Regulation of neonatal liver protein synthesis by insulin and amino acids in pigs

Transcription

1 Am J Physiol Endocrinol Metab 286: E994 E1003, First published February 3, 2004; /ajpendo 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. Davis 1 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 2 Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 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, First published February 3, 2004; /ajpendo 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 kg 0.66 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 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. neonate; growth; translation initiation; nutrition; eukaryotic initiation factor-4e GROWTH AND PROTEIN DEPOSITION RATES 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 concentrations 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-trna i ), 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 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-trna i 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 ( tdavis@bcm.tmc.edu). E994 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.

2 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 ( 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 kg 1 h 1 ) somatostatin (BACHEM, Torrance, CA) infusion. After a 10-min infusion of somatostatin, a continuous infusion of replacement glucagon (150 ng kg 1 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 kg 0.66 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-[4-3 H]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 E995 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 Thr 70. 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 Thr 70. Quantitation of 4E-BP1 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 [ 3 H]GDP bound to eif2 for unlabeled GDP or GTP, as previously described (11, 23). Briefly, an eif2 [ 3 H]GDP binary complex was formed in the absence of magnesium chloride. The eif2 [ 3 H]GDP complex was then stabilized by the addition of magnesium to a final concentration of 2 mm. The eif2 [ 3 H]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 (K s; percentage of protein mass synthesized in a day) was calculated as K s %/day S b /S a 1,440/t 100 where S b (dpm/min) is the specific radioactivity of the protein-bound phenylalanine and S a (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 K s 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.

3 E996 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 (K s) 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 Outcomes AA Groups Baseline Insulin Groups Fasting Fasting Intermediate Fed Insulin Fasting * * * Fed * * * Fasting ND ND ND * TAA Fasting 3, , , , , Fed 3, , , , , Fasting 3, ND ND ND 1, * BCAA Fasting Fed , Fasting ND ND ND 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).

4 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). E997 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 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).

5 E998 ylation of 4E-BP1 causes disassembly of the eif4e 4E-BP1 complex (29). Phosphorylation of 4E-BP1 at the Thr 70 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 Thr 70 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 4E- BP1 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-trna i 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 ), 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 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 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 eif4e content, or eif2b activity and plasma insulin. 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 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,

6 E999 Fig. 4. Correlations of protein synthesis rates (A) and translation initiation factor activity (B-F) in liver with BCAA during hyperinsulinemia. Downloaded from by on December 5, 2017 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 phosphorylation 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

7 E1000 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 eif4e for 4E-BP1 and an enhanced binding of eif4e to eif4g (17). 4E-BP1 phosphorylation at the Thr 70 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 eif4e complex in response to refeeding (11, 28, 57). In the current study, amino acids, but not insulin, decreased the 4E- BP1 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 4E- BP1 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-trna i 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

8 E1001 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 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 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. 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 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

9 E1002 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-trna i 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 (T. A. Davis) and DK (L. S. Jefferson) and by the US Department of Agriculture/Agricultural Research Service under Cooperative Agreement no (T. A. Davis). This research was also supported in part by NIH Training Grant T32 HD 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: , 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, 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, 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, Davis TA, Burrin DG, Fiorotto ML, Reeds PJ, and Jahoor F. Role of insulin and amino acids in the regulation of protein synthesis in the neonate. J Nutr 128: 347S 350S, 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, Davis TA, Fiorotto ML, Burrin DG, Reeds PJ, Nguyen HV, Beckett PR, Vann RC, and O Connor PMJ. Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs. Am J Physiol Endocrinol Metab 282: E880 E890, Davis TA, Fiorotto ML, Nguyen HV, and Reeds PJ. Protein turnover in skeletal muscle of suckling rats. Am J Physiol Regul Integr Comp Physiol 257: R1141 R1146, 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 Regul Integr Comp Physiol 265: R334 R340, Davis TA, Fiorotto ML, and Reeds PJ. Amino acid compositions of body and milk protein change during the suckling period in rats. J Nutr 123: , Davis TA, Nguyen HV, Suryawan A, Bush JA, Jefferson LS, and Kimball SR. Developmental changes in the feeding-induced stimulation of translation initiation in skeletal muscle and liver of neonatal pigs. Am J Physiol Endocrinol Metab 279: E1226 E1234, Denne SC and Kalhan SC. Leucine metabolism in human newborns. Am J Physiol Endocrinol Metab 253: E608 E625, Denne SC, Rossi EM, and Kalhan SC. Leucine kinetics during feeding in normal newborns. Pediatr Res 30: 23 27, Everson WV, Flaim KE, Susco DM, Kimball SR, and Jefferson LS. Effect of amino acid deprivation on initiation of protein synthesis in rat hepatocytes. Am J Physiol Cell Physiol 256: C18 C27, Flaim KE, Liao WSL, Peavy DE, Taylor JM, and Jefferson LS. The role of amino acids in the regulation of protein synthesis in perfused rat liver. II. Effects of amino acid deficiency on peptide chain initiation, polysomal aggregation and distribution of albumin mrna. J Biol Chem 257: , Garlick PJ, McNurlan MA, and Preedy VR. A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [ 3 H] phenylalanine. Biochem J 192: , Gingras AC, Raught B, and Sonenburg N. eif4 initiation factors: effectors of mrna recruitment to ribosomes and regulators of translation. In: Annual Reviews in Biochemistry, edited by Richardson CC. Palo Alto, CA: Annual Reviews, 1999, p Goldspink DF and Kelly FJ. Protein turnover and growth in the whole body, liver and kidney of the rat from the fetus to senility. Biochem J 217: , Hsu CJ, Kimball SR, Antonetti DA, and Jefferson LS. Effects of insulin action on total RNA, poly (A) RNA, and mrna in primary cultures of rat hepatocytes. Am J Physiol Endocrinol Metab 263: E1106 E1112, Iiboshi Y, Papst PJ, Hunger SP, and Terada N. L-Asparaginase inhibits the rapamycin-targeted signaling pathway. Biochem Biophys Res Commun 260: , Jefferson LS, Liao WSL, Peavy DE, Miller TB, Appel MC, and Taylor JM. Diabetes-induced alterations in liver protein synthesis. Changes in the relative abundance of mrnas for albumin and other plasma proteins. J Biol Chem 258: , Jefferson LS, Li JB, and Rannels SR. Regulation by insulin of amino acid release and protein turnover in the perfused rat hemicorpus. J Biol Chem 252: , Kimball SR, Everson WV, Flaim KE, and Jefferson LS. Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes. Am J Physiol Cell Physiol 256: C28 C34, Kimball SR, Farrell PA, Nguyen HV, Jefferson LS, and Davis TA. Developmental decline in components of signal transduction pathways regulating protein synthesis in pig muscle. Am J Physiol Endocrinol Metab 282: E585 E592, Kimball SR, Horetesky RL, and Jefferson LS. Signal transduction pathways involved in the regulation of protein synthesis by insulin in L6 myoblasts. Am J Physiol Cell Physiol 274: C221 C228, 1998.

10 E Kimball SR, Horetesky RL, and Jefferson LS. Implication of eif2b rather than eif4e in the regulation of protein synthesis by amino acids in L6 myoblasts. J Biol Chem 273: , Kimball SR and Jefferson LS. Cellular mechanisms involved in the action of insulin on protein synthesis. Diabetes Metab Res 4: , 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, Kimball SR, Jurasinski CV, Lawrence JC, and Jefferson LS. Insulin stimulated protein synthesis in skeletal muscle by enhancing the association of eif-4e and eif-4g. Am J Physiol Cell Physiol 272: C754 C759, Kimball SR, Karinch AM, Feldhoff RC, and Jefferson LS. Purification and characterization of eukaryotic initiation factor eif-2b from liver. Biochim Biophys Acta 1201: , Kimball SR, Mellor H, Flowers KM, and Jefferson LS. Role of translation initiation factor eif-2b in the regulation of protein in mammalian cells. Prog Nucleic Acid Res 54: , Kimball SR, Shantz LM, Horetsky RL, and Jefferson LS. Leucine regulates translation of specific mrnas in L6 myoblasts through mtormediated changes in availability of eif4e and phosphorylation of ribosomal protein S6. J Biol Chem 274: , Kimball SR, Vary TC, and Jefferson LS. Regulation of protein synthesis by insulin. Annu Rev Physiol 56: , Laemlli UK. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227: , Lin RA, Kong X, Haystead TAJ, Pause A, Belsham G, Soneburg N, and Lawrence JC. PHAS-1 as a link between mitogen-activated protein kinase and translation initiation. Science 266: , Meyuhas O and Hornstein E. Translational control of TOP mrnas. In: Translational Control of Gene Expression, edited by Sonenberg N, Hershey JWB, and Matthews MB. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2000, p Minich WB, Balasta ML, Goss DJ, and Rhoads RE. Chromatographic resolution of in vivo phosphorylated and non phosphoylated eukaryotic initiation factor eif-4e: increased cap affinity of the phosphorylation form. Proc Natl Acad Sci USA 91: , Morley SJ, Rau M, Kay JE, and Pain VM. Increased phosphorylation of eukaryotic initiation factor 4 during early activation of T lymphocytes correlates with increased initiation factor 4F complex formation. Eur J Biochem 218: 39 48, Mosoni L, Houlier ML, Mirand PP, Bayle G, and Grizard J. Effect of amino acids alone or with insulin on muscle and liver protein synthesis in adult and old rats. Am J Physiol Endocrinol Metab 264: E614 E620, O Connor PMJ, Bush JA, Suryawan A, Nguyen HV, and Davis TA. Insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonatal pigs. Am J Physiol Endocrinol Metab 284: E110 E119, O Connor PMJ, Kimball SR, Suryawan A, Bush JA, Nguyen HV, Jefferson LS, and Davis TA. Regulation of translation initiation by insulin and amino acids in skeletal muscle of neonatal pigs. Am J Physiol Endocrinol Metab 285: E40 E53, Pause A, Belsham GJ, Gingras AC, Donze O, Lin TA, Lawrence JC, and Sonenberg N. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5 -cap function. Nature 371: , Perez-Sala D, Parrilla R, and Ayuso MS. Key role of L-alanine in the control of hepatic protein synthesis. Biochem 241: , Proud CG and Denton RM. Molecular mechanisms for the control of translation by insulin. Biochem J 328: , Rhoads RE. Regulation of eukaryotic protein synthesis by initiation factors in the recruitment of mrna to ribosomes. Biochimie 76: , Shah OJ, Antonetti DA, Kimball SR, and Jefferson LS. Leucine, glutamine, and tyrosine reciprocally modulate the translation initiation factors eif4f and eif2b in perfused rat liver. J Biol Chem 274: , Shen W, Malon D, Boyle DW, and Liechty EA. IGF-I and insulin regulate eif4e formation by different mechanisms in muscle and liver in the ovine fetus. Am J Physiol Endocrinol Metab 283: E593 E603, Sonenburg N. Regulation of translation and cell growth by eif4e. Biochimie 76: , 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, Tauveron I, Larbaud D, Champredon C, Debras E, Tesseraud S, Bayle G, Bonnet Y, Thieblot P, and Grizard J. Effect of hyperinsulinemia and hyperaminoacidemia on muscle and liver protein synthesis in lactating goats. Am J Physiol Endocrinol Metab 267: E877 E885, Von Manteuffel SR, Dennis PB, Pullen N, Gingras AC, Sonenberg N, and Thomas G. The insulin-induced signalling pathway leading to S6 and initiation factor 4E-binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70(s6k). Mol Cell Biol 17: , Wang X, Campbell L, Miller CM, and Proud CG. Amino acid availability regulates p70s6 kinase and multiple translation factors. Biochem J 334: , Welsh GI, Stokes CM, Wang X, Sakaue H, Ogawa W, Kasuga M, and Proud CG. Activation of translation initiation factor eif2b by insulin requires phosphatidyl inositol 3-kinase. FEBS Lett 410: , Weng QP, Kozlowski M, Belham C, Zhang A, Comb MJ, and Avruch J. Regulation of the p70 S6 kianase by phosphorylation in vivo. Analysis using site specific antiphosphopeptide antibodies. J Biol Chem 273: , Wray-Cahen D, Beckett PR, Nguyen HV, and Davis TA. Insulinstimulated amino acid utilization during glucose and amino acid clamps decreases with development. Am J Physiol Endocrinol Metab 273: E305 E314, 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, Yoshizawa F, Kimball SR, and Jefferson LS. Modulation of translation initiation in rat skeletal muscle and liver in response to food intake. Biochem Res Commun 240: , 1997.