Insulin and amino acids independently stimulate skeletal
muscle protein synthesis in neonatal pigs
Pamela M. J.
O'Connor,
Jill A.
Bush,
Agus
Suryawan,
Hanh V.
Nguyen, and
Teresa A.
Davis
United States Department of Agriculture, Agricultural
Research Service, Children's Nutrition Research Center and
Section of Neonatology, Department of Pediatrics, Baylor
College of Medicine, Houston, Texas 77030
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ABSTRACT |
Infusion of physiological levels of
insulin and/or amino acids reproduces the feeding-induced stimulation
of muscle protein synthesis in neonates. To determine whether insulin
and amino acids independently stimulate skeletal muscle protein
synthesis in neonates, insulin secretion was blocked with somatostatin
in fasted 7-day-old pigs (n = 8-12/group) while
glucose and glucagon were maintained at fasting levels and insulin was
infused to simulate either less than fasting, fasting, intermediate, or
fed insulin levels. At each dose of insulin, amino acids were clamped
at either the fasting or fed level; at the highest insulin dose, amino
acids were also reduced to less than fasting levels. Skeletal muscle protein synthesis was measured using a flooding dose of
L-[4-3H]phenylalanine. Hyperinsulinemia
increased protein synthesis in skeletal muscle during hypoaminoacidemia
and euaminoacidemia. Hyperaminoacidemia increased muscle protein
synthesis during hypoinsulinemia and euinsulinemia. There was a
dose-response effect of both insulin and amino acids on muscle protein
synthesis. At each insulin dose, hyperaminoacidemia increased
muscle protein synthesis. The effects of insulin and amino acids on
muscle protein synthesis were largely additive until maximal rates of
protein synthesis were achieved. Amino acids enhanced basal protein
synthesis rates but did not enhance the sensitivity or responsiveness
of muscle protein synthesis to insulin. The results suggest that
insulin and amino acids independently stimulate protein synthesis in
skeletal muscle of the neonate.
neonate; insulin action; growth; protein; nutrition
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INTRODUCTION |
THE RELATIVE RATES OF
GROWTH and protein synthesis are higher during the neonatal
period than at any other stage of postnatal life (16, 20, 29, 31,
35). During the neonatal period, more rapid gains in protein
mass occur in skeletal muscle than in other tissues (50).
Previous studies in rats and pigs suggest that neonates utilize their
dietary amino acids efficiently for growth because they are capable of
a greater increase in protein synthesis in response to feeding than
older animals (9, 15, 17, 18, 40, 41). The feeding-induced
stimulation of protein synthesis is more dramatic in skeletal muscle
than in other organs and decreases profoundly with development
(7-9, 17). For example, in response to refeeding,
fractional rates of skeletal muscle protein synthesis in 7-day-old pigs
increase from 15 to 24%/day and in 26-day-old pigs from 4 to 6%/day
(9).
Insulin is recognized as a key factor in the regulation of
skeletal muscle protein synthesis in the neonate. Postprandial changes
in protein synthesis are positively correlated with changes in
circulating insulin concentrations in the neonatal pig
(12). Studies using our novel
hyperinsulinemic-euglycemic-euaminoacidemic clamp technique in neonatal
pigs have shown that insulin stimulates whole body amino acid disposal
in the neonate and that the insulin sensitivity and responsiveness of
amino acid disposal decreases with development (48). The
infusion of insulin at doses achieving fed plasma insulin levels, when
amino acids and glucose are maintained at fasting levels, reproduces
the feeding-induced stimulation of muscle protein synthesis (11,
49). This response of protein synthesis to insulin declines with
development (11, 49) in parallel with the developmental
decline in the stimulation of muscle protein synthesis by feeding
(9).
The postprandial rise in amino acids also plays an important regulatory
role in the stimulation of protein synthesis by feeding in the neonate.
Recently, we demonstrated that the infusion of amino acids at a dose
that reproduces fed-state amino acid levels increases protein synthesis
in skeletal muscle of the neonate (13). This increase in
muscle protein synthesis occurs in the presence of either fasting or
fed insulin levels and is similar to that obtained by insulin
stimulation alone. The infusion of fed levels of either insulin or
amino acids, alone or in combination, increases muscle protein
synthesis to within the range normally found in the fed state. However,
it was not determined whether insulin and amino acids interact at
submaximal doses to stimulate skeletal muscle protein synthesis in the neonate.
In the present study, we wished to determine whether insulin and amino
acids independently regulate skeletal muscle protein synthesis in the
neonate. Specifically, we asked whether 1) the stimulation
of muscle protein synthesis by insulin requires concurrent amino acid
stimulation; 2) the stimulation of muscle protein synthesis by amino acids requires insulin; 3) the basal fasting
insulin level stimulates muscle protein synthesis; 4) there
is a dose-response effect of amino acids on muscle protein synthesis;
5) there is a dose-response effect of insulin on muscle
protein synthesis; and 6) there is an effect of amino acids
on the insulin sensitivity of muscle protein synthesis. To address
these issues, pancreatic glucose-amino acid clamps were used in fasted
neonatal pigs to block insulin secretion while glucose and glucagon
were maintained at fasting levels. Insulin was infused to achieve
levels that simulate less than fasting (~0 µU/ml insulin), fasting
(~2 µU/ml insulin), intermediate (~6 µU/ml insulin), and fed
(~30 µU/ml insulin) levels while at each insulin dose amino acids
were clamped at either the fasting or fed level. At the highest insulin
dose, amino acids were also reduced to less than fasting levels. The results showed that insulin and amino acids act independently to
stimulate protein synthesis in skeletal muscle of the neonate.
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METHODS |
Animals.
Eleven multiparous crossbred (Yorkshire × Landrace × Hampshire × Duroc) sows (Agriculture Headquarters, Texas
Department of Criminal Justice, Huntsville, TX) were housed in
lactation crates in individual, environmentally controlled rooms,
maintained on a commercial diet (5084, PMI Feeds, Richmond, IN), and
provided water ad libitum throughout the lactation period. After
farrowing, piglets remained with the sow and were not given
supplemental creep feed. Male and female piglets were studied at
5-8 days of age (2.1 ± 0.4 kg). Three to five days before
the infusion study, pigs were anesthetized, and catheters were
surgically inserted into a jugular vein and a carotid artery with the
use of sterile techniques as described previously (48).
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 Laboratory Animals.
Pancreatic glucose-amino acid clamps.
Pigs (n = 8-12/treatment group; total of 90) were
placed, unanesthetized, in a sling restraint system following a 12-h
fast. During a 30-min basal period before the clamp procedure was
initiated, blood samples were taken and immediately analyzed for blood
glucose (YSI 2300 STAT Plus, Yellow Springs Instrument, Yellow Springs, OH) to establish the average basal concentration of blood glucose (19). Plasma total branched-chain amino acid (BCAA)
concentrations were determined by rapid enzymatic kinetic assay
(5) to establish the average basal concentration of BCAA
to be used in the subsequent clamp procedure. Pancreatic glucose-amino
acid clamps were performed using techniques similar to those previously
described (45). The clamp was initiated with a
primed (20 µg/kg), continuous (100 µg · kg
1 · h
1)
somatostatin (Bachem, Torrance, CA) infusion. After a 10-min infusion
of somatostatin, a continuous infusion of replacement glucagon (150 ng · kg
1 · h
1; Eli Lilly,
Indianapolis, IN) was initiated and continued to the end of the clamp
period. Insulin was infused at 0, 10, 22, and 110 ng · kg
0.66 · min
1 to
achieve plasma insulin concentrations of ~0, 2, 6, and 30 µU/ml to
simulate less than fasting, fasting, intermediate, and fed insulin
levels, respectively (9). At each dose of insulin, amino
acids were clamped at either the fasting (~500 nmol BCAA/ml) or fed
(~1,000 nmol BCAA/ml) level by adjusting the infusion rate of a
balanced amino acid mixture (see below) to maintain the plasma BCAA
concentration within 10% of the desired level (48). At the highest insulin dose only, amino acids also were allowed to fall
below fasting levels (~250 nmol BCAA/ml) by omitting the amino acid
clamp. The amino acid mixture (13) contained (mmol) arginine (20.1), histidine (12.9), isoleucine (28.6), leucine (34.3),
lysine (27.4), methionine (10.1), phenylalanine (12.1), threonine
(21.0), tryptophan (4.4), valine (34.1), alanine (27.3; 38% provided
as alanyl-glutamine), aspartate (12.0), cysteine (6.2), glutamate
(23.8), glutamine (17.1; 100% provided as alanyl-glutamine), glycine
(54.3; 4% provided as glycyl-tyrosine), proline (34.8), serine (23.8),
taurine (2.0), and tyrosine (7.2; 83% provided as glycyl-tyrosine).
Blood samples were also taken at intervals for later determination of
circulating insulin, glucagon, and individual essential and
nonessential amino acid concentrations.
Tissue protein synthesis in vivo.
The fractional rate of protein synthesis was measured with a flooding
dose of L-[4-3H]phenylalanine (14,
26) injected 90 min after the initiation of the clamp procedure.
Pigs were killed at 2 h, and samples of longissimus dorsi muscle
were collected and rapidly frozen. The specific radioactivity values of
the protein hydrolysate, homogenate supernatant, and blood supernatant
were determined as previously described (16). Previous
studies have demonstrated that, after a flooding dose of tritiated
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
(14).
Plasma hormones and substrates.
The concentrations of individual amino acids from frozen plasma samples
obtained at 0 and 90 min of the insulin infusions were measured with an
HPLC method (PICO-TAG reverse-phase column; Waters, Milford, MA) as
previously described (18). Plasma radioimmunoreactive insulin concentrations were measured using a porcine insulin
radioimmunoassay kit (Linco, St. Louis, MO) that used porcine insulin
antibody and human insulin standards. Plasma radioimmunoreactive
glucagon concentrations were measured using a porcine glucagon
radioimmunoassay kit (Linco) that used porcine glucagon antibody and
human glucagon standards.
Calculations and statistics.
The fractional rate of protein synthesis (Ks;
percentage of protein mass synthesized in a day) was calculated as
where Sb (in dpm/nmol) is the specific radioactivity
of the protein-bound phenylalanine, Sa (in dpm/nmol) is the
specific radioactivity of the tissue free phenylalanine at the time of tissue collection and the linear regression of the blood specific radioactivity of the animal at 5, 15, and 30 min against time, t is the time of labeling in minutes, and 1,440 is the
minutes-to-day conversion.
Analysis of variance (general linear modeling) was used to
assess the effect of insulin, amino acids, and their interaction. If
there was an interaction between insulin and amino acids, Student's t-test was used to test for differences between groups. 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.
Probability values of <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 21 amino
acid comparisons in each of the nine treatment groups would be
significantly different among groups by random chance, a more conservative statistical approach for amino acid comparison was used;
therefore, probability values of <0.01 were considered statistically different. Data are presented as means ± SE. The insulin
sensitivity of muscle protein synthesis was calculated by nonlinear
regression analysis.
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RESULTS |
Infusions, hormones, and substrates.
Fasted 7-day-old pigs were infused with somatostatin (to block insulin
secretion), glucagon (at replacement levels), and glucose (as needed to
maintain fasting levels). Insulin was infused at four doses to achieve
levels that simulated 1) less than fasting, 2)
fasting, 3) intermediate between fasting and fed, and
4) fed conditions. At each dose of insulin, amino acids were
clamped at either the fasting or fed level; at the highest insulin
dose, amino acids were also allowed to fall to less than fasting
levels. Table 1 shows the circulating
insulin, glucagon, and glucose concentrations during the infusion
compared with baseline (0 time) values, when amino acids were clamped
at fasting, fed, and less than fasting levels. Targeted plasma insulin
levels, i.e., 0, 2, 6, and 30 µU/ml, were largely achieved in all
treatment groups. Hyperaminoacidemia or hypoaminoacidemia did not alter
plasma insulin or glucagon concentrations. Circulating glucose
concentrations were maintained at basal fasting levels during the
infusion of somatostatin, glucagon, insulin, and/or amino acids.
Replacement circulating glucagon levels were achieved during the
somatostatin clamp in most groups.
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Table 1.
Plasma insulin, glucagon, and glucose concentrations in response to
insulin and amino acid infusion during pancreatic glucose-amino acid
clamps in 7-day-old pigs
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Circulating essential and nonessential amino acid concentrations
achieved during the infusions are compared with baseline (0 time)
values in Figs. 1 and
2, respectively. The circulating concentrations of both essential and nonessential amino acids were
maintained at the fasting level during the euaminoacidemic clamps. The
exceptions were reductions in serine, taurine, or tyrosine and an
increase in citrulline in one or more groups. Hyperaminoacidemia
increased the circulating concentrations of essential amino acids by
~90% and those of nonessential amino acids by ~50%
(P < 0.001). In the presence of hyperinsulinemia, without amino acid infusion, essential and nonessential amino acids
fell to ~50% of fasting levels (P < 0.001).

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Fig. 1.
Plasma concentrations of essential amino acids at
baseline (0 time) and in the presence of ~0, 2, 6, and 30 µU/ml
plasma insulin levels while amino acids were clamped at 500 (A), 1,000 (B), and 250 (C) nmol
branched-chain amino acids (BCAA)/ml by use of a balanced amino acid
mixture in 7-day-old pigs. In all groups, glucose was clamped at the
fasting level. Values are means ± SE; n = 8-12 per treatment group. * Statistically different from
baseline values (P < 0.01).
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Fig. 2.
Plasma concentrations of nonessential amino acids at
baseline (0 time) and in the presence of ~0, 2, 6, and 30 µU/ml
plasma insulin levels while amino acids were clamped at 500 (A), 1,000 (B), and 250 (C) nmol
BCAA/ml by use of a balanced amino acid mixture in 7-day-old pigs. In
all groups, glucose was clamped at the fasting level. Values are
means ± SE; n = 8-12 per treatment group.
* Statistically different from baseline values (P < 0.01).
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Figure 3 shows the net whole body glucose
disposal and amino acid disposal rates as indicated by the glucose and
amino acid infusion rates during the 2-h infusion period. Amino acids
were not infused in the ~0 and 2 µU/ml plasma insulin level groups, because plasma amino acid concentrations did not fall below 10% of the basal level. Amino acids were also not infused in the
hyperinsulinemic-euglycemic-hypoaminoacidemic group, thereby
allowing plasma amino acid concentrations to fall to ~50% of the
basal level. Insulin, but not amino acids, increased glucose infusion
rates. Both insulin and amino acids increased amino acid infusion
rates.

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Fig. 3.
Whole body net glucose disposal rates (A) and
amino acid disposal rates (B) in the presence of ~0, 2, 6, and 30 µU/ml plasma insulin levels while amino acids were clamped at
500, 1,000, and 250 nmol BCAA/ml by use of a balanced amino acid
mixture. In all groups, glucose was clamped at fasting levels. Values
are means ± SE; n = 8-12 per treatment
group. Amino acid disposal rate was the average rate of infusion of
leucine from a balanced amino acid mixture necessary to maintain
circulating BCAA near either the basal fasting level (500 nmol/ml) or
twofold the fasting level (1,000 nmol/ml). Amino acids were not infused
in the ~0 and 2 µU/ml insulin/fasting amino acid treatment groups
and were allowed to fall to below fasting level in the ~30 µU/ml
insulin/less than fasting amino acid level (250 nmol BCAA/ml) group.
Glucose disposal rate was the average rate of infusion of dextrose
necessary to maintain circulating glucose near the fasting level.
Hyperinsulinemia increased glucose and amino acid disposal rates.
* Significantly different from previous insulin level within the
same amino acid (AA) level (P < 0.05). Significant
effect of amino acids within the same insulin group (P < 0.05).
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Skeletal muscle protein synthesis.
To determine whether insulin and amino acids stimulate skeletal muscle
protein synthesis independently in the neonate, we asked six specific
questions. We asked whether 1) the stimulation of muscle
protein synthesis by insulin requires concurrent amino acid
stimulation; 2) the stimulation of muscle protein synthesis by amino acids requires insulin; 3) the basal fasting
insulin level stimulates muscle protein synthesis; 4) there
is a dose-response effect of amino acids on muscle protein synthesis;
5) there is a dose-response effect of insulin on muscle
protein synthesis; and 6) there is an effect of amino acids
on the insulin sensitivity of muscle protein synthesis. The data from
pigs treated with four different doses of insulin and two to three
doses of amino acids were thus analyzed in subsets, so that each
question could be addressed specifically. Because muscles composed of
primarily fast-twitch, glycolytic fibers predominate in the musculature of the neonatal pig and are the most responsive to anabolic agents (11, 13), we studied the longissimus dorsi muscle, which
contains primarily fast-twitch muscle fibers.
Previous studies have shown that insulin infusion in the neonatal pig
stimulates protein synthesis in skeletal muscle when amino acids are
clamped at the fasting level (11, 48). To determine
whether insulin-stimulated muscle protein synthesis requires
maintenance of fasting amino acid levels, insulin was increased to the
fed level (30 µU/ml) while amino acids were either clamped at the
fasting level (500 nmol BCAA/ml) or allowed to fall below the fasting
level (250 nmol BCAA/ml). Results were compared with those for the
basal condition of fasting insulin (2 µU/ml) and amino acid (500 nmol
BCAA/ml) levels (Fig. 4). Insulin increased the rate of muscle protein synthesis in the absence of amino
acid infusion (P < 0.001), although not to the rate of muscle protein synthesis when amino acids were clamped at the fasting
level (P < 0.001). This suggests that the stimulation of muscle protein synthesis by insulin does not require concurrent amino acid stimulation in the neonate.

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Fig. 4.
Fractional protein synthesis rates
(Ks) in longissimus dorsi muscle of 7-day-old
pigs in the presence of hyperinsulinemia (~30 µU/ml) and either
euaminoacidemia or hypoaminoacidemia (500 or 250 nmol BCAA/ml) compared
with basal fasting condition (~2 µU insulin/ml and 500 nmol
BCAA/ml). Values are means ± SE; n = 8-12
per treatment group. Insulin increased protein synthesis rates during
hypoaminoacidemia (P < 0.001). Protein synthesis rates
were further increased during euaminoacidemia (P < 0.001). * Significantly different from basal fasting condition
(P < 0.05). Significantly different from
hyperinsulinemic- euaminoacidemic group (P < 0.05).
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To determine whether the stimulation of muscle protein synthesis by
amino acids requires insulin, insulin was reduced to nearly zero by
somatostatin infusion, and amino acids were either maintained at the
fasting level (500 nmol BCAA/ml) or raised to the fed level (1,000 nmol
BCAA/ml; Fig. 5). Raising amino acids
from the fasting to the fed level, when insulin was reduced to nearly
zero, increased muscle protein synthesis (P < 0.02).
This suggests that the stimulation of muscle protein synthesis by amino
acids does not require insulin in the neonate.

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Fig. 5.
Fractional protein synthesis rates in longissimus dorsi
muscle of 7-day-old pigs in the presence of hypoinsulinemia (~0
µU/ml) with either euaminoacidemia (500 nmol BCAA/ml) or
hyperaminoacidemia (1,000 nmol BCAA/ml). Values are means ± SE;
n = 8-12 per treatment group. Hyperaminoacidemia
in the presence of hypoinsulinemia increased muscle protein synthesis
(P < 0.02). Significantly different from
hypoinsulinemic-euaminoacidemic group (P < 0.05).
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To determine whether basal fasting insulin levels stimulate muscle
protein synthesis, insulin levels were either reduced to nearly zero or
were maintained at the fasting level (2 µU/ml) while amino acids were
maintained at the fasting level (500 nmol BCAA/ml; Fig.
6). An increase in fractional rates of
protein synthesis from 10.2 ± 0.8 to 13.9 ± 0.6%/day
(P < 0.002) suggests that basal fasting insulin levels
stimulate protein synthesis in neonatal muscle.

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Fig. 6.
Fractional protein synthesis rates in longissimus dorsi
muscle of 7-day-old pigs are compared during hypoinsulinemia (~0
µU/ml) and euinsulinemia (~2 µU/ml) in the presence of
euaminoacidemia (500 nmol BCAA/ml). Values are means ± SE;
n = 8-12 per treatment group. Raising insulin from
0 to 2 µU/ml increased protein synthesis rates (P < 0.002). * Significantly different from
hypoinsulinemic-euaminoacidemic group (P < 0.05).
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To determine whether there is a dose-response effect of amino acids on
muscle protein synthesis, amino acids were allowed to fall below
fasting (250 nmol BCAA/ml), remain at fasting (500 nmol BCAA/ml), or
increase to fed levels (1,000 nmol BCAA/ml; Fig.
7). In each group, insulin was infused at
the fed level (30 µU/ml), because insulin infusion is required to
reduce circulating amino acids below fasting levels by promoting amino
acid disposal (48). Protein synthesis rates increased
progressively as circulating amino acids were increased to fasting
(P < 0.001) and then fed (P = 0.06)
levels. Thus amino acids increase muscle protein synthesis in a
dose-response manner.

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Fig. 7.
Fractional protein synthesis rates in longissimus dorsi
muscle of 7-day-old pigs in the presence of hyperinsulinemia (~30
µU/ml) are compared during hypoaminoacidemia (250 nmol BCAA/ml),
euaminoacidemia (500 nmol BCAA/ml), or hyperaminoacidemia (1,000 nmol BCAA/ml). Values are means ± SE; n = 8-12 per treatment group. Raising amino acids increased protein
synthesis rates progressively (P < 0.05).
* Significantly different from hyperinsulinemic-hypoaminoacidemic
group (P < 0.001).
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Figure 8 compares the dose-response
effect of insulin on muscle protein synthesis in the presence of
euaminoacidemia (500 nmol BCAA/ml) or hyperaminoacidemia (1,000 nmol
BCAA/ml) amino acid levels. There was a progressive increase in protein
synthesis rates as the level of insulin was increased
(P < 0.005). Amino acids increased muscle protein
synthesis (P < 0.05) at each dose of insulin except
the highest dose (30 µU/ml), where there was a tendency for amino
acids to stimulate muscle protein synthesis (P < 0.10). Thus there was a dose-response effect of insulin on muscle
protein synthesis in the presence of fasting or fed amino acid levels.
The effects of insulin and amino acids were additive until maximal
rates of protein synthesis were achieved.

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Fig. 8.
Fractional protein synthesis rates in longissimus dorsi
muscle of 7-day-old pigs at ~0, 2, 6, and 30 µU/ml plasma insulin
levels during euaminoacidemia (500 nmol BCAA/ml) and hyperaminoacidemia
(1,000 nmol BCAA/ml). Protein synthesis rates increased progressively
as the insulin dose was increased (P < 0.005).
Hyperaminoacidemia increased protein synthesis at the ~0, 2, and 6 µU/ml insulin doses (P < 0.05), with a
nonsignificant increase at the ~30 µU/ml insulin dose
(P < 0.10). Values are means ± SE;
n = 8-12 per treatment group. The effects of
insulin and amino acids were additive until maximal rates of protein
synthesis were achieved at the fed insulin and amino acid levels.
* Statistically significantly different from previous insulin level
within the same amino acid level (P < 0.05).
Significantly different from euaminoacidemic group within same
insulin group (P < 0.05).
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Figure 9 shows a nonlinear regression
model of muscle protein synthesis vs. plasma insulin when amino acids
were maintained at fasting levels and when amino acids were increased
to simulate fed levels. The results show a curvilinear relationship
between protein synthesis and insulin that was influenced by amino
acids. Amino acids increased the basal rate of protein synthesis and tended to increase the maximum rate of protein synthesis. Thus the
actual response of muscle protein synthesis to insulin, i.e., the
difference between the maximum response and the baseline rate, did not
change with amino acid supplementation. The half-maximum response of
protein synthesis to insulin was estimated at 2.1 µU/ml when amino
acids were at the fasting level and 1.9 µU/ml when amino acids were
at the fed level. This suggests that amino acids do not change the
sensitivity of muscle protein synthesis to insulin in the neonate.

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Fig. 9.
Nonlinear regression analysis of muscle protein synthesis
vs. plasma insulin in the presence of euaminoacidemia and
hyperaminoacidemia. A curvilinear relationship between protein
synthesis and plasma insulin is demonstrated that is influenced by
amino acid (AA) concentration. The half-maximum response of protein
synthesis to insulin was estimated at 2.1 µU/ml in the presence of
euaminoacidemia and at 1.9 µU/ml in the presence of
hyperaminoacidemia. Values are means ± SE; n = 8-12 per treatment group.
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DISCUSSION |
Previous studies have shown that both insulin and amino acids play
important roles in the feeding-induced stimulation of protein synthesis
in skeletal muscle of the neonate (10, 11, 13, 49).
Reproduction of postprandial protein synthesis rates in skeletal muscle
in the neonate can be achieved by the infusion of insulin to the fed
level when amino acids and glucose are clamped at basal fasting levels
(49). Furthermore, the infusion of amino acids to fed
levels, while glucose and insulin are at fasting levels, can also
reproduce the feeding-induced stimulation of muscle protein synthesis
in the neonate (13). However, due to the achievement of
maximal rates of muscle protein synthesis rates by the infusion of
insulin and/or amino acids to achieve fed levels in previous studies,
it was not discerned whether there are additive effects of insulin and
amino acids at submaximal levels. The results of the present study
showed that both insulin and amino acids stimulated muscle protein
synthesis in a dose-response manner and that the effects of insulin and
amino acids were additive until maximal rates were achieved.
Furthermore, the results suggest that insulin and amino acids act
independently to stimulate the postprandial rise in skeletal muscle
protein synthesis in neonates.
Effectiveness of pancreatic glucose-amino acid clamp.
To isolate the independent effects of insulin and amino acids on
skeletal muscle protein synthesis in the neonate, we used our
previously reported pancreatic glucose-amino acid clamp
(45). We chose this method because it is preferable to the
diabetic animal model, which is fraught with confounding metabolic
effects including aberrations in plasma glucose and ketone body
concentration (15, 32, 33, 38). Using the pancreatic
glucose-amino acid clamp, we largely achieved the targeted insulin and
amino acid levels, which were within the narrow physiological range. We
also reduced insulin and amino acids below fasting levels, which in the
case of insulin was at the detectable limit of the assay and in the
case of amino acids required insulin stimulation to promote amino acid
uptake. The infusion procedure also achieved the maintenance of glucose
and glucagon at fasting levels for the duration of the experiment. The
choice of omitting an amino acid clamp in one insulin-stimulated
treatment group not only served to test the independent effects of
insulin on muscle protein synthesis but also demonstrated the profound
effects of physiological levels of insulin on whole body amino acid
disposal in the neonate by reducing circulating amino acid levels by
one-half. Thus the amino acid clamp is particularly useful in rapidly
growing animals and has been recently used in the fetal lamb model to
examine the effects of hormonal stimulation (42, 44). Use
of a balanced amino acid mixture to clamp amino acids at fasting levels
also largely prevented the reduction in nonessential amino acids that can occur with the use of current commercial formulas (11, 13, 44, 48, 49).
Effect of insulin and amino acids on muscle protein synthesis.
Although we previously demonstrated that muscle protein synthesis rates
can be raised to fed rates by the infusion of insulin when amino acids
are clamped at fasting levels (49), a potential stimulatory effect of the amino acids infused to maintain fasting amino
acid levels could not be ruled out. In the present study, we showed
that infusing insulin to simulate fed insulin levels stimulated muscle
protein synthesis even when amino acids were allowed to fall to
one-half of basal fasting amino acid levels by the omission of an amino
acid clamp. Although the rates of muscle protein synthesis observed
were submaximal, suggesting that insulin's stimulatory effect on
muscle protein synthesis is blunted if basal amino acids levels are not
maintained, the results more importantly indicate that insulin's
stimulatory effect on muscle protein synthesis in the neonate does not
require concurrent amino acid stimulation. This finding further defines
insulin's role as a regulator of the postprandial stimulation of
muscle protein synthesis in the young growing animal, as previously
described in hindlimb of the young lamb (47), and in
just-weaned but growing rats (24). However, studies in
more mature animals (3, 4, 36, 39) and humans (21,
23, 27, 30, 34, 37, 43) found insulin to have little, if any,
effect on muscle protein synthesis in the absence of amino acid
infusion, a finding which contrasts markedly with those studies
conducted during early growth and development. For example, a recent in
vitro study suggests that the postprandial rise in muscle protein
synthesis requires both insulin and amino acids in young adult rats
(3). This suggests that the response of muscle protein
synthesis to insulin wanes and is lost with development.
In a recent study (13), a stimulatory effect of amino
acids on protein synthesis, in the presence of fasting insulin levels, was demonstrated in skeletal muscle of young postnatal pigs.
Furthermore, studies in young, adult, and elderly populations have also
shown that amino acids, either alone or concurrent with insulin
infusion, have a stimulatory effect on muscle protein synthesis and are a primary physiological regulator of protein synthesis in skeletal muscle (6, 28, 46). In this study, we wished to determine whether or not insulin plays a permissive role in amino acid-stimulated muscle protein synthesis in neonates. Our data showed that increasing circulating amino acids to fed levels in the near absence of insulin increased muscle protein synthesis. However, the increase in protein synthesis was less than that which occurred in the presence of fasting
insulin levels due to a reduction in the baseline rate of protein
synthesis. In fact, the rates of muscle protein synthesis achieved by
amino acid stimulation in the near absence of insulin was similar to
that achieved by insulin stimulation when amino acids were below
fasting levels. These results suggest that the stimulation of skeletal
muscle protein synthesis by amino acids does not require insulin in the
neonate. The finding contrasts with the recent study of Anthony et al.
(1), in which somatostatin infusion blocked the
stimulation of skeletal protein synthesis by leucine in mature rats.
This suggests that, although the stimulation of muscle protein
synthesis by insulin and amino acids in the neonate is independent,
with maturation insulin is required to play a permissive role in amino
acid-stimulated muscle protein synthesis. Although leucine increased
muscle protein synthesis in alloxan-induced diabetic rats
(2), in a severely diabetic model (partial
pancreatectomy), the protein synthesis-stimulatory effect of resistance
exercise was blocked by insulin deficiency (22).
The use of somatostatin in the pancreatic glucose-amino acid clamp
allowed us to reduce endogenous insulin to near zero so as to determine
whether basal fasting insulin could stimulate muscle protein synthesis
in the neonate. The results show that the neonatal muscle is so
sensitive to insulin that even basal fasting insulin levels have a
stimulatory effect on protein synthesis. This enhanced sensitivity of
neonatal muscle protein synthesis to insulin may contribute to the high
efficiency rates of protein deposition and the rapid growth rate seen
at this age. Furthermore, this finding exposes the vulnerability of the
young patient with diabetes, in whom muscle protein synthesis rates may
be suppressed due to the absence of insulin.
Having previously shown that fed levels of amino acids stimulate
skeletal muscle protein synthesis (13), we further wished to determine whether there was a dose-response effect of amino acids on
muscle protein synthesis. The results showed a progressive increase in
muscle protein synthesis rates, as circulating amino acid
concentrations were increased by infusion from below-fasting to fasting
levels and finally to fed levels, indicating that amino acids stimulate
skeletal muscle protein synthesis in neonatal pigs in a dose-response manner.
Our previous studies (48) showed that maximal rates of
skeletal muscle protein synthesis were achieved by the infusion of insulin to simulate insulin levels in the fed steady state (10 mU/ml)
with no further increase in muscle protein synthesis at insulin levels
that reproduce the immediate postprandial period (30 µU/ml) or
with pharmacological doses (800 µU/ml). In the present study, four
doses of insulin (~0, 2, 6, and 30 µU/ml) were infused to simulate
below-fasting, fasting, intermediate between fasting and fed, and
postprandial fed insulin levels, to define the dose-response effect of
insulin on muscle protein synthesis in neonatal pigs. We demonstrated a
progressive increase in muscle protein synthesis rates as the insulin
level was increased to maximum physiological fed levels. Furthermore,
at each dose of insulin except the highest dose, an increase in amino
acids from the fasted to the fed level increased muscle protein
synthesis. This suggests that the stimulation of muscle protein
synthesis by insulin and amino acids is additive until maximal rates of
protein synthesis are achieved.
Garlick and Grant (25) previously reported that amino
acids enhanced the sensitivity of skeletal muscle protein synthesis to
insulin in postabsorptive, young, weaned rats, such that maximal rates
of protein synthesis were achieved at lower insulin concentrations when
amino acids were concurrently infused. We observed that, because amino
acids increased the baseline rate of muscle protein synthesis and
tended to increase the maximum rate of muscle protein synthesis, amino
acids did not affect the responsiveness of muscle protein synthesis to
insulin in neonates. Furthermore, we demonstrate that the half-maximum
response of protein synthesis to insulin was similar at both fasting
and fed circulating amino acid levels, indicating that amino acids do
not alter the sensitivity of muscle protein synthesis to insulin in the
neonate. Differences in our results in neonatal pigs and those by
Garlick and Grant in young, weaned rats could be attributed to stage of
development at the time of study. Our findings suggest that insulin and
amino acids independently stimulate muscle protein synthesis in the
neonate and imply independent action of insulin and amino acids on the intracellular signaling pathways that regulate muscle protein synthesis.
Perspectives.
Our previous studies showed that feeding stimulates skeletal muscle
protein synthesis in neonates (9, 17) and that the magnitude of the feeding response can be reproduced by the infusion of
insulin and/or amino acids (11, 13, 49). This suggests that the postprandial rise in insulin and amino acids mediates the
feeding-induced stimulation of muscle protein synthesis in neonates.
Using our pancreatic glucose-amino acid clamps in the present study
allowed us to look in more detail at the individual roles of insulin
and amino acids in this response. The results highlight the exquisite
insulin sensitivity of skeletal muscle protein synthesis in the
neonate, in that the basal fasting insulin level stimulated protein
synthesis and insulin increased protein synthesis in the absence of
amino acid infusion. In addition, the results showed that amino acids
stimulated muscle protein synthesis in the absence of insulin, thereby
increasing the basal rate of muscle protein synthesis, but did not
enhance the sensitivity of muscle protein synthesis to insulin. Thus
the results suggest that insulin and amino acids act independently to
stimulate protein synthesis in skeletal muscle of the neonate. The
ability of skeletal muscle protein synthesis to respond to the
postprandial rise in both insulin and amino acids likely contributes to
the efficient utilization of dietary amino acids for protein deposition
and the rapid gain in skeletal muscle mass in the neonate. These
findings highlight the importance of a protein-containing diet for
skeletal muscle growth in the neonate.
 |
ACKNOWLEDGEMENTS |
We acknowledge with respect and affection the helpful discussions
shared with the recently deceased Dr. Peter J. Reeds.
 |
FOOTNOTES |
We thank Drs. M. Fiorotto and D. Burrin also for helpful discussions,
W. Liu and J. Rosenberger for technical assistance, J. Cunningham, F. Biggs, and J. Stubblefield 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.
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, TX. This project has been funded in part
by the National Institute of Arthritis and Musculoskeletal and Skin
Diseases Institute Grant RO1 AR-44474 and the USDA/ARS under
Cooperative Agreement no. 58-6250-6-001. This research was also supported in part by National Institute of Child Health and Human
Development 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.
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.
September 24, 2002;10.1152/ajpendo.00326.2002
Received 18 July 2002; accepted in final form 20 September 2002.
 |
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