Differential effect of sepsis on ability of leucine and IGF-I to stimulate muscle translation initiation
Charles H. Lang and
Robert A. Frost
Departments of Cellular and Molecular Physiology and Surgery, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Submitted 22 March 2004
; accepted in final form 21 May 2004
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ABSTRACT
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Polymicrobial sepsis impairs skeletal muscle protein synthesis, which results from impairment in translation initiation under basal conditions. The purpose of the present study was to test the hypothesis that sepsis also impairs the anabolic response to amino acids, specifically leucine (Leu). Sepsis was induced by cecal ligation and puncture, and 24 h later, Leu or saline (Sal) was orally administered to septic and time-matched nonseptic rats. The gastrocnemius was removed 20 min later for assessment of protein synthesis and signaling components important in peptide-chain initiation. Oral Leu increased muscle protein synthesis in nonseptic rats. Leu was unable to increase protein synthesis in muscle from septic rats, and synthetic rates remained below those observed in nonseptic + Sal rats. In nonseptic + Leu rats, phosphorylation of eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1) in muscle was markedly increased compared with values from time-matched Sal-treated nonseptic rats. This change was associated with redistribution of eIF4E from the inactive eIF4E·4E-BP1 to the active eIF4E·eIF4G complex. In septic rats, Leu-induced phosphorylation of 4E-BP1 and changes in eIF4E distribution were completely abrogated. Sepsis also antagonized the Leu-induced increase in phosphorylation of S6 kinase 1 and ribosomal protein S6. Sepsis attenuated Leu-induced phosphorylation of mammalian target of rapamycin and eIF4G. The ability of sepsis to inhibit anabolic effects of Leu could not be attributed to differences in plasma concentrations of insulin, insulin-like growth factor I, or Leu between groups. In contrast, the ability of exogenous insulin-like growth factor I to stimulate the same signaling components pertaining to translation initiation was not impaired by sepsis. Hence, sepsis produces a relatively specific Leu resistance in skeletal muscle that impairs the ability of this amino acid to stimulate translation initiation and protein synthesis.
eukaryotic initiation factor 4E-binding protein 1; eukaryotic initiation factor 4G; mammalian target of rapamycin
AMINO ACID AVAILABILITY, especially that of leucine, represents an important nutritional signal responsible for postprandial stimulation of muscle protein synthesis (22, 52, 60). In this regard, amino acid refeeding after short-term starvation increases the protein synthetic rate in skeletal muscle (3, 35, 50). Moreover, physiologically relevant concentrations of dietary amino acids can enhance synthesis in the perfused hindlimb preparation, incubated epitrochlearis muscle, and cultured myocytes (13, 8, 10, 28, 30, 39, 50, 54). In general, the ability of a mixture of amino acids to stimulate protein synthesis appears to be largely mediated by the branched-chain amino acids in general and leucine in particular. Although the pathway by which amino acid and leucine sufficiency is detected and the signal is transduced intracellularly remains controversial (cf. 21, 36, 37, 51, 59), the anabolic effects of this amino acid are clearly mediated by cell signaling pathways that stimulate translational efficiency. The translation of mRNA proceeds via a multistep reaction catalyzed by a number of factors that regulate the processes of initiation, elongation, and termination. The control of protein synthesis by amino acids, especially leucine, occurs most notably by an increased rate of translation initiation in vivo and in vitro (13, 8, 10, 28, 30, 34, 50, 54, 60).
The protein anabolic effects of leucine are mediated by activation of intracellular signaling pathways, the elements of which are also stimulated in response to insulin and insulin-like growth factor (IGF)-I (Fig. 1). The amino acid- and mitogen-stimulated pathways appear to converge at the serine/threonine protein kinase mammalian target of rapamycin (mTOR) (17, 33, 41, 47). In the protein synthetic signal transduction pathway, mTOR represents a point of bifurcation whereby the stimulation of mTOR leads to the phosphorylation of eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) along one pathway and the phosphorylation of ribosomal protein S6 (rpS6) kinase-1 (S6K1) along a parallel pathway (16, 18). The central role of these proteins in regulating protein synthesis has been extensively reviewed (4, 22, 33, 47).

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Fig. 1. Schematic representation of pathways by which nutrition and growth factors regulate translational control of muscle protein synthesis. Linear model shows the central importance of mammalian target of rapamycin (mTOR) as a bifurcation point for stimulation of S6 kinase 1 (S6K1) and formation of the active eukaryotic initiation factor (eIF) 4F complex via phosphorylation of eIF4E-binding protein-1 (4E-BP1). Our data suggest that, in muscle, sepsis specifically impairs the ability of leucine, functioning as a nutrient signal, to stimulate protein synthesis by altering upstream mediators of mTOR activity. TOP, terminal oligopyrimidine tract; PI3K, phosphatidylinositol 3-kinase; IGF-I, insulin-like growth factor I; rp, ribosomal protein.
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Alterations in the phosphorylation state of 4E-BP1 have been observed in muscle cells in response to leucine and growth factors (insulin and IGF-I), and phosphorylation modulates the formation of the functional eIF4F complex (19, 2830, 38). During cap-dependent translation, the eIF4F complex is necessary for recognition and unwinding of the secondary structure in the 5'-untranslated regions of capped mRNA. Additionally, eIF4F promotes the migration and recruitment of the 43S preinitiation complex to the mRNA, thereby enhancing peptide-chain initiation (43). The eIF4F complex is heterotrimeric, with each subunit (e.g., eIF4E, eIF4G, and eIF4A) having a discrete function. Of these subunits, eIF4E is the least abundant in muscle and is considered to be rate limiting in the binding of mRNA to ribosomes (12, 46). eIF4E interacts with the m7GpppX cap structure found on all nuclear-encoded mRNAs to form an eIF4E·mRNA complex. During translation initiation, the eIF4E·mRNA complex binds to eIF4G (which functions as a scaffold protein) and eIF4A (which functions as an RNA helicase) to form the active eIF4F complex, thereby allowing translation to proceed. In skeletal muscle, the assembly of the functional eIF4F complex is controlled in part by 4E-BP1, which functions as a cap-dependent translational repressor. This binding protein obstructs the interaction of eIF4G with eIF4E, limiting assembly of the active eIF4F complex (12, 19, 46). Increased phosphorylation of 4E-BP1 in response to growth factors and mitogens results in the release of eIF4E, its binding to eIF4G, and stimulation of mRNA translation (43).
The proline-directed serine/threonine protein kinase S6K1 is also phosphorylated in response to a complete mixture of amino acids or leucine alone (3, 13, 30, 35). A significant portion of this leucine-induced phosphorylation of S6K1 is inhibited by rapamycin, suggesting the importance of mTOR in the phosphorylation and activation of this downstream kinase (3, 25). Hierarchical multisite phosphorylation of sites in the linker region (e.g., Thr389) leads to full and maximal activation of the kinase. Activation of S6K1 leads to the phosphorylation of rpS6, which facilitates the translation of a class of mRNAs encoding proteins containing a terminal oligopyrimidine tract (e.g., TOP RNAs) downstream of their transcription initiation site (11). Leucine rapidly phosphorylates rpS6 in skeletal muscle (28, 30) and stimulates translation initiation by enhancing the affinity of the ribosome for binding TOP-containing mRNAs.
Negative nitrogen balance and muscle wasting remain among the defining characteristics of a number of catabolic conditions, including sepsis (45). This catabolism is undoubtedly multifactorial and results from a simultaneous increase in protein degradation and decrease in the rate of protein synthesis (20, 49, 5557). The sepsis-induced decrease in muscle protein synthesis results from a proportional decrease in the synthesis of myofibrillar and sarcoplasmic proteins (57) and appears largely restricted to muscle with a predominance of fast-twitch fibers (55). A similar reduction in muscle protein synthesis has been observed in response to endotoxin and the administration of inflammatory cytokines (27, 31, 32). The sepsis-induced decrease in muscle protein synthesis is due to a defect in translational efficiency as opposed to changes in total mRNA or the number of ribosomes (5557). Although the cellular mechanism leading to this decrease is unclear, various catabolic stimuli impair peptide-chain initiation via a decrease in the phosphorylation of 4E-BP1 and/or S6K1 (29, 31, 32, 48, 56). This decrement in the basal rate of translation initiation cannot be ascribed to a decrease in the prevailing plasma concentration of leucine (49). However, the influence of sepsis on the responsiveness of skeletal muscle to exogenous leucine, with regard to its ability to stimulate phosphorylation of S6K1 and alter the formation of the active eIF4E·eIF4G complex, has not been investigated. Therefore, the purpose of the present study was to determine whether sepsis antagonizes the ability of leucine to stimulate protein synthesis and the phosphorylation of S6K1 and/or 4E-BP1 in skeletal muscle (Fig. 1). In addition, because many of the metabolic effects of leucine are mediated via mTOR, the ability of sepsis to alter phosphorylation of this kinase was also assessed. Finally, because leucine and growth factors have been reported to phosphorylate S6K1 and 4E-BP1 by different signaling pathways (35), we also examined whether sepsis altered the ability of IGF-I to activate cell signaling pathways that regulate translation initiation.
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MATERIALS AND METHODS
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Animals.
Male Sprague-Dawley rats (200225 g body wt; Charles River Breeding Laboratories, Cambridge, MA) were acclimated for 1 wk in a light-controlled room (12:12-h light-dark cycle) under constant temperature. Water and standard rat chow were provided ad libitum. All experiments were approved by the Institutional 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.
Experimental protocols.
For experiments where sepsis was induced by cecal ligation and puncture, rats were anesthetized with pentobarbital sodium (5060 mg/kg), and a midline laparotomy was performed. The cecum was ligated at its base and punctured twice with a 20-gauge needle. The cecum was then returned to the peritoneal cavity, and the muscle and skin layers were closed. Rats were resuscitated with 10 ml of 0.9% sterile saline administered subcutaneously. Nonseptic control animals were subjected to a midline laparotomy with intestinal manipulation and then resuscitated with the same volume of saline. During the operation, rats were placed on a warming pad to maintain body temperature. After surgery, food was withheld, but the animals were permitted free access to water for the reminder of the study. Hence, any observed changes between septic and nonseptic rats cannot be attributed to differences in food intake or nutritional status.
Approximately 24 h after induction of sepsis, saline (0.155 mol/l) or leucine (1.35 g/kg body wt, prepared as 54.0 g/l of L-amino acid in distilled water) was administered by oral gavage to animals in the septic and nonseptic groups (30). This dose of leucine was selected because it is equivalent to that consumed in a 24-h period when rats of this age and strain are provided free access to food (1).
On the basis of previously reported results (1), a separate group of septic and nonseptic rats was injected intravenously with IGF-I (25 nmol/kg body wt) or an equivalent volume (0.5 ml) of isotonic saline. This dose of IGF-I was selected because it produces a robust phosphorylation of the IGF-I receptor with only negligible stimulation (e.g., phosphorylation) of the insulin receptor (29).
Protein synthesis and RNA content.
The in vivo rate of protein synthesis was determined for muscle via the flooding-dose technique, as described previously (3032). After oral administration of leucine, rats were loosely restrained in a porous towel and injected with L-[2,3,4,5,6-3H]phenylalanine (150 mM, 30 µCi/ml, 1 ml/100 g body wt) administered via percutaneous puncture of a lateral tail vein 10 min after treatment with leucine. Thereafter, rats were anesthetized with pentobarbital, and an arterial blood sample was collected in a heparinized syringe from the abdominal aorta 10 min after injection of phenylalanine. The gastrocnemius was excised 20 min after administration of vehicle or leucine (or IGF-I in the second study). This time point was selected on the basis of previous studies (29) and preliminary data indicating a maximal response for the various end points determined in this study. A portion of muscle from each animal was homogenized immediately for measurement of protein factors important in the control of mRNA translation, and the remaining tissue was frozen between aluminum clamps precooled in liquid nitrogen. The frozen tissues were powdered under liquid nitrogen with a mortar and pestle. Blood was centrifuged, and plasma was collected. All tissue and plasma samples were stored at 70°C until analyzed. A portion of the powdered muscle was used to estimate the rate of incorporation of [3H]phenylalanine into protein, exactly as described previously (3032).
Changes in the number of ribosomes or in the efficiency of mRNA translation may alter tissue protein synthesis (9). Because
85% of the RNA is ribosomal RNA, changes in total RNA content reflect changes in the number of ribosomes. Total RNA content was measured from muscle homogenates, as previously described (3032). Translational efficiency was subsequently calculated by dividing the rate of protein synthesis for a particular tissue by the RNA content for that tissue.
Plasma determinations.
Blood was collected into heparinized syringes and centrifuged, and the plasma was collected for determination of insulin (Linco Research, St. Charles, MO) or total IGF-I by radioimmunoassay (31). The plasma leucine concentration was determined by derivatization with phenylisothiocyanate followed by high-performance liquid chromatography analysis (32).
Immunoprecipitation and Western blotting.
The tissue preparation was the same as that previously described by our laboratory (2932). Briefly, fresh muscle homogenates were prepared in a 1:7 ratio of ice-cold homogenization buffer (in mM: 20 HEPES, pH 7.4, 2 EGTA, 50 NaF, 100 KCl, 0.2 EDTA, 50
-glycerophosphate, 1 DTT, 0.1 PMSF, 1 benzamidine, and 0.5 sodium vanadate) with a Polytron homogenizer and clarified by centrifugation. The supernatant was aliquoted into microcentrifuge tubes, and 2x sample buffer (2 ml of 0.5 M Tris, pH 6.8, 2 ml of glycerol, 2 ml of 10% SDS, 0.2 ml of
-mercaptoethanol, 0.4 ml of a 4% solution of bromphenol blue, and 1.4 ml of water to a final volume of 8 ml) was added in a 1:1 ratio. The samples were boiled and cooled on ice before being used for Western blot analysis. The samples were subjected to electrophoresis on a 7.5% polyacrylamide gel for S6K1, a 15% polyacrylamide gel for phosphorylated S6 and 4E-BP1, and a 6% polyacrylamide gel for mTOR. Proteins were electrophoretically transferred to nitrocellulose membranes. The blots were incubated with primary antibodies to total S6K1 (catalog no. 230, Santa Cruz Biotechnology, Santa Cruz, CA), phosphospecific S6K1 (Thr421/Ser424 or Thr389; Cell Signaling Technology, Beverly, MA), total 4E-BP1 (Bethyl Laboratories, Montgomery, TX), phosphospecific 4E-BP1 (Thr37; Cell Signaling Technology), total and phosphorylated (Ser2448) mTOR (Bethyl Laboratories), total and phosphorylated (Ser1108) eIF4G (Cell Signaling Technology), or phosphorylated S6 (a generous gift of M. J. Birnbaum, University of Pennsylvania) primary antibodies. The blots were washed with TBST (1x TBS + 0.1% Tween 20) and incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit) at room temperature. The blots were developed with enhanced chemiluminescence Western blotting reagents according to the manufacturer's (Amersham) instructions. The blots were exposed to X-ray film in a cassette equipped with a DuPont Lightning Plus intensifying screen. After it was developed, the film was scanned (Microtek ScanMaker IV) and analyzed using NIH Image 1.6 software.
The 4E-BP1·eIF4E and eIF4G·eIF4E complexes were quantified as described previously (2932). Briefly, eIF4E was immunoprecipitated from aliquots of supernatants with use of an anti-eIF4E monoclonal antibody (kindly provided by L. S. Jefferson and S. R. Kimball, Pennsylvania State University College of Medicine). Antibody-antigen complexes were collected using magnetic beads as described previously (2932) and subjected to electrophoresis using a 7.5% or 15% polyacrylamide gel. Proteins were then electrophoretically transferred to a nitrocellulose membrane. The blots were incubated with a mouse anti-human eIF4E antibody, a rabbit anti-rat 4E-BP1 antibody, or a goat anti-eIF4G antibody. The phosphorylated forms of 4E-BP1 were measured after immunoprecipitation of 4E-BP1 from the tissue homogenates after centrifugation. The various phosphorylated forms of 4E-BP1 were separated by SDS-PAGE and analyzed by protein immunoblotting. The blots were then developed with enhanced chemiluminescence, and the autoradiographs were scanned for analysis as described above.
Statistical analysis.
Experimental data for each condition are summarized as means ± SE, with seven or eight animals in each treatment group. Statistical evaluation of the data was performed using ANOVA followed by Student-Newman-Keuls test to determine treatment effect. Differences between the groups were considered significant when P < 0.05.
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RESULTS
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Sepsis- and leucine-induced changes in muscle protein synthesis.
Sepsis produced by cecal ligation and puncture decreased protein synthesis in the gastrocnemius by 30% (Fig. 2). Oral leucine increased the protein synthetic rate in nonseptic rats by 35%. In contrast, leucine failed to significantly increase protein synthesis in muscle from septic rats, and as a result, the values in the two groups of septic rats were not significantly different. To determine whether a change in the number of ribosomes or the efficiency of mRNA translation was responsible for the sepsis- and leucine-induced changes in muscle protein synthesis, the RNA content of muscle was quantified. There was no sepsis- or leucine-induced change in the total RNA per muscle protein (data not shown), suggesting that the changes in muscle protein synthesis illustrated in Fig. 2 resulted from corresponding changes in the efficiency of translation and not a change in the number of ribosomes.

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Fig. 2. Effect of sepsis on leucine-induced changes in muscle protein synthesis. Protein synthesis was measured by incorporation of [3H]phenylalanine into proteins of gastrocnemius. On the day of the experiment, saline (Sal) or leucine (Leu) was administered to rats by oral gavage. Animals were anesthetized, an arterial catheter was implanted, and radiolabeled phenylalanine was injected 10 min after leucine administration. After 10 min, gastrocnemius was excised and freeze clamped. The 4 groups of rats are as follows: nonseptic + Sal (e.g., controls), nonseptic + Leu, septic + Sal, and septic + Leu. Values are means ± SE for 78 animals per group. Means with different superscripts (a, b, and c) are statistically different from each other (P < 0.05).
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Alterations in eIF4E distribution.
The mechanistic interactions between sepsis and leucine were investigated by analysis of known regulatory steps in the control of translation initiation (43, 47). In this regard, neither sepsis nor leucine significantly altered the total amount of eIF4E in the gastrocnemius (Fig. 3, middle). In contrast, sepsis increased the amount of eIF4E bound to 4E-BP1 (67%; Fig. 3, top and bottom). Because the hyperphosphorylated
-isoform of 4E-BP1 cannot bind to eIF4E, the eIF4E immunoprecipitate contains the two nonphosphorylated (
and
) isoforms of 4E-BP1, which are resolved as a doublet on Western blot analysis. In nonseptic rats, orally administered leucine markedly decreased the amount of the inactive eIF4E·4E-BP1 complex (55%). In contrast to this response, leucine administration failed to significantly alter the amount of eIF4E·4E-BP1 in muscle from septic rats. Hence, values in the septic + leucine group were not significantly different from those in the septic + saline group.

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Fig. 3. Effect of sepsis on leucine-induced changes in binding of 4E-BP1 to eIF4E in skeletal muscle. Top: eIF4E was immunoprecipitated (IP), and amount of 4E-BP1 bound to eIF4E was assessed by immunoblotting (IB). Middle: representative Western blot of total eIF4E. Bottom: densitometric analysis of immunoblots of 4E-BP1 associated with eIF4E, with value from nonseptic rats treated with saline set at 1.0 arbitrary unit (AU). Values are means ± SE; n = 78 per group. Means with different superscripts are statistically different from each other (P < 0.05).
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Sepsis markedly decreased the amount of eIF4E bound to eIF4G (80%) under basal (e.g., nonstimulated) conditions (Fig. 4). In nonseptic rats, leucine increased the amount of the active eIF4E·eIF4G complex (75%). In contrast to this response, leucine failed to alter the amount of eIF4E·eIF4G in muscle from septic rats; therefore, values in the septic + leucine group were not significantly different from those in the septic + saline group.

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Fig. 4. Effect of sepsis on leucine-stimulated association of eIF4G with eIF4E in skeletal muscle. Top: eIF4E was immunoprecipitated, and amount of eIF4G bound to eIF4E was assessed by immunoblotting. Middle: representative Western blot of total eIF4E. Bottom: densitometric analysis of immunoblots of eIF4G associated with eIF4E, with value from nonseptic rats treated with saline set at 1.0 AU. Values are means ± SE; n = 78 per group. Means with different superscripts are statistically different from each other (P < 0.05).
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The interaction between eIF4E and eIF4G can be regulated in part by the phosphorylation of eIF4G. The phosphorylation of this component of the eIF4F complex is enhanced by mitogen and serum stimulation (26, 29, 30) and inhibited by rapamycin (3, 25, 26). In muscle from the nonseptic + saline group, there was a constitutive phosphorylation of eIF4G (S1108; Fig. 5). Sepsis produced a small, albeit statistically significant, decrease of 35% in the content of phosphorylated eIF4G. The extent of eIF4G phosphorylation in muscle was increased almost twofold in leucine-treated nonseptic rats. In contrast, there was no significant leucine-induced increase in eIF4G phosphorylation in septic rats. Finally, the above-mentioned changes in eIF4G phosphorylation were not due to a leucine- or sepsis-induced change in the content of total eIF4G in muscle (Fig. 5, middle).

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Fig. 5. Effect of sepsis on leucine-stimulated phosphorylation of eIF4G. Top: representative immunoblot of Ser1108-phosphorylated (P) and total eIF4G (4G). Bottom: densitometric analysis of eIF4G phosphorylation, with value from nonseptic rats treated with saline set at 1.0 AU. Values are means ± SE; n = 78 per group. Means with different superscripts are statistically different from each other (P < 0.05).
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Phosphorylation of 4E-BP1.
To further define the mechanism through which sepsis modulates eIF4E availability, we examined the phosphorylation state of 4E-BP1. Mitogen activation and amino acids stimulate multisite phosphorylation of 4E-BP1, resulting in the dissociation of 4E-BP1 from eIF4E, and thereby permits binding of the freed eIF4E to eIF4G (15, 43, 47). The above-mentioned changes in eIF4E distribution in the septic + saline group were associated with a 53% decrease in the amount of the hyperphosphorylated
-isoform of 4E-BP1 (Fig. 6). Conversely, the extent of 4E-BP1 phosphorylation was increased
120% in nonseptic leucine-treated rats. Finally, there was little or no increase in the
-isoform of 4E-BP1 in muscle from septic rats (Fig. 6). Phosphorylation of 4E-BP1 is ordered and hierarchical, with Thr36 and Thr47 being the initial residues phosphorylated (15). The sepsis- and/or leucine-induced changes in Thr36 phosphorylation of 4E-BP1 were comparable to those described for the
-isoform of 4E-BP1 (data not shown).

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Fig. 6. Effect of sepsis on leucine-stimulated phosphorylation of 4E-BP1. Top: representative immunoblot for total 4E-BP1. Bottom: densitometric analysis of hyperphosphorylated -isoform of 4E-BP1. Value from nonseptic rats treated with saline was set at 1.0 AU. Values are means ± SE; n = 78 per group. Means with different superscripts are statistically different from each other (P < 0.05).
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S6K1 and S6 phosphorylation.
When S6K1 is subjected to SDS-PAGE, it resolves into multiple bands with different electrophoretic mobilities dependent on the extent of phosphorylation at various serine/threonine sites. In this regard, the most slowly migrating forms represent the heavily phosphorylated, and thus highly active, form of the kinase. There was constitutive S6K1 phosphorylation in muscle from rats in the nonseptic + saline group (Fig. 7A). Sepsis did not appear to appreciably change the mobility of the bands, indicating that the relative phosphorylation of S6K1 was unaltered under basal conditions. In nonseptic control rats, oral leucine decreased the mobility of the electrophoretic bands, suggesting an increased phosphorylation of the kinase. In contrast, in septic rats, the ability of leucine to increase phosphorylation was largely absent (Fig. 7A).

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Fig. 7. Effect of sepsis on leucine-stimulated phosphorylation of S6K1. A: representative immunoblot for effect of sepsis on total S6K1. B: representative immunoblot for phosphorylation of Thr389 of S6K1. C: representative immunoblot for phosphorylation of Thr421/Ser424 of S6K1. Bottom: densitometric analysis of S6K1 phosphorylation of Thr389. Value from nonseptic rats treated with saline was set at 1.0 AU. Values are means ± SE; n = 78 per group. Means with different superscripts are statistically different from each other (P < 0.05).
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Different phosphorylation sites in S6K1 were next examined using phosphospecific antibodies. In muscle from saline-treated nonseptic rats, there was essentially no constitutive phosphorylation of Thr389 (Fig. 7B and bottom). There was a marked increase in the phosphorylation of S6K1 in muscle from leucine-treated nonseptic rats. In contrast, leucine failed to stimulate Thr389 phosphorylation of S6K1 in muscle from septic rats. Leucine was also unable to stimulate the phosphorylation of S6K1 at Thr421/Ser424 (Fig. 7C).
The phosphorylation state of rpS6, a physiologically relevant S6K1 substrate, was also determined. rpS6 is a component of the 40S ribosome, and previous studies report that phosphorylation of rpS6 occurs concomitantly with increased rates of protein synthesis (11, 25). In the present study, rpS6 exhibited a constitutive level of phosphorylation in muscle from nonseptic control rats (Fig. 8). Basal phosphorylation of S6 was decreased 35% in muscle from septic rats compared with time-matched nonseptic rats. In response to leucine, S6 phosphorylation was increased fourfold in nonseptic rats (Fig. 8). In contrast, leucine failed to enhance the phosphorylation of rpS6 in muscle from septic rats.

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Fig. 8. Effect of sepsis on leucine-stimulated phosphorylation of rpS6. Top: representative immunoblot of phosphorylated rpS6 (S6). Bottom: densitometric analysis of rpS6 phosphorylation. Value from nonseptic rats treated with saline was set at 1.0 AU. Values are means ± SE; n = 78 per group. Means with different superscripts are statistically different from each other (P < 0.05).
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mTOR.
The proline-directed serine-threonine protein kinase referred to as mTOR is a common intermediate in the phosphorylation of 4E-BP1 and S6K1 induced by amino acids and growth factors (16, 18). Its activity is regulated in part by phosphorylation. There was constitutive phosphorylation of mTOR (Ser2448) in muscle from rats in the nonseptic + saline group (Fig. 9). Sepsis decreased the basal content of mTOR phosphorylation by 45%. Oral leucine administration doubled the amount of mTOR phosphorylated on Ser2448 in muscle from nonseptic rats. However, in marked contrast, there was no difference in the amount of phosphorylated mTOR between septic + leucine rats and time-matched rats in the septic + saline group. None of the treatments altered the total amount of mTOR in skeletal muscle (Fig. 9, middle).

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Fig. 9. Effect of sepsis on leucine-stimulated phosphorylation of mTOR. Top and middle: representative immunoblots of Ser2448-phosphorylated and total mTOR, respectively. Bottom: densitometric analysis of mTOR phosphorylation. Value from nonseptic rats treated with saline was set at 1.0 AU. Values are means ± SE; n = 78 per group. Means with different superscripts are statistically different from each other (P < 0.05).
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Plasma hormone and leucine concentrations.
Table 1 presents data on selected plasma hormone concentrations as well as the plasma concentration of leucine in the four experimental groups. The prevailing plasma concentration of the anabolic hormones insulin and IGF-I can affect translation initiation via alterations in eIF4E distribution and the phosphorylation of S6K1. Sepsis had no significant effect on the plasma insulin concentration compared with values from nonseptic + saline rats at the time point assessed. However, the plasma insulin concentration was increased approximately threefold in the leucine-treated animals compared with time-matched saline-treated control rats. The elevation in insulin observed in the septic + leucine group was not different from the elevation observed in the nonseptic + leucine group. At 24 h after induction of sepsis, the plasma IGF-I concentration was reduced by 31% compared with values from saline-treated nonseptic control rats. Leucine administration did not significantly change the plasma IGF-I concentration in nonseptic or septic animals. There was no difference in the plasma concentration of leucine between animals in the nonseptic + saline and septic + saline group (Table 1). As anticipated, oral leucine gavage increased the plasma leucine concentration to a similar magnitude in nonseptic and septic rats.
Responsiveness of muscle to IGF-I.
To determine whether skeletal muscle from septic rats was resistant to the actions of other anabolic agents, a separate group of rats was injected with a dose of IGF-I known to increase translation initiation and protein synthesis in muscle (6, 29). IGF-I was capable of stimulating the phosphorylation of 4E-BP1, S6K1, and mTOR in muscle from nonseptic rats (Fig. 10). Furthermore, the ability of IGF-I to enhance phosphorylation of these substrates was not impaired in muscle from septic rats. IGF-I was also able to increase the amount of the eIF4E·eIF4G complex, decrease the amount of eIF4E·4E-BP1, and increase eIF4G phosphorylation to the same extent in muscle from nonseptic and septic rats (data not shown).

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Fig. 10. Effect of IGF-I on phosphorylation of 4E-BP1 (A), S6K1 (B), and mTOR (C) in muscle from septic and nonseptic rats. Rats were injected intravenously with IGF-I, and muscle was sampled 20 min later. AC: densitometric analysis of immunoblots for each protein. Value from nonseptic rats treated with saline was set at 1.0 AU. Values are means ± SE; n = 67 per group. Means with different superscripts are statistically different from each other (P < 0.05).
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DISCUSSION
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Basal sepsis-induced changes.
Numerous forms of stress, when prolonged, lead to the erosion of lean body mass (45). Studies from our laboratory and others demonstrate that sepsis, endotoxin, and the inflammatory cytokines modulate muscle protein balance by decreasing global protein synthesis (5, 31, 32, 42, 49, 55). Moreover, this sepsis-induced reduction leads to an impaired synthetic rate in the sarcoplasmic and myofibrillar protein pools (57). Data from the present study seek to expand understanding of the molecular basis for this sepsis-induced decrease in muscle protein synthesis that is responsible in part for the muscle wasting observed in chronic catabolic conditions.
Under basal conditions (e.g., no exogenous stimulation), a hypermetabolic polymicrobial septic insult decreased the basal phosphorylation of the translational repressor molecule 4E-BP1. This change was associated with an increased amount of the inactive 4E-BP1·eIF4E complex and a reciprocal decrease in the amount of the active eIF4G·eIF4E complex. Prior studies suggest that the association of eIF4E with eIF4G may be rate limiting for protein synthesis in muscle (14, 26, 31, 32, 47, 60); therefore, these results are internally consistent with the observed decrement in muscle protein synthesis. Comparable changes in the formation of the active eIF4F complex in muscle have also been reported in response to endotoxin or TNF-
(27, 31, 32). However, these data are in contrast with those reported by Vary and Kimball (56), who reported no change in the muscle content of phosphorylated 4E-BP1, eIF4E·4E-BP1, or eIF4E·eIF4G in septic rats 5 days after implantation of a fecal-agar pellet containing Escherichia coli and Bacteroides fragilis. The reason for this apparent discrepancy is not known but may be related to the difference in the duration of the septic insult between the previous study and the present study. As previously described, these sepsis-induced changes in protein synthesis and signal transduction are strongly correlated to the reduction in IGF-I concentration in the plasma or muscle per se (31).
The present results extend these original observations and provide some novel insights into the possible mechanism for the sepsis-induced decrease in protein synthesis. Sepsis clearly impaired the basal phosphorylation of protein factors important in the regulation of peptide-chain initiation, including mTOR, eIF4G, and rpS6, and these changes were independent of a change in the total mTOR, eIF4G, or S6 content. Because phosphorylation of mTOR at Ser2448 is considered crucial in the activation of the kinase (41) and because mTOR is believed to represent a bifurcation point for the regulation of 4E-BP1 and S6K1 phosphorylation (18), these data suggest that sepsis suppresses upstream mediators of mTOR activity in muscle under basal conditions (Fig. 1). The sepsis-induced change in Ser1108 phosphorylation of eIF4G is noteworthy, because phosphorylation of serine residues at this site on the protein is associated with enhanced rates of translation and may represent a secondary mechanism regulating the interaction of eIF4E with eIF4G (40). Although eIF4G phosphorylation has been reported to be rapamycin sensitive (e.g., mTOR dependent), the kinase action of mTOR on the phosphorylation of eIF4G (Ser1108) appears to be indirect (44). Finally, sepsis also decreased the constitutive level of S6 phosphorylation. rpS6 is one of the physiological substrates for S6K1 (11). Because of the sepsis-induced decrease in S6 phosphorylation, we expected to see a concomitant reduction in the phosphorylation and activation of S6K1. However, no such reduction was observed, as evidenced by the similar banding pattern on the total S6K1 immunoblot. S6K1 is known to undergo an ordered series of phosphorylation events, only some of which can be detected by one-dimensional SDS-PAGE. Therefore, it is possible that sepsis decreased S6K1 phosphorylation but that we failed to detect such a change. Alternatively, sepsis may impair the activity of kinases other than S6K1 or stimulate phosphatases that are capable of modulating S6 phosphorylation under the basal condition. In this regard, we were unable to detect any significant change in the amount of S6K2 in skeletal muscle in response to sepsis (data not shown). Regardless of the exact mechanism, sepsis clearly decreased S6 phosphorylation in the gastrocnemius. Such a defect would be expected to impair the translation of those mRNAs containing TOP sequences at their 5' end. These messages might account for up to 15% of the total cellular mRNA (43, 47) and partially explain the decreased skeletal muscle protein synthesis produced by sepsis.
Sepsis-induced leucine resistance.
It has been previously demonstrated that a mixture of amino acids or leucine alone is sufficient to increase muscle protein synthesis in fasted naive control rats. The exogenous administration of leucine increases muscle protein synthesis under in vivo conditions (13, 14, 23, 30, 34, 35, 60), in the perfused hindlimb (23, 34), in incubated muscles (10, 34), and in cultured myocytes (28). The enhanced synthetic rate is a result primarily of the enhanced rate of initiation relative to peptide-chain elongation (7). The accelerated rate of protein synthesis in response to leucine is attributable to the concomitant increase in mTOR activity, because it can be largely inhibited by rapamycin (3). The ability of rapamycin to prevent the leucine-induced increase in protein synthesis may also be in part mediated by the diminished phosphorylation of S6K1 and 4E-BP1, the latter of which would be expected to decrease the amount of the active eIF4E·eIF4G complex (19, 43).
In nonseptic control rats, the leucine-induced stimulation of muscle protein synthesis was associated with an altered availability of eIF4E, as evidenced by the increased amount of this initiation factor bound to eIF4G and the decreased amount of eIF4E bound to the translational repressor molecule 4E-BP1. This redistribution of eIF4E was associated with the hyperphosphorylation of 4E-BP1. These leucine-induced changes in eIF4F complex assembly are consistent with results from earlier reports (13, 17, 30). The present study did not assess whether the changes in eIF4E distribution were a direct effect of leucine or occurred secondary to the concomitant increase in circulating insulin. Numerous studies indicate that an increase in the plasma insulin concentration within the physiological range is insufficient to stimulate muscle protein synthesis in fasted adult rats (14). In addition, when fasted rats are given an oral carbohydrate load, which increases the plasma insulin concentration to that seen in leucine-treated rats, muscle protein synthesis was not increased (3, 38). Furthermore, in vivo administration of amino acids and leucine does not increase the phosphorylation of phosphatidylinositol 3-kinase or Akt/PKB (35), two components of the linear signaling pathway known to be activated in response to insulin (29). However, other studies report that blocking the leucine-induced hyperinsulinemia by the infusion of somatostatin was efficacious in preventing the normally observed increase in muscle protein synthesis (2). Finally, preliminary data from our laboratory indicate that the leucine-induced hyperinsulinemia does not stimulate muscle insulin receptor phosphorylation when determined 520 min after leucine administration in control rats (unpublished observation). Therefore, there is no direct evidence that any of the leucine effect on translational control of protein is mediated via activation of the traditional insulin signaling pathway. Hence, although previous studies have demonstrated that septic rats are indeed insulin resistant (23), we do not believe this defect is related to the inability of leucine to stimulate protein synthesis and translation initiation.
The most noteworthy and provocative data presented in the present study pertain to the "leucine resistance" induced by sepsis in skeletal muscle. For example, sepsis blocked the leucine-induced formation of the functional eIF4F complex. That is, in muscle from the septic + leucine rats, there was no increase in the amount of the eIF4E·eIF4G complex, and the amount of the inactive eIF4E·4E-BP1 complex remained elevated. The inability of leucine to redistribute eIF4E in septic rats may result from the inability of this amino acid to stimulate eIF4G phosphorylation (40). Alternatively, or in addition, sepsis prevented the leucine-induced increase in 4E-BP1 phosphorylation in muscle. Furthermore, sepsis abrogated the stimulatory effect of leucine on mTOR phosphorylation, suggesting a role for mTOR in the phosphorylation of 4E-BP1.
Sepsis also prevented the leucine-induced phosphorylation of S6K1 and, thereby, blocked the expected increase in S6 phosphorylation. According to the prevailing model of activation for S6K1, the sites in the autoinhibitory domain (Ser411, Ser418, Thr421, and Ser424) are phosphorylated by an upstream kinase, the identity of which has not been resolved (16). These phosphorylation events disrupt the interaction between the carboxy- and amino-terminal domains, thereby permitting S6K1 to unfurl and expose additional sites in the linker and kinase domains. Subsequently, the Thr389 residue in the linker domain is phosphorylated, and this step has been demonstrated to be necessary for the full and functional activation of S6K1 (58). Our results indicate that sepsis prevented the leucine-induced stimulation of phosphorylation sites in the autoregulatory and linker domains (e.g., Thr421/Ser424 and Thr389, respectively). The ability of sepsis to prevent the leucine-induced changes in translation initiation could not be attributed to differences in the prevailing blood concentration of insulin, IGF-I, or leucine between rats in the nonseptic + leucine and septic + leucine groups. Therefore, the exact cellular mechanism whereby sepsis adversely influences leucine signaling remains to be defined.
In contrast to the above-mentioned sepsis-induced leucine resistance in skeletal muscle, there was no significant reduction in the ability of the anabolic hormone IGF-I to stimulate the various signaling intermediates important in the control of protein synthesis (Fig. 1). IGF-I acutely increased the phosphorylation of mTOR, 4E-BP1, and S6K1 and increased the amount of eIF4E in the active eIF4E·eIF4G complex. These effects of IGF-I are consistent with previous reports in skeletal muscle (24, 29, 53). Furthermore, several different laboratories have reported that IGF-I is capable of stimulating protein synthesis to the same magnitude in muscle from control and septic rats with peritonitis (24, 53). However, these earlier studies were performed under in vitro conditions or in the perfused hindlimb. Our results confirm these initial findings and extend them to the in vivo condition. Thus there is no evidence that sepsis induces an IGF-I resistance in skeletal muscle.
In conclusion, the results presented here demonstrate that sepsis markedly impairs the ability of leucine to stimulate muscle protein synthesis. The ability of sepsis to antagonize the leucine effect on translation initiation appears mediated via a decreased phosphorylation and activation of mTOR, eIF4G, 4E-BP1, and S6K1. As a consequence of these changes, more of the eIF4E remained in the inactive eIF4E·4EBP1 complex, and there was less phosphorylation of rpS6. Finally, the sepsis-induced decrease in nutrient signaling through leucine in muscle was not a generalized response, because IGF-I was capable of stimulating the phosphorylation of regulatory components of the protein synthetic pathway to the same extent in nonseptic and septic rats.
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GRANTS
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This work was supported in part by National Institute of General Medical Sciences Grant GM-38032.
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ACKNOWLEDGMENTS
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We thank Jen McCoy, Danuta Huber, and Nobuko Deshpande for expert technical assistance.
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FOOTNOTES
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Address for reprint requests and other correspondence: C. H. Lang, Dept. of Cellular and Molecular Physiology, H166, Penn State College of Medicine, 500 University 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.
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