INTRODUCTION

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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 · kg¹ · day¹ (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.

	MATERIALS AND METHODS

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Animal Care and Surgical Procedure

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1

Experimental Design

View larger version (24K): [in this window] [in a new window]   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).

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 · min¹ · kg¹ 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 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 (PF_umb, 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 (BF_umb, 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

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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).

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.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Rapid assay fetal arterial plasma lysine concentrations () and Trophamine infusion rates (-×-) during the study.

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 · min¹ · kg¹ control, 24.3 ± 1.2 µmol · min¹ · kg¹ 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 · min¹ · kg¹ control, 48.1 ± 2.7 µmol · min¹ · kg¹ hyperinsulinemia, P < 0.00001).

View larger version (21K): [in this window] [in a new window]   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.

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 · min¹ · kg¹) and insulin infusion periods (2.14 ± 0.56 µmol · min¹ · kg¹; 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 · min¹ · kg¹; the mean infusion rate for the essential amino acids was 1.60 ± 0.31 µmol · min¹ · kg¹, and it was 1.28 ± 0.30 µmol · min¹ · kg¹ 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 · min¹ · kg¹, 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 · day¹ · kg¹. Net nitrogen uptake rates for each amino acid are shown in Table 4.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Fetal arterial plasma amino acid concentrations in control and insulin infusion periods (means ± SE); * P < 0.05, ** P < 0.005.

View larger version (33K): [in this window] [in a new window]   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.

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 · min¹ · kg¹) 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 · min¹ · kg¹) was specific to the leucine concentration in the insulin infusion period, which was higher than in the control period.

View larger version (22K): [in this window] [in a new window]   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.

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 K_m. Representative Michaelis-Menten curves for leucine and lysine are shown in Fig. 7. The mean K_m 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 K_m (12.4 ± 1.5 µU/ml) and individual K_m values of the essential amino acids were in the physiological range of plasma insulin concentrations in late-gestation fetal sheep (11). Additionally, the mean K_s 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.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Michaelis-Menten curves describing the effect of fetal arterial plasma insulin concentration on fetal leucine (top: Leu utilization rate = 9.5 µmol · min1 · kg1 × [I]/11 µU/ml + [I] ± 1.46) and lysine (bottom: Lys utilization rate = 5.2 µmol · min1 · kg1 × [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.

	DISCUSSION

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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, V_max is the asymptote value for amino acid utilization, [I] is an experimental fetal arterial plasma insulin concentration, and K_m is the insulin concentration at V_max/2. An additional parameter, K_s, was calculated as the fetal insulin concentration at which the difference between the amino acid utilization rate predicted by the model and the V_max 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.