1 Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; and 2 United States Department of Agriculture/ Agricultural Research Service, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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Protein synthesis is repressed in both skeletal muscle and liver after a short-term fast and is rapidly stimulated in response to feeding. Previous studies in rats and pigs have shown that the feeding-induced stimulation of protein synthesis is associated with activation of the 70-kDa ribosomal protein S6 kinase (S6K1) as well as enhanced binding of eukaryotic initiation factor eIF4E to eIF4G to form the active eIF4F complex. In cells in culture, hormones and nutrients regulate both of these events through a protein kinase termed the mammalian target of rapamycin (mTOR). In the present study, the involvement of mTOR in the feeding-induced stimulation of protein synthesis in skeletal muscle and liver was examined. Pigs at 7 days of age were fasted for 18 h, and then one-half of the animals were fed. In addition, one-half of the animals in each group were administered rapamycin (0.75 mg/kg) 2 h before feeding. The results reveal that treating 18-h fasted pigs with rapamycin, a specific inhibitor of mTOR, before feeding prevented the activation of S6K1 and the changes in eIF4F complex formation observed in skeletal muscle and liver after feeding. Rapamycin also ablated the feeding-induced stimulation of protein synthesis in liver. In contrast, in skeletal muscle, rapamycin attenuated, but did not prevent, the stimulation of protein synthesis in response to feeding. The results suggest that feeding stimulates hepatic protein synthesis through an mTOR-dependent process involving enhanced eIF4F complex formation and activation of S6K1. However, in skeletal muscle, these two processes may account for only part of the stimulation of protein synthesis, and thus additional steps may be involved in the response.
neonate; growth; eukaryotic initiation factor 4E; 4E-binding protein 1; S6K1
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INTRODUCTION |
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IN BOTH CELLS IN CULTURE and animals in vivo, protein synthesis is stimulated by a variety of hormones and nutrients. In particular, insulin and amino acids independently activate the initiation phase of mRNA translation, an effect that involves enhanced binding of both mRNA and initiator methionyl-tRNA (met-tRNAi) to the 40S ribosomal subunit (reviewed in Ref. 42). The met-tRNAi binding step in translation initiation is mediated by eukaryotic initiation factor eIF2, which binds to the 40S ribosomal subunit as a GTP · eIF2 · met-tRNAi ternary complex (reviewed in Refs. 35, 39). After binding to the 40S ribosomal subunit, the guanosine triphosphate (GTP) bound to eIF2 is hydrolyzed to guanosine diphosphate (GDP), and eIF2 is released from the 40S ribosomal subunit as a binary complex with GDP. Before binding met-tRNAi and participating in another round of initiation, the GDP bound to eIF2 is exchanged for GTP through the action of a second initiation factor, termed eIF2B. However, recent in vivo studies in both rats and pigs suggest that eIF2B activity may not be regulated by changes in nutritional status (15, 50).
The mRNA binding step in translation initiation is mediated by a complex of three initiation factors, termed eIF4F, that is composed of eIF4E, the protein that binds to the m7GTP cap at the 5'-end of the mRNA, eIF4A, an RNA helicase, and eIF4G, a scaffolding protein that, in addition to binding eIF4A and eIF4E, also binds to the ribosome through interactions with eIF3. The best characterized mechanism for regulating mRNA binding to the 40S ribosomal subunit involves the reversible association of eIF4E with the translational repressor, 4E-binding protein 1 (4E-BP1). Binding of 4E-BP1 to eIF4E blocks the association of eIF4E with eIF4G, thereby preventing the binding of mRNA to the 40S ribosomal subunit. 4E-BP1 binding to eIF4E is modulated by phosphorylation of 4E-BP1 on multiple Ser and Thr residues, where hyperphosphorylation precludes and hypophosphorylation is permissive for binding.
In cells in culture, insulin stimulates binding of eIF4E to eIF4G (3, 30, 31, 44), an effect mediated by phosphorylation of 4E-BP1, which results in dissociation of the 4E-BP1 · eIF4E complex, freeing eIF4E to bind to eIF4G (reviewed in Ref. 22). Phosphorylation of 4E-BP1 requires activation of phosphatidylinositol (PI) 3-kinase and protein kinase B (PKB) (21, 43) but additionally involves another protein kinase, referred to as the mammalian target of rapamycin (mTOR) (5, 18). mTOR serves as a bifurcation point in insulin signaling, with both 4E-BP1 and the 70-kDa ribosomal protein S6 kinase (S6K1) being downstream of the kinase. Inhibition of mTOR by the bacterially derived macrolide immunosuppressant, rapamycin, obviates phosphorylation of both 4E-BP1 and S6K1 (5, 18). Like insulin, amino acids, and, in particular Leu, promote phosphorylation of 4E-BP1 and binding of eIF4E to eIF4G (24, 28, 40, 45, 48). However, in contrast to insulin, amino acids do not activate PI 3-kinase but do require mTOR to be active for phosphorylation of 4E-BP1 and S6K1 to occur (24, 40, 45).
In animals subjected to a short (18-h) fast, protein synthesis is repressed in both skeletal muscle and liver (1, 6, 10, 12, 13). The stimulation of protein synthesis that occurs after feeding of fasted animals is associated with phosphorylation of 4E-BP1, release of eIF4E from the inactive 4E-BP1 · eIF4E complex, and increased binding of eIF4E to eIF4G (15, 50). However, no change in eIF2B activity is observed in response to feeding. In the present study, we asked whether activation of S6K1 and binding of eIF4E to eIF4G are required for the stimulation of protein synthesis caused by feeding in the neonate. We chose the neonatal pig as the animal model, because the activation of S6K1, formation of the eIF4F complex, and rates of protein synthesis are highly responsive to feeding, and because these effects are more pronounced the younger the animal (10, 15). We show that, administered 2 h before feeding, rapamycin has differential effectiveness in preventing the feeding-induced stimulation of protein synthesis in skeletal muscle and liver. Thus activation of S6K1 and/or formation of the eIF4F complex is a required component of the feeding-induced stimulation of protein synthesis in liver in neonatal pigs. In contrast, activation of S6K1 and eIF4F complex formation accounts for only part of the stimulation in muscle protein synthesis.
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MATERIALS AND METHODS |
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Animals. Two crossbred (Landrace × Yorkshire × Hampshire × Duroc) pregnant sows (Agriculture Headquarters, Texas Dept. 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 with water ad libitum. After farrowing, piglets remained with the sow and were not given supplemental creep feed. Three days before the study was performed, piglets were anesthetized, and catheters were surgically inserted into a jugular vein as described previously (10). Piglets were returned to the sow until fasting was initiated.
At 7 days of age, 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. The two treatment groups were further subdivided, and one-half of the animals in each subgroup were administered rapamycin (0.75 mg/kg in 5% dimethyl sulfoxide) into the jugular vein catheter. In fasted pigs, rapamycin was injected at the end of the 18-h fasting period, and pigs were killed 2 h later. In fed pigs, rapamycin was injected 2 h before feeding, and pigs were killed 3.5 h later. Pigs were provided with water throughout the fasting period. Pigs that were fed after the 18-h fast were given two gavage feeds 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. Polyvinylidene difluoride (PVDF) membrane was obtained from Bio-Rad. L-[4-3H]phenylalanine was obtained from Amersham Pharmacia Biotech, Piscataway, NJ. Antibodies against S6K1 were purchased from Santa Cruz Biotechnology.
Measurement of protein synthesis in skeletal muscle and liver. The fractional rate of protein synthesis was measured with a flooding dose of L-[4-3H]phenylalanine (19) injected 30 min before the pigs were killed. The specific radioactivities of the protein hydrolysate, homogenate supernatant, and blood supernatant were determined as described previously (14). The fractional rate of protein synthesis (Ks, percentage of protein mass synthesized in a day) was calculated as Ks (%/day) = [(Sb/Sa) × (1,400/t)] × 100, where Sb is the specific radioactivity of the protein-bound phenylalanine, Sa is the specific radioactivity of the tissue-free phenylalanine at the time of the tissue collection and the linear regression of the blood specific radioactivity of the animal at 5, 15, and 30 min against time, and t is the time of labeling in minutes. We have demonstrated (11) that the specific radioactivity of the muscle free phenylalanine, after a flooding dose of the amino acid is administered, is in equilibrium with the aminoacyl tRNA specific radioactivity and therefore provides an equally valid measure of fractional synthesis rate.
Protein immunoblot analysis. Blots were developed using an Amersham ECL Western Blotting Kit as described previously (32). Films were scanned using a Microtek ScanMaker V scanner connected to a Macintosh PowerMac 9600 computer. Images were obtained using the ScanWizard Plugin (Microtek) for Adobe Photoshop and quantitated using NIH Image software.
Quantitation of 4E-BP1 · eIF4E and eIF4G · eIF4E complexes. The association of eIF4E with 4E-BP1 or eIF4G was quantitated by the previously described method (28). Briefly, eIF4E and the 4E-BP1 · eIF4E and eIF4G · eIF4E complexes were immunoprecipitated from aliquots of tissue extracts using an anti-eIF4E monoclonal antibody. The antibody · antigen complex was collected by incubation for 1 h with goat anti-mouse Biomag IgG beads (PerSeptive Diagnostics). 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 on a 15% polyacrylamide gel for quantitation of 4E-BP1 and eIF4E. Proteins were then electrophoretically transferred to a PVDF membrane as described previously (32). 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 for 1 h at room temperature. The blots then were developed using an ECL Western blotting kit as described above.
Analysis of 4E-BP1 phosphorylation state.
Aliquots of tissue extracts 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 using a rabbit anti-human
4E-BP1 antibody as described previously (30). Previous
studies have shown that phosphorylation of 4E-BP1 causes a decrease in
the electrophoretic mobility of the protein on SDS-polyacrylamide gels
(25, 37). Thus 4E-BP1 present in tissue extracts is
separated into multiple electrophoretic forms during SDS-PAGE, with the more slowly migrating forms representing more highly phosphorylated 4E-BP1. Because it is the hyperphosphorylated -form that does not
bind to eIF4E, results are presented as the percentage of 4E-BP1
present in the
-form.
S6K1 phosphorylation. An aliquot of tissue homogenate was combined with an equal volume of SDS sample buffer, and the diluted samples were subjected to electrophoresis on a 7.5% polyacrylamide gel (36). The samples were then analyzed by protein immunoblot analysis using a rabbit anti-rat S6K1 polyclonal antibody as described above.
Statistical analyses. All data were analyzed using a one-way analysis of variance (ANOVA) with fasted non-rapamycin-treated animals as the independent variable. When a significant overall effect was observed, differences among individual means were assessed by the Tukey-Kramer Comparisons Test. The level of significance was set at P < 0.05 for all statistical tests.
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RESULTS |
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As shown in Fig. 1, protein
synthesis in skeletal muscle of pigs fasted for 18 h was
stimulated 62% 1.5 h after feeding. Rapamycin, administered to
fasted animals, had no significant effect on muscle protein synthesis.
However, when administered 2 h before feeding, rapamycin markedly
attenuated the stimulation of protein synthesis caused by feeding,
although it did not completely prevent the increase. Thus the majority
of the stimulation of muscle protein synthesis caused by feeding is
mediated by a rapamycin-sensitive process.
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In a recent study (15), we observed that the stimulation
of protein synthesis in skeletal muscle caused by feeding neonatal pigs
that had been fasted overnight was associated with increased formation
of the eIF4G · eIF4E complex. In the present study, we confirm
the observation that feeding promotes association of eIF4G with eIF4E,
and we extend it to show that, in the presence of rapamycin, feeding no
longer stimulates this association (Fig. 2). Although not statistically
significant, it is noteworthy that, even in the presence of rapamycin,
binding of eIF4G to eIF4E tended to increase after feeding. Rapamycin
had no effect on eIF4G · eIF4E complex formation in fasted
animals, although it should be noted that the amount of eIF4G bound to
eIF4E in muscle from fasted animals is already low and near the
threshold of sensitivity for the assay. Thus a decrease in binding
likely could not have been detected.
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The observed changes in eIF4E binding to eIF4G were inversely
proportional to alterations in 4E-BP1 association with eIF4E (Fig.
3). Thus the amount of 4E-BP1 bound to
eIF4E in muscle extracts from fed animals was ~15% of that in
extracts from fasted animals. Rapamycin prevented the decline in 4E-BP1
binding to eIF4E associated with feeding but did not promote
association beyond that observed in muscle from fasted animals.
Furthermore, rapamycin had no effect on 4E-BP1 · eIF4E binding
in muscle from fasted animals.
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As shown in Fig. 4, the decline in 4E-BP1
binding to eIF4E caused by feeding was associated with phosphorylation
of 4E-BP1. On SDS-polyacrylamide gels, 4E-BP1 can be resolved into
multiple electrophoretic forms (denoted ,
, and
),
representing different phosphorylated forms of the protein
(37). The
- and
-forms of 4E-BP1 both bind to eIF4E
and should therefore both reduce formation of the eIF4F complex. The
-form does not bind to eIF4E and so allows formation of the eIF4F
complex. In particular, in muscle from fasted animals, 4E-BP1 was
predominantly present in the hypophosphorylated
-form, whereas, in
muscle from fed animals, ~80% of the protein was in the
hyperphosphorylated,
-form. As expected, rapamycin prevented
completely the feeding-induced stimulation of 4E-BP1 phosphorylation.
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In addition to promoting eIF4F formation, feeding also stimulates
phosphorylation of S6K1 (20). In the present study, the effect of rapamycin on the feeding-induced phosphorylation of S6K1
phosphorylation was examined. During electrophoresis on
SDS-polyacrylamide gels, S6K1 resolves into multiple electrophoretic
forms based on the amount of phosphate present on the protein.
Similar to 4E-BP1, hyperphosphorylated forms exhibit decreased and
hypophosphorylated forms increased mobility (47). As shown
in Fig. 5, feeding promoted formation of
the more slowly migrating, highly phosphorylated forms of S6K1.
Rapamycin was without effect on basal phosphorylation under fasting
conditions but prevented completely the stimulation of S6K1
phosphorylation caused by feeding.
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In addition to stimulating protein synthesis in skeletal muscle,
feeding a fasted animal promotes protein synthesis in liver (10,
49, 50). To determine whether the stimulation of protein synthesis in liver was blocked by rapamycin, as was shown above for
muscle, 18-h fasted animals were treated with rapamycin before feeding.
As shown in Fig. 6A, in the
absence of rapamycin, feeding caused a significant stimulation in liver
protein synthesis. Rapamycin had no significant effect on liver protein
synthesis in fasted animals, although there was a trend toward lower
rates after treatment. Furthermore, in contrast to skeletal muscle,
where rapamycin did not completely prevent the stimulation of protein
synthesis, in liver, rapamycin obviated the increase in protein
synthesis caused by feeding.
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To establish that the changes in liver protein synthesis were
associated with changes in translation initiation, phosphorylation of
4E-BP1 and S6K1 was examined. As described above for skeletal muscle,
feeding 18-h fasted animals caused a dramatic increase in the amount of
4E-BP1 in the hyperphosphorylated -form (Fig. 6B). Thus,
in fasted animals, 4E-BP1 was almost exclusively in the
hypophosphorylated
-form; no
-form was detected. Feeding caused a
shift in 4E-BP1 distribution such that 50% of the protein was in the
hyperphosphorylated
-form. Rapamycin prevented completely the shift
caused by feeding. In liver of fasted animals, S6K1 was already
partially in hyperphosphorylated forms, and feeding promoted
further phosphorylation (Fig. 5B). Treating fasted animals with
rapamycin caused a shift in S6K1 distribution such that the majority of the protein was present in the fastest migrating, hypophosphorylated form. In addition, rapamycin blocked the
hyperphosphorylation of S6K1 caused by feeding. Overall, the results
suggest that feeding stimulates protein synthesis in liver entirely
through a rapamycin-dependent process.
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DISCUSSION |
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The aim of the present study was to examine the contribution of increased eIF4F complex formation and/or S6K1 activity in controlling protein synthesis after feeding. We chose an animal model that is highly responsive to the stimulatory action of feeding on fractional protein synthesis rates, eIF4F complex formation, and S6K1 activation. One way to examine the contribution of eIF4F and S6K1 is to prevent the changes in these factors and determine whether protein synthesis is still enhanced by feeding. Herein, we used rapamycin in an attempt to specifically block the effects of feeding on protein synthesis in skeletal muscle and liver. Rapamycin is a specific inhibitor of the protein kinase mTOR (reviewed in Ref. 17). However, rapamycin does not directly inhibit mTOR but instead forms an inhibitory complex with the immunophilin referred to as FK506 binding protein (FKBP12) (7, 8, 41). Like rapamycin, FK506 is an immunosuppressant that binds to FKBP12, but unlike rapamycin, the FK506 · FKBP12 complex does not inhibit mTOR.
The importance of mTOR in regulating translation initiation was first demonstrated in yeast, where loss of TOR function was shown to cause an inhibition of translation initiation with characteristics reminiscent of cells subjected to nutrient deprivation (4). Subsequent studies in mammalian cells confirmed the importance of mTOR in mediating the signal generated both by provision of amino acids to amino acid-deprived cells (24, 40, 45, 48) and by treatment of serum-deprived cells with insulin or insulin-like growth factor (IGF) I (3, 23, 29, 43). Other studies have shown that the ability of rapamycin to restrain protein synthesis varies with cell type. Thus, in L6 myoblasts (33), BC3H1 cells (38), and Swiss 3T3 cells (27), rapamycin has little or no effect on protein synthesis in fed cells, whereas in other cases, such as NIH 3T3 cells (5), rapamycin reduces protein synthesis by 50%. In each of these studies, protein synthesis was determined by the incorporation of [35S]methionine into protein, which measures the synthesis of the majority of cellular proteins, i.e., it is a measure of global protein synthesis. In part, the different responses observed in various cell types may reflect the proportion of actively translating mRNAs that have long, highly structured 5'-untranslated regions (5'-UTRs) or that contain a 5'-terminal oligopyrimidine tract (TOP) sequence. Thus, in both L6 myoblasts (33) and Swiss 3T3 cells (27), where global protein synthesis is insensitive to rapamycin, the macrolide obviates the stimulation of translation of mRNAs harboring the TOP sequence. Rapamycin also potently inhibits the translation of ornithine decarboxylase mRNA, which has a long, highly structured 5'-UTR (46). The translation of mRNAs with highly structured 5'-UTRs is regulated by eIF4F, whereas translation of TOP mRNAs is regulated by activation of S6K1 (reviewed in Ref. 16). Because mTOR activity is essential for enhanced formation of the eIF4F complex and activation of S6K1 by amino acids and insulin, inhibition of mTOR by rapamycin would be expected to have a selective effect in modulating translation of such messages. Thus we hypothesize that the differential sensitivity of L6 myoblasts and NIH 3T3 cells may be a result of a greater proportion of mRNAs in NIH 3T3 cells having highly structured 5'-UTRs and/or TOP sequences. Alternatively, eIF4F and/or S6K1 activity may be limiting in NIH 3T3 compared with L6 myoblasts, which would increase the apparent sensitivity to rapamycin.
In the present study, rapamycin completely blocked the feeding-induced stimulation of global protein synthesis in liver. In contrast, in skeletal muscle, rapamycin attenuated but did not completely prevent the stimulation. However, rapamycin was equally effective at preventing changes in eIF4F complex formation and S6K1 phosphorylation in both liver and skeletal muscle. Similarly, Dardevet et al. (9) report that, in incubated muscle preparations, rapamycin attenuates but does not prevent insulin- or IGF-I-stimulated protein synthesis but does block S6K1 activation. These results suggest that feeding stimulates hepatic protein synthesis through activation of eIF4F and/or S6K1. Moreover, although activation of eIF4F and S6K1 accounts for part of the stimulation of muscle protein synthesis, it probably does not account entirely for the effect.
The mechanism responsible for the rapamycin-insensitive stimulation of muscle protein synthesis is unknown. In PC12 pheochromocytoma cells, nerve growth factor- and epidermal growth factor-enhanced binding of eIF4G to eIF4E is prevented by an inhibitor of the mitogen-activated protein (MAP) kinase signaling pathway (34). Likewise, in HEK293 cells, two structurally unrelated inhibitors of the MAP kinase pathway block eIF4G association with eIF4E caused by stimulation of cells with phorbol esters (26). These results suggest that formation of the eIF4F complex cannot be regulated only through the mTOR signaling pathway but also through the MAP kinase pathway. Interestingly, in the present study, there is a trend toward greater binding of eIF4G to eIF4E after feeding in rapamycin-treated pigs, although the difference is not significant. Furthermore, we have recently found that rapamycin attenuates but does not completely prevent the feeding-induced binding of eIF4G to eIF4E in skeletal muscle of fasted rats (2). Thus, in part, feeding may promote eIF4F complex formation through the MAP kinase signaling pathway. It must also be considered that the proportion of mRNAs represented by the TOP family or containing highly structured 5'-UTRs may be greater in liver than in skeletal muscle, which would increase the apparent sensitivity of liver protein synthesis to rapamycin. Overall, it is clear that the mechanism by which feeding stimulates protein synthesis in skeletal muscle through a rapamycin-insensitive pathway is an important topic that should be addressed in future studies.
In summary, in the present study, we show that the feeding-induced stimulation of protein synthesis in liver of neonatal pigs is exquisitely sensitive to rapamycin. The stimulation is associated with increased phosphorylation of 4E-BP1, which promotes dissociation of the 4E-BP1 · eIF4E complex and subsequent binding of eIF4E to eIF4G. The stimulation is also associated with enhanced phosphorylation of S6K1. Furthermore, we show that the stimulation of protein synthesis in skeletal muscle caused by feeding in neonatal pigs is only partially prevented by rapamycin. In that tissue, modulation of eIF4F and S6K1 does not account entirely for the changes in protein synthesis. Thus feeding in the neonate enhances protein synthesis via both shared and tissue-specific mechanisms.
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ACKNOWLEDGEMENTS |
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The authors thank S. Rannels for technical assistance and J. Cunningham and F. Biggs for care of animals.
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FOOTNOTES |
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This work is a publication of the Penn State University College of Medicine and the US Department of Agriculture, Agricultural Research Service (USDA/ARS) Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, TX. This project has been funded in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-15658 (L. S. Jefferson) and DK-13499 (L. S. Jefferson), National Institute of Arthritis and Musculoskeletal and Skin Diseases Institute Grant R01 AR-44474 (T. A. Davis), and the USDA/ARS under Cooperative Agreement number 58-6250-6-001 (T. A. Davis). 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 organizations imply endorsements by the US Government.
Address for reprint requests and other correspondence: S. R. Kimball, Dept. of Cellular and Molecular Physiology, Penn State Univ. College of Medicine, 500 University Dr., Hershey, PA 17033 (E-mail: skimball{at}psu.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 7 April 2000; accepted in final form 6 July 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anthony, JC,
Anthony TG,
Kimball SR,
Vary TC,
and
Jefferson LS.
Orally administered leucine stimulates protein synthesis in skeletal muscle of postabsorptive rats in association with increased eIF4F formation.
J Nutr
130:
139-145,
2000
2.
Anthony JC, Yoshizawa F, Anthony TG, Vary TC, Jefferson LS, and Kimball
SR. Leucine stimulates translation initiation in skeletal muscle
of post-absorptive rats via a rapamycin-sensitive pathway. J
Nutr. In press.
3.
Azpiaza, I,
Saltiel AR,
DePaoli-Roach AA,
and
Lawrence JC.
Regulation of both glycogen synthase and PHAS-I by insulin in rat skeletal muscle involves MAP kinase-independent and rapamycin-sensitive pathways.
J Biol Chem
271:
5033-5039,
1996
4.
Barbet, NC,
Schneider U,
Helliwell SB,
Stansfield I,
Tuite MF,
and
Hall MN.
TOR controls translation initiation and early G1 progression in yeast.
Mol Biol Cell
7:
25-42,
1996[Abstract].
5.
Beretta, L,
Gingras AC,
Svitkin YV,
Hall MN,
and
Sonenberg N.
Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation.
EMBO J
15:
658-664,
1996[Abstract].
6.
Burrin, DG,
Davis TA,
Fiorotto ML,
and
Reeds PJ.
Hepatic protein synthesis in suckling rats: effects of state of development and fasting.
Pediatr Res
31:
247-252,
1992[Abstract].
7.
Choi, J,
Chen J,
Schreiber SL,
and
Clardy J.
Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP.
Science
273:
239-242,
1996[Abstract].
8.
Chung, J,
Kuo CJ,
Crabtree GR,
and
Blenis J.
Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases.
Cell
69:
1227-1236,
1992[ISI][Medline].
9.
Dardevet, D,
Sornet C,
Vary TC,
and
Grizard J.
Phosphatidylinositol 3-kinase and p70 S6 kinase participate in regulation of protein turnover in skeletal muscle by insulin and insulin-like growth factor I.
Endocrinology
137:
4087-4094,
1996[Abstract].
10.
Davis, TA,
Burrin DG,
Fiorotto ML,
and
Nguyen HV.
Protein synthesis in skeletal muscle and jejunum is more responsive to feeding in 7- than in 26-day-old pigs.
Am J Physiol Endocrinol Metab
270:
E802-E809,
1996
11.
Davis, TA,
Fiorotto ML,
Nguyen HV,
and
Burrin DG.
Aminoacyl-tRNA and tissue free amino acid pools are equilibrated after a flooding dose of phenylalanine.
Am J Physiol Endocrinol Metab
277:
E103-E109,
1999
12.
Davis, TA,
Fiorotto ML,
Nguyen HV,
Burrin DG,
and
Reeds PJ.
Response of muscle protein synthesis to fasting in suckling and weaned rats.
Am J Physiol Regulatory Integrative Comp Physiol
261:
R1373-R1380,
1991
13.
Davis, TA,
Fiorotto ML,
Nguyen HV,
and
Reeds PJ.
Enhanced response of muscle protein synthesis and plasma insulin to food intake in suckled rats.
Am J Physiol Regulatory Integrative Comp Physiol
265:
R334-R340,
1993
14.
Davis, TA,
Fiorotto ML,
Ngyuen HV,
and
Reeds PJ.
Protein turnover in skeletal muscle of suckling rats.
Am J Physiol Regulatory Integrative Comp Physiol
257:
R1141-R1146,
1989
15.
Davis TA, Nguyen HV, Suryawan A, Bush J, Jefferson LS, and Kimball
SR. Developmental changes in the stimulation of translation
initiation by feeding in muscle and liver of neonatal pigs.
Am J Physiol Endocrinol Metab. In press.
16.
Dennis, PB,
Fumagalli S,
and
Thomas G.
Target of rapamycin (TOR): balancing the opposing forces of protein synthesis and degradation.
Curr Opin Genet Dev
9:
49-54,
1999[ISI][Medline].
17.
Dufner, A,
and
Thomas G.
Ribosomal S6 kinase signaling and the control of translation.
Exp Cell Res
253:
100-109,
1999[ISI][Medline].
18.
Fadden, P,
Haystead TAJ,
and
Lawrence JC.
Identification of phosphorylation sites in the translational regulator, PHAS-I, that are controlled by insulin and rapamycin in rat adipocytes.
J Biol Chem
272:
10240-10247,
1997
19.
Garlick, PJ,
McNurlan MA,
and
Preedy VR.
A rapid and convenient technique for measuring the rate of protein synthesis in tissue by injection of [3H]phenylalanine.
Biochem J
192:
719-723,
1980[ISI][Medline].
20.
Gautsch, TA,
Anthony JC,
Kimball SR,
Paul GL,
Layman DK,
and
Jefferson LS.
Availability of eIF4E regulates skeletal muscle protein synthesis during recovery from exercise.
Am J Physiol Cell Physiol
274:
C406-C414,
1998
21.
Gingras, AC,
Kennedy SG,
O'Leary MA,
Sonenberg N,
and
Hay H.
4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt (PKB) signaling pathway.
Genes Dev
12:
502-513,
1998
22.
Gingras, AC,
Raught B,
and
Sonenberg N.
eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation.
In: Annual Reviews in Biochemistry, edited by Richardson CC. Palo Alto, CA: Annual Reviews, 1999, p. 913-963.
23.
Graves, LM,
Bornfeldt KE,
Argast GM,
Krebs EG,
Kong X,
Lin TA,
and
Lawrence JC.
cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.
Proc Natl Acad Sci USA
92:
7222-7226,
1995[Abstract].
24.
Hara, K,
Yonezawa K,
Weng QP,
Kozlowski MT,
Belham C,
and
Avruch J.
Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism.
J Biol Chem
273:
14484-14494,
1998
25.
Haystead, TAJ,
Haystead CMM,
Hu C,
Lin TA,
and
Lawrence JC.
Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase. Identification of a site phosphorylated by MAP kinase in vitro and in response to insulin in rat adipocytes.
J Biol Chem
269:
23185-23191,
1994
26.
Herbert, TP,
Kilhams GP,
Batty IH,
and
Proud CG.
Distinct signalling pathways mediate insulin and phorbol ester-stimulated eukaryotic initiation factor 4F assembly and protein synthesis in HEK 293 cells.
J Biol Chem
275:
11249-11256,
2000
27.
Jefferies, HBJ,
Reinhard C,
Kozma SC,
and
Thomas G.
Rapamycin selectively represses translation of the "polypyrimidine tract" mRNA family.
Proc Natl Acad Sci USA
91:
4441-4445,
1994[Abstract].
28.
Kimball, SR,
Horetsky RL,
and
Jefferson LS.
Implication of eIF2B rather than eIF4E in the regulation of global protein synthesis by amino acids in L6 myoblasts.
J Biol Chem
273:
30945-30953,
1998
29.
Kimball, SR,
Horetsky RL,
and
Jefferson LS.
Signal transduction pathways involved in the regulation of protein synthesis by insulin in L6 myoblasts.
Am J Physiol Cell Physiol
274:
C221-C228,
1998
30.
Kimball, SR,
Jefferson LS,
Fadden P,
Haystead TAJ,
and
Lawrence JC.
Insulin and diabetes cause reciprocal changes in the association of eIF-4E and PHAS-I in rat skeletal muscle.
Am J Physiol Cell Physiol
270:
C705-C709,
1996
31.
Kimball, SR,
Jurasinski CV,
Lawrence JC,
and
Jefferson LS.
Insulin stimulates protein synthesis in skeletal muscle by enhancing the association of eIF-4E and eIF-4G.
Am J Physiol Cell Physiol
272:
C754-C759,
1997
32.
Kimball, SR,
Karinch AM,
Feldhoff RC,
Mellor H,
and
Jefferson LS.
Purification and characterization of eukaryotic initiation factor eIF-2B from liver.
Biochim Biophys Acta
1201:
473-481,
1994[ISI][Medline].
33.
Kimball, SR,
Shantz LM,
Horetsky RL,
and
Jefferson LS.
Leucine regulates translation of specific mRNAs in L6 myoblasts through mTOR-mediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6.
J Biol Chem
274:
11647-11652,
1999
34.
Kleijn, M,
and
Proud CG.
Glucose and amino acids modulate translation factor activation by growth factors in PC12 cells.
Biochem J
347:
399-406,
2000[ISI][Medline].
35.
Kleijn, M,
Scheper GC,
Voorma HO,
and
Thomas AAM
Regulation of translation initiation factors by signal transduction.
Eur J Biochem
253:
531-544,
1998[Abstract].
36.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
37.
Lin, TA,
Kong X,
Haystead TAJ,
Pause A,
Belsham G,
Sonenberg N,
and
Lawrence JC.
PHAS-I as a link between mitogen-activated protein kinase and translation initiation.
Science
266:
653-656,
1994[ISI][Medline].
38.
Marx, SO,
and
Marks AR.
Cell cycle progression and proliferation despite 4BP-1 dephosphorylation.
Mol Cell Biol
19:
6041-6047,
1999
39.
Pain, VM.
Initiation of protein synthesis in eukaryotic cells.
Eur J Biochem
236:
747-771,
1996[Abstract].
40.
Patti, ME,
Brambilla E,
Luzi L,
Landaker EJ,
and
Kahn CR.
Bidirectional modulation of insulin action by amino acids.
J Clin Invest
101:
1519-1529,
1998
41.
Price, DJ,
Grove JR,
Calvo V,
Avruch J,
and
Bierer BE.
Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase.
Science
257:
973-977,
1992[ISI][Medline].
42.
Rhoads, RE.
Signal transduction pathways that regulate eukaryotic protein synthesis.
J Biol Chem
274:
30337-30340,
1999
43.
Scott, PH,
Brunn GJ,
Kohn AD,
Roth RA,
and
Lawrence JC.
Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway.
Proc Natl Acad Sci USA
95:
7772-7777,
1998
44.
Von Manteuffel, SR,
Dennis PB,
Pullen N,
Gingras AC,
Sonenberg N,
and
Thomas G.
The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70(S6K).
Mol Cell Biol
17:
5426-5436,
1997[Abstract].
45.
Wang, X,
Campbell LE,
Miller CM,
and
Proud CG.
Amino acid availability regulates p70 S6 kinase and multiple translation factors.
Biochem J
334:
261-267,
1998[ISI][Medline].
46.
Wen, L,
Huang JK,
and
Blackshear PJ.
Rat ornithine decarboxylase gene. Nucleotide sequence, potential regulatory elements, and comparison to the mouse gene.
J Biol Chem
264:
9016-9021,
1989
47.
Weng, QP,
Kozlowski M,
Belham C,
Zhang A,
Comb MJ,
and
Avruch J.
Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies.
J Biol Chem
273:
16621-16629,
1998
48.
Xu, G,
Kwon G,
Marshall CA,
Lin TA,
Lawrence JC,
and
McDaniel ML.
Branched-chain amino acids are essential in the regulation of PHAS-I and p70 S6 kinase by pancreatic -cells. A possible role in protein translation and mitogenic signaling.
J Biol Chem
273:
28178-28184,
1998
49.
Yoshizawa, F,
Endo M,
Ide H,
Yagasaki K,
and
Funabiki R.
Translational regulation of protein synthesis in the liver and skeletal muscle of mice in response to refeeding.
J Nutr Biochem
6:
130-136,
1995[ISI].
50.
Yoshizawa, F,
Kimball SR,
and
Jefferson LS.
Modulation of translation initiation in rat skeletal muscle and liver in response to food intake.
Biochem Biophys Res Commun
240:
825-831,
1997[ISI][Medline].