Endotoxin-induced decrease in muscle protein synthesis is
associated with changes in eIF2B, eIF4E, and IGF-I
Charles H.
Lang,
Robert A.
Frost,
Leonard S.
Jefferson,
Scot R.
Kimball, and
Thomas C.
Vary
Departments of Cellular and Molecular Physiology, and
Surgery, Pennsylvania State University College of Medicine, Hershey,
Pennsylvania 17033
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ABSTRACT |
The present study examined potential mechanisms
contributing to the inhibition of protein synthesis in skeletal muscle
after administration of endotoxin (LPS). Rats implanted with vascular catheters were injected intravenously with a nonlethal dose of Escherichia coli LPS, and samples were collected at 4 and 24 h thereafter; pair-fed control animals were also included. The rate of
muscle (gastrocnemius) protein synthesis in vivo was reduced at both
time points after LPS administration. LPS did not alter tissue RNA
content, but the translational efficiency was consistently reduced at
both time points. To identify mechanisms responsible for regulating
translation, we examined several eukaryotic initiation factors (eIFs).
The content of eIF2
or the amount of eIF2
in the phosphorylated
form did not change in response to LPS. eIF2B activity was decreased in
muscle 4 h post-LPS but activity returned to control values by 24 h. A
decrease in the relative amount of eIF2B
protein was not responsible
for the LPS-induced reduction in eIF2B activity. LPS also markedly
altered the distribution of eIF4E in muscle. Compared with control
values, LPS-treated rats demonstrated 1) a transient increase
in binding of the translation repressor 4E-binding protein-1 (4E-BP1)
with eIF4E, 2) a transient decrease in the phosphorylated
-form of 4E-BP1, and 3) a sustained decrease in the amount
of eIF4G associated with eIF4E. LPS also decreased insulin-like growth
factor (IGF) I protein and mRNA expression in muscle at both times. A
significant linear relationship existed between muscle IGF-I and the
rate of protein synthesis or the amount of eIF4E bound to eIF4G. In
summary, these data suggest that LPS impairs muscle protein synthesis,
at least in part, by decreasing translational efficiency, resulting
from an impairment in translation initiation associated with
alterations in both eIF2B activity and eIF4E availability.
eukaryotic initiation factors; eukaryotic initiation factor 2; eukaryotic initiation factor 4E; peptide-chain initiation; lipopolysaccharide; heart; liver; insulin-like growth factor I; rats
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INTRODUCTION |
NEGATIVE NITROGEN BALANCE and the loss of muscle
protein are hallmarks of gram-negative sepsis (37). The erosion of lean body mass in this condition results from both a decrease in muscle protein synthesis (3, 19, 41, 42, 45) and an increase in proteolysis
(17, 33, 45). Previous work in animal models of sepsis indicates that
the impairment of protein synthesis occurs in muscles with a
predominance of fast-twitch fibers (e.g., gastrocnemius) as opposed to
slow-twitch fibers (e.g., soleus) (41). Moreover, the sepsis-induced
inhibition of protein synthesis is primarily a result of a decrease in
translational efficiency rather than a decrease in the number of
ribosomes (4, 41). Translational efficiency reflects how well the
existing protein synthetic machinery is functioning.
The process of mRNA translation involves the following three steps:
initiation, elongation, and termination. In general, previous work has
demonstrated that impaired muscle protein synthesis induced by chronic
hypermetabolic peritonitis in the rat results from multiple defects in
the initiation phase of translation (5). Initiation is regulated by a
large number of protein factors, termed eukaryotic initiation factors
(eIFs). One of these initiation factors, eIF2, mediates the first step
in initiation, which involves the attachment of the initiator
methionyl-tRNA (met-tRNAmeti) to the 40S
ribosomal subunit to form the 43S preinitiation complex (36). Chronic
peritonitis does not significantly alter the total amount of eIF2
protein or the amount of eIF2 in the phosphorylated form in muscle (5,
39). However, the activity of eIF2 can be modulated by the activity of
another initiation factor, eIF2B (44). Inhibition of eIF2B activity has
been demonstrated in muscle from septic rats (39). A second regulatory
step in initiation involves the binding of mRNA to the 43S
preinitiation complex, which is mediated by eIF4F (36). Although
alterations in this step occur in other catabolic conditions associated
with decreases in protein synthesis (25, 45), its role in sepsis per se
has not been assessed.
The signals responsible for changes in regulating protein synthesis
during sepsis are not fully elucidated. Insulin-like growth factor
(IGF) I is an anabolic hormone that can function in both a classical
endocrine fashion and in a paracrine/autocrine manner to modulate
tissue metabolism (13). Elevations in IGF-I increase muscle protein
synthesis and decrease muscle proteolysis (2, 6). IGF-I concentrations
are depressed in the blood and muscle after induction of peritonitis or
injection of endotoxin (LPS; see Refs. 7, 10, 30). Several lines of
evidence suggest that IGF-I is important in regulating protein
synthesis in muscle during infection. First, we have previously
demonstrated a significant positive linear relationship between the
content of IGF-I protein in muscle and the rate of protein synthesis in
that same tissue (30). Second, acute administration of IGF-I in septic
animals leads to a stimulation of muscle protein synthesis (38).
Finally, inhibition of cytokine expression after induction of sepsis is associated with concomitant increases in both IGF-I and muscle protein
synthesis (7, 30).
LPS is a component of the outer cell wall of gram-negative bacteria and
is believed to be an important mediator of the metabolic sequela
accompanying bacterial invasion (18). The in vivo administration of LPS
reproduces many of the most important components of the metabolic
response to infection. Pertinent to the present study, both sepsis and
LPS have been demonstrated to decrease muscle protein content, in part,
by decreasing the rate of protein synthesis and translational
efficiency (1, 5, 21, 34). As described above, decreases in eIF2B
activity and eIF2B
content are associated with the decrease in
muscle protein synthesis in septic rats (39, 42, 43). However, there
have been no studies designed to investigate the mechanisms by which
LPS decreases muscle protein synthesis. It is commonly assumed, but not
proven, that the decrease in translational efficiency observed in
response to LPS is mediated by mechanisms similar to those reported for
sepsis. The purpose of the present study was to determine whether a
single nonlethal dose of LPS impaired regulation of the initiation
process by modulating eIF2 and/or eIF4F in skeletal muscle. The
concentration of IGF-I in blood and muscle was also determined as a
potential mediator of these changes. Because sepsis and LPS also alter
protein synthesis in liver (3, 11, 21, 40), we examined these same
initiation factors in this tissue for purpose of comparison.
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METHODS AND MATERIALS |
Animal preparation and experimental protocol.
Male Sprague-Dawley rats (375-400 g; Charles River
Breeding Laboratories, Wilmington, MA) were housed in a controlled
environment and were provided water and rat chow ad libitum for 1 wk
before beginning the study. At ~0800, animals were anesthetized with an intramuscular injection of ketamine and xylazine (100 and 1 mg/kg
body wt, respectively), and catheters were implanted in the carotid
artery and jugular vein aseptically (29). After surgery, rats were
returned to individual cages, and food was withdrawn for the remainder
of the protocol. At 1500 on the day of surgery, at which time all
animals were conscious and freely moving within their cages, one group
of rats was injected intravenously with Escherichia coli LPS
(100 µg/100 g body wt; Difco 026:B6, Detroit, MI), and a second group
of animals (i.e., controls) was injected intravenously with a similar
volume of isotonic saline (0.5 ml/100 g body wt). A third group of rats
was left untreated at the 1500 time point but was injected with the LPS
at 1100 the following day. All rats were killed at ~1500 the day
after surgery. Hence, animals in all three experimental groups (24-h
LPS-treated, control, and 4-h LPS- treated) were without food for the
same period of time. All LPS-injected rats survived until the time they
were killed. Experiments were approved by the Animal Care and Use
Committee of The Pennsylvania State University College of Medicine and
adhered to the National Institutes of Health guidelines for the use of
experimental animals.
Protein synthesis.
The rate of protein synthesis in vivo was determined using the
flooding-dose technique, as originally described by Garlick et al. (14)
and modified in our laboratory (4, 41, 45). Ten minutes before animals
were killed, an arterial blood sample (1 ml) was obtained for
determination of plasma concentrations of IGF-I and insulin. After this
blood sample and all others, an equal volume of saline was injected to
maintain blood volume. Next,
L-[2,3,4,5,6-3H]phenylalanine (Phe;
150 mM, 30 µCi/ml; 1 ml/100 g body wt) was injected intravenously. At
2, 6, and 10 min after injection of the radioisotope, blood samples
were collected for measurement of Phe concentration and radioactivity.
Rats were anesthetized with pentobarbital sodium intravenously after
the 6-min blood sample. Immediately after the removal of the 10-min
blood sample, selected tissues were excised, weighed, and frozen
between aluminum blocks precooled to the temperature of liquid
nitrogen. The frozen tissues were later powdered under liquid nitrogen
using a mortar and pestle and were stored at
70°C.
A portion of the powdered tissue was used to estimate the rate of
incorporation of [3H]Phe into protein exactly
as described previously (4, 41, 45). The protein concentration in these
samples was assayed by the biuret method using BSA as a standard. The
specific radioactivity of the plasma Phe was measured by HPLC using
supernatant from TCA extracts of plasma. The specific radioactivity was
calculated by dividing the amount of radioactivity in the peak
corresponding to Phe by the concentration of the amino acid in the same
fraction. The rate of protein synthesis was calculated as described
using the mean plasma Phe specific radioactivity of the three time
points as the precursor pool.
Total RNA.
Total RNA was measured on homogenates of tissue samples. Briefly,
frozen powdered tissue was homogenized in 5 vol of ice-cold 10% TCA.
The homogenate was centrifuged at 10,000 g for 11 min at
4°C. The supernatant was discarded, and the remaining pellet was
mixed in 2.5 ml of 6% perchloric acid (PCA). The sample was centrifuged at 10,000 g for 6 min at 4°C. The supernatant
was discarded, and the procedure was repeated. Next, 1.5 ml of 0.3 N
KOH were added to the pellet, and the samples were placed in a 50°C
water bath for 1 h. Samples were then mixed with 5 ml of 4 N PCA and
centrifuged at 10,000 g for 11 min. The concentration of RNA in
the supernatant was determined by measuring the absorbance at 260 nm
and correcting for the absorbance at 232 nm, as previously described
(40). Total RNA was expressed as micrograms of RNA per gram protein.
Amounts of eIF2 and eIF2B.
The relative amounts of the
-subunit of eIF2 (eIF2
) and the
-subunit of eIF2B (eIF2B
) in various tissues were estimated by
protein immunoblot analysis, as described previously (4, 39, 45). eIF2
and eIF2B were chosen because changes in the expression and/or activity
of these initiation factors correlate with alterations in protein
synthesis (27, 42). eIF2 consists of three subunits of which the
-subunit appears important in regulating protein synthesis (36).
Likewise, eIF2B is a multimeric protein consisting of five subunits,
with the
-subunit being the catalytic subunit (44). Briefly, tissue
was homogenized in 7 vol of buffer composed of (in mM) 20 Tris (pH
7.4), 250 sucrose, 100 KCl, 0.2 EDTA, 1 dithiothreitol (DTT), 50 NaF,
50
-glycerolphosphate, 1 phenylmethylsulfonyl fluoride (PMSF), 1 benzamidine, and 0.5 sodium vanadate. The samples were mixed with
2× Laemmli SDS buffer (60°C), boiled, and centrifuged. Equal
amounts of protein from tissue homogenates were electrophoresed at 60 mA in a 12.5% polyacrylamide gel. After electrophoresis, proteins in
the gel were transferred to nitrocellulose. After blocking with nonfat
milk (5% wt/vol) in 25 mM Tris (pH 7.6)-0.9% saline containing 0.01%
Tween 20 (Tris-NaCl-Tween), the membranes were washed extensively in
Tris-NaCl-Tween. The nitrocellulose was incubated for 1 h at room
temperature with an antibody specific for either eIF2
or eIF2B
(4, 39, 45). Antibodies were visualized using an enhanced
chemiluminescence (ECL) procedure with the secondary antibody linked to
horseradish peroxidase (Amersham). The blots were exposed to X-ray film
in a cassette equipped with a Du Pont Lightning Plus intensifying screen. After development, the film was scanned (Microtek ScanMaker IV)
and quantitated using NIH Image 1.6 software.
Measurement of phosphorylation state of eIF2
.
The relative amount of eIF2
present in the phosphorylated form,
designated eIF2
(P), was estimated by immunological visualization of
proteins after separation using slab-gel isoelectric focusing (IEF; see
Ref. 39). Tissues were homogenized in the same buffer as described
above for eIF2. A 75-µl aliquot of the homogenate was mixed with 42.9 mg urea and 300 µl IEF sample buffer [9.5 M urea, 2% Nonidet
P-40, ampholytes (BHD Resolyte 4-8), and 0.7 M
-mercaptoethanol]. The samples were electrofocused and then transferred electrophoretically to polyvinylidene difluoride membranes. The membranes were subsequently incubated with an eIF2
monoclonal antibody, and eIF2
was visualized as described above. The proportion of eIF2
present in the phosphorylated state was measured by
densitometric scanning of the membranes and is expressed as a
percentage of the total eIF2
content (i.e., phosphorylated + unphosphorylated).
Determination of eIF2B activity.
eIF2B activity in tissue was measured in postmitochondrial supernatants
using a [3H]GDP-GDP exchange assay, as
previously described (4, 39, 45). Fresh tissue was homogenized in
buffer consisting of (in mM) 20 triethanolamine (pH 7.0), 2 magnesium
acetate, 150 KCl, 0.5 DTT, 0.1 EDTA, 250 sucrose, 5 EGTA, and 50
-glycerolphosphate. The homogenate was then centrifuged at 15,000 g for 15 min at 4°C. The supernatant was assayed
immediately for eIF2B activity, as described previously (24). Briefly,
aliquots of the reaction mixture were analyzed for eIF2B activity by
measuring the decrease in
eIF2 · [3H]GDP complex bound
to nitrocellulose filters. The rate of exchange was linear over the
time points measured (data not shown). Under these conditions, ~50%
(0.3 pmol) of the [3H]GDP was released from the
eIF2 · [3H]GDP during the
assay procedure.
Quantification of 4E-BP1 · eIF4E and
eIF4G · eIF4E complexes.
The association of eIF4E with either 4E-BP1 or eIF4G was determined as
previously described (25, 45). Briefly, tissue was homogenized in 7 vol
of 20 mM HEPES, pH 7.4, 100 mM KCl, 0.2 mM EDTA, 2 mM EGTA, 1 mM DTT,
50 mM NaF, 50 mM
-glycerolphosphate, 0.1 mM PMSF, 1 mM benzamidine,
0.5 mM sodium vanadate, and 1 µM microcystin LR using a Polytron
homogenizer. The homogenate was centrifuged at 10,000 g for 10 min at 4°C. eIF4E as well as 4E-BP1 · eIF4E and
eIF4G · eIF4E complexes were immunoprecipitated from aliquots of 10,000-g supernatants using an anti-eIF4E
monoclonal antibody. The antibody-antigen complex was collected by
incubation for 1 h with BioMag goat anti-mouse IgG beads (Perseptive
Biosystems, Framingham, MA). Before use, the beads were washed in 1%
nonfat dry milk in 50 mM Tris · HCl, pH 7.4, 150 mM
NaCl, 5 mM EDTA, 0.1%
-mercaptoethanol, 0.5% Triton X-100, 50 mM
NaF, 50 mM
-glycerolphosphate, 0.1 mM PMSF, 1 mM benzamidine, and
0.5 mM sodium vanadate (buffer B). The beads were captured
using a magnetic sample rack and were washed two times with buffer
B and one time with buffer B containing 500 mM NaCl rather
than 150 mM. Resuspending in SDS-sample buffer and boiling for 5 min
eluted protein bound to the beads. The beads were precipitated by
centrifugation, and supernatants were collected. Supernatants were
subjected to electrophoresis either on a 7.5% polyacrylamide gel for
quantitation of eIF4G or on a 15% polyacrylamide gel for quantitation
of 4E-BP1 and eIF4E. Proteins were then electrophoretically transferred
to nitrocellulose as previously described (25, 45). The membranes were
incubated with a mouse anti-human eIF4E antibody, a rabbit anti-rat
4E-BP1 antibody, or a rabbit anti-eIF4G antibody for 1 h at room
temperature. The blots were then developed using ECL. Films were
scanned and quantitated as described above. The amounts of 4E-BP1 and
eIF4G were normalized by the amount of eIF4E in the immunoprecipitate.
Previous studies have shown that insignificant amounts of either the
4E-BP1 · eIF4E complex or
eIF4G · eIF4E complex are lost in the pellet during
the 10,000-g centrifugation step (26). Thus it is unlikely that
a redistribution of initiation complexes occurs under the conditions
employed during homogenization. In addition, >95% of the eIF4E
present in muscle homogenates is immunoprecipitated using the
monoclonal eIF4E antibody. Preliminary studies, using purified rat
liver proteins or recombinant proteins from Sf9 cells, indicated that
the amount of protein added per lane was sufficient to place
determinations in the linear portion of the detection curve for each
initiation factor (Jefferson and Kimball, unpublished data).
Phosphorylation state of eIF4E and 4E-BP1.
The phosphorylated and nonphosphorylated forms of eIF4E in tissue
extracts were separated by IEF on a slab gel and were quantitated by
protein immunoblot analysis, as previously described (25, 45). The
various phosphorylated forms of 4E-BP1 were measured after
immunoprecipitation of 4E-BP1 from tissue homogenates after centrifugation at 10,000 g (25, 45). 4E-BP1 was
immunoprecipitated as described above for immunoprecipitation of eIF4E.
The immunoprecipitates were solubilized with SDS sample buffer. The
various phosphorylated forms of 4E-BP1 were separated by
electrophoresis, with more slowly migrating forms representing more
highly phosphorylated 4E-BP1, and were quantitated by protein
immunoblot analysis as described above.
IGF-I and insulin.
The concentration of total IGF-I in plasma was determined using a
modified acid-ethanol (0.25 N HCl-87.5% ethanol) procedure with
cyroprecipitation, and muscle was processed using acid homogenization and Sep-Pak (C18) extraction (30). The tissue eluate was
evaporated, and the dried sample was reconstituted with RIA buffer
containing 0.25% BSA for IGF-I determination. IGF-I in plasma and
gastrocnemius was determined by RIA. Recombinant human
[Thr59]IGF-I was used for iodination and
standards (Genentech, South San Francisco, CA). The ED50
for this assay is 0.03-0.08 ng/tube. The protein concentration in
muscle was determined by the biuret method, and tissue IGF-I content
was expressed as nanograms IGF-I per microgram of tissue protein.
Total RNA was isolated from liquid nitrogen-frozen gastrocnemius using
TRI Reagent TR-118 as outlined by the manufacturer (Molecular Research
Center, Cincinnati, OH). Samples of total RNA (20 µg) were run under
denaturing conditions in 1% agarose-6% formaldehyde gels. Northern
blotting occurred via capillary transfer to Zeta-Probe GT blotting
membranes (Bio-Rad Laboratories, Hercules, CA). An 800-bp probe from
rat IGF-I (Peter Rotwein, Portland, OR) was labeled using a Random
Primed DNA Labeling kit (Roche Molecular Biochemicals, Indianapolis,
IN). For normalization of RNA loading, a rat 18S oligonucleotide was
radioactively end labeled using polynucleotide kinase (Amersham
Pharmacia Biotech, Piscataway, NJ). Membranes were prehybridized and
hybridized at 42°C in 50% formamide-6× sodium
chloride-sodium phosphate-EDTA-5× Denhardt's-1% SDS-10%
dextran sulfate-100 µg/ml herring testis DNA. All membranes were
washed at room temperature two times in 2× standard sodium citrate (SSC)-0.1% SDS for 5 min and one time in 0.1× SSC-0.1% SDS for 15 min. Additionally, membranes hybridized with rat IGF-I were
washed at 65°C in 0.1× SSC-0.1% SDS for 15-30 min. All
data were normalized with the signal generated by ribosomal 18S RNA. Finally, membranes were exposed to a phosphoimager screen, and the
resultant data were quantitated using ImageQuant software (Molecular
Dynamics, Sunnyvale, CA).
The plasma insulin concentration was determined using a commercially
available RIA (Diagnostic Products, Los Angeles, CA).
Statistics.
Values are presented as means ± SE. The number of rats per group is
indicated in the legends to Figs. 1-9 and Tables 1 and 2. Data were
analyzed by one-way ANOVA to test for overall differences among groups.
When ANOVA indicated a significant difference, individual means were
compared using the Newman-Keuls test. Statistical significance was set
at P < 0.05.
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RESULTS |
Tissue weight and protein content.
There was no change in the weight of the gastrocnemius, heart, or liver
at 4 or 24 h after injection of LPS compared with values from control
animals (Table 1). However, the protein
content of the gastrocnemius was decreased 16% (P < 0.05) at
24 h post-LPS (Table 1) compared with values from either the 4-h LPS or
control group. As a result of this change, the amount of total protein per whole muscle was decreased 19% in rats 24 h after LPS (Table 1).
There was no statistically significant change in the protein content or
total protein per tissue for either heart or liver in response to LPS
(Table 1).
Protein synthesis and translational efficiency.
Four hours after injection of LPS, protein synthesis in gastrocnemius
was decreased 37% compared with control values (Fig. 1A). Muscle protein synthesis was
slightly less depressed in rats 24 h after LPS. LPS also decreased the
rate of protein synthesis in myocardium (26-33%) and liver (46%)
at both time points examined (Fig. 1, B and C).

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Fig. 1.
Effect of endotoxin (LPS) on in vivo rates of protein synthesis in
gastrocnemius (A), heart (B), and liver (C).
Tissues were collected from rats at 4 or 24 h after injection of LPS
and from pair-fed control animals. Rates of protein synthesis were
measured after intravenous injection of
[3H]phenylalanine (Phe). Values are means ± SE; n = 5-6 rats/group. Values with different letters are
significantly different from each other (P < 0.05). Values
with the same letter were not significantly different.
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Changes in the number of ribosomes or in the efficiency of mRNA
translation may cause a reduction in tissue protein synthesis (5). To
determine which mechanism was responsible for the LPS-induced alterations in protein synthesis, the RNA content and translational efficiency were determined. Because ~80% of the RNA is ribosomal RNA, changes in total RNA content reflect changes in the number of
ribosomes. The RNA content of gastrocnemius, heart, and liver averaged
1,090 ± 18, 1,294 ± 43, and 3,692 ± 79 µg/g protein for control
animals. The RNA contents of tissues from either 4- or 24-h LPS-treated
rats were not significantly different from control values (data not
shown). These data suggest that an alteration in the relative abundance
of ribosomes was not responsible for the LPS-induced change in tissue
protein synthesis.
The efficiency of translation, calculated by dividing the protein
synthetic rate by the total RNA content, provides an index of how
rapidly the existing ribosomes are synthesizing protein (5). Figure
2 illustrates that LPS decreased
translational efficiency in skeletal muscle (~35%), heart (~30%),
and liver (~45%) at 4 and 24 h.

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Fig. 2.
Translational efficiency in tissues from control and LPS-treated rats.
A: gastrocnemius; B: heart; C: liver.
Translational efficiency was calculated by dividing the rate of protein
synthesis in a particular tissue by the RNA content for that tissue.
Values are means ± SE; n = 5-6 rats/group. Values with
different letters are significantly different from each other
(P < 0.05).
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Alterations in the amount of eIF2 and eIF2B, and eIF2B activity.
One possible mechanism for the LPS-induced decrease in translation is
via alterations in the amount and/or activity of specific eIF proteins
(36, 44). With the use of Western blot analysis, there was no
difference detected in the amount of eIF2
relative to total protein
in either gastrocnemius, heart, or liver from control or LPS-treated
rats (Table 2).
The percentage of phosphorylated eIF2
averaged 5 ± 2%
in gastrocnemius and 6 ± 2% in heart from control rats. The
injection of LPS did not produce a significant change in eIF2
phosphorylation in gastrocnemius or heart at either time point (data
not shown). In contrast, the percentage of eIF2
in the
phosphorylated form was increased more than twofold in the liver from
LPS-injected rats at both time points (Fig.
3).

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Fig. 3.
Effect of LPS on the phosphorylation state of eukaryotic initiation
factor (eIF) 2 in liver. Tissue homogenates were subjected to
isoelectric focusing (IEF) slab gel electrophoresis, transferred to
nitrocellulose, and visualized with a eIF2 -specific monoclonal
antibody. Inset: representative immunoblot of liver from
control, 4-h LPS, and 24-h LPS groups (lanes 1, 2 and
3, respectively). Bars indicate proportion of eIF2 in the
phosphorylated (P) state. Values were quantitated by densitometric
analysis of immunoblots of IEF gels. Values are means ± SE; n = 5-6 rats/group. Values with different letters are significantly
different from each other (P < 0.05).
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The effect of LPS on eIF2B activity was also measured in
postmitochondrial supernatants of tissues from control and experimental rats. eIF2B activity was markedly decreased 4 h post-LPS in
gastrocnemius (50%), heart (39%), and liver (66%) compared with
control values (Fig. 4). At 24 h after LPS,
eIF2B activity was not different from control values in gastrocnemius
and heart but was still significantly reduced in liver (49%). The
LPS-induced change in eIF2B activity in these tissues was not produced
by a concomitant change in the amount of eIF2B
protein (Table 2).

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Fig. 4.
Effect of LPS on the activity of eIF2B in various tissues. A:
gastrocnemius; B: heart; C: liver. eIF2B activity was
measured in postmitochondria supernatants by the GDP exchange assay,
and data are expressed as the pmol of GDP
exchanged · min 1 · mg
tissue protein 1. Values are means ± SE; n = 5-6 rats/group. Values with different letters are significantly
different from each other (P < 0.05).
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Regulation of eIF4E.
Another potential mechanism for decreasing initiation and protein
synthesis involves altered regulation of eIF4E (28). Binding of the
translational repressor 4E-BP1 to eIF4E forms an inactive complex. This
is visualized on an immunoblot as an increase in the amount of 4E-BP1
present in an eIF4E immunoprecipitate. Figure 5 shows that the
- and
-forms of
4E-BP1 were detected in immunoprecipitates of muscle and liver from all
groups, and the densitometric analysis of both bands from several
experiments is shown. In gastrocnemius, LPS increased the amount of
4E-BP1 associated with eIF4E by 55% at 4 h, but by the 24-h time point
values were not different from those of control animals. A more
pronounced and sustained increase in association of 4E-BP1 with eIF4E
was observed in liver after LPS (Fig. 5B). The amount of 4E-BP1
combined with eIF4E was increased 3.3-fold at 4 h and remained almost
2-fold higher at 24 h post-LPS. Because of the lack of tissue,
determinants of eIF4E availability were not performed on heart.

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Fig. 5.
Effect of LPS on the amount of 4E-binding protein-1 (4E-BP1) associated
with eIF4E in skeletal muscle and liver. A: gastrocnemius;
B: liver. Insets: representative immunoblots of muscle
and liver from control, 4-h LPS, and 24-h LPS groups (lanes 1,
2, and 3, respectively). The - and -forms of
4E-BP1 in the immunoprecipitate are identified on right. Bars
show densitometric analysis of total 4E-BP1 bound to eIF4E in each
tissue; data are expressed in arbitrary volume units (AU). Values are
means ± SE; n = 5-6 rats/group. Values with different
letters are significantly different from each other (P < 0.05).
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4E-BP1 has at least five potential phosphorylation sites, and the
various phosphorylated forms of the protein are resolved into three
bands by SDS-PAGE (28). For each tissue, the total amount of all three
phosphorylated forms did not differ between control and LPS-treated
rats (data not shown), indicating that the total amount of 4E-BP1 was
not altered. Phosphorylation of 4E-BP1 in the
-form is known to
decrease the association of the binding protein with eIF4E and increase
translation (28). In the present study, the amount of 4E-BP1 in
the
-form in muscle was decreased 41% at 4 h post-LPS, and at 24 h
the amount in the
-form was intermediate between that of
values in control and 4-h LPS-treated rats (Fig.
6A). A comparable decrease
(~35%) in the
-form in liver was observed in LPS-injected rats at
both time points (Fig. 6B).

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Fig. 6.
Effect of LPS on the amount of 4E-BP1 in the -form in muscle and
liver. A: gastrocnemius; B: liver. Insets:
representative immunoblots in which the -, -, and -forms
of 4E-BP1 are identified (left). Bars show densitometric
analysis where data are expressed as the amount of 4E-BP1 in the
-form as a percentage of the total of all phosphorylated and
nonphosphorylated forms of 4E-BP1. Values are means ± SE; n = 5-6 rats/group. Values with different letters are significantly
different from each other (P < 0.05).
|
|
In a similar manner, eIF4E immunoprecipitates were used to measure the
association of eIF4E with eIF4G. The gastrocnemius from LPS-treated
rats showed a 64% decrease in eIF4E with eIF4G at 4 h and a 46%
decrease at 24 h (Fig. 7A). A more
dramatic decrease in the binding of eIF4E with eIF4G was seen in liver
from LPS-treated rats. At both time points examined, the association of
eIF4E with eIF4G in liver was reduced by ~70% (Fig. 7B).
This decrease in muscle and liver was not the result of a reduction in
the amount of eIF4E in the immunoprecipitate between control and
LPS-treated rats (data not shown). Thus these data suggest that the
LPS-induced decrease in translation and protein synthesis in muscle and
liver results in part from a decreased formation of the active
eIF4E · eIF4G complex. To further define the
potential mechanism through which LPS inhibits translation, the
phosphorylation of eIF4E was examined. In both gastrocnemius and liver,
LPS did not alter the percentage of eIF4E in the phosphorylated state
at either time point examined (Table 2).

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Fig. 7.
Effect of LPS on the amount of eIF4G associated with eIF4E in skeletal
muscle and liver. Insets: representative immunoblots for muscle
and liver from control, 4-h LPS, and 24-h LPS groups (lanes 1,
2, and 3, respectively). Bars show densitometric
analysis of eIF4G bound to eIF4E in each tissue; data are expressed as
AU. Values are means ± SE; n = 5-6 rats/group. Values
with different letters are significantly different from each other
(P < 0.05).
|
|
IGF-I and insulin.
The plasma concentration of IGF-I was reduced 37% at 4 h and 25% at
24 h after injection of LPS (Fig.
8A). Moreover, the content of IGF-I
protein in gastrocnemius was also reduced at these two times (59 and
37%, respectively) as was the expression of IGF-I mRNA (62 and 29%,
respectively; Fig. 8, B and C). There was a positive
correlation between the IGF-I protein content in the gastrocnemius and
the IGF-I mRNA expression in the same tissue. Least squares linear
correlation of the data indicated that the slope of the line was
significantly different from zero (y = 0.0137x
0.105; r2 = 0.79; P < 0.05). Likewise,
there was a positive linear relationship between the IGF-I protein
content in gastrocnemius and the rate of protein synthesis in the
tissue (y = 0.95x + 30.0;
r2 = 0.71; P < 0.05). Finally, for
skeletal muscle, there was also a significant positive linear
relationship between the IGF-I content and the amount of eIF4G bound to
eIF4E (Fig. 9).

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Fig. 8.
Effect of LPS on the plasma IGF-I levels, IGF-I peptide content in
muscle, and IGF-I mRNA expression in muscle. Gastrocnemius was
collected from rats at 4 or 24 h after injection of LPS and from
pair-fed control animals. The presence of three IGF-I transcripts was
visualized for control and LPS-treated rats (data not shown). Because
of its prominence, only the ~7.5-kb transcript was used for
quantitation; however, qualitatively all transcripts were coordinately
regulated by LPS. Ribosomal 18S RNA indicated that the amount of RNA
loaded was similar for each lane (data not shown). Inset:
autoradiograph of IGF-I mRNA with lanes 1, 2, and
3 representative of control, 4-h LPS, and 24-h LPS groups,
respectively. Bars in C are densitometric analysis of tissue
IGF-I mRNA for each group. Values are means ± SE; n = 5-6 rats/group. Values with different letters are
significantlydifferent from each other (P < 0.05).
|
|

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Fig. 9.
Correlation between muscle IGF-I protein content and the amount of
eIF4G bound to eIF4E in the same muscle. Equation for the line based on
least squares linear analysis of data is y = 101.6x + 196.8, r2 = 0.66; P < 0.05. , Control;
, 4-h LPS; , 24-h LPS.
|
|
The plasma insulin concentration was 13.5 ± 0.7 µU/ml in control
animals. Insulin levels were not different from control values at 4 h
post-LPS (14.7 ± 2.6 µU/ml) but were significantly elevated at 24 h
(24.8 ± 4.1 µU/ml; P < 0.05).
 |
DISCUSSION |
LPS-induced changes in muscle.
The results of the present study confirm earlier reports indicating
that in vivo administration of LPS decreases protein synthesis in
skeletal and cardiac muscle (1, 21, 34). Furthermore, because the total
amount of RNA was unaltered by LPS, the translational efficiency in
these tissues was also reduced. eIF2, a heterotrimer consisting of
-,
-, and
-subunits, represents a major regulatory control
point for initiation (36). The first step in initiation is the
formation of a ternary complex consisting of eIF2, GTP, and
met-tRNAmeti. eIF2 mediates the binding
of met-tRNAmeti to the 40S ribosomal
subunit to form the 43S preinitiation complex. In other conditions, a
reduction in the tissue content of eIF2 protein is associated with a
decrease in initiation and a concomitant reduction in the rate of
protein synthesis (16, 27). However, similar to the septic condition
(4, 39), LPS did not significantly alter the total amount of eIF2
protein, as assessed by Western blot of eIF2
in gastrocnemius or heart.
The ability of eIF2 to form a ternary complex can also be reduced by
decreasing the activity of another eukaryotic initiation factor, eIF2B
(44). eIF2 is bound to GDP as an inactive complex when it is released
from the ribosome, and this GDP must be exchanged for GTP before
binding another molecule of met-tRNAmeti. This guanine nucleotide exchange is catalyzed by eIF2B and is required
for the recycling and activation of eIF2. Hence, a decrease in eIF2B
activity reduces the amount of eIF2 · GTP that is
available to bind to met-tRNAmeti and
thereby impairs translation initiation and protein synthesis. Such a
decrease in eIF2B activity has been previously reported in muscle of
septic rats (39) and other catabolic conditions (20, 31). Both
gastrocnemius and heart demonstrated a marked decrease in eIF2B
activity at 4 h post-LPS that correlated with the observed decrease in
protein synthesis. However, by 24 h, at which time protein synthesis
was still depressed in these tissues, eIF2B activity had largely
returned to control values. These data indicate that an LPS-induced
decrease in eIF2B activity was not responsible for the decrease in
protein synthesis in skeletal muscle and heart at the latter time
point. However, we cannot exclude the possibility that the early
LPS-induced decrease in protein synthesis was partially due to a
reduction in eIF2B activity.
One mechanism for the regulation of eIF2B activity involves
phosphorylation of the
-subunit of eIF2, which increases the affinity of eIF2 for eIF2B (44). Phosphorylation of eIF2
converts eIF2 from a substrate into a competitive inhibitor limiting the turnover of the eIF2 · GDP complex. Hence, the
formation of the highly stable eIF2
(P) · eIF2B
complex sequesters available eIF2B, leading to an impairment of
initiation. In both skeletal and cardiac muscle, there was no
significant or consistent increase in the phosphorylation status of
eIF2
, in agreement with the lack of a detectable change in muscle
from septic rats (39). A second mechanism by which eIF2B activity can
be regulated is by decreasing the amount of eIF2B protein. However, we
detected no significant change in the relative amount of eIF2B
,
which represents the catalytic subunit of eIF2B, in either
gastrocnemius or heart. This finding differs from that observed in
chronic (5-day) sepsis, where a consistent decrease in the amount of
eIF2B
protein and mRNA expression has been reported (22, 42). We
believe that this apparent difference between endotoxemic and septic
rats occurs because the duration of the insult is much longer in the
latter condition. In this regard, data from our laboratory indicate
that eIF2B
is only slightly decreased at 3 days postinfection,
whereas it is substantially reduced (40%) at 5 days postinfection
(22). The exact mechanism by which LPS decreases eIF2B activity in
muscle remains to be elucidated but appears independent of changes in the amount of eIF2B protein or the phosphorylation state of eIF2.
A second regulatory point in controlling translation initiation is
mediated by eIF4F and involves the binding of mRNA to the 43S
preinitiation complex (28). One component of the eIF4F complex, eIF4E,
binds directly to the m7GTP cap structure present at the
5'-end of most eukaryotic mRNAs and stimulates mRNA binding to
the small ribosomal subunit. During initiation, the
eIF4E · mRNA complex binds eIF4G and eIF4A, forming the functional eIF4F cap-binding complex. LPS administration produced a
marked and sustained decrease in the amount of eIF4E associated with
eIF4G. This decreased association was evident at both 4 and 24 h
postinjection of LPS and is consistent with the depression in muscle
protein synthesis at these time points. Moreover, this response is
consistent with the decreased binding of eIF4E with eIF4G observed in
other conditions in which protein synthesis is diminished (15, 31, 45).
Unlike the eIF2/2B system, sepsis-induced changes in eIF4E have not
been reported previously.
One mechanism for modulating the formation of the
eIF4E · eIF4G complex is by regulating the relative
distribution of eIF4E between inactive and active protein complexes
(32). In this regard, 4E-BP1 functions as a repressor of translation
initiation by binding to amino acid residues in eIF4E that also bind
eIF4G, thereby preventing formation of the active
eIF4E · eIF4G complex. In other catabolic conditions
exhibiting decreases in muscle protein synthesis, the amount of 4E-BP1
bound to eIF4E is increased in muscle (15, 16, 25, 31, 45). However, in
the present study, the amount of 4E-BP1 associated with eIF4E in muscle
was modestly increased by LPS at only the 4-h time point, but not at 24 h. The function of eIF4E can also be regulated by phosphorylation of
either 4E-BP1 or eIF4E (28). Phosphorylation of 4E-BP1 releases eIF4E
from the 4E-BP1 · eIF4E complex. This permits the
eIF4E · mRNA complex to bind eIF4G and stimulate
initiation. Therefore, stimuli that impair initiation and protein
synthesis are often associated with a decreased percentage of 4E-BP1 in
the phosphorylated
-form (26, 31, 45). Consistent with the decrease
in muscle protein synthesis, LPS significantly decreased the amount of
4E-BP1 in the
-form at 4 h, and values at the 24-h time point were
intermediate between those of control and early endotoxemic rats.
Alterations in the phosphorylation state of eIF4E also influence eIF4E
binding to mRNA. Both the phosphorylated and nonphosphorylated forms of eIF4E bind to the mRNA cap structure, but the affinity of the factor
for the m7GTP cap is increased severalfold by
phosphorylation of eIF4E (35). Furthermore, increases in
phosphorylation are proportional to increases in translation in
selected in vitro systems (28). However, the percentage of eIF4E
phosphorylated was unaltered by LPS in the present study.
Our laboratory and others have previously reported that sepsis,
endotoxemia, and inflammation decrease plasma levels of the anabolic
hormone IGF-I (7, 8, 30). This decrease likely results primarily from
an impairment in hepatic IGF-I synthesis as opposed to an increase in
clearance from the circulation (43). Decreases in IGF-I protein and
mRNA expression in skeletal muscle have also been observed after
induction of sepsis or injection of LPS (7, 8, 10, 30). Based on the
known actions of IGF-I on muscle protein balance (38, 40), the
LPS-induced reduction in IGF-I might be expected to decrease muscle
protein synthesis. In the present study, there was a significant linear correlation between the IGF-I content in gastrocnemius and the rate of
muscle protein synthesis. Furthermore, IGF-I was also correlated with
the amount of eIF4E bound to eIF4G. This response is consistent with
stimulation of translation initiation by IGF-I in the perfused hindlimb
(38). Although these correlations do not prove cause and effect, the
relationships are consistent with the known metabolic effects of IGF-I
and suggest a role of IGF-I in the LPS-induced decrease in muscle
protein synthesis by modulating initiation through eIF4G binding to
eIF4E. In contrast, the LPS-induced changes in various eIFs do not
appear to be caused by concomitant changes in the plasma insulin
concentration. A decrease in insulin would be consistent with
essentially all of the alterations observed in muscle from LPS-treated
rats (16, 20, 25, 26). However, at the 4-h time point, insulin levels
were not significantly different between groups. Moreover, at the 24-h
time point, endotoxemic rats were mildly hyperinsulinemic. Although we
can exclude an absolute decrease in plasma insulin as a cause for the
observed changes, it is possible that an impairment in insulin action
might be a participating factor. In this regard, LPS has been reported to produce insulin resistance in skeletal muscle and to impair several
components of the insulin-signaling pathway (9).
LPS-induced changes in liver.
Sepsis, LPS, and a variety of other inflammatory insults are known to
induce a reprioritization of hepatic protein synthesis (3, 11, 21).
This leads to the enhanced synthesis of positive acute-phase proteins
and a concomitant decrease in the synthesis and secretion of other
proteins (3, 11). In chronic models of infection, total hepatic protein
synthesis has been shown to be increased (3). After injection of LPS,
total hepatic protein synthesis has been reported to be either elevated
or unchanged (1, 12, 21). Under the present experimental conditions, LPS decreased total hepatic protein synthesis. In this tissue, there
was a pronounced LPS-induced decrease in eIF2B activity at both time
points, which was in contrast to the relatively transient change
observed in muscle. An inhibition of eIF2B activity would be expected
to limit the exchange of GTP for GDP on eIF2. The net effect of this
defect would be a slower rate of ternary complex formation, thereby
limiting translation initiation. Moreover, in contrast to muscle, the
amount of eIF2
in the phosphorylated form was also consistently
increased. As explained above, an increase in phosphorylated eIF2
would be expected to decrease the guanine nucleotide exchange activity
of eIF2B and decrease initiation. Thus LPS appears to regulate one of
the initial steps in translation initiation in liver. It is also
noteworthy that these LPS-induced changes in hepatic eIF2/2B differ
from the response observed in hypermetabolic septic rats in which
neither eIF2B activity nor phosphorylated eIF2
is significantly
altered (39). The reasons for this difference are not known.
The relative distribution of eIF4E in liver was also greatly influenced
by LPS. There was a sustained two- to threefold increase in the amount
of eIF4E bound to 4E-BP1 in response to LPS. This increase is
consistent with the reduction in 4E-BP1 in the
-form. However, the
most striking LPS-induced change in liver was the marked decrease in
the amount of eIF4G binding to eIF4E, which would be expected to
severely limit translation initiation.
Summary.
Our data indicate that LPS-induced decreases in skeletal muscle protein
synthesis are associated with a transient decrease in eIF2B activity
and a more sustained decrease in binding of eIF4E with eIF4G,
indicating an impairment in eIF4F function. The LPS-induced decrease in
muscle protein synthesis and the amount of eIF4E bound to eIF4G were
correlated with the reduction in tissue IGF-I. LPS also decreased total
hepatic protein synthesis, and this change was associated with
sustained changes in both eIF2 and eIF4E, the most dramatic of which
was a >70% reduction in the amount of eIF4E bound to eIF4G. These
data indicate that in vivo administration of a nonlethal dose of LPS
alters a variety of key regulatory steps in translation initiation that
can potentially account for the decreased rate of tissue protein synthesis.
 |
ACKNOWLEDGEMENTS |
We thank Duanqing Wu, Gerald Nystrom, and Xiaoli Liu for excellent
technical assistance.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
GM-38032, GM-39277, and DK-13499.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. H. Lang,
Dept. of Cell. Molec. Physiology (H166), Penn State College of
Medicine, Hershey, PA 17033-0850 (E-mail: clang{at}psu.edu).
Received 13 August 1999; accepted in final form 22 December 1999.
 |
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