Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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We
reported that the inhibition of protein synthesis in skeletal muscle
during sepsis correlated with reduced eukaryotic initiation factor
eIF2B activity. The present studies define changes in eIF2B phosphorylation in gastrocnemius of septic animals. eIF2B kinase activity was significantly elevated 175% by sepsis compared with sterile inflammation, whereas eIF2B phosphatase activity was
unaffected. Phosphorylation of eIF2B
-Ser535 was
significantly augmented over 2-fold and 2.5-fold after 3 and 5 days and
returned to control values after 10 days of sepsis. Phosphorylation of
glycogen synthase kinase-3 (GSK-3), a potential upstream kinase
responsible for the elevated phosphorylation of eIF2B
, was
significantly reduced over 36 and 41% after 3 and 5 days and returned
to control values after 10 days of sepsis. The phosphorylation of PKB,
a kinase thought to directly phosphorylate and inactivate GSK-3, was
significantly reduced ~50% on day 3, but not on
days 5 or 10, postinfection compared with
controls. Treatment of septic rats with TNF-binding protein prevented
the sepsis-induced changes in eIF2B
and GSK-3 phosphorylation,
implicating TNF in mediating the effects of sepsis. Thus increased
phosphorylation of eIF2B
via activation of GSK-3 is an important
mechanism to account for the inhibition of skeletal muscle protein
synthesis during sepsis. Furthermore, the study presents the first
demonstration of changes in eIF2B
phosphorylation in vivo.
glycogen synthase kinase-3; protein kinase B; tumor necrosis
factor-binding protein; gastrocnemius; psoas; infection; eukaryotic
initiation factor 2B phosphatase; eIF2B kinase
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INTRODUCTION |
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SEPSIS INDUCES PROFOUND ALTERATIONS in whole body protein metabolism. Marked weight loss and accelerated nitrogen excretion characterize the host's response to severe systemic bacterial infection. Nitrogen losses equivalent to 5-17% of total body protein may be observed in septic patients despite aggressive nutritional support. Much of the whole body negative nitrogen balance occurs secondary to a net catabolism of skeletal muscle proteins. Muscle protein wasting in sepsis results from both a prolonged decrease in protein synthesis and an increase in protein degradation (for review see Refs. 2, 40, and 41). In contrast, the magnitude and duration of muscle wasting are usually short-lived in trauma or sterile inflammation, with the restoration of lean body mass and skeletal protein metabolism occurring within days of the insult (2, 3, 31, 40, 41, 47, 51).
Regulation of protein synthesis occurs predominantly through changes in the abundance of ribosomes, translational efficiency, and/or concentration of translatable mRNA. The sepsis-induced inhibition of protein synthesis in skeletal muscle results from a defect in translational efficiency (3, 14, 44, 47) rather than changes in total mRNA (24, 48) or the number of ribosomes (3, 47). Our previous studies have shown that sepsis diminished the translational efficiency secondary to a defect in translation initiation rather than reductions in elongation (47).
Two steps (the binding of met-tRNA
On the basis of in vitro experiments, eIF2B activity is regulated by
both direct and indirect mechanisms involving allosteric binding,
competitive inhibitory processes, and/or phosphorylation (11,
22). However, not all of these mechanisms appear important for
controlling eIF2B activity in skeletal muscle during sepsis. The
best-characterized regulatory mechanism involves the phosphorylation of
eIF2 on its -subunit [eIF2(
P)] (23, 30).
Phosphorylation of eIF2
converts the protein from a substrate into
an inhibitor of eIF2B (7, 23, 30). The resulting
eIF2(
P) · GDP · eIF2B complex is highly stable and
inactive (7, 23, 30). Thus phosphorylation of eIF2
results in a reduction in the amount of eIF2B available for GTP
exchange. Under a variety of conditions in nonmuscle cells, the
proportion of eIF2
in the phosphorylated form inversely correlates
with rates of protein synthesis (16, 23, 30, 44). We could
not detect a change in the extent of eIF2(
P) in skeletal muscle
during chronic sepsis (44). eIF2B is also subject to
allosteric regulation by a variety of effector metabolites (6,
16, 26). Inactivation of eIF2B by NAD+ and
NADP+ is reversed by addition of equimolar amounts of NADH
or NADPH (6, 16). However, we have shown that the
NADPH-to-NADP+ concentration ratio is not significantly
altered in gastrocnemius of septic rats (6, 16, 45). Thus
diminished eIF2B activity does not appear to be regulated by changes in
the redox state during sepsis. These findings suggest that mechanisms
other than the phosphorylation state of eIF2
or cytosolic redox
state regulate eIF2B activity in skeletal muscle during sepsis.
In addition to the above mechanisms for regulating eIF2B
activity, the catalytic -subunit of eIF2B (eIF2B
) is a
substrate for several protein serine/threonine kinases, which either
enhance or inhibit its guanine nucleotide exchange activity after
phosphorylation (7, 26, 32, 33, 56, 57). For example,
phosphorylation of eIF2B by casein kinase (CK)-1 or CK-2 stimulates the
activity of the protein in vitro (7, 32, 33), although one
group has been unable to reproduce the stimulatory effect of CK-1
(27). In contrast, phosphorylation of eIF2B by glycogen
synthase kinase (GSK)-3 in cells in culture results in inactivation of
the guanine nucleotide exchange activity (12, 54-57).
There is no information available concerning the potential role of
altered phosphorylation of eIF2B
in regulating eIF2B activity in any
tissue in vivo.
Here, we define the temporal changes in extent of phosphorylation in
eIF2B in gastrocnemius of septic animals and determine whether
anti-cytokine therapy can prevent the sepsis-induced alterations observed. In addition, we examine the role of GSK-3 as a potential upstream kinase responsible for the sepsis-induced alterations in
eIF2B
phosphorylation. The findings suggest that phosphorylation of
eIF2B
may be an important regulator of eIF2B activity, limiting translation initiation in skeletal muscle during sepsis.
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MATERIALS AND METHODS |
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Animals. Adult male Sprague-Dawley rats weighing 150-225 g were maintained on a 12:12-h light-dark cycle and fed ad libitum. Chronic abdominal sepsis was created by implantation of a fecal-agar pellet (1.5 ml) inoculated with 104 colony-forming units (CFU) of Escherichia coli and 108 CFU of Bacteroides fragilis into the peritoneal cavity as previously described (3, 4, 13-15, 44, 45, 47, 50-53). The animals develop an abdominal abscess resulting in a hyperdynamic, hypermetabolic septic condition. Control animals underwent the intra-abdominal implantation of a sterilized fecal-agar pellet to which sterile saline had been added to allow for the generation of a sterile abscess (14, 46, 47, 50). Both groups of animals consumed the same amount of rat chow over the course of the experiment (49).
Three, five, or ten days after the implantation of the fecal- agar pellet, animals were anesthetized with pentobarbital sodium, and the gastrocnemius and psoas were excised, weighed, and either frozen between clamps precooled to the temperature of liquid nitrogen for immunoblot studies or homogenized for eIF2B kinase assays. The frozen tissue was powdered under liquid nitrogen and stored ateIF2B kinase assay.
Purified eIF2B was used to assay the kinases present in extracts of
psoas from sterile inflammatory and septic rats responsible for
phosphorylating the protein (12). Fresh psoas was excised, minced with scissors, and homogenized at 4°C in 7 vol buffer
A containing (in mM) 50 Tris · HCl (pH 7.4), 150 KCl, 5 magnesium acetate, 6
-mercaptoethanol, 250 sucrose, and 5 EGTA by
use of a Polytron PT10 set to 60% of full power. The homogenate was
centrifuged at 10,000 g (4°C) for 10 min, and the
supernatant was decanted. The supernatant then was centrifuged at
300,000 g (4°C) for 35 min, and the supernatant was used
for eIF2B
kinase assays. eIF2B
kinase activity was assayed in
buffer containing 20 mM HEPES (pH 7.4), 2.5 mM magnesium acetate
supplemented with 10 µl of the 300,000-g supernatant, 5 µg of purified eIF2B
, and 7.5 µCi [
-32P]ATP in
a total volume of 45 µl at 30°C. At 5, 10, and 15 min, 15 µl were
removed from the reaction mixture and placed in a tube containing 15 µl of 2× Laemmli SDS sample buffer warmed to 60°C and heated at
90°C for 2 min. Samples underwent electrophoresis on a 10%
polyacrylamide gel at 60 mA. The polyacrylamide gels were dried by use
of the Easy Breeze gel dryer (Hoffer Scientific, San Francisco, CA)
without heat. The gels were exposed to X-ray film, and the
autoradiographs were developed. After development, the film was scanned
(Microtek ScanMaker IV) and quantified with the use of NIH Image 1.6 software. eIF2B
used as substrate was expressed in Sf9 insect cells
by use of the baculovirus expression system and subsequently purified
to >98% homogeneity using immunoaffinity chromatography
(9).
Phosphorylation or dephosphorylation of eIF2B.
A portion of the purified eIF2B
(15 µg) was dephosphorylated using
-phosphatase (3,440 U; New England BioLabs, Boston, MA) by
incubation at 37°C for 30 min in the buffer supplied by the manufacturer. After incubation, an equal volume of 2× Laemmli SDS
sample buffer was added, and the mixture was heated at 90°C for 5 min. A second portion of the purified protein (15 µg) was phosphorylated using GSK-3 (85 U; New England Biolabs) by incubation at
30°C for 60 min in a solution consisting of (in mM) 35 Tris (pH 7.4),
7
-mercaptoethanol, 7% glycerol, 0.05 PMSF, 0.7 benzamidine, 12 magnesium chloride, and 0.25 ATP. An equal volume of 2× Laemmli SDS
sample buffer was then added, and the mixture was heated at 90°C for
5 min.
Determination of the phosphorylation state of eIF2B, GSK-3,
and PKB.
eIF2B
, GSK-3, and PKB in extracts of gastrocnemius were resolved by
electrophoresis, and the phosphorylated and unphosphorylated forms of
each enzyme were quantified by protein immunoblot analysis. An aliquot
(0.2 g) of the powdered tissue was weighed and homogenized in 7 vol
buffer A [20 mM HEPES (pH 7.4), 100 mM KCl, 0.2 mM EDTA, 2 mM EGTA, 1 mM DTT, 50 mM NaF, 50 mM
-glycerophosphate, 0.1 mM PMSF,
1 mM benzamidine, 0.5 mM sodium vanadate, and 1 µM microcystin LR]
by use of a Polytron homogenizer. The homogenate was centrifuged at
10,000 g for 10 min at 4°C, and the pellet was discarded.
Aliquots of the supernatant were mixed with equal volumes of 2×
Laemmli SDS sample buffer (60°C), boiled for 3 min, and centrifuged.
The samples were subjected to SDS polyacrylamide slab gel
electrophoresis followed by transfer of proteins to polyvinylidene
difluoride (PVDF) membranes (Immobilon-P; Bio-Rad Laboratories,
Hercules, CA), as described previously (17, 18). After
transfer of the proteins to PVDF membranes, phosphorylation of
eIF2B
, GSK-3, or PKB was analyzed by sequential immunoblotting,
first with phosphospecific antibodies that specifically recognized the
phosphorylated forms of eIF2B
(Biosource International, Camarillo,
CA), GSK-3 (Upstate Biotechnology, Lake Placid, NY), or PKB (Cell
Signaling Technologies, Vancouver, Canada). After
development of the blot, the membranes were treated with a solution
containing 62.5 mM Tris · HCl (pH 6.7), 100 mM
-mercaptoethanol, and 2% (wt/vol) SDS to remove antibodies as per
the manufacturer's instructions. Reexposure of the membrane to
secondary antibody revealed that this procedure removed the primary
anti-phospho-antibodies used. The membranes were then blocked with
nonfat dry milk and incubated with an antibody that recognizes eIF2B
(19), GSK-3 (Biosource International), or PKB (Cell
Signaling Technologies) independently of the phosphorylation states
(total eIF2B
, GSK-3, or PKB). All blots were developed using
enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ) and then exposed to film. After development, the film
was scanned (Microtek ScanMaker IV) and quantified using NIH Image 1.6 software. The phosphorylated eIF2B
, GSK-3, or PKB signal densities
were normalized to the respective total eIF2B
, GSK-3, or PKB signal
to reflect the relative ratio of phosphorylated eIF2B
, GSK-3, or PKB
to total eIF2B
, GSK-3, or PKB, respectively.
eIF2B phosphatase assay.
Gastrocnemius was homogenized in 7 vol of buffer B
containing (in mM) 20 HEPES, pH 7.4, 100 KCl, 0.2 EDTA, 2 EGTA, 1 DTT, 0.1 PMSF, and 1 benzamidine, using a Polytron homogenizer, and centrifuged at 1,000 g at 4°C for 3 min. An aliquot (25 µl) of the supernatant was diluted with 25 µl of homogenization
buffer and 60 ng of the phosphorylated 32P-labeled eIF2B
protein at 30°C. At 0, 2, 3, and 5 min, 10 µl of the reaction
mixture were added to 10 µl of 2× Laemmli sample buffer warmed to
60°C and boiled for 3 min. The sample was resolved by electrophoresis
on a 12.5% polyacrylamide gel. The gel was dried and exposed to X-ray
film in a cassette equipped with Du Pont Lightning Plus intensifying
screens. The autoradiographs were scanned and analyzed. Phosphatase
activity was calculated as the loss of radioactivity over time. Rat
eIF2B
was expressed in and purified from Sf9 cells as described
previously (12). A portion of the purified protein (10 µg) was phosphorylated in vitro by incubation of eIF2B
with
purified GSK-3 and [
-32P]ATP. The phosphorylation
reaction was terminated by addition of AMP-PNP to a final concentration
of 1 mM.
Statistical analysis. Values shown are means ± SE. Statistical evaluation of the data was performed using Student's t-test. Differences among the means were considered significant when P < 0.05.
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RESULTS |
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Effect of sepsis on eIF2B kinase activity.
We have previously established that eIF2B activity is reduced in
gastrocnemius and psoas of septic animals (44), but the mechanism responsible for the inhibition remains unknown. One possible
mechanism to account for the decreased eIF2B activity would be an
increased phosphorylation of eIF2B
. Initially, it was important to
establish whether or not sepsis increased eIF2B
kinase activity in
extracts of psoas. Typical autoradiographs of the eIF2B
kinase assay
from psoas of sterile inflammatory and septic rats on day 5 postsurgery are shown in Fig.
1A. A significantly (P < 0.05) greater eIF2B
kinase activity
was detected in extracts of psoas obtained from septic rats [226 ± 21 arbitrary units (AU)/min, n = 5]
compared with rats with a sterile abscess (130 ± 24 AU/min, n = 6).
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Effect of sepsis on eIF2B phosphorylation.
An increase in eIF2B
kinase activity would be expected to alter the
steady-state phosphorylation state of eIF2B
. Therefore, we sought to
evaluate the effect of sepsis on the phosphorylation of eIF2B
by
immunoblot techniques using an antibody raised to a peptide
corresponding to the phosphoserine amino acid residue (Ser535) of eIF2B
phosphorylated by GSK-3
(54). This site was chosen because Jefferson et al.
(12) have provided evidence that GSK-3 is the predominant
kinase present in muscles composed of fast-twitch fibers
(gastrocnemius, psoas) that phosphorylates eIF2B
(12). GSK-3 phosphorylates eIF2B
at a single conserved serine residue (Ser535 in the rat enzyme) and leads to an inhibition in
guanine nucleotide exchange activity (56). The antibody
specifically recognized the GSK-3-dependent phosphorylated form of
eIF2B
[phospho(P)-eIF2B
; Fig. 1B]. In these studies,
exogenous GSK-3 was used to phosphorylate purified eIF2B
in vitro.
Therefore, the anti-phospho-eIF2B
antibody was used to examine the
extent of phosphorylation of eIF2B
in skeletal muscle during sepsis.
The extent of phosphorylation of eIF2B
was increased over 2-fold
(P < 0.01) and 2.5-fold (P < 0.001)
in gastrocnemius after 3 and 5 days of septic abscess formation, respectively, compared with sterile inflammatory animals (Fig. 2). By 10 days of sepsis, the extent of
phosphorylation of eIF2B
returned to control values.
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Effect of sepsis on GSK-3 phosphorylation.
These studies clearly raised the question as to the possible role for
GSK-3 in mediating the sepsis-induced changes in phosphorylation of
eIF2B. Studies performed in cells in culture suggest that phosphorylation of eIF2B
by GSK-3 may result in a decreased activity of the guanine nucleotide exchange function of the protein (33, 56, 57). GSK-3 activity itself is regulated by reversible phosphorylation, where phosphorylation of GSK-3 results in inactivation of the enzyme. We have used changes in phosphorylation (assessed by
immunoblotting techniques) as an indicator of the effect of sepsis on
the activation of GSK-3 in gastrocnemius from sterile inflammatory and
septic rats (Fig. 3). The extent of
phosphorylation of GSK-3 was decreased over 36% (P < 0.001) and 41% (P < 0.01) in gastrocnemius after 3 and 5 days of abscess formation in septic rats, respectively, compared
with sterile inflammatory animals (Fig. 3). By 10 days of sepsis, the
extent of phosphorylation of GSK-3 was not significantly different
between control and septic rats.
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Effect of sepsis on PKB phosphorylation.
The protein kinase PKB has been shown to phosphorylate GSK-3 in
response to insulin in L6 cells in culture (5). PKB itself is activated by phosphorylation. We therefore investigated the phosphorylation state of PKB as a possible upstream mediator of the
changes in phosphorylation of GSK-3 (Fig.
4). The phosphorylation state of PKB was
significantly reduced ~50% (P < 0.001) on day 3 postinfection compared with sterile inflammatory rats. There were no significant differences between the two groups in the phosphorylation state of PKB on day 5 or day 10 postinfection.
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Effect of modulation of cytokine response during sepsis on
phosphorylation of eIF2B, GSK-3, and PKB.
The mediators responsible for the changes in eIF2B
and GSK-3
phosphorylation during sepsis remain unknown. Cytokines, which are
polypeptides produced by cells of the immune system, have been
implicated as potential mediators of the septic response, because they
elicit various and often overlapping effects designed to protect
the host in response to an inflammatory or bacterial insult. The
natural induction of cytokines during inflammation is beneficial, but
overproduction, as occurs in sepsis, is detrimental to the host
(37). Three proinflammatory cytokines, namely, TNF, interleukin (IL)-1, and IL-6 may play a role in mediating the effects
of sepsis on skeletal muscle protein metabolism. One approach to
understanding the role for these cytokines in mediating the inhibition
in skeletal muscle protein synthesis is to modify their release and/or
biological action during a septic insult. In this regard, the
biological activity of TNF is modulated in vivo by the proteolytic
shedding of the extracellular domain of the p55 and p75 TNF receptors.
An increase in soluble TNF receptors in the bloodstream neutralizes
circulating TNF, thereby lowering the biologically active concentration
of TNF in the plasma (10, 21, 28, 34, 38). TNFbp is a
dimeric, polyethylene glycol-linked form of the human p55-soluble TNF
receptor (8, 29, 34). This synthetic TNF antagonist,
TNFbp, is more potent than the native soluble TNF receptor in its
ability to block bioactivity of TNF (8, 29, 34). Blocking
the bioavailability of the proinflammatory cytokine TNF by use of a
specific TNFbp ameliorates the inhibition of skeletal muscle protein
synthesis and translational efficiency during sepsis (4).
If phosphorylation of eIF2B
is an important mechanism in controlling
mRNA translation, then treatment of septic rats with TNFbp should
result in a reduction in the phosphorylation of eIF2B
. Indeed,
treating septic rats with TNFbp for 5 days significantly reduced
(P < 0.05) the phosphorylation of eIF2B
compared
with saline-treated septic rats (Fig.
5A). We next wished to
determine whether TNFbp modulated GSK-3 phosphorylation in septic rats.
Treating septic rats with TNFbp for 5 days significantly increased
(P > 0.05) the phosphorylation of GSK-3 compared with saline-treated septic rats (Fig. 5B). TNFbp did not have any
significant effect on the extent of phosphorylation of PKB in septic
rats (Fig. 4C).
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Effect of sepsis on eIF2B phosphatase activity.
The increase in the phosphorylation state of eIF2B
during sepsis may
also result from a diminished eIF2B
phosphatase activity. Therefore,
we measured phosphatase activity toward eIF2B
by use of homogenates
from gastrocnemius in vitro. No significant differences in eIF2B
phosphatase activity were observed between sterile inflammatory and
septic rats either on day 3 (sterile 31 ± 11 vs.
sepsis 30 ± 14 AU/mg protein) or on day 5 (sterile
33 ± 9 vs. sepsis 32 ± 9 AU/mg protein) postinfection.
Because no differences were observed on day 3 and day
5 postinfection, we did not examine eIF2B
phosphatase activity
on day 10 postinfection, when no differences in eIF2B
phosphorylation between sterile abscess and septic abscess rats were observed.
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DISCUSSION |
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In the present study, we have investigated whether sepsis
modulates the phosphorylation state of eIF2B in skeletal muscle during any of three different phases of the septic response. The first
phase (day 3) is characterized by recovery from abdominal surgery and initiation of a septic focus. During this phase, both sterile inflammation and sepsis are associated with elevations in white
cell counts (39). Moreover, the gastrocnemius displays a
marked protein wasting induced in part by an inhibition of protein synthesis that occurs in sepsis but not sterile inflammation
(36). In the present study, phosphorylation of eIF2B
was noticeably enhanced in septic rats during this period. During the
second phase (day 5), the weight of the abscess is greater
in septic rats compared with sterile inflammatory rats
(39). Likewise, septic animals manifest a twofold
elevation in white cell counts, a low-grade fever, and a hyperdynamic
cardiovascular state, indicating a differential host response to sepsis
compared with sterile inflammatory abscess rats (25, 39,
50). During this phase, gastrocnemius from septic rats exhibits
a striking muscle wasting characterized by a 50% inhibition in rates
of protein synthesis (3, 4, 13-15, 43, 47, 48, 51,
53). As observed on day 3 postinfection, phosphorylation of eIF2B
is noticeably enhanced in septic rats compared with sterile inflammatory rats 5 days after the induction of
sepsis. The third phase (day 10) is characterized by a
waning of the hypermetabolic septic state. During this phase, there is recovery from the septic insult, as evidenced by a 40% drop in the
size of the abscess compared with day 5 postsurgery, a 50% fall in the white blood cell count, and a restoration of rates of
protein synthesis to values observed in nonoperated animals (14). In the present study, there were no significant
differences in phosphorylation of eIF2B
between the two groups at
this time. Taken together, these observations indicate that alterations
in the phosphorylation state of eIF2B
appear to correlate with the changes of protein synthesis and translation efficiency after induction
and recovery from the septic insult.
GSK-3 was originally identified as the enzyme responsible for
phosphorylating glycogen synthase, leading to its inactivation and
thereby reducing glycogen synthesis. More recently, it has become
evident that GSK-3 can also phosphorylate a number of proteins involved
in the regulation of the other metabolic processes, including eIF2B
(56, 57). GSK-3 itself is regulated by phosphorylation, where phosphorylation of GSK-3 leads to inactivation (for review see
Refs. 35 and 58). In the present study, the extent of phosphorylation of GSK-3 was decreased in gastrocnemius after 3 and 5 days of abscess formation in septic rats compared with sterile
inflammatory animals but returned to control values by 10 days of
sepsis. Thus alterations in phosphorylation of GSK-3 during sepsis were
inversely related to those of eIF2B
. Hence, sepsis may cause an
enhanced phosphorylation of eIF2B
with an associated inhibition of
eIF2B activity through activation of GSK-3 kinase activity secondary to
decreased phosphorylation of GSK-3.
In recent years, PKB has emerged as the most likely candidate to phosphorylate and inactivate GSK-3 (5). PKB, like GSK-3, undergoes reversible phosphorylation, with high levels of PKB activity associated with an increased phosphorylation of PKB. In the present studies, we provide evidence of a differential response in the phosphorylation of PKB over the course of the septic episode. Initially, the extent of phosphorylation of PKB is depressed, but beyond day 3 postinfection, the phosphorylation of PKB returns to values observed in sterile inflammatory rats. Hence, reductions in PKB phosphorylation may explain the decrease in GSK-3 phosphorylation on day 3 postinfection. However, PKB phosphorylation is not altered in septic rats on day 5 postinfection, a time when GSK-3 phosphorylation is depressed compared with sterile inflammatory rats.
The studies described herein provide strong evidence supporting the
hypothesis that phosphorylation of eIF2B by GSK-3 is intimately involved in the downregulation of eIF2B activity during initiation and progression of the septic process. Administration of TNFbp reduced phosphorylation of eIF2B
in gastrocnemius of septic
rats. Thus the reduction in the phosphorylation of eIF2B
correlates
with our previous observation that attenuating the TNF response to
infection prevents the sepsis-induced inhibition in protein synthesis
(1, 14, 42). In addition, administration of TNFbp to
septic rats increased the phosphorylation state of GSK-3, suggesting
that TNF may be modifying the phosphorylation state of GSK-3. Hence,
the changes in phosphorylation of both eIF2B
and GSK-3 may be
mediated by a TNF-dependent event initiated by the septic insult.
Activation of GSK-3 during the initiation phase of the septic response
correlates with changes in PKB activation by phosphorylation. The
finding that the time course of changes in PKB phosphorylation differed
from that observed for eIF2B
and GSK-3, coupled with the observation
that administration of TNFbp reversed the sepsis-induced changes in
eIF2B
and GSK-3 phosphorylation but not PKB phosphorylation,
suggests that PKB may not play an important role in the regulation of
GSK during the hypermetabolic phase of sepsis. Further studies will be
required to delineate the mechanisms responsible for the regulation of GSK-3 activity during the progression of sepsis.
Although the correlation between extent of phosphorylation of eIF2B
and protein synthesis in skeletal muscle of septic rats does not
necessarily prove cause and effect, the relationship is consistent with
the inhibition of translation initiation observed in skeletal muscle of
septic rats and suggests a potential role of eIF2B
phosphorylation
in the regulation of protein synthesis during sepsis. Although we have
used a specific anti-phospho-eIF2B
antibody with which we can assess
the relative level of phosphorylation of Ser535, this
approach does not give absolute levels of phosphorylation of the
protein. Despite this limitation, the data suggest that changes in
phosphorylation of eIF2B
may be an important regulatory mechanism
controlling eIF2B activity during sepsis.
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ACKNOWLEDGEMENTS |
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Anti-phospho-eIF2B antibody was generously provided by Biosource
International. TNFbp was kindly provided by Dr. C. Edwards from Amgen
(Boulder, CO).
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FOOTNOTES |
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This work was supported in part by National Institute of General Medical Sciences Grant GM-39277.
Address for reprint requests and other correspondence: T. C. Vary, Dept. of Cellular and Molecular Physiology, Rm. C4710, Penn State Univ. College of Medicine, 500 University Dr., Hershey, PA 17033 (E-mail:tvary{at}psu.edu).
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. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00171.2002
Received 23 April 2002; accepted in final form 15 July 2002.
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