Stimulation of Protein Synthesis by Both Insulin and Amino Acids Is Unique to Skeletal Muscle in...

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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, TX 77030

Short title: Protein synthesis in neonates

Correspondence and reprint requests:

T. A. Davis, Ph.D.

USDA/ARS Children's Nutrition Research Center

Baylor College of Medicine

1100 Bates Street

Houston, TX 77030

Phone: (713) 798-7169

Fax: (713) 798-7171

E-mail: [email protected]

Copyright 2002 by the American Physiological Society.

AJP-Endo Articles in PresS. Published on January 2, 2002 as DOI 10.1152/ajpendo.00517.2001

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ABSTRACT

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 current 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.

Key Words: Neonate, insulin action, amino acids, protein synthesis, nutrition

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INTRODUCTION

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

(18, 12). 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 which

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, using 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 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

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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 predominately 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 (36, 37, 53).

A recent study from this laboratory suggests that the feeding-induced stimulation of visceral

tissue protein synthesis in the neonate cannot be attributed to insulin (14). 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 (2, 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

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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

Animals and surgery. Multiparous crossbred (Yorkshire x Landrace x Hampshire x 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 Inc., Richmond, IN) throughout a 28-day lactation period. Sows

delivered 8-12 piglets and these remained with the sow 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 5 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 sow 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

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Laboratory Animals.

Infusions. Pigs were placed, unanesthetized, in a sling restraint system following 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), using 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 Corp., 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, in order 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

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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 TrophAmine 10% (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 (mmolar) 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,

skin, liver, jejunum, pancreas, kidney, and spleen were collected, rapidly frozen in liquid nitrogen,

and stored at -70oC.

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).

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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 of protein mass

synthesized in a day) were calculated as:

Ks (%/day) = [(Sb/Sa) x (1440/t)] x 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 amino-acyl 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-

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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 ± SEM.

RESULTS

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 �U/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).

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 to baseline (0 time) values in Figure

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1. During the 4-hr 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 current 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 using 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 about 50% (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.

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

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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).

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 among the two protein fractions in any

treatment group.

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

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than the effect of hyperinsulinemia alone (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).

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).

DISCUSSION

Regulation of skeletal muscle protein synthesis by both 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, insulin infusion, when amino acids and glucose are

maintained near fasting levels, 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.

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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, 37, 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 current 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, though, the stimulation of skeletal muscle protein synthesis by insulin was

similar in the current 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

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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-hour 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 current study further show that the stimulation of muscle protein synthesis

by amino acids and/or insulin was greater in those muscles containing predominately 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, 35), demonstrating that protein

synthesis is more responsive to anabolic agents in muscles composed of predominantly fast fibers

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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

current study is consistent with our previously reported findings in pig and rat muscles (18, 14).

Because the skeletal muscles we studied in the neonatal pig have predominately fast-twitch

contractile characteristics, but different oxidative and glycolytic properties (1, 47, 54), the differences

in protein synthesis rates between muscles which we observed are most likely attributable to the

distinct differences in the muscles metabolic rather than twitch characteristics.

The results of the current 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 that myofibrillar and sarcoplasmic protein synthesis rates are similar, as in the

current study, and that the age-associated decline in the synthesis rates of the two protein fractions

is proportional over this phase of development (14).

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 current

study, infusing insulin at a dose that reproduced plasma insulin levels in the fed state, while

maintaining glucose, essential amino acids, and nonessential amino acids at the fasting level,

stimulated protein synthesis in cardiac muscle, skin, and spleen. However, in a previous study,

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

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in cardiac muscle and skin, but not spleen (14). 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 current study. Amino acid infusion has no effect

on cardiac muscle protein synthesis in the adult rat (41), as in the neonatal pigs of the current study,

suggesting that cardiac muscle may be resistant to the anabolic effects of amino acids throughout the

life cycle.

Our previous study 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 (14). In the current study, the infusion of physiological

levels of insulin, when nonessential amino acids, 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 current study. These responses to

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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 (3, 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 (2, 44) animals,

consistent with the developmental decline in the amino acid-induced stimulation of liver protein

synthesis in the current study.

In the current 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 current 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 have shown that in the fed

state, dietary amino acids make a greater contribution than arterial amino acids to jejunal protein

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synthesis (52).

Whole-body amino acid and glucose disposal. Previous studies 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 (61). The current 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, 36). 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

R1-E-00517-2001 17

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 current

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.

R1-E-00517-2001 18

ACKNOWLEDGMENTS

The authors 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 Co. for the generous donation of porcine insulin.

This work is a publication of the USDA/ARS 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 Institute Grant R01 AR44474 and the U.S. Department of Agriculture, Agricultural

Research Service under Cooperative Agreement number 58-6250-6-001. This research was also

supported in part by NIH training grant T32 HD07445. The contents of this publication do not

necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of

trade names, commercial products or organizations imply endorsement by the U.S. Government.

Present address of P. J. Reeds, Ph.D.: University of Illinois, Department of Animal Sciences,

1207 W. Gregory, Room 432, Urbana, IL 61801. Present address of R. Vann, Ph.D.: CMREC,

Mississippi State University, 1320 Seven Springs Road, Raymond, MS 39154.

R1-E-00517-2001 19

REFERENCES

1. Anapol F, Herring, SW. Ontogeny of histochemical fiber types and muscle function in the

masseter muscle of miniature swine. Am J Anthropol 112:595-613,2000.

2. 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.

3. Ahlman B, Charlton M, Fu A, Berg C, O’Brian P, and Nari KS. Insulin’s effect on

synthesis rates of liver proteins. A swine model comparing various precursors of protein

synthesis. Diabetes 50: 947-954, 2001.

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, 1991.

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.

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.

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.

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.

R1-E-00517-2001 20

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.

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.

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.

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.

14. Davis TA, Fiorotto ML, Beckett PR, Burrin DG, Reeds PJ, Wray-Cahen D, 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.

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 Metabol 272: E877-E884, 1997.

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 Metabol 277: E103-E109, 1999.

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: R1373-1380,1991.

R1-E-00517-2001 21

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.

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.

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.

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: E1226-1234, 2000.

22. DeFronzo RA, Tobin JD, and Andres R. Glucose clamp technique: a method for

quantifying insulin secretion and resistance. Am J Physiol Endocrinol Metab 237 : E214-

E223, 1979.

23. Denne SC, and Kalhan SC. Leucine metabolism in human newborns. Am J Physiol


24. Denne SC, Rossi EM, and Kalhan SC. Leucine kinetics during feeding in normal

newborns. Pediatr Res 30: 23-27, 1991.

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.

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.

27. Flaim KE, Peavy DE, Everson WV, and Jefferson LS. The role of amino acids in the

R1-E-00517-2001 22

regulation of protein synthesis in perfused rat liver. J Biol Chem 257: 2932-2938, 1982.

28. Garlick P J, 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.

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.

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.

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: 1448-124494, 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.

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.

34. Jefferson LS, Liao WSL, Peavy DE, Miller TB, Appel MC, 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.

35. Kimball SR, and Jefferson LS. Cellular mechanisms involved in the action of insulin on

protein synthesis. Diabetes Metab Rev 4: 773:787, 1988.

36. 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

R1-E-00517-2001 23

process. Am J Physiol Endocrinol Metab 279: E1080-E1087, 2000.

37. 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, In press.

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.

39. Louard RJ, Fryburg DA, R. Gelfand A, and Barrett EJ. Insulin sensitivity of protein and

glucose metabolism in human forearm skeletal muscle. J Clin Invest 90: 2348-2354, 1992.

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 8-32d. Br J Nutr 43: 289-304, 1980.

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.

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.

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.

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

R1-E-00517-2001 24


45. Moughan PJ, and Rowan AM. The pig as a model animal for human nutrition research.

Proc Nutr Soc N Z 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.

47. Ouali A, Talmant A. Calpains and calpastatin distribution in bovine, porcine and ovine

skeletal muscle. Meat Science 28:331-348, 1990.

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.

49. Preedy VT, Garlick PJ. Response of muscle protein synthesis to parenteral administration

of amino acid mixtures in growing rats. J Parenter Enter Nutr 17: 113-118, 1993.

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.

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.

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.

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.

R1-E-00517-2001 25

54. Suzuki A, Cassens RG. A histochemical study of myofiber types in muscle of the growing

pig. J Anim Sci 51:1449-1461, 1980.

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.

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.

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.

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

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.

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.

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


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.

63. Young VR. The role of skeletal and cardiac muscle in the regulation of protein metabolism.

R1-E-00517-2001 26

In: Mammalian Protein Metabolism, edited by HN Munro. New York:Academic, 1970, vol.

4, p. 585-674.

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Figure Legends

Fig. 1: Plasma concentrations of essential amino acids 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).

Fig. 2: Whole-body net amino acid and glucose disposal rates during control,

hyperinsulinemic-euglycemic-euaminoacidemic (Insulin), euinsulinemic-euglycemic-


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 BCAA 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)

Fig. 3: Fractional protein synthesis rates in longissimus dorsi, gastrocnemius, and masseter

muscles in 7- and 26-day-old pigs during control, hyperinsulinemic-euglycemic-euaminoacidemic

(Insulin), euinsulinemic-euglycemic-hyperaminoacidemic (AA), and hyperinsulinemic-euglycemic-

hyperaminoacidemic (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).

Fig. 4: Fractional synthesis rates in total (TP), myofibrillar (MP), and sarcoplasmic (SP)

R1-E-00517-2001 28

proteins in longissimus dorsi muscle of 7-day-old pigs during control, hyperinsulinemic-euglycemic-

euaminoacidemic (Insulin), euinsulinemic-euglycemic-hyperaminoacidemic (AA), and

hyperinsulinemic-euglycemic-hyperaminoacidemic (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).

Fig. 5: Fractional protein synthesis rates in cardiac muscle, skin, and spleen in 7- and 26-day-

old pigs during control, hyperinsulinemic-euglycemic-euaminoacidemic (Insulin), euinsulinemic-

euglycemic-hyperaminoacidemic (AA), and hyperinsulinemic-euglycemic-hyperaminoacidemic

(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).

Fig. 6: Fractional protein synthesis rates in liver, kidney, and pancreas of 7- and 26-day-old

pigs during control, hyperinsulinemic-euglycemic-euaminoacidemic (Insulin), euinsulinemic-

euglycemic-hyperaminoacidemic (AA), and hyperinsulinemic-euglycemic-hyperaminoacidemic

(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).

Fig. 7: Fractional protein synthesis rates in jejunum of 7- and 26-day-old pigs during control,

hyperinsulinemic-euglycemic-euaminoacidemic (Insulin), euinsulinemic-euglycemic-


R1-E-00517-2001 29

clamps. Values are means ± SE; n = 7-12 per age and treatment group. Protein synthesis decreased

with age in jejunum (P < 0.001).

R1-E-00517-2001 30

Table 1. Plasma insulin and glucose concentrations in response to insulin and/or amino acid infusion

in 7- and 26-day-old pigs

Age Basal Control Insulin AA Insulin + AA

Insulin 7 d 2.6 ± 0.2 2.2 ± 0.2 27.2 ± 2.1* 2.7 ± 0.1 28.3 ± 1.7*26 d 3.2 ± 0.3 2.3 ± 0.2 27.3 ± 1.9* 3.4 ± 0.2 35.0 ± 1.2*

Glucose 7 d 76 ± 2 75 ± 2 78 ± 3 81 ± 3 78 ± 326 d 72 ± 2 71 ± 3 77 ± 3 70 ± 3 72 ± 3

Plasma concentrations of insulin in �U/ml and glucose in mg/dl are means ± SE; n = 7-12

per age and treatment group. * Insulin values in Insulin and Insulin+AA groups, but not Control or

AA groups, were significantly greater than basal, pre-infusion values (P < 0.001). Glucose values

in all treatment groups were not statistically different from basal values (P > 0.05).

R1-E-00517-2001 31

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ALA ASN ASP CIT GLU GLN GLY ORN PRO SER TAU TYR

Nonessential

Baseline Control Insulin AA Insulin + AA

ALA ASN ASP CIT GLU GLN GLY ORN PRO SER TAU TYR0

200

400

600

800

1000

1200

(nm

ol/m

l)

Nonessential

26-day-oldPlasma Amino Acids

ARG HIS ILE LEU LYS MET PHE THR TRP VAL

Essential

7-day-oldPlasma Amino Acids

ARG HIS ILE LEU LYS MET PHE THR TRP VAL0

100

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Figure 1

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Amino Acid Disposal Rate

0.0

1.0

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(mm

ol T

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Glucose Disposal Rate

7-day-old 26-day-old0

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ose/

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Figure 2

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ControlInsulin AAInsulin + AA

Longissimus Dorsi

Gastrocnemius

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Figure 3

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TP MP SP0

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Intestine


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Figure 7

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