1 Departments of Cellular and Molecular Physiology and Surgery, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; and 2 Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021
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ABSTRACT |
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This study examined potential mechanisms
contributing to the inhibition of protein synthesis in skeletal muscle
and heart after administration of tumor necrosis factor (TNF)-. Rats
had vascular catheters implanted, and TNF-
was infused continuously for 24 h. TNF-
decreased in vivo-determined rates of global
protein synthesis in gastrocnemius (39%) and heart (25%). The
TNF-
-induced decrease in protein synthesis in the gastrocnemius
involved a reduction in the synthesis of both myofibrillar and
sarcoplasmic proteins. To identify potential mechanisms responsible for
regulating mRNA translation, we examined several eukaryotic initiation
factors (eIFs) and elongation factors (eEFs). TNF-
decreased the
activity of eIF-2B in muscle (39%) but not in heart. This diminished
activity was not caused by a reduction in the content of eIF-2B
or
the content and phosphorylation state of eIF-2
. Skeletal muscle and heart from TNF-
-treated rats demonstrated 1) an increased
binding of the translation repressor 4E-binding protein-1 (4E-BP1) with eIF-4E, 2) a decreased amount of eIF-4E associated with
eIF-4G, and 3) a decreased content of the
hyperphosphorylated
-form of 4E-BP1. In contrast, the infusion of
TNF-
did not alter the content of eEF-1
or eEF-2, or the
phosphorylation state of eEF-2. In summary, these data suggest that
TNF-
impairs skeletal muscle and heart protein synthesis, at least
in part, by decreasing mRNA translational efficiency resulting from an
impairment in translation initiation associated with alterations in
eIF-4E availability.
eukaryotic initiation factors-2B and -4E; 4E-binding protein-1; elongation factors-1 and -2; rats; tumor necrosis factor-
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INTRODUCTION |
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TUMOR NECROSIS FACTOR
(TNF)- is a pleiotropic cytokine, the overexpression of which
appears responsible for many of the metabolic alterations observed in
response to various catabolic conditions (4, 11, 22, 58).
One of the metabolic hallmarks produced by TNF-
is the erosion of
lean body mass as evidenced by a decrease in total carcass nitrogen and
a pronounced impairment in muscle protein balance (1, 37, 48,
50). This imbalance was observed in early studies where the
acute administration of TNF-
to naive control animals or humans led
to an increased urinary nitrogen excretion and efflux of amino acids
from muscle (15, 16, 46, 55). Moreover, acute in vivo
administration of TNF-
has been shown to impair amino acid uptake by
muscle (2, 57). However, subsequent investigations
generally failed to detect significant alterations in either the rate
of protein synthesis or protein degradation in response to the acute
injection of TNF-
(8, 25, 40, 46). More recently, Zamir
et al. (58) was able to demonstrate a significant increase
in proteolysis and a tendency for a decrease in protein synthesis in
skeletal muscle incubated in vitro 2 h after a single injection of
TNF-
. These investigators postulated that the apparent discrepancy
between their work and earlier studies may be related to the interval
between the time of cytokine injection and tissue removal. That is,
TNF-
was shown to increase proteolysis when muscle was examined
2 h post-TNF-
(58) but not when protein balance
was determined after 6-8 h (19). The limited ability
of TNF-
to impair muscle protein balance at these later time points
is consistent with the rapid clearance of TNF-
from the systemic
circulation (5). However, in previous studies a primed,
constant intravenous infusion of this cytokine for 24 h has been
demonstrated to produce a severe insulin resistance in skeletal muscle
(29) and to markedly decrease the concentration of the
anabolic hormone insulin-like growth factor (IGF) I in both the
circulation and muscle (12). These data suggest that a
more prolonged exposure of tissues to TNF-
may be a prerequisite for
producing detectable cytokine-induced changes in muscle protein
synthesis in vivo.
The decrease in muscle protein synthesis observed in a number of cachectic conditions is caused by a decreased translation efficiency rather than a reduction in the number of ribosomes (10, 30, 45). The translation of mRNA into protein is often divided into the following three stages: initiation, elongation, and termination. In sepsis and other catabolic conditions, the decrease in muscle protein synthesis is associated with defects in mRNA translation initiation, the first rate-determining phase of protein synthesis (10, 31, 44). Translation initiation is regulated by a large number of protein factors termed eukaryotic initiation factors (eIFs). One of these initiation factors, eIF-2, mediates the first step in initiation and promotes the attachment of the initiator methionyl-tRNA (met-tRNAi) to the 40S ribosomal subunit to form the 43S preinitiation complex (10, 42). A second critical point of translational regulation involves the binding of the 5'-end of cellular mRNA to the 43S preinitiation complex, which is mediated by the cap-binding protein complex eIF-4F (42, 44). Although the decrease in muscle protein synthesis in various catabolic conditions has been generally linked to inhibition of peptide-chain initiation, the exact site of the molecular lesion often varies (10, 31, 44).
Although translational controls operate most frequently during the
initiation phase, some studies have also implicated alterations in
elongation as a control point in the translation of specific mRNAs
(38). Both eukaryotic elongation factor (eEF)-1 and -2 are
critical for catalyzing the sequential addition of amino acid residues
to the COOH-terminal end of the nascent peptide. eEF-1 promotes the
GTP-dependent recruitment of aminoacyl-tRNAs to the A-site of
ribosomes. eEF-2 is also important for normal mRNA translation and
catalyzes the translocation of the peptidyl-tRNA from the A-site to the
P-site on the ribosome. Alterations in the amount and/or
phosphorylation state of eEF-1 and eEF-2 have been linked to changes in
protein synthesis (35, 53).
The purpose of the present study was to determine whether a prolonged
continuous infusion of TNF- would decrease skeletal muscle protein
synthesis and, if so, to determine whether this response was associated
with defects in peptide-chain initiation, via alterations in eIF-2
and/or eIF-4F, or an impairment in elongation. Because defects in
myocardial performance are often observed in response to TNF-
and
various catabolic conditions (28, 41), we also examined
the mechanism by which the infusion of TNF-
impairs protein
synthesis, translation initiation, and elongation in heart. Finally,
the TNF-
-induced change in global protein synthesis in various
nonmuscle tissues was also determined and compared with that observed
in different striated muscles.
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METHODS AND MATERIALS |
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Animal preparation and experimental protocol. Adult specific pathogen-free male Sprague-Dawley rats (285-310 g; Charles River Breeding Laboratories, Cambridge, MA) were housed at a constant temperature, exposed to a 12:12-h light-dark cycle, and maintained on standard rodent chow and water ad libitum for at least 1 wk before experiments were performed. All experiments were approved by the Animal Care and Use Committee at the Pennsylvania State University College of Medicine and adhered to the National Institutes of Health guidelines for the use of experimental animals.
Rats were anesthetized with an intramuscular injection of ketamine and xylazine (90 and 9 mg/kg, respectively), and sterile surgery was performed to implant catheters in the carotid artery and jugular vein, as previously described (12, 29). Briefly, the venous catheter was passed through a tightly coiled stainless steel spring and was fixed to a freely rotating swivel (Instech, Plymouth, PA); the arterial catheter was coiled and secured with tape on the dorsal surface of the rat. The intravenous infusion of TNF-Protein synthesis.
After the start of the TNF- infusion (24 h), the in vivo rate of
global protein synthesis was determined for various tissues using the
flooding-dose technique, as originally described by Garlick et al.
(17). Animals were injected intraperitoneally with
L-[2,3,4,5,6-3H]phenylalanine (Phe; 150 mM,
30 µCi/ml; 1 ml/100 g body wt). Later (10 min), a blood sample was
collected from the arterial catheter into heparinized syringes. The
gastrocnemius, psoas, soleus, heart, diaphragm, liver, small intestine,
kidney, spleen, and brain were rapidly excised and weighed. A portion
of the gastrocnemius and heart from each animal was taken for
measurement of eIF-2B activity and analysis of the eIF-4E system, and
the remaining tissue was 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. Blood was
centrifuged (13,000 g for 1 min at 4°C), and plasma was
collected. All tissue and plasma samples were stored at
70°C until analyzed.
Total RNA. Because some catabolic conditions can lead to a decrease in total RNA within 24 h (43), the total RNA was measured from muscle homogenates, as previously described (30). Briefly, frozen powdered tissue was homogenized in 5 vol of ice-cold 10% TCA. After centrifugation, the supernatant was discarded, and the remaining pellet was mixed with 6% 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, 0.3 N KOH was added to the pellet, and the samples were placed in a 50°C water bath for 1 h. Samples were then mixed with 4 N PCA and centrifuged. The concentration of RNA in the supernatant was determined by measuring the absorbance at 260 nm and correcting for the absorbance at 232 nm. These data were then used to calculate translational efficiency, which equals the ks for a particular tissue divided by the RNA content for that tissue.
Isolation of ribosomal subunits. Fresh muscle tissue (psoas and heart) was used to isolate 40S and 60S ribosomal subunits by sucrose density gradient centrifugation (51). Briefly, muscles were homogenized in a motor-driven glass-on-glass homogenizer in 4 vol of homogenization buffer [14 mM triethanolamine (pH 7.0), 2 mM magnesium acetate, 250 mM KCl, 0.5 mM dithiothreitol (DTT), 0.08 mM EDTA, 5 mM EGTA, 250 mM sucrose, and 1 mg nagarse (protease, type XXVII); Sigma Chemical, St. Louis, MO]. The homogenate was centrifuged at 10,000 g for 15 min, and the supernatant was recovered. Aliquots of the samples (0.7 ml), to which 0.1 vol of 10% (wt/vol) Triton X-100 and deoxycholate solution had been added, were then layered on 0.44-2.0 M exponential sucrose gradients. The samples were centrifuged at 167,000 g in a SW41 rotor (Beckman Instruments) for 20 h to resolve the 40S and 60S ribosomal subunits. The absorbance of the gradients was monitored at 254 nm, and fractions were collected using a density gradient fractionator (Instrumentation Specialties, Lincoln, NE). These data provide information pertaining to changes in peptide-chain initiation relative to changes in elongation/termination.
Amount of eIF-2 and eIF-2B.
The relative amounts of the -subunit of eIF-2 (eIF-2
), the
phosphorylated form of eIF-2
, and the
-subunit of eIF-2B
(eIF2-B
) in gastrocnemius and heart were estimated by protein
immunoblot analysis, as described previously (30,32, 54).
eIF-2 and eIF-2B were chosen because change in the content and/or
activity of these initiation factors correlates with alterations in
protein synthesis (10, 42). eIF-2 consists of three
subunits of which the
-subunit appears important in regulating
protein synthesis (42). Likewise, eIF-2B is a multimeric
protein consisting of five subunits, with the
-subunit being the
catalytic subunit (42). Previous studies have established
that the expression of the
-subunit is representative of other
subunits (54). Therefore, the relative abundance of eIF-2B
is believed to be representative of the eIF-2B holoenzyme. 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 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, boiled, and centrifuged. Equal amounts of protein
from tissue homogenates were electrophoresed in a 12.5% polyacrylamide
gel. After electrophoresis, proteins in the gel were transferred to
nitrocellulose. After blocking for 30 min 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 antibodies specific for either eIF-2
,
Ser51-phosphorylated eIF-2
, or eIF-2B
. 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 DuPont Lightning Plus intensifying screen. After development,
the film was scanned (Microtek ScanMaker IV) and analyzed using
National Institutes of Health Image 1.6 software.
Determination of eIF-2B activity.
eIF-2B activity in tissue was measured in postmitochondrial
supernatants using a [3H]GDP-GDP exchange assay, as
previously described (30, 32). 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 centrifuged at
15,000 g for 15 min at 4°C. The supernatant was assayed
immediately for eIF-2B activity by measuring the decrease in the
eIF-2 · [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
eIF-2 · [3H]GDP during the assay procedure.
Measurement of phosphorylation state of eIF-2.
The relative amount of eIF-2
present in the phosphorylated form,
designated eIF-2
(P), was estimated by immunological visualization of
proteins after separation using slab-gel isoelectric focusing (IEF; see
Refs. 30, 32, and 54). Tissues were
homogenized in the same buffer as described above for eIF-2. A 75-µl
aliquot of the homogenate was mixed with 42.9 mg of urea and 300 µl
of 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 (PVDF) membranes (Immobilon-P; Bio-Rad
Laboratories, Hercules, CA). The membranes were subsequently incubated
with an eIF-2
monoclonal antibody that recognizes both
phosphorylated and nonphosphorylated forms of the protein. eIF-2
was
visualized as described above. The proportion of eIF-2
present in
the phosphorylated state was measured by densitometric scanning of the
membranes and was expressed as a percentage of the total eIF-2
content (i.e., phosphorylated + unphosphorylated).
Analysis of 4E-binding protein-1 · eIF-4E and
eIF-4G · eIF-4E complexes.
The association of eIF-4E with either 4E-binding protein-1 (BP1) or
eIF-4G was determined as previously described (30, 32, 45). Briefly, tissue was homogenized in 7 vol of 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 -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. eIF-4E, 4E-BP1 · eIF-4E, and
eIF-4G · eIF-4E complexes were immunoprecipitated from aliquots
of 10,000-g supernatants using an anti-eIF-4E 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 buffer B (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). 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. Protein bound to the beads was eluted by boiling in SDS-sample
buffer for 5 min. The beads were collected by centrifugation, and the
supernatants were subjected to electrophoresis either on a 7.5%
polyacrylamide gel for analysis of eIF-4G or on a 15% polyacrylamide
gel for quantitation of 4E-BP1 and eIF-4E. Proteins were then
electrophoretically transferred to nitrocellulose. The membranes were
incubated with a mouse anti-human eIF-4E antibody, a rabbit anti-rat
4E-BP1 antibody, or a rabbit anti-eIF-4G antibody for 1 h at room
temperature. The blots were developed using ECL, and autoradiographs
were scanned and analyzed as described above.
Phosphorylation state of eIF-4E and 4E-BP1. The phosphorylated and nonphosphorylated forms of eIF-4E in tissue extracts were separated by IEF on a slab gel and analyzed by protein immunoblot analysis, as previously described (30, 32). The phosphorylated forms of 4E-BP1 were measured after immunoprecipitation of 4E-BP1 from tissue homogenates after centrifugation at 10,000 g. 4E-BP1 was immunoprecipitated as described above for immunoprecipitation of eIF-4E. The various phosphorylated forms of 4E-BP1 were separated by SDS-PAGE and analyzed by protein immunoblotting as described above.
Analysis of elongation factors eEF-1 and eEF-2. Muscles were homogenized in 7 vol of buffer A. The samples were mixed with equal volumes of 2× Laemmli SDS sample buffer (60°C), boiled for 3 min, and centrifuged. Equal amounts of protein were subjected to SDS-PAGE using Criterion Precast 10-20% Tris · HCl gradient gels (Bio-Rad Laboratories) followed by transfer of proteins to PVDF membranes as described previously (53). After blocking with nonfat dry milk, the membranes were incubated with antibodies specific for eEF-1A (Santa Cruz Antibodies, Santa Cruz, CA) or eEF-2 (35, 53). The phosphorylation status of eEF-2 in the tissue homogenate was analyzed by sequential immunoblotting first with an antibody that specifically recognized eEF-2 phosphorylated on Thr56 (35). After development of the blot as described above, the membranes were treated with SDS as per the manufacture's instruction to remove antibodies. The membranes were then blocked with milk and probed with an antibody that recognizes eEF-2 independent of its phosphorylation state. The blots were developed using ECL, and the autoradiographs were scanned and analyzed as described above.
Hemodynamic and metabolic determinations. Mean arterial blood pressure (MABP) and heart rate (HR) were determined by a pressure transducer attached to the arterial catheter before the injection of [3H]Phe. Colonic temperature was determined immediately after determination of MABP and HR, as previously described (29). The plasma concentrations of insulin, IGF-I, and corticosterone were determined by RIA (12, 29). Leptin concentrations were also determined by RIA (Linco Research, St. Charles, MO). Amino acid concentrations were determined using reverse-phase HPLC after precolumn derivatization of amino acids with phenylisothicyanate, as previously described (34). Plasma free fatty acid (FFA) concentrations were determined colorimetrically (Waco Chemicals, Richmond, VA).
Statistics.
Values are presented as means ± SE. The number of rats in each
group is indicated in Figs. 1-6 and Tables 1-4. Data were
analyzed by Student's t-test to determine the treatment
effect. Statistical significance was set at P < 0.05.
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RESULTS |
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In vivo rates of protein synthesis and translational efficiency.
Figure 1A shows that after
24 h of TNF- infusion the in vivo rate of protein synthesis was
decreased in the predominantly fast-twitch gastrocnemius by 39%
compared with values from time-matched control animals. Similarly,
TNF-
also decreased ks in the slow-twitch soleus muscle (23%) and in heart (ventricle only) by 25%. In contrast to other muscles, there was no difference in the rate of protein synthesis for the diaphragm between TNF-
-infused and time-matched control animals. Changes in the number of ribosomes or in the efficiency of mRNA translation may cause a reduction in tissue protein
synthesis (10). To determine which mechanism was
responsible for the TNF-
-induced decrease in
ks, the RNA content and translational efficiency
were determined. Because ~85% of the RNA is ribosomal RNA, changes
in total RNA content reflect changes in the number of ribosomes. We
could not detect any significant TNF-
-induced change in total RNA in
any muscle examined (Fig. 1B). These data suggest that an
alteration in the relative abundance of ribosomes was not responsible
for the TNF-
-induced reduction in muscle ks.
Hence, the efficiency of translation, which provides an index of
how rapidly the existing ribosomes are synthesizing protein, was
also decreased in gastrocnemius, soleus, and heart in response to the
infusion of TNF-
(Fig. 1C).
Polysome profiles.
Insight into whether alterations in initiation or
elongation-termination were mediating the TNF--induced decrease in
protein synthesis was obtained by examining polysome profiles from
skeletal muscle and heart. The relative rate of mRNA translation
initiation vs. elongation-termination can be assessed by isolating
nonpolysome-associated ("free") 40S and 60S ribosomal subunits
using sucrose gradient centrifugation. Figure
3A shows that TNF-
significantly increased the RNA content of fractions containing both
the free 40S and 60S subunits isolated from the psoas (fast-twitch)
muscle by ~35%. In general, the distribution of 40S and 60S
ribosomal subunits between polysome and nonpolysome fractions is
indicative of the balance between the rates of mRNA translation
initiation and elongation-termination. That is, when the rate of
initiation is decreased relative to elongation-termination, free
ribosomal subunits are binding to mRNA at a slower rate (initiation)
than they are moving along mRNA (elongation) and exiting (termination).
The net result of this defect is an increased abundance of free 40S and
60S ribosomal subunits in skeletal muscle in response to TNF-
. In
contrast, there was no difference in free ribosomal subunits in cardiac muscle from TNF-
-infused rats compared with control animals (Fig. 3B).
Relative amounts of eIF-2 and eIF-2B and eIF-2B activity.
One possible mechanism for the TNF--induced decrease in
translational efficiency is altering the amount, availability, or activity of specific eIF proteins. The effect of TNF-
on eIF-2B activity was measured in postmitochondrial supernatant of muscles from
control and experimental rats. eIF-2B activity was decreased 39% in
gastrocnemius from TNF-
-infused rats compared with control values
(Fig. 4A). However, this
decreased activity could not be explained by a concomitant decrease in
the amount of eIF-2B
protein (control = 224 ± 28 vs.
TNF-
= 266 ± 35 AU; P = not significant). In contrast to skeletal muscle, there was no detectable difference in
eIF-2B activity in the heart between control and TNF-
-infused rats
(Fig. 4).
Availability of eIF-4E.
A second potential site for control of peptide-chain initiation
involves the regulation of eIF-4E (10, 42). Western blot analysis indicated there was no significant difference in the tissue
content of eIF-4E between control and TNF--infused rats for either
gastrocnemius (control = 1,124 ± 68 AU vs. TNF-
= 1,089 ± 102 AU) or heart (control = 667 ± 45 vs.
713 ± 61 AU). However, eIF-4E can be sequestered into an inactive
complex through binding of the translational repressor molecule 4E-BP1.
This is visualized on an immunoblot as an increase in the amount of
4E-BP1 present in the eIF-4E immunoprecipitate. Therefore, we examined the ability of TNF-
to modify the distribution of eIF-4E in both gastrocnemius and heart. Figure
5A shows that the
- and
-forms of 4E-BP1 were detected in immunoprecipitates of both tissues from all groups. TNF-
increased the relative content of the inactive eIF-4E · 4E-BP1 complex by 46% in gastrocnemius and by 33% in heart. When eIF-4E is bound to 4E-BP1, it is unable to interact with
eIF-4G to form the active eIF-4E · eIF-4G complex. Therefore, we determined whether the TNF-
-induced increase in
eIF-4E · 4E-BP1 complex formation resulted in a decreased
association of eIF-4E with eIF-4G. As shown in Fig. 5B,
TNF-
also dramatically decreased the tissue content of the active
eIF-4E · eIF-4G complex in muscle and heart (66 and 43%, respectively).
Phosphorylation of 4E-BP1 and eIF-4E.
4E-BP1 has at least five potential phosphorylation sites, and the
various phosphorylated forms of the protein are resolved into three
bands by one-dimensional SDS-PAGE (44). These bands have
been designated ,
, and
in order of their decreasing electrophoretic mobility (Fig.
6A, inset). For
each tissue, the total amount of all three bands combined did not
differ between control and TNF-
-infused rats (data not shown),
indicating that the total amount of 4E-BP1 was not altered.
Hyperphosphorylation of 4E-BP1 is known to decrease the association of
the binding protein with eIF-4E and increase translation. In the
present study, the amount of 4E-BP1 in the hyperphosphorylated
-form
was decreased in both gastrocnemius (64%) and heart (50%) from
TNF-
-treated rats compared with tissues from time-matched control
animals (Fig. 6A).
eEF-1 and -2.
Compared with time-matched control values, the infusion of TNF- did
not significantly alter the content of eEF-1
, eEF-2, or
phosphorylated eEF-2 in either gastrocnemius or heart (Table 2).
Hemodynamic and metabolic characteristics.
TNF--infused rats had an MABP and body temperature that was not
significantly different from control values (Table
3). TNF-
-treated rats demonstrated a
significant tachycardia (21%). These data indicate that
TNF-
-infused rats were hemodynamicly stable and not preterminal at
the time measurements of protein synthesis and translation control were performed.
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DISCUSSION |
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In the present study, TNF- decreased the rate of protein
synthesis in the gastrocnemius, soleus, and heart between 20 and 40%.
Moreover, at least for gastrocnemius, TNF-
decreased synthesis of
both myofibrillar and sarcoplasmic proteins. The rate of total mixed
protein synthesis was approximately equal to that of the myofibrillar
proteins. The relative abundance of contractile proteins in muscle may
in part explain this observation. A similar response pattern occurs in
septic rats (54). In contrast, no alteration in protein
synthesis was observed in diaphragms from TNF-
-infused rats. The
ability of TNF-
to decrease protein synthesis in both the
gastrocnemius and soleus is in contrast to the selective decrease in
synthesis observed in fast-twitch skeletal muscle (e.g., gastrocnemius) in other catabolic conditions, including sepsis, endotoxemia, alcohol
intoxication, and glucocorticoid excess (30, 32, 43, 45).
Previous studies have generally failed to detect consistent alterations
in the in vivo rate of muscle protein synthesis in response to a bolus
injection of TNF-
(1, 8, 25, 46, 58). This difference
between the current investigation and these previous studies may be
related to the sustained increase in blood TNF-
levels produced by
the primed constant infusion of the cytokine for 24 h as opposed
to the transient elevations produced by protocols using bolus
injections. Additionally, even though our control animals underwent the
same degree of surgical trauma (e.g., vascular catheterization) as the
TNF-
-infused rats, we cannot exclude the possibility that this
preceding trauma may have sensitized or primed animals to the catabolic
effects of TNF-
. A similar situation as been reported whereby
implantation of vascular catheters augmented the metabolic effects
induced by endotoxin (3).
The infusion of TNF- also decreased protein synthesis in the small
intestine, but whether this reduction was restricted to the smooth
muscle layer and/or the mucosa was not assessed. This TNF-
-induced
decrease in protein synthesis in muscle and gut is not the result of a
generalized suppression of synthesis in all tissues. In this regard,
rates of protein synthesis in the kidney, lung, and brain were
unaltered by TNF-
. Moreover, TNF-
increased protein synthesis in
liver and spleen by 25-35%. A stimulation of hepatic and splenic
protein synthesis has been previously reported in response to a
short-term (~3 h) infusion of TNF-
(21).
The cellular content of ribosomes may be rate limiting for protein
synthesis under some conditions (10, 31). Thus the TNF--induced decrease in tissue protein synthesis may occur through a reduction in the number of ribosomes and/or a decrease in
translational efficiency (10, 31). In the present study,
the abundance of ribosomes, as estimated by the total RNA content, was
not significantly altered by TNF-
in any of the tissues examined.
Therefore, our data suggest that the TNF-
-induced decrease in muscle
protein synthesis results primarily from an impairment in translational efficiency and not from a diminished capacity for protein synthesis. Likewise, the TNF-
-induced increase in hepatic and splenic protein synthesis appears to result from a corresponding increase in
translational efficiency.
The impaired translational efficiency observed in muscle from
TNF--infused rats may result from a defect in either peptide-chain initiation and/or elongation-termination. In the present study, analysis of the distribution of ribosomal subunits between free subunits and polysomes was used to estimate the rate of initiation relative to elongation. TNF-
increased the number of
nonpolysome-associated 40S and 60S subunits in psoas muscle. A
comparable increase has been previously observed in other catabolic
states, such as infection, diabetes, and starvation (20, 27,
52). This increase indicates a decrease in peptide-chain
initiation relative to elongation-termination in skeletal muscle. This
conclusion is consistent with our current data demonstrating a
TNF-
-induced change in eIF-4E availability but no change in either
eEF-1
or eEF-2 content. In contrast to skeletal muscle, TNF-
did
not alter the distribution in ribosomal subunits in heart.
The first step in initiation is the formation of a ternary complex
consisting of eIF-2, GTP, and met-tRNAi (42).
The binding of met-tRNAi to the 40S subunit to form the 43S
preinitiation complex is mediated by eIF-2, which is a heterotrimer
consisting of -,
- and
-subunits. Previous studies have
indicated that a reduction in eIF-2 is associated with a concomitant
reduction in global rates of protein synthesis (27).
However, the relative content of eIF-2
was not altered in either
skeletal muscle or heart of TNF-
-infused rats. Similarly, the
wasting that accompanies sepsis, endotoxemia, and alcohol ingestion was
also not associated with a change in eIF-2
content (10, 30,
44). The ability of eIF-2 to form a ternary complex can also be
decreased via a reduction in the activity of another eukaryotic
initiation factor, eIF-2B (42). eIF-2 is bound to GDP as
an inactive complex when it is released from the ribosome, and this GDP
must be exchanged for GTP before a new round of initiation may proceed.
eIF-2B catalyzes this guanine nucleotide exchange and is required to
regenerate the active eIF-2 · GTP complex. Under certain
conditions, the rate of protein synthesis is directly proportional to
eIF-2B activity in muscle (20, 24). In the present study,
TNF-
decreased eIF-2B activity is skeletal muscle but not in heart.
Hence, in skeletal muscle, the decrease in eIF-2B activity would be
expected to reduce the amount of eIF-2 · GTP that is available
to bind to met-tRNAi, thereby decreasing protein synthesis
by impairing translation initiation. A similar mechanism does not
appear to be responsible for the TNF-
-induced decrease in myocardial
protein synthesis.
The activity of eIF-2B can be regulated by the amount of eIF-2B
protein. However, we detected no significant change in the relative
amount of eIF-2B, which represents the catalytic subunit of this
protein. Alternatively, eIF-2B activity can be regulated by changing
the phosphorylation state of the
-subunit of eIF-2, which increases
the affinity of eIF-2 for eIF-2B (42). Phosphorylation of
Ser51 on eIF-2
converts eIF-2 from a substrate into a
competitive inhibitor limiting the regeneration of the
eIF-2 · GDP complex. Hence, initiation is impaired by the
formation of the highly stable eIF-2
(P) · eIF-2B complex
that sequesters available eIF-2B. Although eIF-2B activity and
initiation can be strongly inhibited by the phosphorylation of only a
fraction of the eIF-2, we were unable to detect a significant
alteration in the phosphorylation state of eIF-2
in either
gastrocnemius or heart in response to TNF-
. Therefore, the exact
mechanism by which TNF-
decreases eIF-2B activity in gastrocnemius
remains to be elucidated but is independent of changes of the amount of
eIF-2B protein and the phosphorylation state of eIF-2. These data are
consistent with the lack of a change in these eIFs in skeletal muscle
and heart in response to other types of relatively acute (
24 h)
catabolic stimuli (24, 30, 32).
A second rate control point in translation initiation is mediated by
eIF-4F, which regulates the binding of mRNA to the 43S preinitiation
complex (42). The eIF-4F holoprotein is a three-subunit complex composed of eIF-4A, eIF-4E, and eIF-4G. One of these protein components, eIF-4E, binds directly to the m7GTP cap
structure present at the 5'-end of the large majority of eukaryotic
mRNA and plays a critical role in maintaining protein synthesis. During
translation initiation, the eIF-4E · mRNA complex binds to
eIF-4G and eIF-4A to form the active eIF-4F complex. One mechanism for
modulating the formation of the eIF-4F complex is by regulating the
relative distribution of eIF-4E between inactive and active complexes
with other proteins. The eIF-4E · eIF-4G interaction can be
inhibited by small regulatory proteins, eIF-4E binding proteins, that
bind to eIF-4E (33). In the present study, the total
amount of eIF-4E was not altered by TNF-; however, cytokine infusion
did increase the amount of eIF-4E bound to 4E-BP1 in both gastrocnemius
and heart. The binding of 4E-BP1 to eIF-4E is known to inhibit
cap-dependent translation (42, 44). Because both 4E-BP1
and eIF-4G contain a similar eIF-4E binding site, the binding of these
two proteins to eIF-4E is mutually exclusive. In this regard, there was
a pronounced concomitant decrease in the amount of eIF-4E bound to
eIF-4G in muscle and heart from TNF-
-infused rats. These data
strongly suggest that TNF-
decreases initiation, at least in part,
by an impairment in eIF-4F function secondary to decreased eIF-4E
availability. Similar changes in the relative distribution of eIF-4E
have been seen in other wasting conditions (30, 31, 44,
45).
The function of eIF-4E can also be regulated by phosphorylation of
4E-BP1 or eIF-4E. Phosphorylation of 4E-BP1 greatly decreases the
affinity of 4E-BP1 for eIF-4E and leads to dissociation of the
4E-BP1 · eIF-4E complex (23). This permits the
formation of a competent eIF-4F complex and the stimulation of
initiation. Therefore, stimuli that impair initiation and protein
synthesis are often associated with a decreased amount of the
hyperphosphorylated -form of this protein (26, 31, 45).
Consistent with the decrease in protein synthesis, TNF-
markedly
decreased the percentage of 4E-BP1 in the phosphorylated
-form in
both gastrocnemius and heart. A comparable decrease in the
phosphorylation state of 4E-BP1 has previously been described in other
catabolic states (26, 30, 31). In addition, changes in the
phosphorylation state of eIF-4E itself can also influence eIF-4E
availability. Although both phosphorylated and nonphosphorylated eIF-4E
bind to the mRNA cap structure, phosphorylation of eIF-4E enhances the
affinity of the factor for the m7GTP cap by severalfold
(39). Furthermore, in vitro studies have demonstrated that
increases in eIF-4E phosphorylation are proportional to increases in
the rate of translation (23). Conversely, a decrease in
eIF-4E phosphorylation has been correlated with a reduction in protein
synthesis after viral infection (13). In this regard,
quite unexpectedly, TNF-
resulted in a small, but statistically
significant, increase in the phosphorylation of eIF-4E in the gastrocnemius.
Elongation is the phase of the protein synthetic pathway that is
responsible for the growth of nascent polypeptide chains (38). Previous studies have demonstrated that an
impairment in elongation is, at least in part, responsible for the
decreased rate of protein synthesis in various muscle and nonmuscle
tissues under certain situations (35, 53). In mammals, the
elongation process is facilitated by two protein factors, eEF-1 and
eEF-2. Our data indicate that the relative content of these two
elongation factors in both skeletal muscle and heart was not
significantly altered by the infusion TNF-. Furthermore, the extent
of eEF-2 phosphorylation, which has been demonstrated to be inversely
proportional to the rate of protein synthesis (35), was
also not affected by TNF-
. These results are consistent with data
from a previous study that indicated rates of peptide-chain elongation
were not altered in skeletal muscle in response to polymicrobial
peritonitis (52). Collectively, these data suggest that
the abovementioned changes in translation initiation are the primary
cause for the TNF-
-induced decrease in muscle protein synthesis.
The physiological mediators of the TNF--induced decrease in
translation have not been established. The decreased muscle protein synthesis could not be ascribed to a reduction in the plasma insulin or
total amino acid concentrations in the blood. Euaminoacidemia has also
been reported in rats infused with TNF-
for only 3.5 h
(21). However, we cannot exclude the possibility that a
resistant state to insulin and/or amino acids produced by TNF-
was
at least partially responsible for the diminished rate of synthesis
(29). Elevations in plasma FFAs have been shown to be
capable of modulating protein balance in vivo (47).
However, no significant alterations in plasma FFA concentrations were
detected in TNF-
-infused rats. Our data extend previous work
indicating that the acute injection of TNF-
increases plasma leptin
concentrations (14). Because leptin is known to impair
insulin signaling, we cannot exclude the hyperleptinemia as a possible
mediator for the TNF-
-induced decrease in muscle protein synthesis.
Finally, this study confirms our earlier work by demonstrating
that TNF-
markedly decreases circulating levels of the
anabolic hormone IGF-I (18). Alterations in muscle protein
synthesis and initiation (e.g., eIF-4E availability) have been reported
to be proportional to the prevailing concentration of IGF-I (44,
45).
In summary, our data show that a relative sustained infusion of TNF-
is capable of decreasing the rate of protein synthesis in heart and
skeletal muscle. This inflammatory insult decreases both myofibrillar
and sarcoplasmic protein synthesis via a reduction in translational
efficiency. The TNF-
-induced impairment in mRNA translation appears
to result primarily from a redistribution of eIF-4E into the inactive
eIF-4E · 4E-BP1 complex, but not from a change in peptide-chain elongation.
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ACKNOWLEDGEMENTS |
---|
We thank Xiaoli Liu, Duanqing Wu, and Gina Deiter for technical assistance. We also acknowledge Drs. Leonard S. Jefferson and Scot R. Kimball for supplying some of the reagents used in this study.
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FOOTNOTES |
---|
This work was supported in part by National Institutes of Health Grants AA-11290, AA-12814, GM-38032, GM-39277, and HL-66443.
Address for reprint requests and other correspondence: C. H. Lang, Dept. of Cell Molec. Physiology (H166), Penn. State College of Medicine, 500 Univ. Dr., Hershey, PA 17033 (E-mail: clang{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.00366.2001
Received 16 August 2001; accepted in final form 18 September 2001.
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