Developmental changes in the feeding-induced stimulation of translation initiation in muscle of neonatal pigs

Teresa A. Davis1, Hanh V. Nguyen1, Agus Suryawan1, Jill A. Bush1, Leonard S. Jefferson2, and Scot R. Kimball2

1 Department of Pediatrics, United States Department of Agriculture/Agricultural Research Service, Children's Nutrition Research Center, Baylor College of Medicine, Houston, Texas 77030; and 2 Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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The rapid gain in skeletal muscle mass in the neonate is associated with a marked elevation in skeletal muscle protein synthesis in response to feeding. The feeding-induced response decreases with development. To determine whether the response to feeding is regulated at the level of translation initiation, the expression, phosphorylation, and function of a number of eukaryotic initiation factors (eIF) were examined. Pigs at 7 and 26 days of age were either fasted overnight or fed porcine milk after an overnight fast. In muscle of 7-day-old pigs, the hyperphosphorylated form of the eIF4E repressor protein, 4E-binding protein 1 (4E-BP1), was undetectable in the fasting state but rose to 60% of total 4E-BP1 after feeding; eIF4E phosphorylation was unaffected by feeding status. The amount of eIF4E in the inactive 4E-BP1 · eIF4E complex was reduced by 80%, and the amount of eIF4E in the active eIF4E · eIF4G complex was increased 14-fold in muscle of 7-day-old pigs after feeding. The amount of 70-kDa ribosomal protein S6 (p70S6) kinase in the hyperphosphorylated form rose 2.5-fold in muscle of 7-day-old pigs after feeding. Each of these feeding-induced responses was blunted in muscle of 26-day-old pigs. eIF2B activity in muscle was unaffected by feeding status but decreased with development. Feeding produced similar changes in eIF characteristics in liver and muscle; however, the developmental changes in liver were not as apparent as in skeletal muscle. Thus the results demonstrate that the developmental change in the acute stimulation of skeletal muscle protein synthesis by feeding is regulated by the availability of eIF4E for 48S ribosomal complex formation. The results further suggest that the overall developmental decline in skeletal muscle protein synthesis involves regulation by eIF2B.

protein synthesis; growth; insulin; eukaryotic initiation factor 4E; liver


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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THE NEONATAL PERIOD is characterized by rapid growth accompanied by high rates of protein turnover (8, 11, 16). As the neonate develops, fractional rates of growth and protein synthesis decline. During the neonatal period, the gain in protein mass is more rapid in skeletal muscle than in the body as a whole (57). The fractional rate of protein synthesis in skeletal muscle is very high at birth and declines with age, particularly during the 1st mo of life in both the rat and the pig (5, 8). The developmental decline in skeletal muscle protein synthesis is accompanied by a decrease in ribosome number.

Dietary protein is utilized efficiently for protein deposition in the neonate; however, this efficiency declines with development (10, 38). The high efficiency of dietary protein utilization in the neonate is likely due to the enhanced stimulation of protein synthesis in response to feeding (5, 9, 12). The postprandial rise in protein synthesis and the developmental decline in the response to feeding are more pronounced in skeletal muscle than in other organs (2, 3, 5, 9). For example, in response to refeeding, fractional rates of skeletal muscle protein synthesis in 7-day-old pigs increase from 15 to 24%/day and in 26-day-old pigs increase from 4 to 6%/day. However, fractional rates of protein synthesis in liver increase from 76 to 93%/day at 7 days of age and from 63 to 72%/day at 26 days of age (5). The feeding-induced stimulation of protein synthesis is due to an increase in the efficiency of the translation process, i.e., the amount of protein synthesized per unit RNA. Using hyperinsulinemic-euglycemic-amino acid clamps, we have shown that the postprandial elevation in skeletal muscle protein synthesis in neonatal pigs is mediated by insulin (54).

Acute regulation of protein synthesis is achieved in part through changes in the rate of translation of mRNA via alterations in peptide-chain initiation (17, 28, 34). The first important regulatory step in translation initiation is the binding of initiator methionyl-tRNA (met-tRNAi) to the 40S ribosomal subunit, a reaction that is mediated by eukaryotic initiation factor (eIF)2 and results in the formation of the 43S preinitiation complex (31, 45). The eIF2-mediated binding of met-tRNAi to the 40S subunit is regulated by modulation of the activity of eIF2B, a factor required for the exchange of GDP for GTP on eIF2 (32). The activity of this factor can be regulated by phosphorylation of the alpha -subunit of eIF2 and the epsilon -subunit of eIF2B.

The second step critical to the regulation of translation initiation is the binding of mRNA to the 43S preinitiation complex, which is mediated by the eIF4F complex of proteins (34, 46, 48). One of the components of the eIF4F complex, eIF4E, plays a particularly important role because it binds to the m7GTP cap at the 5'-end of mRNA to form an eIF4E · mRNA complex, which binds to eIF4G and eIF4A to form the active eIF4F complex. The active eIF4F complex binds to the 43S preinitiation complex, resulting in formation of the 48S preinitiation ribosomal complex (42). The function of eIF4E may be influenced by either its phosphorylation state (40) or its availability (44). Phosphorylation of eIF4E stimulates translation initiation by increasing its association with eIF4G and eIF4A and/or its affinity for the m7GTP cap of mRNA (39, 40). Availability of eIF4E for formation of the eIF4F complex is limited by its binding to 4E-binding protein 1 (4E-BP1), a protein that competes with eIF4G for binding to eIF4E to form an inactive 4E-BP1 · eIF4E complex (44). Phosphorylation of 4E-BP1 results in a decrease in affinity of 4E-BP1 for eIF4E, and thus an enhancement in formation of the active eIF4E · eIF4G complex. These changes in initiation factor function may be modulated through the phosphatidylinositide 3-hydroxy kinase/mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 (p70S6) kinase signal transduction pathway (26).

The role of initiation factors in the stimulation of translation initiation by anabolic agents has been defined primarily by studies performed in cell culture. These include the ability of insulin both to enhance formation of the 43S preinitiation complex by stimulating eIF2B activity and to promote binding of mRNA to the 43S preinitiation complex by increasing the phosphorylation of eIF4E and 4E-BP1 (14, 44, 52). Less is known of the regulation of translation initiation in the whole animal. Recent studies indicate that some of the initiation factors known to be stimulated by insulin in vitro are not activated by nutrient intake in the mature rodent (49, 55). Because the stimulation of protein synthesis by both feeding and insulin is markedly elevated in the neonate and decreases with development (5, 9, 54), it seems plausible that fundamental differences may exist between the mechanisms that regulate translation initiation in the mature individual and the neonate.

The purpose of the current study was to examine the mechanism(s) by which feeding regulates translation initiation in skeletal muscle of the neonatal pig. We further sought to determine whether the feeding-induced activation of specific translation initiation factors in skeletal muscle decreases with development, in parallel with the previously observed (5) developmental decline in the stimulation of muscle protein synthesis by feeding in the neonatal pig. For comparison, the postprandial modulation of specific initiation factors was also examined in liver. The results show marked upregulation of specific initiation factors in both skeletal muscle and liver in response to the consumption of a meal in the neonatal pig and a dramatic developmental decline in this response in skeletal muscle.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Animals. Eight crossbred (Landrace × Yorkshire × Hampshire × Duroc) pregnant sows (Agriculture Headquarters, Texas Department of Criminal Justice, Huntsville, TX) were housed in lactation crates in individual, environmentally controlled rooms 2 wk before farrowing. Sows were fed a commercial diet (5084, PMI Feeds, Richmond, IN) and provided water ad libitum. After farrowing, piglets remained with the sow and were not given supplemental creep feed. Piglets from 11 litters were studied at 7 days of age (2.2 ± 0.5 kg, n = 47) and 26 days of age (7.6 ± 1.7 kg, n = 43).

Pigs within each litter were randomly assigned to one of two treatment groups and were either 1) fasted for 18 h or 2) fed for 1.5 h after an 18-h fast. Pigs were provided water throughout the fasting period. Pigs that were fed after the 18-h fast were given two gavage administrations of 30 ml/kg body wt of porcine mature milk (University of Nebraska, Lincoln, NE) at 60-min intervals. Pigs were killed, and samples of longissimus dorsi and liver were rinsed in ice-cold saline and rapidly frozen. The protocol was approved by the Animal Care and Use Committee of Baylor College of Medicine and was conducted in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals.

Materials. Enhanced chemiluminescence (ECL) detection reagents and horseradish peroxidase-conjugated sheep antimouse immunoglobulin G (IgG) and donkey anti-rabbit IgG were purchased from Amersham Life Sciences (Arlington Heights, IL). Polyvinylidene difluroide (PVDF) membrane was obtained from Bio-Rad (Hercules, CA). Antibodies against p70S6 kinase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Measurement of eIF2B activity. eIF2B activity in muscle and liver postmitochondrial supernatants was measured as the exchange of [3H]GDP bound to eIF2 for unlabeled GDP or GTP, as previously described (25). Briefly, an eIF2 · [3H]GDP binary complex was formed in the absence of magnesium chloride. The eIF2 · [3H]GDP complex was then stabilized by the addition of magnesium to a final concentration of 2 mM. The eIF2 · [3H]GDP complex was incubated with samples containing eIF2B in the presence of a 100-fold molar excess of unlabeled, HPLC-purified GTP at 30°C for various times. The reaction mixture was filtered through a nitrocellulose filter, the filters were washed, and radioactivity bound to the filter was quantitated using a liquid scintillation counter.

Protein immunoblot analysis. Proteins in polyacrylamide gels were electrophoretically transferred to a PVDF membrane as described previously (31). The membranes were then incubated with primary antibody for 1 h at room temperature. Blots were developed using an Amersham ECL Western Blotting Kit, and films were analyzed by densitometric analysis as described previously (29, 31).

Examination of eIF2alpha phosphorylation and eIF2 content. The relative amount of eIF2alpha in the phosphorylated form was quantified by protein immunoblot analysis with an affinity-purified antibody that specifically recognizes eIF2alpha phosphorylated at Ser51 [eIF2(alpha P)] (35), kindly provided by Drs. Gary S. Krause and Donald J. DeGracia, Wayne State University School of Medicine. For this analysis, samples were resolved by SDS-PAGE on a 12.5% polyacrylamide gel, and the proteins in the gel were electrophoretically transferred to a PVDF membrane as described previously (27). The membranes were incubated with the anti-eIF2(alpha P) antibody, and the blots were developed using an ECL Western blotting kit as described above. The horseradish peroxidase coupled to the anti-rabbit secondary antibody was then inactivated by incubating the blot in 15% H2O2 for 30 min at room temperature. The total amount of eIF2alpha in the samples was determined by reprobing the blot with a monoclonal antibody (kindly provided by Dr. Richard Panniers, National Institutes of Health) that recognizes equally the phosphorylated and unphosphorylated forms of eIF2alpha (36), followed by an anti-mouse secondary antibody. Values obtained using the anti-eIF2(alpha P) antibody were normalized for the total amount of eIF2alpha present in the sample.

Quantitation of 4E-BP1 · eIF4E and eIF4G · eIF4E complexes. The association of eIF4E with 4E-BP1 or eIF4G was quantitated as described previously (26). Briefly, eIF4E and the 4E-BP1 · eIF4E and eIF4G · eIF4E complexes were immunoprecipitated using an anti-eIF4E monoclonal antibody. The immunoprecipitates were resuspended in SDS sample buffer, and the samples were boiled for 5 min. The samples were then centrifuged, and supernatants were subjected to electrophoresis either on a 7.5% polyacrylamide gel for quantitation of eIF4G or on a 15% polyacrylamide gel for quantitation of 4E-BP1 and eIF4E. Proteins were then electrophoretically transferred to a PVDF membrane as described above. The membranes were incubated with a mouse anti-human eIF4E antibody, a rabbit anti-rat 4E-BP1 antibody, or a rabbit anti-human eIF4G antibody. The blots were then developed using an ECL Western blotting kit as described above.

Examination of 4E-BP1 phosphorylation. Aliquots of muscle homogenates were heated at 100°C for 10 min, cooled to room temperature, and then centrifuged at 10,000 g for 10 min at 4°C. The supernatants were diluted with SDS sample buffer and then subjected to protein immunoblot analysis, as described previously (31). Previous studies have shown that phosphorylation of 4E-BP1 causes a decrease in the electrophoretic mobility of the protein on SDS-polyacrylamide gels (18, 36). Thus 4E-BP1 present in tissue extracts was separated into multiple electrophoretic forms during SDS-PAGE, with the more slowly migrating forms representing more highly phosphorylated 4E-BP1.

Measurement of eIF4E phosphorylation and content. The phosphorylated and unphosphorylated forms of eIF4E in tissue extracts were separated by isoelectric focusing on a slab gel and were quantitated by protein immunoblot analysis with a monoclonal antibody against eIF4E, as previously described (26).

Determination of p70S6 kinase phosphorylation. Muscle homogenates were combined with an equal volume of SDS sample buffer, and the diluted samples were subjected to electrophoresis on a 7.5% polyacrylamide gel (35). The samples were then analyzed by protein immunoblot analysis by use of rabbit anti-rat p70S6 kinase polyclonal antibodies, as described above.

Statistics. Analysis of variance (general linear modeling) was used to assess the effects of feeding, age, and their interaction. If there was an interaction between feeding and age, Student's t-test was used to test for differences between treatment groups. Probability values of <0.05 were considered statistically significant. Data are presented as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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eIF2/eIF2B in skeletal muscle. The eIF2-mediated binding of met-tRNAi to the 40S subunit is regulated by modulation of the activity of eIF2B, a factor required for the exchange of GDP for GTP on eIF2 (23). eIF2B-mediated GDP-GTP exchange activity in skeletal muscle was analyzed by the ability of postmitochondrial supernatants of tissue homogenates to exchange radiolabeled GDP for unlabeled GDP or GTP from a preformed eIF2-[3H]GDP binary complex. Tissues were excised, and postmitochondrial supernatants were immediately prepared and assayed for eIF2B activity. Feeding had no effect on eIF2B activity in skeletal muscle (Fig. 1). However, eIF2B activity in muscle was 70% higher in 7- than in 26-day-old pigs (P < 0.001).


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Fig. 1.   Eukaryotic initiation factor 2B (eIF2B) activity in skeletal muscle of fasted and fed pigs at 7 and 26 days of age. eIF2B-mediated GDP-GTP exchange activity was quantitated by the ability of postmitochondrial supernatants of tissue homogenates to exchange radiolabeled GDP for unlabeled GDP or GTP from a preformed eIF2-[3H]GDP binary complex. Values are means ± SE, n = 11-14. eIF2B activity was higher in 7- than in 26-day-old pigs (P < 0.001). Feeding had no effect on eIF2B activity. *Different from value in 7-day-old pigs (P < 0.05).

The activity of eIF2B can be reduced by phosphorylation of the alpha -subunit of eIF2 (23). The amount of eIF2alpha present in the phosphorylated form in skeletal muscle was analyzed by protein immunoblot analysis with an anti-phosphopeptide antibody specific for eIF2alpha phosphorylated on Ser51. Only a small proportion of eIF2alpha was present in the phosphorylated form. There was no effect of feeding or age on the amount of phosphorylated eIF2alpha relative to the total amount of eIF2 in skeletal muscle [7 day, fasted: 1.2 ± 0.3 arbitrary units (U); 7 day, fed: 1.5 ± 0.2 U; 26 day, fasted: 1.9 ± 0.7 U; and 26 day, fed: 3.1 ± 1.2 U]. The total amount of eIF2alpha in muscle was quantitated by reprobing the blot with an antibody that recognizes equally the phosphorylated and unphosphorylated forms of the protein. The total amount of eIF2alpha in both the phosphorylated and unphosphorylated forms in skeletal muscle was unaffected by feeding (7 day, fasted: 1,363 ± 159 U; 7 day, fed: 1,639 ± 93 U; 26 day, fasted: 1,263 ± 160 U; and 26 day, fed: 1,267 ± 171 U). However, the total amount of eIF2alpha tended to be higher in 7- than in 26-day-old pigs in the fed state (+30%, P = 0.08).

eIF4E function in skeletal muscle. One of the subunits of the eIF4F complex, eIF4E, plays a critical role in the binding of mRNA to the 43S preinitiation complex (46, 48). The function of eIF4E in cell culture may be influenced by either its phosphorylation state or its availability (40, 44). To determine whether feeding affected eIF4E phosphorylation status, phosphorylated and unphosphorylated forms of eIF4E were separated by isoelectric focusing slab gel electrophoresis, and the amount of eIF4E present in the phosphorylated form, as a percentage of the total eIF4E in the immunoprecipitate, was analyzed. The amount of phosphorylated eIF4E in skeletal muscle was not significantly affected by feeding or age (Fig. 2). However, there was a tendency for a decrease in eIF4E phosphorylation with feeding (-30%, P = 0.13).


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Fig. 2.   eIF4E phosphorylation in skeletal muscle of fasted and fed pigs at 7 and 26 days of age. Phosphorylated (p) and unphosphorylated forms of eIF4E were separated by isoelectric focusing slab gel electrophoresis, and the amount of eIF4E present in the phosphorylated form, as a percentage of the total eIF4E in the immunoprecipitate, was determined. Inset: results of a representative immunoblot. Values are means ± SE, n = 16-18. Feeding and age had no significant effect on eIF4E phosphorylation.

The availability of eIF4E can be regulated through changes in the amount of eIF4E bound to 4E-BP1 (36). Phosphorylation of 4E-BP1 causes disassembly of the 4E-BP1 · eIF4E complex (30). A means of determining the changes in 4E-BP1 phosphorylation is by examination of the alteration in migration of the protein during SDS-PAGE. Thus 4E-BP1 can be resolved by SDS-PAGE into multiple electrophoretic bands, alpha , beta , and gamma , representing different phosphorylated forms of the protein (36). The most highly phosphorylated and slowest migrating form of the protein, the gamma -form, does not bind eIF4E. The gamma -form of 4E-BP1 was not detected in skeletal muscle of either 7- or 26-day-old fasted pigs (Fig. 3). Feeding differentially increased the amount of 4E-BP1 in the most highly phosphorylated form to 60% of the total 4E-BP1 present at 7 days but only 10% at 26 days of age (P < 0.001).


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Fig. 3.   4E-binding protein 1 (4E-BP1) phosphorylation in skeletal muscle of fasted and fed pigs at 7 and 26 days of age. Tissue extracts were subjected to SDS-PAGE, and the phosphorylation state of 4E-BP1 was determined. Positions of alpha -, beta -, and, gamma -forms are shown on representative immunoblot (inset). Positions of alpha -, beta -, and gamma -forms of 4E-BP1 are denoted (left). Values are means ± SE, n = 12-17. The most highly phosphorylated form of 4E-BP1, the gamma -form, was not detected in either 7- or 26-day-old pigs. Feeding markedly stimulated 4E-BP1 phosphorylation (P < 0.001), and the response was greater in 7- than in 26-day-old pigs (P < 0.001). *Different from value in 7-day-old pigs (P < 0.05); dagger different from values in fasted group (P < 0.05).

Association of eIF4E with 4E-BP1 and eIF4G in skeletal muscle. Phosphorylation of 4E-BP1 in cell culture and in situ decreases the association of 4E-BP1 with eIF4E, thereby allowing eIF4E to bind to eIF4G (30, 36). To determine the amount of 4E-BP1 associated with eIF4E, eIF4E was immunoprecipitated with an anti-eIF4E antibody, followed by immunoblot analysis with an anti-4E-BP1 antibody. Feeding decreased the amount of 4E-BP1 present in the eIF4E immunoprecipitate (P < 0.001; Fig. 4). This feeding-induced reduction in the association of eIF4E with 4E-BP1 was greater (P < 0.001) in 7-day-old (20% of fasted) than in 26-day-old pigs (60% of fasted).


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Fig. 4.   Association of eIF4E with 4E-BP1 in skeletal muscle of fasted and fed pigs at 7 and 26 days of age. eIF4E was immunoprecipitated with a monoclonal anti-eIF4E antibody, and immunoprecipitates were analyzed by protein immunoblot analysis with an anti-4E-BP1 antibody. Inset: results of a representative immunoblot. Values are means ± SE, n = 16-19. Feeding decreased the amount of 4E-BP1 present in the eIF4E immunoprecipitate, and reduction was greater in 7- than in 26-day-old pigs (P < 0.001). *Different from value in 7-day-old pigs (P < 0.05); dagger different from values in fasted group (P < 0.05).

The amount of eIF4G bound to eIF4E was measured in the same eIF4E immunoprecipitates used to quantitate 4E-BP1 bound to eIF4E. Feeding increased the amount of eIF4G associated with eIF4E by 14-fold (P < 0.001; Fig. 5). Moreover, the effect of feeding was greater in 7- than in 26-day-old pigs (P < 0.005).


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Fig. 5.   Association of eIF4E with eIF4G in skeletal muscle of fasted and fed pigs at 7 and 26 days of age. eIF4E was immunoprecipitated with a monoclonal anti-eIF4E antibody, and immunoprecipitates were analyzed by protein immunoblot with an anti-eIF4G antibody. Inset: results of a representative immunoblot. Values are means ± SE, n = 21-22. Feeding increased the amount of eIF4G associated with eIF4E (P < 0.001). The effect of feeding was greater in 7- than in 26-day-old pigs (P < 0.001). *Different from value in 7-day-old pigs (P < 0.05); dagger different from values in fasted group (P < 0.05).

p70S6 kinase. Studies using cells in culture suggest that changes in 4E-BP1 phosphorylation occur through (1), or in parallel with (50), activation of the p70S6 kinase pathway. Phosphorylation of p70S6 kinase is associated with its activation. Therefore, the phosphorylation state of p70S6 kinase in skeletal muscle extracts was determined by protein immunoblot analysis. Feeding decreased the electrophoretic mobility of p70S6 kinase and resulted in the appearance of multiple electrophoretic forms of the kinase, which indicate phosphorylation of the protein (Fig. 6). There was a marked increase in the amount of p70S6 kinase in the most hyperphosphorylated form after feeding (P < 0.001). The increase in p70S6 kinase phosphorylation with feeding was greater at 7 days (+140%) than at 26 days of age (+70%) (P < 0.002).


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Fig. 6.   Phosphorylation state of p70S6 kinase (p70S6k) in skeletal muscle of fasted and fed pigs at 7 and 26 days of age. The phosphorylation state of p70S6 kinase was assessed by protein immunoblot analysis. Inset: results of a representative immunoblot. Values represent the proportion of the protein present in hyperphosphorylated (HP) forms as a percentage of total protein and are means ± SE, n = 12-17. Feeding increased the phosphorylation of the protein (P < 0.001). Increase in p70S6 kinase phosphorylation was greater in 7- than in 26-day-old pigs (P < 0.002). *Different from value in 7-day-old pigs (P < 0.05); dagger different from values in fasted group (P < 0.05).

Initiation factor function in liver. Previous studies in pigs have shown that the stimulation of protein synthesis by feeding in the neonate and the developmental decline in this response are more profound in skeletal muscle than in liver (5). Therefore, we compared the results in skeletal muscle with those in liver for initiation factors eIF2B and 4E-BP1, each of which is critical to the regulation of the two main steps of translation initiation. We also compared the results in the two tissues for p70S6 kinase, the signaling protein immediately upstream of ribosomal protein S6.

eIF2B activity was higher in liver (P < 0.001; Fig. 7) than in skeletal muscle, consistent with the higher rates of fractional protein synthesis in liver than in skeletal muscle that we previously reported in neonatal pigs (5). eIF2B activity in liver was unaffected by feeding, similar to the lack of effect of feeding on eIF2B activity in skeletal muscle. eIF2B activity in liver was 35% higher in 7- than in 26-day-old pigs (P < 0.05), in contrast to the 70% higher eIF2B activity in muscle of 7- than 26-day-old pigs.


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Fig. 7.   eIF2B activity in liver of fasted and fed pigs at 7 and 26 days of age. eIF2B-mediated GDP-GTP exchange activity was determined as described in Fig. 1. Values are means ± SE, n = 11-13. eIF2B activity was higher in liver than in skeletal muscle (P < 0.001; see Fig. 1 for comparison). eIF2B activity in liver was unaffected by feeding but was higher in 7- than in 26-day-old pigs (P < 0.05). *Different from values in 7-day-old pigs (P < 0.05).

Similar to the observation reported above for skeletal muscle, the proportion of 4E-BP1 present in the hyperphosphorylated gamma -form in liver was lower in fasted than in fed pigs (Fig. 8). However, unlike muscle of fasted pigs, where 4E-BP1 was predominantly in the hypophosphorylated alpha -form, in liver of fasted pigs, 4E-BP1 was distributed between the alpha - and beta -forms. Furthermore, feeding increased 4E-BP1 phosphorylation in liver (P < 0.001), although the response appeared to be of a lower magnitude than that in skeletal muscle. In contrast to the results in skeletal muscle, 4E-BP1 phosphorylation increased with development (P < 0.05).


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Fig. 8.   4E-BP1 phosphorylation in liver of fasted and fed pigs at 7 and 26 days of age. Tissue extracts were prepared as described in Fig. 3 legend. Inset: results of a representative immunoblot. Values are means ± SE, n = 8-16. 4E-BP1 phosphorylation was higher in liver than in skeletal muscle (P < 0.05). 4E-BP1 phosphorylation increased with feeding (P < 0.001) and increased with development (P < 0.05). *Different from values in 7-day-old pigs (P < 0.05); dagger different from values in fasted group (P < 0.05).

Similar to 4E-BP1, the proportion of p70S6 kinase in highly phosphorylated forms was higher in liver than in skeletal muscle (P < 0.01; Fig. 9). Feeding increased the phosphorylation of p70S6 kinase in liver (P < 0.001), although the response appeared to be of a lower magnitude than that in skeletal muscle. However, p70S6 kinase phosphorylation in liver did not differ in 7- and 26-day-old pigs, in contrast to the developmental decline in p70S6 kinase phosphorylation in skeletal muscle.


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Fig. 9.   Phosphorylation state of p70S6 kinase in liver of fasted and fed pigs at 7 and 26 days of age. p70S6 kinase phosphorylation was determined as described in Fig. 6. Insert: results of a representative immunoblot. Values represent the proportion of the protein present in hyperphosphorylated (HP) forms as a percentage of total protein and are means ± SE, n = 7-9. Feeding increased the phosphorylation of p70S6 kinase (P < 0.001). dagger Different from values in fasted group (P < 0.05).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ethical considerations preclude the measurement of tissue protein synthesis in the human infant; therefore, we have utilized the neonatal pig as an animal model to examine the role of nutrition and hormones in the regulation of early postnatal growth. The neonatal pig is similar to the human infant in anatomy, developmental physiology, and metabolism (41) but has higher rates of protein synthesis due to its more rapid rate of growth (5, 11). The results of the current study indicate that the postprandial increase in skeletal muscle protein synthesis in the neonate is regulated by the eIF4F complex of proteins. In muscle of the neonatal pig, feeding increased the binding of eIF4G to eIF4E because of the release of eIF4E from the inactive 4E-BP1 · eIF4E complex after the phosphorylation of 4E-BP1. The p70S6 kinase signal transduction pathway likely modulates these changes in the activation of the eIF4F complex. This feeding-induced activation of the eIF4F complex in skeletal muscle decreased with development, in parallel with the developmental decline in the feeding-induced changes in fractional rates of protein synthesis in muscle that we have previously reported (5). The stimulation of liver protein synthesis by feeding also involves regulation by the eIF4F complex, but the response does not decrease with development. The results further suggest that eIF2 and eIF2B are not critical regulatory factors in the stimulation of protein synthesis in muscle and liver by food intake. However, eIF2 and eIF2B may play important roles in regulating the overall decrease in muscle and liver protein synthesis with development.

Effects of feeding and development on eIF2/eIF2B. Numerous studies performed in cell culture have demonstrated that eIF2 plays a critical role in the regulation of translation initiation (23). eIF2 binds GTP and met-tRNAi, and this ternary complex then binds to the 40S ribosomal subunit to form the 43S preinitiation complex. At the end of the initiation process, the GTP bound to eIF2 is hydrolyzed to GDP, and the GDP bound to eIF2 is exchanged for GTP by a reaction mediated by eIF2B. Phosphorylation of the alpha -subunit of eIF2 inhibits the activity of eIF2B. In the current study, feeding of neonatal pigs had no effect on the relative amount of eIF2 present in skeletal muscle or liver. Furthermore, feeding did not alter the activity of eIF2B or the phosphorylation of the alpha -subunit of eIF2.

Previous studies have shown that neither the activity of eIF2B nor the phosphorylation of eIF2alpha in mature rats changes with feeding (4, 55). However, we hypothesized that the initiation factors involved in the formation of the 43S preinitation complex might be activated by feeding in immature animals, because the stimulation of muscle protein synthesis by feeding is markedly elevated in the neonate (5, 7, 9). In addition, the postprandial rise in circulating insulin levels is 20% higher in 7- than in 26-day-old pigs (5). Moreover, the ability of insulin (54) and amino acids (6) to stimulate protein synthesis is also greater the younger the animal. Therefore, we anticipated that initiation factors that are activated in cell culture by insulin or amino acids would also be activated in vivo in muscle of immature animals in response to feeding. Studies performed in cell culture suggest that insulin increases eIF2B activity, albeit without a change in eIF2alpha phosphorylation (52, 53). The reduction in eIF2B activity in muscle of diabetic animals is also suggestive of a role for insulin in this process (21, 22). In addition, studies in cell culture and in perfused liver suggest that amino acids regulate eIF2B activity and that these changes are modulated by alterations in the phosphorylation of eIF2alpha and eIF2Bepsilon (13, 24, 27). Thus the lack of effect of feeding on eIF2B activity in muscle and liver of the neonatal pig is somewhat surprising and suggests that the normal physiological changes in insulin and/or amino acids after food consumption are unlikely to affect protein synthesis via changes in eIF2B activity or eIF2alpha phosphorylation.

Because the decline in the fractional rate of muscle protein synthesis with development largely parallels the decline in ribosome number (5, 8), we hypothesized that other components of the translation initiation system might also change with development. We found that eIF2B activity in muscle of fasted pigs declined by 55% from 7 to 26 days, consistent with the 75% decline in protein synthesis rates and 55% decline in ribosome number previously reported (5). Total eIF2 content of muscle also tended to decrease with development. The 35% decline in eIF2B activity in liver was similar to the 20% reduction in liver protein synthesis rates, although ribosome number did not change during this stage of development (5). Thus the age-associated decline in eIF2B activity was greater in skeletal muscle than in liver, consistent with the greater developmental decline in protein synthesis rates in skeletal muscle compared with liver. Previous studies have shown that eIF2B activity and/or eIF2 content in spleen, liver, kidney, lung, and brain decreases between 1 and 10 mo of age in rats, in parallel with the decline with age in protein synthesis rates (33). However, eIF2B activity and eIF2 content of skeletal muscle were not determined in those studies. Together, our studies indicate that the decline with age in fractional rates of protein synthesis is regulated through changes in both eIF2/eIF2B and ribosome number.

Effects of feeding and development on the eIF4F complex. The second major site of regulation of translation initiation, i.e., the binding of mRNA to the 40S ribosomal subunit, is regulated by the eIF4F complex, which consists of eIF4E, eIF4G, and eIF4A (46, 48). The least abundant initiation factor, eIF4E, plays a particularly important role, because it binds to the 5' end of the GTP cap of mRNA, and this is preceded by the association of eIF4E with the scaffolding protein, eIF4G (42). eIF4F function may be modulated through changes in the phosphorylation and/or the availability of eIF4E.

Studies performed in cell culture suggest that hormones, growth factors, and mitogens promote the binding of eIF4E to the mRNA by increasing the phosphorylation of eIF4E (39, 46, 48). However, recent reports of both in vivo and in situ studies do not support the results of in vitro studies. eIF4E phosphorylation is unaffected by diabetes, replacement insulin, or feeding in vivo in mature rats (29, 55, 49) but is reduced by insulin infusion in situ (30) and protein feeding in vivo in mature rats (56). In the current study, feeding of the neonatal pig tended to reduce eIF4E phosphorylation in skeletal muscle, although the response did not achieve statistical significance. It has been proposed that it is the turnover of phosphate on eIF4E, rather than its net phosphorylation, that is important in regulating the rate of protein synthesis (47). Thus the modest reduction in the amount of eIF4E in the phosphorylated form in the current study may reflect a greater activation of phosphatase than protein kinase in response to feeding in the neonate.

Changes in the availability of eIF4E to participate in translation initiation are modulated via the binding of eIF4E to a translational repressor protein, 4E-BP1 (44). eIF4E binds to either 4E-BP1 or eIF4G, but not to both proteins simultaneously, indicating that 4E-BP1 competes with eIF4G for association with eIF4E (37). The binding of eIF4E to 4E-BP1 is regulated by phosphorylation of 4E-BP1, with phosphorylation being associated with a decrease in the inactive 4E-BP1 · eIF4E complex. Insulin and amino acids stimulate the phosphorylation of 4E-BP1, resulting in a dissociation of the inactive 4E-BP1 · eIF4E complex and an increase in the formation of the active eIF4E · eIF4G complex (15, 27, 29, 36). In mature rodents, feeding results in a two- to threefold increase in the phosphorylation of 4E-BP1 in skeletal muscle and a comparable change in the dissociation of eIF4E from 4E-BP1 and the association of eIF4E with eIF4G (49, 55).

In the neonatal pig in the current study, essentially all of the 4E-BP1 was present in the hypophosphorylated form in muscle in the fasted state. The amount of hyperphosphorylated 4E-BP1 in muscle markedly increased to 60% of the total 4E-BP1 in response to feeding in 7-day-old pigs. However, in fed 26-day-old pigs, hyperphosphorylated 4E-BP1 represented only 10% of the total 4E-BP1. Likewise, the association of 4E-BP1 with eIF4E in muscle decreased to 20% of fasted values in 7-day-old pigs, but to only 60% of fasted values in 26-day-old pigs, in response to feeding. These feeding-induced changes were associated with a 14-fold increase in the active eIF4E · eIF4G complex in muscle of the neonatal pig, and again, the response decreased with development. The results in skeletal muscle of neonatal pigs, together with the results of studies in mature rodents, strongly indicate that there is a progressive age-related decline in the phosphorylation of 4E-BP1, the dissociation of 4E-BP1 from eIF4E, and the association of eIF4E with eIF4G, in response to the ingestion of a meal. These changes in the phosphorylation of 4E-BP1 and the formation of the inactive 4E-BP1 · eIF4E and active eIF4E · eIF4G complexes parallel the developmental decline in the feeding-induced stimulation of protein synthesis and translational efficiency in muscle (5, 9).

Feeding also stimulated the phosphorylation of 4E-BP1 in liver of the neonatal pig in the current study. This suggests that the stimulation of liver protein synthesis by feeding in the neonate (2, 3, 5, 9) involves activation of the eIF4F complex after a meal. Although we have previously shown that liver protein synthesis decreases modestly between 7 and 26 days of age in the pig (5), 4E-BP1 phosphorylation increased with development in the current study. This suggests that factors other than eIF4F complex formation, such as the reduction in eIF2B activity, may be involved in the developmental decline in liver protein synthesis.

Effects of feeding and development on p70S6 kinase. Studies using cells in culture suggest that the phosphorylation of 4E-BP1 is regulated by a signal transduction pathway involving phosphatidylinositide 3-hydroxy kinase, mTOR, and p70S6 kinase (1, 26, 50). Phosphorylation of p70S6 kinase is associated with its activation and results in the phosphorylation of ribosomal S6 protein. Activation of p70S6 kinase is associated with preferential translation of mRNAs encoding specific proteins (20). The signaling pathway that leads to phosphorylation of both 4E-BP1 and p70S6 kinase appears to bifurcate immediately upstream of the two proteins, likely at mTOR. Both insulin and amino acids, when applied to cells in culture, increase the phosphorylation of p70S6 kinase (15, 19, 26, 43). In the current study, feeding of neonatal pigs markedly increased the phosphorylation of p70S6 kinase in both skeletal muscle and liver. This response decreased with development in skeletal muscle but not in liver. The results suggest that the stimulation of protein synthesis by feeding in the neonate involves a signaling pathway that activates p70S6 kinase.

Perspectives. Our results indicate that the enhanced stimulation of skeletal muscle protein synthesis by food intake in the neonate is not regulated by eIF2 or eIF2B. However, eIF2 and eIF2B may play important roles in the long-term changes in muscle protein synthesis that occur over the course of development. The stimulation of muscle protein synthesis by feeding, and the developmental decline in this response, appear to involve regulation by the eIF4F complex. In the neonatal pig, feeding increased the phosphorylation of 4E-BP1, resulting in dissociation of the inactive 4E-BP1 · eIF4E complex and increased association of the active eIF4E · eIF4G complex in skeletal muscle. These changes in eIF4F activation occurred in parallel with increased phosphorylation of p70S6 kinase, suggesting the involvement of the phosphatidylinositide 3-hydroxy kinase/mTOR signaling pathway in this process.

Our results further suggest that the developmental decline in liver protein synthesis, although more modest than that in skeletal muscle, is likely regulated, at least in part, by eIF2B. The stimulation of liver protein synthesis by feeding, like that in skeletal muscle, involves regulation by the eIF4F complex and activation of p70S6 kinase. The lack of effect of development on the stimulation of liver protein synthesis by feeding is likely due to the absence of a developmental change in the activation of translation initiation factors by feeding.

Thus, it seems likely that the enhanced activation of the eIF4F complex after food consumption contributes to the more efficient use of dietary amino acids for growth in the neonate. Furthermore, our study suggests that the enhanced activation of specific eIFs in skeletal muscle of the neonate plays a crucial role in determining the high rate of protein deposition in muscle of the neonate. Further study is required to identify the specific signaling proteins that regulate the developmental changes in the activation of translation initiation by feeding and the potential role of insulin and amino acids as mediators of this process.


    ACKNOWLEDGEMENTS

We thank S. Rannels, P. O'Connor, and W. Liu for technical assistance, J. Cunningham and F. Biggs for care of animals, and L. Loddeke for editorial review.


    FOOTNOTES

This work is a publication of the USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, TX and the Penn State University College of Medicine, Hershey, PA. This project has been funded in part by National Institute of Arthritis and Musculoskeletal and Skin Diseases Institute Grant R01 AR-44474 (TAD) and the US Department of Agriculture, Agricultural Research Service, under Cooperative Agreement number 58-6250-6-001 (TAD). The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organization imply endorsements by the US Government.

Address for reprint requests and other correspondence: T. A. Davis, USDA/ARS Children's Nutrition Research Center, Dept. of Pediatrics, Baylor College of Medicine, 1100 Bates St., Houston, TX 77030 (E-mail: tdavis{at}bcm.tmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 March 2000; accepted in final form 13 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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