Amino acids and insulin are both required to regulate assembly of the eIF4E · eIF4G complex in rat skeletal muscle

Michele Balage1, Sandrine Sinaud1, Magali Prod'Homme1, Dominique Dardevet1, Thomas C. Vary2, Scot R. Kimball2, Leonard S. Jefferson2, and Jean Grizard1

1 Institut National de la Recherche Agronomique et Centre de Recherche en Nutrition Humaine d'Auvergne, Unité de Nutrition et Métabolisme Protéique, 63122 Saint Genes Champanelle, France; and 2 Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The respective roles of insulin and amino acids in regulation of skeletal muscle protein synthesis and degradation after feeding were examined in rats fasted for 17 h and refed over 1 h with either a 25 or a 0% amino acid/protein meal. In each nutritional condition, postprandial insulin secretion was either maintained (control groups: C25 and C0) or blocked with diazoxide injections (diazoxide groups: DZ25 and DZ0). Muscle protein metabolism was examined in vitro in epitrochlearis muscles. Only feeding the 25% amino acid/protein meal in the presence of increased plasma insulin concentration (C25 group) stimulated protein synthesis and inhibited proteolysis in skeletal muscle compared with the postabsorptive state. The stimulation of protein synthesis was associated with increased phosphorylation of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1), reduced binding of eIF4E to 4E-BP1, and increased assembly of the active eIF4E · eIF4G complex. The p70 S6 kinase (p70S6k) was also hyperphosphorylated in response to the 25% amino acid/protein meal. Acute postprandial insulin deficiency induced by diazoxide injections totally abolished these effects. Feeding the 0% amino acid/protein meal with or without postprandial insulin deficiency did not stimulate muscle protein synthesis, reduce proteolysis, or regulate initiation factors and p70S6k compared with fasted rats. Taken together, our results suggest that both insulin and amino acids are required to stimulate protein synthesis, inhibit protein degradation, and regulate the interactions between eIF4E and 4E-BP1 or eIF4G in response to feeding.

protein synthesis; feeding; insulin deficiency; eukaryotic initiation factors


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

REGULATION OF PROTEIN ANABOLISM in response to oral feeding involves both a stimulation of protein synthesis and a suppression of protein degradation (reviewed in Refs. 13, 27, 28). These changes are mediated by feeding-induced increases in plasma concentrations of both nutrients and hormones. Many studies suggest that amino acids and insulin play major roles in promoting postprandial protein anabolism. However, their respective and relative contributions in the regulation of protein anabolism in response to feeding remain unclear. Although in vitro studies show that insulin and amino acids can regulate skeletal muscle protein synthesis and degradation (4, 12, 23) independently, the assessment of their respective roles in vivo is complicated by the concomitant changes in their concentrations that generally accompany experimental changes in plasma insulin and amino acid concentrations. The potential contribution of amino acids and insulin in regulating protein anabolism after feeding has been the subject of many investigations. For example, feeding a protein-free diet in rodents (33, 36) or humans (31) did not induce any stimulation of skeletal muscle or whole body protein synthesis, despite a significant rise in plasma insulin, suggesting that amino acids, but not insulin, are essential in postprandial stimulation of protein synthesis. In accordance with this lack of an effect of insulin, refeeding accelerated skeletal muscle protein synthesis in both diabetic mice and control animals despite there being no changes in postprandial plasma insulin concentrations (24, 26). Likewise, provision of exogenous insulin to freely fed rodents did not increase muscle protein synthesis beyond the effect of refeeding (5, 26). Conversely, provision of anti-insulin antibodies before refeeding prevented (5, 17, 33) or attenuated (14, 26) the stimulation of skeletal muscle protein synthesis in response to food intake in rodents. Moreover, we recently showed (21) that an acute insulin deficiency (due to diazoxide injection) in fed rats significantly decreased muscle protein synthesis. These data suggested that insulin plays a major role in the stimulation of protein synthesis after a meal. Despite these various studies, the relative contribution of amino acids and insulin to postprandial anabolism remains unresolved.

The acute increase in muscle protein synthesis in response to feeding has been shown to occur through a stimulation of translation initiation (6, 17, 33). Initiation is regulated by specific proteins, termed eukaryotic initiation factors (eIFs) (reviewed in Refs. 18-20). The mechanism through which insulin and amino acids regulates translation initiation involves phosphorylation/dephosphorylation of eIFs, especially the translational regulator eIF4E binding protein-1 (4E-BP1). Unphosphorylated 4E-BP1 binds to eIF4E and sequesters it into an inactive complex, 4E-BP1 · eIF4E complex. Insulin and amino acids increase the phosphorylation state of 4E-BP1, which results in dissociation of the 4E-BP1 · eIF4E complex, freeing eIF4E to participate in assembly of the active eIF4E · eIF4G complex.

The stimulation of translation initiation in skeletal muscle in response to feeding a complete diet was associated with a decreased assembly of the inactive 4E-BP1 · eIF4E complex and an increased assembly of the active eIF4E · eIF4G complex (24, 35). Concomitantly, an increase in the most highly phosphorylated form of 4E-BP1 was observed. In contrast, an acute insulin deficiency in rats fed a complete diet involved an inhibition in translation initiation associated with an increase in 4E-BP1 bound to eIF4E and a dissociation of the eIF4E · eIF4G complex (21). Moreover, increased activity of the p70 S6 kinase (p70S6k) was implicated in the stimulation of protein synthesis under conditions that promoted 4E-BP1 phosphorylation (22). It was shown previously that refeeding starved animals enhanced the phosphorylation state of p70S6k (25).

The aim of the study described herein was to assess the respective roles played by insulin and/or amino acids in the regulation of skeletal muscle protein metabolism after feeding. For these studies, rats were starved for 17 h and then refed for 1 h with either a 25% amino acid/protein diet or a protein-free diet. In each nutritional condition, postprandial plasma insulin either was allowed to increase or was blocked with diazoxide injections, as previously described (21). This experimental model allows for the separation of responses resulting from an increase in plasma insulin compared with those for amino acids after feeding. Protein metabolism was then examined in vitro in epitrochlearis muscles taken in the postabsorptive state or 2 h after food intake. The results demonstrate that amino acids and insulin are required in combination to stimulate in vitro protein synthesis after feeding as well as to reduce protein degradation. The stimulation of protein synthesis is associated with an increase in the phosphorylation of 4E-BP1, leading to an increase in the dissociation of the inactive 4E-BP1 · eIF4E complex and an increased binding of eIF4E to eIF4G. In response to feeding, it appears that both insulin and amino acids are required to stimulate protein synthesis and regulate the interactions between eIF4E and 4E-BP1 or eIF4G.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Animals and experimental design. Forty-one male Wistar rats weighing 72.5 ± 0.5 g were housed individually under controlled environmental conditions (temperature 22°C; 12-h dark period starting at 9:00 AM) and were fed ad libitum with a 15% casein diet (Table 1); water was provided freely. Rats were acclimated to their surroundings for 10 days until they reached a mean body weight of 143.9 ± 0.6 g. Food was removed at 4:00 PM on the day before the experiment. The animals were then divided into three groups. One group did not receive food anymore [17-h-fasted, or postabsorptive (PA) group], whereas the two other groups were refed with either a 25 or 0% amino acid/protein meal for 1 h (i.e., from 9:00 to 10:00 AM) on the next morning (Fig. 1). The composition of the two experimental meals is given in Table 1. In the 25% amino acid/protein meal, amino acids originated from both casein (10%) and a specific amino acid mixture (15%) whose composition is given in the legend of Table 1. A combination of protein and a mixture of free amino acids was used to obtain a rapid postprandial increase in plasma amino acids after meal intake. In the 0% amino acid/protein meal, wheat starch was used as a substitute for protein and amino acids; agar-agar (indigestible) was included in the meal to obtain the same consistency between the two types of meals.

                              
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Table 1.   Composition of the diet and experimental meals



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Fig. 1.   Experimental design. At the end of a 17-h fasting period (at the beginning of the dark period), 41 rats were fed either a 25% amino acid/protein meal (n = 17) or a 0% amino acid/protein meal (n = 16) over 1 h. An additional group (n = 8) did not receive any food and constituted the postabsorptive (PA) group. At the end of feeding, each refed group (25 or 0% amino acid/protein meal) was divided into 2 groups. Control (C) and diazoxide-injected (DZ) groups were injected with vehicle or diazoxide, respectively, as described in MATERIALS AND METHODS. Skeletal muscle protein synthesis and degradation were measured in vitro in epitrochlearis muscles 2 h after the end of feeding as well as plasma insulin, glucose, and amino acids.

At the end of the meal, one-half of each refed group received two intraperitoneal injections of diazoxide [25 and 20 mg/100 g body wt in 0.05 N NaOH, respectively, at 10:00 AM (t0) and 11:30 AM]. These groups were named, respectively, DZ25 and DZ0 for the 25% amino acid/protein and the 0% amino acid/protein refed animals. At the same time, the other one-half of the animals received the vehicle and were used as controls (C25 and C0, respectively). The animals were anesthetized with pentobarbital sodium (18 mg/100 g body wt) at 12:00 (2 h after meal, t2). Blood was rapidly collected and centrifuged at 2,000 g for 10 min. The epitrochlearis and gastrocnemius muscles were excised and used for measurement of protein synthesis and protein degradation and analysis of eukaryotic initiation factors, respectively.

Measurement of protein synthesis and degradation. Epitrochlearis muscles were dissected intact for the in vitro measurement of skeletal muscle protein metabolism. They were immediately preincubated for 30 min at 37°C in Krebs-Henseleit buffer (in mM: 120 NaCl, 4.8 KCl, 25 NaHCO3, 2.5 CaCl2, 1.2 KH2PO4, and 1.2 MgSO4, pH 7.4) supplemented with 5 mM HEPES, 5 mM glucose, and 0.1% BSA (99% fatty acid free) and saturated with a 95% O2-5% CO2 gas mixture, as previously described (2). Muscles were then transferred to fresh medium of the same composition containing 0.5 mM L-[14C]phenylalanine (0.15 µCi/ml) and incubated for an additional 75-min period.

At the end of the incubation, muscles were blotted, weighed, and homogenized in 10% trichloroacetic acid (TCA). Samples were centrifuged at 10,000 g for 10 min at 4°C, and TCA-insoluble material was washed three times with 10% TCA. The resultant pellet was solubilized in 1 M NaOH at 37°C for determination of protein and radioactivity incorporated into muscle protein. Tissue protein content was determined using the bicinchoninic acid procedure (Pierce Chemical, Rockford, IL) with crystalline BSA as a standard. Protein-bound radioactivity was measured using scintillation counting. Protein synthesis was calculated by dividing the protein-bound radioactivity by the specific radioactivity of the phenylalanine in the incubation medium. It was expressed as nanomoles phenylalanine incorporated per milligram of protein per 75 min.

Rates of protein degradation were measured by the accumulation of tyrosine in the incubation medium, as described elsewhere (2). Because tyrosine is neither synthesized nor degraded by muscle, except for use by protein synthesis, the release of this amino acid from muscle into the incubation medium reflects net protein balance. Total net protein degradation was estimated simultaneously with protein synthesis as the sum of the accumulation of tyrosine in the incubation buffer over the 75-min incubation period plus the amount of tyrosine equivalents incorporated into proteins via protein synthesis during the same time. To obtain the amount of tyrosine incorporated into muscle proteins, values of incorporated phenylalanine into proteins were multiplied by the molar ratio of tyrosine to phenylalanine in mixed skeletal muscle proteins (0.77) (2, 29). Tyrosine in the incubation medium was assessed fluorometrically as described previously (32). Proteolysis was expressed as nanomoles of tyrosine per milligram of protein per 75 min.

Analysis of eukariotic initiation factors. Gastrocnemius was rapidly excised for analysis of initiation factors as previously described (1, 21). One portion was weighed and homogenized in 7 volumes of buffer consisting of (in mM) 20 HEPES, pH 7.4, 100 KCl, 0.2 EDTA, 2 EGTA, 1 dithiothreitol, 50 NaF, 50 beta -glycerophosphate, 0.1 phenylmethylsulfonyl fluoride, 1 benzamidine, and 0.5 Na3VO4, and 1 µM microcystin LR by use of a Polytron homogenizer. The homogenate was centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was stored at -80°C until analysis.

Phosphorylation state of eIF4E and 4E-BP1. The phosphorylated and unphosphorylated forms of eIF4E in gastrocnemius were separated by isoelectric focusing on a slab gel and quantitated by protein immunoblot analysis, as described previously (1). 4E-BP1 was immunoprecipitated from aliquots of 10,000-g supernatants by use of an anti-4E-BP1 monoclonal antibody and was subjected to protein immunoblot analysis as described previously (1).

Quantification of 4E-BP1 · eIF4E and eIF4G · eIF4E complexes. eIF4E, 4E-BP1 · eIF4E, and eIF4G · eIF4E complexes were immunoprecipitated from aliquots of 10,000-g supernatants by means of an anti-eIF4E monoclonal antibody. Samples were subjected to immunoblot analysis by use of either a monoclonal antibody to 4E-BP1 or a polyclonal antibody to eIF4G to determine the association of 4E-BP1 and eIF4G with eIF4E, as previously described (1).

Phosphorylation of p70S6k. Phosphorylation of the 70-kDa protein p70S6k was determined in the 10,000-g supernatants of gastrocnemius by protein immunoblot analysis, as previously described (1).

Plasma insulin, glucose, and amino acid measurements. Plasma insulin concentrations were analyzed using a commercial RIA kit (ERIA Diagnostics Pasteur, Sanofi, France). Plasma glucose was determined enzymatically using a glucose oxidase kit (Boeringer Mannheim). Plasma amino acids were determined by ion-exchange chromatography after protein precipitation: 500 µl of plasma were added to 125 µl of a sulfosalicylic acid solution (1 M in ethanol with 0.5 M thiodiglycol) previously evaporated to dryness. Norleucine was added as an internal standard. Samples were incubated on ice for 1 h and then centrifuged at 3,500 g for 1 h at 4°C. An aliquot (250 µl) of the supernatant was combined with 125 µl of 0.1 M lithium acetate buffer, pH 2.2. Amino acid concentrations were determined on these extracts by means of an automated amino acid analyzer (Biotronic LC 3000, Roucaire, Velizy, France, with BTC 2410 resin).

Statistical analysis. Values presented are means ± SE. Statistical evaluation of the data was performed by ANOVA to test for overall differences among the groups, followed by the Fisher's protected least significant difference test for multiple comparisons to determine significance between means when ANOVA indicated a significance difference among the group means. Differences among the means were considered significant when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Over the 1-h feeding period, food intake was not significantly different among the groups whatever the meal consummed (4.36 ± 0.50, 4.45 ± 0.26, 3.53 ± 0.33, and 4.02 ± 0.38 g of dry matter in C25, DZ25, C0, and DZ0, respectively).

As shown in Fig. 2, control refed animals (C groups) exhibited a marked increase in plasma insulin compared with postabsorptive (PA group) rats (P < 0.05). This increase was greater with the 25% than with the 0% amino acid/protein meal. As previously observed (21), diazoxide injections dramatically reduced plasma insulin levels in refed animals (DZ groups). Plasma insulin values in diazoxide-injected refed rats were lower than postabsorptive values but not significantly.


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Fig. 2.   Effect of refeeding a 25 or 0% amino acid (AA)/protein meal without (C25 and C0 groups) or with (DZ25 and DZ0 groups) diazoxide treatement on plasma insulin concentration in 17-h-fasted rats. Plasma insulin concentrations were determined by RIA with an insulin assay kit. Results are means ± SE for 7-8 determinations/group. Values not sharing the same superscript letter are significantly different (P < 0.05) by ANOVA.

The postabsorptive plasma glucose concentration was 1.02 ± 0.02 g/l. It was significantly increased in control refed animals (2.01 ± 0.39 and 1.86 ± 0.31 g/l in C25 and C0 groups, respectively, P < 0.05 vs. PA group). Diazoxide injections induced a similar hyperglycemia in both refed groups (4.54 ± 0.28 and 4.93 ± 0.12 g/l in DZ25 and DZ0 groups, respectively, P < 0.05 vs. C25, C0, and PA groups).

Refeeding the 25% amino acid/protein meal markedly increased most of the essential plasma amino acids (except arginine, which did not show any significant increase) compared with the postabsorptive period (Table 2 ). The nonessential amino acids alanine, asparagine, and serine were also increased (P < 0.05). In contrast, refeeding the 0% amino acid/protein meal did not modify the plasma free amino acids compared with the postabsorptive group. The diazoxide injections had only a modest effect on plasma free amino acids in each refed group: a significant increase in arginine, phenylalanine, and glycine in the 25% amino acid/protein refed group and a decrease in glutamic acid and an increase in glycine and serine in the 0% amino acid/protein refed group.

                              
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Table 2.   Plasma amino acid concentrations

As shown in Fig. 3A, refeeding a 25% amino acid/protein meal significantly increased protein synthesis by 20% in epitrochlearis (0.502 ± 0.018 in C25 group vs. 0.421 ± 0.009 nmol phenylalanine · mg protein-1 · 75 min-1 in the PA group, respectively; P < 0.05, n = 8). In contrast, refeeding the 0% amino acid/protein meal had no effect on protein synthesis (0.413 ± 0.006 nmol phenylalanine · mg protein-1 · 75 min-1 in the C0 group). The stimulation of muscle protein synthesis by the 25% amino acid/protein meal was completely abolished when postprandial insulin secretion was blocked by diazoxide injection (DZ25). Moreover, protein synthesis was significantly lower in each diazoxide-treated refed group (0.369 ± 0.017 and 0.356 ± 0.012 nmol phenylalanine · mg protein-1 · 75 min-1 in DZ25 and DZ0 groups, respectively) than in PA and Control groups (Fig. 3A).


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Fig. 3.   Effect of refeeding a 25 or 0% AA/protein meal without (C25 and C0 groups) or with (DZ25 and DZ0 groups) diazoxide treatment on muscle protein synthesis and proteolysis in 17-h-fasted rats. Epitrochlearis muscles were excised and incubated in vitro, as described in MATERIALS AND METHODS. A: rates of protein synthesis were measured by determining incorporation of [14C]phenylalanine into muscle proteins. B: rates of proteolysis were estimated simultaneously with the rates of protein synthesis as the sum of net release of tyrosine into the incubation medium and the amount of tyrosine equivalents incorporated into muscle protein over the same period. Results are means ± SE for 7-8 determinations/group. Values not sharing the same superscript letter are significantly different (P < 0.05) by ANOVA.

Conversely, refeeding rats the 25% amino acid/protein meal decreased protein degradation in epitrochlearis (1.625 ± 0.054 and 1.342 ± 0.032 nmol tyrosine · mg protein-1 · 75 min-1 in PA and C25 groups, P < 0.05) (Fig. 3B). Such an inhibition was totally suppressed when postprandial plasma insulin secretion was prevented by diazoxide injections. Indeed, muscle proteolysis in the DZ25 group was dramatically higher than that in C25 group (1.784 ± 0.064 nmol tyrosine · mg protein-1 · 75 min-1 in the DZ25 group, P < 0.001 vs. C25 group). It was also significantly higher than in postabsorptive fasted rats (DZ25 group vs. PA group). In contrast, refeeding the 0% amino acid/protein meal did not inhibit protein degradation. Insulin deficiency induced by diazoxide injections in that group resulted in a significant increase in proteolysis (P < 0.05 vs. the C0 group).

To examine potential mechanisms regulating protein synthesis, the translation initiation pathway was examined in gastrocnemius. It is now generally acknowledged that phosphorylation of 4E-BP1 regulates translation initiation and protein synthesis by inducing dissociation of the 4E-BP1 · eIF4E. Figure 4A represents the proportion of 4E-BP1 present in the gamma -form (the most highly phosphorylated form) in the five experimental groups. Only refeeding the 25% amino acid/protein meal without insulin suppression (C25 group) caused a dramatic increase (6.8-fold) in the amount of 4E-BP1 present in the gamma -form when compared with the PA group. Indeed, this increase was completely abolished when insulin secretion was blocked by diazoxide injections (DZ25 group). Refeeding the 0% amino acid/protein meal had no effect on the phosphorylation state of 4E-BP1 despite a rise in plasma insulin compared with postabsorptive animals. The hyperphosphorylated form of 4E-BP1 became undetectable when rats fed the 0% amino acid/protein meal were injected with diazoxide (DZ0 group).


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Fig. 4.   Effect of refeeding a 25 or 0% AA/protein meal without (C25 and C0 groups) or with (DZ25 and DZ0 groups) diazoxide treatment on phosphorylation of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1) and amount of 4E-BP1 bound to eIF4E in PA group. A: phosphorylation of 4E-BP1 was determined in extracts of gastrocnemius by immunoblot analysis as described in MATERIALS AND METHODS. Amount of 4E-BP1 in gamma -phosphorylated form is expressed as a percentage of the sum of total amount of 4E-BP1 in each condition. B: binding of eIF4E to 4E-BP1 was determined by immunoprecipitating eIF4E from 10,000-g-centrifuged supernatants of gastrocnemius with an anti-eIF4E monoclonal antibody, as described in MATERIALS AND METHODS. Immunoprecipitates were then subjected to immunoblot analysis using 4E-BP1 antibody. Results are expressed as means ± SE for 6-9 determinations/group, (n.d., nondetectable). Values not sharing the same superscript letter are significantly different (P < 0.05) by ANOVA.

Because hyperphosphorylation of 4E-BP1 is generally inversely proportional to the amount of 4E-BP1 bound to eIF4E, we examined the amount of 4E-BP1 associated with eIF4E. The amount of 4E-BP1 bound to eIF4E was measured by immunoprecipitating eIF4E with an anti-eIF4E antibody, followed by immunoblot analysis with an anti-4E-BP1 antibody. As shown in Fig. 4B, refeeding the 25% amino acid/protein meal resulted in a decrease in the amount of 4E-BP1 associated with eIF4E compared with the PA group. Diazoxide-induced hypoinsulinemia abolished this decrease (DZ25 vs. C25 groups). Refeeding the 0% amino acid/protein meal did not change the amount of 4E-BP1 bound to eIF4E compared with PA animals (Fig. 4B). Moreover, when diazoxide was injected in 0% amino acid/protein refed rats, the amount of 4E-BP1 associated with eIF4E was higher than that in PA and C0 groups. As previously observed, the phosphorylation state of 4E-BP1 closely mirrored the association of 4E-BP1 with eIF4E.

Dissociation of 4E-BP1 from eIF4E results in an increased eIF4E availability for binding to eIF4G. Therefore, we investigated the effect of refeeding in combination with diazoxide-induced insulin deficiency on the amount of eIF4E bound to eIF4G (Fig. 5A). Refeeding the 25% amino acid/protein meal without diazoxide treatment (C25 group) resulted in an increase in the association of eIF4E with eIF4G compared with postabsorptive rats (PA group). Diazoxide injections in 25% amino acid/protein refed animals (DZ25 group) totally suppressed this increase. Insulin-deficiency, as well as 0% amino acid/protein refeeding (in both C0 and DZ0 groups) significantly decreased association of eIF4E with eIF4G compared with the PA group.


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Fig. 5.   Effect of refeeding a 25 or 0% AA/protein meal without (C25 and C0 groups) or with (DZ25 and DZ0 groups) diazoxide treatement on amount of 4E-BP1 bound to eIF4G and on phosphorylation of eIF4E in PA group. A: binding of eIF4G to 4E-BP1 was determined as described in Fig. 4 legend. Immunoprecipitates were then subjected to immunoblot analysis with the use of an eIF4G antibody. B: amount of eIF4E present in phosphorylated form in skeletal muscle was determined by protein immunoblot analysis after slab isoelectric focusing, as described in MATERIALS AND METHODS. Results are expressed as means ± SE for 6-9 determinations/group. Values not sharing the same superscript letter are significantly different (P < 0.05) by ANOVA.

To further define the effects of refeeding on translation initiation, the phosphorylation state of eIF4E was examined (Fig. 5B). Refeeding the 25% amino acid/protein diet (C25 group) resulted in partial dephosphorylation of eIF4E compared with the PA group that did not occur when rats were injected with diazoxide (DZ25 group). Refeeding the 0% amino acid/protein meal (C0 group) had no effect on the phosphorylation state of eIF-4E compared with the PA group. Amino acid and insulin deficiencies (DZ0 group) impaired eIF4E phosphorylation.

We also examined the extent of phosphorylation of p70S6k in extracts of gastrocnemius from each group (Fig. 6). Only refeeding the 25% amino acid/protein meal caused a decrease in the electrophoretic mobility of p70S6k and the appearance of multiple phosphorylated forms indicative of hyperphosphorylation. Refeeding the meal lacking protein and amino acid (C0 group) as well as diazoxide injections had no effect on the phosphorylation state of p70S6k (DZ groups).


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Fig. 6.   Effect of refeeding a 25 or 0% AA/protein meal without (C25 and C0 groups) or with (DZ25 and DZ0 groups) diazoxide treatement on phosphorylation state of p70 S6 kinase (p70S6k) in gastrocnemius of PA group. Phosphorylation state of p70S6k was assessed by protein immunoblot analysis, as described in MATERIALS AND METHODS. Arrows indicate the multiple electrophoretic forms of p70S6k. Data shown are representative of 7-9 rats/group.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies showed that feeding a complete diet increases both plasma insulin and amino acid concentrations. It also stimulates muscle protein synthesis and inhibits protein degradation, resulting in postprandial anabolism. However, the respective and relative contributions of amino acids and insulin to postprandial protein anabolism are not completely elucidated. In the present study, the specific effect of amino acids and insulin in the postprandial regulation of skeletal muscle protein metabolism was assessed using a model of postprandial insulin suppression that we previously developed (21). In this model, 17-h-fasted rats are refed either a 25 or 0% amino acid/protein meal over 1 h, with each group being or not being subjected to a specific inhibitor of insulin secretion (diazoxide injections). Compared with previous models (24, 26, 31, 36), our experimental design allowed for the isolation of the contribution of prandial hyperinsulinemia alone (C0 group), hyperaminoacidemia alone (DZ25 group), and the combination of insulin and amino acids (C25 group) in the regulation of muscle protein metabolism after feeding. Only one other study has investigated simultaneously the role of amino acids and insulin on postprandial skeletal muscle protein synthesis regulation in the same experiment by use of anti-insulin antibodies (33).

As expected (33, 36), the present results show that refeeding an amino acid/protein-rich diet to overnight-starved rats stimulated muscle protein synthesis. The present study clearly reveals that these effects were completely abolished when postprandial insulin increment was suppressed by diazoxide injections. The lack of effect was not a result of different food intake, because animals from different groups consumed similar amounts of the diet over the 1-h feeding period and plasma amino acid concentrations were similar in the two groups. Thus the difference was reflective of the changes in plasma insulin concentrations. With feeding the amino acid/protein-free meal, plasma insulin was significantly increased compared with postabsorptive state, but muscle protein synthesis was not stimulated. These data suggest that insulin per se is not able to stimulate skeletal muscle postprandial protein synthesis.

However, we should note that the increment in the plasma insulin concentration was lower in the 0% than in the 25% amino acid/protein meal-refed rats. Nevertheless, it seems to be unlikely that the concentration of plasma insulin in the 0% amino acid/protein-refed animals is insufficient to regulate protein synthesis, because it was elevated threefold compared with the PA group. Such an increase in plasma insulin concentrations observed after feeding is associated with a stimulation of skeletal muscle protein synthesis (1, 26, 31). However, we cannot exclude the possibility that the significant increase in skeletal muscle protein synthesis observed in the 25%- compared with the 0% amino acid/protein-refed rats resulted in the additional increase in postprandial plasma insulin in the C25 group.

In addition to insulin, amino acids are also involved in the regulation of skeletal muscle protein synthesis. Insulin infusion without concomitant administration of exogenous amino acids resulted in a decrease in plasma amino acids. Under such conditions, the absence or reduction of skeletal muscle protein synthesis resulted from the decreased amino acid availability. In the present experiment, and contrary to a previous study in mice (33), feeding the protein-free diet induced only a moderate decrease in plasma amino acid concentrations compared with postabsorptive values (-17% for essential amino acids, not statistically significant) despite the increase in plasma insulin. Thus the failure of insulin to stimulate protein synthesis in animals fed the 0% amino acid/protein meal cannot be explained by a decrease in plasma amino acid availability compared with the postabsorptive state. Actually, the lack of increment of plasma amino acid concentration in 0% amino acid/protein-refed animals likely accounts for the lack of stimulation in postprandial skeletal muscle protein synthesis.

Overall, these results clearly demonstrate that the stimulation of skeletal muscle protein synthesis in response to food intake requires an increase in both insulin and amino acid concentrations. Indeed, neither insulin alone nor amino acids alone were able to induce a significant increase in muscle protein synthesis in response to feeding. Similar results have been reported by Yoshizawa et al. (33) in mice subjected or not to treatment with anti-insulin serum before refeeding either a 25% casein or a 0% protein diet.

In agreement, Garlick et al. (5) and Preedy and Garlick (17) showed that intravenous administration of anti-insulin serum to food-deprived rats before refeeding suppressed the stimulation of protein synthesis in muscle, suggesting that insulin was necessary for the postprandial stimulation of protein synthesis. Conversely, feeding a diet lacking amino acid and protein in humans (31) or in rodents (33, 36) did not stimulate whole body or skeletal muscle protein synthesis, despite a significant rise in plasma insulin. These data, in accord with ours, demonstrate that insulin per se is not able to stimulate skeletal muscle protein synthesis postprandially.

In the present study, diazoxide injections in rats fed either the 25% or the 0% amino acid/protein meal tended to have plasma insulin concentrations below the postabsorptive level (Fig. 2), inducing a significant decrease in muscle protein synthesis compared with the postabsorptive group (Fig. 3A). Therefore, when the correlations between plasma insulin and muscle protein synthesis with all individual values are compared (Fig. 7), it appears that decreasing the postprandial plasma insulin level below the postabsorptive level (by diazoxide treatment) resulted in an impairement of protein synthesis compared with postabsorptive protein synthesis. Therefore, insulin seems to be essential for maintaining basal protein synthesis independently of plasma amino acid. Such a hypothesis is in agreement with that of Fedele et al. (3), who showed that there is a critical concentration of insulin below which rates of protein synthesis begin to decline in vivo.


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Fig. 7.   Relationship between plasma insulin and rates of protein synthesis from 39 rats of the present experiment. Plasma insulin and protein synthesis were determined as described in MATERIALS AND METHODS. triangle , PA rats; , 25% AA/protein-refed rats; , 0% AA/protein-refed rats.

The stimulation of skeletal muscle protein synthesis caused by feeding a complete diet has been shown to be mediated by an increase in the initiation of mRNA translation (6, 17, 33). One of the most tightly regulated steps in translation initiation is the binding of mRNA to the 40S subunit (16, 19, 20). This step involves the binding of eIF4E to the m7 GTP cap at the 5' end of the mRNA and the subsequent binding of the eIF4E-mRNA complex to eIF4G, which is a critical step in the formation of the 48S preinitiation complex. Two major mechanisms have been described that contribute to the regulation of the assembly of these complexes, each of which involves reversible phosphorylation of proteins implicated in the process.

Previous studies in vitro have reported that increased phosphorylation of eIF4E is observed in cultures of cells in response to a variety of stimuli (growth factors, hormones) and is positively correlated with changes in protein synthesis (18). However, in vivo studies do not support these results. Indeed, the phosphorylation state of eIF4E was not changed in diabetic or insulin-treated diabetic rats (9); it also was not changed in skeletal muscle of overnight-fasted or refed animals (35). In the present study, refeeding the 25% amino acid/protein diet resulted in a decrease in the phosphorylation state of eIF4E. Similarly, Yoshizawa et al. (36) previously reported a decrease in eIF4E phosphorylation in muscle of rats fed with a 20% protein diet in which protein synthesis was clearly stimulated. Therefore, it is unlikely that phosphorylation of eIF4E is involved in postprandial stimulation of protein synthesis. In perfused muscle preparations (10) or in L6 myoblasts (7), the stimulation of protein synthesis by insulin is associated with a decrease, rather than an increase, in the proportion of phosphorylated eIF4E. The consequence of the decrease in eIF4E phosphorylation is not known, but it has been proposed that the rate of phosphate turnover on eIF4E rather than its net phosphorylation is important in regulating protein synthesis (see DISCUSSION in Ref. 7).

Another mechanism through which translation initiation can be regulated involves phosphorylation of the eIF4E binding protein 4E-BP1. Indeed, eIF4E bound to 4E-BP1 can bind to the m7 GTP cap stucture, but cannot bind to eIF4G, the active complex to stimulate translation initiation. Thus 4E-BP1 competes with eIF4G for association to eIF4E. The ability of 4E-BP1 to bind to eIF4E is largely dependent on the phosphorylation state of 4E-BP1. Phosphorylation of 4E-BP1 releases eIF4E from the 4E-BP1 · eIF4E complex, which in turn is available to bind to eIF4G.

The results of the present study show that the stimulation of protein synthesis that occurs after feeding the 25% amino acid/protein meal resulted in an increase in the proportion of 4E-BP1 in the most highly phosphorylated form with a concomitant decrease in the amount of 4E-BP1 bound to eIF4E and an increase in the amount of eIF4G bound to eIF4E. Similar results were obtained in rodents fed a complete diet (24, 35, 36). As previously observed (36), the increased plasma insulin level observed in rats fed the protein free diet (C0 group) had no effect on 4E-BP1 phosphorylation or association of eIF4E with 4E-BP1 or eIF4G, suggesting that postprandial increment of plasma insulin without concomitant increase in plasma amino acids does not regulate 4E-BP1 · eIF4E or eIF4E · eIF4G complexes . Moreover, our study clearly demonstrates that increased aminoacidemia in response to feeding without concomitant increase in plasma insulin concentrations (DZ25 group) had no additional effect on 4E-BP1 phosphorylation or association of eIF4E with 4E-BP1 or eIF4G, which is in accordance with the absence of protein synthesis stimulation. These data are, however, surprising because several studies in vitro have shown that amino acids alone or insulin alone regulates translation initiation (11, 30). Similarly, in vivo, oral administration of leucine increased eIF4F formation in skeletal muscle of postabsorptive rats independently of increases in plasma insulin (1). Unfortunately, we did not investigate eIF2B activity, which has been identified as an essential regulator of protein synthesis by amino acids in L6 myoblasts (8).

The p70S6k signaling pathway is considered an important mechanism for regulating stimulation of protein synthesis. Indeed, numerous studies have shown that phosphorylation of 4E-BP1 by insulin or amino acids occurs in conjunction with the activation of p70S6k (1, 7, 8). As previously observed (34), the increase in plasma insulin after refeeding the 0% amino acid/protein meal was not sufficient to increase the phosphorylation state of p70S6k. However, insulin is required to activate the p70S6k, because feeding the 25% amino acid/protein meal had no effect on the phosphorylation state of the kinase when insulin was reduced by diazoxide injections.

Measurement of muscle protein metabolism with the use of epitrochlearis incubation allowed the determination of protein degradation. In the present study, inhibition of proteolysis in response to feeding depended on both increased plasma insulin and amino acid concentrations, as observed for stimulation of protein synthesis. In contrast, Svanberg et al. (25) showed that elevation of plasma amino acids induced by intravenous infusions resulted in a dose-response decrease in muscle protein breakdown despite unchanged plasma insulin in humans. However, intravenous infusion of amino acids does not completely represent the real effect of a meal. Additional studies will be required to define the proteolytic pathways involved in the regulation of muscle proteolysis after feeding.

In conclusion, the results of the present study demonstrate that amino acids in combination with insulin are required to stimulate muscle protein synthesis and to reduce protein degradation in response to feeding. Indeed, neither selective increment in plasma amino acids nor insulin alone in postprandial conditions was able to regulate protein metabolism. The stimulation of muscle protein synthesis was associated with an increase in the phosphorylation state of 4E-BP1, leading to a dissociation of the inactive 4E-BP1 · eIF4E complex and an increase in the formation of the active eIF4G · eIF4E complex. Furthermore, activation of p70S6k may be involved in the stimulation of protein synthesis after refeeding. It is important to note that these regulatory steps always required the presence of amino acids plus insulin.


    ACKNOWLEDGEMENTS

We thank Sharon Rannels and Claire Sornet for outstanding technical expertise and Gerard Bayle for amino acid analysis.


    FOOTNOTES

This work was supported in part by the Nestle Research Center, Lausanne, Switzerland, and by US Public Health Service (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.

Address for reprint requests and other correspondence: M. Balage, Unité de Nutrition et Métabolisme Protéique, INRA, 63122 Saint Genes Champanelle, France (E-mail: balage{at}clermont.inra.fr).

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.

Received 29 January 2001; accepted in final form 23 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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