Regulation of neonatal liver protein synthesis by insulin and amino ...

Am J Physiol Endocrinol Metab 286: E994 –E1003, 2004. First published February 3, 2004; 10.1152/ajpendo.00391.2003.

Regulation of neonatal liver protein synthesis by insulin and amino acids in pigs Pamela M. J. O’Connor,1 Scot R. Kimball,2 Agus Suryawan,1 Jill A. Bush,1 Hanh V. Nguyen,1 Leonard S. Jefferson,2 and Teresa A. Davis1 1

United States Department of Agriculture/Agricultural Research Service, Children’s Nutrition Research Center, and Section of Neonatology, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030; and 2Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 Submitted 28 August 2003; accepted in final form 31 January 2004

O’Connor, Pamela M. J., Scot R. Kimball, Agus Suryawan, Jill A. Bush, Hanh V. Nguyen, Leonard S. Jefferson, and Teresa A. Davis. Regulation of neonatal liver protein synthesis by insulin and amino acids in pigs. Am J Physiol Endocrinol Metab 286: E994 – E1003, 2004. First published February 3, 2004; 10.1152/ajpendo. 00391.2003.—The high efficiency of protein deposition during the neonatal period is driven by high rates of protein synthesis, which are maximally stimulated after feeding. Infusion of amino acids, but not insulin, reproduces the feeding-induced stimulation of liver protein synthesis. To determine whether amino acid-stimulated liver protein synthesis is independent of insulin in neonates, and to examine the role of amino acids and insulin in the regulation of translation initiation in neonatal liver, we performed pancreatic glucose-amino acid clamps in overnight-fasted 7-day-old pigs. Pigs (n ⫽ 9 –12/ group) were infused with insulin at 0, 10, 22, and 110 ng䡠kg⫺0.66 䡠 min⫺1 to achieve 0, 2, 6, and 30 ␮U/ml insulin, respectively. At each insulin dose, amino acids were maintained at fasting or fed levels or, in conjunction with the highest insulin dose, allowed to fall to below fasting levels. Insulin had no effect on the fractional rate of protein synthesis in liver. Amino acids increased fractional protein synthesis rates in liver at each dose of insulin, including the 0 ␮U/ml dose. There was a dose-response effect of amino acids on liver protein synthesis. Amino acids and insulin increased protein S6 kinase and 4E-binding protein 1 (4E-BP1) phosphorylation; however, only amino acids decreased formation of the inactive 4E-BPI䡠eukaryotic initiation factor-4E (eIF4E) complex. The results suggest that amino acids regulate liver protein synthesis in the neonate by modulating the availability of eIF4E for 48S ribosomal complex formation and that this response does not require insulin.

are more rapid during the neonatal period than at any other stage of postnatal life (8, 12, 18). Dietary protein is utilized with high efficiency in neonates (10), and this is most likely due to the enhanced rates of protein synthesis that occur in response to feeding (4, 9, 13). Studies in suckling pigs have shown that protein synthesis is high in the neonate and declines with development (4, 5). Feeding stimulates protein synthesis in the liver, as it does in other tissues of the neonate (4, 5). Amino acids play a key role in the regulation of liver protein synthesis in the neonate (7). Studies using our novel hormonesubstrate clamps have shown that the infusion of amino acids at a dose that reproduces fed-state plasma amino acid concen-

trations increases protein synthesis in liver of the neonatal pig, and the magnitude of the amino acid-stimulated increase in protein synthesis is similar to that which occurs in response to feeding (4, 7). Although insulin stimulates whole body amino acid disposal and skeletal muscle protein synthesis in the neonate, in neonatal liver, protein synthesis is unresponsive to insulin infusion, and this effect persists with development (6, 7, 56). Acute modulation of tissue protein synthesis rates, including the rate due to amino acid and hormonal stimulation, is regulated by changes in the rate of translation of mRNA via alterations in the rate of peptide chain initiation (15, 27, 33). Previous studies in our laboratory have shown that the postprandial increase in protein synthesis in liver and in skeletal muscle of the neonate is associated with an increase in the efficiency of the translation process, i.e., the amount of protein synthesized per unit RNA (4). This increase in translational efficiency is due primarily to increases in the activation of translation initiation factors involved in the binding of mRNA, and not initiator methionyl-tRNA (met-tRNAi), to the 40S ribosomal subunit (11, 24, 28). Thus, in the neonate, feeding increases the phosphorylation of the 70-kDa ribosomal protein S6 kinase (S6K1) and the eukaryotic initiation factor-4E (eIF4E)-binding protein 4E-BP1, thus enhancing eIF4E availability by releasing it from the inactive 4E-BP1䡠eIF4E complex and increasing the association of eIF4E with eIF4G. These responses to feeding are more pronounced in skeletal muscle than in liver. The postprandial changes in translation initiation in the neonate are dependent on the activation of the protein kinase termed mammalian target of rapamycin (mTOR) and are associated with activation of upstream signaling proteins, including PKB and phosphatidylinositol 3-kinase (28, 49). However, in vivo studies utilizing neonatal pigs suggest that feeding does not alter eIF2B activity, which regulates the binding of met-tRNAi to the 40S ribosomal subunit (11). Recently, we demonstrated that insulin and amino acids independently stimulate muscle protein synthesis in the neonate and that this effect involves modulation of translation factors that regulate mRNA binding to the ribosomal complex (40, 41). In the current study, we wished to determine whether 1) amino acid-induced stimulation of liver protein synthesis is independent of insulin, 2) amino acids exert a dose-response effect on liver protein synthesis, and 3) amino acid-induced liver protein synthesis is regulated by changes in the activation

Address for reprint requests and other correspondence: T. A. Davis, USDA/ ARS Children’s Nutrition Research Center, Baylor College of Medicine, 1100 Bates St., Suite 9064, Houston, TX 77030 (E-mail: [email protected]).

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.

neonate; growth; translation initiation; nutrition; eukaryotic initiation factor-4E

GROWTH AND PROTEIN DEPOSITION RATES

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PROTEIN SYNTHESIS IN NEONATAL PIGS

of specific translation initiation factors that modulate availability of eIF4E for 48S ribosomal complex formation. METHODS

Animals. Multiparous sows (n ⫽ 11; crossbred Yorkshire ⫻ Landrace ⫻ Hampshire ⫻ Duroc; Agriculture Headquarters, Texas Department of Criminal Justice, Huntsville, TX) were housed in lactation crates in individual, environmentally controlled rooms, maintained on a commercial diet (5084, PMI Feeds, Richmond, IN), and provided water ad libitum throughout the lactation period. After farrowing, piglets remained with the sow but were not given supplemental creep feed. Piglets were studied at 5– 8 days of age (2.1 ⫾ 0.4 kg). Three to five days before the infusion study, pigs were anesthetized, and catheters were surgically inserted into a jugular vein and a carotid artery with sterile techniques, as described previously (55). Piglets were returned to the sow until studied. The previously described protocol (40) was approved by the Animal Care and Use Committee of Baylor College of Medicine. The study was conducted in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. Pancreatic glucose-amino acid clamps. After an overnight fast, pigs were placed unanesthetized in a sling restraint system, as previously described by O’Connor et al. (40). The average basal concentration of blood glucose (YSI 2300 STAT Plus, Yellow Springs Instruments, Yellow Springs, OH) and plasma branched-chain amino acid (BCAA) concentrations (3) 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 ␮g/kg), continuous (100 ␮g䡠kg⫺1䡠h⫺1) somatostatin (BACHEM, Torrance, CA) infusion. After a 10-min infusion of somatostatin, a continuous infusion of replacement glucagon (150 ng䡠kg⫺1䡠h⫺1; 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䡠kg⫺0.66䡠min⫺1 to achieve plasma insulin concentrations of ⬃0, 2, 6, or 30 ␮U/ml to simulate below fasting, fasting, intermediate, or fed insulin levels, respectively (4). At each dose of insulin, amino acids were clamped at either the fasting (500 nmol BCAA/ml) or fed (1,000 nmol BCAA/ml) levels by monitoring plasma BCAA concentrations every 5 min and adjusting the infusion rate of a balanced amino acid mixture (7) to maintain its concentration within 10% of the desired level (7, 55). At the highest insulin dose only, amino acids were also allowed to fall below fasting levels by omitting the amino acid clamp. Blood glucose concentrations were measured at 5-min intervals, and the dextrose infusion rate was adjusted to maintain blood glucose at a constant value. Blood samples also were taken at intervals for later determination of circulating insulin, glucagon, and individual essential and nonessential amino acid concentrations. Tissue protein synthesis in vivo. The fractional rate of protein synthesis was measured with a flooding dose of L-[4-3H]phenylalanine (16) injected 90 min after the initiation of the clamp procedure. Pigs were killed at 2 h, samples of liver were collected and rapidly frozen, and fractional rates of protein synthesis were determined as previously described (8). Plasma hormones and substrates. The concentrations of individual amino acids from frozen plasma samples obtained at 0 and 90 min after the start of the insulin infusions were measured with an HPLC method (PICO-TAG reverse-phase column, Waters, Milford, MA) as previously described (10). With a porcine insulin radioimmunoassay kit (Linco, St. Louis, MO) that used porcine insulin antibody and human insulin standards, plasma radioimmunoreactive insulin concentrations were measured. Plasma radioimmunoreactive glucagon concentrations were measured using a porcine glucagon radioimmunoassay kit (Linco, St. Louis, MO) that used porcine glucagon antibody and human glucagon standards. Protein immunoblot analysis. Blots were developed using an Amersham enhanced chemiluminescence (ECL) Western Blotting Kit, as AJP-Endocrinol Metab • VOL

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described previously (30). 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. Examination of 4E-BP1 phosphorylation on Thr70. Aliquots of liver homogenates (supernatants) were heated at 100°C for 10 min, cooled to room temperature, and then centrifuged at 10,000 g for 10 min at 4°C. The supernatants were diluted with SDS sample buffer and then subjected to protein immunoblot analysis, as described previously (11, 30). The membranes were incubated with a polyclonal antibody that specifically recognizes phosphorylation of 4E-BP1 at Thr70. Quantitation of 4E-BP1䡠eIF4E complex. The association of eIF4E with 4E-BP1 was quantitated as described previously (11, 31). Briefly, eIF4E was immunoprecipitated from 10,000-g supernatants of liver homogenates with a monoclonal antibody to eIF4E (26). Next, proteins in the immunoprecipitate were resolved by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were probed with an anti-4E-BP1 antibody and then developed using an ECL Western Blotting Kit (Amersham Pharmacia Biotech). The horseradish peroxidase coupled to the anti-rabbit secondary antibody was then inactivated by incubating the blot in 15% hydrogen peroxide for 30 min at room temperature, and the membranes were reprobed with the monoclonal anti-eIF4E antibody (29). Values obtained using the anti-4E-BP1 antibody were normalized for the amount of eIF4E in the immunoprecipitates. Measurement of S6K1 phosphorylation. Liver homogenates were combined with an equal volume of SDS sample buffer, and the diluted samples were subjected to electrophoresis on a 7.5% polyacrylamide gel (34). The samples were then analyzed by protein immunoblot analysis by use of rabbit anti-rat S6K1 polyclonal antibodies, as previously described (11). Measurement of eIF2B activity. eIF2B activity in fresh liver supernatants was measured as the exchange of [3H]GDP bound to eIF2 for unlabeled GDP or GTP, as previously described (11, 23). Briefly, an eIF2䡠[3H]GDP binary complex was formed in the absence of magnesium chloride. The eIF2䡠[3H]GDP complex was then stabilized by the addition of magnesium to a final concentration of 2 mM. The eIF2䡠[3H]GDP complex was incubated with samples containing eIF2B in the presence of a 100-fold molar excess of unlabeled, HPLC-purified GTP at 30°C for various times. The reaction mixture was filtered through a nitrocellulose filter, the filters were washed, and radioactivity bound to the filter was quantitated using a liquid scintillation counter. Calculations and statistics. The fractional rate of protein synthesis (Ks; percentage of protein mass synthesized in a day) was calculated as K s 共%/day兲 ⫽ 关共Sb/Sa ⫻ 共1,440/t兲兴 ⫻ 100 where Sb (dpm/min) is the specific radioactivity of the protein-bound phenylalanine and Sa (dpm/min) is the specific radioactivity of the tissue-free phenylalanine at the time of 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. Analysis of variance (ANOVA; general linear modeling) was used to assess the effect of insulin, amino acids, and their interaction, and to determine whether there was a linear and/or quadratic relationship between BCAA and translation initiation factor activity, and between Ks and translation initiation factor activity. Student’s t-test was used to test for specific differences between groups. To determine the effectiveness of the clamp procedure, amino acid and insulin concentrations in each treatment group were compared with their basal concentrations by use of t-tests. Probability values of P ⬍ 0.05 were considered statistically significant.

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RESULTS

Infusions, hormones, and substrates. Pancreatic glucoseamino acid clamps were performed in fasted 7-day-old pigs by infusion of somatostatin (to block insulin secretion), glucagon (at replacement levels), and glucose (as needed to maintain fasting levels). Insulin was infused at four doses to achieve levels that simulated 1) less than fasting, 2) fasting, 3) intermediate between fasting and fed, or 4) fed conditions. Amino acids were clamped by monitoring plasma BCAA concentrations and adjusting the infusion rate of a balanced amino acid mixture to maintain these concentrations at fasting or fed levels. This resulted in BCAA of ⬃500 or 1,000 nmol/ml, respectively; at the highest insulin dose, amino acids were also allowed to fall to less than the fasting levels of ⬃250 nmol/ml. As shown in Table 1, targeted plasma insulin levels, i.e., ⬃0, 2, 6, and 30 ␮U/ml, and BCAA levels were largely achieved in all treatment groups. Concentrations of total amino acids from previously reported data are also shown for comparison (40). Circulating glucose and glucagon concentrations were largely maintained at basal fasting levels during the infusion of somatostatin, glucagon, insulin, and/or amino acids (data not shown). Liver protein synthesis. Previous studies have shown that amino acid infusion in the neonatal pig stimulates protein synthesis in liver when insulin is clamped at the fasting level (2 ␮U/ml) (7). To examine the interaction of insulin and amino acids in the stimulation of liver protein synthesis, we used the pancreatic-substrate clamp technique to achieve four different insulin doses of ⬃0, 2, 6, or 30 ␮U/ml while amino acids were either clamped at the fasting level (500 BCAA nmol/ml) or increased to the fed level (1,000 BCAA nmol/ml). Raising amino acids from the fasting to the fed level increased the fractional rate of protein synthesis in liver (P ⬍ 0.001; Fig. 1). The magnitude of the effect of amino acids on liver protein synthesis was similar at each dose of insulin (10%–20%), including when insulin was reduced to nearly zero by somatostatin infusion (P ⬍ 0.05, Fig. 1). Insulin had no effect on the fractional rate of protein synthesis in the liver, and there was no interaction of insulin and amino acids on liver protein synthesis, as determined by ANOVA. However, examination of the individual treatment effects by Student’s t-test revealed that protein synthesis was increased only in response to the highest

Fig. 1. Fractional protein synthesis rates (Ks) in liver of 7-day-old pigs at ⬃0, 2, 6, and 30 ␮U/ml plasma insulin levels during euaminoacidemia [500 nmol branched-chain amino acids (BCAA)/ml] and hyperaminoacidemia (1,000 nmol BCAA/ml). Pigs were infused with somatostatin (to block insulin secretion), glucagon (at replacement levels), and glucose (as needed to maintain fasting levels) while insulin was infused to simulate less than fasting, fasting, intermediate, or fed conditions. Amino acids (AA) were clamped by monitoring plasma BCAA concentrations and adjusting the infusion rate of a balanced AA mixture to maintain concentrations at fasting or fed levels. Values are means ⫾ SE; n ⫽ 8 –12 per treatment group. Plasma insulin levels are in ␮U/ml, and BCAA levels are in nmol/ml. Hyperaminoacidemia, but not hyperinsulinemia, increased protein synthesis rates (P ⬍ 0.001). †Significantly different from euaminoacidemic group within same insulin group (P ⬍ 0.001); *significantly different from ⬃0 ␮U/ml insulin dose within the same AA level (P ⬍ 0.005).

insulin dose in the presence of hyperaminoacidemia (P ⬍ 0.005). To determine whether there is a dose-response effect of amino acids on liver protein synthesis, insulin was infused to simulate the fed level while amino acids were clamped at either the fasting level (500 BCAA nmol/ml) or the fed level (1,000 BCAA nmol/ml) or allowed to fall to below fasting levels (250 BCAA nmol/ml). A progressive increase in liver protein synthesis occurred as amino acids were raised from below fasting to fasting levels (P ⬍ 0.05) and from fasting to fed amino acid levels (P ⬍ 0.001; Fig. 2). Effect of amino acids and insulin on translation initiation factors. To determine whether amino acids and insulin interact in the translational control of liver protein synthesis, we determined the effect of raising amino acid levels from the fasting to the fed level, in the presence of insulin doses ranging from

Table 1. Plasma insulin and amino acid levels in response to insulin and amino acid infusion during pancreatic glucose-amino acid clamps in 7-day-old pigs Insulin Groups Outcomes

Insulin TAA BCAA

AA Groups

Baseline

⬍Fasting

Fasting

Intermediate

Fed

Fasting Fed ⬍Fasting Fasting Fed ⬍Fasting Fasting Fed ⬍Fasting

1.5⫾0.2 1.7⫾0.2 1.8⫾0.2 3,245⫾53 3,172⫾74 3,354⫾134 544⫾19 560⫾25 601⫾52

0.6⫾0.1* 0.7⫾0.2* ND 3,014⫾156 5,546⫾497 ND 507⫾47 888⫾55 ND

2.4⫾0.2 2.7⫾0.3 ND 3,079⫾241 5,227⫾232 ND 506⫾64 1,072⫾74 ND

5.6⫾0.6* 6.2⫾0.2* ND 3,060⫾149 4,649⫾392 ND 534⫾58 873⫾58 ND

30.8⫾4.2* 29.0⫾3.4* 24.0⫾2.0* 2,947⫾123 4,697⫾117 1,851⫾167* 510⫾23 887⫾37 259⫾25

Values are means ⫾ SE; n ⫽ 8 –12 per group. Plasma concentrations of insulin are in ␮U/ml, and amino acids [AA; total (TAA), branched chain (BCAA), essential, and nonessential] are in nmol/ml. ND, not determined. *Significantly different from baseline (time 0) values (P ⬍ 0.05).

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Fig. 2. Fractional protein synthesis rates in liver of 7-day-old pigs during hypoaminoacidemia (250 nmol BCAA/ml), euaminoacidemia (500 nmol BCAA/ml), or hyperaminoacidemia (1,000 nmol BCAA/ml, respectively) in the presence of hyperinsulinemia (⬃30 ␮U/ml). Pigs were infused with somatostatin (to block insulin secretion), glucagon (at replacement levels), and glucose (as needed to maintain fasting levels) while insulin was infused to simulate fed conditions. AA were either allowed to fall to less than fasting levels or clamped at fasting or fed levels by monitoring plasma BCAA concentrations and adjusting the infusion rate of a balanced AA mixture. Values are means ⫾ SE; n ⫽ 8 –12 per treatment group. AA increased liver protein synthesis rates (P ⬍ 0.05). *Significantly different from hyperinsulinemic-hypoaminoacidemic group (P ⬍ 0.05); †significantly different from hyperinsulinemic-euaminoacidemic group (P ⬍ 0.001).

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below fasting to fed levels, on translation initiation factor content and/or phosphorylation. Both 4E-BP1 and S6K1 are downstream of mTOR and thus dependent on the activity of this kinase for activation. Through protein immunoblot analysis, we determined the phosphorylation state of S6K1. Hyperphosphorylation of the protein results in decreased mobility during SDS-polyacrylamide gel electrophoresis (54). Amino acids (P ⬍ 0.001) increased S6K1 phosphorylation (Fig. 3A). The stimulation of S6K1 phosphorylation by amino acids occurred at the basal insulin dose (P ⬍ 0.001) and tended to occur at the zero, intermediate, and highest insulin doses (P ⬍ 0.10). Insulin also increased S6K1 phosphorylation (P ⬍ 0.001); specifically, an effect of insulin occurred in the presence of hyperaminoacidemia at the basal and high insulin doses (P ⬍ 0.05). Studies in cell culture have shown that eIF4E plays a critical role in the binding of mRNA to the 43S preinitiation complex (45, 48) and that the function of eIF4E may be influenced by either its phosphorylation state or its availability for formation of the active eIF4E䡠eIF4G complex (38, 42). To determine whether insulin and/or amino acids affected eIF4E phosphorylation status, the amount of eIF4E present in the phosphorylated form as a percentage of the total eIF4E was determined. There was no significant effect of insulin or amino acids on eIF4E phosphorylation (data not shown). The availability of eIF4E can be regulated through changes in the amount of eIF4E bound to 4E-BP1 such that phosphor-

Fig. 3. Translation initiation factor activation states (A-D) in liver of 7-day-old pigs at ⬃0, 2, 6, and 30 ␮U/ml plasma insulin levels during euaminoacidemia (500 nmol BCAA/ ml) and hyperaminoacidemia (1,000 nmol BCAA/ml). S6K1, protein S6 kinase; eIF4E or eIF2B, eukaryotic initiation factors 4E or 2B, respectively; 4E-BP1, eIF4E-binding protein 1. Values are means ⫾ SE; n ⫽ 8 –12 per treatment group. †Significantly different from euaminoacidemic group within same insulin group (P ⬍ 0.01); *significantly different from ⬃0 ␮U/ml insulin dose within the same AA level (P ⬍ 0.05).


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ylation of 4E-BP1 causes disassembly of the eIF4E䡠4E-BP1 complex (29). Phosphorylation of 4E-BP1 at the Thr70 site has been shown to be important in regulating its association with eIF4E when phosphorylated (28, 29). Amino acids (P ⬍ 0.001) stimulated 4E-BP1 phosphorylation at the Thr70 site (Fig. 3B). Examination of specific differences among individual groups revealed that the effect of amino acids on 4E-BP1 phosphorylation occurred at the low, basal (P ⬍ 0.05), and highest doses (P ⬍ 0.001) and tended to occur at the intermediate insulin dose (P ⬍ 0.10). Insulin increased 4E-BP1 phosphorylation (P ⬍ 0.05), with an effect of insulin occurring in the presence of euaminoacidemia at the intermediate dose of insulin (P ⬍ 0.01), and in the presence of hyperaminoacidemia, at the intermediate and high doses of insulin (P ⬍ 0.005) compared with the zero insulin dose. Phosphorylation of 4E-BP1 in cell culture and in situ decreases the association of 4E-BP1 with eIF4E, thereby allowing eIF4E to bind to eIF4G (11, 29, 35). To determine the combined effect of amino acids and insulin on 4E-BP1 association with eIF4E, eIF4E was immunoprecipitated with an anti-eIF4E antibody, followed by immunoblot analysis with an anti-4E-BP1 antibody. Amino acids (P ⬍ 0.001) decreased the amount of 4E-BP1 present in the eIF4E immunoprecipitate (Fig. 3C). Examination of specific differences among individual groups revealed that the effect of amino acids on 4EBP1䡠eIF4E content occurred at the basal (P ⬍ 0.001), intermediate (P ⬍ 0.005), and high doses (P ⬍ 0.01) of insulin and tended to occur at the zero insulin dose (P ⬍ 0.10). There was no effect of insulin on the association of 4E-BP1 with eIF4E. The binding of met-tRNAi to the 40S ribosomal subunit is primarily regulated by changes in eIF2B activity (31). The response of guanine nucleotide exchange activity of eIF2B to amino acids in the presence of insulin doses of ⬃0, 2, 6, and 30 ␮U/ml was measured. There were no differences in eIF2B activity regardless of insulin or amino acid dose (Fig. 3D). Correlation of liver protein synthesis or translation initiation factor activation with BCAA. There was a significant positive linear correlation of protein synthesis rates (Fig. 4A; P ⬍ 0.0001), 4E-BP1 phosphorylation (Fig. 4C; P ⬍ 0.001), and S6K1 phosphorylation (Fig. 4B; P ⬍ 0.001) with plasma BCAA in the presence of hyperinsulinemia. A negative linear correlation occurred between the 4E-BP1䡠eIF4E complex and BCAA (Fig. 4E; P ⬍ 0.005). No significant correlation existed between eIF2B activity and BCAA or between eIF4E phosphorylation and BCAA. Correlation of liver protein synthesis rate with translation initiation factor activation. There was a positive linear relationship between protein synthesis rates and phosphorylation of 4E-BP1 (Fig. 5A; P ⬍ 0.001) and S6K1 (Fig. 5C; P ⬍ 0.01). There was a negative linear relationship between liver protein synthesis and 4E-BP1䡠eIF4E content (Fig. 5B; P ⬍ 0.01). There was no relationship between protein synthesis rates and eIF2B activity. Correlation of liver protein synthesis or translation initiation factor activation with insulin. There was a significant quadratic correlation of S6K1 phosphorylation (Fig. 6B; P ⬍ 0.01) and 4E-BP1 phosphorylation (Fig. 6C; P ⬍ 0.05) with plasma insulin. No significant correlation existed between protein synthesis rates, 4E-BP1䡠eIF4E content, or eIF2B activity and plasma insulin. AJP-Endocrinol Metab • VOL

DISCUSSION

Studies in our laboratory and others suggest that amino acids are a primary stimulator of liver protein synthesis (7, 15, 43, 46). The current study was performed to further define the role of amino acids in the regulation of liver protein synthesis in the neonate and whether insulin plays a role, albeit permissive, in amino acid-stimulated liver protein synthesis. By using our previously described pancreatic glucose-amino acid clamps (40), we demonstrated that amino acid infusion, which simulates fed amino acid levels, can stimulate fractional rates of protein synthesis in liver regardless of the circulating insulin level present. We also showed a dose-response effect of amino acids on liver protein synthesis. On the other hand, insulin was shown to have little effect on fractional rates of protein synthesis in liver of the neonate. At the cellular level, our results demonstrate that amino acids regulate global rates of protein synthesis in liver of the neonate by mechanisms associated with increased 4E-BP1 and S6K1 phosphorylation and decreased formation of the inactive 4E-BP1䡠eIF4E complex that allows eIF4E availability for 48S preinitiation complex formation. Liver protein synthesis in response to amino acids. We have previously demonstrated that amino acid infusion to the fed level, in the presence of fasted insulin levels, can reproduce the feeding-induced stimulation of liver protein synthesis in the neonate (7). Amino acid-stimulated liver protein synthesis has also been demonstrated in growing animals (15, 46) but not in adults (1, 39). A developmental decline in amino acid-stimulated liver protein synthesis has also been previously demonstrated in our clamp studies performed in growing pigs (7). However, whether there was a potentially permissive effect of fasting insulin levels on amino acid-stimulated liver protein synthesis was not ruled out in our previous study (7). In the current study, by using pancreatic glucose-amino acid clamps, we show the stimulatory effect of amino acids to be independent of insulin in that the effect of amino acids occurred at each dose of insulin, including the zero insulin dose. In the presence of fed insulin levels, we also delineated a dose-response effect of amino acids on neonatal liver protein synthesis. Furthermore, we demonstrate that neonatal liver is so sensitive to amino acids that even increasing plasma amino acid concentrations from below fasting to fasting levels stimulated protein synthesis. These findings highlight the importance of protein in the diet of the young animal. Liver protein synthesis in response to insulin. There are conflicting reports on the role of insulin in the regulation of liver protein synthesis. Some cell culture studies and studies in vivo in diabetic rats suggest a role of insulin in the regulation of liver protein synthesis (19, 21). However, most animal studies have shown an inability of insulin to stimulate liver protein synthesis (39, 47, 50). Our previous studies (6, 7) and this current study find no effect of insulin on the fractional rate of protein synthesis in liver of the neonate. This suggests that the postprandial rise in amino acids, independent of the postprandial rise in insulin, mediates the feeding-induced stimulation of liver protein synthesis in the neonate. It could be argued that the small amount of insulin (0.6 ␮U/ml) present in the pigs infused with somatostatin without insulin replacement was sufficient to play a permissive role in the stimulation of liver protein synthesis by insulin. However, this seems unlikely,

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Fig. 4. Correlations of protein synthesis rates (A) and translation initiation factor activity (B-F) in liver with BCAA during hyperinsulinemia.

given that the lowest insulin level was less than the fasting level (⬃2 ␮U/ml) and was at the lowest level of detection of the insulin assay (0.5 ␮U/ml). Furthermore, the possibility that fed levels of insulin may enhance the stimulation of liver protein synthesis by amino acids should not be overlooked. Effects of amino acids and insulin on translation initiation factors. Our previous feeding studies, with rapamycin as a blocker of mTOR, revealed that the feeding-induced stimulation of liver protein synthesis in neonates is mTOR dependent (28). The phosphorylation of S6K1 is primarily mTOR dependent (25, 51), and previous studies in neonatal pigs have shown that feeding, through its activation of mTOR, causes hyperphosphorylation of S6K1 (11, 28). Increases in the phosphorAJP-Endocrinol Metab • VOL

ylation of S6K1 result in hyperphosphorylation of ribosomal protein S6 and thus facilitate the translation of mRNAs containing terminal oligopyrimidine tracts at the 5⬘ end, which encode elements of the translation apparatus, including ribosomal proteins and elongation factors (32, 36). Therefore, activation of S6K1 appears to increase the synthesis of proteins involved in mRNA translation. In the present study, amino acids increased S6K1 phosphorylation. Previous in vitro and in vivo studies have shown similar findings (2, 20). Amino acid deprivation studies (52) have shown a decrease in S6K1 phosphorylation; however, conclusions drawn as to the regulatory role of amino acids from these substrate deprivation studies are not equivalent to those conclusions derived from

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Fig. 5. Correlations of liver protein synthesis rates with translation initiation factor activity (A-D).

substrate supply studies. Although an effect of insulin on S6K1 phosphorylation was unexpected, the change occurred only in the presence of hyperaminoacidemia and did not result in an increase in the global rate of liver protein synthesis. Stimulation of S6K1 phosphorylation in liver by insulin has been reported recently in the fetus (47). A key step in translation initiation is the binding of mRNA to the 43S preinitiation complex, which is mediated by eIF4F, a complex of proteins that includes eIF4E. Assembly of the eIF4F complex can be regulated by the phosphorylation (38) or availability (44) of eIF4E. The eIF4E phosphorylation status, as a percentage of the total eIF4E, was examined in the current study, as it has been shown in cell culture studies to influence mRNA binding to the 43S preinitiation complex, with ultimate increases in protein synthesis (37, 38, 48). However, no effect of insulin and/or amino acids on eIF4E phosphorylation was observed in this study. These results extend the findings of our previous study that show that feeding has no effect on eIF4E phosphorylation in the neonate. This implies that, in vivo, modulation of eIF4E phosphorylation status is unlikely to be a key regulatory step in the feeding-induced stimulation of liver protein synthesis by amino acids in neonatal pigs. Alternatively, an effect of insulin and/or amino acids on eIF4E phosphorylation could have occurred at an earlier point in time. An important regulatory step in the mRNA binding process is the reversible association of eIF4E with the translational repressor 4E-BP1, which competes with eIF4G to bind to eIF4E. Phosphorylation of 4E-BP1 results in a decreased affinity of AJP-Endocrinol Metab • VOL

eIF4E for 4E-BP1 and an enhanced binding of eIF4E to eIF4G (17). 4E-BP1 phosphorylation at the Thr70 site was stimulated by both amino acids and insulin. In contrast, global rates of protein synthesis in liver were stimulated by amino acids but were unaffected by insulin dose. This finding suggests that amino acid-stimulated 4E-BP1 phosphorylation differs in functionality from insulin-stimulated 4E-BP1 phosphorylation. In a study of fetal lambs, an elevation in insulin also increased 4E-BP1 phosphorylation in liver (47). In mature rats, BCAA enhanced 4E-BP1 phosphorylation with no increase in global rates of protein synthesis in liver (1). The mRNA binding process is primarily regulated by the association of eIF4E with eIF4G, following the dissociation of 4E-BP1 from eIF4E. This results in active eIF4F complex assembly, thus enabling mRNA binding to the 43S preinitiation complex (17). Previous studies in mature rats and neonatal pigs have shown a decrease in the inactive 4E-BP1䡠eIF4E complex in response to refeeding (11, 28, 57). In the current study, amino acids, but not insulin, decreased the 4EBP1䡠eIF4E complex assembly. The effect of amino acids occurred in the presence of the basal, intermediate, and higher doses of insulin. No effect of insulin on the 4EBP1䡠eIF4E complex formation in liver of the ovine fetus has been reported (47). A major site in the regulation of translation initiation is the binding of met-tRNAi to the 40S ribosomal subunit, an event mediated by eIF2 (30, 44). This step is regulated by eIF2B, which exchanges GTP for GDP on eIF2 (31). Studies in cell

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Fig. 6. Correlations of protein synthesis rates (A) and translation initiation factor activity (B-E) with plasma insulin. Continuous line, correlation during hyperaminoacidemia; dotted line, correlation during euaminoacidemia.

culture suggest that insulin and amino acids increase eIF2B activity (14, 26, 53). In situ and in vivo studies suggest that amino acid imbalance induces a decrease in eIF2B activity and protein synthesis in rat liver (1, 46). Studies in diabetic animals suggest that insulin plays an important role in increasing eIF2B activity (22). In the current study, neither insulin nor amino acids had any effect on eIF2B activity. However, this was not unexpected, as previous in vivo studies in both mature rats and neonatal pigs suggest that eIF2B activity is not regulated by changes in nutritional status (11, 28, 57). Correlations. The linear relationships of plasma BCAA concentrations with S6K1 phosphorylation, 4E-BP1 phosphorylation, 4E-BP1䡠eIF4E complex content, and protein synthesis in liver of the neonate in the current study suggest that a maximal response of protein synthesis and translation initiation factor activation to BCAA was not achieved. This raises the AJP-Endocrinol Metab • VOL

possibility that higher concentrations of amino acids may further increase translation initiation factor activity and global rates of protein synthesis. This conclusion is supported by the linear relationship between the activity of these translation initiation factors and protein synthesis. Because we infused amino acids to raise circulating amino acids to levels similar to those of pigs fed sow’s milk, this suggests that the feeding of a higher protein diet could further increase protein synthesis. Furthermore, the positive linear correlation between global protein synthesis rates and translation initiation factor modulation (S6K1 and 4E-BP1 phosphorylation) and a negative linear relationship between global protein synthesis rates and 4E-BP1䡠eIF4E content imply a direct response of this mTORdependent translation initiation pathway and global protein synthesis rates in the liver. In contrast, eIF2B activity showed no relationship with amino acid levels or with protein synthesis

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rates, consistent with our previous findings that feeding does not alter eIF2B activity in neonatal pigs. Thus the results suggest that the postprandial rise in amino acids, by modulation of mRNA binding and not met-tRNAi binding, to the 40S ribosomal subunit plays an important role in the feedinginduced stimulation of liver protein synthesis in the neonate. Insulin’s lack of effect on and correlation with liver protein synthesis is not surprising. However, the quadratic correlation between S6K1 and 4E-BP1 phoshorylation and plasma insulin suggests an mTOR-mediated effect of insulin on these initiation factors that does not appear to modulate global rates of liver protein synthesis. Perspectives. Our previous studies have demonstrated that feeding stimulates protein synthesis in virtually all tissues of the body of the neonate and that the magnitude of the feeding response can be reproduced in skeletal muscle by the infusion of insulin or amino acids and in liver by the infusion of amino acids but not insulin. Recent studies using pancreatic glucoseamino acid clamps demonstrated the independent stimulatory roles of insulin and amino acids on global rates of protein synthesis in skeletal muscle by mechanisms that are associated with enhanced 4E-BP1 and S6K1 phosphorylation and eIF4F complex formation, and by mechanisms independent of this pathway. The results of the current study, also using pancreatic glucose-amino acid clamps, suggest that both amino acids and insulin stimulate S6K1 and 4E-BP1 phosphorylation in the liver of the neonate by an mTOR-dependent process. Amino acids increase global rates of protein synthesis in liver by modulating the availability of eIF4E for 48S ribosomal complex formation, and this response is independent of insulin. ACKNOWLEDGMENTS We thank M. Fiorotto and D. Burrin for helpful discussions; W. Liu, S. Rannels, S. Nguyen, and J. Rosenberger for technical assistance; J. Cunningham, F. Biggs, and J. Stubblefield for care of animals; E. O. Smith for statistical assistance; A. Gillum for graphics; and L. Weiser for secretarial assistance. We acknowledge Eli Lilly Co. for the generous donation of porcine insulin. GRANTS This work is a publication of the United States Department of Agriculture/ Agricultural Research Service 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 Institutes of Health (NIH) Grants AR-44474 (T. A. Davis) and DK-15658 (L. S. Jefferson) and by the US Department of Agriculture/Agricultural Research Service under Cooperative Agreement no. 6250510000-33 (T. A. Davis). This research was also supported in part by NIH Training Grant T32 HD-07445. 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 endorsement by the US Government. REFERENCES 1. Anthony TG, Anthony JC, Yoshizawa F, Kimball SR, and Jefferson LS. Oral administration of leucine stimulates ribosomal protein mRNA translation but not global rates of protein synthesis in the liver of rats. J Nutr 31: 1171–1176, 2001. 2. Anthony TG, Reiter AK, Anthony JC, Kimball SR, and Jefferson LS. Deficiency of dietary EAA preferentially inhibits mRNA translation of ribosomal proteins in liver of meal-fed rats. Am J Physiol Endocrinol Metab 281: E430 –E439, 2001. 3. Beckett PR, Hardin DS, Davis TA, Nguyen HV, Wray-Cahen D, and Copeland KC. Spectrophometric assay for measuring branched chain amino acid concentrations: application for measuring the sensitivity of protein metabolism to insulin. Anal Biochem 240: 48 –53, 1996. AJP-Endocrinol Metab • VOL

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