Effect of hyperinsulinemia on amino acid utilization in the
ovine fetus
Patti J.
Thureen1,
Bryan
Scheer3,
Susan M.
Anderson1,
Janet A.
Tooze2,
David A.
Young2, and
William W.
Hay Jr.1
1 Perinatal Research Center and Departments of Pediatrics
and 2 Preventive Medicine and Biometrics, University of
Colorado Health Sciences Center, Denver, Colorado, 80262; and
3 Department of Surgery, University of Oklahoma College of
Medicine, Oklahoma City, Oklahoma, 73190
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ABSTRACT |
We studied the effect of an acute 4-h period of
hyperinsulinemia (H) on net utilization rates (AAURnet)
of 21 amino acids (AA) in 17 studies performed in 13 late-gestation
fetal sheep by use of a novel fetal
hyperinsulinemic-euglycemic-euaminoacidemic clamp. During H [84 ± 12 (SE) µU/ml H, 15 ± 2 µU/ml control (C), P < 0.00001], euglycemia was maintained by glucose
clamp (19 ± 0.05 µmol/ml H, 1.19 ± 0.04 µmol/ml C), and
euaminoacidemia (mean 4.1 ± 3.3% increase for all amino acid
concentrations [AA], nonsignificantly different from zero) was
maintained with a mixed amino acid solution adjusted to keep lysine
concentration constant and other [AA] near C values. H produced a
63.7% increase in AAURnet (3.29 ± 0.66 µmol · min
1 · kg
1 H,
2.01 ± 0.55 µmol · min
1 · kg
1 C,
P < 0.001), accounting for a 60.1% increase in fetal
nitrogen uptake rate (2,064 ± 108 mg · day
1 · kg
1 H,
1,289 ± 73 mg · day
1 · kg
1 C,
P < 0.001). Mean AA clearance rate
(AAURnet/[AA]) increased by 64.5 ± 18.9%
(P < 0.001). Thus acute physiological H increases net
amino acid and nitrogen utilization rates in the ovine fetus independent of plasma glucose and [AA].
glucose; insulin; amino acids; sheep; nitrogen
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INTRODUCTION |
INSULIN IS REGARDED
AS a major fetal growth factor, based primarily on evidence of
decreased fetal growth rates during periods of decreased fetal plasma
insulin concentration. Much less certain are the effects of increases
in fetal plasma insulin concentrations on fetal growth. It has been
difficult, for example, to assess the independent effect of increased
insulin concentrations on protein metabolism, because independent and
interactive effects of plasma glucose and amino acid concentrations,
which also change in response to insulin, simultaneously affect amino
acid uptake by tissues, thereby contributing in complex ways to protein
metabolism. Not surprisingly, conflicting results from initial studies
have been obtained. For example, Taslikian and Hamilton
(28) were unable to demonstrate any change in total carbon
flux during experimental conditions of hyperinsulinemic euglycemia in
fetal sheep. Also, Liechty et al. (16, 17) determined that
glucose and insulin each enhance protein accretion by decreasing
leucine oxidation but neither inhibits proteolysis. These studies were
conducted without maintaining normal plasma concentrations of amino
acids, and their results differ from those by Milley (24),
who performed tracer leucine studies during insulin infusion while both
glucose and leucine concentrations were clamped at normal levels.
Milley showed that, in the presence of normal leucine concentrations, the effect of insulin on leucine metabolism was to promote net leucine
accretion by decreasing protein breakdown. Milley's results are
similar to studies in adult humans (4, 9) that have shown
that the principal anabolic effect of insulin during euglycemic euinsulinemic conditions is to inhibit protein breakdown.
The aforementioned investigations were designed to measure the effect
of insulin on fetal metabolism of a single amino acid, leucine, without
measuring or assuring normal fetal plasma concentrations of the other
amino acids. Alterations in the concentration of other amino acids
could affect overall amino acid metabolism and insulin secretion.
Because several amino acids compete for the same transporter,
uncontrolled and different patterns of amino acid concentrations could
produce different fluxes of individual amino acids out of the plasma
and into tissues. The present study was designed to more rigorously
determine the independent effect of physiological increases of fetal
plasma insulin concentrations on fetal amino acid utilization rates by
quantification of total fetal amino acid uptake rate of all 21 physiologically normal amino acids under conditions of euaminoacidemia
as well as euglycemia. Studies were conducted in fetal sheep in third
trimester, when the fetal amino acid utilization rate is
~3.6-4.8
g · kg
1 · day
1 (15,
20). Total net uptake or net utilization rates of each amino
acid, defined as the sum of net umbilical uptake rate plus the rate of
intravenous infusion necessary to maintain control period
concentrations of each amino acid, were measured during fetal
hyperinsulinemia and euglycemia. Hyperinsulinemia was produced by fetal
intravenous insulin infusion, and euglycemia was maintained by the
glucose clamp technique. The purpose of this study was to test the
hypothesis that an acute (4-h) increase in fetal plasma insulin
concentration would increase the net utilization rate of all 21 of the
physiologically normal amino acids and produce a similar increase in
net fetal nitrogen utilization.
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MATERIALS AND METHODS |
Animal Care and Surgical Procedure
Studies were performed in late-gestation Columbia-Rambouillet
pregnant sheep obtained from a commercial breeder (Nebeker Ranch, Santa
Monica, CA). Pregnancies were time dated, and all were known singleton
pregnancies. After a 24-h fast, each ewe was prepared for surgery with
intravenous pentobarbital sodium sedation (5 mg/kg initial dose
followed by as-needed infusion) and lumbar intrathecal tetracaine
hydrochloride anesthesia (6 mg in hypertonic glucose). Ampicillin (500 mg) and gentamicin (80 mg) were given intramuscularly at the time of
surgery. After hysterotomy, fetal catheters for blood sampling were
inserted directly into an umbilical vein at the base of the cord and
advanced into the common umbilical vein and into the abdominal aorta
via hindlimb arteries. Catheters for infusions were inserted into the
femoral veins via hindlimb veins. Ampicillin (500 mg) was injected into
the amniotic fluid just before closing the uterus. Maternal catheters
were placed via a single groin incision into the femoral artery for
sampling and into the femoral vein for infusions. All catheters were
tunneled subcutaneously through a maternal skin incision and were
maintained within a plastic pouch secured to the ewe's flank.
Catheters were flushed every other day with heparinized saline (150 U
heparin · ml of 0.9% wt
1 · vol NaCl in
water
1). All animals recovered from surgery and were
standing, eating, and drinking by 6-8 h after surgery. The ewes
were maintained in a temperature-controlled environment (18 ± 2°C) and were allowed ad libitum access to alfalfa pellets, water,
and a mineral block. Ewes were kept in a standard cart next to
similarly housed sheep. All studies were approved by the University of
Colorado Health Sciences Center (UCHSC) Animal Care and Use Committee.
Studies were performed at the UCHSC Perinatal Research Facility, which is accredited by the National Institutes of Health, the US Department of Agriculture, and the American Association for the Accreditation of
Laboratory Animal Care.
Experimental Design
The experimental study was conducted after a postoperative
recovery period of
5 days. Seventeen studies were performed in 13 animals. For animals studied more than once, the second study was at a
different insulin infusion rate, and there was a minimum 5-day interval
between studies. Figure 1 shows the
overall study design. A primed constant infusion of
3H2O of 16.8 µCi/h into the fetal femoral
vein was started at time 0 to measure umbilical blood flow
by the transplacental steady-state diffusion technique
(21). Control period blood samples were obtained at 90, 95, 100, and 105 min of the 3H2O infusion
during the control period conditions of euinsulinemia, euglycemia, and
euaminoacidemia.

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Fig. 1.
Study design. Each animal was studied during the control period
(normal fetal concentrations of insulin, glucose, and amino acids) and
after 4 h of hyperinsulinemia, euglycemia, and eulysinemia
(insulin infusion period).
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Insulin (pure pork insulin, Eli Lilly, Indianapolis, IN) was prepared
by diluting insulin in 0.9% (wt/vol) sodium chloride in water. At 110 min, the fetus was given a 1.0 mU/kg insulin bolus followed by a
continuous insulin infusion of 1.0 mU · min
1 · kg
1 estimated
fetal weight to produce fetal hyperinsulinemia. This primed infusion
rate has previously been shown to increase fetal plasma insulin
concentration approximately fivefold (11). Blood (0.3 ml)
was sampled every 10-15 min for the remainder of the study for
fetal arterial plasma glucose concentration, and a fetal intravenous
infusion of D25W (25% wt/vol dextrose in water) was adjusted to maintain the hyperinsulinemic euglycemic clamp. Thirty minutes after start of the glucose clamp infusion, an amino acid mixture (Trophamine, Kendall-McGaw Laboratories, Irvine, CA) was infused at 1.5 ml/h. Euaminoacidemia was achieved by amino acid clamp
with lysine as an indicator amino acid. Blood (0.3 ml) was sampled
every 10-15 min for rapid measurement of fetal lysine concentration (1), and the infusion rate of Trophamine was adjusted to maintain lysine within 5-10% of control period
concentration and to approximate control period concentrations of other
amino acids.
For most animals, steady-state glucose and lysine concentrations,
clamped to control period concentrations, were achieved ~4.5 h into
the study (range 220-400 min after steady state was established).
A second series of four blood draws at 5-min intervals was obtained at
the end of the insulin infusion period of hyperinsulinemia, euglycemia,
and euaminoacidemia. Blood was sampled simultaneously from fetal
arterial and umbilical venous catheters. Samples were analyzed for
hematocrit, blood oxygen content, oxygen saturation, and concentrations
of plasma glucose, lactate, insulin, amino acids, and
3H2O.
Maternal blood equal to the total volume of blood removed from the
fetus during steady-state draws was transfused into the fetus at a
relatively constant rate, with one-half of the volume given during the
20 min before the steady-state draws and the other one-half at the end
of the steady-state draw (for the first steady state only, because the
animal was euthanized immediately after the final blood samples were
obtained). Isovolumetric transfusions also were given approximately
every 30 min during the glucose clamp between the two sampling periods.
Approximately 12% of fetal blood volume was replaced during the study.
At the end of the study, euthanasia solution was injected into the
mother (12 ml intravenously) and fetus (2 ml intracardiac; Sleepaway,
pentobarbital sodium in 10% alcohol, Fort Dodge Laboratories, Fort
Dodge, IA). At autopsy, the fetus, uterus, uterine membranes, and
cotyledons were removed and weighed separately. Fetal study weight was
extrapolated from gestational age at study and from weight and
gestational age at autopsy according to ovine in utero growth curves
developed in our laboratory for the breed of sheep used in this study.
Blood Sampling Technique and Analytical Methods
Fetal arterial (2.5 ml) and venous (1.8 ml) blood samples for
measurement of plasma glucose, lactate, and insulin concentrations were
collected in plastic syringes lined with EDTA and in heparin-coated capillary syringes for determination of hemoglobin concentration and
oxygen saturation. Plasma was separated within 5 min of sampling in a
refrigerated centrifuge. Samples were processed immediately for plasma
glucose and lactate (YSI Glucose and Lactate Analyzer, Yellow Springs
Instruments, Yellow Springs, OH) and blood oxygen content (OSM III
Hemoximeter, Radiometer, Copenhagen, Denmark, calibrated for fetal
ovine hemoglobin). Fetal arterial plasma for glucose and lysine clamp
assays (0.6 ml) was collected in plastic syringes lined with EDTA.
Lysine concentration during the lysine clamp was measured by a 5-min
enzymatic determination of plasma lysine concentration, as described by
Beckett et al. (1). In the presence of the enzyme
saccharopine dehydrogenase (SaDH, EC 1.5.1.7) and NADH, lysine combines
with
-ketoglutarate to form saccharopine. The rate of conversion of
NADH to NAD in the early reaction is proportional to the lysine
concentration. The rate of change in spectrophotometric absorbance at
340 nm during this reaction (Beckman DU-7 Spectrophotometer,
Beckman Instruments, Fullerton, CA) was used to calculate plasma lysine concentrations from rate of change in absorbance obtained from lysine standards.
Fetal arterial plasma for insulin concentration was frozen immediately
and stored at
70°C until analysis; concentrations were determined
with a radioimmunoassay kit (Binax, South Portland, ME) using ovine
standards provided by Eli Lilly. 3H2O was
measured in 0.1-ml plasma samples that were solubilized in 1.0 ml of
Soluene-350 (quaternary ammonium hydroxide in toluene, Packard) and
then mixed with 15 ml Hionic Fluor (Packard). The 3H
radioactivity was measured in a Packard Tri-Carb 460 C liquid scintillation counter. Blood samples for determination of plasma amino
acid concentrations were collected in EDTA-coated syringes and
centrifuged, and the plasma was stored at
70°C until analysis. Plasma concentrations were measured using a Dionex 300 model 4500 amino
acid analyzer (Dionex, Sunnyvale, CA) after deproteinization with
sulfosalicyclic acid.
Calculations
Plasma and blood flows and net fetal uptakes of amino acids,
glucose, and oxygen.
Umbilical plasma flows (PFumb, ml/min) were calculated from
3H2O samples by use of the steady-state
transplacental diffusion method with tritiated water as the flow
indicator (21). Umbilical blood flows (BFumb,
ml/min) were calculated as (5)
According to Chung et al. (5), amino acid transport
out of the plasma is restricted primarily to plasma amino acids, and plasma concentration differences across organs are ~50% greater than
whole blood concentration differences. Thus Chung et al. have shown
that net umbilical amino acid uptakes are more accurately determined by
measuring plasma uptakes. Therefore, net umbilical amino acid uptake
rates (µmol/min) were determined by
where [
AA]v-a is the plasma amino acid
concentration difference between the umbilical venous (v) and fetal
arterial (a) vessels. Net umbilical glucose uptake rates were
calculated in the same manner, but net umbilical oxygen uptake rate was
determined by blood flow rather than by plasma flow.
Net fetal amino acid utilization and clearance rates.
The net fetal utilization rate of each amino acid during the
hyperinsulinemic clamp was calculated as the sum of the net umbilical uptake rate of each amino acid plus the steady-state rate of infusion of each amino acid in Trophamine into the fetus. Clearance rate for
each amino acid was calculated as the net amino acid utilization rate
divided by the arterial plasma concentration of the amino acid.
Clearance rate was used to account for the effect of changes in fetal
amino acid concentration produced by the Trophamine infusion on total
fetal amino acid uptake rate, assuming an imperfect clamp of each amino
acid and, arbitrarily, a linear relationship between net fetal amino
acid utilization rate and fetal arterial plasma amino acid
concentration over the range of amino acid concentrations achieved
during the study.
Statistical Analysis
Results are expressed as means ± SE. Differences in
measurements between control and insulin infusion periods were assessed by two-tailed paired t-tests. A mixed-effects modeling
approach was employed to determine the effect of fetal insulin
concentration on fetal amino acid utilization rate for each amino acid.
The latter model accounts for the variability between multiple
experimental runs on sheep, the variability within measurements of a
single experimental run on a sheep, and variability between sheep.
Best-fitting models were selected using Akaike's Information Criterion
(14). The linear model and the nonlinear Michaelis-Menten
model employed were chosen not only because they fit the data well, but
also because of the interpretability of their parameters (12,
19). Although use of a Michaelis-Menten model hints at knowledge
of kinetics, its use here is purely empirical. The Michaelis-Menten constant (Km) for the model, defined as the
insulin concentration required to achieve the half-maximal amino acid
utilization rate (Vmax/2), was estimated for each amino
acid. Within the framework of the above model, Scheffé-type
simultaneous confidence bands were employed to determine the substrate
dissociation constant (Ks) for each amino acid,
the fetal insulin concentration above which there is no significant
increase in net fetal amino acid utilization rate (30).
Ks was determined for each amino acid by holding
Km constant and determining the concentration of
insulin at which the difference between the measured asymptote and the model calculation values of net fetal amino acid utilization rate were
not significantly different from zero (31).
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RESULTS |
Fetal age and study weights for 17 experimental studies are shown
in Table 1. There were no differences in
fetal weight specific umbilical plasma or blood flow rates between the
control and insulin infusion periods (Table
2). There were small but significant differences in fetal arterial blood oxygen content between the two
periods, but there were no differences in fetal oxygen or lactate
uptake rates between periods (Table 2).
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Table 2.
Mean umbilical and uterine plasma and blood flow rates, fetal arterial,
plasma oxygen, and lactate concentrations, and oxygen and lactate
umbilical uptake rates during control and insulin infusion periods
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Figure 2 shows the mean (±SE) values
among all studies for fetal arterial plasma rapid assay lysine
concentrations and Trophamine infusion rates during the study. The
Trophamine infusion rate was increased in response to a rapid early
decrease during the first 15-30 min of the insulin infusion and
was then adjusted up or down in response to subsequent rapid assay
lysine concentrations. The final sampling period mean rapid assay
lysine concentration, 61.6 ± 6.1 µU/ml, was 11.6% higher than,
but not statistically different from, the control period concentration
of 55.2 ± 5.0 µU/ml.

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Fig. 2.
Rapid assay fetal arterial plasma lysine concentrations
(  ) and Trophamine infusion rates (-×-) during the
study.
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As shown in Fig. 3, the insulin infusion
increased the mean fetal arterial plasma insulin concentration during
the final sampling period of the insulin clamp by 5.2 ± 0.8-fold
(P < 0.0001) from 15 ± 2 µU/ml control to
84 ± 12 µU/ml hyperinsulinemia. Mean fetal arterial plasma
glucose (1.19 ± 0.04 µmol/ml control, 1.19 ± 0.05 µmol/ml hyperinsulinemia) and lysine concentrations (57.5 ± 6.4 µmol/l control, 57.9 ± 5.8 µmol/l hyperinsulinemia) during
the final sampling period were not different from those in the control period. Mean umbilical glucose uptake rate increased minimally from
control to hyperinsulinemia (21.1 ± 1.0 µmol · min
1 · kg
1
control, 24.3 ± 1.2 µmol · min
1 · kg
1
hyperinsulinemia, P = 0.018), whereas mean fetal
glucose utilization rate (i.e., umbilical glucose uptake rate plus the
steady-state glucose infusion rate into the fetus) increased much more
significantly, by 2.4 ± 1.8-fold (21.1 ± 1.0 µmol · min
1 · kg
1
control, 48.1 ± 2.7 µmol · min
1 · kg
1
hyperinsulinemia, P < 0.00001).

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Fig. 3.
Fetal arterial plasma insulin, lysine, and glucose concentrations
and fetal glucose utilization rates in control and insulin infusion
periods (means ± SE); * P < 0.00001.
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Fetal arterial amino acid concentrations are shown in Fig.
4. Significantly higher essential amino
acid concentrations, except for threonine, were noted during the
insulin infusion period (mean increase 15.8 ± 4.9%) and lower
concentrations for most of the nonessential amino acids (mean change
3.0 ± 3.2%); the mean change for all amino acids was +4.1 ± 3.3% (P < 0.05). There was no change in mean
umbilical venoarterial plasma concentration difference (data not shown)
or fetal weight specific umbilical blood flow rate (Table 2) between
the control and the insulin infusion periods. As a result, mean net
umbilical uptake rates were not different between control (2.01 ± 0.55 µmol · min
1 · kg
1)
and insulin infusion periods (2.14 ± 0.56 µmol · min
1 · kg
1; Fig.
5). However, there was an overall
tendency for umbilical uptake rates of each amino acid to be slightly
higher in the insulin infusion period, except for histidine and
arginine, which were slightly lower, and aspartate, which was less
negative. The infusion rate of each amino acid, calculated as the
product of the Trophamine amino acid concentration and the Trophamine
infusion rate, is shown in Table 3. The
mean infusion rate for all amino acids contained in Trophamine was
1.43 ± 0.21 µmol · min
1 · kg
1; the
mean infusion rate for the essential amino acids was 1.60 ± 0.31 µmol · min
1 · kg
1, and it
was 1.28 ± 0.30 µmol · min
1 · kg
1 for the
nonessential amino acids. Of note, Trophamine does not contain the
nonessential amino acids ornithine, glutamine, asparagine, or
citrulline. The net fetal utilization rate for each amino acid was
significantly increased in the insulin infusion period for all amino
acids except serine, glutamate, citrulline, and taurine. Serine and
citrulline ended up with a positive net utilization rate in the insulin
infusion period, and the net utilization rate of taurine decreased,
whereas glutamate ended up with a less negative net utilization rate
(Fig. 5). The mean net utilization rate for all amino acids in the
insulin infusion period was 3.29 ± 0.67 µmol · min
1 · kg
1,
representing a 63.7 ± 17.1% increase above the mean control period rate (P < 0.00005). This net increase in net
amino acid utilization rate accounted for a 60.1% increase in net
fetal nitrogen uptake rate of 775 mg · day
1 · kg
1. Net
nitrogen uptake rates for each amino acid are shown in Table 4.

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Fig. 4.
Fetal arterial plasma amino acid concentrations in control and
insulin infusion periods (means ± SE); * P < 0.05, ** P < 0.005.
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Fig. 5.
Fetal umbilical amino acid uptake rates in control and insulin
infusion periods and amino acid utilization rates in the insulin
infusion period (means ± SE) for those amino acids contained in
Trophamine (excludes asparagine, glutamine, citrulline, and ornithine).
Significant differences were noted between control and insulin infusion
period values of amino acid utilization rate (considered equal to
umbilical uptake rate in the control period and the sum of umbilical
uptake rate plus Trophamine infusion rate in the insulin infusion
period) for all essential and most nonessential amino acids.
* P < 0.05, ** P < 0.001.
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Fetal amino acid clearance rate increased during the insulin infusion
period for all essential amino acids and for all nonessential amino
acids except serine and taurine. The mean percent increase in clearance
rate for all amino acids was 64.5 ± 18.9% (P < 0.001, Fig. 6). Leucine was selected as a
representative amino acid to assess the effects of a relatively large
increase in amino acid concentration on clearance rate and the increase
in fetal amino acid utilization rate during the insulin infusion
period. Leucine concentration during the insulin infusion was 18.0%
above the control period concentration despite a near-perfect lysine
clamp. Leucine clearance rate, as a measure of insulin effect on total fetal leucine uptake rate independent of leucine concentration, was
enhanced by 45.4% in response to increased insulin concentration. At
the same time, the fetal leucine utilization rate was increased by
68.5% during the insulin infusion period. Thus 66.3% of the increase
in fetal leucine utilization rate (2.27 µmol · min
1 · kg
1) was
insulin specific, calculated as the 45.4% increase in leucine clearance rate divided by the 68.5% increase in fetal leucine utilization rate. The remaining 33.7% increase in fetal leucine utilization rate (1.15 µmol · min
1 · kg
1) was
specific to the leucine concentration in the insulin infusion period,
which was higher than in the control period.

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Fig. 6.
Amino acid clearance rates in control and insulin infusion periods
(means ± SE). Clearance rate was increased for all of the
essential amino acids except histidine and for most of the nonessential
amino acids. * P < 0.05, ** P < 0.001.
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The nonlinear random mixed-effects model based on a Michaelis-Menten
analysis provided a best fit of the data for all amino acids except
valine and glycine, which were best represented by a simple
least-squares linear regression model, and tyrosine, which was equally
well represented by a linear or a nonlinear model. The best-fit models
for tyrosine demonstrated that there was no significant increase in
amino acid utilization rate, as insulin concentration increased above
the control period insulin concentrations. Serine, glutamate, and
citrulline had negative control period umbilical uptake rates. The
slope or "sensitivity" of the insulin effect on fetal amino acid
utilization rate was greater for the essential amino acids than for the
nonessential amino acids, i.e., they had a lower
Km. Representative Michaelis-Menten curves for
leucine and lysine are shown in Fig. 7.
The mean Km for all amino acids was 11 ± 2 µU/ml, not different from the mean control period insulin
concentration of 15 ± 2 µU/ml. As shown in Table
5, the mean Km
(12.4 ± 1.5 µU/ml) and individual Km values of the essential amino acids were in the physiological range of
plasma insulin concentrations in late-gestation fetal sheep
(11). Additionally, the mean Ks for
all of the amino acids was 36.5 ± 4.6 µU/ml, and for the
essential amino acids (Table 5) it was 40.2 ± 5.0 µU/ml.

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Fig. 7.
Michaelis-Menten curves describing the effect of fetal
arterial plasma insulin concentration on fetal leucine (top:
Leu utilization rate = 9.5 µmol · min 1 · kg 1 × [I]/11 µU/ml + [I] ± 1.46) and lysine (bottom:
Lys utilization rate = 5.2 µmol · min 1 · kg 1 × [I]/17 µU/ml + [I] ± 0.66) utilization rates, where [I]
is the experimental steady-state fetal arterial plasma insulin
concentration. * Km fetal insulin
concentration, which is the insulin concentration required to achieve
the half-maximal total amino acid utilization rate.
** Ks value, the fetal plasma insulin
concentration above which there is no further measurable increase of
amino acid utilization rate.
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Table 5.
Michaelis-Menten constants for the effect of fetal arterial plasma
insulin concentration on the fetal utilization rate of the essential
amino acid
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DISCUSSION |
We used a novel approach with a
hyperinsulinemic-euglycemic-euaminoacidemic clamp in late-gestation
fetal sheep to measure the effect of an acute (4-h) increase in fetal
plasma insulin concentration on net umbilical (fetal) uptake and
utilization rates of all 21 physiologically normal amino acids,
independent of insulin-induced changes in fetal plasma amino acid and
glucose concentrations. There were no significant changes in net
umbilical uptake rates for any amino acid during hyperinsulinemia. Thus the amino acid infusion rates during the lysine clamp period accounted for essentially all of the effect of the hyperinsulinemia on increased amino acid utilization. Increased fetal plasma insulin concentrations for 4 h increased fetal utilization rates of all essential and most nonessential amino acids by ~64%, producing a similar percent increase in the net fetal uptake rate of nitrogen derived from amino
acids. Clearance rate, which accounts for the effect of a change in
fetal arterial plasma amino acid concentration on fetal amino acid
utilization rate, also was increased for nearly all of the amino acids;
the mean increase was ~65%. The mean change in concentration of all
amino acids was a positive 4.1%; thus ~96% of the increase in fetal
amino acid and nitrogen utilization rates was due to the independent
effect of an increase in fetal arterial plasma insulin concentration.
We also used a nonlinear random mixed-effects model based on a
Michaelis-Menten analysis to determine the impact of insulin concentration on the fetal utilization rate for each amino acid. The
general equation for this model follows the form
where y is the rate of amino acid utilization,
Vmax is the asymptote value for amino acid utilization,
[I] is an experimental fetal arterial plasma insulin concentration,
and Km is the insulin concentration at
Vmax/2. An additional parameter, Ks,
was calculated as the fetal insulin concentration at which the
difference between the amino acid utilization rate predicted by the
model and the Vmax for each amino acid was not
significantly different from zero. The effect of insulin to increase
amino acid utilization rates was highly significant over the normal
range of fetal arterial plasma insulin concentrations and those that
occur after a physiological increase in fetal plasma glucose
concentration (11). Furthermore, the effect of insulin was
maximal within the high physiological range of insulin concentrations
in fetal sheep (11). These observations define a
relatively high sensitivity of the effect of plasma insulin concentration, independent of changes in plasma glucose and amino acid
concentrations, on amino acid utilization in the late-gestation ovine
fetus. Normal dietary variations in maternal and fetal glucose concentrations, as well as experimental manipulations of fetal glucose
concentrations, produce a similar magnitude of change in fetal insulin
concentrations (11, 26). Thus it is reasonable to conclude
that physiological increases in insulin concentration in late-gestation
fetal sheep have the capacity to promote acute increases in fetal amino
acid and nitrogen utilization. Thus fetal plasma insulin is clearly an
acute regulator of amino acid metabolism.
Only two previous studies of the effect of insulin on fetal amino acid
metabolism used an amino acid infusion to try to maintain certain amino
acid concentrations relatively constant (21, 24, 28). Our
approach using lysine as an indicator amino acid to estimate clamping
of all amino acids was remarkably effective, producing near-control
period concentrations of all of the amino acids and very small or no
changes in net umbilical uptake rate for any amino acid between the
control and the insulin infusion periods. This allowed the rate of
amino acid infusion to represent most of the effect of hyperinsulinemia
on net fetal amino acid utilization. Thus it is reasonable to conclude
that the increase in net amino acid utilization that we measured during
the hyperinsulinemia period represented an effect of hyperinsulinemia
for all of the amino acids that was relatively independent of plasma
amino acid concentrations. For the purposes of this study, we
arbitrarily assumed a linear relationship between net fetal amino acid
utilization rate and fetal arterial plasma amino acid concentration
over the range of amino acid concentrations achieved during the study. Clearly, this reasonable but unproven assumption needs to be
experimentally validated.
The present study was limited to measurements of amino acid uptake and
utilization rates for the whole fetus. Individual organ amino acid
utilization rates might vary considerably, both for total amino acid
utilization and for the balance or pattern of utilization among the
individual amino acids. Furthermore, the present studies were acute,
representing only 4 h of a change in fetal plasma insulin
concentration. Not determined, therefore, are the potential adaptations
of amino acid utilization for the whole fetus and/or for individual
organs in response to sustained changes in fetal insulin concentration.
In comparison, studies of insulin and glucose effects on fetal glucose
metabolism have shown marked time-dependent metabolic adaptations to
chronic vs. acute changes in fetal plasma insulin and glucose
concentrations, both at the physiological level and at the level of
glucose metabolic enzyme and transporter expression and activity
(2, 3, 6, 7, 25). It is reasonable to consider, therefore,
that similar adaptations might occur in amino acid uptake and
metabolism and the regulatory mechanisms responsible for such
adaptations in response to chronic changes in fetal insulin
concentration; such adaptations and their regulation ought to be
defined in future studies.
The present study was also limited to measurements of amino acid uptake
and utilization rates. Specifically, we did not determine the extent to
which experimental hyperinsulinemia promotes net amino acid accretion
in protein, amino acid oxidation in exchange for glucose and lactate
oxidation, or both. Tracers would be necessary to determine the effects
of insulin on specific aspects of amino acid and protein metabolism,
including turnover, synthesis, breakdown, oxidation, and accretion.
Such studies have been performed in postnatal conditions primarily with
the use of leucine as a representative amino acid. From such studies in
postnatal life, it appears that insulin plays a significant anabolic
role, primarily by decreasing protein breakdown to a greater extent
than synthesis. In both adult humans (4, 8, 9, 10, 27) and
canines (13), hyperinsulinemia produced by insulin
infusion decreases leucine turnover, oxidation, nonoxidative disposal,
and appearance from protein breakdown, with a minimal effect on net
balance in protein accretion. In the one other study most comparable to
ours, insulin infusion into the chronically catheterized ovine fetus
under conditions of euglycemia and euleucinemia did not alter leucine
decarboxylation rate (24). These results support the
hypothesis that insulin-induced increase in fetal amino acid nitrogen
uptake rates, as documented in the present study, do lead, at least to
some extent, to increased protein accretion. Proof of this hypothesis
will require similar studies using carbon-labeled isotopes of essential
and nonessential amino acids to quantify amino acid oxidation,
synthesis, and breakdown and nitrogen-labeled isotopes to quantify
nitrogen accretion and excretion in urea.
Wray-Cahen et al. (29) studied the independent effect of
insulin on whole body amino acid disposal rate in neonatal pigs. Amino
acid disposal was significantly sensitive to insulin in the early (7 days old) vs. late (26 days old) postnatal period. Additional studies
in the fetus, therefore, are indicated to determine potential
developmental changes during gestation on the sensitivity of insulin to
affect fetal amino acid utilization.
Although this study did achieve relative euaminoacidemia and euglycemia
during a period of fetal hyperinsulinemia, interpretation of the
results specific to hyperinsulinemia still is incomplete, because the
effect of increased fetal glucose utilization rate on total fetal amino
acid uptake rate was not accounted for. Based on the data of Liechty et
al. (18), it is possible that the simultaneously increased
glucose utilization rate seen in this study would have decreased amino
acid oxidation, thereby contributing indirectly to a greater likelihood
of an anabolic effect of insulin. In short- and long-term ovine fetal
hypoglycemia induced by maternal insulin infusion, however, the
resulting decreased fetal glucose utilization rate was not associated
with increased amino acid oxidation (3, 22). Thus it is
not clearly predictable what the relative rates of oxidative and
nonoxidative amino acid metabolism would be in the presence of
hyperinsulinemia with no change in simultaneous rates of glucose utilization.
In summary, this study uniquely defines the effect of acute (4-h)
increased fetal plasma insulin concentrations on umbilical (fetal)
amino acid uptake and utilization rates of all amino acids independent
of changes in fetal plasma concentrations of amino acids as well as
glucose. This effect of insulin occurs at physiological insulin
concentrations, indicating that changes in insulin concentration in the
fetus that are likely to occur in response to normal dietary variations
in the mother, particularly for glucose, should lead to direct and
immediate regulation of fetal amino acid metabolism. These observations
and the relatively steep slope of the increase in fetal amino acid
utilization rate with physiological increases in fetal plasma insulin
concentration define a relatively high sensitivity to the effect of
insulin on amino acid utilization. The effect of increased fetal plasma
insulin concentrations on amino acid metabolism independent of
insulin-induced increased rates of glucose utilization remains to be
determined, as do interactive effects of insulin, amino acid, and
glucose concentrations on amino acid metabolism.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
HD-20761 and DK-52138. Dr. Scheer was supported by National Institutes
of Health/National Institute of Child Health and Human Development
Student Research Training Grant HD-07446, sponsored by the Society for
Pediatric Research and the American Pediatric Society.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: P. J. Thureen, Campus Box B-195, Univ. of Colorado Health Sciences Center,
4200 East 9th Ave., Denver, CO 80262 (E:mail:
patti.thureen{at}uchsc.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.
Received 20 January 2000; accepted in final form 24 July 2000.
 |
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