Developmental decline in components of signal transduction pathways regulating protein synthesis in pig muscle

Scot R. Kimball1, Peter A. Farrell2, Hahn V. Nguyen3, Leonard S. Jefferson1, and Teresa A. Davis3

1 Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey 17033; 2 Noll Physiology Research Center, The Pennsylvania State University, University Park, Pennsylvania 16802; and 3 Department of Pediatrics, United States Department of Agriculture, Agricultural Research Service, Children's Nutritional Research Center, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our previous studies showed that the feeding-induced stimulation of protein synthesis in skeletal muscle of neonatal pigs is accompanied by enhanced phosphorylation of the eukaryotic initiation factor (eIF)4E-binding protein (4E-BP1) and the ribosomal protein S6 kinase (S6K1). These effects of feeding are substantially reduced with development. The goal of the present investigation was to delineate the basis for the reduced responsiveness to feeding observed in the older animals. In these studies, the content and activity of protein kinases located upstream of S6K1 and 4E-BP1 in signal transduction pathways activated by amino acids, insulin, and insulin-like growth factor I were examined in 7- and 26-day-old pigs that were either fasted overnight or fed porcine milk after an overnight fast. Feeding stimulated phosphatidylinositol (PI) 3-kinase activity to the same extent in muscle of 7- and 26-day-old pigs, suggesting that PI 3-kinase is not limiting in muscle of older animals. In contrast, protein kinase B (PKB) activity was significantly less in muscle from 26- vs. 7-day-old pigs, regardless of nutritional status, suggesting that its activity is regulated by mechanisms distinct from PI 3-kinase. In part, the reduced PKB responsiveness can be attributed to a developmental decline in PKB content. Likewise, muscle content of the protein kinase termed mammalian target of rapamycin (mTOR) in 26-day-old pigs was <25% of that in 7-day-old animals. Finally, in agreement with our earlier work showing that S6K1 phosphorylation is reduced in older animals, S6K1 activity was stimulated to a lesser extent in 26- compared with 7-day-old pigs. Overall, the results suggest that the blunted protein synthetic response observed in 26- vs. 7-day-old neonatal pigs is due in part to decreased content and/or activity of signaling components downstream of PI 3-kinase, e.g., PKB, mTOR, and S6K1.

phosphatidylinositol 3-kinase; protein kinase B; mammalian target of rapamycin; protein 70 S6 kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SKELETAL MUSCLE PROTEIN SYNTHESIS is particularly high during the early neonatal period, resulting in rapid rates of muscle growth (11, 14, 21). Both muscle growth and the rate of synthesis of muscle proteins decline as the neonate develops. These developmental changes can be largely attributed to a reduced response of muscle to the stimulatory action of feeding on protein synthesis (7, 12, 15). Our studies in neonatal pigs and rats have shown that muscle protein synthesis is repressed after 10-18 h of fasting and maximally stimulated 1-3 h after feeding (7, 10, 12). These responses decrease with development. For example, in response to feeding, 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 (7). This suggests that the ability of muscle to respond to feeding is impaired at an early point in development.

To identify the mechanism responsible for the developmental decline in the postprandial stimulation of muscle protein synthesis, our recent studies examined the activation by feeding of factors that regulate translation initiation (13). It was found that the stimulation is, in part, a result of enhanced phosphorylation of both the ribosomal protein S6 kinase (S6K1) and the eukaryotic initiation factor (eIF)4E-binding protein (4E-BP)1. These responses to feeding were reduced in muscle of 26- compared with 7-day-old pigs. Phosphorylation of S6K1 on multiple serine and threonine residues results in its activation, leading to phosphorylation of S6 and ultimately to a preferential increase in the translation of mRNAs containing a terminal oligopyrimidine (TOP) sequence adjacent to the m7GTP cap structure at the 5' end of the message (19, 30). Phosphorylation of 4E-BP1 leads to dissociation of the inactive 4E-BP1 · eIF4E complex and increased assembly of the active eIF4F complex. In the initiation of mRNA translation, the eIF4F complex mediates binding of mRNA to the 40S ribosomal subunit (see review in Ref. 20). The heterotrimeric eIF4F complex is comprised of eIF4E, the protein that binds to the m7GTP cap structure at the 5' end of the mRNA; eIF4A, an RNA helicase; and eIF4G, a scaffolding protein that assembles into an active complex the proteins required for mRNA binding to 40S ribosome subunits. Assembly of the eIF4F complex is regulated, in part, by the binding of eIF4E to 4E-BP1. When eIF4E is bound to 4E-BP1, its interaction with eIF4G is precluded, and the eIF4F complex cannot be assembled. The association of 4E-BP1 with eIF4E is regulated by phosphorylation of 4E-BP1, where hypophosphorylated forms of the protein bind to eIF4E and hyperphosphorylated forms do not.

A possible basis for the decline in the responsiveness of muscle protein synthesis to feeding observed in 26-day-old neonatal pigs could be a reduction during development of the amounts of key translation initiation factors. However, this explanation is unlikely, because no difference in initiation factor content is found when muscles from 26- and 7-day-old animals are compared (13). An alternative explanation for the decreased responsiveness in 26-day-old animals would be a developmental decline in the circulating levels of factors that stimulate protein synthesis. Amino acids, insulin, and insulin-like growth factor I (IGF-I) are anabolic agents that have been shown to both stimulate protein synthesis and increase in response to food ingestion (8, 9, 37). However, our previous studies have shown that the developmental decline in protein synthesis is largely unrelated to changes in the circulating concentrations of these anabolic agents (7). Recent evidence suggests that the developmental change in the responsiveness of muscle protein synthesis to insulin may be due to a developmental decline in the capacity of skeletal muscle to sense or transduce the feeding-induced stimulus. For example, the developmental decline in the feeding-induced stimulation of protein synthesis can be reproduced by the infusion of insulin or amino acids in 7- and 26-day-old pigs (8, 9, 37), suggesting that an altered responsiveness to insulin or amino acids is a primary determinant of the developmental decline in the stimulation of protein synthesis by feeding. Furthermore, the developmental decline in the postprandial stimulation of protein synthesis is paralleled by comparable changes in the activation of early steps in the insulin signaling pathway (36).

In further support of a regulatory role for both insulin and/or amino acids in the developmental change in the feeding-induced stimulation of protein synthesis, both stimuli promote eIF4F assembly and activate the ribosomal protein S6K1 (see review in Ref. 25). Importantly, inhibition of the protein kinase termed mammalian target of rapamycin (mTOR; also known as RAFT or FRAP) strongly attenuates the feeding-induced assembly of both eIF4F and S6K1 activation in neonatal pigs (26). mTOR is downstream of phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB) in the insulin signal transduction pathway (see review in Ref. 16). It is activated by insulin (34) and is absolutely required for amino acid signaling to 4E-BP1 and S6K1 (28). Thus the PI 3-kinase/mTOR signal transduction pathway represents an important control point in the feeding-induced stimulation of protein synthesis.

In the present study, we examined the content and/or activity of a number of components of the signal transduction pathway that lie between the insulin receptor and 4E-BP1 and S6K1, to ascertain their roles in the decreased responsiveness of muscle protein synthesis to feeding in 26- compared with 7-day-old neonatal pigs. We found that muscle PI 3-kinase content is stimulated by feeding to the same extent, regardless of age. In contrast, PKB activity exhibited an age-related decline that is due, in part, to a reduction in PKB content. Furthermore, mTOR content in muscle from 26-day-old pigs was only 20% of the value observed in 7-day-old animals. Overall, the results suggest that the developmental decline in the responsiveness of muscle protein synthesis to feeding may be a result of a reduction in the capacity of intracellular signaling pathways to transduce to the translational apparatus the stimulus provided by feeding.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. The kit for measuring PKB activity and the anti-insulin receptor substrate (IRS)-1 antibody were purchased from Upstate Biotechnology. Both the anti-S6K1 and anti-phospho-Thr389 S6K1 antibodies were purchased from Santa Cruz Biotechnology. The anti-PKB, anti-phospho-Ser473 PKB, and anti-phospho-Thr70 4E-BP1 antibodies were obtained from Cell Signaling Technology. Protein A-Sepharose and protein G-Sepharose were obtained from Pharmacia, and the anti-mTOR antibody and protein G-Agarose were from Calbiochem.

Animals. The neonatal pig was used as an animal model because of its similarity to the human infant in anatomy, developmental physiology, and metabolism (6). Pigs were studied at 7 days of age to avoid the complications of endocytosis of ingested protein in the newborn. Pigs were studied at 26 days of age for comparison to a more advanced stage of development but before diet composition changes. At either 7 or 26 days of age, pigs within a 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 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 muscle were removed, 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.

Protein immunoblot analysis. Blots were developed using an Amersham enhanced chemiluminescence (ECL) Western Blotting Kit, exactly as described previously (27). 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 National Institutes of Health Image software. Results are expressed as arbitrary units, which represent the integrated pixel intensity of the band being analyzed.

Measurement of mTOR and PKB content. Muscle samples were homogenized in seven volumes of buffer A [consisting of 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, pH 7.4), 100 mM KCl, 0.2 mM EDTA, 2 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM dithiothreitol, 50 mM sodium fluoride, 50 mM beta -glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 0.5 mM sodium vanadate] and then centrifuged at 10,000 g for 10 min at 4°C. Samples were mixed with an equal volume of SDS-sample buffer and heated at 100°C for 3 min; mTOR and PKB content was measured by protein immunoblot analysis, as described in Protein immunoblot analysis.

Determination of PKB phosphorylation on Ser473, S6K1 phosphorylation on Thr389, and 4E-BP1 phosphorylation on Thr70. Samples were prepared as described in the previous paragraph, and the phosphorylation of PKB on Ser473, S6K1 on Thr389, and 4E-BP1 on Thr70 was determined by protein immunoblot analysis with antibodies that recognize the proteins only when they are phosphorylated on those residues.

Measurement of PI 3-kinase activity. PI 3-kinase activity was measured in IRS-1 immunoprecipitates from muscle homogenates, as described previously (23). Briefly, homogenate containing 1 mg of protein was immunoprecipitated using an anti-IRS-1 polyclonal antibody (5 µg); the immunoprecipitates were collected using protein A-Sepharose beads. The beads were thoroughly washed and incubated with a lipid mixture and [gamma -32P]ATP. The reaction was stopped by the addition of 1 N HCl, and phospholipid products were resolved by thin-layer chromatography. Radioactivity incorporated into lipids phosphorylated at the 3-position was quantitated using a Bio-Rad phosphoimager.

Measurement of PKB activity. PKB activity was measured using a kit from Upstate Biotechnology (no. 17-188). Briefly, anti-Akt/PKB-alpha antibody (4 µg) was incubated overnight with protein G-agarose, and on the next morning the antibody · protein G complex was washed to remove unbound antibody. Muscle samples were homogenized in seven volumes of buffer B [consisting of 50 mM Tris · HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3VO4, 0.1% 2-mercaptoethanol, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM beta -glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 µM microcystin, and 1 µg/ml each of aprotinin, pepstatin, and leupeptin] and then centrifuged at 10,000 g for 10 min at 4°C. Protein content was measured using a kit (Bio-Rad Detergent Compatible Assay), and muscle supernatant containing 1 mg of protein was incubated with the washed antibody · protein G complex for 90 min at 4°C. The immunoprecipitate was washed and then incubated with [gamma -32P]ATP and the peptide substrate RPRAAATF for 10 min at 30°C. The reaction mixture was centrifuged, and an aliquot (40 µl) of supernatant was added to a tube containing 20 µl of 40% trichloroacetic acid. The acidified sample was adsorbed onto phosphocellulose filter disks, and the disks were sequentially washed with 0.75% phosphoric acid and acetone and then dried. 32P incorporation into peptide bound to the filter disks was quantitated by liquid scintillation spectrometry.

Measurement of S6K1 activity. The method used to measure S6K1 activity was similar to that described above for PKB, except that an anti-S6K1 antibody was used for immunoprecipitation, and the substrate peptide was RRRLSSLRA (23).

Statistical analysis. Data were analyzed using Prism for Macintosh, version 3 (Graphpad Software), and are presented as means ± SE. Analysis was initially performed by two-way ANOVA, with animal age and feeding status as independent variables, and was followed by unpaired t-tests if a significant effect of either age or feeding was detected by ANOVA. The level of significance was set at P < 0.05 for all statistical tests.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we investigated the hypothesis that the basis for the decreased responsiveness of muscle protein synthesis to feeding in 26- compared with 7-day-old animals is a reduction in activity and/or content of one or more of the components of the insulin signal transduction pathway. After an 18-h fast, PI 3-kinase activity was not significantly different in muscle from 26- compared with 7-day-old pigs (Fig. 1). Likewise, in muscle from fed animals, PI 3-kinase activity was the same in both 26- and 7-day-old animals, suggesting that neither PI 3-kinase nor components of the signal transduction pathway upstream of it are limiting in 26-day-old pigs. Thus the reduced responsiveness observed in 26-day-old animals is not caused by a limitation in PI 3-kinase activity.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Phosphatidylinositol 3 (PI 3)-kinase activity in skeletal muscle of food-deprived or fed pigs at 7 and 26 days of age. PI 3-kinase activity was measured in extracts of skeletal muscle, as described in MATERIALS AND METHODS. Animals were either fasted for 18 h (open bars) or fasted for 18 h and then fed (solid bars). Results are presented as pmol of 32Pi incorporated into lipid substrate per mg protein per min and represent means ± SE of 7-8 animals per condition. Effect of feeding on PI 3-kinase activity was significant (P < 0.0001) as assessed by two-way ANOVA. *P = 0.006 vs. fasted 7-day-old pigs; dagger P < 0.0001 vs. fasted 26-day-old pigs.

To examine signaling downstream of PI 3-kinase activity, the activity of PKB was measured. As shown in Fig. 2, PKB activity was significantly less in muscle from 26- than from 7-day-old pigs, regardless of nutritional status. Moreover, although the magnitude of the stimulation of PKB activity was dramatically reduced in muscle from 26- compared with 7-day-old animals, proportionally, the feeding-induced increase in activity was greater than fivefold in animals of both ages, suggesting that the increase is independent of age.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Protein kinase B (PKB) activity in skeletal muscle of food-deprived or fed pigs at 7 and 26 days of age. PKB activity was measured in extracts of skeletal muscle as described in MATERIALS AND METHODS. Animals were either fasted for 18 h (open bars) or fasted for 18 h and then fed (solid bars), as described in the legend to Fig. 1. Results are presented as pmol of 32Pi incorporated per mg muscle protein assayed and represent means ± SE of 7-9 animals per condition. Effects of both age and feeding on PKB activity were significant (P = 0.016 and P = 0.005, respectively) as assessed by two-way ANOVA. *P = 0.015 vs. fasted 7-day-old pigs; Dagger P = 0.0002 vs. fasted 7-day-old pigs, and P = 0.013 vs. fed 7-day-old pigs; dagger P = 0.028 vs. fasted 7-day-old pigs, P = 0.0076 vs. fasted 26-day-old pigs.

One possible explanation for the reduction in PKB activity that was observed between 7 and 26 days of age is that the content of the kinase declines during this period. To assess this possibility, muscle PKB content was measured by Western blot analysis. As shown in Fig. 3, the amount of PKB in muscle of 26-day-old neonatal pigs was significantly reduced compared with that of 7-day-old animals. In contrast, there was no decrease in PKB content in fasted compared with fed animals. Although the age-related decline in PKB content may account for part of the decrease in PKB activity, it is of insufficient magnitude to account entirely for the observed change. An alternative explanation for the reduction in PKB activity is that differential phosphorylation of PKB occurs at the different ages. The activity of PKB is modulated by phosphorylation at two key amino acid residues, Thr308 and Ser473 (1). In the present study, the phosphorylation state of Ser473 was examined by Western blot analysis by use of an anti-phosphopeptide antibody that recognizes PKB only when it is phosphorylated at Ser473. The signal obtained with the anti-phosphopeptide antibody was normalized for total PKB content. As shown in Fig. 4, basal PKB phosphorylation (i.e., phosphorylation in fasted animals) was similar in 26- and 7-day-old pigs. Moreover, PKB phosphorylation was stimulated to a similar extent by feeding in animals of both ages. Interestingly, changes in PKB activity (Fig. 2) did not correlate with changes in phosphorylation of Ser473. The basis for this discrepancy is unknown but may be explained by alterations in phosphorylation at Thr308. Unfortunately, our attempts to measure phosphorylation of Thr308 have been unsuccessful, possibly due to failure of the anti-mouse PKB antibody to recognize the pig protein.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   PKB content in skeletal muscle of food-deprived or fed pigs at 7 and 26 days of age. PKB content was measured by Western blot analysis, as described in MATERIALS AND METHODS. Results from a representative blot are shown as an inset. Each lane on the blot represents an individual animal. Moreover, samples from each condition were analyzed on every blot to minimize blot-to-blot variations in the results. Animals were either fasted for 18 h (open bars) or fasted for 18 h and then fed (solid bars), as described in the legend to Fig. 1. Results represent means ± SE of 12-19 animals per condition. Effect of age on PKB content was significant (P = 0.0001) as assessed by two-way ANOVA. *P = 0.018 vs. fasted 7-day-old pigs, and P = 0.041 vs. fed 7-day-old pigs; dagger P = 0.0003 vs. fasted 7-day-old pigs, and P = 0.0008 vs. fed 7-day-old pigs.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Phosphorylation status of Ser473 on PKB in skeletal muscle of food-deprived or fed pigs at 7 and 26 days of age. PKB phosphorylation on Ser473 was measured by Western blot analysis using an antibody that recognizes PKB only when Ser473 is phosphorylated. Results from a representative blot are shown as an inset. Each lane on the blot represents an individual animal. Moreover, samples from each condition were analyzed on every blot to minimize blot-to-blot variations in the results. Animals were either fasted for 18 h (open bars) or fasted for 18 h and then fed (solid bars), as described in the legend to Fig. 1. Results were normalized for PKB content and represent means ± SE of 16-19 animals per condition. Effect of feeding on Ser473 phosphorylation was significant (P < 0.0001) as assessed by 2-way ANOVA. *P < 0.0001 vs. fasted 7-day-old pigs; dagger P < 0.0001 vs. fasted 26-day-old pigs.

In both cells in culture and animals in vivo, signaling from insulin and amino acids to protein synthesis is prevented by inhibitors of mTOR (see review in Ref. 25), suggesting that this protein kinase plays a key role in the response. To establish whether or not mTOR expression exhibits developmental alterations, muscle mTOR content was measured by Western blot analysis. As shown in Fig. 5, mTOR content was significantly reduced in muscle from 26- compared with 7-day-old pigs. No change in mTOR content was observed in response to feeding in animals of either age.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   Mammalian target of rapamycin (mTOR) content in skeletal muscle of food-deprived or fed pigs at 7 and 26 days of age. mTOR content was measured by Western blot analysis, as described in MATERIALS AND METHODS. Results from a representative blot are shown as an inset. Each lane on the blot represents an individual animal. Moreover, samples from each condition were analyzed on every blot to minimize blot-to-blot variations in results. Animals were either fasted for 18 h (open bars) or fasted for 18 h and then fed (solid bars), as described in legend to Fig. 1. Results represent means ± SE of 8 animals per condition. Effect of age on mTOR content was significant (P < 0.0001) as assessed by 2-way ANOVA. *P = 0.003 vs. fasted 7-day-old pigs, and P = 0.0003 vs. fed 7-day-old pigs; dagger P = 0.003 vs. fasted 7-day-old pigs, and P = 0.0002 vs. fed 7-day-old pigs.

Our previous studies have shown that hyperphosphorylation of S6K1 in response to feeding, as assessed by decreased mobility during SDS-polyacrylamide gel electrophoresis, is reduced in 26- compared with 7-day-old pigs (13) and that this phosphorylation is prevented by pretreatment of the animals with rapamycin (26). In the present study, we extend the earlier observations to examine the phosphorylation of S6K1 on a specific amino acid residue, i.e., Thr389. In in vitro studies, phosphorylation of Thr389, in conjunction with phosphorylation of Thr229, results in maximal activation of S6K1 activity (18). We found that the phosphorylation of S6K1 on Thr389 was dramatically increased by feeding in 7- but not in 26-day-old pigs (Fig. 6). To determine whether phosphorylation of Thr389 in muscle of fed pigs results in activation of the kinase, S6K1 activity was measured in muscle extracts of fed and fasted pigs. As shown in Fig. 7, S6K1 activity was increased in skeletal muscle in response to feeding. In agreement with the observed changes in Thr389 phosphorylation, S6K1 was activated to a lesser extent in muscle of 26- compared with 7-day-old pigs.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6.   Phosphorylation status of Thr389 on S6K1 in skeletal muscle of food-deprived or fed pigs at 7 and 26 days of age. S6K1 phosphorylation on Thr389 was measured by Western blot analysis using an antibody that recognizes S6K1 only when Thr389 is phosphorylated. Results from a representative blot are shown as an inset. Each lane on the blot represents an individual animal. Moreover, samples from each condition were analyzed on every blot to minimize blot-to-blot variations in results. Animals were either fasted for 18 h (open bars) or fasted for 18 h and then fed (solid bars), as described in legend to Fig. 1. Results are means ± SE of 4-5 animals per condition. Effects of both age and feeding on Thr389 phosphorylation were significant (P < 0.0001 for both) as assessed by two-way ANOVA. *P < 0.0001 vs. fasted 7-day-old pigs; dagger P = 0.0002 vs. fed 7-day-old pigs.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   S6 kinase 1 (S6K1) activity in skeletal muscle of food-deprived or fed pigs at 7 and 26 days of age. S6K1 activity was measured in extracts of skeletal muscle, as described in MATERIALS AND METHODS. Animals were either fasted for 18 h (open bars) or fasted for 18 h and then fed (solid bars), as described in legend to Fig. 1. Results are presented as pmol of 32Pi incorporated per mg muscle protein assayed per min, and they represent means ± SE of 8-10 animals per condition. Effects of both age and feeding on S6K1 activity were significant (P = 0.031 and P < 0.0001, respectively) as assessed by 2-way ANOVA. *P < 0.0001 vs. fasted 7-day-old pigs; Dagger P = 0.0003 vs. fed 7-day-old pigs; dagger P < 0.0001 vs. fasted 7-day-old pigs, P = 0.0356 vs. fed 7-day-old pigs, and P < 0.0001 vs. fasted 26-day-old pigs.

Our previous studies showed that feeding increased the amount of 4E-BP1 in the most highly phosphorylated form from 0 to 60% at 7 days of age, but from 0 to only 10% at 26 days of age. In the current study, we determined whether the previously observed phosphorylation patterns corresponded to changes in the phosphorylation of 4E-BP1 at Thr70, a site that has been shown to be important in regulating its association with eIF4E when phosphorylated. We found that feeding increased 4E-BP1 phosphorylation at Thr70 in 7-day-old pigs but not in 26-day-old pigs (Fig. 8).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 8.   Phosphorylation status of Thr70 on eukaryotic initiation factor (eIF)4E-binding protein (BP)-1 in skeletal muscle of food-deprived or fed pigs at 7 and 26 days of age. 4E-BP1 phosphorylation on Thr70 was measured by Western blot analysis with an antibody that recognizes 4E-BP1 only when Thr70 is phosphorylated. Results from a representative blot are shown as an inset. Each lane on the blot represents an individual animal. Moreover, samples from each condition were analyzed on every blot to minimize blot-to-blot variations in results. Animals were either fasted for 18 h (open bars) or fasted for 18 h and then fed (solid bars), as described in legend to Fig. 1. Results are means ± SE of 7-10 animals per condition. Effects of both age and feeding on Thr70 phosphorylation were significant (P = 0.0054 and P = 0016, respectively) as assessed by 2-way ANOVA. *P = 0.002 vs. fasted 7-day-old pigs, P < 0.0001 vs. fasted 26-day-old pigs, and P = 0.0034 vs. fed 26-day-old pigs.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

During the period before weaning, skeletal muscle protein synthesis in fasted pigs exhibits a rapid decline from 15.6 to 4.0%/day between 7 and 26 days of age, respectively (7). Furthermore, the stimulation of protein synthesis that occurs in response to feeding is blunted in muscle from 26- compared with 7-day-old neonatal pigs. In a previous study, we showed that the feeding-induced stimulation of muscle protein synthesis in pigs of both ages is associated with enhanced phosphorylation of 4E-BP1, leading to increased assembly of the active eIF4F complex, as well as hyperphosphorylation of S6K1 (13). However, the feeding-induced phosphorylation of both proteins is reduced in muscle from the older animals. We also found that phosphorylation of 4E-BP1 and S6K1 is prevented by administration of the mTOR inhibitor rapamycin before feeding (26), suggesting that mTOR activity is essential for phosphorylation of these proteins. Similarly, in rat skeletal muscle, oral administration of leucine to fasted animals promotes phosphorylation of both 4E-BP1 and S6K1 (3), and rapamycin blocks the effects (4). The results of these studies, in combination with results obtained using cells in culture, have led to the hypothesis that both insulin and amino acids cause 4E-BP1 and S6K1 phosphorylation by activation of mTOR.

Mechanisms governing mTOR activation are incompletely defined. However, it seems clear that phosphorylation is one important method for modulating mTOR activity. For example, phosphorylation of mTOR by the protein-tyrosine kinase c-Abl inhibits mTOR kinase activity toward a peptide substrate based on the amino acid sequence surrounding Thr389 in S6K1 (29). In addition, overexpression of c-Abl results in inhibition of cap-dependent translation comparable in magnitude to that caused by rapamycin. In contrast to the inhibition of mTOR activity associated with phosphorylation by c-Abl, phosphorylation by PKB is thought to stimulate mTOR activity (34). In particular, recent studies have shown that PKB directly phosphorylates mTOR on Thr2446 and Ser2448; however, of these two sites, only Ser2448 is phosphorylated in vivo (31, 35). A curious finding in the study of Sekulic et al. (35) is that, although insulin enhances phosphorylation of Ser2448 in mTOR, phosphorylation of the site is dispensable for insulin-stimulated phosphorylation of 4E-BP1 and activation of S6K1, suggesting that additional, possibly overlapping, mechanisms exist for regulating mTOR activity. This suggestion is supported by the observation that, although amino acid-stimulated phosphorylation of 4E-BP1 and S6K1 is inhibited by rapamycin, amino acids do not activate PI 3-kinase or PKB (22, 32, 33). Thus amino acids and insulin may share a PKB-independent signaling pathway that activates mTOR. A second, but thus far unexplored, possibility is that amino acids do not activate mTOR but instead regulate an unidentified kinase. In such a model, mTOR activity would limit protein phosphatase activity, as has been shown to occur in yeast (17, 24). Inhibition of mTOR would disinhibit the phosphatase, resulting in dephosphorylation of 4E-BP1 and S6K1.

In the present study, the finding that mTOR content is dramatically lower in muscle from 26- compared with 7-day-old neonatal pigs would imply that phosphorylation of its two downstream targets, 4E-BP1 and S6K1, might likewise be reduced in older animals. In vitro, mTOR phosphorylates S6K1 on Thr389 (5), a residue whose phosphorylation is necessary for maximal S6K1 activity. In the studies presented herein, S6K1 phosphorylation on Thr389 was almost undetectable in fasted animals of either age, suggesting that mTOR activity is minimal under these conditions. In contrast, both Thr389 phosphorylation and S6K1 activity were increased after feeding, and the increase was lesser in magnitude in 26- than in 7-day-old pigs. In addition, 4E-BP1 phosphorylation at Thr70 was increased by feeding in 7-day-old pigs but not in 26-day-old pigs. Thus the reduced abundance of mTOR in 26-day-old animals might hinder transduction of upstream signals to 4E-BP1 and S6K1, leading to diminished responsiveness of translation initiation during development. The basis for the decrease in mTOR content and S6K1 phosphorylation on Thr389 between day 7 and day 26 being greater in magnitude than the changes in S6K1 activity is unknown. However, S6K1 activity is regulated by phosphorylation not only on Thr389 but also on a number of other residues that were not examined in the present study (19).

Another factor that probably contributes to the developmental decline in responsiveness is the blunted stimulation of PKB that occurs after feeding 26-day-old pigs. Reduced PKB content contributes to the decreased activity but cannot account entirely for the effect. Instead, decreased activation of the kinase may play an important role. Activation of PKB occurs through multi-site phosphorylation, where phosphorylation of Thr308 and Ser473 is particularly important (see review in Ref. 18). Thr308 is phosphorylated by a kinase termed phosphatidylinositol-dependent protein kinase-1 (PDK-1), which is thought to be constitutively active, and phosphorylation of PKB by PDK-1 occurs only when both proteins become localized to the plasma membrane. Co-localization is mediated by association of the pleckstrin homology domain present in each protein with phosphatidylinositol-(3,4)-bis-phosphate or phosphatidylinositol-(3,4,5)-trisphosphate, which is generated by phosphorylation by PI 3-kinase of phosphoinositides resident in the plasma membrane. The finding that PI 3-kinase basal activity, as well as its activation by feeding, is unaffected during development suggests that synthesis of 3-phosphoinositides, and thus co-localization of PDK-1 and PKB, are unimpaired during the preweaning period. If this theory is true, then phosphorylation of Thr308 would be stimulated to the same extent by feeding either 7- or 26-day-old pigs. In combination with the observation that Ser473 phosphorylation is unaffected by development, an argument can be made that the reduction in PKB activation found in 26-day-old pigs must occur through a mechanism distinct from phosphorylation at these two sites.

The finding that the activation of PI 3-kinase by feeding was similar in animals of both ages is surprising. Recently we showed that the postprandial stimulation of skeletal muscle protein synthesis in neonatal pigs is associated with enhanced phosphorylation of the insulin receptor and IRS-1 and -2 (36). These changes in the activation of early steps in the insulin signaling pathway were reduced in 26- compared with 7-day-old pigs. On the basis of these results, it would be expected that the signaling components downstream of the insulin receptor, IRS-1, and IRS-2 are activated by feeding, and that these responses decrease as the animal develops. Thus the next step in the signaling pathway, the binding of IRS-1 and -2 to the p85 subunit of PI 3-kinase, was increased by feeding in our previous study, and the response decreased with development (36). Although the binding of IRS-1 or IRS-2 to the p85 subunit of PI 3-kinase is thought to stimulate PI 3-kinase activity (2), we did not find a developmental decline in PI 3-kinase activation in IRS-1 immunoprecipitates. Nonetheless, the signaling components downstream of PI 3-kinase show a developmental pattern of activation by feeding that is similar to the association of PI 3-kinase with IRS-1 and IRS-2 and to the phosphorylation of IRS-1, IRS-2, and the insulin receptor.

Overall, the results of the present study suggest that the developmental decline in the stimulation of muscle protein synthesis by feeding during the preweaning period is due to a reduction in the capacity to transduce the signal emanating from the plasma membrane to the kinases that are proximal to and that regulate the components of the protein synthetic apparatus. In addition to a decrease in PKB and mTOR content, an as-yet-unidentified mechanism attenuates PKB activation in 26-day-old pigs. Finally, it is shown that previously reported changes in 4E-BP1 and S6K1 hyperphosphorylation are associated with modulation of Thr70 and Thr389 phosphorylation, respectively, as well as alterations in S6 kinase activity.


    ACKNOWLEDGEMENTS

We thank Sharon Rannels, Marlin Druckenmiller, William Liu, Agus Suryawan, and Jill Bush for technical support.


    FOOTNOTES

This project was funded in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-15658 (L. S. Jefferson), National Institute of Arthritis and Musculoskeletal and Skin Diseases Institute Grants AR-43127 (P. A. Farrell) and AR-44474 (T. A. Davis), and the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement number 58-6250-6-001 (T. A. Davis).

Address for reprint requests and other correspondence: S. R. Kimball, Dept. of Cellular and Molecular Physiology, The Pennsylvania State Univ. College of Medicine, 500 Univ. Drive, 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.

10.1152/ajpendo.00269.2001

Received 18 December 2000; accepted in final form 30 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alessi, DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, and Hemmings BA. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15: 6541-6551, 1996[Abstract].

2.   Alessi, DR, and Downes CP. The role of PI 3-kinase in insulin action. Biochem Biophys Acta 1436: 151-164, 1998[ISI][Medline].

3.   Anthony, JC, Anthony TG, Kimball SR, Vary TC, and Jefferson LS. Orally administered leucine stimulates protein synthesis in skeletal muscle of post-absorptive rats in association with increased eIF4F formation. J Nutr 130: 139-145, 2000[Abstract/Free Full Text].

4.   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 130: 2413-2419, 2000[Abstract/Free Full Text].

5.   Burnett, PE, Barrow RK, Cohen NA, Snyder SH, and Sabatini DM. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci USA 95: 1432-1437, 1998[Abstract/Free Full Text].

6.   Burrin, DG. Nutrient requirements and metabolism. In: Biology of the Domestic Pig, edited by Pond WG, and Mersmann HJ.. Ithaca, NY: Cornell University Press, 2001, p. 309-389.

7.   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[Abstract/Free Full Text].

8.   Davis, TA, Burrin DG, Fiorotto ML, Reeds PJ, and Jahoor F. Roles of insulin and amino acids in the regulation of protein synthesis in the neonate. J Nutr 128: 347S-350S, 1998[ISI][Medline].

9.   Davis, TA, Fiorotto ML, Beckett PR, Burrin DG, Reeds PJ, Wray-Cahen D, and Nguyen HV. Differential effects of insulin on peripheral and visceral tissue protein synthesis in neonatal pigs. Am J Physiol Endocrinol Metab 280: E770-E779, 2001[Abstract/Free Full Text].

10.   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[Abstract/Free Full Text].

11.   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[Abstract/Free Full Text].

12.   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[Abstract/Free Full Text].

13.   Davis, TA, Nguyen HV, Suryawan A, Bush J, Jefferson LS, and Kimball SR. Developmental changes in the feeding-induced stimulation of translation initiation in muscle of neonatal pigs. Am J Physiol Endocrinol Metab 279: E1226-E1234, 2000[Abstract/Free Full Text].

14.   Denne, SC, and Kalhan SC. Leucine metabolism in human newborns. Am J Physiol Endocrinol Metab 253: E608-E615, 1987[Abstract/Free Full Text].

15.   Denne, SC, Rossi EM, and Kalhan SC. Leucine kinetics during feeding in normal newborns. Pediatr Res 30: 23-27, 1991[Abstract].

16.   Dennis, PB, Fumagalli S, and Thomas G. Target of rapamycin (TOR): balancing the opposing forces of protein synthesis and degradation. Curr Opinion Cell Reg 9: 49-54, 1999.

17.   Di Como, CJ, and Arndt KT. Nutrients, via the TOR proteins, stimulate the association of Tap42 with type 2A phosphatases. Genes Develop 10: 1904-1916, 1996[Abstract].

18.   Dufner, A, and Thomas G. Ribosomal S6 kinase signaling and the control of translation. Exptl Cell Res 253: 100-109, 1999[Medline].

19.   Fumagalli, S, and Thomas G. S6 phosphorylation and signal transduction. In: Translational Control of Gene Expression, edited by Sonenberg N, Hershey JWB, and Mathews MB.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2000, p. 695-717.

20.   Gingras, AC, Raught B, and Sonenberg N. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. In: Annu Rev Biochem, edited by Richardson CC.. Palo Alto, CA: Annual Reviews, 1999, p. 913-963.

21.   Goldspink, DF, and Kelly FJ. Protein turnover and growth in the whole body, liver, and kidney of the rat from the foetus to senility. Biochem J 217: 507-516, 1984[ISI][Medline].

22.   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[Abstract/Free Full Text].

23.   Hernandez, JM, Fedele MJ, and Farrell PA. Time course evaluation of protein synthesis and glucose uptake after acute resistance exercise in rats. J Appl Physiol 88: 1142-1149, 2000[Abstract/Free Full Text].

24.   Jiang, Y, and Broach JR. Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. EMBO J 18: 2782-2792, 1999[Abstract/Free Full Text].

25.   Kimball, SR, and Jefferson LS. Regulation of translation initiation in mammalian cells by amino acids. In: Translational Control of Gene Expression, edited by Sonenberg N, Hershey JWB, and Mathews MB.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2000, p. 561-579.

26.   Kimball, SR, Jefferson LS, Nguyen HV, Suryawan A, Bush JA, and Davis TA. Feeding stimulates protein synthesis in muscle and liver of neonatal pigs through an mTOR-dependent process. Am J Physiol Endocrinol Metab 279: E1080-E1087, 2000[Abstract/Free Full Text].

27.   Kimball, SR, Karinch AM, Feldhoff RC, Mellor H, and Jefferson LS. Purification and characterization of eukaryotic initiation factor eIF-2B from liver. Biochem Biophys Acta 1201: 473-481, 1994[ISI][Medline].

28.   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[Abstract/Free Full Text].

29.   Kumar, V, Sabatini D, Pandey P, Gingras AC, Majumder PK, Kumar M, Yuan ZM, Carmichael G, Weichselbaum R, Sonenberg N, Kufe D, and Kharbanda S. Regulation of the rapamycin and FKBP-target 1/mammalian target of rapamycin and cap-dependent initiation of translation by the c-Abl protein-tyrosine kinase. J Biol Chem 275: 10779-10787, 2000[Abstract/Free Full Text].

30.   Meyuhas, O, and Hornstein E. Translational control of TOP mRNAs. In: Translational Control of Gene Expression, edited by Sonenberg N, Hershey JWB, and Mathews MB.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2000, p. 671-693.

31.   Nave, BT, Ouwens DM, Withers DJ, Alessi DR, and Shepherd PR. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino acid deficiency on protein translation. Biochem J 344: 427-431, 1999[ISI][Medline].

32.   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[Abstract/Free Full Text].

33.   Pham, PT, Heydrick SJ, Fox HL, Kimball SR, Jefferson LS, and Lynch CJ. Assessment of cell signaling pathways in the regulation of mTOR by amino acids in rat adipocytes. J Cell Biochem 79: 427-441, 2000[ISI][Medline].

34.   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[Abstract/Free Full Text].

35.   Sekulic, A, Hudson CC, Homme JL, Yin P, Otterness DM, Karnitz LM, and Abraham RT. A direct linkage between the phosphoinositide 3-kinase-Akt signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res 60: 3504-3523, 2000[Abstract/Free Full Text].

36.   Suryawan, A, Nguyen HV, Bush JA, and Davis TA. Developmental changes in the feeding-induced activation of the insulin-signaling pathway in neonatal pigs. Am J Physiol Endocrinol Metab 281: E908-E915, 2001[Abstract/Free Full Text].

37.   Wray-Cahen, D, Nguyen HV, Burrin DG, Beckett PR, Fiorotto ML, Reeds PJ, Wester TJ, and Davis TA. Response of skeletal muscle protein synthesis to insulin in suckling pigs decreases with development. Am J Physiol Endocrinol Metab 275: E602-E609, 1998[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 282(3):E585-E592
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society