Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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The effect of dietary protein on the initiation of mRNA translation was examined in rats starved for 18 h and then fed isocaloric diets containing either 20% protein (20P) or no added protein (0P). Feeding the 20P diet, but not the 0P diet, stimulated protein synthesis in skeletal muscle and liver by 38 and 41%, respectively. The stimulation was associated with reduced binding of eukaryotic initiation factor (eIF) 4E to the translational repressor 4E-BP1, increased formation of the active eIF4E-eIF4G complex, and increased phosphorylation of 4E-BP1. In contrast, feeding a 0P diet had no effect on any of these parameters. Feeding a 20P diet resulted in partial dephosphorylation of eIF4E in both tissues. In liver, refeeding a 0P diet also resulted in partial eIF4E dephosphorylation, suggesting that the phosphorylation state of eIF4E is not important in the stimulation of protein synthesis under these conditions. Finally, plasma insulin concentrations were the same in rats fed either diet (14.8 ± 4.9 vs. 15.5 ± 4.5 µU/ml for 20P and 0P groups, respectively), suggesting that feeding-induced changes in plasma insulin are not sufficient to stimulate protein synthesis. Instead, a combination of dietary protein and insulin may be required to stimulate translation initiation.
insulin; eukaryotic initiation factor eIF4E; eukaryotic initiation factor eIF4G; 4E-BP1
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INTRODUCTION |
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RATES OF PROTEIN SYNTHESIS in growing rodents are highly sensitive to the nutritional status of the animal (10, 26, 35, 38). In particular, previous studies show that protein synthesis is reduced in a variety of tissues after a period of food deprivation as short as 18 h. Refeeding rapidly restores protein synthesis to values observed in freely fed animals. The signal(s) that causes the stimulation of protein synthesis in response to refeeding has been the subject of investigation for decades, with the majority of studies reporting that an increase in either insulin or amino acids is responsible for the stimulation. In vitro studies using incubated or perfused muscle preparations have confirmed the importance of both insulin and amino acids in regulating protein synthesis (4, 7, 8, 20, 33). However, although insulin and amino acids can independently regulate protein synthesis in vitro, recent in vivo studies suggest that a combination of insulin and amino acids is required to stimulate protein synthesis in skeletal muscle in response to feeding (36, 38).
In both skeletal muscle (15, 20, 26) and liver (5, 6), the stimulation of protein synthesis caused by amino acids and insulin is mediated by an increase in the initiation of mRNA translation. The process of initiation is comprised of numerous steps and is mediated by 12 proteins referred to as eukaryotic initiation factors (eIFs). Of the many steps, two appear to be particularly important in the physiological regulation of translation. These steps are the binding of initiator methionyl-tRNAi to the 40S ribosomal subunit, mediated by eIF2, and the binding of the 40S ribosome to the 5' end of mRNA, mediated by eIF4F. One of the eIF4F family members, eIF4E, plays an important role in the binding of mRNA to the 40S ribosomal subunit because it is the initiation factor that binds the m7GTP cap present at 5'-end of eukaryotic mRNAs (27, 31). During translation initiation, the eIF4E-mRNA complex binds to eIF4G and eIF4A to form the active eIF4F complex (27, 28, 32). Alterations in either the phosphorylation state or availability of eIF4E modulate the function of the eIF4F complex; specifically, eIF4E phosphorylation stimulates initiation through increased association with eIF4G and eIF4A (24) and/or increased mRNA cap-binding affinity (23). In addition, the availability of eIF4E for eIF4F complex formation can be modulated by changes in the association of eIF4E with an eIF4E-binding protein termed 4E-BP1 (25). 4E-BP1 competes with eIF4G for binding to eIF4E and sequesters eIF4E into an inactive complex. The binding of eIF4E to 4E-BP1 is regulated by phosphorylation of the binding protein, which results in a decrease in the affinity of eIF4E for 4E-BP1.
In a previous study (39), we showed that the increase in translation initiation in both skeletal muscle and liver in response to feeding a diet composed of carbohydrate, lipid, and protein was not associated with any detectable change in the activity of eIF2B or in the phosphorylation state of eIF2B, both regulators of the binding of initiator methionyl-tRNAi to the 40S ribosomal subunit. Instead, changes in initiation were associated with alterations in the phosphorylation state of eIF4E and/or the association of eIF4E with eIF4G and 4E-BP1. It was not clear from the earlier study whether the stimulation of protein synthesis resulted from a simple increase in caloric intake or whether one key component of the diet was the stimulus. The objective of the present study was to examine the role of the protein component of the diet in the stimulation of protein synthesis after feeding. In these studies, rats were starved for 18 h and then refed isocaloric diets containing either 20% protein or no added protein. It was found that the protein component of the diet is necessary for stimulating protein synthesis in both skeletal muscle and liver. Furthermore, the stimulation of protein synthesis caused by refeeding a diet containing 20% protein could be attributed to an increase in eIF4E availability. Finally, the effect of dietary protein on eIF4E availability and the phosphorylation state of eIF4E was not a result of changes in plasma insulin concentration. Instead, the results indicate that amino acid availability in combination with increased plasma insulin regulates translation initiation.
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MATERIALS AND METHODS |
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Animals. Male Sprague-Dawley rats (~200 g) were maintained on a 12:12-h light-dark cycle with food and water provided ad libitum. On the day before experimentation, rats were fasted for 18 h and randomly divided into three groups: fasted, 20P, and 0P. Animals were allowed free access to water throughout the experiment. Animals in the fasted group were killed without refeeding. Animals in the 20P group were fed a complete diet containing 20% protein (Harlan Teklad, Madison, WI) for 1 h before being killed. Animals in the 0P group were fed an isocaloric protein-free diet (Harlan Teklad) for 1 h before being killed. On average, animals consumed ~25% of their daily food intake during the hour before being killed (4.5 g diet, 20P group; 3.5 g diet, 0P group).
Measurement of protein synthesis. Rates of protein synthesis were estimated with a slight modification of the flooding-dose method as described previously (17). Each rat was restrained and injected with radioisotope via a lateral tail vein. For the flooding dose, 1.0 ml of L-[3H]phenylalanine (100 µCi/ml; 150 mM in 0.9% NaCl) was injected per 100 g body weight. Five minutes after injection of radioisotope, rats were anesthetized with pentobarbital sodium. Ten minutes after injection of radioisotope, a venous blood sample (1 ml) was withdrawn from the inferior vena cava for measurement of plasma phenylalanine concentration and radioactivity. Immediately after removal of the blood sample, both gastrocnemius muscles and the liver were excised in that order and rinsed in ice-cold saline. One gastrocnemius and approximately one-half of the liver were frozen between aluminum blocks precooled to the temperature of liquid N2. The other gastrocnemius and the remainder of the liver were reserved for analysis of eIF4E association with 4E-BP1 and eIF4G as described in Quantitation of eIF4E, 4E-BP1-eIF4E, and eIF4G-eIF4E complexes. The exact time of removal of each tissue was recorded. Fractional synthesis rates were calculated as described previously.
Quantitation of eIF4E, 4E-BP1-eIF4E, and eIF4G-eIF4E
complexes. Tissues were excised as described in
Measurements of protein synthesis and
immediately weighed and homogenized in 7 volumes of
buffer
A [20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), 100 mM KCl, 0.2 mM EDTA, 2 mM ethylene
glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, 1 mM dithiothreitol, 50 mM NaF, 50 mM
-glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 0.5 mM sodium
vanadate] with either a Dounce (liver) or a Polytron homogenizer
(muscle). The association of 4E-BP1 and eIF4G with eIF4E was
quantitated in 10,000 g supernatants by protein immunoblot analysis after immunoprecipitation of the eIF4E-4E-BP1 and eIF4E-eIF4G complexes with a monoclonal antibody to
eIF4E exactly as described previously (34).
Quantitation of phosphorylated and unphosphorylated eIF4E in extracts of skeletal muscle and liver. To examine the phosphorylation state of eIF4E in extracts of gastrocnemius muscle or liver, the phosphorylated and unphosphorylated forms of the protein were separated by isoelectric focusing on a slab gel and quantitated by protein immunoblot analysis as described previously (34).
Examination of 4E-BP1 phosphorylation state in extracts of skeletal muscle and liver. 4E-BP1 was immunoprecipitated from 10,000 g supernatants of skeletal muscle and liver with an anti-4E-BP1 monoclonal antibody and then was subjected to protein immunoblot analysis as described previously (34). Previous studies showed that phosphorylation of 4E-BP1 causes a decrease in the electrophoretic mobility of the protein on SDS-polyacrylamide gels (14, 21). Thus 4E-BP1 was separated into multiple electrophoretic forms during SDS-polyacrylamide gel electrophoresis, with the more slowly migrating forms representing more highly phosphorylated 4E-BP1.
Statistical analyses. Data are means ± SE. ANOVA was performed to determine whether there were significant (P < 0.05) differences among the groups. When an ANOVA indicated a significant difference among the means, the Tukey-Kramer Multiple Comparisons Test was used to determine which means were significantly different.
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RESULTS |
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Previous studies demonstrate that feeding after an overnight fast stimulates protein synthesis in skeletal muscle and liver (26, 38). In the present study, the mechanism through which protein synthesis is regulated by the protein component of a complete diet was examined. As shown in Fig. 1, 1 h after 18 h-fasted rats were refed a 20P diet, protein synthesis significantly increased in both muscle and liver. In contrast, refeeding an isocaloric diet lacking protein had no effect on protein synthesis in either tissue. The lack of effect of the 0P diet on protein synthesis was not a result of a differential intake of the two diets, because the animals consumed similar amounts of both diets over the 1-h feeding period. Instead, the difference was reflective of the amount of protein consumed.
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Numerous studies show that insulin stimulates protein synthesis both in vivo and in vitro (2, 7-9, 11, 12, 33). To determine whether or not plasma insulin concentrations were proportional to protein synthesis, the concentration of the hormone in plasma from animals fed either a 20P or a 0P diet was measured. As shown in Fig. 2, refeeding either a 20P or 0P diet increased plasma insulin concentration ~2.5-fold. Thus the rise in plasma insulin appears dependent on caloric intake but independent of protein ingestion. Furthermore, an increase in plasma insulin was not sufficient to stimulate protein synthesis in either muscle or liver in rats fed a 0P diet. However, this result does not exclude the possibility that an increase in plasma insulin may be required for the stimulation of protein synthesis by the 20P diet.
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In an earlier study (39), we found that feeding a complete diet to rats
starved for 18 h stimulated protein synthesis; concomitantly, eIF4E
redistributed from an inactive complex with 4E-BP1 to the active
eIF4E-eIF4G complex. Therefore, in the present study, the role of
dietary protein in regulating the eIF4E distribution between the
eIF4E-4E-BP1 and eIF4E-eIF4G complexes was examined. As shown in Fig.
3, two forms of 4E-BP1, referred to as and
, were associated with eIF4E in both muscle and liver. Refeeding
a 20P diet to postabsorptive rats significantly decreased the amount of
4E-BP1 associated with eIF4E in both tissues (Fig. 3). In contrast,
refeeding a 0P diet had no effect on the association of 4E-BP1 and
eIF4E. Thus the increased plasma insulin caused by refeeding a 0P diet
was not sufficient to alter the association of 4E-BP1 and eIF4E.
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The association of 4E-BP1 and eIF4E is regulated by changes in the
phosphorylation state of 4E-BP1 where the most highly phosphorylated or
-form does not bind to eIF4E (21, 25). In the present study,
refeeding the rats a 20P diet caused a significant increase in the
amount of 4E-BP1 in the
-form in both muscle and liver compared with
fasted or 0P animals (Fig. 4). In contrast,
refeeding the rats a 0P diet had no effect on 4E-BP1 distribution among the phosphorylated forms. In muscle, the increase in 4E-BP1 in the
-form was associated with a decrease in the amount in the
-form
with no change in the amount in the
-form. In liver, refeeding the
rats a 20P diet caused a reduction in the amount of 4E-BP1 in both the
- and
-forms. The functional significance, if any, of the
differential change in 4E-BP1 distribution between the
- and
-forms in muscle and liver is unknown. However, as shown in Fig. 3,
both forms bind to eIF4E and presumably prevented binding of eIF4G to
eIF4E.
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The effect of dietary protein on the binding of eIF4G to eIF4E was examined by measuring the amount of eIF4G in the eIF4E immunoprecipitates. As noted previously (18, 37), eIF4G resolves into multiple electrophoretic forms migrating at 200-220 kDa (Fig. 5). Refeeding a 20P diet caused an increase in the amount of eIF4G present in the eIF4E immunoprecipitate in both muscle and liver, whereas refeeding a 0P diet was without effect in either tissue (Fig. 5). The results suggest that refeeding a 20P diet stimulated protein synthesis in both skeletal muscle and liver in part by promoting formation of the eIF4F complex.
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To further define the effects of refeeding dietary protein on translation initiation, the phosphorylation state of eIF4E was examined. Refeeding a 20P diet resulted in partial dephosphorylation of eIF4E in both tissues (Fig. 6). In liver, but not muscle, refeeding a 0P diet also resulted in partial dephosphorylation of eIF4E. The results suggest that, at least in liver, phosphorylation of eIF4E was independent of changes in protein synthesis after refeeding.
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DISCUSSION |
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Previous studies show that refeeding a complete diet to starved animals stimulates protein synthesis in skeletal muscle (26, 38) and liver (38). In the present study, the role of dietary protein in the stimulation of protein synthesis was examined. Refeeding a 20P diet stimulated protein synthesis in both muscle and liver. In contrast, refeeding a 0P diet did not stimulate protein synthesis in either tissue despite the fact that a 0P diet was as effective at raising plasma insulin concentration as was a 20P diet. However, it is important to note that one of us [Yoshizawa et al.(38)] has shown that, although refeeding a protein-containing diet increased total plasma amino acid concentration 220%, refeeding a protein-free diet resulted in a decrease to ~60% of the 18 h-fasted value. Thus it is possible that, in part, the failure of insulin to stimulate protein synthesis in animals fed a 0P diet may be due to a decrease in amino acid availability. Overall, the results suggest that the stimulation of protein synthesis in response to food intake is not necessarily mediated by insulin alone but instead is regulated by the increase in plasma amino acid concentration that occurs after feeding a 20P diet in combination with increased plasma insulin concentrations.
The stimulation of protein synthesis caused by refeeding a complete diet is associated with activation of the mRNA cap-binding protein eIF4E [(40); present study]. There are currently two characterized mechanisms through which the activity of eIF4E can be regulated. In the first case, phosphorylation of eIF4E promotes binding of the protein to the m7GTP cap structure (23). The phosphorylation state of eIF4E is increased in cells in culture in response to a variety of stimuli, including growth factors, mitogenes, hormones, and cytokines, and is positively correlated with changes in protein synthesis (27, 32). However, in vivo studies do not support the results obtained with cells in culture. The proportion of phosphorylated eIF4E is the same in skeletal muscle from control, diabetic, or insulin-treated diabetic rats (16). Likewise, the proportion of phosphorylated eIF4E in muscle is unchanged by an overnight fast or refeeding (34, 39). Interestingly, insulin-stimulated protein synthesis in perfused muscle preparations is associated with a decrease, rather than an increase, in the proportion of phosphorylated eIF4E (16). In the present study, refeeding a 20P diet also resulted in net dephosphorylation of eIF4E. The basis for the decrease in eIF4E phosphorylation is unknown. However, in insulin-treated 3T3 cells in culture, the magnitude of the increase in 32Pi into eIF4E caused by insulin is much greater than the increase in the proportion of the protein in the phosphorylated form (29, 30). On the basis of these studies, it has been proposed that the rate of phosphate turnover on eIF4E rather than its net phosphorylation is important in controlling protein synthesis. Thus the decrease in the amount of eIF4E in the phosphorylated form observed in the present study may be a result of stimulation of both protein kinase and phosphatase activities, with the latter predominating.
The second mechanism through which the activity of eIF4 regulates translation initiation involves modulation of the availability of eIF4E. In vitro studies with recombinant proteins suggest that either 4E-BP1 or eIF4G can individually bind to eIF4E, but both proteins cannot bind simultaneously (13, 22). Thus 4E-BP1 competes with eIF4G for association with eIF4E. The binding of 4E-BP1 to eIF4E does not prevent the binding of eIF4E to mRNA but does prevent binding of the eIF4E-mRNA complex to eIF4G (19). The results of the present study were in agreement with the in vitro studies. In both skeletal muscle and liver from rats refed a 20P diet, the amount of 4E-BP1 bound to eIF4E decreased, whereas the amount of eIF4G bound to eIF4E increased. The changes in binding of eIF4E to 4E-BP1 and eIF4G in both skeletal muscle and liver were associated with alterations in the phosphorylation state of 4E-BP1. Refeeding a 20P diet caused a significant increase in the amount of 4E-BP1 present in the most phosphorylated form. Phosphorylation of 4E-BP1 in vitro by either mitogen-activated protein kinase (21) or the mammalian target of rapamycin (mTOR) kinase (3) greatly reduces the affinity of eIF4E for 4E-BP1. Whether feeding activates one of these two protein kinases or an as yet unidentified 4E-BP1 protein kinase remains to be determined. However, a recent study shows that refeeding starved mice activates the signaling pathway of which mTOR is a component in muscle from both control and diabetic animals (34). This finding suggests that activation of mTOR by exogenous signals distinct from insulin, such as dietary amino acids, may be responsible for the observed increase in 4E-BP1 phosphorylation in response to refeeding.
In both incubated diaphragm muscles (1) and in perfused hindlimb preparations (16), insulin causes an increase in the proportion of 4E-BP1 in the most highly phosphorylated form with a concomitant decrease in the amount of 4E-BP1 and increase in the amount of eIF4G bound to eIF4E. Furthermore, in the present study an increase in plasma insulin was positively correlated with increased 4E-BP1 phosphorylation in rats fed a 20P diet. In contrast, the increased plasma insulin concentration observed in rats fed a 0P diet had no effect on 4E-BP1 phosphorylation or association of eIF4E with 4E-BP1 or eIF4G. However, in the earlier studies, the concentration of insulin used was pharmacological rather than the physiological concentrations of the present study. Thus physiological concentrations of insulin may not be sufficient to activate the kinases responsible for phosphorylating 4E-BP1.
In summary, the results reported herein demonstrate that the protein component of the diet is necessary for stimulating translation initiation in both skeletal muscle and liver. Refeeding a 20P diet to 18 h-fasted rats for 1 h causes an increase in protein synthesis in both skeletal muscle and liver, whereas refeeding a 0P diet does not. The stimulation of protein synthesis that occurs in both skeletal muscle and liver is associated with an increase in the proportion of 4E-BP1 in the most highly phosphorylated form, leading to a decrease in formation of the inactive 4E-BP1-eIF4E complex and an increase in formation of the active eIF4G-eIF4E complex. Plasma insulin concentrations were the same after refeeding either a 20P or 0P diet, suggesting that a combination of dietary protein and increased plasma insulin concentration may be required to stimulate translation initiation in muscle and liver.
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ACKNOWLEDGEMENTS |
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We are grateful to Sharon Rannels, Lynne Pletcher, and Rebecca Eckman for technical assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Diabetes and Digestive and Kidney Disease Grants DK-13499 and DK-15658 (to L. S. Jefferson) and National Institute of General Medical Services Grant GM-39277 (to T. C. Vary).
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: L. S. Jefferson, Dept. of Cellular and Molecular Physiology, Penn. State Univ., College of Medicine, PO Box 850, Hershey, PA 17033.
Received 29 April 1998; accepted in final form 17 July 1998.
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