Amino acid-induced stimulation of translation initiation in rat skeletal muscle

Thomas C. Vary, Leonard S. Jefferson, and Scot R. Kimball

Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Amino acids stimulate protein synthesis in skeletal muscle by accelerating translation initiation. In the two studies described herein, we examined mechanisms by which amino acids regulate translation initiation in perfused skeletal muscle hindlimb preparation of rats. In the first study, the effects of supraphysiological amino acid concentrations on eukaryotic initiation factors (eIF) 2B and 4E were compared with physiological concentrations of amino acids. Amino acid supplementation stimulated protein synthesis twofold. No changes were observed in eIF2B activity, in the amount of eIF4E associated with the eIF4E-binding protein (4E-BP1), or in the phosphorylation of 4E-BP1. The abundance of eIF4E bound to eIF4G and the extent of phosphorylation of eIF4E were increased by 800 and 20%, respectively. In the second study, we examined the effect of removing leucine on translation initiation when all other amino acids were maintained at supraphysiological concentrations. Removal of leucine from the perfusate decreased the rate of protein synthesis by 40%. The inhibition of protein synthesis was associated with a 40% decrease in eIF2B activity and an 80% fall in the abundance of eIF4E · eIF4G complex. The fall in eIF4G binding to eIF4E was associated with increased 4E-BP1 bound to eIF4E and a reduced phosphorylation of 4E-BP1. In contrast, the extent of phosphorylation of eIF4E was unaffected. We conclude that formation of the active eIF4E · eIF4G complex controls protein synthesis in skeletal muscle when the amino acid concentration is above the physiological range, whereas removal of leucine reduces protein synthesis through changes in both eIF2B and eIF4E.

leucine; eukaryotic initiation factors 4E, 4G, and 2B; 4E-binding protein 1


    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
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AMINO ACIDS play a regulatory role in modulating both protein synthesis and protein degradation in a wide range of tissues. Much evidence indicates that a major portion of the increase in skeletal muscle protein synthesis in vivo by refeeding after a brief starvation is independent of changes in anabolic hormones (insulin) and is attributable to amino acids themselves (44, 53). During in vitro perfusion or incubation of skeletal muscles, inclusion of amino acids at 5 or 10 times normal plasma concentrations stimulates protein synthesis (16, 19, 28, 33). Likewise, infusion of amino acids to achieve a relative hyperaminoacidemia stimulates protein synthesis in muscle in vivo (1, 2, 15, 30, 43-45). Besides amino acids functioning as precursors, they also appear to have a regulatory role for protein synthesis. Amino acids increase rates of protein synthesis by enhancing translation efficiency (19, 28), through stimulating peptide-chain initiation relative to elongation (28). Of all the amino acids, the branched-chain amino acids (leucine, isoleucine, and valine) have a unique role in this process. A mixture of the branched-chain amino acids can support protein synthesis in muscle as well as the full complement of amino acids (16, 19, 28, 33). Of the branched-chain amino acids, leucine appears to be the specific effector on protein synthesis and translation initiation in muscle (7, 8, 10, 28). However, the mechanisms by which amino acids stimulate translation initiation are presently unknown.

The process of peptide-chain initiation involves essentially four major steps (26, 47): 1) dissociation of the 80S ribosome into 40S and 60S ribosomal subunits; 2) formation of the 43S preinitiation complex with binding of initiator methionyl-tRNA to the 40S subunit; 3) binding of mRNA to the 43S preinitiation complex; and 4) association of the 60S ribosomal subunit to form an active 80S ribosome. Two of the steps involved in peptide-chain initiation appear important as major regulatory points in the overall control of protein synthesis in vivo. The first step controlling peptide-chain initiation is the binding of initiator methionyl-tRNA to the 40S ribosomal subunit to form the 43S preinitiation complex. This reaction is mediated by eukaryotic initiation factor 2 (eIF2) and is regulated by the activity of another initiation factor, eIF2B. The second regulatory step involves the binding of mRNA to the 43S preinitiation complex, which is mediated by eIF4F, a complex of several subunits. One of the subunits, eIF4E, binds the 7-methylguanosine 5'-triphosphate cap structure present at the 5'-end of eukaryotic mRNAs to form an eIF4E · mRNA complex (40). During translation initiation, the eIF4E · mRNA complex binds to eIF4G and eIF4A to form the active eIF4F complex (40-42). The binding of eIF4E to eIF4G is controlled, in part, by the translation repressor protein 4E-binding protein 1 (4E-BP1; PHAS-I). Binding of 4E-BP1 to eIF4E is hypothesized to limit eIF4E availability for formation of active eIF4E · eIF4G complex. The binding of 4E-BP-1 to eIF4E is regulated by phosphorylation of 4E-BP1 (for review see Ref. 13).

The effect of amino acids on each of these regulatory steps is presently being investigated. Our knowledge of the mechanisms by which amino acids modulate components of the translation apparatus has been garnered from studies with various cell culture systems. In either RINm5F or Fao hepatoma cells in culture, increasing amino acid concentrations above those normally contained in MEM culture medium increase phosphorylation of 4E-BP1 (38, 52). Furthermore, this response could be mimicked by addition of 10 mM branched-chain amino acids or leucine (38, 52). On the other hand, incubation of Chinese hamster ovary cells or L6 myoblasts with medium lacking individual amino acids results in dephosphorylation of 4E-BP1 and eIF4E, which is rapidly reversed by resupplying the deprived amino acids (18, 21, 51). There is very little information available concerning whether amino acids regulate protein synthesis by a similar mechanism in intact organs.

The present set of investigations was designed to examine the mechanisms by which amino acid availability regulates translation initiation in perfused skeletal muscle. Specifically, the effects of amino acids on initiation factors controlling formation of 43S preinitiation complex and mRNA binding to the ribosome were investigated using perfused hindlimb preparations. To further define the ability of amino acids to regulate translation initiation, the effect of removal of leucine from supraphysiological concentrations of amino acids on eIF2B and eIF4E was examined. The use of the perfused hindlimb allowed direct examination of the effect of amino acids on the regulation of translation initiation in skeletal muscle independent of alterations in hormones and/or metabolites that occur in vivo after infusion of amino acids. The results indicate that increasing amino acids in the perfusate to supraphysiological concentrations (10× plasma levels) stimulated protein synthesis through enhanced binding of eIF4E to eIF4G but not through increased activity of eIF2B. In contrast, deletion of leucine from the perfusate lowered rates of protein synthesis by decreasing both eIF2B activity and the amount of eIF4E bound to eIF4G. The reduced binding of eIF4E to eIF4G was associated with a reciprocal increase in the amount of eIF4E associated with 4E-BP1 and a corresponding decrease in the extent of phosphorylation of 4E-BP1.


    MATERIALS AND METHODS
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Experimental protocols. Adult male Sprague-Dawley rats weighing 150-225 g were maintained on a 12:12-h light-dark cycle and were fed ad libitum. All experiments were performed with the isolated perfused hindlimb preparation as described in Hindlimb perfusions. Food was not withdrawn before perfusion of the hindlimb. All perfusions were initiated between 10 AM and 12 PM. In the first set of experiments, we examined the effect of increasing perfusate amino acids from normal plasma concentrations (1×) to 10× those concentrations. Previous studies by our laboratories indicated that protein synthesis and translational efficiency were significantly augmented after perfusion with buffer containing amino acids at 10× their normal plasma concentration (19, 28). In the second set of experiments, we investigated the effect of removal of one amino acid, leucine, with other amino acids maintained 10× the normal plasma concentrations. Leucine was chosen because a mixture of the branched-chain amino acids can support protein synthesis in muscle as well as the full complement of amino acids (16, 19, 28, 33). Of the branched-chain amino acids, leucine appears to be the specific effector branched-chain amino acid on protein synthesis in muscle (7, 8, 10, 28).

Hindlimb perfusions. Hindlimb perfusions were carried out according to the method described by Bylund-Fellenius et al. (9) as modified in Ref. 19. Rats were anesthetized with pentobarbital sodium (50 mg/kg body wt), and the skin covering the right and left hindlimbs was removed. A mid-line incision was made, and both the inferior vena cava and the abdominal aorta were exposed. The abdominal aorta was cannulated, and the perfusate was immediately delivered via the abdominal aorta at a rate of 0.32 ml · min-1 · g-1 (9) to the hindlimb musculature. The inferior vena cava was then cannulated. The first 50 ml of perfusate passing through the hindlimb were discarded. The inferior vena cava cannula was then connected to the perfusion system, and recirculation of the perfusate was begun. After perfusion for an additional 5 min, L-[3H]phenylalanine was introduced into the perfusate to 2 µCi/ml and perfusion was continued for 60 min. After perfusion, gastrocnemius muscles were dissected from the soleus and plantaris and were excised and rapidly (within 2 s of excision) frozen between aluminum blocks precooled to the temperature of liquid nitrogen or used directly for analysis of eIFs. A sample of perfusate was centrifuged, and the plasma was decanted. The plasma samples were stored at -20°C until analyzed for phenylalanine specific radioactivity as described previously (19).

The perfusate (250 ml/hindlimb) consisted of a modified Krebs-Henseleit bicarbonate buffer containing 30% (vol/vol) washed bovine erythrocytes, 4.5% (wt/vol) BSA (BSA fraction V), 11 mM glucose, 1 mM phenylalanine, and all other amino acids at normal rat plasma concentration as previously described (19). In one set of experiments, amino acids were raised to 10× their plasma concentrations (19). In another set of experiments, leucine was deleted (0 mM) from the perfusate, whereas all other amino acids were maintained at 10× concentrations. The medium was maintained at 37°C and gassed with humidified 95% O2-5% CO2.

Measurement of synthesis of total mixed proteins. Rates of protein synthesis were estimated by the incorporation of [3H]phenylalanine into muscle proteins (19, 25). Frozen tissue was powdered under liquid nitrogen. A portion (0.5 g) was homogenized in 5 vol of ice-cold 10% (wt/vol) TCA and centrifuged at 10,000 g for 11 min at 4°C. The supernatant was decanted, and the pellet was washed five times with 10% TCA to remove any acid-soluble radioactivity. After being washed with acetone and then with water, the pellet was dissolved in 0.1 N NaOH. Aliquots were assayed for protein by use of the Biuret method with crystalline BSA as a standard. Another aliquot was assayed for radioactivity by liquid scintillation spectrometry with corrections for quenching (disintegrations/min).

The rate of protein synthesis, expressed as nanomoles of phenylalanine incorporated into protein per hour per gram of muscle, was determined by dividing the disintegrations per hour incorporated into proteins by the perfusate phenylalanine specific radioactivity. The perfusate phenylalanine concentrations averaged 1,237 ± 108 µM in experiments with 1× amino acids, 1,429 ± 163 µM in experiments with 10× amino acids, and 1,299 ± 97 µM in experiments with 10× amino acids minus leucine. At perfusate concentrations above 800 µM, the specific radioactivity of tRNA bound phenylalanine is the same as that of the extracellular and intracellular free phenylalanine (9). The intracellular free phenylalanine concentration was calculated to be 1,123 ± 218 µM in gastrocnemius perfused with 1× amino acids, 1,486 ± 207 µM in gastrocnemius perfused with 10× amino acids, and 1,344 ± 69 µM in gastrocnemius perfused with 10× amino acids minus leucine, indicating equilibration of the perfusate and intracellular phenylalanine concentrations. Therefore, the specific radioactivity of perfusate phenylalanine provides an accurate estimate of the specific radioactivity of tRNA bound phenylalanine.

Measurement of synthesis of myofibrillar and nonmyofibrillar (sarcoplasmic) proteins. Another portion (1 g) of the frozen muscle was used to separate the myofibrillar and sarcoplasmic proteins according to the procedures described previously (50). Muscle samples were homogenized in 7.5 ml of ice-cold buffer consisting of 10 mM KH2PO4 (pH 7.4) with a motor- driven glass-on-glass homogenizer. The samples were centrifuged at 3,000 g at 4°C for 20 min. The pellet contains the myofibrillar proteins, and the supernatant fraction contains the sarcoplasmic proteins. The pellet containing the myofibrillar proteins was washed twice with 2 ml of buffer containing 0.1 mM KH2PO4 (pH 7.4). The supernatant fractions from the washes were pooled and added to the nonmyofibrillar fraction. The myofibrillar pellet was dissolved in 2 ml of 0.6 M NaCl and centrifuged for 30 s at 500 g at 4°C. The supernatant was saved for measurement of radioactivity in myofibrillar proteins, and the pellet was discarded. Both the myofibrillar and sarcoplasmic protein fractions were treated with an equal volume of 10% (wt/vol.) TCA. The supernatants were discarded, and the precipitated protein fractions were processed as described in Measurement of synthesis of total mixed proteins for measurement of incorporation of radioactivity in total protein.

Total RNA content and measurement of translational efficiency. Total RNA was measured from muscle homogenized in 5 vol of ice-cold 10% TCA. The homogenates were centrifuged at 10,000 g for 11 min at 4°C. The supernatants were discarded, and the remaining pellets were suspended in 2.5 ml of 6% (wt/vol) perchloric acid (PCA). The samples were centrifuged at 10,000 g for 6 min at 4°C, the supernatants were discarded, and the procedure was repeated. Then, 1.5 ml of 0.3 N KOH was added to the pellet, and the samples were placed in a 50°C water bath for 1 h. Samples were then mixed with 5 ml of 4 N PCA and centrifuged at 10,000 g for 11 min. The concentration of RNA was determined as previously described (19, 48, 49). Total RNA was expressed as milligrams of RNA per gram tissue. Translational efficiency was calculated by dividing the rate of protein synthesis by the total tissue RNA content (9, 19, 48, 49).

eIF2B activity. eIF2B activity in supernatants prepared from perfused muscles was measured by the GDP-exchange assay as previously described (20, 23). eIF2B activity was measured as the decrease in eIF2 · [3H]GDP complex bound to nitrocellulose filters after incubation with unlabeled GTP (20, 23). The rate of exchange of GTP for [3H]GDP was linear over the time points examined. Under the experimental conditions utilized, ~50% (0.3 pmol) of the [3H]GDP is released from the eIF2 · [3H]GDP complex during the 3-min assay period. eIF2B activity is expressed as picomoles of GDP exchanged per minute per milligram of protein.

Quantification of 4E-BP1 · eIF4E and eIF4G · eIF4E complexes. The association of eIF4E with 4E-BP1 and eIF4G was determined as previously described in our laboratory (25, 43). Gastrocnemius muscles were rapidly removed, immediately weighed, and homogenized in 7 volumes of buffer A (20 mM HEPES, pH 7.4, 100 mM KCl, 0.2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 50 mM NaF, 50 mM beta -glycerolphosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.5 mM sodium vanadate, and 1 µM microcystin LR) with a Polytron homogenizer. The homogenate was centrifuged at 10,000 g for 10 min at 4°C, and the pellet was discarded. eIF4E and 4E-BP1 · eIF4E and eIF4G · eIF4E complexes were immunoprecipitated from aliquots of 10,000 g supernatants with an anti-eIF4E monoclonal antibody. The antibody · antigen complex was collected by incubation for 1 h with goat anti-mouse Biomag IgG beads (PerSeptive Diagnostics). Before use, the beads were washed in 1% nonfat dry milk in buffer B (50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% beta -mercaptoethanol, 0.5% Triton X-100, 50 mM NaF, 50 mM beta -glycerolphosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 0.5 mM sodium vanadate). The beads were captured with a magnetic stand and washed twice with buffer B and once 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 collected by centrifugation, and the 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 a polyvinylidene difluoride (Immobilon P) membrane as previously described (25, 43). 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 with an enhanced chemiluminescence Western blotting kit as per the instructions of the manufacturer (Amersham). Films were scanned with a Microtek ScanMaker III scanner equipped with a transparent media adapter connected to a Macintosh computer. Images were obtained with the ScanWizard Plugin (Microtek) for Adobe Photoshop and quantitated with National Institutes of Health Image 1.60 software.

Determination of the phosphorylation state of eIF4E. Phosphorylated and unphosphorylated forms of eIF4E in extracts of hindlimb muscles were separated by isoelectric fousing on a slab gel and quantitated by protein immunoblot analysis, as previously described (25, 43).

Determination of phosphorylation state of 4E-BP1. The various phosphorylated forms of 4E-BP1 were measured after immunoprecipitation of 4E-BP1 from muscle homogenates after centrifugation at 10,000 g (25, 43). 4E-BP1 was immunoprecipitated from the supernatants as described in Quantification of 4E-BP1 · eIF4E and eIF46 · eIF4E complexes for immunoprecipitation of eIF4E. The immunoprecipitates were solubilized with SDS sample buffer. The various phosphorylated forms of 4E-BP1 were separated by electrophoresis and quantitated by protein immunoblot analysis as described previously (22, 24, 25).

Determination of phosphorylation state of p70 S6 kinase. Gastrocnemius was homogenized in 7 vol of ice-cold extraction buffer A with a Polytron PT10. The homogenate was centrifuged at 10,000 g for 10 min at 4°C, and the pellet was discarded. The supernatant was aliquoted and quickly frozen to the temperature of liquid nitrogen. Frozen aliquots were thawed, mixed with 2× Laemmli SDS sample buffer, and subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Proteins were then electrophoretically transferred onto polyvinylidene difluoride (Immobilon P) membranes and blocked with Tris-buffered saline containing 5% (wt/vol) nonfat powdered dry milk. Membranes were incubated with a polyclonal antibody that recognizes p70 S6 kinase (Santa Cruz Biotechnology, Santa Cruz, CA). The blots were developed with an enhanced chemiluminescence Western blotting kit according to the instructions of the manufacturer (Amersham). After development, the radiographic films were subjected to densitometric scanning as described in Quantification of 4E-BP1 · eIF4E and eIF4G · eIF4E complexes.

Statistical analysis. Values shown are means ± SE. Statistical evaluation of the data was performed with ANOVA to test for overall differences among groups, followed by the Student-Newman-Keuls test for multiple comparisons to determine significance between means when ANOVA indicated a significant difference among the group means. Differences among the means were considered significant when P < 0.05.


    RESULTS
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Effect of amino acids on skeletal muscle protein synthesis. Elevating perfusate amino acids to 10× plasma concentrations augmented the synthesis of total mixed proteins in gastrocnemius muscle compared with values obtained with 1× amino acids (Fig. 1A). The contribution of the myofibrillar and sarcoplasmic proteins to the rate of total mixed protein synthesis was investigated (Table 1). In muscles perfused with 1× amino acids, the rate of synthesis of sarcoplasmic proteins was faster than of myofibrillar proteins. The rate of synthesis of both myofibrillar and sarcoplasmic proteins was significantly increased during perfusion with 10× amino acids (Table 1).


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Fig. 1.   Effect of amino acid supplementation and leucine deprivation from 10× amino acid supplementation on protein synthesis in gastrocnemius. Hindlimbs of rats were perfused as described in MATERIALS AND METHODS in the presence of amino acids at 1 or 10× the concentrations found in normal rat plasma. In another group (10× - Leu), leucine was omitted from buffer supplemented with 10× amino acids. Rates of protein synthesis (A) were estimated in hindlimb preparations perfused in vitro by measuring incorporation of [3H]Phe into mixed proteins of gastrocnemius as described in MATERIALS AND METHODS. Translational efficiency (B) was calculated by dividing rate of protein synthesis by RNA content. Results are means ± SE for 10-15 animals in each group. A: ANOVA, F = 13.72, P < 0.001; * P < 0.001 vs. 1 or 10× - Leu. B: ANOVA, F = 10.47, P < 0.001; * P < 0.01 vs. 1 or 10× - Leu.


                              
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Table 1.   Synthesis of myofibrillar and sarcoplasmic proteins: effects of amino acids

In liver, cell swelling associated with augmented influx of amino acids is thought to promote anabolism (4, 31). Therefore, we measured the total tissue water in gastrocnemius after perfusion with buffer containing 1 or 10× plasma concentrations of amino acids to determine whether or not cell water increased during perfusion with 10× amino acids. Increasing amino acid availability to 10× plasma concentrations did not significantly increase the total tissue water content (0.827 ± 0.008 ml/g at 1× amino acids vs. 0.827 ± 0.008 ml/g at 10× amino acids). Hence, the rise in protein synthesis is probably not related to changes in tissue water.

To investigate other mechanisms responsible for stimulation of protein synthesis, the total tissue RNA and translational efficiency were measured. Increasing amino acid availability to 10× plasma concentrations did not significantly alter total RNA content (data not shown) but did enhance translational efficiency approximately twofold (Fig. 1B).

To investigate potential causes of the increased translational efficiency, the effect of amino acid supplementation on eIF2B activity was examined. No significant differences in eIF2B activity were observed between muscles perfused with 1 or 10× amino acids (Fig. 2).


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Fig. 2.   Effect of amino acid supplementation and leucine deprivation from 10× amino acid supplementation on eukaryotic initiation factor (eIF) 2B activity. Extracts of gastrocnemius, obtained from muscles perfused as described in Fig. 1, were assayed for eIF2B activity as described in MATERIALS AND METHODS. Results are means ± SE for 5-6 animals in each group (ANOVA, F = 5.87, P < 0.05; * P < 0.05 vs. 1× or 10× + Leu).

Because amino acid supplementation did not enhance eIF2B activity, we examined the effect of amino acids on eIF4E and the translation repressor 4E-BP1. Initially, we investigated whether or not changes in the phosphorylation of 4E-BP1 could account for stimulation of protein synthesis by amino acids in perfused skeletal muscle (Fig. 3A). Under a variety of conditions, 4E-BP1 undergoes multiple phosphorylations, which are characterized by reduced mobility after electrophoresis. 4E-BP1 can be resolved into multiple electrophoretic bands termed the alpha -, beta -, and gamma -forms, representing differentially phosphorylated forms of the protein (29, 39). The most highly phosphorylated form of the protein, the gamma -form, does not bind eIF4E (29, 39). In the present study, raising the perfusate amino acid concentration from 1 to 10× did not affect the abundance of 4E-BP1 in the gamma -form (Fig. 3A).


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Fig. 3.   Effect of amino acid supplementation and leucine deprivation from 10× amino acid supplementation on phosphorylation of 4E-binding protein (4E-BP1) and eIF4E, amount of 4E-BP1 bound to eIF4E, and amount of eIF4G bound to eIF4E in gastrocnemius. A: phosphorylation of 4E-BP1 in extracts of gastrocnemius obtained from perfusions described in Fig. 1 was determined by protein immunoblot analysis after SDS-PAGE. Amount of 4E-BP1 in gamma -phosphorylated form is expressed as a percentage of the sum of phosphorylated and nonphosphorylated form (ANOVA, F = 5.12 P < 0.015; * P < 0.05 vs. 1 or 10× + Leu). To determine amount of 4E-BP1 (B) or eIF4G (C) bound to eIF4E, eIF4E was immunoprecipitated from 10,000 g supernatants of hindlimb muscle extracts by use of an anti-eIF4E monoclonal antibody. Immunoprecipitates were subsequently subjected to protein immunoblot analysis with either anti-4E-BP1 or anti-eIF4G antibodies (B: ANOVA, F = 45.0 P < 0.0001, ** P < 0.01 vs. 1 or 10× - Leu) (C: ANOVA, F = 8.99 P < 0.005; § P < 0.01 vs. 1 or 10× - Leu). D: amount of phosphorylated and nonphosphorylated eIF4E in extracts of gastrocnemius was determined by protein immunoblot analysis after slab gel isoelectric focusing electrophoresis. Amount of eIF4E in phosphorylated form is expressed as the percentage of the sum of phosphorylated and nonphosphorylated forms. Results are means ± SE for 5-10 animals in each group.

To investigate the effect of amino acids on the association of 4E-BP1 with eIF4E, eIF4E immunoprecipitates were analyzed for 4E-BP1 content (Fig. 3B). Raising the perfusate amino acid from 1× to 10× had no significant effect on the amount of 4E-BP1 associated with eIF4E. Thus altered binding of eIF4E to 4E-BP1 does not appear responsible for stimulation of translation initiation by supraphysiological concentrations of amino acids.

In a similar manner, eIF4E immunoprecipitates were also used to measure association of eIF4E with eIF4G (Fig. 3C). Elevating the amino acid concentration from 1× to 10× caused an eightfold increase in the amount of eIF4G that was immunoprecipitated with eIF4E. This observation was not the result of an increased amount of eIF4E in the immunoprecipitates (data not shown). This finding indicates that amino acids stimulate translation efficiency, and hence protein synthesis, in skeletal muscle, in part by promoting the formation of the active eIF4E · eIF4G complex.

To further define potential mechanisms responsible for the amino acid-induced acceleration of protein synthesis, the effect of increasing the amino acid concentration from 1× to 10× on phosphorylation of eIF4E was examined (Fig. 3D). In these studies, slab gel isoelectric focusing was used to separate phosphorylated and nonphosphorylated forms of eIF4E followed by immunoblot analysis to quantitate the amount of eIF4E in the two forms. In muscles perfused at 1× amino acids, ~86% of the eIF4E is in its phosphorylated form. Inclusion of amino acids at 10× plasma concentrations did not significantly affect the proportion of eIF4E in the phosphorylated form. Thus the ability of amino acid supplementation to stimulate translation efficiency in skeletal muscle probably does not involve increasing phosphorylation of eIF4E.

We examined the extent of phosphorylation of p70 S6 kinase to determine whether this was an important regulatory mechanism in skeletal muscle after amino acid supplementation. p70 S6 kinase is activated by multisite phosphorylation that results in phosphorylated forms exhibiting retarded electrophoretic mobility when subjected to SDS-PAGE (11, 27). We used this property (assessed by immunoblotting techniques) as an indicator of the effect of amino acids on the activation of the kinase (Fig. 4). Amino acid supplementation did not appreciably increase the prominence of the electrophoretically retarded bands compared with homogenates obtained from muscles perfused with 1× amino acid concentrations. The percentage of p70 S6 kinase in either the least phosphorylated form (50 ± 4 vs. 56 ± 4%) or most phosphorylated form (9 ± 1 vs. 10 ± 1%) was not significantly different in muscles perfused with 10× amino acids compared with muscles perfused with 1× amino acids. Thus the phosphorylation state of p70 S6 kinase was not altered in skeletal muscle after perfusion with elevated amino acid concentrations.


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Fig. 4.   Effect of amino acid supplementation and leucine deprivation from 10× amino acid supplementation on p70 S6 kinase phosphorylation. A: homogenates were obtained from muscles of 1st set of experiments perfused with medium containing either 1 or 10× amino acid concentrations. B: homogenates were obtained from muscles of 2nd set of experiments perfused with medium containing either 10× amino acid concentrations with (+) or without (-) leucine added. All homogenates were subjected to SDS-PAGE, and proteins were transferred electrophoretically to polyvinylidene difluoride membranes. Membranes were exposed to a polyclonal antibody that specifically recognizes p70 S6 kinase, and antibodies were visualized with a chemiluminesence detection system. Arrows indicate gel retardation of p70 S6 kinase induced by amino acids. Immunoblots are representative of 7 perfusions with 1× amino acids and 5 perfusions with 10× amino acid for A and 4 perfusions with 10× amino acids + Leu and 5 perfusions of 10× amino acids - Leu for B.

Effect of omission of leucine on protein synthesis in skeletal muscle at supraphysiological perfusate concentrations of all other amino acids. Studies in perfused skeletal muscle (28), incubated diaphragm (7, 8, 16, 46), and perfused heart (10, 46) have provided evidence that leucine can substitute for a complete mixture of amino acids in stimulating protein synthesis. In the present series of experiments, we examined the effect of removing leucine from the 10× mixture of amino acids on protein synthesis, translational efficiency, eIF2B activity, and regulation of eIF4E in perfused gastrocnemius. When leucine was removed from the perfusate, rates of protein synthesis declined by ~40%, despite all other amino acids being present at supraphysiological concentrations (Fig. 1A). Removal of leucine from the perfusate lowered the intracellular leucine concentration compared with values obtained in muscles perfused with either 1× amino acids (79 ± 3 vs. 173 ± 5 µM, respectively, P < 0.001) or 10× amino acids plus leucine (79 ± 3 vs. 409 ± 122 µM, respectively, P < 0.05).

The effect of omission of leucine from the perfusate on the synthesis of myofibrillar and sarcoplasmic proteins was also determined (Table 1). The rate of synthesis of both myofibrillar and sarcoplasmic proteins was significantly diminished during perfusion with 10× amino acids without leucine (Table 1). Thus diminished leucine availability limited the synthesis of myofibrillar and sarcoplasmic proteins.

Removal of leucine from the perfusate did not alter total RNA content but did inhibit translational efficiency ~50% (Fig. 1B). To understand the mechanisms responsible for the inhibition of translational efficiency caused by omission of leucine from the perfusate, we examined eIF2B and eIF4E. Removing leucine from the perfusate was associated with a significant 40% reduction in eIF2B activity (Fig. 2). Thus diminished eIF2B activity may be responsible, in part, for the decreased rate of protein synthesis after perfusion with buffer lacking leucine.

To further define the effect of omission of leucine from the perfusate on the regulation of protein synthesis, components of the eIF4E system were examined. The effect of removal of leucine on 4E-BP1 phosphorylation is shown in Fig. 3A. The proportion of 4E-BP1 in the gamma -form was reduced 43% compared with perfusion with leucine. Previous reports have suggested that a decreased phosphorylation of 4E-BP1 correlates with increased association of eIF4E with 4E-BP1 (24, 25, 29). Removal of leucine from the medium caused a 3.6-fold increase in the amount of 4E-BP1 that was immunoprecipitated with eIF4E, indicative of an increase in the proportion of eIF4E in the inactive 4E-BP1 · eIF4E complex (Fig. 3B).

The amount of eIF4G bound to eIF4E was measured in the same immunoprecipitates used to quantitate 4E-BP1 bound to eIF4E (Fig. 3C). Removal of leucine from the medium reduced the amount of eIF4G that was immunoprecipitated with eIF4E by 80% compared with values obtained with leucine in the perfusate, indicative of a decreased formation of the eIF4G · eIF4E complex. To further investigate effects of omission of leucine from the perfusate, the extent of phosphorylation of eIF4E was examined (Fig. 3D). There was no significant change in eIF4E phosphorylation when leucine was removed from the perfusate.

We also examined the extent of phosphorylation of p70 S6 kinase in gastrocnemius after leucine deprivation. Omission of leucine from the perfusate reduced the prominence of the more electrophoretically retarded bands, indicating a net decrease in the phosphorylation state of the protein (Fig. 4). The percentage of p70 S6 kinase in the least phosphorylated form increased from 62 ± 2% in muscles perfused with 10× amino acids to 78 ± 2% in muscles perfused with 10× amino acids minus leucine (P < 0.005). Conversely, the percentage of p70 S6 kinase in the most phosphorylated form decreased from 10 ± 1% in muscles perfused with 10× amino acids plus leucine to 2 ± 0.5% in muscles perfused with 10× amino acids minus leucine (P < 0.001).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, addition of amino acids to the perfusate enhanced the synthesis of mixed proteins in skeletal muscle. In addition, we also demonstrated that amino acid supplementation stimulates the synthesis of both myofibrillar and sarcoplasmic proteins. Because myofibrillar proteins make up the bulk of protein in skeletal muscle, this observation suggests that amino acids increase the synthesis of these proteins to a greater extent than sarcoplasmic proteins. An accelerated rate of protein synthesis, secondary to a stimulation of translation initiation, is a consistent response to an increased supply of amino acids in skeletal muscle (19, 28, 33). However, the mechanisms responsible for the accelerated translation initiation remain unknown. In the present study, we examined the ability of amino acids to modulate eIF2B activity and binding of eIF4E to eIF4G, two crucial steps controlling translation initiation in skeletal muscle. eIF2B activity was unaffected when the amino acid concentration was elevated from 1× to 10×, suggesting that eIF2B is not involved in the stimulation of protein synthesis under these conditions. Instead, amino acid supplementation increased the amount of eIF4E bound to eIF4G.

During translation initiation, mRNA binds either directly to eIF4E already associated with 40S ribosomes or to free eIF4E with subsequent binding of the mRNA · eIF4E · eIF4G complex to the ribosome (40, 42). With either scenario, the increased amount of eIF4E associated with eIF4G after amino acid supplementation would augment the association of mRNA with the ribosome. Thus supraphysiological concentrations of amino acids may stimulate skeletal muscle protein synthesis by improving the ability of eIF4E to bind mRNA to ribosomes through an increased abundance of the active eIF4E · eIF4G complex.

Phosphorylation of eIF4E enhances the affinity of the factor for 7-methylguanosine 5'-triphosphate cap analogs of mRNA (32) and for eIF4G and eIF4A (35). These effects may contribute to activation of mRNA translation with supraphysiological concentrations of amino acids. In this regard, increased phosphorylation of eIF4E correlates with enhanced rates of protein synthesis in cells in culture stimulated with mitogens, growth factors, or serum (36, 37, 40) or transformed with ras or src oncogenes (12, 42). However, increased phosphorylation of eIF4E does not always correlate with enhanced rates of protein synthesis (25, 41). In the present study, no significant increase in the phosphorylation of eIF4E was observed after inclusion of amino acids at 10× the plasma concentration. Therefore, it is unlikely that the stimulation of protein synthesis by amino acids occurred through increased phosphorylation of eIF4E.

A second mechanism for modulating eIF4E involves its relative distribution between inactive and active complexes with other proteins. eIF4E binds to small, acid- and heat-labile proteins, termed 4E-BP1 (PHAS-I), 4E-BP2 (PHAS-II), and 4E-BP3 to form an inactive complex (29, 39). In rat skeletal muscle, the predominant form is 4E-BP1. Unphosphorylated 4E-BP1 binds to eIF4E to form an inactive 4E-BP1 · eIF4E complex. When eIF4E is bound to 4E-BP1, eIF4E binds to mRNA but cannot form an active eIF4E · eIF4G complex (17). Thus formation of 4E-BP1 · eIF4E complex prevents binding of mRNA to the ribosome. 4E-BP1 binding to eIF4E essentially limits cap-dependent mRNA translation by physically sequestering eIF4E into an inactive complex. We previously reported that both diabetes and starvation increase the amount of eIF4E found in the inactive 4E-BP1 · eIF4E complex in skeletal muscle, with a concomitant decrease in the association of eIF4E with eIF4G (24, 25). Augmenting amino acid availability did not alter the abundance of eIF4E associated with the translation repressor 4E-BP1.

The interaction between 4E-BP1 and eIF4E is regulated by the extent of 4E-BP1 phosphorylation. Phosphorylation of 4E-BP1 releases eIF4E from the 4E-BP1 · eIF4E complex and allows the eIF4E · mRNA complex to bind to eIF4G and then to the 40S ribosome (29). Refeeding of starved rats or insulin treatment of diabetic rats increases the phosphorylation of 4E-BP1, causing a dissociation of the 4E-BP1 · eIF4E complex, thereby promoting translation initiation (24, 25, 43). Presumably, the release of eIF4E from the 4E-BP1 · eIF4E complex secondary to increased phosphorylation of 4E-BP1 allows eIF4E to bind to eIF4G and form the active eIF4E · eIF4G complex. In fact, stimulation of protein synthesis in response to acute administration of insulin is associated with a 12-fold increase in the amount of eIF4G bound to eIF4E in perfused skeletal muscle (25). In the present set of experiments, elevating the perfusate amino acids beyond 1× did not influence the phosphorylation of 4E-BP1. In contrast to previous reports concerning the regulation of eIF4E by hormones (24, 25, 29) or amino acids (51), no reciprocal relationship between eIF4E found in the inactive 4E-BP1 · eIF4E complex and the active eIF4G · eIF4E complex was observed. However, at least two reports (14, 34) indicate that the association of eIF4E with eIF4G can be modulated independent of eIF4E binding to 4E-BP1. In both National Institutes of Health 3T3 cells (34) and Xenopus kidney cells (14) in culture, serum promotes protein synthesis and increased association of eIF4E with eIF4G without any alteration in the amount of eIF4E bound to 4E-BP1. Indeed, rapamycin or wortmannin did not prevent the insulin-stimulated formation of eIF4G · eIF4E complex, whereas the decrease in 4E-BP1 bound to eIF4E was reduced by these compounds (22). Hence, the binding of eIF4E to eIF4G can be modulated by mechanisms distinct from 4E-BP1. Moreover, several studies have shown a poor temporal correlation between diminished phosphorylation of 4E-BP1, the inhibition of general translation initiation, and eIF4E binding to eIF4G (3, 14, 21). Thus amino acids appear to augment protein synthesis without reducing the abundance of 4E-BP1 · eIF4E complex. Furthermore, the findings indicate that formation of the active 4E-BP1 · eIF4E complex is more complicated than simply a release of eIF4E from the inactive 4E-BP1 · eIF4E complex.

Another potential mechanism to account for a modulation of translation initiation by amino acids is through regulation of p70 S6 kinase (11, 25). Phosphorylation of ribosomal S6 protein enhances translation of mRNA into protein (for review see Ref. 11). Ribosomal S6 protein is uniquely positioned to regulate translation by its location at the tRNA binding site on the 40S ribosome. Ribosomal S6 protein is phosphorylated by a family of 70-kDa protein kinases referred to as p70 S6 kinase (11). Phosphorylation of ribosomal S6 protein results in a modest increase in the overall rate of protein synthesis. The p70 S6 kinase is, in turn, activated by phosphorylation catalyzed by the FK506 rapamycin associated protein (FRAP)/mammalian target of rapamycin (mTOR) signaling pathway (5). Linkage of FRAP/mTOR pathway with changes in phosphorylation of p70 S6 kinase would define potential downstream regulators of responses to amino acid supplementation. We anticipated that the phosphorylation state would be shifted to the slower migrating species by amino acids if phosphorylation of p70 S6 kinase were important in regulating protein synthesis. However, no significant differences were observed in the prominence of the phosphorylated bands of the p70 S6 kinase between muscles perfused with medium containing 1× or 10× amino acids. These results suggest that p70 S6 kinase does not play a significant role in the amino acid-induced stimulation of synthesis of mixed skeletal muscle proteins.

Numerous studies have reported that stimulation of protein synthesis does not require provision of all amino acids. The branched-chain amino acids can accelerate protein synthesis to the same extent as the full complement of amino acids. (7, 8, 10, 16, 19, 28, 46). Of the branched-chain amino acids, leucine alone stimulates translation initiation in the absence of other amino acids (28), indicating that it plays an important role in the regulation of protein synthesis. Because of the unique role of leucine in controlling protein synthesis, we investigated the effect of removing leucine from the perfusion medium on protein synthesis when the concentration of other amino acids was maintained at elevated (10×) levels. Omission of leucine from the perfusate resulted in a 40% inhibition of protein synthesis. Thus the stimulation of protein synthesis by 10× amino acid concentrations could not be maintained when leucine was omitted from the perfusate. Removal of leucine most likely induces an amino acid imbalance that prevents the complete upregulation of protein synthesis by the remaining amino acids.

In contrast to the studies where all amino acids are raised to 10×, removal of leucine from the perfusate resulted in inhibitory effects on both eIF2B and eIF4E. These results indicate that mechanisms responsible for regulating protein synthesis in the two experimental conditions may be slightly different. The alterations in eIFs in muscles perfused with 10× amino acids minus leucine were characterized by a 40% decrease in eIF2B activity and an 80% fall in the amount of the active eIF4E · eIF4G complex. Similar effects on eIF2B and eIF4E were previously reported after removal of leucine from incubation medium of L6 myoblasts in culture (21). In contrast, the extent of phosphorylation of eIF4E was unaffected by removal of leucine from the perfusate. When leucine was removed from the medium, the fall in eIF4G binding to eIF4E was associated with a corresponding increase in the amount of 4E-BP1 bound to eIF4E. The binding of 4E-BP1 to eIF4E is regulated, in part, by phosphorylation of 4E-BP1. When 4E-BP1 becomes dephosphorylated, its binding to eIF4E is enhanced. In the present study, removal of leucine from the perfusate reduced the percentage of 4E-BP1 in the gamma -phosphorylated form. This was associated with a concomitant increase in the amount of 4E-BP1 bound to eIF4E. Likewise, Xu et al. (52) showed that leucine stimulates PHAS-I phosphorylation in a concentration-dependent manner in pancreatic RINm5F cells in cultures. Hence, in the presence of amino acids, removal of leucine caused reciprocal changes in the binding of eIF4E to eIF4G and 4E-BP1. The increase in 4E-BP1 bound to eIF4E was the result of reduced phosphorylation of 4E-BP1. The inhibition of protein synthesis associated with removal of leucine from the perfusate correlated with increased binding of 4E-BP1 to eIF4E and dissociation of eIF4E · eIF4G complex, as well as a fall in eIF2B activity.

FRAP/mTOR phosphorylates not only 4E-BP1 but p70 S6 kinase as well (3, 6). Because leucine deprivation lowered the proportion of 4E-BP1 in the gamma -phosphorylated form, we anticipated that removing leucine from the perfusate would reduce the phosphorylation of p70 S6 kinase as well. The prominence of the phosphorylated bands of the p70 S6 kinase appeared lessened after removal of leucine from the perfusate. Thus reduced phosphorylation of p70 S6 kinase correlated with reduced rates of protein synthesis in skeletal muscle perfused with medium lacking leucine. These results suggest that inactivation of p70 S6 kinase by dephosphorylation may also play a role in the inhibition of skeletal muscle protein synthesis after removal of leucine from the perfusate. Moreover, the data suggest that the site affected by leucine either lies uptream of FRAP/mTOR or is a phosphatase with dual specificity for 4E-BP1 and p70 S6 kinase.

In summary, raising the concentrations of all amino acids from physiological to supraphysiological levels stimulates protein synthesis primarily by enhancing the binding of eIF4E to eIF4G. The magnitude of the change in the binding of eIF4E to eIF4G (8×) is more than sufficient to account for the stimulation of protein synthesis (2×). Keeping amino acids at supraphysiological concentrations and eliminating leucine from the perfusate lowered the rate of protein synthesis. Removal of leucine from the perfusate resulted in a 40% decrease in eIF2B activity and an 80% fall in the amount of active eIF4E · eIF4G complex, whereas the extent of phosphorylation of eIF4E was unaffected. When leucine was removed from the medium, the fall in eIF4G binding to eIF4E was associated with a corresponding increase in the amount of 4E-BP1 bound to eIF4E secondary to a reduced percentage of 4E-BP1 in the gamma -phosphorylated form. By comparing the responses of eIF2B and eIF4E with supraphysiological concentrations of amino acids and during leucine deprivation, these results suggest that eIF2B is not further stimulated by amino acids at supraphysiological concentrations. It appears that elevating amino acids enhances the binding of eIF4E to eIF4G, thereby promoting translation initiation. In contrast, leucine deprivation causes eIF2B activity and eIF4G binding to eIF4E to become rate controlling.


    ACKNOWLEDGEMENTS

We are grateful for the outstanding technical support provided by Rebecca Eckman, Sharon Rannels, and Lynne Hugendubler.


    FOOTNOTES

This work was supported by National Institute of General Medical Sciences Grant GM-39277 (T. C. Vary) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-15658 (L. S. Jefferson) awarded by the National Institutes of Health.

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: T. C. Vary, Dept. of Cellular and Molecular Physiology, Penn State Univ. College of Medicine, 500 Univ. Drive, Hershey, PA 17033 (E-mail: tvary{at}psghs.edu).

Received 8 December 1998; accepted in final form 28 July 1999.


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