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Modulation of Translation Initiation in Rat Skeletal Muscle and Liver in Response to Food Intake
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
240, 825–831 (1997)
RC977652
Modulation of Translation Initiation in Rat Skeletal Muscle and Liver in Response to Food Intake Fumiaki Yoshizawa, Scot R. Kimball, and Leonard S. Jefferson Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Received October 6, 1997
Protein synthesis is altered in both skeletal muscle and liver in response to nutritional status with food deprivation being associated with an inhibition of mRNA translation. In the present study, the effect of food-intake on the initiation of mRNA translation was examined in rats fasted for 18-h and then refed a complete diet. Fasting and refeeding caused alterations in translation initiation in both skeletal muscle and liver that were not associated with any detectable changes in the activity of eIF2B or in the phosphorylation state of eIF2a. Instead, alterations in initiation were associated with changes in the phosphorylation state of eIF4E and/or the association of eIF4E with eIF4G as well as the eIF4E binding protein, 4E-BP1. In muscle from fasted rats, the amount of eIF4E present in an inactive complex with 4E-BP1 was increased 5-fold compared to freely fed control animals. One hour after refeeding a complete diet, the amount of 4E-BP1 bound to eIF4E was reduced to freely fed control values. Reduced association of the two proteins was the result of increased phosphorylation of 4E-BP1. Refeeding a complete diet also stimulated the binding of eIF4E to eIF4G to form the active eIF4F complex. In liver, the amount of eIF4E associated with eIF4G, but not the amount of eIF4E associated with 4E-BP1, was altered by fasting and refeeding. Furthermore, in liver, but not in skeletal muscle, fasting and refeeding resulted in modulation of the phosphorylation state of eIF4E. Overall, the results suggest that protein synthesis may be differentially regulated in muscle and liver in response to fasting and refeeding. In muscle, protein synthesis is regulated through modulation of the binding of eIF4E to eIF4G and in liver through modulation of both phosphorylation of eIF4E as well as binding of eIF4E to eIF4G. q 1997 Academic Press
Starvation is associated with an inhibition of protein synthesis in both skeletal muscle and liver (1, 2). Feeding a diet containing a mixture of carbohydrate, lipid, and protein to starved rats rapidly restores protein syn-
thesis both tissues to values observed in freely-fed animals (3, 4). The acute increase in protein synthesis in response to refeeding occurs through a stimulation of translation of mRNA. Recent studies (4-6) have shown that refeeding stimulates translation by increasing the rate at which the synthesis of new polypeptide chains is initiated as opposed to a stimulation of polypeptide elongation or termination. Translation initiation is a complex process comprising a set of reactions leading to formation of an 80S initiation complex (7). At least 12 proteins termed eukaryotic initiation factors (abbreviated eIF) are involved in the process. Two particular steps in the initiation pathway appear to be important in the physiological regulation of translation, i.e. the binding of initiator methionyl-tRNAi (met-tRNAi) to the 40S ribosomal subunit, a reaction mediated by eIF2, and the initial binding of the 40S ribosome to the 5* end of mRNA, a reaction mediated by eIF4E. The first step involves formation of a ternary complex consisting of eIF2, GTP, and met-tRNAi (8). Formation of the ternary complex is controlled by the interaction of eIF2 with a second initiation factor, eIF2B, which catalyzes the exchange of GDP bound to eIF2 for free GTP. Modulation of the activity of eIF2B is an important mechanism for regulating protein synthesis (8). In many cases, the activity of eIF2B is regulated by changes in the phosphorylation state of the a-subunit of eIF2 (8, 9, 10). The second step involves the binding of mRNA to the 40S ribosomal subunit through the interaction of a family of proteins collectively refered to as eIF4. One of the eIF4 family members, eIF4E, plays an important role in the binding of mRNA to the 40S ribosomal subunit because it is the first initiation factor to bind to the m7GTP cap present at the 5*-end of eukaryotic mRNAs. It is also reported to be the least abundant of the known initiation factors (11), and increasing or decreasing the cellular content of eIF4E results in a stimulation or reduction, respectively, in protein synthesis (12, 13). During translation initiation, the eIF4ErmRNA complex binds to eIF4G and eIF4A to form the active
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eIF4F complex (12-14). The eIF4FrmRNA complex then binds to the 40S ribosomal subunit through the interaction between eIF4G and eIF3 that is already bound to the 40S ribosomal subunit. Recent studies have shown that the activity of eIF4E is regulated by an eIF4E binding protein termed 4E-BP1. eIF4E bound to 4E-BP1 cannot bind to eIF4G and thus cannot bind to the 40S ribosomal subunit. The binding of eIF4E to 4E-BP1 is regulated in part by phosphorylation of 4EBP1 with hyperphosphorylation resulting in dissociation of the 4E-BP1reIF4E complex. Finally, several studies have shown a direct correlation between protein synthesis and the phosphorylation state of eIF4E (12, 13). In the present study, the mechanism through which food intake stimulates translation initiation was examined in both skeletal muscle and liver in vivo. It was found that the amount of eIF4G bound to eIF4E was decreased following an 18-h fast in both skeletal muscle and liver. The binding of eIF4E to eIF4G was rapidly stimulated in both tissues in response to refeeding. In muscle, but not liver, the changes in eIF4E binding to eIF4G were associated with reciprocal changes in the binding of 4E-BP1 to eIF4E. The changes in 4E-BP1 binding to eIF4E were the result of alterations in the phosphorylation state of 4E-BP1. In liver, but not muscle, fasting and refeeding caused a decrease in the amount of eIF4E in the phosphorylated form, suggesting that the activity of an eIF4E kinase and/or phosphatase is regulated in liver in response to food intake.
GreIF4E and 4E-BP1reIF4E complexes were immunoprecipitated from aliquots of 10,000 g supernatants of gastrocnemius muscle and liver using an anti-eIF4E monoclonal antibody (18). The recovery of eIF4E in the immunoprecipitate was greater than 90% using this protocol. The antibody-antigen complex was collected by incubation for 1h with Biomag goat anti-mouse immunoglobulin G beads (Per-Septive Diagnostics). Before use, the beads were washed in 0.1% nonfat dry milk in buffer B [50mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.4, 150mM NaCl, 5mM EDTA, 0.1%b-mercaptoethanol, 0.5% Triton X-100, 50mM NaF, 50mM b-glycerophosphate, 0.1mM phenylmethylsulfonyl fluoride, 1mM benzamidine, and 0.5mM sodium vanadate]. The beads were captured using a magnetic stand and were washed twice with buffer B and once with buffer B containing 500mM NaCl rather than 150mM. Protein bound to the beads was eluted by resuspending the beads in SDS sample buffer and boiling the sample for 5 min. 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 a 15% polyacrylamide gel for quantitation of eIF4E and 4E-BP1. Proteins in the gels were then electrophoretically transferred to polyvinylidene difluoride membranes, and the membranes were incubated with either an anti-eIF4E monoclonal antibody, a rabbit anti-rat eIF4G polyclonal antibody, or a rabbit anti-rat 4E-BP1 polyclonal antibody for 1 h at room temperature or overnight at 47C. The blots were then developed using an ECL Western blotting kit as described previously (18).
MATERIALS AND METHODS
Examination of 4E-BP1 phosphorylation state in extracts of liver and skeletal muscle. 4E-BP1 was immunoprecipitated from 10,000 1 g supernatants of skeletal muscle and liver using an anti4E-BP1 monoclonal antibody, and then was subjected to protein immunoblot analysis as described above. Previous studies have shown that phosphorylation of 4E-BP1 causes a decrease in the electrophoretic mobility of the protein on SDS-polyacrylamide gels (19, 20). Thus, 4E-BP1 present in tissue extracts was separated into multiple electrophoretic forms during SDS-polyacrylamide gel electrophoresis, with the more slowly migrating forms representing more highly phosphorylated 4E-BP1.
Animals. Male Sprague-Dawley rats ( Ç200g ) were maintained on a 12:12 h light-dark cycle with food and water provided ad libitum. On the day before experimentation, rats were randomly divided into three groups. The first group was provided standard rat chow and water ad libitum. The second group was fasted for 18 hours prior to sacrifice; the animals were allowed free access to water during the fast. The third group was similarly fasted, but in addition, were fed standard rat chow 1 h prior to sacrifice. Rats were anesthetized with sodium pentobarbital and the gastrocnemius muscle and liver were removed and rinsed in ice-cold saline. Tissues were immediately weighed and homogenized in 7 volumes of buffer A [in mM: 20 N-2hydroxyethylpiperazine-N*-2-ethanesulfonic acid (pH 7.4), 100 KCl, 0.2 EDTA, 2 ethylene glycol-bis(B-aminoethyl ether)-N,N,N*,N*-tetraacetic acid, 1 dithiothreitol (DTT), 50 NaF, 50 B-glycerophosphate, 0.1 phenylmethylsulfonyl fluoride, 1 benzamidine, and 0.5 sodium vanadate] using either a Dounce homogenizer (liver) or a Polytron homogenizer (muscle). The homogenates were centrifuged at 10,000g for 10 min at 47C.
Quantitation of phosphorylated and unphosphorylated eIF4E in extracts of liver and skeletal muscle. 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 were quantitated by protein immunoblot analysis as described previously (17). Films were scanned using a Microtek ScanMaker III scanner equipped with a transparent media adapter connected to a Power Macintosh 7100/ 80 computer. Images were obtained using Scan Wizard Plugin (Microtek) for Adobe Photoshop and quantitated using NIH Image 1.60 software.
Statistical analyses. The data are expressed as means { SE. ANOVA was performed to determine whether there were significant (põ0.05) differences among the groups. When an ANOVA indicated any significant difference among the means, Tukey-Kramer Multiple comparisons Test was also used to determine which means were significantly different. Student’s t test was also used to determine the differences between the fasted and refed groups.
Measurement of eIF2B activity. eIF2B activity in skeletal muscle (15) and liver (16) were measured exactly as previously described.
RESULTS
Quantitation of eIF4E, 4E-BP1reIF4E, and eIF4GreIF4E complexes. Aliquots of 10,000 g supernatants of muscle or liver were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and eIF4E, eIF4G, and 4E-BP1 were detected by protein immunoblot analysis as described previously (17). The association of 4E-BP1 and eIF4G with eIF4E was quantitated by the method described previously (17). Briefly, eIF4E and the eIF4-
Previous studies have shown that translation initiation is altered in skeletal muscle and liver in response to the availability of essential amino acids (21,22) and insulin (23), respectively. In both tissues in vitro, translation initiation is regulated in part through changes in the activity of eIF2B. In the present study, the activ-
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and b-forms) were found associated with eIF4E in extracts of either skeletal muscle or liver. In skeletal muscle of 18-h fasted rats, the amount of eIF4E present in an inactive complex with 4E-BP1 was significantly increased compared to freely fed animal. One hour after refeeding, the amount of 4E-BP1 bound to eIF4E was reduced to control values. In liver there was a trend toward increased binding of 4E-BP1 to eIF4E in response to fasting, however, the difference between freely fed and fasted animals was not significant. In order to examine the effect of fasting and refeeding on the phosphorylation state of 4E-BP1, extracts of skeletal muscle and liver were immunoprecipitated with an anti-4E-BP1 monoclonal antibody. The immunoprecipitates were then subjected to SDS-polyacrylamide gel electrophoresis to resolve the phosphorylated forms of the protein. As shown in Figure 3, in muscle, an 18-h fast caused a significant decrease in the amount of 4E-BP1 in the most phosphorylated, or g, form with a corresponding increase in the amount in the least phosphorylated, or a, form. One hour after FIG. 1. Effect of fasting and refeeding on eIF2B activity. eIF2B activity was measured in 10,000 1 g supernatants by the GDP exchange assay as described under Materials and Methods. Results are expressed as the amount of [3H]GDP bound to eIF2 exchanged for nonradiolabeled GDP with time and are normalized for the amount of tissue protein added to the assay. The results represent the average of samples from five animals per condition.
ity of eIF2B was measured in extracts of muscle and liver from starved and refed rats and compared to the activity in tissue extracts from control animals. As shown in Figure 1, the activity of eIF2B was unaffected by either an 18-h fast or refeeding. Likewise, the amount of eIF2a in the phosphorylated form was unchanged in response to fasting or refeeding (results not shown). A second step in translation initiation that has been shown to be subject to acute regulation is the binding of mRNA to the 40S ribosomal subunit, a reaction mediated by eIF4E. In cells in culture treated with insulin, the activity of eIF4E is regulated through changes in the phosphorylation state of eIF4E as well as by modulation of the amount of eIF4E bound to 4E-BP1 (24). The association of eIF4E and 4E-BP1 is controlled through changes in the phosphorylation state of 4EBP1. The phosphorylation state of 4E-BP1 in cell or tissue extracts can be easily assessed by protein immunoblot analysis following SDS-polyacrylamide gel electrophoresis. During electrophoresis, 4E-BP1 is resolved into multiple electrophoretic forms with changes in electrophoretic mobility being inversely correlated with the number of phosphate groups on the protein. As shown in Figure 2, only the two forms exhibiting the greatest electrophoretic mobility (designated the a-
FIG. 2. Effect of fasting and refeeding on the association of 4EBP1 and eIF4E in skeletal muscle and liver. eIF4E was immunoprecipitated from 10,000 g supernatants of skeletal muscle and liver with an anti-eIF4E monoclonal antibody. Immunoprecipitates were then subjected to protein immunoblot analysis using a 4E-BP1 monoclonal antibody. Results represent means { SE for 5 animals per condition. Values not sharing the same superscript letter are significantly different (põ0.05) by Tukey-Kramer Multiple comparisons Test. * indicates significant differences from fasting groups (põ0.05) by Student’s t test. Insets: results of representative immunoblots. Lanes represent samples from freely fed (Fe), 18-h fasted (Fa), and refed (Re) animals.
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tein synthesis in both skeletal muscle and liver in part by promoting formation of the eIF4F complex. To further define the effects of fasting and refeeding on translation initiation, the phosphorylation state of eIF4E was examined. In muscle, the amount of eIF4E in the phosphorylated form was unaffected by either an 18-h fast or refeeding (Fig. 5). In contrast, in liver, an 18-h fast resulted in a partial dephosphorylation of eIF4E. Refeeding caused a further decrease in the proportion of eIF4E in the phosphorylated form. DISCUSSION Protein synthesis is regulated by changes in the activity of eIF2B under a wide variety of conditions (8). In cells in culture, the activity of eIF2B is regulated by changes in the phosphorylation state of a-subunit of eIF2 (8,25,26). However, little information is available concerning the role of phosphorylation of eIF2a in controlling translation in whole tissues or the intact animal. In one study, protein synthesis was shown to be
FIG. 3. Effect of fasting and refeeding on the phosphorylation state of 4E-BP1 in skeletal muscle and liver. Tissue extracts were subjected to SDS-polyacrylamide gel electrophoresis and the phosphorylation state of 4E-BP1 was determined by protein immunoblot analysis as described under Materials and Methods. Results represent means { SE for five animals per condition. Values not sharing the same superscript letter are significantly different (põ0.05) by Tukey-Kramer Multiple comparisons Test. Insets: results of representative immunoblots. Lanes represent samples from freely fed (Fe), 18-h fasted (Fa), and refed (Re) animals.
refeeding, the distribution of 4E-BP1 among the phosphorylated forms returned to the control pattern. In contrast to skeletal muscle, no change in the amount of 4E-BP1 in the g-form was observed in liver following an 18-h fast. However, fasting did result in an increase in the proportion of the protein in the a-form. Surprisingly, refeeding caused both a decrease in the amount of 4E-BP1 in the a-form as well as an increase in the amount in the g-form. The effect of food-intake on the binding of eIF4G to eIF4E was also examined by measuring the amount of eIF4G associated with eIF4E when eIF4E was immunoprecipitated using a monoclonal anti-eIF4E antibody. Fasting caused a decrease in the amount of eIF4G present in the eIF4E immunoprecipitate in both muscle and liver compared to the freely-fed, control condition (Fig. 4), although the difference was not significant in muscle. Refeeding significantly increased the amount of eIF4G bound to eIF4E in both muscle and liver. These findings suggest that food-intake stimulates pro-
FIG. 4. Effect of fasting and refeeding on association of eIF4G and eIF4E in skeletal muscle and liver. eIF4E was immunoprecipitated from 10,000 1 g supernatants of skeletal muscle and liver as described in the legend to Fig. 2. Immunoprecipitates were then subjected to protein immunoblot analysis using an anti-eIF4G polyclonal antibody. Results represent means { SE for five animals per condition. Values not sharing the same superscript letter are significantly different (põ0.05) by Tukey-Kramer Multiple comparisons Test. * indicates significant differences from fasting groups (põ0.05) by Student’s t test. Insets: results of representative immunoblots. Lanes represent samples from freely fed (Fe), 18-h fasted (Fa), and refed (Re) animals.
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FIG. 5. Effect of fasting and refeeding on the phosphorylation state of eIF4E in skeletal muscle and liver. The amount of eIF4E present in the phosphorylated form in skeletal muscle and liver was determined by protein immunoblot analysis after slab gel isoelectric focusing. Results represent means { SE for five animals per condition. Values not sharing the same superscript letter are significantly different (põ0.05) by Tukey-Kramer Multiple comparisons Test. Insets: results of representative immunoblots. Lanes represent samples from freely fed (Fe), 18-h fasted (Fa), and refed (Re) animals.
inhibited in perfused rat livers deprived of single amino acids (27). The probable basis for the inhibition of protein synthesis in response to amino acid deprivation was an increase in phosphorylation of eIF2a with a concomitant decrease in eIF2B activity (28). In contrast, it was reported that 48-h of starvation had no effect on the activity of eIF2B or on phosphorylation of eIF2a in muscle even though protein synthesis was decreased (29). In the present study, neither fasting nor refeeding had any effect on the activity of eIF2B (Fig. 1) or on the proportion of eIF2a in the phosphorylated form (data not shown). Therefore, the effects of fasting or refeeding on translation initiation in both skeletal muscle and liver involve alterations in other components of the translation machinery. The binding of mRNA to the 40S ribosomal subunit is another tightly regulated step in the initiation of translation (30). This step involves the binding of eIF4E to the m7GTP cap at the 5* end of mRNA and the subsequent binding of the eIF4ErmRNA complex to a second initiation factor, eIF4G. Two different mechanisms for controlling the binding of mRNA to the 40S ribosome subunit have been reported, each of which involves reversible phosphorylation of the pro-
teins involved in the process. In the first case, phosphorylation of the eIF4E binding protein, 4E-BP1, regulates translation initiation by increasing the availability of eIF4E. eIF4E bound to 4E-BP1 can bind to mRNA through the m7GTP cap structure, but the eIF4Er4E-BP1 complex cannot bind to eIF4G (20,31). Thus, 4E-BP1 competes with eIF4G for association to eIF4E. In the second case, phosphorylation of eIF4E promotes binding of the protein to the m7GTP cap structure (32). Previous studies have reported that increased incorporation of 32P into eIF4E is observed in response to a variety of stimuli, including with growth factor, mitogenes, hormone, and cytokines (12,13) and is positively correlated with changes in protein synthesis (12,13). In particular, insulin stimulates the incorporation of 32P into eIF4E, although the magnitude of the increase in 32P incorporation is much greater than the increase in the proportion of the protein in the phosphorylated form (33,34). On the basis of these studies, it has been proposed that the rate of eIF4E phosphorylation rather than its net phosphorylation state is important in controlling protein synthesis. In the present study, the finding in liver that refeeding caused a significant decrease in eIF4E phosphorylation may reflect an increase in the overall rate of phosphate turnover on the protein with the stimulation of eIF4E phosphatase activity being greater than the increase in eIF4E kinase activity. In contrast to the changes in eIF4E phosphorylation observed in liver, no change in eIF4E phosphorylation was observed in muscle, suggesting that protein synthesis in muscle is regulated by mechanism(s) distinct from changes in eIF4E phosphorylation. Instead, in muscle, fasting and refeeding caused changes in the association of eIF4E with 4E-BP1 and eIF4G. In vitro studies using recombinant proteins have suggested that either 4E-BP1 or eIF4G can individually bind to eIF4E, but both proteins cannot bind simultaneously (31,35). The results of the present study using skeletal muscle are in agreement with the in vitro results. In muscle from fasted rats, the amount of 4E-BP1 bound to eIF4E increases whereas the amount of eIF4G bound to eIF4E decreases. Conversely, refeeding results in a reduction in the amount of 4E-BP1 bound to eIF4E with a concomitant increase in the amount of eIF4G associated with the protein. The changes in binding of eIF4E to 4E-BP1 and eIF4G in muscle are associated with alterations in the phosphorylation state of 4EBP1. Phosphorylation of 4E-BP1 in vitro by either mitogen-activated protein (MAP) kinase (20) or the mammalian target of rapamycin (mTOR) kinase (36) 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 is unknown. However, studies in mice have shown that refeeding starved animals activates the signaling pathway of which mTOR is a component, suggesting that activa-
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tion of mTOR may be responsible for the observed increase in 4E-BP1 phosphorylation in response to refeeding. In contrast to the results in muscle, in liver the amount of eIF4G bound to eIF4E changes in response to fasting and refeeding in the absence of changes in the amount of 4E-BP1 bound to eIF4E. This result suggests that in liver there is a pool of eIF4E that is not associated with either 4E-BP1 or eIF4G and that starvation causes a dissociation of the eIF4GreIF4E complex with a resulting increase in the amount of ‘free’ eIF4E. The mechanism involved in the dissociation of eIF4E from eIF4G in the absence of changes in the association of 4E-BP1 and eIF4E is unknown. However, it has been suggested that alterations in the phosphorylation state of eIF4E might modulate the interaction of eIF4E and eIF4G (37,38). In addition, the finding that insulin stimulates the phosphorylation of eIF4G (39) presents the possibility that changes in phosphorylation of eIF4G in response to starvation and refeeding might play an important role in regulating the association of the two proteins. Alternatively, although the amount of eIF4E associated with 4E-BP1 does not change in liver during fasting, the amount of eIF4E bound to a second eIF4E binding protein, 4E-BP2, may. The predominant form of eIF4E binding protein in muscle is 4E-BP1 (40). In contrast, liver has both 4E-BP1 and 4E-BP2 (40). Thus, in liver, fasting and refeeding may modulate the amount of eIF4E bound to 4E-BP2, but not 4E-BP1. Further studies will be required to examine this possibility. The signal that stimulates protein synthesis and modulates the phosphorylation state of eIF4E and 4EBP1 after refeeding is unknown. A number of studies have shown that both insulin and amino acids can play important roles in the regulation of protein synthesis (41-46). In particular, it has been postulated that the response of muscle protein synthesis to food intake is mediated primarily by increases in the plasma concentration of insulin and amino acids which act cooperatively to stimulate protein synthesis (3,4). However, the role of insulin in stimulating protein synthesis in muscle after refeeding has been questioned in a recent study using diabetic mice (17). In that study, refeeding starved mice stimulated protein synthesis to a similar extent in muscle from both control and diabetic animals. Thus, in muscle, amino acids may be the primary mediators of the increase in protein synthesis in response to refeeding. It has also been suggested that the stimulation of liver protein synthesis in response to food intake is not necessarily mediated by insulin, but instead is regulated by the increase in plasma amino acid concentration that occurs after feeding (4). Further studies will be needed to elucidate the role of insulin and amino acids in regulating translation initiation in response to food intake. In summary, the changes in translation initiation in
both skeletal muscle and liver with nutrition state are not associated with any detectable changes in the activity of eIF2B or in the phosphorylation state of eIF2a. Instead, the changes result from alternations in the phosphorylation state of eIF4E and 4E-BP1. In liver, fasting and refeeding modulate both the phosphorylation state of eIF4E as well as the amount of eIF4G bound to eIF4E. In contrast, in muscle the inhibition of protein synthesis that is observed in fasted and refed animals is independent of changes in eIF4E phosphorylation. Instead, changes in protein synthesis are associated with reciprocal changes in the binding of 4E-BP1 and eIF4G to eIF4E. In muscle the changes in the amount of eIF4E bound to 4E-BP1 are associated with alterations in the phosphorylation state of 4E-BP1. Each of these effects is reversed by refeeding for 1 hour. ACKNOWLEDGMENTS We are grateful to Sharon Rannels and Lynne Pletcher for technical assistance. This work was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grants DK-15658 and DK-13499.
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20. Lin, T.-A., Kong, X., Haystead, T. A. J., Pause, A., Belsham, G., Sonenberg, N., and Lawrence, J. C. (1994) Science 266, 653–656. 21. Flaim, K. E., Peavy, D. E., Everson, W. V., and Jefferson, L. S. (1982) J. Biol. Chem. 257, 2932–2938. 22. Flaim, K. E., Liao, W. S. L., Peavy, D. E., Taylor, J. M., and Jefferson, L. S. (1982) J. Biol. Chem. 257, 2939–2946. 23. Kelly, F. J., and Jefferson, L. S. (1985) J. Biol. Chem. 260, 6677– 6683. 24. Flynn, A., and Proud, C. G. (1996) Cancer Surveys 27, 293–310. 25. Proud, C. G. (1992) Current Topics Cell. Reg. 32, 243–369. 26. Hershey, J. W. B. (1989) J. Biol. Chem. 264, 20823–20826. 27. Everson, W. V., Flaim, K. E., Susco, D. M., Kimball, S. R., and Jefferson, L. S. (1989) Am. J. Physiol. 256, C18–C27. 28. Kimball, S. R., Antonetti, D. A., Brawley, R. M., and Jefferson, L. S. (1991) J. Biol. Chem. 266, 1969–1976. 29. Cox, S., Redpath, N. T., and Proud, C. G. (1988) FEBS Lett. 239, 333–338. 30. Pain, V. M. (1996) Eur. J. Biochem. 236, 747–771. 31. Haghighat, A., Mader, S., Pause, A., and Sonenberg, N. (1995) EMBO J. 14, 5701–5709. 32. Minich, W. B., Balasta, M. L., Goss, D. J., and Rhoads, R. E. (1994) Proc. Natl. Acad. Sci. USA 91, 7668–7672. 33. Rinker-Schaeffer, C. W., Austin, V., Zimmer, S., and Rhoads, R. E. (1992) J. Biol. Chem. 267, 10659–10664.
34. Rychlik, W., Rush, J. S., Rhoads, R. E., and Waechter, C. J. (1990) J. Biol. Chem. 265, 19467–19471. 35. Mader, S., Lee, H., Pause, A., and Sonenberg, N. (1995) Mol. Cell. Biol. 15, 4990–4997. 36. Brunn, G. J., Hudson, C. C., Sekulic, A., Williams, J. M., Hosoi, H., Houghton, P. J., Lawrence, J. C., and Abraham, R. T. (1997) Science 277, 99–101. 37. Morley, S. J., Dever, T. E., Etchison, D., and Traugh, J. A. (1991) J. Biol. Chem. 266, 4669–4672. 38. Morley, S. J., and Pain, V. M. (1995) J. Cell. Sci. 108, 1751– 1760. 39. Morley, S. J., and Traugh, J. A. (1990) J. Biol. Chem. 265, 10611–10616. 40. Lin, T.-A., and Lawrence, J. C. (1996) J. Biol. Chem. 271, 30199– 30204. 41. Garlick, P. J., and Grant, I. (1988) Biochem. J. 254, 579–584. 42. Stirewalt, W. S., and Low, R. B. (1983) Biochem. J. 210, 323– 330. 43. Frayn, K. N., and Maycock, P. F. (1979) Biochem. J. 184, 323– 330. 44. Fulks, R. M., Li, J. B., and Goldberg, A. L. (1975) J. Biol. Chem. 250, 290–298. 45. Garlick, P. J., Fern, M., and Preedy, V. R. (1983) Biochem. J. 210, 669–676. 46. Reeds, P. J., Hay, S. M., Glennie, R. T., Mackie, W. S., and Garlick, P. J. (1985) Biochem. J. 227, 255–261.
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Modulation of Translation Initiation in Rat Skeletal Muscle and Liver in Response to Food Intake Fumiaki Yoshizawa, Scot R. Kimball, and Leonard S. Jefferson Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Received October 6, 1997
Protein synthesis is altered in both skeletal muscle and liver in response to nutritional status with food deprivation being associated with an inhibition of mRNA translation. In the present study, the effect of food-intake on the initiation of mRNA translation was examined in rats fasted for 18-h and then refed a complete diet. Fasting and refeeding caused alterations in translation initiation in both skeletal muscle and liver that were not associated with any detectable changes in the activity of eIF2B or in the phosphorylation state of eIF2a. Instead, alterations in initiation were associated with changes in the phosphorylation state of eIF4E and/or the association of eIF4E with eIF4G as well as the eIF4E binding protein, 4E-BP1. In muscle from fasted rats, the amount of eIF4E present in an inactive complex with 4E-BP1 was increased 5-fold compared to freely fed control animals. One hour after refeeding a complete diet, the amount of 4E-BP1 bound to eIF4E was reduced to freely fed control values. Reduced association of the two proteins was the result of increased phosphorylation of 4E-BP1. Refeeding a complete diet also stimulated the binding of eIF4E to eIF4G to form the active eIF4F complex. In liver, the amount of eIF4E associated with eIF4G, but not the amount of eIF4E associated with 4E-BP1, was altered by fasting and refeeding. Furthermore, in liver, but not in skeletal muscle, fasting and refeeding resulted in modulation of the phosphorylation state of eIF4E. Overall, the results suggest that protein synthesis may be differentially regulated in muscle and liver in response to fasting and refeeding. In muscle, protein synthesis is regulated through modulation of the binding of eIF4E to eIF4G and in liver through modulation of both phosphorylation of eIF4E as well as binding of eIF4E to eIF4G. q 1997 Academic Press
Starvation is associated with an inhibition of protein synthesis in both skeletal muscle and liver (1, 2). Feeding a diet containing a mixture of carbohydrate, lipid, and protein to starved rats rapidly restores protein syn-
thesis both tissues to values observed in freely-fed animals (3, 4). The acute increase in protein synthesis in response to refeeding occurs through a stimulation of translation of mRNA. Recent studies (4-6) have shown that refeeding stimulates translation by increasing the rate at which the synthesis of new polypeptide chains is initiated as opposed to a stimulation of polypeptide elongation or termination. Translation initiation is a complex process comprising a set of reactions leading to formation of an 80S initiation complex (7). At least 12 proteins termed eukaryotic initiation factors (abbreviated eIF) are involved in the process. Two particular steps in the initiation pathway appear to be important in the physiological regulation of translation, i.e. the binding of initiator methionyl-tRNAi (met-tRNAi) to the 40S ribosomal subunit, a reaction mediated by eIF2, and the initial binding of the 40S ribosome to the 5* end of mRNA, a reaction mediated by eIF4E. The first step involves formation of a ternary complex consisting of eIF2, GTP, and met-tRNAi (8). Formation of the ternary complex is controlled by the interaction of eIF2 with a second initiation factor, eIF2B, which catalyzes the exchange of GDP bound to eIF2 for free GTP. Modulation of the activity of eIF2B is an important mechanism for regulating protein synthesis (8). In many cases, the activity of eIF2B is regulated by changes in the phosphorylation state of the a-subunit of eIF2 (8, 9, 10). The second step involves the binding of mRNA to the 40S ribosomal subunit through the interaction of a family of proteins collectively refered to as eIF4. One of the eIF4 family members, eIF4E, plays an important role in the binding of mRNA to the 40S ribosomal subunit because it is the first initiation factor to bind to the m7GTP cap present at the 5*-end of eukaryotic mRNAs. It is also reported to be the least abundant of the known initiation factors (11), and increasing or decreasing the cellular content of eIF4E results in a stimulation or reduction, respectively, in protein synthesis (12, 13). During translation initiation, the eIF4ErmRNA complex binds to eIF4G and eIF4A to form the active
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eIF4F complex (12-14). The eIF4FrmRNA complex then binds to the 40S ribosomal subunit through the interaction between eIF4G and eIF3 that is already bound to the 40S ribosomal subunit. Recent studies have shown that the activity of eIF4E is regulated by an eIF4E binding protein termed 4E-BP1. eIF4E bound to 4E-BP1 cannot bind to eIF4G and thus cannot bind to the 40S ribosomal subunit. The binding of eIF4E to 4E-BP1 is regulated in part by phosphorylation of 4EBP1 with hyperphosphorylation resulting in dissociation of the 4E-BP1reIF4E complex. Finally, several studies have shown a direct correlation between protein synthesis and the phosphorylation state of eIF4E (12, 13). In the present study, the mechanism through which food intake stimulates translation initiation was examined in both skeletal muscle and liver in vivo. It was found that the amount of eIF4G bound to eIF4E was decreased following an 18-h fast in both skeletal muscle and liver. The binding of eIF4E to eIF4G was rapidly stimulated in both tissues in response to refeeding. In muscle, but not liver, the changes in eIF4E binding to eIF4G were associated with reciprocal changes in the binding of 4E-BP1 to eIF4E. The changes in 4E-BP1 binding to eIF4E were the result of alterations in the phosphorylation state of 4E-BP1. In liver, but not muscle, fasting and refeeding caused a decrease in the amount of eIF4E in the phosphorylated form, suggesting that the activity of an eIF4E kinase and/or phosphatase is regulated in liver in response to food intake.
GreIF4E and 4E-BP1reIF4E complexes were immunoprecipitated from aliquots of 10,000 g supernatants of gastrocnemius muscle and liver using an anti-eIF4E monoclonal antibody (18). The recovery of eIF4E in the immunoprecipitate was greater than 90% using this protocol. The antibody-antigen complex was collected by incubation for 1h with Biomag goat anti-mouse immunoglobulin G beads (Per-Septive Diagnostics). Before use, the beads were washed in 0.1% nonfat dry milk in buffer B [50mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.4, 150mM NaCl, 5mM EDTA, 0.1%b-mercaptoethanol, 0.5% Triton X-100, 50mM NaF, 50mM b-glycerophosphate, 0.1mM phenylmethylsulfonyl fluoride, 1mM benzamidine, and 0.5mM sodium vanadate]. The beads were captured using a magnetic stand and were washed twice with buffer B and once with buffer B containing 500mM NaCl rather than 150mM. Protein bound to the beads was eluted by resuspending the beads in SDS sample buffer and boiling the sample for 5 min. 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 a 15% polyacrylamide gel for quantitation of eIF4E and 4E-BP1. Proteins in the gels were then electrophoretically transferred to polyvinylidene difluoride membranes, and the membranes were incubated with either an anti-eIF4E monoclonal antibody, a rabbit anti-rat eIF4G polyclonal antibody, or a rabbit anti-rat 4E-BP1 polyclonal antibody for 1 h at room temperature or overnight at 47C. The blots were then developed using an ECL Western blotting kit as described previously (18).
MATERIALS AND METHODS
Examination of 4E-BP1 phosphorylation state in extracts of liver and skeletal muscle. 4E-BP1 was immunoprecipitated from 10,000 1 g supernatants of skeletal muscle and liver using an anti4E-BP1 monoclonal antibody, and then was subjected to protein immunoblot analysis as described above. Previous studies have shown that phosphorylation of 4E-BP1 causes a decrease in the electrophoretic mobility of the protein on SDS-polyacrylamide gels (19, 20). Thus, 4E-BP1 present in tissue extracts was separated into multiple electrophoretic forms during SDS-polyacrylamide gel electrophoresis, with the more slowly migrating forms representing more highly phosphorylated 4E-BP1.
Animals. Male Sprague-Dawley rats ( Ç200g ) were maintained on a 12:12 h light-dark cycle with food and water provided ad libitum. On the day before experimentation, rats were randomly divided into three groups. The first group was provided standard rat chow and water ad libitum. The second group was fasted for 18 hours prior to sacrifice; the animals were allowed free access to water during the fast. The third group was similarly fasted, but in addition, were fed standard rat chow 1 h prior to sacrifice. Rats were anesthetized with sodium pentobarbital and the gastrocnemius muscle and liver were removed and rinsed in ice-cold saline. Tissues were immediately weighed and homogenized in 7 volumes of buffer A [in mM: 20 N-2hydroxyethylpiperazine-N*-2-ethanesulfonic acid (pH 7.4), 100 KCl, 0.2 EDTA, 2 ethylene glycol-bis(B-aminoethyl ether)-N,N,N*,N*-tetraacetic acid, 1 dithiothreitol (DTT), 50 NaF, 50 B-glycerophosphate, 0.1 phenylmethylsulfonyl fluoride, 1 benzamidine, and 0.5 sodium vanadate] using either a Dounce homogenizer (liver) or a Polytron homogenizer (muscle). The homogenates were centrifuged at 10,000g for 10 min at 47C.
Quantitation of phosphorylated and unphosphorylated eIF4E in extracts of liver and skeletal muscle. 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 were quantitated by protein immunoblot analysis as described previously (17). Films were scanned using a Microtek ScanMaker III scanner equipped with a transparent media adapter connected to a Power Macintosh 7100/ 80 computer. Images were obtained using Scan Wizard Plugin (Microtek) for Adobe Photoshop and quantitated using NIH Image 1.60 software.
Statistical analyses. The data are expressed as means { SE. ANOVA was performed to determine whether there were significant (põ0.05) differences among the groups. When an ANOVA indicated any significant difference among the means, Tukey-Kramer Multiple comparisons Test was also used to determine which means were significantly different. Student’s t test was also used to determine the differences between the fasted and refed groups.
Measurement of eIF2B activity. eIF2B activity in skeletal muscle (15) and liver (16) were measured exactly as previously described.
RESULTS
Quantitation of eIF4E, 4E-BP1reIF4E, and eIF4GreIF4E complexes. Aliquots of 10,000 g supernatants of muscle or liver were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and eIF4E, eIF4G, and 4E-BP1 were detected by protein immunoblot analysis as described previously (17). The association of 4E-BP1 and eIF4G with eIF4E was quantitated by the method described previously (17). Briefly, eIF4E and the eIF4-
Previous studies have shown that translation initiation is altered in skeletal muscle and liver in response to the availability of essential amino acids (21,22) and insulin (23), respectively. In both tissues in vitro, translation initiation is regulated in part through changes in the activity of eIF2B. In the present study, the activ-
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and b-forms) were found associated with eIF4E in extracts of either skeletal muscle or liver. In skeletal muscle of 18-h fasted rats, the amount of eIF4E present in an inactive complex with 4E-BP1 was significantly increased compared to freely fed animal. One hour after refeeding, the amount of 4E-BP1 bound to eIF4E was reduced to control values. In liver there was a trend toward increased binding of 4E-BP1 to eIF4E in response to fasting, however, the difference between freely fed and fasted animals was not significant. In order to examine the effect of fasting and refeeding on the phosphorylation state of 4E-BP1, extracts of skeletal muscle and liver were immunoprecipitated with an anti-4E-BP1 monoclonal antibody. The immunoprecipitates were then subjected to SDS-polyacrylamide gel electrophoresis to resolve the phosphorylated forms of the protein. As shown in Figure 3, in muscle, an 18-h fast caused a significant decrease in the amount of 4E-BP1 in the most phosphorylated, or g, form with a corresponding increase in the amount in the least phosphorylated, or a, form. One hour after FIG. 1. Effect of fasting and refeeding on eIF2B activity. eIF2B activity was measured in 10,000 1 g supernatants by the GDP exchange assay as described under Materials and Methods. Results are expressed as the amount of [3H]GDP bound to eIF2 exchanged for nonradiolabeled GDP with time and are normalized for the amount of tissue protein added to the assay. The results represent the average of samples from five animals per condition.
ity of eIF2B was measured in extracts of muscle and liver from starved and refed rats and compared to the activity in tissue extracts from control animals. As shown in Figure 1, the activity of eIF2B was unaffected by either an 18-h fast or refeeding. Likewise, the amount of eIF2a in the phosphorylated form was unchanged in response to fasting or refeeding (results not shown). A second step in translation initiation that has been shown to be subject to acute regulation is the binding of mRNA to the 40S ribosomal subunit, a reaction mediated by eIF4E. In cells in culture treated with insulin, the activity of eIF4E is regulated through changes in the phosphorylation state of eIF4E as well as by modulation of the amount of eIF4E bound to 4E-BP1 (24). The association of eIF4E and 4E-BP1 is controlled through changes in the phosphorylation state of 4EBP1. The phosphorylation state of 4E-BP1 in cell or tissue extracts can be easily assessed by protein immunoblot analysis following SDS-polyacrylamide gel electrophoresis. During electrophoresis, 4E-BP1 is resolved into multiple electrophoretic forms with changes in electrophoretic mobility being inversely correlated with the number of phosphate groups on the protein. As shown in Figure 2, only the two forms exhibiting the greatest electrophoretic mobility (designated the a-
FIG. 2. Effect of fasting and refeeding on the association of 4EBP1 and eIF4E in skeletal muscle and liver. eIF4E was immunoprecipitated from 10,000 g supernatants of skeletal muscle and liver with an anti-eIF4E monoclonal antibody. Immunoprecipitates were then subjected to protein immunoblot analysis using a 4E-BP1 monoclonal antibody. Results represent means { SE for 5 animals per condition. Values not sharing the same superscript letter are significantly different (põ0.05) by Tukey-Kramer Multiple comparisons Test. * indicates significant differences from fasting groups (põ0.05) by Student’s t test. Insets: results of representative immunoblots. Lanes represent samples from freely fed (Fe), 18-h fasted (Fa), and refed (Re) animals.
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tein synthesis in both skeletal muscle and liver in part by promoting formation of the eIF4F complex. To further define the effects of fasting and refeeding on translation initiation, the phosphorylation state of eIF4E was examined. In muscle, the amount of eIF4E in the phosphorylated form was unaffected by either an 18-h fast or refeeding (Fig. 5). In contrast, in liver, an 18-h fast resulted in a partial dephosphorylation of eIF4E. Refeeding caused a further decrease in the proportion of eIF4E in the phosphorylated form. DISCUSSION Protein synthesis is regulated by changes in the activity of eIF2B under a wide variety of conditions (8). In cells in culture, the activity of eIF2B is regulated by changes in the phosphorylation state of a-subunit of eIF2 (8,25,26). However, little information is available concerning the role of phosphorylation of eIF2a in controlling translation in whole tissues or the intact animal. In one study, protein synthesis was shown to be
FIG. 3. Effect of fasting and refeeding on the phosphorylation state of 4E-BP1 in skeletal muscle and liver. Tissue extracts were subjected to SDS-polyacrylamide gel electrophoresis and the phosphorylation state of 4E-BP1 was determined by protein immunoblot analysis as described under Materials and Methods. Results represent means { SE for five animals per condition. Values not sharing the same superscript letter are significantly different (põ0.05) by Tukey-Kramer Multiple comparisons Test. Insets: results of representative immunoblots. Lanes represent samples from freely fed (Fe), 18-h fasted (Fa), and refed (Re) animals.
refeeding, the distribution of 4E-BP1 among the phosphorylated forms returned to the control pattern. In contrast to skeletal muscle, no change in the amount of 4E-BP1 in the g-form was observed in liver following an 18-h fast. However, fasting did result in an increase in the proportion of the protein in the a-form. Surprisingly, refeeding caused both a decrease in the amount of 4E-BP1 in the a-form as well as an increase in the amount in the g-form. The effect of food-intake on the binding of eIF4G to eIF4E was also examined by measuring the amount of eIF4G associated with eIF4E when eIF4E was immunoprecipitated using a monoclonal anti-eIF4E antibody. Fasting caused a decrease in the amount of eIF4G present in the eIF4E immunoprecipitate in both muscle and liver compared to the freely-fed, control condition (Fig. 4), although the difference was not significant in muscle. Refeeding significantly increased the amount of eIF4G bound to eIF4E in both muscle and liver. These findings suggest that food-intake stimulates pro-
FIG. 4. Effect of fasting and refeeding on association of eIF4G and eIF4E in skeletal muscle and liver. eIF4E was immunoprecipitated from 10,000 1 g supernatants of skeletal muscle and liver as described in the legend to Fig. 2. Immunoprecipitates were then subjected to protein immunoblot analysis using an anti-eIF4G polyclonal antibody. Results represent means { SE for five animals per condition. Values not sharing the same superscript letter are significantly different (põ0.05) by Tukey-Kramer Multiple comparisons Test. * indicates significant differences from fasting groups (põ0.05) by Student’s t test. Insets: results of representative immunoblots. Lanes represent samples from freely fed (Fe), 18-h fasted (Fa), and refed (Re) animals.
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FIG. 5. Effect of fasting and refeeding on the phosphorylation state of eIF4E in skeletal muscle and liver. The amount of eIF4E present in the phosphorylated form in skeletal muscle and liver was determined by protein immunoblot analysis after slab gel isoelectric focusing. Results represent means { SE for five animals per condition. Values not sharing the same superscript letter are significantly different (põ0.05) by Tukey-Kramer Multiple comparisons Test. Insets: results of representative immunoblots. Lanes represent samples from freely fed (Fe), 18-h fasted (Fa), and refed (Re) animals.
inhibited in perfused rat livers deprived of single amino acids (27). The probable basis for the inhibition of protein synthesis in response to amino acid deprivation was an increase in phosphorylation of eIF2a with a concomitant decrease in eIF2B activity (28). In contrast, it was reported that 48-h of starvation had no effect on the activity of eIF2B or on phosphorylation of eIF2a in muscle even though protein synthesis was decreased (29). In the present study, neither fasting nor refeeding had any effect on the activity of eIF2B (Fig. 1) or on the proportion of eIF2a in the phosphorylated form (data not shown). Therefore, the effects of fasting or refeeding on translation initiation in both skeletal muscle and liver involve alterations in other components of the translation machinery. The binding of mRNA to the 40S ribosomal subunit is another tightly regulated step in the initiation of translation (30). This step involves the binding of eIF4E to the m7GTP cap at the 5* end of mRNA and the subsequent binding of the eIF4ErmRNA complex to a second initiation factor, eIF4G. Two different mechanisms for controlling the binding of mRNA to the 40S ribosome subunit have been reported, each of which involves reversible phosphorylation of the pro-
teins involved in the process. In the first case, phosphorylation of the eIF4E binding protein, 4E-BP1, regulates translation initiation by increasing the availability of eIF4E. eIF4E bound to 4E-BP1 can bind to mRNA through the m7GTP cap structure, but the eIF4Er4E-BP1 complex cannot bind to eIF4G (20,31). Thus, 4E-BP1 competes with eIF4G for association to eIF4E. In the second case, phosphorylation of eIF4E promotes binding of the protein to the m7GTP cap structure (32). Previous studies have reported that increased incorporation of 32P into eIF4E is observed in response to a variety of stimuli, including with growth factor, mitogenes, hormone, and cytokines (12,13) and is positively correlated with changes in protein synthesis (12,13). In particular, insulin stimulates the incorporation of 32P into eIF4E, although the magnitude of the increase in 32P incorporation is much greater than the increase in the proportion of the protein in the phosphorylated form (33,34). On the basis of these studies, it has been proposed that the rate of eIF4E phosphorylation rather than its net phosphorylation state is important in controlling protein synthesis. In the present study, the finding in liver that refeeding caused a significant decrease in eIF4E phosphorylation may reflect an increase in the overall rate of phosphate turnover on the protein with the stimulation of eIF4E phosphatase activity being greater than the increase in eIF4E kinase activity. In contrast to the changes in eIF4E phosphorylation observed in liver, no change in eIF4E phosphorylation was observed in muscle, suggesting that protein synthesis in muscle is regulated by mechanism(s) distinct from changes in eIF4E phosphorylation. Instead, in muscle, fasting and refeeding caused changes in the association of eIF4E with 4E-BP1 and eIF4G. In vitro studies using recombinant proteins have suggested that either 4E-BP1 or eIF4G can individually bind to eIF4E, but both proteins cannot bind simultaneously (31,35). The results of the present study using skeletal muscle are in agreement with the in vitro results. In muscle from fasted rats, the amount of 4E-BP1 bound to eIF4E increases whereas the amount of eIF4G bound to eIF4E decreases. Conversely, refeeding results in a reduction in the amount of 4E-BP1 bound to eIF4E with a concomitant increase in the amount of eIF4G associated with the protein. The changes in binding of eIF4E to 4E-BP1 and eIF4G in muscle are associated with alterations in the phosphorylation state of 4EBP1. Phosphorylation of 4E-BP1 in vitro by either mitogen-activated protein (MAP) kinase (20) or the mammalian target of rapamycin (mTOR) kinase (36) 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 is unknown. However, studies in mice have shown that refeeding starved animals activates the signaling pathway of which mTOR is a component, suggesting that activa-
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tion of mTOR may be responsible for the observed increase in 4E-BP1 phosphorylation in response to refeeding. In contrast to the results in muscle, in liver the amount of eIF4G bound to eIF4E changes in response to fasting and refeeding in the absence of changes in the amount of 4E-BP1 bound to eIF4E. This result suggests that in liver there is a pool of eIF4E that is not associated with either 4E-BP1 or eIF4G and that starvation causes a dissociation of the eIF4GreIF4E complex with a resulting increase in the amount of ‘free’ eIF4E. The mechanism involved in the dissociation of eIF4E from eIF4G in the absence of changes in the association of 4E-BP1 and eIF4E is unknown. However, it has been suggested that alterations in the phosphorylation state of eIF4E might modulate the interaction of eIF4E and eIF4G (37,38). In addition, the finding that insulin stimulates the phosphorylation of eIF4G (39) presents the possibility that changes in phosphorylation of eIF4G in response to starvation and refeeding might play an important role in regulating the association of the two proteins. Alternatively, although the amount of eIF4E associated with 4E-BP1 does not change in liver during fasting, the amount of eIF4E bound to a second eIF4E binding protein, 4E-BP2, may. The predominant form of eIF4E binding protein in muscle is 4E-BP1 (40). In contrast, liver has both 4E-BP1 and 4E-BP2 (40). Thus, in liver, fasting and refeeding may modulate the amount of eIF4E bound to 4E-BP2, but not 4E-BP1. Further studies will be required to examine this possibility. The signal that stimulates protein synthesis and modulates the phosphorylation state of eIF4E and 4EBP1 after refeeding is unknown. A number of studies have shown that both insulin and amino acids can play important roles in the regulation of protein synthesis (41-46). In particular, it has been postulated that the response of muscle protein synthesis to food intake is mediated primarily by increases in the plasma concentration of insulin and amino acids which act cooperatively to stimulate protein synthesis (3,4). However, the role of insulin in stimulating protein synthesis in muscle after refeeding has been questioned in a recent study using diabetic mice (17). In that study, refeeding starved mice stimulated protein synthesis to a similar extent in muscle from both control and diabetic animals. Thus, in muscle, amino acids may be the primary mediators of the increase in protein synthesis in response to refeeding. It has also been suggested that the stimulation of liver protein synthesis in response to food intake is not necessarily mediated by insulin, but instead is regulated by the increase in plasma amino acid concentration that occurs after feeding (4). Further studies will be needed to elucidate the role of insulin and amino acids in regulating translation initiation in response to food intake. In summary, the changes in translation initiation in
both skeletal muscle and liver with nutrition state are not associated with any detectable changes in the activity of eIF2B or in the phosphorylation state of eIF2a. Instead, the changes result from alternations in the phosphorylation state of eIF4E and 4E-BP1. In liver, fasting and refeeding modulate both the phosphorylation state of eIF4E as well as the amount of eIF4G bound to eIF4E. In contrast, in muscle the inhibition of protein synthesis that is observed in fasted and refed animals is independent of changes in eIF4E phosphorylation. Instead, changes in protein synthesis are associated with reciprocal changes in the binding of 4E-BP1 and eIF4G to eIF4E. In muscle the changes in the amount of eIF4E bound to 4E-BP1 are associated with alterations in the phosphorylation state of 4E-BP1. Each of these effects is reversed by refeeding for 1 hour. ACKNOWLEDGMENTS We are grateful to Sharon Rannels and Lynne Pletcher for technical assistance. This work was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grants DK-15658 and DK-13499.
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