Maternal high-protein supplements designed to increase birth weight have not been successful. We recently showed that maternal amino acid infusion into pregnant sheep resulted in competitive inhibition of amino acid transport across the placenta and did not increase fetal protein accretion rates. To bypass placental transport, singleton fetal sheep were intravenously infused with an amino acid mixture (AA, n = 8) or saline [control (Con), n = 10] for ∼12 days during late gestation. Fetal leucine oxidation rate increased in the AA group (3.1 ± 0.5 vs. 1.4 ± 0.6 μmol·min−1·kg−1, P < 0.05). Fetal protein accretion (2.6 ± 0.5 and 2.2 ± 0.6 μmol·min−1·kg−1 in AA and Con, respectively), synthesis (6.2 ± 0.8 and 7.0 ± 0.9 μmol·min−1·kg−1 in AA and Con, respectively), and degradation (3.6 ± 0.6 and 4.5 ± 1.0 μmol·min−1·kg−1 in AA and Con, respectively) rates were similar between groups. Net fetal glucose uptake decreased in the AA group (2.8 ± 0.4 vs. 3.9 ± 0.1 mg·kg−1·min−1, P < 0.05). The glucose-O2 quotient also decreased over time in the AA group (P < 0.05). Fetal insulin and IGF-I concentrations did not change. Fetal glucagon increased in the AA group (119 ± 24 vs. 59 ± 9 pg/ml, P < 0.05), and norepinephrine (NE) also tended to increase in the AA group (785 ± 181 vs. 419 ± 76 pg/ml, P = 0.06). Net fetal glucose uptake rates were inversely proportional to fetal glucagon (r2 = 0.38, P < 0.05), cortisol (r2 = 0.31, P < 0.05), and NE (r2 = 0.59, P < 0.05) concentrations. Expressions of components in the mammalian target of rapamycin signaling pathway in fetal skeletal muscle were similar between groups. In summary, prolonged infusion of amino acids directly into normally growing fetal sheep increased leucine oxidation. Amino acid-stimulated increases in fetal glucagon, cortisol, and NE may contribute to a shift in substrate oxidation by the fetus from glucose to amino acids.
- fetal protein metabolism
- fetal growth
previous attempts to improve fetal outcomes with use of maternal high-protein supplementation have actually increased the risk for small-for-gestational-age birth and infant mortality (26, 36). Similarly, supplementation of pregnant sheep with intravenous amino acids for several days at the end of gestation demonstrated adverse fetal effects, including hypoxia and respiratory and metabolic acidosis (34). The mechanisms that explain poor fetal outcomes in response to maternal protein supplementation during pregnancy are unknown.
Experiments using normal pregnant sheep have led to some testable explanations for how high-protein supplementation during pregnancy might adversely affect fetal outcomes (4). Short- and long-term maternal amino acid infusions using mixtures of amino acids with a higher essential-nonessential amino acid balance increased fetal concentrations of branched-chain amino acids (BCAA; leucine, isoleucine, and valine) but decreased fetal concentrations of other amino acids, including the essential amino acid threonine (4, 20, 34). These findings indicate that maternal amino acid supplementation results in competition among coinfused amino acids for transport across the placenta. Disruption of normal rates of amino acid transport into the fetus and, thus, abnormal fetal plasma amino acid concentrations could limit fetal protein synthesis and/or lead to abnormal amino acid metabolism, even to the point of producing metabolic acidosis. Also, despite increased fetal BCAA concentrations, lack of concurrent increases in fetal anabolic hormones such as insulin might minimize potential increases in protein accretion rates (34). Furthermore, prolonged maternal amino acid infusion increased net transport of leucine to the fetus, fetal leucine concentrations, and fetal leucine oxidation rates, indicating that the fetus uses excess amino acids for oxidative metabolism, rather than protein synthesis and accretion (34). It is not known whether these observations are specific to the mode of delivery of supplemental amino acids to the fetus (via the mother and placenta vs. a direct fetal infusion) or to the excess amino acids themselves. These questions become particularly relevant when testing interventions that might improve fetal growth after chronic exposure to placental insufficiency and improve organ-specific sensitivity to nutrients and hormones (e.g., insulin) later in life.
To determine fetal responses to prolonged, exogenous amino acid supplementation under normal conditions, an intravenous mixture of amino acids was infused directly into the ovine fetus for 10–14 days near the end of gestation. We hypothesized that a direct infusion of amino acids into a normally growing fetus, bypassing potential competitive inhibition of amino acid transport across the placenta, would promote increased rates of fetal protein synthesis and accretion. However, contrary to our hypothesis, our results showed that only fetal leucine oxidation was increased during the fetal amino acid infusion compared with saline-infused, control fetuses. We also compared fetal gas exchange, glucose and lactate uptake rates, and hormone profiles between amino acid- and saline-infused groups.
MATERIALS AND METHODS
Studies were conducted in 18 Columbia-Rambouillet ewes carrying singleton pregnancies during late gestation (full term = 147 days gestational age). Indwelling catheters were surgically placed into the vasculature of the mother and fetus at ∼120 days gestational age, as previously described (5). Catheterized vessels included the maternal femoral artery and vein, fetal abdominal aorta, fetal femoral veins, and umbilical vein. All animal procedures were in compliance with the guidelines of the US Department of Agriculture, the National Institutes of Health, and the American Association for the Accreditation of Laboratory Animal Care. The animal care and use protocols were approved by the University of Colorado Institutional Animal Care and Use Committee.
Ewes were randomly assigned to a fetal amino acid infusion (AA) group (n = 8) or a fetal saline infusion control (Con) group (n = 10). Because of umbilical venous catheter failure, complete metabolic studies, including fetal substrate uptake measurements and amino acid metabolic rates, were completed in seven AA and six Con sheep. Animals were allowed to recover from surgery for ≥5 days prior to the start of the infusions. The AA group received a continuous, intravenous infusion of Trophamine, an amino acid mixture enriched with essential amino acids (University of Colorado Central Admixture Pharmacy, Aurora, CO). The rate of infusion was adjusted daily to achieve a 25–50% increase in fetal plasma BCAA concentrations (3). The Con group received a fetal intravenous infusion of 0.9% NaCl at a rate adjusted to match the rate of sodium delivery from Trophamine in the AA group. These infusions were administered directly into the fetal femoral vein. Prior to the start of the infusion, fetal arterial plasma was sampled for glucose, lactate, and amino acid concentrations, and fetal arterial whole blood was sampled for measurement of pH, Pco2, Po2, blood hemoglobin-O2 saturation, and O2 content. During the infusion period, fetal arterial plasma was sampled daily for glucose and lactate concentrations. On days 2, 5, 8, and 11 and on the final day of the infusion, fetal arterial blood was sampled for plasma amino acid concentrations, blood gas measurements, and plasma insulin concentrations. On the final day of experimental infusion, fetal arterial plasma was also sampled for IGF-I, norepinephrine (NE), cortisol, and glucagon concentrations. Maternal arterial blood was sampled prior to the start of the infusion, on day 8, and on the final day of the infusion for blood gas measurements, plasma glucose and lactate concentrations, and plasma amino acid concentrations.
On the final day of the infusion (mean duration of infusion was 12 ± 0.4 days and similar between groups), a metabolic study was performed to measure net umbilical (fetal) substrate uptakes and fetal leucine metabolism, as previously described (7). After samples were collected at time 0 for naturally occurring isotopic enrichments, a solution containing ethanol (300 mg bolus followed by 12.8 mg/min; AAPER Alcohol and Chemical, Shelbyville, KY) was infused into the fetal venous circulation to measure umbilical blood flow using the transplacental diffusion method (27). Each fetus also was infused with l-[1-13C]leucine (45.7 μmol/ml, starting bolus of 137 μmol followed by constant infusion at 1.5 μmol/min; Cambridge Isotope Laboratories, Woburn, MA) for measurement of fetal leucine metabolism. After 180 min of tracer infusion, four steady-state blood samples were drawn simultaneously from the umbilical vein and fetal abdominal aorta. Fetal arterial plasma was used to measure steady-state concentrations of amino acids, α-ketoisocaproic acid (KIC), glucose, lactate, and stable isotopic enrichments of [1-13C]leucine and [1-13C]KIC. Fetal whole blood was used to measure ethanol concentrations, fetal O2 content, and 13CO2.
Blood gas measurements, O2 content, hematocrit, and insulin, plasma amino acid, glucose, lactate, cortisol, and glucagon concentrations were measured as previously described (25, 34, 35). IGF-I concentrations were measured by ELISA (ALPCO Immunoassays, Salem, NH; intra- and interassay coefficients of variation = 5–7% and 2–7%, respectively). NE concentrations were measured using the following methods. After addition of the internal standard (3,4-dihydroxynorephedrine), plasma NE was purified using the diphenylborate extraction method. The extracted NE was reacted with benzylamine to form a fluorescent derivative (14, 43, 47, 48). NE and the internal standard derivatives were separated on HPLC using a reverse-phase column (XBridge C-18, Waters) and the Alliance HPLC system equipped with a fluorescence detector (model 2475, Waters). A standard curve was constructed using varying amounts of NE and a constant amount of 3,4-dihydroxynorephedrine. The ratio of the areas of NE to 3,4-dihydroxynorephedrine peaks was used to determine the amount of NE in plasma (intra-assay coefficient of variation = 16%).
Isotopic enrichments were determined using a gas chromatograph-mass spectrometer (model 5975, Agilent Technologies, Wilmington, DE) equipped with a HP5-MS column (30 m × 0.25 mm × 0.25 μm). Plasma KIC isotopic enrichment was measured after conversion to the α-hydroxyisocaproic acid tert-butyldimethylsilyl (t-BDMS) derivative. Plasma (0.05 ml) was mixed with 0.1 ml of water, and sodium borohydride (0.025 ml, 10 mg/ml in water) was added to reduce KIC to α-hydroxyisocaproic acid. The reduction reaction was carried out at ambient temperature for 15 min. The reaction was terminated by addition of 0.025 ml of 4 N HCl. Ethyl acetate (1 ml) was added to the acidified solution, and the sample was mixed vigorously for 1 min using a vortex. The ethyl acetate layer was washed with 0.5 ml of 0.04 M HCl, and the organic solvent was removed under reduced pressure. α-Hydroxyisocaproic acid was converted to the t-BDMS derivative by addition of 0.05 ml of t-BDMS reagent (50% N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tert-butyldimethylchlorosilane in anhydrous acetonitrile) to the ethyl acetate extract residue, and the reaction was carried out at 85°C for 20 min. The isotopic enrichment was determined using the selective ion monitoring method at mass-to-charge ratio of 304:303. Plasma leucine isotopic enrichment also was measured after conversion to the t-BDMS derivative. For purification of leucine, plasma (0.05 ml) was mixed with 0.05 ml of the internal standard, norleucine (10 μg/ml), and acidified by addition of 0.15 ml of 50% acetic acid. Cation exchanger (AG-50, H+ form, 50 mg) was added to the mixture. After centrifugation (7,000 g for 1 min), the supernatant was discarded. The ion exchanger was washed three times using 1 ml of 70% isopropanol. The amino acid was eluted using 0.6 ml of 5 N ammonium hydroxide. The ammonium hydroxide solution was transferred to a glass sample vial and dried under reduced pressure. Leucine was derivatized by addition of 0.05 ml of t-BDMS reagent (50% N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tert-butyldimethylchlorosilane in anhydrous acetonitrile) at 65°C for 1 h. The isotopic enrichment was determined using the selective ion monitoring method at mass-to-charge ratio of 303:302.
For determination of whole blood 13CO2 enrichment, CO2 was isolated from the blood by addition via syringe of 0.1 ml of saturated citric acid solution and measured by isotope ratio mass spectrometry, as previously described (5). Ethanol concentrations for umbilical blood flow were determined in triplicate aliquots with a quantitative enzymatic UV determination method (alcohol 332-UV, Sigma, St. Louis, MO) (10).
Umbilical blood flows were calculated by dividing umbilical plasma flow by (1 − fractional fetal hematocrit). Umbilical venous-fetal arterial plasma concentration differences in glucose, lactate, and individual amino acids were multiplied by the umbilical plasma flow (Fick principle) to calculate net fetal substrate uptake by the fetus from the placenta. The total fetal amino acid uptake rate was calculated by summing all the individual fetal amino acid uptake rates. Fetal O2 consumption rate was calculated by multiplying the umbilical venous-arterial blood O2 content difference by umbilical blood flow rate. Individual fetal amino acid delivery rates were determined by adding the net fetal uptake rate and the exogenous infusion rate of the amino acid from the Trophamine infusion. Similarly, the total fetal amino acid delivery rate was calculated by summing all the individual fetal amino acid delivery rates. Net fetal carbon uptake was calculated by multiplying the number of carbon atoms in the amino acids, glucose, and lactate by their respective net fetal uptake rates. [1-13C]leucine tracer fluxes between the placenta and fetal plasma and between fetal plasma and fetal tissues were calculated as previously described (Table 1) (7). The exogenous fetal leucine infusion rate from the Trophamine infusion was accounted for when fetal protein breakdown and accretion rates were calculated (Table 1). The glucose-O2 quotient was calculated by dividing the difference between the umbilical venous and fetal arterial glucose concentrations by the difference in the umbilical venous and fetal arterial O2 contents and multiplying that result by 6.
Organ isolation and measurements were performed at the completion of the physiological studies, as previously described (25, 34). Biopsies of the fetal biceps femoris muscle were taken under steady-state experimental conditions, snap-frozen in liquid nitrogen, and stored at −70°C until further analysis. Fetal liver was also collected under steady-state conditions and snap-frozen in liquid nitrogen.
Western blot analysis.
Protein was extracted from pulverized skeletal muscle (100 mg) and prepared for Western blot analysis, as previously described (6). Membranes were incubated with antibodies detecting phosphorylated (Ser473) protein kinase B (AKT), total AKT, phosphorylated (Thr421/Ser424) p70 S6 kinase (p70S6k), total p70S6k, phosphorylated (Ser235/236) ribosomal protein S6 (rpS6), total rpS6, phosphorylated (Thr37/46) eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), total 4EBP1, phosphorylated (Thr56) eukaryotic elongation factor 2 (eEF2), total eEF2, phosphorylated (Ser52) eIF2α, total eIF2α, phosphorylated (Ser2448) mammalian target of rapamycin (mTOR), and total mTOR [all diluted 1:500 in Tris-buffered saline + Tween 20 (TBST) with 5% BSA, except phosphorylated rpS6 (diluted 1:5,000) and total rpS6 (diluted 1:10,000); Cell Signaling Technology and Stressgen]. β-Actin (diluted 1:100,000 in TBST; MP Biomedical, Solon, OH) was used to control for loading differences, and reference samples were analyzed on every membrane to control for differences in transfer efficiency. Results from the densitometry are presented in arbitrary units and as fold change relative to control fetuses.
RNA isolation and real-time PCR.
RNA from fetal liver was isolated, reverse-transcribed, and used for real-time PCR (42). Real-time PCR assays for phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), and 18S rRNA (18S) were performed as previously described (42). Results for PEPCK and G6Pase expression are expressed relative to 18S expression.
Data analysis and statistics.
Statistical analysis was performed using SAS version 9.1 (SAS Institute). Values are means ± SE. A mixed-model ANOVA was performed to determine effects of treatment group (AA or Con), duration of the infusion had been running, and interaction between treatment group and the day of infusion to determine whether changes over time were different between the two treatment groups. A random animal term was included to account for repeat measurements made in the same animal. When ANOVA was significant, posttest comparisons were made using Fisher's least-squares difference. Measurements made once were compared by Student's t-test or Mann-Whitney U-test. P < 0.05 was considered significant; P < 0.1 was noted as a trend. The relationship between hormones and glucose uptake rate was analyzed by linear regression.
Fetal arterial plasma amino acid concentrations and delivery rates.
Fetal arterial plasma BCAA increased by day 2 of the infusion in the AA group (group × day interaction, P < 0.05; Fig. 1, A–C). This pattern of increase was representative of all other essential amino acid concentrations during the infusion, except for threonine, which did not differ between groups on any day during the infusion (data not shown). To achieve this, the exogenous fetal amino acid infusion rate was increased in a step-wise fashion each day of the infusion period, so that the AA group received an average of 7.4 ± 0.3 g/day of exogenous amino acids by the end of the infusion. On the final day of infusion, fetal arterial plasma concentrations of all essential amino acids were increased in the AA group compared with the Con group, except for histidine and threonine, which were similar (Fig. 1D). Most nonessential amino acid concentrations were similar between groups, except for taurine, proline, tryptophan, and ornithine, which were increased in the AA group (Fig. 1E).
Individual net fetal (umbilical) amino acid uptake rates from the placenta were similar between the groups (Fig. 2). However, the total fetal amino acid uptake rate (sum of the individual amino acid uptake rates) tended to be lower in the AA group (Table 2). The delivery rates (umbilical uptake + exogenous infusion) of the essential amino acids leucine, isoleucine, phenylalanine, and lysine were increased in the AA group on the final day of infusion (Fig. 2A). The delivery rates for the nonessential amino acids arginine, aspartate, and proline also were increased (Fig. 2B). Additionally, the total amino acid delivery rate (sum of the individual amino acid delivery rates) tended to be higher in the AA group (Table 2). The total amount of carbon delivered to the fetus from amino acids, glucose, and lactate combined was similar in both groups, as less carbon from glucose and more carbon from amino acids was delivered to the fetus in the AA group (Table 2).
Fetal acid-base balance and oxygenation.
Fetal arterial pH, Pco2, Po2, blood hemoglobin-O2 saturation, and blood O2 content did not change over time in the AA or Con group, nor did they differ between groups (Fig. 3). Fetal arterial hematocrit changed over time similarly in both groups (P < 0.05; Fig. 3F).
Fetal arterial plasma glucose concentrations decreased in the AA group (22.7 ± 1.5 mg/dl at baseline vs. 18.7 ± 0.8 mg/dl on the final day of infusion) but not in the Con group [20.2 ± 0.6 mg/dl at baseline vs. 19.0 ± 1.2 mg/dl on the final day of infusion, P < 0.005 (group × day interaction); Fig. 4A]. The fetal glucose-O2 quotient decreased in the AA group, but not in the Con group [P < 0.05 (group × day interaction); Fig. 4B]. Fetal arterial plasma lactate concentrations increased in the AA group initially (2.1 ± 0.1 mmol/l at baseline vs. 4.4 ± 1.3 mmol/l on day 7) and then returned to baseline [P < 0.0005 (group × day interaction); Fig. 4C].
Fetal arterial plasma insulin concentrations were measured during the infusion and on the final day of the infusion and were not statistically different between the groups (Table 2). Fetal plasma arterial IGF-I concentrations were measured at the end of the infusion period and also were similar between groups (Table 2). Fetal glucagon increased by 100% in the AA group compared with the Con group (Table 2). Although mean fetal plasma NE concentrations tended to be higher in the AA group (P = 0.06) and mean cortisol concentrations were not statistically different (Table 2), there was more variability in NE and cortisol concentrations in the AA group (P < 0.05, by F-test).
Fetal metabolic study.
On the final infusion day, net fetal (umbilical) substrate uptake and fetal leucine metabolic rates were determined under steady-state conditions. Umbilical blood and plasma flow rates did not differ between groups, nor did net fetal O2 consumption or lactate uptake rates (Table 2). Net fetal glucose uptake rate was 28% lower in the AA group than the Con group (P < 0.05, Table 2). Net fetal glucose uptake rate was inversely proportional to fetal cortisol, NE, and glucagon (Fig. 5). Calculated fetal leucine flux rates to determine protein metabolism are shown in Table 1. There were no differences in protein degradation, protein accretion, or protein synthesis rates between the groups. Fetal leucine oxidation rate was increased by 120% in the AA group (P < 0.05) compared with the Con group.
Fetal characteristics, organ weights, and tissue analysis.
Fetal weight, gestational age, crown-rump length, and weights for fetal organs, including liver, heart, lungs, kidneys, spleen, brain, and placentomes, were similar between the groups (Table 3). For seven proteins involved in the mTOR signaling pathway (4EBP1, eEF2, p70S6k, eIF2α, rpS6, AKT, and mTOR), there was no difference in the ratio of phosphorylated to total amount of each protein or in the total amount of each protein normalized to actin, with the exception of total 4EBP1 expression, which was decreased in the AA group (Fig. 6, Table 4). PEPCK and G6Pase mRNA abundance in the liver did not differ significantly between groups (Fig. 4, D and E).
Maternal parameters during treatment.
Maternal feed (1.5 ± 0.3 and 1.4 ± 0.2 kg/day for AA and Con, respectively) and water (6.0 ± 0.6 and 5.4 ± 0.3 l/day for AA and Con, respectively) intakes were similar between groups. Maternal glucose and lactate concentrations were similar between groups and did not change during the infusion period (maternal glucose = 71.8 ± 6.4 and 73.2 ± 2.5 mg/dl in AA and Con, respectively, on the final day; maternal lactate = 0.74 ± 0.06 and 0.84 ± 0.15 mmol/l in AA and Con, respectively, on the final day). Maternal blood gas measurements also were similar between groups and did not change during the infusion period (data not shown). Maternal amino acid concentrations remained similar between groups (data not shown), except for tryptophan, which was lower in the Con group on the final day [55 ± 6 vs. 35 ± 3 μmol/l, P < 0.05 (group effect)], and ornithine, which increased in the AA group only during the infusion [97 ± 8 and 107 ± 13 μmol/l at baseline and on the final day, respectively, P < 0.05 (group × day interaction)].
Previous studies showed that maternal high-protein supplementation during human pregnancy results in poor fetal outcomes, including fetal growth restriction. Using late-gestation pregnant sheep, this study aimed to determine responses to prolonged and exogenous fetal amino acid supplementation under normal conditions to understand the mechanisms that might explain how fetal growth is compromised when excess amino acids are delivered chronically to the fetus. The novel findings of this study are that a chronic infusion of mixed amino acids directly into a normally growing sheep fetus did not simply replace placental transport of amino acids to the fetus, as amino acid concentrations and delivery rates into the fetus were maintained or increased. The amino acid infusion increased fetal leucine oxidation but did not affect protein synthesis or accretion rates. Furthermore, there was a decrease in fetal glucose uptake from the placenta, indicating that exogenous amino acids were used as alternative oxidative substrates to glucose when provided in excess of normal rates of utilization (including synthesis into proteins and oxidation). Unlike maternal protein or amino acid supplementation, the fetal amino acid infusion was well tolerated by the fetus without causing acidemia or hypoxia.
Direct infusion of amino acids into the fetus during this study avoided competitive inhibition of coinfused amino acids across the placenta. Several previous studies showed that chronic or acute maternal infusion of amino acids into the pregnant sheep decreased fetal concentrations of several amino acids, despite increased or maintained maternal concentrations (18–20, 34). Most notably, fetal concentrations of the essential amino acid threonine appear to be particularly vulnerable under these experimental conditions (18–20, 34). Multiple amino acids share affinity for several transporter systems (32, 33). Specifically, the β°+ and ASC amino acid transporters share affinity for BCAA and threonine, which could explain unbalanced transport of amino acids across the placenta to the fetus when maternal amino acid concentrations are manipulated (8). Greater increases in maternal plasma BCAA than threonine would be expected to promote BCAA transport to the fetus and reduce threonine transport, producing outcomes for these amino acids similar to those found during the maternal Trophamine infusions (20, 34). Reduced fetal uptake of even one essential amino acid by competitive inhibition would contribute to reduced net protein synthesis throughout the fetal body and, thus, net protein balance and growth rate. In the present study, a direct fetal amino acid infusion increased fetal plasma concentrations of almost all essential amino acids and maintained fetal threonine concentrations. The direct fetal Trophamine infusion did not, however, increase fetal concentrations of all nonessential amino acids, some of which might be conditionally essential for maximal protein synthesis rates in the fetus and neonate.
Although the total delivery of amino acids (exogenous infusion + umbilical uptake rate) was higher to the AA than to the Con fetus, the total net fetal amino acid uptake rate by the fetus from the placenta tended to be lower in this group. Thus a direct fetal amino acid infusion might have decreased placental transport of amino acids. The mechanism for this remains unknown. However, previous studies in the sheep fetus demonstrated that a direct fetal glucagon infusion experimentally increasing glucagon concentrations inhibited umbilical amino acid uptake (19, 34, 41). In the present study, the amino acid infusion increased fetal plasma glucagon concentrations, indicating that the decreased umbilical uptake of amino acids in the AA group might be due to elevated glucagon concentrations. Another possible explanation for the lower net uptake of placental amino acids in the AA group is increased flux of amino acids back to the placenta. In fact, leucine flux from the fetus into the placenta was increased in the AA group; however, this difference was not statistically significant (Table 1).
Supplemental amino acids were not, however, used to increase fetal protein accretion or synthesis in the normally growing fetus, and excess leucine was oxidized. A similar increase in substrate oxidation has been observed when glucose is infused into the fetus (15). There are likely several mechanisms that explain the lack of increased protein synthesis or accretion in response to a direct amino acid infusion observed in our study. The major substrates for fetal oxidative metabolism are glucose, lactate, and amino acids (2, 45). In the present study, the rate of leucine oxidation was increased in the fetus, indicating that the fetus used the excess amino acids for oxidative, rather than anabolic, metabolism. We used leucine to estimate cumulative amino acid oxidation rates, although the metabolic fate of other specific amino acids may differ. Since O2 consumption did not change, it is likely that the fetus preferentially used leucine and other amino acids, instead of another substrate such as glucose or lactate, for oxidative metabolism. In fact, we observed a decreased glucose-O2 quotient in the AA group throughout the infusion period. Thus the fraction of the total fetal O2 consumption required to completely oxidize glucose acquired by the fetus from the umbilical circulation was less when amino acids were infused into the fetus (17). Furthermore, there was no difference in total carbon delivery to the fetus. Carbon delivery from amino acids increased, but carbon delivery from glucose decreased. Thus the AA group remained in relative metabolic balance by using less of the total O2 consumed to oxidize glucose and more to oxidize carbon from excess amino acid delivery, rather than increasing protein accretion rates.
Mechanisms that explain the placental changes in substrate delivery and fetal oxidative substrate switching in response to the amino acid infusion have yet to be determined. Net fetal glucose uptake from the placenta was decreased in the AA group. Fetal glucose uptake is directly related to the transplacental glucose gradient (16, 45). The maternal-fetal glucose gradients were similar between the two groups, indicating that other mechanisms are mediating changes in placental glucose transport and fetal glucose utilization. We found that cortisol, NE, and glucagon concentrations were inversely related to glucose uptake by the fetus. Previous studies in pregnant sheep showed that fetal hypercortisolemia reduced uteroplacental delivery of glucose and lactate to the fetus (12, 28, 29, 45). Increased NE concentrations also have been associated with reduced glucose oxidation rates and increased lactate production from glycolysis in states of chronic hypoglycemia as a result of placental insufficiency (46). Although the animals in the present study were not growth-restricted or subjected to hypoglycemia, the exogenous infusion of amino acids perturbed their normal metabolic milieu, resulting in adaptive responses that might be similar to the chronically stressed animal. Alternatively, it is possible that counterregulatory hormones were stimulated to maintain euglycemia in the fetus and increase fetal hepatic gluconeogenesis. We did not observe a significant increase in the expression of PEPCK and G6Pase, genes that encode enzymes that are rate-limiting for gluconeogenesis, although gene expression among animals was variable. Conversely, excess carbon delivery from the amino acid infusion might have driven cycling among alanine, pyruvate, and lactate without directly stimulating gluconeogenesis, as fetal lactate concentrations increased during the midportion of the infusion. We speculate that elevated fetal cortisol and NE concentrations facilitated changes in fetal substrate availability for oxidative metabolism to maintain O2 consumption rates, although an understanding of exact mechanisms by which these changes occur in the context of fetal nutrient manipulation requires further investigation.
Fetal insulin and IGF-I concentrations did not change in response to a chronic fetal amino acid infusion. The anabolic hormones insulin and IGF-I likely affect the fetal response to a direct amino acid infusion and have been shown to promote fetal protein synthesis and accretion (22, 37–39). Insulin promotes fetal amino acid utilization, oxidation, and protein synthesis in a variety of animal models, both independently and synergistically with supplemental amino acids (5, 9, 30, 40). For example, protein synthesis rates in neonatal piglets are maximal in response to an amino acid load only with concurrent increases in insulin or IGF-I concentrations (30, 31, 40). Furthermore, acute amino acid infusion into the normal fetus increased mTOR signaling to promote translation initiation in fetal skeletal muscle only when insulin was concurrently increased (6). In the present study, 4EBP1, eEF2, p70S6k, eIF2α, rpS6, eEF2, and mTOR expression and activation were similar between AA and Con groups. As insulin is a significant regulator of skeletal muscle protein synthesis via the mTOR signaling pathway, it is possible that, without a concurrent increase in insulin, amino acids are shuttled toward oxidative, rather than anabolic, pathways in the normally growing fetus.
Data from this study serve as a basis to develop novel approaches to increasing nutrition and promoting anabolic conditions in pregnancies where growth might be compromised. This study was limited to the evaluation of direct fetal amino acid infusion into normally growing fetuses, which we presume were growing at maximal rates (11). Response to fetal amino acid supplementation in a fetus affected by intrauterine growth restriction (IUGR) cannot be predicted on the basis of the results of this study. Fetuses affected by IUGR have alterations in mRNA levels of key regulatory genes, likely due to epigenetic modifications (1, 13, 21, 23), which may alter their response to amino acid supplementation. Furthermore, substrate metabolic rates and hormone concentrations differ dramatically in the IUGR fetus compared with a normally grown fetus and may change differently following fetal nutrient supplementation (24, 42, 44). Investigation into the most optimal maternal or fetal amino acid supplementation protocols is needed to develop nutritional strategies to more positively affect growth in the IUGR fetus.
In conclusion, we found that, in response to a direct infusion of amino acids, the late-gestation ovine fetus remains in relative metabolic balance by using amino acids (in place of glucose) for oxidative metabolism, rather than protein synthesis or accretion. We speculate that the mechanisms driving this involve complex interactions between placental transport and fetal anabolic and regulatory hormones.
This work was supported by National Institutes of Health (NIH) Building Interdisciplinary Careers in Women's Health Scholar Award K12-HD-057022 (L. D. Brown, Principal Investigator) and the Children's Hospital Colorado Research Institute (L. D. Brown, Principal Investigator). P. J. Rozance was supported by a Pilot and Feasibility Award from the Diabetes and Endocrinology Research Center, University of Colorado (NIH Grant P30-DK-57516), as well as NIH Grants R01-DK-088139 and K08-HD-060688. S. R. Thorn was supported by NIH Grant K01-DK-090199.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Diabetes and Digestive and Kidney Diseases or National Institute of Child Health and Human Development.
No conflicts of interest, financial or otherwise, are declared by the authors.
A.M.M., P.J.R., and L.D.B. are responsible for conception and design of the research; A.M.M., M.M.G., M.C.O., S.R.T., P.J.R., and L.D.B. performed the experiments; A.M.M., M.M.G., M.C.O., S.R.T., P.J.R., and L.D.B. analyzed the data; A.M.M., S.R.T., P.J.R., and L.D.B. interpreted the results of the experiments; A.M.M. prepared the figures; A.M.M. drafted the manuscript; A.M.M., S.R.T., P.J.R., and L.D.B. edited and revised the manuscript; A.M.M., M.M.G., S.R.T., P.J.R., and L.D.B. approved the final version of the manuscript.
We thank Karen Trembler, David Caprio, Alex Cheung, and Gates Roe for technical support and Giacomo Meschia, William W. Hay, and Randall Wilkening for intellectual support.
- Copyright © 2012 the American Physiological Society