Glucose stimulates protein synthesis in skeletal muscle of neonatal pigs through an AMPK- and mtor-independent process

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1 Am J Physiol Endocrinol Metab 293: E595 E603, First published June 5, 2007; doi: /ajpendo Glucose stimulates protein synthesis in skeletal muscle of neonatal pigs through an AMPK- and mtor-independent process Asumthia S. Jeyapalan, Renan A. Orellana, Agus Suryawan, Pamela M. J. O Connor, Hanh V. Nguyen, Jeffery Escobar, Jason W. Frank, and Teresa A. Davis United States Department of Agriculture/Agricultural Research Service Children s Nutrition Research Center and Pediatric Critical Care Section, Department of Pediatrics, Baylor College of Medicine, Houston, Texas Submitted 22 February 2007; accepted in final form 30 May 2007 Jeyapalan AS, Orellana RA, Suryawan A, O Connor PM, Nguyen HV, Escobar J, Frank JW, Davis TA. Glucose stimulates protein synthesis in skeletal muscle of neonatal pigs through an AMPK- and mtor-independent process. Am J Physiol Endocrinol Metab 293: E595 E603, First published June 5, 2007; doi: /ajpendo Skeletal muscle protein synthesis is elevated in neonates in part due to an enhanced response to the rise in insulin and amino acids after eating. In vitro studies suggest that glucose plays a role in protein synthesis regulation. To determine whether glucose, independently of insulin and amino acids, is involved in the postprandial rise in skeletal muscle protein synthesis, pancreatic-substrate clamps were performed in neonatal pigs. Insulin secretion was inhibited with somatostatin and insulin was infused to reproduce fasting or fed levels, while glucose and amino acids were clamped at fasting or fed levels. Fractional protein synthesis rates and translational control mechanisms were examined. Raising glucose alone increased protein synthesis in fast-twitch glycolytic muscles but not in other tissues. The response in muscle was associated with increased phosphorylation of protein kinase B (PKB) and enhanced formation of the active eif4e eif4g complex but no change in phosphorylation of AMP-activated protein kinase (AMPK), tuberous sclerosis complex 2 (TSC2), mammalian target of rapamycin (mtor), 4E-binding protein-1 (4E-BP1), ribosomal protein S6 kinase (S6K1), or eukaryotic elongation factor 2 (eef2). Raising glucose, insulin, and amino acids increased protein synthesis in most tissues. The response in muscle was associated with phosphorylation of PKB, mtor, S6K1, and 4E-BP1 and enhanced eif4e eif4g formation. The results suggest that the postprandial rise in glucose, independently of insulin and amino acids, stimulates protein synthesis in neonates, and this response is specific to fast-twitch glycolytic muscle and occurs by AMPK- and mtor-independent pathways. neonates; adenosine 5 -monophosphate-activated protein kinase; tuberous sclerosis complex 2; mammalian target of rapamycin; eukaryotic initiation factor 4E RATES OF GROWTH ARE HIGHER in the neonatal period than during any other stage of postnatal life (5, 15). During the neonatal period, the gain in protein mass in skeletal muscle is more rapid than in other tissues of the body (13, 35). Previous studies (7, 8) performed in neonatal pigs show that neonates efficiently utilize their dietary amino acids for growth due to their enhanced capability to increase protein synthesis in response to feeding. The feeding-induced stimulation of protein synthesis is greatest in skeletal muscle compared with other tissues in the body and decreases profoundly with development. Insulin has been recognized as a key factor in the stimulation of skeletal muscle protein synthesis after a meal in growing animals (10, 20, 35, 38). Insulin stimulates whole body protein synthesis in the fetal sheep, hindlimb protein synthesis in the lamb, and skeletal muscle protein synthesis in weaned rats, and this can be blocked by anti-insulin serum (41, 48). Previous studies (10, 35) have shown that increasing insulin to fed levels, while maintaining glucose and amino acids at fasting levels, stimulates protein synthesis in neonatal pigs, and this response is specific to skeletal muscle. This response to insulin declines with development in parallel with the developmental decline in the response of muscle protein synthesis to feeding (7, 9, 10, 50). The postprandial rise of amino acids is also critical to the upregulation of skeletal muscle protein synthesis in growing and maturing individuals (10, 46). Previous studies (10) have demonstrated that raising amino acids to the fed state, while maintaining insulin and glucose at fasting levels, increases protein synthesis in the neonatal pig, and this response occurs in most tissues. Furthermore, pancreatic-substrate clamp studies (8, 35) demonstrated that insulin and amino acids act independently to stimulate protein synthesis in skeletal muscle of the neonate. Carbohydrates are the major component of milk and commercially available formula and are largely responsible for the rise in circulatory glucose and insulin after a meal. Studies in mature individuals (4) suggest that muscle protein synthesis is unaffected by changes in blood glucose levels. In the postweaned rat, glucose stimulates muscle protein synthesis, and it has been presumed (20) that this is due to the glucosestimulated rise in insulin levels. However, these studies did not determine whether glucose plays a role, independent of insulin, in the stimulation of protein synthesis in skeletal muscle after a meal. In vitro studies in the Chinese hamster ovarian cell line and in cardiac myocytes (39, 51) suggest that a rise in glucose stimulates protein synthesis independently of insulin. A potential mechanism by which glucose could regulate protein synthesis is via an AMP-activated protein kinase (AMPK) pathway. AMPK, a sensor of cellular energy, is activated by rising AMP levels as a result of energy starvation, resulting in activation of tuberous sclerosis complex 1/2 (TSC1/2) (19, 23, 25). TSC1/TSC2 acts as an inhibitor of the mammalian target of rapamycin (mtor) (24, 29). mtor regulates mrna translation by phosphorylation of the 70-kDa S6 kinase-1 (S6K1) and eukaryotic initiation factor (eif)4e- Address for reprint requests and other correspondence: T. A. Davis, USDA/ ARS Children s Nutrition Research Center, Baylor College of Medicine, 1100 Bates Ave., Houston, TX ( tdavis@bcm.edu). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. E595

2 E596 binding protein 1 (4E-BP1) (26, 43, 47). S6K1 activation results in phosphorylation of the ribosomal subunit, ribosomal protein (rp)s6, which may lead to an increase in the translation of mrnas that encode proteins found in the protein synthetic machinery (31, 33). Phosphorylation of the repressor protein, 4E-BP1, permits the dissociation of eif4e, allowing it to bind to eif4g. This active complex of eif4e eif4g mediates the binding of mrna to the 40S ribosomal complex in the initiation of mrna translation (30, 31, 33, 36, 44). However, whether changes in circulating glucose levels alter the activation of these signaling proteins and translation initiation factors has not yet been determined. The purpose of this study was 1) to determine whether glucose, independently of insulin and amino acids, stimulates muscle protein synthesis in skeletal muscle and other tissues of the neonate and 2) to identify the nutrient-signaling pathway by which this occurs. To address these questions, pancreaticsubstrate clamps were performed in fasted neonatal pigs with somatostatin infusion to block endogenous insulin secretion, glucagon provided at replacement levels, glucose and amino acids clamped at either fasting or fed levels, and insulin infused to reproduce fasting or fed levels. The results suggest that the postprandial rise in glucose, independently of changes in insulin or amino acids, stimulates protein synthesis in fast-twitch glycolytic muscle only, and this response occurs via AMPKindependent and mtor-independent pathways. METHODS Animals. Four multiparous crossbred (Yorkshire Landrace Hampshire Duroc) sows (Agriculture Headquarters, Texas Department of Criminal Justice, Huntsville, TX) were housed in lactation crates in individual, environmentally controlled rooms and maintained on a commercial diet (5084; PMI Feeds, Richmond, IN) throughout the lactation period. After farrowing, piglets remained with the sow and were not supplemented with creep feed. Male and female piglets were studied at 4 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 (17). Piglets were returned to the sows until they were studied. The protocol was approved by the Animal Care and Use Committee of Baylor College of Medicine and was conducted in accordance with the National Research Council s Guide for the Care and Use of Laboratory Animals. Pancreatic-substrate clamps. Pigs (n 8/group, total of 24) were placed unanesthetized in a sling restraint system after an overnight fast. Pigs were assigned to one of three treatment groups: 1) fasting glucose, fasting insulin, fasting amino acid clamp (control); 2) fed glucose, fasting insulin, fasting amino acid clamp (glucose); and 3) fed glucose, fed insulin, fed amino acid clamp (glucose insulin amino acid). The fasting glucose, insulin, and amino acid clamp group served as a negative control, and the fed glucose, insulin, and amino acid group served as a positive control. During a 30-min basal period before the clamp procedure was initiated, blood samples were taken and immediately analyzed for blood glucose (YSI 2300 STAT Plus; Yellow Springs Instruments, Yellow Springs, OH) to establish the average basal concentration of blood glucose. Plasma total branchedchain amino acid (BCAA) concentrations were determined by a rapid enzymatic kinetic assay to establish the average basal concentration of BCAA to be used in the substrate clamp procedure (1). The clamp was initiated with a primed (600 g/kg), continuous (600 g kg 1 h 1 ) infusion of somatostatin (Bachem, Torrance, CA). After a 30-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 10 and 110 ng kg 0.66 min 1 to achieve plasma insulin concentrations of 2 and 30 U/ml, respectively, to simulate the fasting and fed insulin states. Amino acids were clamped at either the fasting ( 500 nmol BCAA/ml) or fed ( 1,000 nmol BCAA/ml) levels by adjusting the infusion rate of a balanced amino acid mixture to maintain the plasma BCAA concentration within 10% of the desired level (9, 10). The amino acid mixture contained (in mmol/l) arginine (20.1), histidine (12.9), isoleucine (28.6), leucine (34.3), lysine (27.4), methionine (10.1), phenylalanine (12.1), threonine (21.0), tryptophan (4.4), valine (34.1), alanine (27.3; 38% provided as alanyl-glutamine), aspartate (12.0), cysteine (6.2), glutamate (23.8), glutamine (17.1; 100% provided as alanyl-glutamine), glycine (54.3; 4% provided as glycyl-tyrosine), proline (34.8), serine (23.8), taurine (2.0), and tyrosine (7.2; 83% provided as glycyl-tyrosine). Blood samples were also taken at 5-min intervals for later determination of circulating insulin and individual essential and nonessential amino acid concentrations. Tissue protein synthesis in vivo. The fractional rate of protein synthesis was measured with 10 ml/kg body wt of a flooding dose (1.5 mmol/kg body wt) of L-[4-3 H]phenylalanine (0.5 mci/kg body wt; Amersham Biosciences, Piscataway, NJ) injected 90 min after the initiation of the clamp procedure (21). Pigs were killed at 2 h, and samples of longissimus dorsi, gastrocnemius, masseter, diaphragm, heart muscle, liver, jejunum, spleen, pancreas, lung, and kidney were collected and rapidly frozen in liquid nitrogen. The specific radioactivity of the protein hydrolysate, homogenate supernatant, and blood supernatant was determined as previously described (13). Previous studies (12) have demonstrated that, after a flooding dose of [ 3 H]phenylalanine is administered, the specific radioactivity of tissue free phenylalanine is in equilibrium with the aminoacyl-trna specific radioactivity, and therefore, the tissue free phenylalanine is a valid measure of the tissue precursor pool specific radioactivity. Protein immunoblot analysis. Proteins from longissimus dorsi muscle homogenates (14) were separated on polyacrylamide gels. For each assay, all samples were run at the same time in triple-wide gels (CBS Scientific, Del Mar, CA) to eliminate interassay variation. Proteins were electrophoretically transferred to polyvinylidene difluoride transfer membranes (Bio-Rad, Hercules, CA), which were incubated with appropriate antibodies, washed, and exposed to an appropriate secondary antibody as previously described (14). For normalization, immunoblotting performed with antiphosphospecific antibodies were stripped in stripping buffer (Pierce Biotechnology, Rockford, IL) and reprobed with the corresponding nonphosphospecific antibodies. Blots were developed using an enhanced chemiluminescence kit (Amersham Life Sciences, Arlington Heights, IL), visualized, and analyzed using a ChemiDoc-It Imaging System (UVP, Upland, CA). Primary antibodies that were used in the immunoblotting were PKB (total and Ser 473 ; Cell Signaling Technology, Beverly, MA), AMPK (total and Thr 172 ; Cell Signaling Technology), TSC2 (total and Thr 1462 ; Cell Signaling), mtor (total and Ser 2448 ; Cell Signaling), S6K1 (total and Thr 389 ; Cell Signaling), and 4E-BP1 (total; Bethyl Laboratories, Montgomery, TX, and Thr 70 ; Cell Signaling). Quantification of eif4e 4E-BP1 and eif4e eif4g complexes. These complexes were immunoprecipitated using an anti-eif4e monoclonal antibody (gift of Dr. Leonard Jefferson, Pennsylvania State University College of Medicine, Hershey, PA) from aliquots of fresh tissue homogenates (17). Briefly, samples were homogenized in 7 volumes of buffer (in mm: 20 HEPES, 2 EGTA, 50 NaF, 100 KCl, and 0.2 EDTA, ph 7.4) containing Sigma P3840 Protease Inhibitor Cocktail (Sigma Chemical, St. Louis, MO) and centrifuged at 10,000 g for 10 min at 4 C. Supernatants were incubated overnight at 4 C with constant rocking with anti-eif4e antibody. Immunoprecipitates were recovered with goat anti-rabbit IgG magnetic beads (Polysciences, Warrington, PA), washed and resuspended in sample buffer as described elsewhere (17), and immediately subjected to protein immunoblot analysis using rabbit anti-4e-bp1 (Cell Signaling) antibody or

3 E597 rabbit anti-eif4g (Bethyl Laboratories). Amounts of 4E-BP1 and eif4g were corrected by the eif4e recovered from the immunoprecipitate. Analysis of insulin receptor phosphorylation. Insulin receptors were immunoprecipitated (46) using an anti-insulin receptor antibody (Santa Cruz Biotechnology, Santa Cruz, CA). To determine the phosphorylation of the insulin receptor on tyrosine residues, samples were subjected to immunoblotting with an anti-phosphotyrosine antibody (Santa Cruz). Values were normalized for insulin receptor abundance in the precipitates. Calculations and statistics. The fractional rate of protein synthesis (K s; %protein mass synthesized in a day) was calculated as K s (%/day) [(S b/s a) (1,440/t)] 100, where S b (in dpm/nmol) is the specific radioactivity of the protein bound phenylalanine, S a (in dpm/nmol) 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, t is the time of labeling in minutes, and 1,440 is the number of minutes per day. Analysis of variance (general linear models) was used to determine the overall effect of treatment. If there was an effect of treatment, then post hoc pairwise comparisons were done using Tukey s procedure. To determine the effectiveness of the clamp procedure, amino acids, glucose, and insulin concentrations were compared with basal concentrations within each treatment group using paired t-tests. Probability values of P 0.05 were considered statistically significant. RESULTS Infusions. Pigs were infused with glucose, insulin, and amino acids to reproduce 1) fasting glucose, fasting insulin, and fasting amino acid levels (control), 2) fed glucose, fasting insulin, and fasting amino acid levels (glucose), and 3) fed glucose, fed insulin, and fed amino acid levels (glucose insulin amino acid). The fasting glucose, insulin, and amino acid clamp group served as a negative control, and the fed glucose, insulin, and amino acid group served as a positive control. Figure 1 shows that glucose was increased by infusion without affecting the circulating insulin levels in those animals where only glucose was to be raised to the fed state. Circulating insulin levels that are found in the neonatal pig during the fasting and postprandial conditions (2 and 30 U/ml, respectively) were reproduced largely by infusion. Similarly, circulating amino acid concentrations that are found normally during fasting and postprandial conditions (500 and 1,000 M BCAA) were also reproduced. Tissue protein synthesis. Figure 2 shows the fractional rates of protein synthesis in skeletal muscles of different fiber types in neonatal pigs. Raising glucose alone to fed levels while maintaining insulin and amino acids at fasting levels increased the fractional rates of protein synthesis in the longissimus dorsi muscle by 45% and in the gastrocnemius muscle by 35% (P 0.05). Infusion of glucose, insulin, and amino acids to raise concentrations to fed levels increased the fractional rates of protein synthesis in the longissimus dorsi muscle by 89% and in the gastrocnemius muscle by 94% compared with that in control animals (P 0.01). Raising glucose, insulin, and amino acids to the fed state increased protein synthesis by 83% in the diaphragm (P 0.01) and by 52% in the masseter muscle (P 0.01). There was no effect of raising glucose alone on protein synthesis in the diaphragm or masseter muscle. Figure 3 shows the K s in visceral tissues. Raising glucose alone to fed levels did not change the rates of protein synthesis in liver, gut, spleen, pancreas, or kidney. However, raising Fig. 1. Circulating glucose, insulin, and branched-chain amino acids (BCAA) during pancreatic-substrate clamps in neonatal pigs. Pigs were infused to reproduce 1) fasting glucose, fasting insulin, and fasting amino acid levels (F; control), 2) fed glucose, fasting insulin, and fasting amino acid levels ( ; glucose), and 3) fed glucose, fed insulin, and fed amino acid levels (E; glucose insulin amino acid) during pancreatic-substrate clamps. *Different from control group (P 0.05); different from glucose group (P 0.05). glucose, insulin, and amino acids to fed levels increased protein synthesis in the liver by 22%, pancreas by 48%, spleen by 20%, kidney by 24%, and the lung by 46% (P 0.05). Protein signaling and translation initiation. To study the mechanism by which glucose stimulates protein synthesis in fast-twitch glycolytic skeletal muscle, Western blot analysis was performed in the longissimus dorsi muscle. Figure 4 shows that there was no difference in the phosphorylation of AMPK on the Thr 172 residue in muscle among any of the experimental groups. PKB phosphorylation on Ser 473 was increased (P 0.05) when glucose was raised to the fed level and insulin and amino acids were maintained at fasting levels. Raising glucose, insulin, and amino acids to postprandial levels further increased PKB phosphorylation on Ser 473 (P 0.05). Because an elevation in glucose alone increased PKB phosphorylation, insulin receptor phosphorylation on tyrosine residues was measured. Tyrosine phosphorylation of the insulin receptor was unaffected by a rise in glucose, when insulin and amino acids were maintained at fasting levels. However, raising insulin, amino acids, and glucose to fed levels increased insulin receptor phosphorylation on tyrosine residues (P 0.05). Figure 5 shows the phosphorylation of TSC2 on its Thr 1462 residue. There was no effect of raising glucose alone; however, there was an increase in phosphorylation of TSC2 when glucose, insulin, and amino acids were raised to fed levels in

4 E598 Fig. 2. Fractional rates of protein synthesis (K s) in different muscles of neonatal pigs during pancreatic-substrate clamps. Open bar, control group; gray bar, glucose group; black bar, glucose insulin amino acid group. LD, longissimus dorsi; gastroc, gastrocnemius; mass, masseter. Values are means SE; n 8/treatment group. *Different from control group (P 0.05); different from glucose group (P 0.05). muscle. Phosphorylation of mtor on Ser 2448 was increased in the presence of fed levels of glucose, insulin, and amino acids (P 0.05) but not with the elevation of glucose alone. S6K1 phosphorylation on its Thr 389 residue was increased in muscle when glucose, insulin, and amino acids were raised to the fed state (P 0.05; Fig. 6). However, raising glucose alone had no effect on S6K1 phosphorylation. Phosphorylation of 4E-BP1 on Thr 70 was increased after raising glucose, insulin, and amino acids to the fed state (P 0.05). There was no change in 4E-BP1 phosphorylation when glucose alone was raised to fed levels. The formation of the inactive 4E-BP1 eif4e complex was reduced in animals infused with glucose, insulin, and amino acids to fed levels (P 0.05; Fig. 7). However, raising glucose alone had no effect on the abundance of the inactive 4E- BP1 eif4e complex. The formation of the active eif4e eif4g complex was increased in neonatal piglets whose glucose was increased to the fed level while insulin and amino acids were maintained at fasting levels (P 0.05). Raising glucose, insulin, and amino acids to fed levels further increased the formation of the active eif4e eif4g complex (P 0.05). Furthermore, there was no treatment effect on eef2 phosphorylation on Thr 56 (Fig. 8). DISCUSSION Protein synthesis is elevated in the neonate, in part due to an enhanced response to the rise in insulin and amino acids after Fig. 3. K s in various visceral organs of neonatal pigs during pancreaticsubstrate clamps. Open bar, control group; gray bar, glucose group; black bar, glucose insulin amino acid group. Values are means SE; n 8/treatment group. *Different from control group (P 0.05); different from glucose group (P 0.05). Fig. 4. Phosphorylation of adenosine monophosphate kinase (AMPK) on Thr 172 residue (A), protein kinase B (PKB) on Ser 473 residue (B), and insulin receptor (IR) on tyrosine residues (C) in LD muscle of neonatal pigs during pancreatic-substrate clamps. Values of the phosphorylation of AMPK were normalized for AMPK content in samples; values of the phosphorylation of PKB were normalized for PKB content in samples; values of the phosphorylation of the IR were normalized for IR content in the samples. Open bar, control group; gray bar, glucose group; black bar, glucose insulin amino acid (AA) group. Values are means SE in arbitrary densitometric units; n 8/treatment group. There was no significant difference among any of the treatment groups for AMPK phosphorylation. *Different from control group (P 0.05); different from glucose group (P 0.05). eating (7, 9, 10, 35). Our previous studies using an insulinamino acid-glucose clamp technique (35) have demonstrated that protein synthesis in most visceral tissues is responsive to stimulation by amino acids but that protein synthesis in skeletal muscle of the neonate is regulated by both insulin and amino acids, albeit independently. Since protein synthesis in skeletal muscle of the neonate is highly sensitive to a number of anabolic agents, including feeding, amino acids, leucine, insulin, and insulin-like growth factor I (IGF-I), we wished to determine whether the postprandial rise in glucose also plays a role in the regulation of protein synthesis (7, 10, 11, 17, 35). Our results show that protein synthesis is stimulated by a rise in glucose alone, and this response occurs in skeletal muscle containing primarily fast-twitch glycolytic fibers. Role of glucose in the regulation of protein synthesis. In vitro studies performed in the Chinese hamster ovarian cell line as well as in cardiomyocytes (39, 51) have shown that

5 E599 In the present study, raising glucose alone in neonatal pigs had no effect on protein synthesis in visceral tissues, including the liver, intestine, pancreas, spleen, kidney, and lung. Although the pattern of tissue response to glucose, i.e., sensitive in skeletal muscle but insensitive in visceral tissue, is similar to the pattern of tissue response to insulin (9), it seems unlikely that the rise in muscle protein synthesis with glucose infusion was related to insulin. A high dose of somatostatin was infused to block insulin secretion, and this was initiated 30 min prior to the infusion of glucose. As a consequence, plasma levels of insulin did not increase in response to glucose administration. This suggests that the postprandial rise in glucose stimulates protein synthesis in the neonate and that this is specific to fast-twitch glycolytic muscles and is independent of insulin. Although IGF-I can stimulate skeletal muscle protein synthesis (11), it seems unlikely that the acute rise in glucose in the present study stimulated muscle protein synthesis through an IGF-I-mediated process. Whether the glucose-stimulated rise in muscle protein synthesis can be maintained with chronic administration of glucose alone has not been determined but seems unlikely because amino acids are expected to become a limiting factor as they are utilized for protein synthesis (16). Fig. 5. Phosphorylation (p) of tuberous sclerosis complex 2 (TSC2) on Thr 1462 (A) and mammalian target of rapamycin (mtor) on Ser 2448 (B) in LD muscle of neonatal pigs during pancreatic-substrate clamps. Values of the phosphorylation of TSC2 were normalized for TSC2 content in samples; values of the phosphorylation of mtor were normalized for mtor content in samples. Open bar, control group; gray bar, glucose group; black bar, glucose insulin AA group. Values are means SE in arbitrary densitometric units; n 8/treatment group. *Significantly different from control group (P 0.05); different from glucose group (P 0.05). glucose can stimulate protein synthesis. In the weaned rat, glucose stimulates muscle protein synthesis, but it has been presumed (20) that this rise is secondary to a glucose-induced rise in insulin levels. To the best of our knowledge, the present work is the first study to examine the effect of glucose on protein synthesis in vivo independently of the effects of amino acids and insulin. Our results show that glucose stimulates protein synthesis in longissimus dorsi and gastrocnemius muscles, which contain primarily fast-twitch glycolytic fibers, but not in a muscle that contains primarily oxidative fibers, such as the masseter. The increase in muscle protein synthesis in these muscles is likely attributable to their unique metabolic properties. Glycolytic, fast-twitch muscles (white skeletal muscles) use glucose as their primary energy source during contraction, whereas red muscle fibers are richer in mitochondria and utilize various nutrients as an energy source (27, 40). Moreover, the stimulatory effect of glucose alone in muscles containing fast-twitch glycolytic fibers is consistent with previous studies demonstrating that protein synthesis is more responsive to anabolic agents in muscles that contain primarily fast-twitch glycolytic fibers than in those that contain primarily slowtwitch oxidative fibers (7, 9, 10, 17, 38). Fig. 6. Phosphorylation of protein S6 kinase (S6K1) on Thr 389 (A) and eukaryotic initiation factor (eif)4e-binding protein (4E-BP1) on Thr 70 (B) in LD muscle of neonatal pigs during pancreatic-substrate clamps. Values of the phosphorylation of S6K1 were normalized for S6K1 content in samples; values of the phosphorylation of 4E-BP1 were normalized for 4E-BP1 content in samples. Open bar, control group; gray bar, glucose group; black bar, glucose insulin AA group. Values are means SE in arbitrary densitometric units; n 8/treatment group. *Different from control group (P 0.05); different from glucose group (P 0.05).

6 E600 Fig. 7. Association of 4E-BP1 with eif4e (A) and association of the eif4g with eif4e to form the active eif4g eife complex (B) in LD muscle of neonatal pigs during pancreatic-substrate clamps. Open bar, control group; gray bar, glucose group; black bar, glucose insulin AA group. Values are means SE in arbitrary densitometric units; n 8/treatment group. *Different from control group (P 0.05); different from glucose group (P 0.05). Previous studies (7, 9, 35, 37) have shown that raising both insulin and amino acids to fed levels increases protein synthesis in several visceral tissues, such as pancreas, kidney, spleen, and liver, as well as in skeletal muscle. In the present study, raising glucose, together with insulin and amino acids, also increased protein synthesis in these tissues, and the rise in protein synthesis was similar to that which occurred with feeding. Our findings suggest that the postprandial rise in insulin, amino acids, and glucose independently mediates the increase in tissue protein synthesis after a meal in neonates. Regulation of muscle protein synthesis. To identify the mechanism involved in the stimulation of muscle protein synthesis by glucose alone, the activation of a number of signaling proteins that may be involved in nutrient signaling pathways was measured. Because in vitro studies have suggested that AMPK acts as an energy sensor within the cell (23, 25), we postulated that the glucose-induced increase in muscle protein synthesis may be mediated through AMPK. Studies conducted in C 2 C 12 myotubes have shown that an increase in cellular AMP levels increases AMPK activation by increasing AMPK phosphorylation on its Thr 72 residue (23, 25, 49). AMPK activation downregulates mtor signaling by phosphorylating TSC2 (49). TSC1/2 is a tumor suppressor complex that assimilates many signaling cascades to inactivate Rheb, a small GTPase, and thereby inhibits mtor-dependent cell growth (2, 28, 29). Cell culture studies (29) indicate that the TSC1/2 complex is regulated by both growth factors and amino acids. In the present study, we found that raising glucose alone to the fed state, or in the presence of raised amino acids and insulin levels, did not change the phosphorylation of AMPK on its Thr 72 residue. The lack of change in AMPK phosphorylation on Thr 72 in response to glucose stimulation raises the question as to whether AMPK acts as an energy sensor during periods of fasting (22, 29). Previous studies conducted in our laboratory (43) have shown that there is no change in AMPK activity in skeletal muscle in response to feeding and fasting in neonatal pigs. Thus, it is unclear at this time what role that AMPK has in the regulation of skeletal muscle protein synthesis during normal physiological conditions. In the present study, we also found that there was no change in the phosphorylation of TSC2 on Thr 1462 in response to a rise in glucose alone. However, in the presence of raised insulin, glucose, and amino acid levels, TSC2 phosphorylation on Thr 1462 was increased. This supports our previous studies (43), which showed that feeding stimulates phosphorylation of TSC2 in skeletal muscle of neonatal pigs. Phosphorylation of PKB on Ser 473 in response to growth factor stimulation has been demonstrated (45) to increase the phosphorylation of TSC2 on Thr 1462, resulting in the downregulation of TSC1/2 complex activity and the upregulation of mtor activity. In the present study, there was an increase in phosphorylation of PKB in those animals whose glucose was raised to fed levels while maintaining insulin and amino acids at fasting levels. This increase in PKB phosphorylation was not mediated by insulin because there was no change in phosphorylation of tyrosine residues on the insulin receptor in those animals in which glucose alone was raised to fed levels. The increase in PKB phosphorylation with glucose stimulation was further enhanced when insulin and amino acids were also raised to fed levels, consistent with our previous finding that feeding and insulin increases PKB phosphorylation in skeletal muscle of neonatal pigs (18, 42, 44). Fig. 8. Phosphorylation of eukaryotic elongation factor 2 (eef2) on Thr 56 in LD muscle of neonatal pigs during pancreatic-substrate clamps. Values of the phosphorylation of eef2 were normalized for eef2 content in samples. Open bar, control group; gray bar, glucose group; black bar, glucose insulin AA group. Values are means SE in arbitrary densitometric units; n 8/treatment group.

7 E601 mtor is a major protein kinase that modulates translation initiation components (32, 47). The phosphorylation of mtor on Ser 2448 activates the kinase and is stimulated by both insulin and amino acids (34, 36). In this study, there was no change in mtor phosphorylation on Ser 2448 in those animals whose circulating glucose concentrations were raised to fed levels. These results suggest that mtor does not play a role in the regulation of skeletal muscle protein synthesis by glucose in fast-twitch glycolytic muscles. However, in response to a rise in circulating glucose, amino acids, and insulin levels, phosphorylation of mtor on Ser 2448 increased, consistent with our previous findings of enhanced mtor activation in muscle of neonatal pigs in response to feeding (43). Factors downstream of mtor. Phosphorylation status of S6K1 is primarily mtor dependent, as is that for the repressor protein, 4E-BP1 (31, 47). Previous studies done by our laboratory (36) have shown that raising insulin alone increases phosphorylation of S6K1 through activation of mtor. Phosphorylation of S6K1 results in hyperphosphorylation of the ribosomal protein rps6. This hyperphosphorylation of rps6 (31, 47) assists in the translation of mrna containing terminal oligopyrimidine tracts at the 5 end, which encodes translation apparatus. In this study, we supported our previous work (36) showing that insulin and amino acids increase S6K1 phosphorylation; however, there was no effect on phosphorylation of S6K1 in those animals whose glucose alone was raised. This supports the assertion that the increase in protein synthesis in fast-twitch glycolytic muscle by glucose is not mediated by an mtor-dependent pathway. The binding of the translation repressor protein, 4E-BP1, to eif4e acts as an important regulator of the mrna-binding process (30, 47). Phosphorylation of 4E-BP1 on its Thr 70 site decreases its affinity for eif4e, thus freeing eif4e to bind with eif4g, and reduces the inactive 4E-BP1 eif4e complex formation. In the present study, raising glucose alone did not alter the phosphorylation of 4E-BP1 or the association of 4E-BP1 with eif4e. However, raising glucose, insulin, and amino acid levels increased 4E-BP1 phosphorylation and promoted the dissociation of 4E-BP1 from eif4e. These findings are consistent with our previous work demonstrating that the postprandial rise in insulin and amino acids mediate the feedinginduced increase in 4E-BP1 phosphorylation and dissociation of 4E-BP1 from eif4e. Formation of the active eif4e eif4g complex enables mrna to bind to the 43S preinitiation complex, thereby enhancing translation initiation (47). In the present study, a rise in glucose alone increased the formation of the active eif4e eif4g complex, although there was no change in 4E-BP1 or S6K1 phosphorylation on Thr 389. The increased eif4e eif4g formation with glucose stimulation was further enhanced when insulin and amino acids were also raised in the present study. Previous work from our laboratory (14, 17, 35, 36) also showed that feeding, amino acids, or insulin also promote eif4e eif4g assembly. Furthermore, we (17) recently demonstrated that the infusion of leucine to fed levels increases eif4e eif4g formation. Increased muscle protein synthesis rates and eif4g eif4e formation without changes in 4E-BP1 and S6K1 phosphorylation has also been reported by others in response to leucine in vivo (3). Together, these results suggest that nutrient-induced increases in muscle protein synthesis can be regulated independently of mtor-mediated signaling. Collectively, our work suggests that the feeding-induced increase in muscle protein synthesis in the neonate is mediated by the postprandial rise in insulin, amino acids, and glucose and involves both mtor-dependent and mtor-independent pathways. To examine whether stimulation of elongation factors plays a role in the stimulation of protein synthesis by glucose, phosphorylation of eukaryotic elongation factor 2 (eef2) was examined. Phosphorylation of eef2 on its Thr 56 site inhibits the elongation step of translation (6). mtor and S6K1 are thought to modulate the activity of eef2 kinase by phosphorylation. Phosphorylation of eef2 kinase increases phosphorylation of eef2 on its Thr 56 residue, thus downregulating eef2 and ultimately decreasing ribosomal translocation during peptide elongation. Interestingly, there was no change in phosphorylation of eef2 in response to glucose, amino acid, and insulin stimulation, suggesting that the initiation rather than the elongation step in translation plays the primary role in regulating protein synthesis in response to feeding. Perspectives. Our previous work has shown that insulin and amino acids act independently to stimulate protein synthesis in skeletal muscle of the neonate, when glucose is maintained at fasting levels. Herein, we demonstrate for the first time in vivo that glucose can also act independently of amino acids and insulin to stimulate muscle protein synthesis. This effect occurs in fast-twitch glycolytic muscles and is enhanced with the addition of insulin and amino acids. Furthermore, the glucoseinduced increase in muscle protein synthesis appears to occur via mtor- and AMPK-independent pathways that increase the formation of the active eif4e eif4g complex. The results highlight the multiple nutrient factors that affect protein synthesis and thus growth in young animals. Our results also emphasize the importance of a balanced carbohydrate- and protein-containing diet in the growth of skeletal muscle in the neonate. ACKNOWLEDGMENTS We thank W. Liu, J. Fleming, and M. Ng for technical assistance, D. Miller and J. Stubblefield for care of animals, E. O. Smith for statistical assistance, A. Gillum for graphics, and L. Weiser for secretarial assistance. Present address for J. Escobar: Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA GRANTS This work is a publication of the US 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 Institute of Arthritis and Musculoskeletal and Skin Diseases Grant R01-AR and the US Department of Agriculture, Agricultural Research Service, under Cooperative Agreement No This research was also supported in part by National Institute of Child Health and Human Development Training Grants T32-HD and K12-HD and National Institute of Arthritis and Musculoskeletal and Skin Diseases-mentored award K08-AR A1. 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