Fetal hepatic and umbilical uptakes of glucogenic substrates during a glucagon-somatostatin infusion

Cecilia Teng, Frederick C. Battaglia, Giacomo Meschia, Michael R. Narkewicz, and Randall B. Wilkening

Departments of Pediatrics and Physiology, University of Colorado School of Medicine, Aurora, Colorado, 80045-0508


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To test the hypothesis that fetal hepatic glutamate output diverts the products of hepatic amino acid metabolism from hepatic gluconeogenesis, ovine fetal hepatic and umbilical uptakes of glucose and glucogenic substrates were measured before and during fetal glucagon-somatostatin (GS) infusion and during the combined infusion of GS, alanine, glutamine, and arginine. Before the infusions, hepatic uptake of lactate, alanine, glutamine, arginine, and other substrates was accompanied by hepatic output of pyruvate, aspartate, serine, glutamate, and ornithine. The GS infusion induced hepatic output of 1.00 ± 0.07 mol glucose carbon/mol O2 uptake, an equivalent reduction in hepatic output of pyruvate and glutamate carbon, a decrease in umbilical glucose uptake and placental uptake of fetal glutamate, an increase in hepatic alanine and arginine clearances, and a decrease in umbilical alanine, glutamine, and arginine uptakes. The latter result suggests that glucagon inhibits umbilical amino acid uptake. We conclude that fetal hepatic pyruvate and glutamate output is part of an adaptation to placental function that requires the fetal liver to maintain both a high rate of catabolism of glucogenic substrates and a low rate of gluconeogenesis.

fetal liver; placenta; glutamate; fetal gluconeogenesis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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UNDER NORMAL PHYSIOLOGICAL CONDITIONS, the liver of the ovine fetus releases glutamate in the fetal circulation (18, 23, 25). About one-third of the glutamate released is produced from hydrolysis of plasma glutamine (25), and the remainder is produced via hepatic oxidative metabolism. The fetal infusion of uniformly labeled [13C]lactate results in the appearance of hepatic venous glutamate, which is labeled mostly with either one or two 13C carbons per molecule (T. S. Guyton, P. V. Fennessey, F. C. Battaglia, G. Meschia, and R. B. Wilkening, unpublished observations). Glutamate output is an important aspect of fetal hepatic metabolism, as demonstrated by the calculation that hepatic O2 consumption would have to increase ~65% if the liver were to retain and oxidize the glutamate that it normally excretes (25).

Glutamate is only one of several glucogenic substrates that are exchanged between the fetal liver and its circulation (3, 8, 18). The relative importance of each substrate in determining the balance of this exchange has not been evaluated, because different substrates have been studied at different times. Therefore, one aim of the present study was to measure simultaneously and under normal physiological conditions the fetal hepatic uptakes of glucose, lactate, pyruvate, and all of the main glucogenic amino acids.

In fetal life, hepatic glutamate output may depend on a low rate of hepatic gluconeogenesis (8) that prevents the utilization of the products of hepatic amino acid oxidation for glucose synthesis (19). Pharmacological doses of glucagon have been shown to increase in the fetal rat the activity of hepatic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32), a rate-limiting enzyme of hepatic gluconeogenesis (7), and to stimulate the appearance of fetal glucose in fetal lamb plasma (6). This evidence suggests that the infusion of large doses of glucagon in the fetus could be used as a test of the hypothesis that stimulation of fetal hepatic gluconeogenesis reduces fetal hepatic glutamate output. In preliminary experiments, we infused glucagon for 3 days at a constant rate in the fetal circulation. In apparent agreement with the predicted effect, we observed an increase in fetal plasma glucose that was accompanied by a decline in fetal plasma glutamate concentration. However, we also observed a decline in the plasma concentrations of several other glucogenic amino acids, notably glutamine, alanine, and arginine. Normally, these amino acids are delivered by the placenta to the umbilical circulation and are taken up by the fetal liver (18). All of these changes persisted for the duration of the infusion and were reversed by its termination. In addition, the glucagon infusion caused an increase of fetal plasma insulin that could be suppressed by the coinfusion of somatostatin.

On the basis of these preliminary observations, we designed the present study to measure fetal hepatic and umbilical uptakes of amino acids, glucose, lactate, and pyruvate under the following three conditions: control, during a fetal glucagon-somatostatin infusion, and during the combined infusion of glucagon, somatostatin, alanine, glutamine, and arginine. The latter condition was meant to provide information about the effect of glucagon on fetal hepatic glutamate output when there is no restriction of the supply of glucogenic amino acids to the liver.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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All animal experimentation was performed according to the Helsinki convention for the care and use of animals and was approved by the local Institutional Animal Care and Use Committee. Columbia-Rambouillet ewes, each carrying a single fetus, were studied. Three animals were used to collect preliminary data and to develop the following procedure, which was then applied to five sheep.

Animal preparation. At gestational ages ranging between 118 and 123 days, the ewes were placed under general anesthesia with pentobarbital sodium (20 mg/kg initial dose) and spinal anesthesia with tetracaine hydrochloride (20 mg). Under sterile surgical conditions, the uterus was exposed via a midline laparotomy incision. Through an 8-cm uterine incision, 20-gauge polyvinyl catheters were introduced into the fetal abdominal aorta via a hindlimb pedal artery, the fetal inferior vena cava via a hindlimb pedal vein, the two fetal axillary veins via brachial veins of the forelimbs, and the common umbilical vein via one of the two umbilical veins. Next, the fetal left hepatic vein was catheterized via a right thoracotomy, as previously described (25). After a catheter was placed in the amniotic cavity and the uterine and abdominal incisions were closed, a catheter was inserted into a maternal femoral artery. All catheters were secured at their point of insertion with cyanoacrylate adhesive and tunneled subcutaneously to a pouch in the flank of the ewe.

Analgesics were given to the ewe in the first two postoperative days. Ampicillin (500 mg) was administered via the amniotic catheter at the end of surgery and for the first three postoperative days. After surgery, the ewe was kept in a rectangular 1.5 × 1.0-m cart at 18 ± 2°C room temperature, with ad libitum access to alfalfa pellets, water, and mineral block. Two ewes were kept in adjacent carts at all times to create a nonstressful environment. Each day, the cart was cleaned, food intake was recorded, and all catheters were flushed with a solution of heparin in isotonic saline (30 IU/ml). The experimental protocol was carried out 5-11 days (mean 8 days) after surgery. At the end of the study, both mother and fetus were injected intravenously with an overdose of pentobarbital sodium. Samples of fetal liver were obtained within 5 min after the pentobarbital sodium injection and were analyzed for PEPCK activity.

Experimental protocol. The experiment required the collection of fetal blood during three separate sampling periods: the control period, during which the fetus was infused with 3H2O, the first experimental period (E1), during which the fetus was infused with 3H2O, glucagon, and somatostatin, and the second experimental period (E2), during which the fetus was infused with 3H2O, glucagon, somatostatin, alanine, glutamine, and arginine. The 3H2O infusion was via one of the two fetal axillary veins. Its purpose was to estimate umbilical blood flow by means of the steady-state transplacental diffusion method (24) and to estimate the contribution of umbilical venous blood to blood flow of the left hepatic lobe (25). The glucagon-somatostatin infusion was via the fetal inferior vena cava. It started 7 h after completion of the control sampling and lasted 20 h. The amino acid infusion was via one of the two axillary veins. It started immediately after collection of the last set of E1 samples and lasted 3 h. The timing of infusions and sampling is summarized in Fig. 1. It was planned so all of the blood samplings could be completed in the morning hours. An additional purpose of the overnight glucagon-somatostatin infusion was to deplete the liver of glycogen so that, in interpreting the subsequent measurements of hepatic uptake, we could rely on the assumption that the net hepatic glucose output included glucose produced by gluconeogenesis. Information about the infusates is given in Table 1. In each sampling period, four sets of simultaneously drawn blood samples were obtained at 20-min intervals from the maternal artery, the fetal artery, the umbilical vein, and the left hepatic vein. Each fetal sample was analyzed for hematocrit, hemoglobin content, O2 saturation, 3H2O, glucose, lactate, pyruvate, and amino acids. In addition, fetal arterial blood was analyzed for plasma insulin and glucagon. The total amount of fetal blood drawn in each sampling period was 28 ml. To compensate for blood loss, the fetus was transfused two times with 14 ml of maternal blood at the beginning and at the end of each sampling period. Given an estimated fetal blood volume of 250 ml, the transfusions were aimed at maintaining fetal blood volume within ±6% of normal. Maternal arterial blood was analyzed for plasma glucose and amino acid concentrations.


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Fig. 1.   Summary of experimental protocol, showing time of blood sampling and fetal infusions. E1, first experimental period; E2, second experimental period.


                              
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Table 1.   Composition and infusion rates of the three fetal infusates

Analytical methods. Hematocrit was measured after a 3-min centrifugation in a microcapillary centrifuge (model MB; International Equipment, Needham Heights, MA). Hemoglobin and O2 content were measured spectrophotometrically (OSM3; Radiometer, Copenhagen, Denmark). Blood O2 content was calculated as the product of hemoglobin content, expressed as O2 capacity, times O2 saturation. Plasma glucose and lactate concentrations were measured in duplicate with a glucose-lactate analyzer (YSI model 2700 Select and Dual Standard). Blood pyruvate was measured in duplicate by an assay using the oxidation of NADH to NAD+ by pyruvate in the presence of lactate dehydrogenase (Sigma Diagnostics, St. Louis, MO). Plasma 3H2O concentration was measured in triplicate aliquots in a scintillation counter.

Plasma samples for amino acid analysis were stored at -70°C until the day of measurement. At that time, the samples were thawed quickly and deproteinized with 15% sulfosalicylic acid containing 0.3 µmol/ml norleucine as an internal standard. The pH was brought to 2.2 by titration with 1.5 N LiOH. After centrifugation, the supernatant was analyzed with a Dionex HPLC amino acid analyzer (Dionex, Sunnyvale, CA). Each set of simultaneously drawn samples was loaded on the analyzer to run within 12 h, and the same column was used for all samples from an individual animal. Reproducibility within the same column had a mean value of ±2%. Amino acid absorbances were measured after reaction with ninhydrin at 570 nm, except proline, which was measured at 440 nm wavelength. Plasmal insulin and glucagon were measured in duplicate using a rat insulin RIA kit (catalog no. R1-13K) and a Glucagon RIA kit (catalog no. GL-32K) from Linco Research (St. Charles, MO). Cross-reactivity of the rat insulin test with sheep insulin was virtually 100%. According to manufacturer specifications, the glucagon kit was specific for pancreatic glucagon. Glucagon was measured in blood samples protected from proteolysis by the addition of aprotinin.

For the PEPCK assay, cytosolic fractions were prepared from fresh liver according to the method of Chang and Lane (4). PEPCK was assayed by the [14C]bicarbonate fixation assay, as previously reported (20). Results were expressed as nanomoles of product fixed per minute per milligram of cytosolic protein. Protein was determined by the method of Lowry as modified by Hartree (11). The cytosolic fraction was assayed routinely for mitochondrial contamination by measuring citrate synthase (21) and was found to be <1%.

Calculations. Umbilical blood flow was calculated by the steady-state transplacental diffusion method, using tritiated water as the blood flow indicator (24).

The umbilical uptake of the two metabolic substrates that were measured in whole blood (O2 and pyruvate) was calculated by means of the equation
umbilical uptake<SUB><IT>x</IT></SUB><IT>=Q</IT>(<IT>&ggr;−&agr;</IT>)<SUB>b<IT>,x</IT></SUB>
where Q is umbilical blood flow (ml · min- · kg fetus-1) and (gamma  - alpha )b,x is the concentration difference of substrate x per milliliter of blood (b) between umbilical venous (gamma ) and umbilical arterial (alpha ) blood.

The umbilical uptake of substrates that were measured in plasma was calculated by means of the equation
umbilical uptake<SUB><IT>x</IT></SUB><IT>=Q</IT>(<IT>&ggr;−&agr;</IT>)<SUB>p<IT>,x</IT></SUB>(1<IT>−&phgr;<SUB>x</SUB></IT>Ht)
where (gamma  - alpha )p,x is the concentration difference of substrate x per milliliter of plasma (p) across the umbilical circulation, Ht is the fractional hematocrit, and phi x is a coefficient whose value is inversely related to the contribution of erythrocytes to uptake. For lactate, phi x was set to zero on the assumption that there is rapid lactate exchange between plasma and erythrocytes and because fetal plasma and blood lactate concentrations are approximately equal (2). For each amino acid, phi x was set to one, because in the sheep fetus the amino acid exchange between plasma and erythrocytes is quite slow compared with the exchange between plasma and body organs (5). For glucose, phi x was set equal to 0.24 on the basis of paired analysis of blood and plasma glucose concentrations in the sheep fetus (unpublished observations).

The uptake of metabolic substrates by the left lobe of the fetal liver was normalized for hepatic O2 uptake by calculating a substrate-to-O2 uptake molar ratio. For pyruvate
hepatic pyruvate-to-O<SUB>2</SUB> uptake ratio

<IT>=</IT>(h<SUB>i</SUB><IT>−</IT>h)<SUB>b,Pyr</SUB><IT>/</IT>(h<SUB>i</SUB><IT>−</IT>h)<SUB>b,O<SUB>2</SUB></SUB>
where (hi - h)b represents the concentration difference between the blood entering the circulation of the left hepatic lobe (hi) and the blood exiting the liver via the left hepatic vein (h).

For all of the other substrates, the substrate-to-O2 uptake molar ratio was calculated according to the equation
hepatic substrate<SUB><IT>x</IT></SUB><IT>-</IT>to-O<SUB>2</SUB> uptake ratio

<IT>=</IT>(h<SUB>i</SUB><IT>−</IT>h)<SUB><IT>p,x</IT></SUB>(1<IT>−&phgr;<SUB>x</SUB></IT>Ht)<IT>/</IT>(h<SUB>i</SUB><IT>−</IT>h)<SUB>b,O<SUB>2</SUB></SUB>
where (hi - h)p,x represents the input - output plasma concentration difference of substrate x across the left hepatic lobe circulation.

The hi concentration of each substrate was calculated from the measured values of umbilical venous and fetal arterial concentration, according to the equation
h<SUB>i</SUB><IT>=</IT>(F<IT>&ggr;</IT>)<IT>+</IT>(1<IT>−</IT>F)<IT>&agr;</IT>
where F represents the fractional contribution of umbilical venous blood to the total blood flow of the left hepatic lobe (25). In each fetus, the F factor was calculated (25) using the tritiated water concentration (t) in gamma , alpha , and h
F<IT>=</IT>(h<IT>−&agr;</IT>)<SUB><IT>p,</IT>t</SUB><IT>/</IT>(<IT>&ggr;−&agr;</IT>)<SUB><IT>p,</IT>t</SUB>

Statistical analysis. For each of the concentration and uptake measurements, the data were subjected to two-way ANOVA (control, E1, and E2 studies in 5 sheep). Sheep were considered a random effect, and the four repeated measurements during a study on a sheep were averaged before analysis. Comparison-specific P values were calculated for the three paired studies (control vs. E1, control vs. E2, and E1 vs. E2). P values were considered significant at P < 0.05.


    RESULTS
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The experimental procedure had no significant effect on umbilical blood flow and O2 uptake (Table 2). Umbilical venous blood contributed most of the blood perfusing the left hepatic lobe (F = 0.92 ± 0.027), in agreement with previous studies (8, 13, 25).

                              
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Table 2.   Gestational age, fetal and placental weights, umbilical blood flows, and O2 uptakes in 5 sheep

Carbohydrate metabolism. In the control period, the liver showed no glucose output and a relatively large uptake of lactate. Hepatic pyruvate output accounted for ~40% of the hepatic lactate uptake. The placenta delivered into the umbilical circulation glucose and lactate and had a small uptake of pyruvate (Table 3).

                              
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Table 3.   Glucose, lactate, and pyruvate concentrations and uptakes and glucagon and insulin concentrations in control and two experimental periods

The glucagon-somatostatin infusion stimulated glucose output by the fetal liver, increased fetal plasma glucose concentration, and decreased umbilical glucose uptake. In addition, it decreased the concentrations of both lactate and pyruvate in fetal plasma and decreased the hepatic output of pyruvate. The alanine-glutamine-arginine infusion was associated with an increase in plasma lactate, pyruvate, and glucose concentrations and a decrease in hepatic lactate uptake. Plasma insulin remained virtually constant throughout the experiment.

Amino acid metabolism. Most of the amino acids had positive hepatic and umbilical uptakes. Aspartate, serine, glutamate, and ornithine had negative hepatic uptakes (i.e., hepatic output). Glutamate also had a large negative umbilical uptake (i.e., placental uptake of fetal glutamate), whereas aspartate, serine, and ornithine had virtually no umbilical uptake (Tables 4-6).

                              
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Table 4.   Fetal arterial plasma concentrations of amino acids in the control period and in experimental periods


                              
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Table 5.   Amino acid-to-O2 uptake molar ratios (×103) across the fetal hepatic circulation in the control period and two experimental periods


                              
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Table 6.   Umbilical uptakes of amino acids in the control period and the two experimental periods

The glucagon-somatostatin infusion significantly decreased the fetal plasma concentrations of all amino acids, with the exception of phenylalanine. For the glucogenic amino acids aspartate, threonine, glutamate, glutamine, proline, alanine, ornithine, and arginine, the plasma concentration decreased to <50% of control values. The alanine-glutamine-arginine infusion restored the plasma concentrations of the infused amino acids to normal values. The infusion also returned plasma ornithine concentration to normal. The decrease in fetal plasma glutamate was associated with a decrease in fetal hepatic glutamate output and placental uptake of fetal glutamate to ~20% of control values. Among the amino acids that are normally taken up by the fetal liver, their decrease in plasma concentration during the glucagon-somatostatin infusion was not associated with a significant decrease in hepatic uptake, with the exception of glutamine. By contrast, the increase in plasma alanine, glutamine, and arginine concentrations caused by the alanine-glutamine-arginine infusion was associated with a significant increase in the hepatic uptake of all three amino acids (E2 vs. E1 comparison). The relation between the hepatic uptake and hepatic input concentration for alanine, glutamine, and arginine is presented in Fig. 2. Figure 2 shows that hormonal stimulation increased the hepatic uptake per unit plasma concentration of alanine and arginine and may have had a similar but smaller effect on glutamine uptake.


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Fig. 2.   Hepatic uptake vs. hepatic plasma input concentration for alanine, glutamine, and arginine. E1 and E2 points, representing measurements during the glucagon-somatostatin infusion, are joined by a solid straight line. Linear extrapolation (dashed lines) is used to show that, at equal concentration values, hepatic uptake was less in the control period (C). Mean uptake (Table 5) and concentration values for the 5 sheep were used to construct the graph.

The umbilical uptakes of alanine, glutamine, and arginine were significantly less than control during the alanine-glutamine-arginine infusion. For each amino acid, the decrease in uptake was not associated with a significant increase in fetal plasma concentration above control and could not be accounted for by a decrease in maternal concentration, which remained virtually constant throughout the experiment (data not shown). Figure 3 presents a semilogarithmic plot of umbilical uptake vs. the maternal-to-fetal plasma concentration ratio for the three amino acids. It is apparent that, at comparable values of maternal and fetal concentrations, the umbilical uptakes were less than normal during the glucagon-somatostatin infusion.


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Fig. 3.   Umbilical uptake vs. the base 10 logarithm of the maternal arterial (A)-to-fetal arterial (alpha ) plasma concentration ratio for alanine, glutamine, and arginine. The E1 and E2 points, representing measurements during the glucagon-somatostatin infusion, are joined by a solid straight line. Linear extrapolation (dashed lines) is used to show that, at equal maternal-to-fetal concentration ratios, umbilical uptake was greater in the control period. Mean uptake values (Table 6) and ratios of the mean concentrations for the 5 sheep were used to construct the graph.

At the end of the study, hepatic cytosolic PEPCK activity was significantly higher than in seven fetuses of comparable age that had undergone experiments that did not involve stimulation of fetal gluconeogenesis (28.7 ± 7.1 vs. 7.5 ± 1.8 nmol · min-1 · mg protein-1, P < 0.05).


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The control data for hepatic and umbilical uptakes are in good agreement with previous measurements (3, 5, 8, 12, 13, 18, 23, 25). However, this is the first study in which the fetal hepatic uptakes of glucose and all major glucogenic substrates have been measured in the same experimental preparation. The study demonstrates that, under normal physiological conditions, the fetal liver has an uptake of glucogenic substrates that is large compared with its rate of oxidative metabolism. According to the data summarized in Fig. 4, the combined normal fetal hepatic uptake of lactate and the glucogenic amino acids glutamine, alanine, arginine, threonine, valine, methionine, glycine, proline, histidine, and asparagine represents a hepatic uptake of 2.47 ± 0.33 mol substrate carbon/mol O2 uptake. Contrary to what might be predicted from knowledge of postnatal hepatic metabolism (14), this uptake does not result in the hepatic output of glucose. It results in the hepatic output of five other glucogenic substrates, namely pyruvate, glutamate, serine, aspartate, and ornithine for a total output of 1.53 ± 0.23 mol substrate carbon/mol O2 uptake. The two major components of this output are glutamate and pyruvate (1.30 ± 0.23 substrate carbon-to-O2 uptake molar ratio).


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Fig. 4.   Fetal hepatic uptake and output of glucose carbon and glucogenic substrate carbon under normal physiological conditions (control) and during a glucagon-somatostatin infusion into the fetal circulation. Each number represents a substrate carbon-to-O2 uptake molar ratio.

The hepatic and umbilical uptakes reported here are not directly comparable because they are expressed in different units. However, a rough estimate of hepatic uptake as micromoles per minute per kilogram fetus can be obtained by using the glutamate data. Placental glutamate uptake is ~60% of the fetal plasma glutamate disposal rate (19). On the assumption that the fetal liver is virtually the only net producer of fetal plasma glutamate, we can estimate normal hepatic glutamate output as umbilical uptake divided by 0.6. Therefore, we estimate that the control hepatic glutamate output was 8.8 µmol · min-1 · kg fetus-1 (i.e., 5.28/0.6) and that all of the hepatic uptakes in Tables 3 and 5 should be multiplied by 0.048 (i.e., 8.8/183) to estimate hepatic uptakes in the same units as the umbilical uptakes. The 0.048 conversion factor represents hepatic O2 uptake in millimoles per minute per kilogram fetus. Given the complexity of the fetal hepatic circulation, the O2 consumption of the fetal liver has been difficult to measure, and its true value is uncertain. In a recent study of fetal hepatic blood flow (13), fetal O2 consumption was estimated to be 0.058 mmol · min-1 · kg fetus-1. Despite this uncertainty, the comparison of hepatic and umbilical uptakes allows two interesting conclusions. First, normal hepatic glucose uptake is virtually zero compared with umbilical uptake. Second, among the essential amino acids, hepatic uptake of the branched-chain amino acids (leucine, isoleucine, and valine) is a small fraction of umbilical uptake, whereas the hepatic uptake of threonine, methionine, phenylalanine, and histidine is approximately one-half the umbilical uptake. The essential amino acids for which the fetal liver is the main site of catabolism would be expected to show a relatively high hepatic-to-umbilical uptake ratio.

The present study demonstrates that a glucagon-somatostatin infusion markedly alters the metabolic exchange between the fetal liver and its circulation. Approximately 16 h after the start of the infusion, the fetal liver had switched from glucose uptake to glucose release. Inspection of Table 3 shows that this switch amounted to a loss of 1.00 ± 0.07 mol glucose carbon/mol O2 uptake [i.e., 6 × (0.132 + 0.035)]. Tables 3 and 5 show that this deficit was compensated for primarily by a decline in the hepatic release of glucogenic substrates. Concomitant with the loss of glucose carbon, there was a similar decrease in the combined output of pyruvate, aspartate, serine, glutamate, and ornithine carbon from 1.53 ± 0.23 to 0.52 ± 0.13 mol substrate carbon/mol O2 uptake. The most important components of this decrease were glutamate and pyruvate (from 1.30 ± 0.23 to 0.34 ± 0.09 mol substrate carbon/mol O2 uptake). By contrast, none of the glucogenic substrates that are normally taken up by the fetal liver had a significant increase in uptake. Their aggregate uptake showed a small, statistically insignificant decrease (from 2.47 ± 0.33 to 2.34 ± 0.11 mol substrate carbon/mol O2 uptake). Note that, in principle, a decrease in the substrate-to-O2 uptake molar ratio may be the result of an increase in O2 uptake. However, the relative changes in carbohydrate and amino acid uptake would remain the same if each uptake were to be expressed per gram of liver or any other parameter. The effect of the glucagon-somatostatin infusion on the fetal hepatic exchange of glucose and glucogenic substrates is summarized in Fig. 4.

The most likely explanation for the decrease in hepatic pyruvate and glutamate output is that glucagon stimulation increased the flux of glucogenic amino acids and lactate carbon into hepatic glucose production and decreased the availability of pyruvate and glutamate for hepatic excretion. In favor of this explanation is the finding that the glucagon-somatostatin infusion significantly increased the activity of cytosolic PEPCK. The result that hepatic uptake of glucogenic amino acids was not greater than normal is explained by the decrease in their plasma concentrations. When the fetal infusion of alanine, glutamine, and arginine restored the plasma concentrations of these amino acids to the normal range, their hepatic uptakes increased significantly. Concomitant with this increase, there was a decrease in lactate uptake, suggesting competition for hepatic uptake between lactate and the infused amino acids. A plot of the hepatic uptake vs. plasma concentration (Fig. 2) shows that hormonal stimulation had actually increased alanine and arginine uptake per unit plasma concentration; i.e., it had increased their hepatic clearance. The increase in clearance is likely to represent a change in the activity of hepatic amino acid transporters. Alanine uptake by the liver is mediated in part by the sodium-dependent "system A" transporters, the activity of which can be increased by glucagon (16). In this context, it is important to note that the fetal infusion of the glucocorticoid dexamethasone decreases hepatic glutamate output primarily via inhibition of the hepatic uptakes of glutamine and alanine and does not result in the output of glucose (23). Together, the glucagon-somatostatin and dexamethasone experiments point to the conclusion that the high glutamate output of the fetal liver is sustained by combining a low rate of gluconeogenesis with a high rate of catabolism of glucogenic amino acids taken up from the fetal circulation.

The decrease in umbilical glucose uptake associated with the glucagon-somatostatin infusion is a predictable effect of the significant increase in fetal plasma glucose. The transport rate of maternal glucose to the fetus is a function of the glucose concentration difference between maternal and fetal plasma (12). We surmise that fetal hepatic glucose production was the main cause of the increase in fetal plasma glucose, which in turn caused a decrease in the transplacental glucose concentration gradient that drives maternal glucose into the umbilical circulation. Similarly, the decrease in placental uptake of fetal glutamate was a consequence of the decrease in hepatic glutamate output, which caused the decrease in fetal arterial plasma glutamate and reduced the availability of glutamate for placental uptake.

The data about alanine, glutamine, and arginine umbilical uptakes require a different interpretation. The glucagon-somatostatin infusion inhibited the umbilical uptake of these amino acids, and the inhibition could not be accounted for by changes in maternal and fetal plasma concentrations. Tracer studies have shown that, for any given amino acid, umbilical uptake is the difference of two opposite fluxes, i.e., from placenta to fetus and from fetus to placenta (3, 9, 25). The flux of fetal alanine into the placenta is mediated by system A transporters (22). This suggests that glucagon may have acted similarly on hepatic and placental alanine transport by increasing the alanine flux mediated by these transporters. An increase of alanine flux into the placenta would result in a decrease of alanine umbilical uptake. The significant decrease in proline umbilical uptake may have also been the result of a change in system A activity. It is uncertain whether the suggested mechanism for the decrease in alanine uptake could be applied to glutamine. There is no information about the role of system A transporters in mediating glutamine exchange between the fetus and placenta. Furthermore, a decrease in the placental uptake of fetal glutamate could, by itself, decrease placental glutamine output via a decrease in placental glutamine production. Concomitant with the decrease in arginine umbilical uptake, there was a decrease in lysine uptake. Because arginine and lysine are basic amino acids, this finding suggests a change in the placental transport system that mediates the fetoplacental exchange of these amino acids. The unexpected finding of umbilical uptake inhibition suggests that fetal glucagon plays a role in regulating placental amino acid transport. However, the effect on umbilical uptake was produced by infusing the high dose of glucagon that is required to stimulate fetal hepatic glucose output. Experiments with much lower doses would be necessary to verify this suggestion. An additional implication for which there is no direct evidence is that glucagon receptors are present on the fetal surface of the placenta.

It is apparent that one important function of the low rate of fetal hepatic gluconeogenesis is to promote the uptake of maternal glucose by the fetus. The absence of fetal hepatic glucose output maintains the glucose concentration in fetal plasma at a much lower level than in maternal plasma and determines the steep transplacental glucose concentration gradient that drives glucose from mother to fetus. Although, via this mechanism, maternal glucose represents a major metabolic fuel for the fetus, the transport rate of maternal glucose across the placenta is not sufficient, by itself, to sustain fetal energy metabolism. In addition to glucose, fetal oxidative metabolism uses lactate and amino acids supplied by the placenta (1).

The net flux of amino acids from placenta to fetus includes all of the major neutral and basic glucogenic amino acids, with the exception of the neutral amino acid serine (5). Adaptation of the liver to prenatal life requires the ability to contribute to the rapid disposal of the glucogenic substrates supplied by the placenta without utilizing these metabolites for glucose synthesis. The present study indicates that, within the fetal liver, the routing of glucogenic substrates into gluconeogenesis is blocked at the formation of pyruvate and glutamate and results in their hepatic output. Fetal hepatic glutamate output is likely to depend on the activity of a specialized glutamate transport system that is normally expressed much more in fetal than in postnatal life. A candidate for this role is the sodium-independent glutamate transporter described by Makowske and Christensen (17). It is present in fetal hepatocytes and hepatoma cells but is virtually absent in the adult hepatocyte.

Glutamate uptake by the placenta is mediated by a sodium-dependent transport system (22). Glutamate is used by placental mitochondria in steroidogenesis (15). It is not yet clear what other functions placental glutamate uptake and metabolism may serve. The lactate uptake-pyruvate output of the fetal liver is matched by a reciprocal lactate output-pyruvate uptake by the fetal hindlimbs (2).

Hepatic serine output is another interesting aspect of the integration of placental and fetal hepatic function. Tracer studies have shown large and opposite serine exchange fluxes between placenta and fetus, which result in virtually zero uptake or a small net uptake of fetal serine by the placenta (3). The placenta converts maternal and fetal serine to fetal glycine, which is then used by the fetal liver to produce some of the hepatic serine output (3). A study of amino acid uptake by the fetal hindlimb has demonstrated a significant serine uptake by this organ (26). It is apparent that fetal hepatic serine output has the function of supplying serine to other fetal organs and to the placenta. The glucagon-somatostatin infusion significantly decreased fetal plasma serine concentration but had no detectable effect on hepatic serine output. Therefore, it is not clear from the present study whether a low rate of fetal gluconeogenesis contributes to fetal hepatic serine output.


    ACKNOWLEDGEMENTS

We are grateful to Professor Gary Zerbe for help in statistical analysis and to Susan Anderson for technical assistance.


    FOOTNOTES

This work was supported by National Institutes of Health Grants R29 HD-31648, PO1 HD-20761, and RO1 HD-34837.

Address for reprint requests and other correspondence: F. C. Battaglia, Fitzsimons, Bldg. 260, POB 6508, MS F441, 13243 E. 23rd Ave., Aurora, CO 80045-0508.

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.

10.1152/ajpendo.00248.2001

Received 7 July 2001; accepted in final form 7 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Battaglia, FC, and Meschia G. An Introduction to Fetal Physiology. Orlando, FL: Academic, 1986.

2.   Boyle, DW, Meschia G, and Wilkening RB. Metabolic adaptation of the fetal hind limb to severe, non-lethal hypoxia. Am J Physiol Regulatory Integrative Comp Physiol 263: R1130-R1135, 1992[Abstract/Free Full Text].

3.   Cetin, I, Fennessey PV, Sparks JW, Meschia G, and Battaglia FC. Fetal serine fluxes across fetal liver, hindlimb, and placenta in late gestation. Am J Physiol Endocrinol Metab 263: E786-E793, 1992[Abstract/Free Full Text].

4.   Chang, HC, and Lane MD. The enzymatic carboxylation of phosphoenolpyruvate. II. Purification and properties of liver mitochondrial phosphoenolpyruvate carboxykinase. J Biol Chem 241: 2413-2420, 1996[Abstract/Free Full Text].

5.   Chung, M, Teng C, Timmerman M, Meschia G, Wilkening RB, and Battaglia FC. Production and utilization of amino acids by ovine placenta in vivo. Am J Physiol Endocrinol Metab 274: E13-E22, 1998[Abstract/Free Full Text].

6.   Devaskar, SU, Ganguli S, Styer D, Devaskar UP, and Sperling MA. Glucagon and glucose dynamics in sheep: evidence for glucagon resistance in the fetus. Am J Physiol Endocrinol Metab 246: E256-E265, 1984[Abstract/Free Full Text].

7.   Girard, J, and Sperling M. Glucagon in the fetus and the newborn. In: Handbook of Experimental Pharmacology, edited by Lefebvre PJ.. Berlin: Springer-Verlag, 1983, p. 251-273.

8.   Gleason, CA, and Rudolph AM. Gluconeogenesis by the fetal sheep liver in vitro. J Dev Physiol (Eynsham) 7: 185-194, 1985[ISI][Medline].

9.   Guyton, TS, deWilt H, Fennessey PV, Meschia G, Wilkening RB, and Battaglia FC. Alanine umbilical uptake disposal rate, and decarboxylation rate in the fetal lamb. Am J Physiol Endocrinol Metab 265: E497-E503, 1993[Abstract/Free Full Text].

11.   Hartree, EF. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem 48: 122-127, 1972.

12.   Hay, WW, Jr, Molina RA, Digiacomo JE, and Meschia G. Model of placental glucose consumption and glucose transfer. Am J Physiol Regulatory Integrative Comp Physiol 258: R569-R577, 1990[Abstract/Free Full Text].

13.   Holcomb, RG, and Wilkening RB. Fetal hepatic oxygen consumption under normal conditions in the fetal lamb. Biol Neonate 75: 310-318, 1999[ISI][Medline].

14.   Jungas, RL, Halperin ML, and Brosnan JT. Quantitative analysis of amino acid oxidation and related gluconeogenesis in humans. Physiol Rev 72: 419-448, 1992[Abstract/Free Full Text].

15.   Klimek, J, Makarewicz W, Swierczynski J, Bossy-Bukato G, and Zelewski L. Mitochondrial glutamine and glutamate metabolism in human placenta and its possible link with progesterone biosynthesis. Trophoblast Res 7: 77-86, 1993.

16.   Lim, SK, Cynober L, DeBandt JP, and Aussel C. A Na+-dependent system A and ASC-independent amino acid transport system stimulated by glucagon in rat hepatocytes. Cell Biol Int 23: 7-12, 1999[ISI][Medline].

17.   Makowske, M, and Christensen HN. Contrasts in transport systems for anionic amino acids in hepatocytes and a hepatoma cell line HTC. J Biol Chem 257: 5663-5670, 1982[Free Full Text].

18.   Marconi, AM, Battaglia FC, Meschia G, and Sparks JW. A comparison of amino acid arteriovenous differences across the liver and placenta of the fetal lamb. Am J Physiol Endocrinol Metab 257: E909-E915, 1989[Abstract/Free Full Text].

19.   Moores, RR, Vaughn PR, Battaglia FC, Fennessey PV, Wilkening RB, and Meschia G. Glutamate metabolism in the fetus and placenta of late gestation sheep. Am J Physiol Regulatory Integrative Comp Physiol 267: R89-R96, 1994[Abstract/Free Full Text].

20.   Narkewicz, MR, Carver TD, and Hay WW, Jr. Induction of cytosolic phosphoenolpyruvate carboxykinase in the ovine fetal liver by chronic fetal hypoglycemia and hypoinsulinemia. Pediatr Res 33: 493-496, 1993[Abstract].

21.   Shepherd, D, and Garland PB. Kinetic properties of citrate synthase from rat liver mitochondria. Biochem J 114: 597-610, 1969[ISI][Medline].

22.   Smith, CH, Moe AJ, and Ganapathy V. Nutrient transport pathways across the epithelium of the placenta. Annu Rev Nutr 12: 183-206, 1992[ISI][Medline].

23.   Timmerman, M, Teng C, Wilkening RB, Fennessey PV, Battaglia FC, and Meschia G. Effect of dexamethasone on fetal hepatic glutamine-glutamate exchange. Am J Physiol Endocrinol Metab 278: E839-E845, 2000[Abstract/Free Full Text].

24.   Van Veen, LC, Hay WW, Jr, Battaglia FC, and Meschia G. Fetal CO2 kinetics. J Dev Physiol (Eynsham) 6: 359-365, 1984[ISI][Medline].

25.   Vaughn, PR, Lobo C, Battaglia FC, Fennessey PV, Wilkening RB, and Meschia G. Glutamine-glutamate exchange between placenta and fetal liver. Am J Physiol Endocrinol Metab 268: E705-E711, 1995[Abstract/Free Full Text].

26.   Wilkening, RB, Boyle DW, Teng C, Meschia G, and Battaglia FC. Amino acid uptake by the fetal ovine hind limb under normal and euglycemic hyperinsulinemic states. Am J Physiol Endocrinol Metab 266: E72-E78, 1994[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 282(3):E542-E550
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society




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