abscess. Gavage with Leu stimulated protein synthesis and enhanced the assembly of the
active eukaryotic initiation factor (eIF)4G-eIF4E complex. Increased assembly of the active eIF4G-eIF4E complex was
associated with a robust rise in phosphorylation of eIF4G(Ser1108) and a decreased assembly of inactive eIF4E binding
protein-1 (4E-BP1)-eIF4E complex in both sterile inflammatory and septic rats. The reduced assembly of 4E-BP1-eIF4E
complex was associated with an increase in phosphorylation of 4E-BP1 in the g-form following Leu gavage. Phosphorylation
of 70-kDa ribosomal protein S6 kinase on Thr389 was also increased following Leu gavage, as well as the phosphorylation of
mammalian target of rapamycin on Ser2448 or Ser2481. In contrast, phosphorylation of protein kinase B (PKB) on Thr308 or
Ser473 was not augmented following Leu gavage in septic rats. We conclude that Leu stimulates a PKB-independent signal
pathway elevating the eIF4G-eIF4E complex assembly through increased phosphorylation of eIF4G and decreased
association of 4E-BP1 with eIF4E in skeletal muscle during sepsis. J. Nutr. 137: 2074–2079, 2007.

Introduction
Sepsis, the systemic response to bacterial infection, remains a
serious complication contributing to a high (20–45%) mortality
rate. A major complication contributing to the morbidity and
mortality in septic patients is the development of diffuse tissue
injury referred to as multiple organ system dysfunction. Multiple
organ system dysfunction can lead to the failure of major organ
systems of the body to maintain homeostasis and is manifested
in skeletal muscle by a loss of skeletal muscle mass producing
diminished muscle strength (1–3). Muscle weakness in septic
patients contributes to a continued dependence on mechanical
respirators, an increased risk of pneumonia, and the complica-
tions associated with an inability for ambulation. These clinical
complications prolong hospitalization and convalescence,
thereby increasing health-care costs (4).
Protein wasting during sepsis results from an imbalance
between the rate of protein synthesis and protein degradation
(5). The relative contribution of protein synthesis and proteol-
1
Supported in part by NIH General Medical Sciences Award GM39277.
2
Author disclosure: T. C. Vary, no conflicts of interest.
* To whom correspondence should be addressed. E-mail: tvary@psu.edu.
ysis to the overall net catabolic state in muscle varies depending
upon the severity of the septic insult. Protein degradation varies
and becomes accelerated as the septic episode worsens. In con-
trast, rates of protein synthesis are reduced to a similar extent
regardless of the severity of the septic insult (6,7). Furthermore,
the synthesis of both myofibrillar and sarcoplasmic proteins is
equally diminished, indicating that sepsis affects the overall
process of protein synthesis (8). As the septic process wanes,
skeletal muscle protein mass and protein synthesis return toward
control values (9). In contrast to sepsis, the decrease in protein
synthesis and increase in proteolysis in skeletal muscle is short-
lived in nonseptic, traumatic insults and restoration of positive
nitrogen balance and lean body mass occurs quickly (9–11).
The sepsis-induced inhibition in protein synthesis results
from a restraint in the process of initiation of messenger RNA
(mRNA)3 translation. Translation initiation is a complex process
in which initiator transfer RNA, 40S, and 60S ribosomal subunits
3
Abbreviations used: eIF, eukaryotic initiation factor; 4E-BP1, eIF4E binding
protein-1; mTOR, mammalian target of rapamycin; PAGE, polyacrylamide gel
electrophoresis; PKB, protein kinase B; PKC, protein kinase C; S6K1, 70-kDa
ribosomal protein S6 kinase; SP, septic; ST, sterile.

2074 0022-3166/07 $8.00 ª 2007 American Society for Nutrition.

Manuscript received 16 March 2007. Initial review completed 12 April 2007. Revision accepted 20 June 2007.
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 are assembled by eukaryotic initiation factors (eIF) into an 80S
ribosome at the initiation codon of mRNA. In eukaryotes,
protein synthesis initiates with the binding of the multimeric
translation initiation complex eIF4F to the monomethylated cap
present on the 5# end of most mRNAs (12).
Translation initiation appears regulated through the forma-
tion of the eIF4G-eIF4E complex. A positive linear relationship
exists between rates of protein synthesis and the amount of
eIF4G associated with eIF4E in muscle in vivo (13). Consistent
with this relationship, assembly of the eIF4G-eIF4E complex
is significantly diminished in skeletal muscle from septic rats
(13,14). The diminished assembly of eIF4G-eIF4E complex is
not the result of a reduced amount of eIF4G in muscles from
septic rats (13). However, eIF4G-eIF4E complex assembly ap-
pears regulated through the availability of eIF4E for binding to
eIF4G in part through the association of eIF4E with a family of
translational repressor proteins [eIF4E binding proteins (4E-
BP)] (13) and phosphorylation of eIF4G (15) during chronic
hypermetabolic, hyperdynamic sepsis. Endotoxin administra-
tion (16–18) or infusion of cytokines (15,19,20) cause similar
changes. Reduced amounts of eIF4E associated with eIF4G
following chronic sepsis would be expected to diminish the
association of mRNA with the ribosome and, hence, limit
protein synthesis.
Amino acids, Leu (Leu) in particular, have the ability to
regulate protein synthesis (21,22). Emerging evidence indicates
that amino acids acting as nutrient signals themselves modulate
cellular processes leading to an acceleration of protein synthesis
through augmented mRNA translation initiation (12). Formation
of the active eIF4E-eIF4G complex is dependent upon amino
acids in postprandial state (23). Oral administration of Leu en-
hances skeletal muscle protein synthesis through changes in the
regulation of proposed effectors of mRNA translation initiation,
as evidenced by upregulated phosphorylation of the translational
repressor, 4E-BP1, the association of eIF4G with eIF4E, and the
phosphorylation of the 70-kDa ribosomal protein S6 kinase
(S6K1) (24). Likewise, protein synthesis in gastrocnemius is
stimulated by short-term (30 min) perfusion with buffer sup-
plemented with amino acids at 10 times greater than the plasma
concentration by enhancing peptide-chain initiation (25).
The purpose of this study was to test the hypothesis that Leu
stimulates protein synthesis in gastrocnemius of septic rats by
increasing the formation of the active eIF4E-eIF4G complex.
Materials and Methods
Animals and experimental design. Adult male Sprague-Dawley rats
weighing 150–225 g were maintained on a 12-h-light:12-h-dark cycle
and maintained on a standard rodent chow (Teklad Rodent Diet no.
2018) and water ad libitum. Chronic abdominal sepsis was created by
implantation of a fecal-agar pellet (1.5 mL) inoculated with 104 colony
forming units of Escherichia coli and 108 colony forming units of
Bacteroides fragilis into the peritoneal cavity as previously described
(10,13,14,25,26). Replacing the bacterial inoculum with an equal
volume of sterile saline generated the nonseptic inflammation abscess
(10,13,14,25,26). After recovery from the surgical procedures, the intra-
abdominal abscess was allowed to develop for 5 d.
Both septic and nonseptic inflammatory rats developed an abdominal
abscess and showed leukocytosis; however, the magnitude of the leuko-
cytosis was greater in the septic rats (27). Bacteremia was not observed in
either nonseptic abscess or control rats (27). Introduction of the infected
fecal-agar pellet resulted in a hyperdynamic, hypermetabolic septic con-
dition, which was not observed in rats with the nonseptic abscess (28).
We previously established that both nonseptic and septic rats have re-
duced food intake in the first 2 d post-surgery compared with control rats
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that consumed food ad libitum (9,29). Thereafter, sterile inflammatory
and septic rats ate the same amount of food on a daily basis as rats that
consumed the diet ad libitum. Furthermore, food intake did not differ
between the sterile inflammatory and septic rats over the course of the
experiment.
The chronic septic model used in the studies described herein mimics
the human septic condition in that rats develop a chronic stable
intraperitoneal abscess and exhibit a hyperdynamic cardiovascular state,
allowing us to examine the host response to infection in rats that are
hemodynamically stable. Our septic, but not sterile, abscess rats show
many of the signs and symptoms of the human septic condition (30,31),
including increased cardiac output and decreased peripheral resistance
(indicative of a hyperdynamic cardiovascular state), a low-grade fever,
leukocytosis (27,32), bacteremia (27), and weight loss despite normal
food intake (9,10,29). The septic rats show hormonal alterations
characteristic of septic patients, including insulin resistance, reduced
plasma IGF-I concentrations, and an elevated growth hormone concen-
tration (33). This septic rat model shows a growth hormone resistance
similar to that observed in human septic patients (34).
On the day before the experiment, food was removed from the rat
cages. On the day of the experiment (time 0), the sterile inflammatory
and septic rats were randomly divided into 2 groups and were given 1 of
the following solutions by oral gavage (2.5 mL/100 g body weight)
according to their designated treatment group: sterile (ST, 0.155 mol
NaCl/L); septic (SP, 0.155 mol NaCl/L); ST 1 Leu (ST1Leu, 54.0 g/L
L-Leu); or SP 1 Leu (SP1Leu, 54.0 g/L L-Leu) as described previously
(15,35–37). The dose of Leu gavaged is equivalent to the amount of Leu
eaten by rats of this age and strain during a 24-h period (35,38,39).
Experiments were approved by the Institutional Animal Care and Use
Committee of Pennsylvania State University College of Medicine and
adhered to NIH guidelines for the use of experimental animals.
Rates of protein synthesis. Twenty minutes after receiving a gavage
containing either saline or Leu, rats were anesthetized (Nembutal 50
mg/kg body weight). A catheter was inserted into the carotid artery and a
blood sample was taken for measurement of plasma Leu concentrations
(Table 1). Rates of protein synthesis were determined in vivo following
injection with L-[2,3,4,5,6-3H]phenylalanine (Phe) (150 mmol/L, 30 mCi/L;
1 mL/100 g body weight) via the jugular vein 30 min after gavage, as
originally described by Garlick et al. (40) and modified in our laboratory
(8,10,11,41,42). Muscle lysates were prepared as described below and a
portion of the gastrocnemius was frozen between blocks precooled to the
temperature of liquid nitrogen for measurement of incorporation of
radioactive Phe into muscle proteins. Rates of protein synthesis were
measured as the incorporation of [3H]Phe into protein as described
previously (8,10,11,41,42).
Preparation of muscle lysates. The gastrocnemius was excised,
weighed, and prepared for analysis of the phosphorylation state of
eIF4G, mammalian target of rapamycin (mTOR), protein kinase C
(PKC)e, S6K1, PKB, and 4E-BP1, as well as the association of eIF4G with
eIF4E and the association of 4E-BP1 with eIF4E, as described previously
(15,43,44).
Determination of extent of eIF4G or mTOR phosphorylation. The
relative extent of phosphorylation of eIF4G or mTOR proteins was
determined following separation by 7.5% SDS-polyacrylamide gel
electrophoresis (PAGE) as described previously using phospho-specific
TABLE 1 Effect of oral Leu gavage on plasma Leu
concentrations and rates of protein synthesis in
chronic (5 d) sterile inflammatory and septic rats1
ST ST1Leu SP SP1Leu
Plasma Leu, mmol/L 153 6 34 1539 6 129 165 6 21 1824 6 224
Protein synthesis, %/d 20 6 2 34 6 4 11 6 12 33 6 3
1
Values are means 6 SEM, n ¼ 8–12. Gavage with Leu (1Leu) was significant for all
variables, P , 0.0001.
2
P , 0.05 vs. ST.
Leucine increases protein synthesis during sepsis 2075
 antibodies that recognize either phospho-eIF4G (Ser1108), phospho-
mTOR(Ser2448), or phospho-mTOR(Ser2481) (Cell Signaling Technol-
ogy) (15,43,44). The phosphorylated eIF4G or mTOR signal densities
were normalized to the respective total eIF4G or mTOR signal to reflect
the relative ratio of phosphorylated eIF4G or mTOR to total eIF4G or
mTOR, respectively.
Determination of extent of 4E-BP1 phosphorylation. The various
phosphorylated forms of 4E-BP1 (designated a, b, and g) were separated
by SDS-PAGE electrophoresis and quantitated by protein immunoblot
analysis as described previously (37,43,44).
Determination of extent of S6K1 and PKB phosphorylation. Ly-
sates from gastrocnemius muscle were mixed with 23 Laemmli-SDS
sample buffer and subjected to electrophoresis on 12.5% SDS-PAGE
Criterion gels (Biorad) as described previously using phospho-specific
antibodies that recognize either phospho-S6K1(Thr389), phospho-PKB
(Thr308), or phospho-PKB(Ser473) (Cell Signaling Technology) and quanti-
fied as described above for eIF4G phosphorylation (13,39,43,44). Results
are presented as the ratio of the densitometric analysis of blot for phos-
phorylated form divided by total (phosphorylated 1 unphosphorylated)
on same gel.
Statistical analysis. Values shown are means 6 SEM. Data were
analyzed using a 2-way ANOVA with condition (ST vs. SP) and Leu
gavage ST1Leu vs. SP1Leu as factors (Graphpad Prism4 Software). We
performed a Sidak post hoc test to determine differences between means
for all significant interactions and main effects. For all analyses, dif-
ferences were considered significant at P , 0.05.
Results
Rates of protein synthesis. Gavage with a solution containing
Leu raised the plasma concentration to 10-fold that in rats with
either a sterile or septic intra-abdominal abscess (Table 1). The
plasma Leu concentration did not differ between ST and SP rats
following either saline or Leu gavage. Sepsis depressed rates of
protein synthesis by 40% (Table 1), consistent with our previous
findings in gastrocnemius (8,10,13,45). Elevating the plasma
Leu concentration enhanced muscle protein synthesis in rats
with a sterile abscess. Likewise, gavage with a solution com-
posed of Leu increased protein synthesis SP and the response was
similar to that observed in ST (Table 1).
Assembly of active eIF4G-eIF4E complex. The mechanistic
interactions between sepsis and Leu were investigated by
analysis of known regulatory steps controlling mRNA transla-
tion initiation. We previously posited that translation initiation
is limited through assembly of active eIF4G-eIF4E complex in
sepsis (13). Consistent with this hypothesis, the abundance of
eIF4G-eIF4E complex was significantly reduced during sepsis
(Table 2). In the ST groups, the abundance of eIF4G associated
with eIF4E was 2.2-fold greater following administration of Leu
than those administered saline (Table 2). A similar increase in
the assembly of an eIF4G-eIF4E complex was observed in septic
rats gavaged with a solution containing Leu.
Assembly of active eIF4G-eIF4E complex is dependent upon
both the availability of eIF4E and the extent of eIF4G
phosphorylation. The extent of eIF4G phosphorylation was
1.5-fold greater in ST1Leu compared with ST. Likewise, there
was an approximate 2.6-fold increase in eIF4G phosphorylation
in SP1Leu rats compared with SP rats (Table 2). Finally, changes
in eIF4G phosphorylation were not a result of Leu- or sepsis-
induced change in the content of total eIF4G (data not shown).
The abundance of eIF4E associated with 4E-BP1 did not
differ between ST and SP rats gavaged with a solution containing
saline. There was a marked decreased in the amount of the
inactive eIF4E-4E-BP1 complex in ST1Leu or SP1Leu rats com-
pared with their corresponding saline gavaged groups (Table 2).
The amount of the inactive eIF4E-4E-BP1 complex in SP1Leu
did not differ from ST1Leu.
Phosphorylation of 4E-BP1. When phosphorylated, 4E-BP1
resolves into distinct electrophoretic forms (a, b, and g) with the
g-form representing the highest degree of phosphorylation (46).
To further define the mechanism that modulates eIF4E binding
with 4E-BP1, we examined the phosphorylation state of 4E-BP1
(Table 2). Gavaging with Leu increased the proportion of the
hyperphosphorylated g-isoform of 4E-BP1 in ST1Leu 7.2-fold
and SP1Leu rats 47-fold compared with ST and SP, respectively
(Table 2).
Phosphorylation of S6K1. Avruch et al. (47) have provided
evidence that following phosphorylation, S6K1 activity in vivo
is most closely related to the phosphorylation of residue
Thr389. Therefore, we examined phosphorylation of the Thr389
residue as a measure of S6K1. Oral Leu administration robustly
increased the phosphorylation of S6K1 4.6-fold compared with
muscles from ST rats. Likewise, gavage with a solution con-
taining Leu increased the extent of S6K1 phosphorylation
5.8-fold in SP rats (Table 2).
Phosphorylation of mTOR. mTOR, a Pro-directed Ser-Thr
protein kinase, is posited as a common upstream kinase in a
signaling cascade leading to phosphorylation of 4E-BP1 and

TABLE 2 Effect of oral Leu gavage on phosphorylation of eIF4G and association of eIF4E with eIF4G
or 4E-BP1 in chronic (5 d) sterile inflammatory and septic rats1

ST ST1Leu SP SP1Leu
a
eIF4G associated with eIF4E, eIF4G/eIF4E 18 67 58 6 18 26 1 33 6 8
eIF4G(Ser1108) phosphorylation, phospho-eIF4G(Ser1108)/total eIF4G 36 68 91 6 11 41 6 6 146 6 19
4E-BP1 associated with eIF4E, 4EBP-1/eIF4E 17 62 8 6 3 21 6 3 36 1
4E-BP1 phosphorylation, g-form 4E-BP1/total 4E-BP1 5 62 41 6 6 11 6 3 63 6 5
S6K1(Thr389) phosphorylation, phospho-S6K1(Thr389)/total S6K1 5 61 28 6 5 66 1 41 6 6
mTOR(Ser2448) phosphorylation, phospho-mTOR(Ser2448)/total mTOR 11 62 40 6 4 14 6 2 37 6 2
mTOR(Ser2481) phosphorylation, phospho-mTOR(Ser2481)/total mTOR 7 61 12 6 1 86 1 15 6 1
PKB(Ser473)phosphorylation, phopsho-PKB(Ser473)/total PKB 10 61 15 6 3 76 12 66 12
PKB(Thr308)phosphorylation, phospho-PKB(Thr408)/total PKB 5 61 8 6 1 46 1 46 1
1
Values are means 6 SEM, n ¼ 7–14. Gavage with Leu (1Leu) was significant for all variables, P , 0.005, with exception of PKB
phosphorylation, which did not show a Leu gavage effect.
2
P , 0.05 for effect of SP vs. ST.

2076 Vary
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 S6K1 induced by Leu (12). Its activity is regulated in part by
phosphorylation. Oral Leu administration increased the extent
of mTOR phosphorylated on Ser2448 or Ser2481 from ST rats
(Table 2). Likewise, gavaging SP rats with a solution containing
Leu increased the extent of mTOR phosphorylated on Ser2448
or Ser2481(Table 2). None of the treatments altered the total
amount of mTOR in skeletal muscle (data not shown).
Phosphorylation of PKB. PKB, also known as AKT, is a Ser/
Thr kinase hypothesized to enhance mTOR activity. PKB itself
is activated by reversible phosphorylation. Consequently, we
also examined the response of PKB phosphorylation to Leu
gavage following sterile inflammation or sepsis. The level of
PKB phosphorylation on Thr308 was not significantly affected by
sepsis and did not increase with a gavage containing Leu
(Table 2). Sepsis decreased (234%) the phosphorylation of PKB
on Ser473 and the extent of phosphorylation did not recover
to values observed in sterile inflammatory rats when gavaged
with a solution containing Leu (Table 2).
Discussion
The results of this study indicate that raising the plasma Leu
concentration 10 times fasting Leu concentrations following
gavage with a solution containing Leu enhanced factors asso-
ciated with accelerating translational control in gastrocnemius
of rats possessing a chronic, intra-abdominal septic abscess. Con-
trol of mRNA translation initiation posits assembly of active
eIF4G-eIF4E complex as a central regulator of protein synthesis.
Indeed, assembly of active eIF4G-eIF4E complex positively cor-
relates with rates of protein synthesis in skeletal muscle (13). In
this study, raising the plasma Leu enhanced the assembly of an
active eIF4G-eIF4E complex and stimulated protein synthesis
during sepsis.
Assembly of an active eIF4G-eIF4E complex depends upon
both the availability of eIF4E and on phosphorylation of eIF4G.
Phosphorylation of eIF4G on residues in the C-terminal region
of the eIF4G protein including Ser1108 fully actives eIF4G (48).
Increased phosphorylation of eIF4G on Ser1108 enhances
formation of active eIF4G-eIF4E complex in cells in culture
(48,49), leading to an increased rate of protein synthesis.
Likewise, elevated phosphorylation of eIF4G correlates with
mRNA translation in skeletal muscle during perfusion of hind
limb with buffer containing Leu (50) or following oral admin-
istration of a single bolus of Leu (24). In this study, Leu gavage
increased the extent of eIF4G phosphorylation in septic rats.
Enhancing the availability of eIF4E to bind with eIF4G
represents another mechanism for increasing assembly of eIF4G-
eIF4E complex. eIF4E availability is controlled through inter-
actions with 4E-BP1 (46,51,52) that compete with eIF4G for a
common binding site on eIF4E (53,54). Hence, reduced binding
of eIF4E with 4E-BP1 would be expected to enhance availability
for eIF4E to associate with eIF4G. Indeed, gavage with a
solution containing Leu markedly reduced the binding of the
eIF4E to 4E-BP1 in rats with either a sterile or septic abscess.
Taken together, the results are consistent with a combined role of
eIF4G phosphorylation and increased availability of eIF4E as a
means to increase the assembly of active eIF4G-eIF4E complex
following Leu gavage.
Amino acids, and Leu in particular, consistently activate the
S6K1 and the translation repressor 4E-BP1 through enhanced
phosphorylation both in vitro (55,56) and in vivo in control rats
(23,35,38,39,57,58). Phosphorylating S6K1 and 4E-BP1 are as-
sociated with an acceleration of mRNA translation initiation,
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leading to a stimulation of protein synthesis. Enhanced phos-
phorylation of 4E-BP1 lowers the interactions between eIF4E
and 4E-BP1, allowing increased availability of eIF4E. On the
other hand, phosphorylation of S6K1 promotes translation of
mRNA containing 5#-terminal oligopyrimidine tract sequences.
In both sterile inflammatory and septic rats, Leu was able to
cause an increase in phosphorylation of S6K1 and 4E-BP1. The
S6K1 and 4E-BP1 are phosphorylated by a common upstream
kinase, mTOR, suggesting a role for mTOR in mediating in part
the effects of Leu to phosphorylate these 2 proteins (37,59).
Indeed, in this investigation, Leu increased the phosphorylation
of mTOR in gastrocnemius of septic rats, consistent with Leu’s
role in stimulating mTOR.
Lastly, an upstream activator of both mTOR and S6K1
phosphorylation appears to be PKB (12). PKB is activated by
phosphorylation of 2 critical Ser/Thr residues (i.e. Thr308 and
Ser473). Gavage with a solution containing Leu did not signif-
icantly increase PKB phosphorylation in sterile inflammatory
rats. During sepsis, PKB(Ser473) phosphorylation was reduced
30%, a defect in cell signaling that was not corrected by gavage
with a solution containing Leu. Studies by Orellana et al. (18)
did not observe a significant reduction in phosphorylation of
PKB(Ser473) during a 20-h endotoxin infusion in 5–6-d-old
piglets provided amino acids and dextrose for 2 h prior to
sampling of the longissimus dorsi. The differences between that
study and our set of investigations may relate to the relative lack
of insulin resistance in muscle of neonatal pigs (18) compared
with adult rats (13). Similarly, Lang (17) observed a defect in the
ability of Leu to enhance phosphorylation of PKB in adult rats.
Therefore, increased phosphorylation of mTOR, S6K1, 4E-BP1,
and eIF4G induced by Leu administration during sepsis occurred
without evidence of activation of the PKB pathway. These ob-
servations are consistent with Leu acting downstream of PKB.
The effects of Leu on translational control reported in this
study are in contrast to previous reports in the literature showing
a blunted response to oral Leu gavage in fasted rats subjected to
perforation of the intestinal tract (cecal ligation and puncture)
for 24 h in cardiac (20) or skeletal muscle (60). Likewise, Lang
et al. (17) showed that LPS administration partially or com-
pletely abrogated the Leu-induced phosphorylation of 4E-BP1,
S6K1, ribosomal S6, and mTOR and changes in eIF4E distribu-
tion after 4 h. There are several potential reasons for these
discrepancies. First, our model shows a defined bacteremia (27)
contrasting with the endotoxin model (17) where a sustained
(chronic) bacteremia cannot be induced. Models of sepsis
induced by endotoxin administration, while allowing for quan-
tifiable administration, represent an acute insult initiating a
depression of cardiac index, mean arterial pressure, and total
leukocyte count (61), which contrasts with the human septic
condition characterized by an increased cardiac output (30).
Second, models involving perforation of the intestinal tract
(cecal ligation and puncture) suffer in that the bacterial insult is
dependent upon the organisms present in the gastrointestinal
tract, leading to variability in the bacterial insult. The variability
of the response is shown by the studies of Hasselgren et al. (62–
64), who reported that sepsis had either no effect or resulted in
decreased rate of protein synthesis in muscle, and our studies,
showing the metabolic response appears dependent upon the
invading organism (27). The cecal ligation and puncture model
reflects an acute peritonitis rather than a chronic septic process.
In this regard, animals subjected to cecal ligation and puncture
are anorexic, become dehydrated, and expire within 72 h. In
contrast, all rats implanted with an infected fecal agar pellet that
survive the abscess formation stage (48 h) live. Hence, both the
Leucine increases protein synthesis during sepsis 2077
 LPS injection model and cecal ligation and puncture model
produce preterminal septic shock rather than a hemodynami-
cally stable chronic sepsis.
In summary, oral administration of a solution composed of
Leu raised plasma Leu concentrations ;10 times more than
plasma concentrations. Oral administration of Leu to septic rats
stimulated phosphorylation of both 4E-BP1 and eIF4G, maxi-
mizing the assembly of active eIF4G-eIF4E complex. The
increased formation of active eIF4G-eIF4E complex and activa-
tion of S6K1 pathway was associated with accelerated rates of
protein synthesis to values observed in rats with a sterile,
nonseptic abscess. Thus, an enteral Leu treatment modality
apparently overcomes the inhibition of protein synthetic process
through acutely augmenting eIF4G-eIF4E during chronic sepsis.
Acknowledgments
The author thanks Gina Deiter, Beth Gren, and Rachel Eicher
for their expert technical contributions and Dr. Leonard S.
Jefferson from our institution for kindly providing the eIF4E
antibodies used in this study.
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