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U.S. Department of Agriculture/Agriculture Research Service, Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 and * Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033
2 To whom correspondence and reprint requests should be addressed. E-mail: tdavis{at}bcm.tmc.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0, 17, 52, and 255 pmol/L (0, 2, 6, 30 µU/mL), and amino acids were clamped at food-deprived or fed levels. In skeletal muscle, insulin increased the activation of IR, IRS-1, PI 3-kinase, PKB and S6 and stimulated protein synthesis. In liver, insulin increased the activation of IR, IRS-1, PI 3-kinase, PKB and S6, but had no effect on protein synthesis. Raising amino acids from the food-deprived to the fed level did not alter the insulin-induced activation of IR, IRS-1, PI 3-kinase and PKB but increased S6 phosphorylation and protein synthesis in skeletal muscle and liver. The results suggest that the stimulation of protein synthesis in muscle by insulin involves activation of insulin signaling components, and the stimulation of protein synthesis in muscle and liver by amino acids occurs by mechanisms independent of the early steps of this pathway. Furthermore, amino acids do not alter the insulin-stimulated activation of early steps in the insulin signaling pathway.
KEY WORDS: • insulin receptor • protein kinase B • translation initiation • skeletal muscle • liver • protein synthesis • muscle
Rapid rates of growth and protein deposition, particularly in skeletal muscle, are accompanied by high rates of protein synthesis in the neonate (1–4). Skeletal muscle protein synthesis is uniquely sensitive to stimulation by feeding during the neonatal period, and this is mediated by the postprandial rise in insulin and amino acids (5–9). Recently, we found that the enhanced feeding-induced stimulation of muscle protein synthesis in the neonate is associated with increased activation of insulin signaling components leading to translation initiation (10–12). However, the individual roles of insulin and amino acids in eliciting this response were not determined.
One of the major metabolic effects of insulin in mammalian cells is the stimulation of protein synthesis (13). Insulin stimulates protein synthesis by activation of the insulin receptor tyrosine kinase and phosphorylation of intracellular substrates of the insulin receptor substrate (IRS)2 family (13,14). Tyrosine-phosphorylated IRS proteins propagate the signal to Src homology 2 domain–containing proteins such as the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, which activates its p110 catalytic subunit (15). Furthermore, insulin also stimulates the activation of downstream effectors of PI 3-kinase, including protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR), and translational components, such as 70-kDa ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E-binding protein (4E-BP1) (16,17). Activation of S6K1 leads to hyperphosphorylation of ribosomal protein S6 (18). Furthermore, activation of the translational repressor, 4E-BP1, by hyperphosphorylation will increase the availability of eukaryotic initiation factor 4E (eIF4E) for binding eIF4G to form the eIF4F initiation complex (19). Thus, both S6K1 and 4E-BP1 activation are crucial for the regulation of protein synthesis by insulin.
Considerable evidence suggests that amino acids directly stimulate protein synthesis (20,21). Furthermore, evidence from in vitro studies suggests that amino acids stimulate protein synthesis independently of insulin (22–25). Studies using in vitro and cell culture systems have shown that amino acids initiate a signal by activation of mTOR, followed by phosphorylation of 4E-BP1 and S6K1 and subsequent activation of components leading to translation initiation (21,25,26).
We recently demonstrated, using pancreatic-glucose-amino acid clamps, that the postprandial elevations in insulin and amino acids independently mediate the feeding-induced stimulation of skeletal muscle protein syntheses in neonatal pigs (9). However, in vitro studies using pharmacologic doses of insulin and amino acids suggest that amino acids downregulate early steps in the insulin signaling pathway, leading to translation initiation (23). Therefore, it was the purpose of the current study to identify whether the postprandial rise in amino acids modifies the insulin-stimulated activation of signaling components leading to translation initiation in skeletal muscle of the neonate. Pancreatic-glucose-amino acid clamps were utilized in which the state of insulin was as follows: 1) absent, 2) at food-deprived levels, 3) intermediate between food-deprived and fed levels or 4) at fed levels, while amino acids were maintained at either food-deprived or fed levels. Results in skeletal muscle were compared with that in liver because amino acids, but not insulin, stimulate liver protein synthesis in neonates (8). The results suggest that the physiologic rise in amino acids does not alter the insulin-stimulated activation of early steps in the insulin-signaling pathway but that amino acids activate signaling components downstream of PKB in skeletal muscle of the neonate.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Materials. BioMag goat anti-mouse IgG and goat anti-rabbit IgG magnetic beads were obtained from Polysciences (Warrington, PA), and the magnetic sample rack was from Promega (Madison, WI). Reagents for SDS-PAGE were from BioRad Laboratories (Richmond, CA). The protein assay kit was purchased from Pierce (Rockford, IL). Anti-phosphotyrosine (PY) antibody, insulin receptor and IRS-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PI 3-kinase (p85) was purchased from Mbl International (Watertown, MA). The enhanced chemiluminescence Western blotting detection kit (ECL-Plus) was obtained from Amersham (Arlington Heights, IL). Other chemicals and reagents were from Sigma Chemical (St. Louis, MO).
Pancreatic-glucose-amino acid clamps.
The clamp procedure was described previously by O’Connor et al. (9). Briefly, pigs were placed awake in a sling restraint system after overnight food deprivation. The average basal concentration of blood glucose (YSI 2300 STAT Plus, Yellow Springs Instruments, Yellow Springs, OH) and plasma BCAA concentrations (28) to be used in the subsequent pancreatic-glucose-amino acids clamp procedure were established during a 30-min basal period. The clamp was initiated with a primed (20 µg/kg), continuous [100 µg/(kg · h)] somatostatin (BACHEM, Torrance, CA) infusion. After a 10-min infusion of somatostatin, a continuous infusion of replacement glucagon [150 ng/(kg · h)]; Eli Lilly, Indianapolis, IN) was initiated and continued to the end of the clamp period. Insulin was infused at 0, 10, 22, or 110 ng/(kg0.66 · min) to achieve plasma insulin concentrations of 0, 17, 52, and 255 pmol/L (0, 2, 6, 30 µU/mL) to simulate less than food-deprived, food-deprived, intermediate and fed insulin levels (4). At each dose of insulin, amino acids were clamped at either the food-deprived (500 µmol/L BCAA) or fed (1000 µmol/L BCAA) level by monitoring the BCAA every 5 min and adjusting the infusion rate of a balanced amino acid mixture (8) to maintain the plasma BCAA concentration within 10% of the desired level (7,8). Blood glucose concentrations were measured at 5-min intervals, and the dextrose infusion rate was adjusted to maintain the glucose concentration at a constant value (29). Blood samples also were taken at intervals for later determination of circulating insulin, glucagon and individual essential and nonessential amino acid concentrations (30).
Hormone and substrate assay. The concentrations of individual amino acids from frozen plasma samples obtained at 0 and 90 min of the insulin infusion were measured with an HPLC method (PICO-TAG reverse-phase column; Waters, Milford, MA) as previously described (30). Plasma radioimmunoreactive insulin concentrations were measured using a porcine insulin RIA kit (Linco, St. Charles, MO) that used porcine insulin antibody and human insulin standards. The lowest level of detection of the insulin assay was 5 pmol/L (0.5 µU/ml).
Tissue protein synthesis in vivo. The fractional rate of protein synthesis was measured with a flooding dose of L-[4-3H]phenylalanine (31) injected 90 min after the initiation of the clamp procedure for a 30-min labeling period. Pigs were killed at 2 h, samples of longissimus dorsi were collected and rapidly frozen and fractional rates of protein synthesis were determined as previously described (3).
Immunoprecipitation and Western blot analysis. To measure IR and IRS-1 abundance, the tissue samples were immunoprecipitated using anti-IR or anti-IRS-1 antibodies as previously described (11). To measure IR or IRS-1 tyrosine phosphorylation, equal amounts of IR and IRS-1 immunoprecipitate were subjected to SDS-PAGE. The proteins were then transferred to a polyvinylidene difluoride membrane and incubated with anti-phosphotyrosine antibody in a Tris-buffered saline-Tween 20 solution (TBS-T) containing 5 g/L nonfat dried milk for 1 h at room temperature. Membranes were then incubated with secondary antibody diluted 1:2000 in TBS-T. The membranes were washed in TBS-T three times for 10 min and developed with an enhanced chemiluminescence detection kit (ECL-Plus, Amersham Pharmacia, Piscataway, NJ) before exposure onto Kodak-X-Omat film. To measure the association of IRS-1 with PI 3-kinase, equal amounts of IRS-1 immunoprecipitate were subjected to SDS-PAGE followed by incubation with anti-PI 3-kinase (p85) antibody. The blots were quantified by computerized densitometry (Molecular Dynamics Pharmacia, Piscataway, NJ). To determine PKB phosphorylation on Ser473, samples were subjected to SDS-PAGE, and the phosphorylation of PKB on Ser473 was determined by protein immunoblot analysis with antibodies that recognize the protein only when it is phosphorylated on that residue. Phosphorylation of ribosomal protein S6 was determined in 10,000 x g supernatants by protein immunoblot analysis, as previously described (32). Membranes were incubated with an anti-phosphopeptide antibody specific for phosphorylated S6 (a kind gift from Dr. Morris J. Birnbaum, Department of Medicine, University of Pennsylvania).
Statistics. ANOVA (general linear modeling) was used to assess the effects of insulin, amino acids and their interaction. The existence of simple linear or quadratic relationships between insulin or amino acids and insulin signaling component activity, and between insulin signaling component activity and protein synthesis was tested by fitting first- and second-degree polynomials. Probability values of <0.05 were considered significant. Data are presented as means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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500 and 1000 µmol/L, respectively (Table 1). Circulating glucose and glucagon concentrations were largely maintained at baseline food-deprived levels during the infusion of somatostatin, glucagon, insulin and/or amino acids (data not shown).
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0, 17, and 52 pmol/L (0, 2, 6 µU/mL) plasma insulin levels (P < 0.05) and tended to increase protein synthesis at the 255 pmol/L (30 µU/mL) plasma insulin level (P < 0.10).
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The phosphorylation of IRS-1 was determined by immunoprecipitating the protein sample using anti-IRS-1, followed by immunoblotting with anti-PY antibody. Insulin infusion increased IRS-1 phosphorylation in a dose-responsive manner (P < 0.05; Fig. 1C). Raising amino acids to the fed level did not affect phosphorylation of IRS-1.
The activation of PI 3-kinase was determined by measuring the association of PI 3-kinase with phosphorylated IRS-1. The protein samples were immunoprecipitated using anti-IRS-1 antibody, followed by immunoblotting with PI 3-kinase p85 subunit antibody. There was a dose-response effect of insulin on the association of PI 3-kinase with IRS-1 (P < 0.05; Fig. 1D). Fed levels of amino acids did not alter the effect of insulin on PI 3-kinase activation.
Insulin has been shown to stimulate the activation of PKB (13). In contrast, studies using cell culture systems indicate that amino acids either inhibit or do not affect PKB activation (21,23). In this study, we determined the activation of PKB by measuring the phosphorylation of PKB at Ser473. Insulin increased PKB phosphorylation, and the phosphorylation of PKB was greater at 52 pmol/L (6 µU/mL) than at 17 pmol/L (2 µU/mL) and at 255 pmol/L (30 µU/mL) than at 52 pmol/L (6 µU/mL) (P < 0.05) (Fig. 1E). Amino acids did not alter the insulin-induced phosphorylation of PKB, although there was a tendency at the highest insulin level for amino acids to blunt PKB phosphorylation.
Because insulin and amino acids have been shown to activate S6K1, followed by the phosphorylation of ribosomal protein S6 (18,25), we determined the activation of S6 by measuring its phosphorylation. The phosphorylation of S6 was increased only at the highest insulin level (255 pmol/L, 30 µU/mL), and amino acids had a synergistic effect on S6 phosphorylation (Fig. 1F; P < 0.05). At lower levels of insulin (17 and 52 pmol/L or 2 and 6 µU/mL, respectively), neither insulin nor amino acids had any effect on the phosphorylation of S6.
Effect of insulin and amino acids on protein synthesis and insulin signaling components in neonatal liver. We recently demonstrated that amino acid, but not insulin infusion in the neonatal pig stimulates protein synthesis in liver (O’Connor, P. M., Kimball, S. R., Suryawan, A., Bush, J. A., Nguyen, H. V., Jefferson, L. S. & Davis, T. A., unpublished results). For the purpose of comparison, we have included the liver protein synthesis data herein. Raising amino acids from the food-deprived to the fed level increased liver protein synthesis (P < 0.001; Fig. 2A), and the effect of amino acids on liver protein synthesis was present at each dose of insulin. Liver protein synthesis was unaffected by insulin, although a stimulatory effect was seen at the high dose of insulin in the presence of hyperaminoacidemia (P < 0.005).
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We further investigated the effect of insulin and amino acids of the phosphorylation of PKB and S6 in the liver. Insulin increased PKB phosphorylation (P < 0.05), and this effect was seen at 52 and 255 pmol/L (6 and 30 µU/mL) (Fig. 2E). Amino acid infusion did not affect the insulin-induced phosphorylation of PKB. In the liver, raising both insulin and amino acids to fed levels increased the phosphorylation of S6 (P < 0.05) (Fig. 2F).
Correlation of insulin signaling component activity with plasma insulin and amino acid levels. In the current study, we determined in muscle and liver of neonatal pigs the relationship between insulin signaling component activation and circulating amino acid or insulin levels. In skeletal muscle (Fig. 3), there were positive linear relationships between insulin concentration and the activation of IR (P < 0.001), IRS-1 (P < 0.001) and PI 3-kinase (P < 0.005), and curvilinear relationships between insulin concentration and PKB phosphorylation (P < 0.006) and protein synthesis (P < 0.005). However, there was no significant relationship between insulin concentration and phosphorylation of S6. In the liver, all insulin signaling components were positively correlated (P < 0.001) with insulin concentration in a quadratic manner, but there was no relationship between protein synthesis and insulin concentration (Fig. 4). There was no significant relationship between insulin signaling component activation and amino acids levels in skeletal muscle (data not shown). However, there was a tendency for PKB phosphorylation (P < 0.06) and S6 phosphorylation (P < 0.10) in the liver to be correlated with circulating amino acid levels (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The results of this study indicate that in skeletal muscle and liver of neonatal pigs, insulin stimulates the tyrosine phosphorylation of the insulin receptor and IRS-1, the association of IRS-1 with PI 3-kinase and serine phosphorylation of PKB. These effects of insulin were largely progressive at each of the 4 doses of insulin, ranging from less than food-deprived to fed levels. However, S6 phosphorylation in skeletal muscle was stimulated only by the presence of fed levels of insulin with a further increase in the presence of fed levels of both insulin and amino acids. In liver, the presence of fed levels of both insulin and amino acids were required to stimulate S6 phosphorylation.
The effect of amino acids on early steps of the insulin signaling pathway has been controversial. Studies using CHO-IR cell cultures indicate that amino acids have no effect on insulin-stimulated activation of IR, IRS-1 and PI 3-kinase (22). However, hepatocyte and L6 skeletal muscle cell culture studies suggest that amino acids blunt the activation of PI 3-kinase (23,26), despite normal tyrosine phosphorylation of IR and IRS-1 (26). Furthermore, an in vivo study using adult human skeletal muscle showed that plasma amino acid elevation induces skeletal muscle insulin resistance in humans (33). The current results suggest that in neonates, early steps of the insulin signaling pathway are resistant to the inhibitory effect of amino acids. This is consistent with studies showing that amino acids reduce glucose disposal in adults (34) but not in neonates (9). Because neonatal muscle is highly sensitive to insulin and this response decreases with development, we speculate that any inhibitory effect of amino acids could not overcome the enhanced insulin-stimulated activity of early steps of the insulin signaling pathway in the neonate (10,11). Alternatively, a 100% increase in amino acids from the food-deprived to the fed level may not be sufficient to inhibit insulin signaling.
In skeletal muscle of the neonate, the lack of effect of low insulin doses (17 and 52 pmol/L or 2 and 6 µU/mL) on S6 phosphorylation contrasts with the stimulatory effect of low doses of insulin on muscle protein synthesis (9). However, our current findings are supported by our previous report that insulin levels of 52 pmol/L (6 µU/mL) or lower are insufficient for insulin-stimulated S6K1 phosphorylation (35). Additionally, our findings are consistent with the report by Anthony et al. (36) indicating that administration of a low dose of insulin [(4 pmol/(min · kg)] to diabetic rats did not stimulate S6 phosphorylation but a higher dose of insulin [(20 pmol/(min · kg)] significantly induced S6 phosphorylation. Furthermore, our finding that amino acids increased S6 phosphorylation only in the presence of a fed level of insulin is also consistent with the results on S6 phosphorylation in diabetic rats (36) and our previous results in neonatal pigs for the kinase, S6K1, that phosphorylates S6 (35).
The results of the current study indicate that in neonatal pigs, there are tissue-specific effects of insulin and amino acids on the signaling pathway leading to protein synthesis. Insulin increased the activation of IR, IRS-1, PI 3-kinase and PKB in liver, as it did in muscle, in a dose-responsive manner. Only the highest dose of insulin stimulated S6 phosphorylation in muscle and this was further enhanced in the presence of fed levels of amino acids. However, in liver, the presence of fed levels of both insulin and amino acids was required to stimulate S6 phosphorylation. Furthermore, both insulin and amino acids stimulated protein synthesis in muscle, but not liver, whereas only amino acids, but not insulin, stimulated protein synthesis in liver. The stimulation of protein synthesis in muscle and liver by amino acids occurred at each dose of insulin. Because amino acids can stimulate protein synthesis in hepatocytes (37) and in the liver of neonatal pigs (O’Connor, P. M., Kimball, S. R., Suryawan, A., Bush, J. A., Nguyen, H. V., Jefferson, L. S. & Davis, T. A., unpublished results) in the absence of insulin, this suggests that liver is very sensitive to amino acid stimulation. Furthermore, it could be argued that the liver is also highly sensitive to insulin and that the small amount of insulin (5 pmol/L; 0.6 µU/mL) present in the pigs infused with somatostatin without insulin replacement was sufficient to maintain the basal level of liver protein synthesis. However, this seems unlikely given than the lowest insulin level (5 pmol/L; 0.5 µU/mL) was less than the food-deprived level (17 pmol/L; 2 µU/mL) and was at the lowest level of detection of the insulin assay. Further, there is evidence of tissue-specific effects of insulin and amino acids from our recent study, which demonstrated that rapamycin, a specific inhibitor of mTOR, completely blocked the feeding-induced stimulation of S6K1 phosphorylation and protein synthesis in liver, while only partially blocking muscle protein synthesis (38). This suggests a tissue-specific effect of rapamycin on the feeding-induced stimulation of protein synthesis. The current study also suggests that at insulin concentrations below the feeding level, dietary amino acids stimulate protein synthesis through a mechanism other than S6 phosphorylation.
In this study, the relationships among insulin signaling activity and insulin level, amino acid level or protein synthesis rate were determined. In skeletal muscle, the results showed that there was no relationship between the activity of insulin signaling components and amino acid concentrations ranging from the food-deprived to the fed level. On the other hand, the activation of all insulin signaling components except S6 in muscle was positively correlated with plasma insulin concentrations in a linear or curvilinear fashion. Furthermore, there was a linear or curvilinear relationship between protein synthesis rate in muscle and the activity of all insulin signaling components measured. These data indicate that insulin, but not amino acids, activates the insulin signaling pathway leading to the stimulation of protein synthesis in skeletal muscle of neonatal pigs. For comparison, we also examined another insulin-sensitive tissue, liver. There was a curvilinear relationship between circulating insulin and the activity of all insulin signaling components, suggesting that higher insulin levels would have no further effect on the activation of insulin signaling components in liver. In addition, although there was no relationship between circulating amino acid levels and the activation of IR, IRS-1 and PI 3-kinase, there was a tendency for amino acid level to be correlated with phosphorylation of both PKB and S6. In contrast to skeletal muscle, there was no significant correlation between protein synthesis rate and insulin signaling component activity in the liver of neonatal pigs. Thus, the activation of insulin signaling components likely affected other insulin-induced metabolic events in liver, but not liver protein synthesis.
In summary, the data presented herein indicate that insulin infusion within the physiologic range activates the insulin signaling components, IR, IRS-1, PI 3-kinase and PKB in a dose-responsive manner in skeletal muscle and liver of neonatal pigs. S6 phosphorylation in muscle and liver is less sensitive to insulin than the upstream signaling components and requires the presence of fed levels of amino acids in liver. Because insulin infusion stimulates protein synthesis in skeletal muscle but not in liver (8), this suggests the presence of factors that inhibit the insulin signaling pathway downstream of PKB in liver, but not in muscle. Alternatively, liver protein synthesis may be highly sensitive to less than food-deprived levels of insulin. Although amino acids did not activate the insulin signaling pathway in either skeletal muscle or liver, amino acids did increase the phosphorylation of S6 in both tissues. This suggests that amino acids stimulate protein synthesis by a PI 3-kinase/PKB independent pathway in the neonate. In both tissues, postprandial levels of insulin seem to be required for amino acids to effectively stimulate S6 phosphorylation. Furthermore, our data also indicate that fed levels of amino acids do not downregulate the early steps of the insulin signaling pathway in the neonate, perhaps due to their high activity of insulin signal transduction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; eIF4E, eukaryotic initiation factor 4E; eIF4F, eukaryotic initiation factor 4F; eIF4G, eukaryotic initiation factor 4G; IR, insulin receptor; IRS-1, insulin receptor substrate 1; mTOR, mammalian target of rapamycin; PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; S6K1, 70-kDa ribosomal protein S6 kinase 1.
Manuscript received 28 August 2003. Initial review completed 15 September 2003. Revision accepted 13 October 2003.
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Different from the euaminoacidemic group within the same insulin group, P < 0.05.
