Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs

Teresa A. Davis, Marta L. Fiorotto, Douglas G. Burrin, Peter J. Reeds, Hanh V. Nguyen, Philip R. Beckett, Rhonda C. Vann, and Pamela M. J. O'Connor

United States Department of Agriculture/Agricultural Research Service, Children's Nutrition Research Center, and Endocrinology and Metabolism Section, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In neonatal pigs, the feeding-induced stimulation of protein synthesis in skeletal muscle, but not liver, can be reproduced by insulin infusion when essential amino acids and glucose are maintained at fasting levels. In the present study, 7- and 26-day-old pigs were studied during 1) fasting, 2) hyperinsulinemic-euglycemic-euaminoacidemic clamps, 3) euinsulinemic-euglycemic-hyperaminoacidemic clamps, and 4) hyperinsulinemic-euglycemic-hyperaminoacidemic clamps. Amino acids were clamped using a new amino acid mixture enriched in nonessential amino acids. Tissue protein synthesis was measured using a flooding dose of L-[4-3H]phenylalanine. In 7-day-old pigs, insulin infusion alone increased protein synthesis in various skeletal muscles (from +35 to +64%), with equivalent contribution of myofibrillar and sarcoplasmic proteins, as well as cardiac muscle (+50%), skin (+34%), and spleen (+26%). Amino acid infusion alone increased protein synthesis in skeletal muscles (from +28 to +50%), also with equivalent contribution of myofibrillar and sarcoplasmic proteins, as well as liver (+27%), pancreas (+28%), and kidney (+10%). An elevation of both insulin and amino acids did not have an additive effect. Similar qualitative results were obtained in 26-day-old pigs, but the magnitude of the stimulation of protein synthesis by insulin and/or amino acids was lower. The results suggest that, in the neonate, the stimulation of protein synthesis by feeding is mediated by either amino acids or insulin in most tissues; however, the feeding-induced stimulation of protein synthesis in skeletal muscle is uniquely regulated by both insulin and amino acids.

insulin action; nutrition; growth; liver; translation initiation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DURING THE NEONATAL PERIOD, the gain in protein mass in skeletal muscle is more rapid than in other tissues of the body (63). Fractional rates of protein synthesis in skeletal muscle of the newborn are high and decline more rapidly than in other tissues of the body during the early postnatal period (12, 18). This developmental decline in skeletal muscle protein synthesis varies with fiber type and is accompanied by a decrease in ribosome number and a specific accumulation of myofibrillar proteins (18, 25).

The efficiency with which dietary amino acids are utilized for protein deposition is also high in the neonate and declines markedly with development (20, 40). This high efficiency is likely due to the enhanced stimulation of protein synthesis in response to feeding (12, 15, 17, 19). The feeding-induced stimulation of protein synthesis occurs in virtually all tissues of the neonate; however, the postprandial rise in protein synthesis and the developmental decline in the response to feeding are most pronounced in skeletal muscle, particularly in those muscles that are primarily fast twitch, glycolytic (8-10, 12, 15, 17, 19). The developmental decline in the response to feeding in muscle parallels the marked fall in the nutrient-induced activation of translation initiation factors that regulate the binding of mRNA to the 40S ribosomal subunit (21).

Insulin is likely a central regulatory factor in the elevated rate of muscle protein deposition in the neonate. Results of studies, by use of the hyperinsulinemic-euglycemic-euaminoacidemic clamp technique originally developed in our laboratory, show that insulin stimulates whole body amino acid disposal in the neonate and that both the insulin sensitivity and the responsiveness of amino acid disposal are attenuated with the advancement of development (61). When insulin is infused at doses reproducing plasma insulin levels observed under fed conditions and essential amino acids and glucose are clamped at fasting levels, skeletal muscle protein synthesis increases to values observed in the fed state (62). The stimulation of protein synthesis by insulin, like the response to feeding, is greater in muscles that are predominantly fast twitch glycolytic and is not specific to myofibrillar proteins (14). This response to insulin decreases with development (14, 62), in parallel with the developmental decline in the stimulation of muscle protein synthesis by feeding (12). Recent studies suggest that the developmental decline in the response of skeletal muscle protein synthesis to food consumption results from a reduction in the effectiveness of the insulin-signaling pathway to transduce to the translation apparatus the stimulus provided by feeding (35, 37, 53).

A recent study from this laboratory (14) suggests that the feeding-induced stimulation of visceral tissue protein synthesis in the neonate cannot be attributed to insulin. We found that the infusion of insulin in the neonate stimulates protein synthesis in skeletal muscle, cardiac muscle, and skin, but insulin did not stimulate, and in fact tended to lower, protein synthesis in liver, intestine, pancreas, and kidney (14). The ability of amino acids to stimulate liver protein synthesis is controversial, but limited studies suggest that amino acids increase protein synthesis in the liver of immature (27, 51) but not mature (3, 44) animals. In contrast, amino acids appear to stimulate protein synthesis in the skeletal muscle of young, adult, and elderly populations (6, 41, 49, 58, 59). Therefore, we hypothesized that protein synthesis in all tissues of the neonate is responsive to the postprandial rise in amino acid concentrations.

During the hyperinsulinemic-euglycemic-euaminoacidemic clamps in previous studies, we infused Trophamine, an amino acid mixture designed for use in neonates (14, 61, 62). However, in these studies, detailed examination of amino acid concentrations during "euaminoacidemic"-hyperinsulinemic-euglycemic clamps showed that the circulating concentrations of some of the nonessential amino acids were lower at the highest insulin concentrations, which could potentially limit the availability of substrate for protein synthesis. Therefore, we developed a new amino acid clamp mixture designed specifically to keep the concentrations of all amino acids constant during insulin stimulation. We determined, in 7- and 26-day-old pigs, the response of tissue protein synthesis to 1) the infusion of insulin at a dose that reproduces insulin levels in the fed state while circulating amino acids and glucose are maintained at fasting levels, 2) the infusion of amino acids at a dose that reproduces fed-state amino acid levels while insulin and glucose are kept at fasting levels, and 3) the infusion of insulin and amino acids at doses that reproduce insulin and amino acid levels in the fed state while glucose is kept at fasting level.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and surgery. Multiparous crossbred (Yorkshire × Landrace × Hampshire × Duroc) sows (n = 8; 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 a 28-day lactation period. Sows delivered 8-12 piglets, and these remained with the sows for nursing. Piglets were not given supplemental creep feed. Piglets weighing 2.0 ± 0.1 kg were studied at 7 days of age (n = 35), and piglets weighing 7.2 ± 0.1 kg were studied at 26 days of age (n = 39). Three to five days before the insulin infusion study, pigs were anesthetized, and catheters were inserted surgically into a jugular vein and a carotid artery (61). Piglets were returned to the sows until studied. The protocol 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.

Infusions. Pigs were placed unanesthetized in a sling restraint system after a 12-h fast. Pigs were assigned to one of four treatment groups: 1) fasting (Control), 2) hyperinsulinemic-euglycemic-euaminoacidemic clamp (Insulin), 3) euinsulinemic-euglycemic-hyperaminoacidemic clamp (AA), and 4) hyperinsulinemic-euglycemic-hyperaminoacidemic clamp (Insulin+AA). Body weights were similar in each treatment group at each age studied. During a 30-min basal period, blood samples were obtained and immediately analyzed for glucose (YSI 2300 STAT Plus, Yellow Springs Instruments, Yellow Springs, OH) to establish the average basal concentration of blood glucose to be used in the subsequent euglycemic clamp procedure (22). Plasma samples were analyzed for total branched-chain amino acids (BCAA), by use of a rapid enzymatic kinetic assay (5) to establish the average basal concentration of BCAA to be used in the subsequent euaminoacidemic or hyperaminoacidemic clamp procedure. The clamps were initiated with a primed, constant (12 ml/h) infusion of insulin (Eli Lilly, Indianapolis, IN) at either 0 or 100 ng · kg-0.66 · min-1. Venous blood samples (0.2 ml) were acquired every 5 min and immediately analyzed for glucose and BCAA concentrations. The infusion rate of dextrose (Baxter Healthcare, Deerfield, IL) was adjusted as necessary to maintain the blood glucose concentration within ±10% of the average basal concentration. Euaminoacidemia was obtained by adjusting the infusion rate of an amino acid mixture to maintain the plasma BCAA concentration within 10% of the fasting level. Hyperaminoacidemia was obtained by infusing an amino acid mixture to raise plasma BCAA concentrations to twofold the fasting level to reproduce the level of amino acids present in the fed state (12).

A new amino acid mixture was developed for use as an infusate during the euaminoacidemic and hyperaminoacidemic clamp procedures. The composition of the new amino acid mixture was based on the amino acid composition of body protein (20) and our previous observations on the changes in plasma amino acid concentrations when 10% Trophamine (McGaw, Irvine, CA) was infused during hyperinsulinemic-euglycemic-euaminoacidemic clamp studies (14, 61, 62). The new mixture utilized two dipeptides, glycyl-tyrosine and alanyl-glutamine, to avoid the problem of poor solubility of tyrosine and instability of glutamine and contained higher concentrations of nonessential amino acids than commercial amino acid mixtures. The new amino acid mixture contained (millimolar) 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).

Tissue protein synthesis in vivo. Fractional rates of protein synthesis were measured with a flooding dose of L-[4-3H]phenylalanine (29) injected 3.5 h after the initiation of the clamp. Blood samples were taken at 5, 15, and 30 min after the injection for measurement of the specific radioactivity of the extracellular free pool of phenylalanine. Pigs were killed at 4 h after the initiation of the clamp. Samples of longissimus dorsi, gastrocnemius, masseter, and cardiac muscles and skin, liver, jejunum, pancreas, kidney, and spleen were collected, rapidly frozen in liquid nitrogen, and stored at -70°C.

Total protein was isolated from all tissues sampled, and sarcoplasmic and myofibrillar proteins were isolated in longissimus dorsi muscle of 7-day-old pigs as described previously (18, 25). The specific radioactivities of the protein hydrolysates, homogenate supernatants, and blood supernatants were determined as previously described (18).

Plasma insulin and amino acids. Plasma immunoreactive insulin concentrations were measured using a porcine insulin radioimmunoassay kit (Linco, St. Louis, MO). Plasma amino acid concentrations were measured with a high-performance liquid chromatography method (PICO-TAG reverse-phase column, Waters, Milford, MA) as previously described (19).

Calculations and statistics. Fractional rates of protein synthesis (Ks; percent protein mass synthesized in a day) were calculated as
K<SUB>s</SUB> (<IT>%/</IT>day)<IT>=</IT>[(S<SUB>b</SUB>/S<SUB>a</SUB>)<IT>×</IT>(1,440<IT>/t</IT>)]<IT>×</IT>100
where Sb is the specific radioactivity of the protein-bound phenylalanine, Sa is the specific radioactivity of the tissue free phenylalanine for the labeling period determined from the value of the animal at the time of the tissue collection, corrected by the linear regression of the blood specific radioactivity of the animal against time, and t is the time of labeling in minutes. We have demonstrated that, after a flooding dose of 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 (16).

Three-way analysis of variance was used to assess the effect of insulin, amino acids, age, and their interaction on tissue protein synthesis. When significant interactions were detected, the value in each treatment group for each age was compared with the control value by use of t-tests. To determine the effectiveness of the clamp procedure, individual amino acid, glucose, and insulin concentrations in each treatment group were compared with their basal concentrations by use of t-tests. Differences of P < 0.05 were considered statistically significant for all comparisons except those for plasma amino acid concentrations. Because there was an increased probability that one of the 22 amino acid comparisons in each of the four treatment groups would be significantly different among groups by random chance, a more conservative statistical approach for amino acid comparisons was used; therefore, probability values of <0.01 were considered statistically different. Results are presented as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Infusions. Pigs at 7 and 26 days of age were infused with insulin, glucose, amino acids, and/or saline to reproduce 1) fasting insulin, glucose, and amino acid levels (Control group); 2) fed insulin, fasting glucose, and fasting amino acid levels (hyperinsulinemic-euglycemic-euaminoacidemic clamp; Insulin group); 3) fasting insulin, fasting glucose, and fed amino acid levels (euinsulinemic-euglycemic-hyperaminoacidemic clamp; AA group); and 4) fed insulin, fasting glucose, and fed amino acid levels (hyperinsulinemic-euglycemic-hyperaminoacidemic clamp; Insulin+AA group). Table 1 shows that the circulating insulin concentrations normally found in the neonatal pig in the fasting condition and after a meal (3 and 30 µU/ml, respectively ) were largely reproduced in both 7- and 26-day-old pigs by the infusion of 0 and 100 ng · kg-0.66 · min-1. Hyperaminoacidemia did not alter circulating insulin concentrations. Plasma glucose concentrations were maintained at basal fasting levels during the infusion of insulin and/or amino acids in 7- and 26-day-old pigs (Table 1).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Plasma insulin and glucose concentrations in response to insulin and/or amino acid infusion in 7- and 26-day-old pigs

Circulating essential and nonessential amino acid concentrations in control and in insulin- and/or amino acid-infused 7- and 26-day-old pigs are compared with baseline (0 time) values in Fig. 1. During the 4-h saline infusion period in control pigs, plasma amino acid concentrations remained stable in 7-day-old pigs, although leucine, isoleucine, and lysine increased and alanine and glutamate decreased in 26-day-old pigs (P < 0.01). Because circulating nonessential amino acid concentrations decreased in our previous hyperinsulinemic-euglycemic-euaminoacidemic clamp studies, which utilized Trophamine as the amino acid infusate (14, 62), in the present study, we developed a new amino acid mixture, which contains higher concentrations of nonessential amino acids than Trophamine. The circulating concentrations of both nonessential and essential amino acids were maintained largely at the fasting level during the hyperinsulinemic-euglycemic-euaminoacidemic clamp with the new amino acid infusate. The exceptions were elevations in histidine and tryptophan and a reduction in asparagine (P < 0.01). Hyperaminoacidemic clamps in the presence of euinsulinemia or hyperinsulinemia increased the circulating concentrations of essential amino acids about twofold and those of nonessential amino acids ~50% (P < 0.01).


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 1.   Plasma concentrations of essential and nonessential amino acids during Control, hyperinsulinemic-euglycemic-euaminoacidemic (Insulin), euinsulinemic-euglycemic-hyperaminoacidemic (AA), and hyperinsulinemic-euglycemic-hyperaminoacidemic (Insulin+AA) clamps in 7- and 26-day-old pigs. Values are means ± SE; n = 7-12 per age and treatment group. * Amino acid values statistically significantly different from basal values (P < 0.01).

Figure 2 shows the net whole body amino acid and glucose disposal rates, as indicated by the amino acid and glucose infusion rates during the last hour of the infusion period. Amino acids were not infused in the control animals, because plasma amino acid concentrations did not fall below 10% of the basal level. During the infusion of either insulin or amino acids, the amino acid disposal rates were about twofold greater in 7- than in 26-day-old pigs (P < 0.01), but the glucose disposal rates did not differ significantly with age. The effects of insulin and amino acids on amino acid disposal were additive.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2.   Whole body net amino acid (A) and glucose (B) disposal rates during Control, Insulin, AA, and Insulin+AA clamps in 7- and 26-day-old pigs. Values are means ± SE; n = 7-12 per age and treatment group. Amino acid disposal rate was the average rate of infusion of total amino acids (TAA) from an amino acid mixture necessary to maintain circulating branched-chain amino acids near either the basal fasting level or twofold the fasting level from 180 to 240 min of infusion. Amino acids were not infused in control animals. Amino acid disposal rates were greater in 7- than in 26-day-old pigs (P < 0.01). * Disposal rates statistically significantly different from control values (P < 0.05).

Tissue protein synthesis. Fractional rates of protein synthesis in skeletal muscles of different fiber types in 7- and 26-day-old pigs are shown in Fig. 3. Under fasting conditions, the fractional rates of protein synthesis in longissimus dorsi, gastrocnemius, and masseter muscles were about threefold, twofold, and 50% higher in 7- than in 26-day-old pigs, respectively (P < 0.001). Hyperinsulinemia increased (P < 0.005) protein synthesis in longissimus dorsi (57%), gastrocnemius (64%), and masseter (36%) muscles, and these responses decreased with age (P < 0.001). Hyperaminoacidemia also increased (P < 0.01) protein synthesis in longissimus dorsi (48%), gastrocnemius (50%), and masseter (28%) muscles (P < 0.01). The amino acid response also decreased with age (P < 0.05). In contrast to whole body amino acid disposal, the combination of hyperinsulinemia and hyperaminoacidemia did not exert an additive effect on muscle protein synthesis (P > 0.05).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Fractional protein synthesis rates (Ks) in longissimus dorsi (A), gastrocnemius (B), and masseter (C) muscles in 7- and 26-day-old pigs during Control, Insulin, AA, and Insulin+AA clamps. Values are means ± SE; n = 7-12 per age and treatment group. Protein synthesis decreased with age in longissimus dorsi, gastrocnemius, and masseter muscles (P < 0.001). Hyperinsulinemia increased protein synthesis in all muscles (P < 0.005), and the response decreased with age (P < 0.001). Hyperaminoacidemia increased protein synthesis in all muscles (P < 0.01), and the response decreased with age (P < 0.05). * Protein synthesis rates statistically significantly different from control rates (P < 0.05).

To determine whether the stimulation of skeletal muscle protein synthesis by insulin and/or amino acids is specific to myofibrillar proteins, protein synthesis rates in myofibrillar and sarcoplasmic proteins were determined in longissimus dorsi muscle of 7-day-old pigs in the four treatment groups. Hyperinsulinemia and hyperaminoacidemia, either alone or in combination, increased protein synthesis in both myofibrillar and sarcoplasmic protein fractions to a similar extent (60-80%; Fig. 4). Rates of protein synthesis did not differ between the two protein fractions in any treatment group.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4.   Ks in total (TP), myofibrillar (MP), and sarcoplasmic proteins (SP) in longissimus dorsi muscle of 7-day-old pigs during Control, Insulin, AA, and Insulin+AA clamps. Values are means ± SE; n = 7-12 per age and treatment group. Hyperinsulinemia and hyperaminoacidemia increased protein synthesis in all fractions (P < 0.05). * Protein synthesis rates statistically significantly different from control rates (P < 0.05).

Fractional rates of protein synthesis in cardiac muscle, skin, and spleen of 7- and 26-day-old pigs are shown in Fig. 5. The fractional rates of protein synthesis in cardiac muscle, skin, and spleen in the basal fasting condition were 15, 90, and 30% higher in 7- than in 26-day-old pigs, respectively (P < 0.01). Hyperinsulinemia increased protein synthesis in cardiac muscle (50%; P < 0.001), skin (34%; P < 0.001), and spleen (26%; P < 0.05), and these responses to insulin decreased with age (P < 0.05). Hyperaminoacidemia had no effect on protein synthesis in these tissues. The combination of hyperinsulinemia and hyperaminoacidemia had no greater effect on protein synthesis than the effect of hyperinsulinemia alone (P > 0.05).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.   Ks in cardiac muscle (A), skin (B), and spleen (C) in 7- and 26-day-old pigs during Control, Insulin, AA, and Insulin+AA clamps. Values are means ± SE; n = 7-12 per age and treatment group. Protein synthesis decreased with age in cardiac muscle, skin, and spleen (P < 0.001). Hyperinsulinemia increased protein synthesis in cardiac muscle (P < 0.001), skin (P < 0.001), and spleen (P < 0.05), and the responses in cardiac muscle (P < 0.005), skin (P < 0.05), and spleen (P < 0.05) decreased with age. * Protein synthesis rates statistically significantly different from control rates (P < 0.05).

Fractional rates of protein synthesis in liver, kidney, and pancreas of 7- and 26-day-old pigs are shown in Fig. 6. The fractional rate of protein synthesis in liver in the basal fasting condition was 28% higher in 7- than in 26-day-old pigs (P < 0.001). Hyperinsulinemia had no effect on protein synthesis in any of these organs (P > 0.05). Nevertheless, an elevation in circulating amino acids increased protein synthesis in liver (27%; P < 0.001), pancreas (28%; P < 0.05), and kidney (10%; P < 0.005), and the response in liver decreased with age (P < 0.05). The combination of hyperinsulinemia and hyperaminoacidemia had no greater effect on protein synthesis in these tissues than the effect of hyperaminoacidemia alone (P > 0.05).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 6.   Ks in liver (A), kidney (B), and pancreas (C) of 7- and 26-day-old pigs during Control, Insulin, AA, and Insulin+AA clamps. Values are means ± SE; n = 7-12 per age and treatment group. Protein synthesis decreased with age in liver (P < 0.001). Hyperaminoacidemia increased protein synthesis in liver (P < 0.001), kidney (P < 0.005), and pancreas (P < 0.05), and the response in liver decreased with age (P < 0.05). * Protein synthesis rates statistically significantly different from control rates (P < 0.05).

The fractional rates of protein synthesis in the jejunum of 7- and 26-day-old pigs are shown in Fig. 7. The fractional rate of protein synthesis in jejunum was higher in 7- than in 26-day-old pigs (P < 0.001). Neither hyperinsulinemia nor hyperaminoacidemia significantly altered the rate of protein synthesis in the jejunum (P > 0.05).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 7.   Ks in jejunum of 7- and 26-day-old pigs during Control, Insulin, AA, and Insulin+AA clamps. Values are means ± SE; n = 7-12 per age and treatment group. Protein synthesis decreased with age in jejunum (P < 0.001).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Regulation of skeletal muscle protein synthesis by insulin and amino acids. Current and previous studies suggest that insulin plays a key role in the elevated rate of protein deposition and the increased response of skeletal muscle protein synthesis to feeding in the neonate. First, postprandial changes in protein synthesis in neonatal pigs are positively correlated with changes in circulating insulin concentrations (13). Second, when amino acids and glucose are maintained near fasting levels, insulin infusion increases amino acid disposal in the neonatal pig and ovine fetus (57, 61). The insulin sensitivity and responsiveness of amino acid disposal decrease with development. Third, raising insulin concentrations in the neonatal pig to levels typical of the fed state increases the rate of skeletal muscle protein synthesis to within the range normally present in the fed state, even when essential amino acids and glucose are maintained at fasting levels (21, 62). This response to insulin, like the response to feeding, is attenuated with development. Fourth, insulin infusion stimulates whole body and skeletal muscle protein synthesis in the fetal sheep, young lamb, and weaned rat (28, 38, 60) but has little, if any, effect in the adult human or rat (4, 30, 32, 39, 41). These findings suggest that the ability of skeletal muscle protein synthesis to respond to the postprandial rise in insulin wanes with development. Fifth, the developmental decline in the feeding-induced activation of the insulin-signaling pathway leading to translation initiation parallels the developmental decline in feeding- and insulin-stimulated protein synthesis (21, 35, 53). This suggests that the high activity of the insulin-signaling pathway contributes to the increased response of muscle protein synthesis rates to feeding in the neonate.

In the present study, we used an amino acid clamp technique to prevent the drop in circulating amino acid concentrations that occurs when amino acids are not infused concurrently with insulin. Because the circulating concentrations of many nonessential amino acids decreased at the highest insulin concentrations in our previous studies (14, 62), in the present study, we developed a new amino acid mixture that contained higher concentrations of nonessential amino acids than the Trophamine used previously. Using this new amino acid infusate, we maintained almost all nonessential amino acids, as well as essential amino acids, at the fasting level during the infusion of insulin. Critically, however, the stimulation of skeletal muscle protein synthesis by insulin was similar in the present and previous studies (14, 62) and, indeed, was similar to the response to feeding (12). This suggests that maintenance of nonessential amino acid concentrations is not required for the insulin-induced stimulation of skeletal muscle protein synthesis.

To determine the role of amino acids in the postprandial rise in protein synthesis, the new amino acid mixture was infused to raise circulating amino acid concentrations to near the fed level. Importantly, circulating concentrations of insulin, measured every 30 min during the 4-h infusion, did not increase in response to amino acid infusion. This suggests that the physiological rise in amino acid concentrations stimulated protein synthesis in skeletal muscle in the absence of a rise in insulin concentration. Although the combined infusion of amino acids and insulin increased protein synthesis, the increase was similar to that obtained during amino acid or insulin stimulation alone. This implies that insulin and amino acids may be interacting with the same signaling pathway within skeletal muscle. This idea is supported by the observation that the muscle protein synthesis response to insulin and amino acids decreased with development, in parallel with the developmental decline in the feeding-induced stimulation of skeletal muscle protein synthesis. In young, adult, and elderly populations, amino acid infusion, either alone or concurrent with insulin infusion, stimulates protein synthesis in skeletal muscle (6, 41, 49, 58, 59). This suggests that the ability of amino acids to stimulate skeletal muscle protein synthesis wanes, but is not lost, with age.

The results of the present study further show that the stimulation of muscle protein synthesis by amino acids and/or insulin was greater in those muscles containing predominantly fast-twitch, glycolytic muscle fibers. The response to both anabolic agents was greater in longissimus dorsi and gastrocnemius muscles, which contain fast-twitch fibers that are predominantly glycolytic, than in the masseter muscle, which is composed entirely of oxidative fibers. These findings are consistent with our previously reported results (19, 21) and those of others (4, 36), demonstrating that protein synthesis is more responsive to anabolic agents in muscles composed of predominantly fast fibers than in muscles of slow oxidative fibers. Furthermore, the greater developmental decline in fractional protein synthesis rates in fast-twitch glycolytic than in slower oxidative muscles in the present study is consistent with our previously reported findings in pig and rat muscles (18, 14). Because the skeletal muscles that we studied in the neonatal pig have predominantly fast-twitch contractile characteristics but different oxidative and glycolytic properties (1, 47, 54), the differences in protein synthesis rates between the muscles that we observed are most likely attributable to the distinct differences in the muscles' metabolic rather than twitch characteristics.

The results of the present study demonstrate that hyperinsulinemia and hyperaminoacidemia, alone or in combination, stimulate both myofibrillar and sarcoplasmic protein synthesis. In addition, the degree of stimulation by these agents was proportional. These results are consistent with our previous studies, which showed that the synthesis of both protein fractions is increased similarly by milk feeding (26) and insulin infusion (14) in neonatal pigs. Furthermore, we have previously shown in the neonatal pig (14) that myofibrillar and sarcoplasmic protein synthesis rates are similar, as in the present study, and that the age-associated decline in the synthesis rates of the two protein fractions is proportional over this phase of development.

Regulation of tissue protein synthesis by either insulin or amino acids alone. Feeding increases protein synthesis in virtually all tissues of the neonatal pig (7, 8, 12, 15). In the present study, infusing insulin at a dose that reproduced plasma insulin levels in the fed state while maintaining glucose and essential and nonessential amino acids at the fasting level stimulated protein synthesis in cardiac muscle, skin, and spleen. However, in a previous study (14), infusing insulin at doses reproducing fed-state plasma insulin levels while keeping glucose and essential amino acids, but not nonessential amino acids, at fasting levels stimulated protein synthesis in cardiac muscle and skin but not spleen. This suggests that keeping nonessential amino acid concentrations at fasting levels is not required for the insulin-induced stimulation of protein synthesis in cardiac muscle or skin, although it may play a permissive role in spleen. Furthermore, an elevation in amino acids in the presence of either euinsulinemia or hyperinsulinemia had no effect on protein synthesis in cardiac muscle, skin, or spleen. Thus the results are consistent with the hypothesis that the postprandial rise in insulin, but not amino acids, largely mediates the stimulation of protein synthesis by feeding in the cardiac muscle, skin, and spleen of the neonate.

Insulin also appears to regulate protein synthesis in the cardiac muscle of young (48) but not adult (41) rats, consistent with the developmental decline in the insulin-induced stimulation of cardiac muscle protein synthesis observed in the present study. Amino acid infusion has no effect on cardiac muscle protein synthesis in the adult rat (41), as in the neonatal pigs of the present study, suggesting that cardiac muscle may be resistant to the anabolic effects of amino acids throughout the life cycle.

Our previous study (14) showed that insulin infusion does not stimulate, and in some cases reduces, protein synthesis in liver, intestine, pancreas, and kidney, despite maintenance of glucose and essential amino acids at fasting levels. In the present study, the infusion of physiological levels of insulin, when nonessential and essential amino acids and glucose were clamped at fasting levels, did not stimulate, but also did not reduce, protein synthesis in these tissues. This suggests that the reduction in protein synthesis in liver and intestine during the infusion of insulin in the previous study (14) was due to a reduction in substrate, i.e., nonessential amino acids or nitrogen. Moreover, the infusion of amino acids at doses reproducing fed-state amino acid levels increased protein synthesis in liver, pancreas, and kidney in the present study. These responses to amino acid infusion occurred in the presence of either euinsulinemia or hyperinsulinemia. The results are suggestive of an important role of amino acids, but not insulin, in the regulation of protein synthesis in the liver, pancreas, and kidney of the neonate.

Many (1, 44, 55), but not all (33, 34), studies report an inability of insulin to stimulate liver protein synthesis. However, none have reported a reduction in liver protein synthesis with insulin infusion. The reduction in liver protein synthesis in the previous insulin infusion study (14) may have been due to the more profound effect of insulin on circulating amino acids in neonatal animals, which have higher amino acid turnover rates than older animals (23, 61). Indeed, amino acids appear to stimulate liver protein synthesis in growing (27, 51), but not adult or elderly (3, 44), animals, consistent with the developmental decline in the amino acid-induced stimulation of liver protein synthesis in the present study.

In the present study, we found that the physiological elevation of insulin, amino acids, or both had no effect on protein synthesis in the jejunum. Insulin infusion in healthy adults (42) and in diabetics (46) reduces protein synthesis in splanchnic tissues, consisting primarily of gut and liver. However, insulin deprivation modestly reduces, and insulin treatment restores, protein synthesis rates in the small intestinal mucosa of diabetics (11), suggesting that insulin may be required for the maintenance of mucosal protein synthesis. Feeding stimulates intestinal protein synthesis in growing animals (9, 12, 15), but in the present study, amino acids were infused peripherally rather than enterally. Thus we postulate that supplying amino acids to the mucosa via the luminal rather than the arterial route is likely the most important modulator of the feeding-induced stimulation of intestinal protein synthesis. Indeed, previous studies in neonatal pigs (52) have shown that, in the fed state, dietary amino acids make a greater contribution than arterial amino acids to jejunal protein synthesis.

Whole body amino acid and glucose disposal. Previous studies (61) showed that insulin stimulates the utilization of amino acids for protein deposition in the whole body of the neonatal pig and that both the insulin sensitivity and responsiveness of amino acid disposal decrease with development. The present study extends our previous work to show a marked developmental decline in insulin-stimulated amino acid disposal in the presence of either fasted- or fed-state amino acid levels. Furthermore, the amount of amino acids required to raise amino acid concentrations to near the fed state in the presence of either fasting- or fed-state insulin levels also decreased with development. This developmental decrease in amino acid disposal in response to hyperinsulinemia and/or hyperaminoacidemia paralleled the developmental decline in the ability of these anabolic agents to stimulate protein synthesis in most tissues. We have recently shown a marked developmental decline in both insulin receptor abundance and the feeding-induced activation of the insulin receptor in skeletal muscle of the neonate (53). This developmental change in the activation of the insulin receptor is propagated down the insulin-signaling pathway to many of the eukaryotic initiation factors that regulate translation (21, 35). Furthermore, the feeding-induced stimulation of muscle protein synthesis is dependent on activation of a protein kinase, mammalian target of rapamycin or mTOR (37), which has been shown in vitro to be important in mediating the signal generated by both amino acids and insulin (31, 50). The lack of developmental decline in insulin-stimulated glucose disposal in the presence of either euaminoacidemia or hyperaminoacidemia is surprising and implies differential developmental regulation of these two insulin-stimulated metabolic responses.

Perspectives. Feeding stimulates whole body protein synthesis in the newborn infant, but the effect is much smaller in the adult (24, 43, 56), implying that different mechanisms regulate protein synthesis in adults and neonates. Studies using the neonatal pig as an animal model of the human infant (45) have shown that feeding stimulates protein synthesis in virtually all tissues of the body of the neonate, a response that decreases with development (12). The results of the present study strongly support the hypothesis that the feeding-induced stimulation of protein synthesis in skeletal muscle is mediated by the postprandial rise in both insulin and amino acids. Protein synthesis in cardiac muscle, skin, and spleen is responsive to insulin but not amino acid stimulation, whereas liver, kidney, and pancreas are responsive to amino acid but not insulin stimulation. Because neither insulin nor amino acid infusion altered intestinal protein synthesis, we postulate that amino acid stimulation by the luminal rather than arterial route may be required for the feeding-induced stimulation of intestinal protein synthesis. Thus it appears that the stimulation of protein synthesis by feeding is mediated by either insulin or amino acids in most tissues of the neonate, but in skeletal muscle, the feeding-induced stimulation of protein synthesis is regulated by both insulin and amino acids. The ability of skeletal muscle to respond to the postprandial rise in two anabolic stimuli likely contributes to the efficient utilization of nutrients for growth in the neonate and the more rapid gain in protein mass in skeletal muscle than in other tissues of the body in the neonate.


    ACKNOWLEDGEMENTS

We thank W. Liu, J. Rosenberger, J. Henry, and X. Chang for technical assistance, J. Cunningham and F. Biggs for care of animals, E. O. Smith for statistical assistance, L. Loddeke for editorial assistance, A. Gillum for graphics, and J. Croom for secretarial assistance. We acknowledge Eli Lilly for the generous donation of porcine insulin.


    FOOTNOTES

This work is a publication of the US Department of Agriculture, Agricultural Research Service (USDA/ARS) Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, Texas. This project has been funded in part by National Institute of Arthritis and Musculoskeletal and Skin Diseases Institute Grant R01 AR-44474 and the USDA/ARS under Cooperative Agreement no. 58-6250-6-001. This research was also supported in part by National Institutes of Health 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.

Present address of P. J. Reeds: University of Illinois, Dept. of Animal Sciences, 1207 W. Gregory, Rm. 432, Urbana, IL 61801. Present address of R. Vann: Central Mississippi Research and Extension Center, Mississippi State University, 1320 Seven Springs Rd., Raymond, MS 39154.

Address for reprint requests and other correspondence: T. A. Davis, USDA/ARS Children's Nutrition Research Center, Baylor College of Medicine, 1100 Bates St., Houston, TX 77030 (E-mail: tdavis{at}bcm.tmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00517.2001

Received 16 November 2001; accepted in final form 18 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahlman, B, Charlton M, Fu A, Berg C, O'Brian P, and Nair KS. Insulin's effect on synthesis rates of liver proteins. A swine model comparing various precursors of protein synthesis. Diabetes 50: 947-954, 2001[Abstract/Free Full Text].

2.   Anapol, F, and Herring SW. Ontogeny of histochemical fiber types and muscle function in the masseter muscle of miniature swine. Am J Anthropol 112: 595-613, 2000[ISI][Medline].

3.   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 131: 1171-1176, 2001[Abstract/Free Full Text].

4.   Baillie, AGS, and Garlick PJ. Attenuated responses of muscle protein synthesis to fasting and insulin in adult female rats. Am J Physiol Endocrinol Metab 262: E1-E5, 1992[Abstract/Free Full Text].

5.   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[ISI][Medline].

6.   Bennet, WM, Connacher AA, Scrimgeour CM, and Rennie MJ. The effect of amino acid infusion on leg protein turnover assessed by L-[15N]phenylalanine and L-[1-13C]leucine exchange. Eur J Clin Invest 20: 41-50, 1990[ISI][Medline].

7.   Burrin, DG, Davis TA, Ebner S, Schoknecht PA, Fiorotto ML, and Reeds PJ. Colostrum enhances the nutritional stimulation of vital organ protein synthesis in neonatal pigs. J Nutr 127: 1284-1289, 1997[Abstract/Free Full Text].

8.   Burrin, DG, Davis TA, Ebner S, Schoknecht PA, Fiorotto ML, Reeds PJ, and McAvoy S. Nutrient-independent and nutrient-dependent factors stimulate protein synthesis in colostrum-fed newborn pigs. Pediatr Res 37: 593-599, 1995[Abstract].

9.   Burrin, DG, Davis TA, Fiorotto ML, and Reeds PJ. Stage of development and fasting affect protein synthetic activity in the gastrointestinal tissues of suckling rats. J Nutr 121: 1099-1108, 1991[ISI][Medline].

10.   Burrin, DG, Davis TA, Fiorotto ML, and Reeds PJ. Hepatic protein synthesis in suckling rats: effects of stage of development and fasting. Pediatr Res 31: 247-252, 1992[Abstract].

11.   Charlton, M, Ahlman B, and Nair KS. The effect of insulin on human small intestinal mucosal protein synthesis. Gastroenterology 188: 299-306, 2000.

12.   Davis, TA, Burrin DG, Fiorotto ML, and Nguyen HV. Protein synthesis in skeletal muscle and jejunum is more responsive to feeding in 7- than in 26-day-old pigs. Am J Physiol Endocrinol Metab 270: E802-E809, 1996[Abstract/Free Full Text].

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

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

15.   Davis, TA, Fiorotto ML, Burrin DG, Pond WG, and Nguyen HV. Intrauterine growth restriction does not alter response of protein synthesis to feeding in newborn pigs. Am J Physiol Endocrinol Metab 272: E877-E884, 1997[Abstract/Free Full Text].

16.   Davis, TA, Fiorotto ML, Nguyen HV, and Burrin DG. Aminoacyl-tRNA and tissue free amino acid pools are equilibrated after a flooding dose of phenylalanine. Am J Physiol Endocrinol Metab 277: E103-E109, 1999[Abstract/Free Full Text].

17.   Davis, TA, Fiorotto ML, Nguyen HV, Burrin DG, and Reeds PJ. Response of muscle protein synthesis to fasting in suckling and weaned rats. Am J Physiol Regulatory Integrative Comp Physiol 261: R1373-R1380, 1991[Abstract/Free Full Text].

18.   Davis, TA, Fiorotto ML, Nguyen HV, and Reeds PJ. Protein turnover in skeletal muscle of suckling rats. Am J Physiol Regulatory Integrative Comp Physiol 257: R1141-R1146, 1989[Abstract/Free Full Text].

19.   Davis, TA, Fiorotto ML, Nguyen HV, and Reeds PJ. Enhanced response of muscle protein synthesis and plasma insulin to food intake in suckled rats. Am J Physiol Regulatory Integrative Comp Physiol 265: R334-R340, 1993[Abstract/Free Full Text].

20.   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: 947-956, 1993[ISI][Medline].

21.   Davis, TA, Nguyen HV, Suryawan A, Bush JA, Jefferson LS, and Kimball LS. 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, 2000[Abstract/Free Full Text].

22.   DeFronzo, RA, Tobin JD, and Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol Endocrinol Metab Gastrointest Physiol 237: E214-E223, 1979[Abstract/Free Full Text].

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

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

25.   Fiorotto, ML, Davis TA, and Reeds PJ. Regulation of myofibrillar protein turnover during maturation in normal and undernourished rat pups. Am J Physiol Regulatory Integrative Comp Physiol 278: R845-R854, 2000[Abstract/Free Full Text].

26.   Fiorotto, ML, Davis TA, Reeds PJ, and Burrin DG. Nonnutritive factors in colostrum enhance myofibrillar protein synthesis in the newborn pig. Pediatr Res 48: 511-517, 2000[Abstract/Free Full Text].

27.   Flaim, KE, Peavy DE, Everson WV, and Jefferson LS. The role of amino acids in the regulation of protein synthesis in perfused rat liver. J Biol Chem 257: 2932-2938, 1982[Abstract/Free Full Text].

28.   Garlick, PJ, Fern M, and Preedy VR. The effect of insulin infusion and food intake on muscle protein synthesis in postabsorptive rats. Biochem J 210: 669-676, 1983[ISI][Medline].

29.   Garlick, PJ, McNurlan MA, and Preedy VR. A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H]phenylalanine. Biochem J 192: 719-723, 1980[ISI][Medline].

30.   Gelfand, RA, and Barrett EJ. Effect of physiologic hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man. J Clin Invest 80: 1-6, 1987[ISI][Medline].

31.   Hara, K, Yonezawa K, Weng QP, Kozlowski MT, Belham C, and Avruch J. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem 273: 1448-1494, 1998.

32.   Heslin, MJ, Newman E, Wolf RF, Pisters PWT, and Brennan MF. Effect of hyperinsulinemia on whole body and skeletal muscle leucine carbon kinetics in humans. Am J Physiol Endocrinol Metab 262: E911-E918, 1992[Abstract/Free Full Text].

33.   Hsu, C-J, Kimball SR, Antonetti DA, and Jefferson LS. Effects of insulin on total RNA, poly(A)+ RNA, and mRNA in primary cultures of rat hepatocytes. Am J Physiol Endocrinol Metab 263: E1106-E1112, 1992[ISI][Medline].

34.   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: 1369-1375, 1983[Abstract/Free Full Text].

35.   Kimball, SR, Farrell PA, Nguyen HV, Jefferson LS, and Davis TA. Developmental decline in components of signal transduction pathways regulating muscle protein synthesis in neonatal pigs. Am J Physiol Endocrinol Metab 282: E585-E592, 2002[Abstract/Free Full Text].

36.   Kimball, SR, and Jefferson LS. Cellular mechanisms involved in the action of insulin on protein synthesis. Diabetes Metab Rev 4: 773-787, 1988[ISI][Medline].

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

38.   Liechty, EA, Boyle DW, Moorehead H, Liu YM, and Denne SC. Effect of hyperinsulinemia on ovine fetal leucine kinetics during prolonged maternal fasting. Am J Physiol Endocrinol Metab 263: E696-E702, 1992[Abstract/Free Full Text].

39.   Louard, RJ, Fryburg DA, Gelfand RA, and Barrett EJ. Insulin sensitivity of protein and glucose metabolism in human forearm skeletal muscle. J Clin Invest 90: 2348-2354, 1992[ISI][Medline].

40.   McCracken, KJ, Eddie SM, and Stevenson WG. Energy and protein nutrition of early-weaned pigs. Effect of energy intake and energy:protein on growth, efficiency and nitrogen utilization of pigs between 8-32 d. Br J Nutr 43: 289-304, 1980[ISI][Medline].

41.   McNulty, PA, Young LH, and Barrett EJ. Response of rat heart and skeletal muscle protein in vivo to insulin and amino acid infusion. Am J Physiol Endocrinol Metab 264: E958-E965, 1993[Abstract/Free Full Text].

42.   Meek, SE, Persson M, Ford GC, and Nair KS. Differential regulation of amino acid exchange and protein dynamics across splanchnic and skeletal muscle beds by insulin in healthy human subjects. Diabetes 47: 1824-1835, 1998[Abstract].

43.   Melville, S, McNurlan MA, McHardy KC, Broom J, Milne E, Calder AG, and Garlick PJ. The role of degradation in the acute control of protein balance in adult man: failure of feeding to stimulate protein synthesis as assessed by L-[1-13C]leucine infusion. Metabolism 38: 248-255, 1989[ISI][Medline].

44.   Mosoni, L, Houlier M-L, 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: E613-E620, 1993.

45.   Moughan, PJ, and Rowan AM. The pig as a model animal for human nutrition research. Proc Nutr Soc NZ 14: 116-123, 1989.

46.   Nair, KS, Ford GC, Ekberg K, Fernqvist-Forbes E, and Wahren J. Protein dynamics in whole body and in splanchnic and leg tissues in type I diabetic patients. J Clin Invest 95: 2926-2937, 1995[ISI][Medline].

47.   Ouali, A, and Talmant A. Calpains and calpastatin distribution in bovine, porcine and ovine skeletal muscle. Meat Sci 28: 331-348, 1990[ISI].

48.   Pain, V, Albertse EC, and Garlick PJ. Protein metabolism in skeletal muscle, diaphragm, and heart of diabetic rats. Am J Physiol Endocrinol Metab 245: E604-E610, 1983[Abstract/Free Full Text].

49.   Preedy, VT, and Garlick PJ. Response of muscle protein synthesis to parenteral administration of amino acid mixtures in growing rats. J Parenter Enteral Nutr 17: 113-118, 1993[Abstract].

50.   Scott, PH, Brunn GJ, Kohn AD, Roth RA, and Lawrence JC, Jr. Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. Proc Natl Acad Sci USA 95: 7772-7777, 1998[Abstract/Free Full Text].

51.   Shah, OJ, Antonetti DA, Kimball SR, and Jefferson LS. Leucine, glutamine, and tyrosine reciprocally modulate the translation initiation factors eIF4F and eIF2B in perfused liver. J Biol Chem 274: 36168-36175, 1999[Abstract/Free Full Text].

52.   Stoll, B, Burrin DG, Henry JG, Jahoor F, and Reeds PJ. Dietary and systemic phenylalanine utilization for mucosal and hepatic constitutive protein synthesis in pigs. Am J Physiol Gastrointest Liver Physiol 276: G49-G57, 1999[Abstract/Free Full Text].

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

54.   Suzuki, A, and Cassens RG. A histochemical study of myofiber types in muscle of the growing pig. J Anim Sci 51: 1449-1461, 1980[ISI][Medline].

55.   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, 1994[Abstract/Free Full Text].

56.   Tessari, P, Zanetti M, Barazzoni R, Vettore M, and Michielan F. Mechanism of postprandial protein accretion in human skeletal muscle. J Clin Invest 98: 1361-1372, 1996[Abstract/Free Full Text].

57.   Thureen, PJ, Scheer B, Anderson SM, Tooze JA, Young DA, and Hay WW. Effect of hyperinsulinemia on amino acid utilization in the ovine fetus. Am J Physiol Endocrinol Metab 279: E1294-E1304, 2000[Abstract/Free Full Text].

58.   Vary, TC, Jefferson LS, and Kimball SR. Amino acid-induced stimulation of translation initiation in rat skeletal muscle. Am J Physiol Endocrinol Metab 277: E1077-E1086, 1999[Abstract/Free Full Text].

59.   Volpi, E, Ferrando AA, Yeckel CW, Tipton KD, and Wolfe RR. Exogenous amino acids stimulate net muscle protein synthesis in the elderly. J Clin Invest 101: 2000-2007, 1998[Abstract/Free Full Text].

60.   Wester, TJ, Lobley GE, Birnie LM, and Lomax MA. Insulin stimulates phenylalanine uptake across the hind limb in fed lambs. J Nutr 130: 608-611, 2000[Abstract/Free Full Text].

61.   Wray-Cahen, D, Beckett PR, Nguyen HV, and Davis TA. Insulin-stimulated amino acid utilization during glucose and amino acid clamps decreases with development. Am J Physiol Endocrinol Metab 273: E305-E314, 1997[Abstract/Free Full Text].

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

63.   Young, VR. The role of skeletal and cardiac muscle in the regulation of protein metabolism. In: Mammalian Protein Metabolism, edited by Munro HN. New York: Academic, 1970, vol. 4, p. 585-674.


Am J Physiol Endocrinol Metab 282(4):E880-E890