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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

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 eIF2alpha or the amount of eIF2alpha 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 eIF2Balpha 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 gamma -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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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
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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 eIF2Balpha 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.


    METHODS AND MATERIALS
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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 alpha -subunit of eIF2 (eIF2alpha ) and the epsilon -subunit of eIF2B (eIF2Bepsilon ) 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 alpha -subunit appears important in regulating protein synthesis (36). Likewise, eIF2B is a multimeric protein consisting of five subunits, with the epsilon -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 beta -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 eIF2alpha or eIF2Bepsilon (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 eIF2alpha . The relative amount of eIF2alpha present in the phosphorylated form, designated eIF2alpha (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 beta -mercaptoethanol]. The samples were electrofocused and then transferred electrophoretically to polyvinylidene difluoride membranes. The membranes were subsequently incubated with an eIF2alpha monoclonal antibody, and eIF2alpha was visualized as described above. The proportion of eIF2alpha present in the phosphorylated state was measured by densitometric scanning of the membranes and is expressed as a percentage of the total eIF2alpha 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 beta -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 alpha -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% alpha -mercaptoethanol, 0.5% Triton X-100, 50 mM NaF, 50 mM alpha -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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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).

                              
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Table 1.   Effect of LPS on tissue weight and protein content

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.

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).

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 eIF2alpha relative to total protein in either gastrocnemius, heart, or liver from control or LPS-treated rats (Table 2).

                              
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Table 2.   Effect of LPS on the relative amount of total eIF2alpha , eIF2Bepsilon , and phosphorylated eIF4E in selected tissues

The percentage of phosphorylated eIF2alpha averaged 5 ± 2% in gastrocnemius and 6 ± 2% in heart from control rats. The injection of LPS did not produce a significant change in eIF2alpha phosphorylation in gastrocnemius or heart at either time point (data not shown). In contrast, the percentage of eIF2alpha 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) 2alpha in liver. Tissue homogenates were subjected to isoelectric focusing (IEF) slab gel electrophoresis, transferred to nitrocellulose, and visualized with a eIF2alpha -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 eIF2alpha 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).

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 eIF2Bepsilon 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).

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 alpha - and beta -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 alpha - and beta -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).

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 gamma -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 gamma -form in muscle was decreased 41% at 4 h post-LPS, and at 24 h the amount in the gamma -form was intermediate between that of values in control and 4-h LPS-treated rats (Fig. 6A). A comparable decrease (~35%) in the gamma -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 gamma -form in muscle and liver. A: gastrocnemius; B: liver. Insets: representative immunoblots in which the alpha -, beta -, and gamma -forms of 4E-BP1 are identified (left). Bars show densitometric analysis where data are expressed as the amount of 4E-BP1 in the gamma -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; black-triangle, 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
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

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 alpha -, beta -, and gamma -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 eIF2alpha 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 alpha -subunit of eIF2, which increases the affinity of eIF2 for eIF2B (44). Phosphorylation of eIF2alpha converts eIF2 from a substrate into a competitive inhibitor limiting the turnover of the eIF2 · GDP complex. Hence, the formation of the highly stable eIF2alpha (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 eIF2alpha , 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 eIF2Bepsilon , 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 eIF2Bepsilon 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 eIF2Bepsilon 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 gamma -form (26, 31, 45). Consistent with the decrease in muscle protein synthesis, LPS significantly decreased the amount of 4E-BP1 in the gamma -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 eIF2alpha in the phosphorylated form was also consistently increased. As explained above, an increase in phosphorylated eIF2alpha 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 eIF2alpha 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 gamma -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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