Main

Studies in the fetal lamb have demonstrated a unique metabolic relationship between serine and glycine. In the fetal lamb, neither serine nor glycine are transported to the fetus across the placenta from the mother in significant quantities when compared with other amino acids that have been studied(1–3). Cetin et al.(4) have recently confirmed these findings for glycine in human pregnancies. In vivo studies in the fetal lamb have demonstrated that serine is produced in significant quantities by the fetal liver, whereas glycine is produced in the placenta(1–3). This interrelationship between serine and glycine in the fetus has led to the hypothesis of the existence of an interorgan hepatoplacental shuttle of serine and glycine(1, 2). Studies in growth-retarded human fetuses have shown that plasma serine and glycine concentrations are low(5, 6). Thus, an understanding of the enzymatic site of regulation of serine and glycine interconversion may provide data as to the potential site of dysregulation of serine and glycine metabolism in intrauterine growth-retarded infants.

Previous studies in our laboratory have suggested that, in vivo in the late gestation fetal ovine liver, serine is produced from glycine by the combined action of SHMT (EC 2.1.2.1) and the GCS (EC 2.1.1.10)(1). Studies in primary cultures of hepatocytes from late gestation fetal lambs have similarly suggested that SHMT and GCS are functioning in a coupled manner in the production of serine(7). Both Pitts and Rowsell have also suggested that, in the kidney, it is the coupled action of SHMT and GCS that is responsible for serine production from glycine(8, 9). However, because the activities of both isoforms of SHMT are high in both the fetal ovine liver in late gestation and in rat kidney, it is unclear whether it is the cytosolic, the mitochondrial isoform of SHMT, or both that are functioning with GCS. In the fetal lamb, it has been suggested that mSHMT activity is present at mid gestation, whereas cSHMT activity is low (R. Moores, personal communication). Thus, if this pattern persists in hepatocytes in culture, it may allow clarification of the relative roles of hepatic mSHMT and cSHMT in serine and glycine interconversion in the fetal ovine liver.

Studies in the late gestation ovine fetal liver and fetal ovine hepatocytes have suggested that glycine is the precursor for approximately 30% of the observed hepatic serine production(1, 7). The metabolic precursor(s) that serve as the source(s) for the remainder of fetal hepatic serine synthesis are unknown. Likely candidates would include synthesis via the phosphorylated or the nonphosphorylated pathway of serine biosynthesis or intermediaries of glucose metabolism via glycerol (Fig. 1). Cheung et al.(10) have suggested that, in the guinea pig, the phosphorylated pathway for serine biosynthesis is active in fetal liver, whereas it is the nonphosphorylated pathway that is active in adult liver. These data have not been confirmed in other species. Further characterization of the metabolic sources of fetal hepatic serine biosynthesis are important as the first step in the investigation of potential abnormalities in serine and glycine metabolism in the human fetus.

Figure 1
figure 1

Major metabolic pathways for serine biosynthesis.

Full size image

The goal of this study was to investigate the metabolism of serine and glycine via SHMT, GCS, and the combined action of the phosphorylated and nonphosphorylated pathways of serine biosynthesis in primary culture of hepatocytes isolated from midgestation fetal lambs to determine the relative roles of these various pathways in fetal hepatic serine and glycine metabolism.

METHODS

Materials. Tetrahydrofolate, L-serine, and pyridoxyl 5′-phosphate were obtained from Fluka (Ronkonkoma, NY). L-[3-14C]Serine was from ICN (Costa Mesa, CA). MEMα (glucose, arginine free) supplemented with 0.4 mM ornithine and 2 mM glutamine was purchased from Life Technologies, Inc. (Grand Island, NY). 1-[13C1]Glycine, 2-[13C1]glycine, 1-[13C1]serine, 2,5-[15N2]glutamine, 2-[15N1]glutamine, and 1,2-[13C2]glutamine were from Cambridge Isotope Laboratories (Woburn, MA). Collagenase B was from Boerhinger Mannheim (Indianapolis, IN), and FCS from Hyclone (Logan, UT). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) and were of the purest form available.

Hepatocyte isolation and culture. Hepatocytes were isolated from mid-gestation fetal lambs with modifications of our previously described method(11). In brief, after delivery by cesarean section, the fetus was dried, anesthetized with pentobarbital (40 mg/kg) and given i.v. heparin (1000 U). The umbilical vein was catheterized with an 18 gauge catheter, and the liver was perfused in a retrograde fashion for 5-10 min with HEPES perfusion solution (10 mM HEPES, pH 7.4, 138 mM NaCl, 3 mM KCl, 0.7 mM Na2HPO4) at 37°C. The solution was then modified by supplementation with 1.7 mM CaCl2 and 0.025% collagenase and perfusion continued for 10 min. After further digestion with stirring for 10 min in a sterile beaker containing the HEPES/CaCl2/collagenase at 37°C, the cells were filtered through nylon mesh and isolated by low speed centrifugation (50 × g for 2 min). Hepatocytes were resuspended in MEMα supplemented with 1.1 mM glucose, 2 mM lactate, and 0.2% albumin, decanted for 20 min to remove hematopoietic cells, collected by centrifugation, and washed two times in medium. Cells were plated at a density of 1 million cells/2-cm well or 6 million cells/10-cm plate in MEMα supplemented with 1.1 mM glucose, 2 mM lactate (fetal ovine glucose and lactate concentrations), 0.2% albumin, and 10% FCS and allowed to adhere overnight. After adherence, the medium was changed to MEMα supplemented with 1.1 mM glucose, 2 mM lactate, and 0.2% albumin with either no additional serine (standard medium, serine ≅ 300 nmol/mL), or additional serine above that found in MEMα (high serine medium, serine ≅ 650 nmol/mL) whereas the concentration of all other amino acids was not changed. Hepatocytes, were cultured in 95% humidity in a 5% CO2 incubator and medium was changed every 24 h.

Studies of serine and glycine interconversion. For studies of serine metabolism and serine to glycine flux, 1-[13C1]serine was selected as the tracer. For studies of glycine metabolism and glycine to serine flux, both 1-[13C1]glycine and 2-[13C1]glycine were used as tracers. In a typical experiment, the stable isotope of interest was added to the medium (t0) to achieve a 10-20% enrichment above baseline. Cells were then incubated in the presence of this enriched medium for 24 h, after which the medium was aspirated with a sterile pipette, cellular debris removed by centrifugation, and medium was snap frozen and stored at -80°C until subsequent analysis.

Studies of transamination of serine from glutamine. The contribution of nitrogen from glutamine via transamination reactions to serine synthesis can be estimated from the enrichment in serine by 2,5-[15N2]glutamine. This can be used to estimate the relative contribution to serine biosynthesis from phosphoserine aminotransferase and serine pyruvate aminotransferase (Fig. 1). For the studies of the role of transamination reactions (which include the phosphorylated and nonphosphorylated pathways of serine biosynthesis), we determined the incorporation of labeled nitrogen from glutamine into serine. This is based on the transamination reactions of L-phosphoserine aminotransferase (EC 2.6.1.52), a glutamate-dependent transamination, and L-serine aminotransferase (EC 2.6.1.51), an alanine-dependent transamination in both pathways (Fig. 1). For this, both 2-[15N1]glutamine and 2,5-[15N2]glutamine were used as tracers to determine the contribution from the amide(2-[15N1]glutamine) or amine (2,5-[15N2]glutamine) nitrogen. Medium was harvested as above and analyzed as outlined below. The medium amino acid concentrations and enrichments (MPE) in M + 1 and M + 2 serine and glutamine, and M + 1 glycine, alanine, glutamate, and tyrosine were determined at 0 and 24 h. Alanine and glutamate are proximate end products of transamination with the number 5 nitrogen of glutamine by a variety of transaminases. Because tyrosine aminotransferase is not present in fetal hepatocytes, tyrosine was analyzed to be sure that there was a fetal pattern to the transamination reactions(12). Production and utilization rates for glutamine nitrogen were determined as outlined below. The percent contribution to serine synthesis was determined as below. In one experiment, 1,2-[13C2]glutamine was used as the tracer to control for any incorporation of the glutamine carbon skeleton into serine.

Medium amino acid concentrations. The concentration of medium amino acids at 0 and 24 h was determined on a JOEL amino acid analyzer with norleucine as the internal standard as previously reported(11, 13, 14). Preliminary experiments demonstrated that changes in medium seine and glycine concentrations were linear for 24 h.

Stable isotope studies. Incorporation of 13C-label into glycine (using 1-[13C1]serine as the tracer) or serine (using either 1-[13C1]glycine, 2-[13C1]glycine, or 1,2-[13C2]glutamine as the tracer) and 15N-label into glycine, serine, glutamine, glutamate, alanine, and tyrosine (using either 2,5-[15N2]glutamine or 2-[15N1]glutamine as the tracer) was determined at 0 and 24 h by gas chromatography/mass spectrometry as we have previously described(13, 14). Preliminary experiments demonstrated that changes in medium serine and glycine enrichments were linear for 24 h. Change in MPE was determined by monitoring at mass = 247/246 for enrichment of 1 amu for glycine and at mass = 391/390 and 392/390 for 1 and 2 amu for serine. When indicated, change in MPE was determined by monitoring at mass = 432/431 and 433/431 for enrichments of 1 and 2 amu for glutamine, 433/432 for 1 amu for glutamate, 261/260 for 1 amu for alanine, and 467/466 for 1 amu for tyrosine.

Calculations. Glycine or serine production and utilization rates were determined by stable isotope dilution methods using 1-[13C1]glycine for glycine and 1-[13C1]serine for serine by the following formula: where MPEaat0 or 24 = MPE at 0 or 24 h of the amino acid of interest and AAt0 or 24 = the nanomoles present of the amino acid of interest at 0 or 24 h.

The percent of the tracer used for synthesis of the tracee was determined by: Equation:

The percent of tracee synthesis from the tracer was calculated by:equations:

Glycine can be used for serine biosynthesis either directly via SHMT or via the coupled reaction of GCS and SHMT (Fig. 2). To determine the relative contribution to serine biosynthesis of the coupled action of SHMT and GCS, both 1-[13C1]glycine and 2-[13C1]glycine were used as tracers in parallel experiments. Any increase in serine enrichment from 2-[13C1]glycine compared with 1-[13C1]glycine is due to the action of GCS and labeling of serine via the number 2 carbon of glycine labeling the methylene tetrahydrofolate pool. The degree of coupling of SHMT and GCS was expressed as: Equation where 1 is no coupling and 2 is complete coupling.

Figure 2
figure 2

Metabolic pathway for serine and glycine interconversion with SHMT and the GCS demonstrating the incorporation of the number 2 carbon from glycine (outlined “[stylized C]”) into the number 3 position of serine with the combined action of GCS and SHMT.

Full size image

Enzyme activity assays. For enzyme activity determination, cells from 6-10 10-cm plates were pooled for analysis. Enzyme activity was determined at isolation and after 24 h of culture in the presence of 10 nM dexamethasone (standard medium) and 10 nM dexamethasone and 100 nM insulin. The determination of enzyme activity after culture in the presence of insulin was performed to determine whether there were significant differences in the activity of SHMT with or without insulin. Cells were washed two times with ice-cold PBS and harvested by scraping with a rubber policeman into PBS. After isolation by low speed centrifugation and homogenization in 0.25 M sucrose, 10 mM HEPES (pH 7.4), and 0.1 mM EGTA, using 10 strokes of a glass/glass homogenizer followed by 10 strokes with a glass/Teflon homogenizer, mitochondrial and cytosolic fractions were prepared by differential centrifugation. Before assay, mitochondria were sonicated and the 100,000× g supernatant was used for the SHMT and citrate synthase assays. Cytosolic and mitochondrial extracts were assayed for SHMT activity according to the method of Geller and Kotb(15) by measuring the rate of formation of N5,10[14C]methylene tetrahydrofolate from 3-[14C1]serine and tetrahydrofolate and were expressed as nanomoles of product formed/mg (cytosolic or mitochondrial) protein/min. Citrate synthase activity (a marker of mitochondrial purity) was determined spectrophotometrically according to Shepherd and Garland(16). Protein concentration was determined by the method Lowry(17), as modified by Hartree(18). Cellular DNA was determined in individual cell wells used for the stable isotope studies on cellular tricholoracetic acid precipitates following the method of Burton(19).

Statistics. Data are expressed as mean ± SEM. All stable isotope experiments were carried out in duplicate/culture with triplicate analyzes of each sample. Comparison between t0 andt24 values is by the paired t test with significance taken at p < 0.05.

All aspects of the animal care and use were reviewed and approved by the UCHSC institutional animal care and use committee.

RESULTS

Hepatocytes were isolated from six mid-gestation fetal lambs with a mean age of 81 ± 6 d (73-86 d, normal gestation = 145 d). The yield of the perfusions varied from 0.3 × 109 to 1.5 × 109 hepatocytes with a mean viability of 97% (95-99%).

Change in medium amino acid concentration. The change in specific medium amino acid concentrations after 24 h of culture in two different medium serine concentrations is shown in Table 1. Initial medium serine and glycine concentrations in standard medium were 293 ± 33 and 662 ± 16 nmol/mL, respectively. Over 24 h there was a net increase in medium serine (2.58 ± 1.70 μmol/24 h/mg of DNA or 67 ± 32 nmol/mL/24 h) and a net decrease in medium glycine (-5.44± 2.03 μmol/24 h/mg of DNA or -114 ± 24 nmol/mL/24 h). The initial serine and glycine concentrations in high serine medium were 644± 88 nmol/mL and 506 ± 45 nmol/mL. Over 24 h, there was significant variability between cultures in the change in medium serine and glycine that was not significantly different from zero.

Table 1 Changes in medium amino acid concentrations over 24 h in cultures of midgestation fetal ovine hepatocytes
Full size table

In standard medium, there was net production of glutamate and alanine in addition to serine with net utilization of all other amino acids except valine and taurine, which were not significantly different from zero. Thus, mid-gestation fetal ovine hepatocytes exhibit a net production of medium serine, glutamate, and alanine, similar to that of late gestation fetal ovine hepatocytes(11) and similar to the pattern seenin vivo across the fetal ovine liver(2, 20). In the high serine medium, serine production ceased, glutamate and alanine production continued, and there was a sparing of the net utilization of most of the other amino acids (Table 1).

Serine metabolism. The rates of serine production and utilization for cells cultured in standard and high serine media are shown inTable 2. In standard medium, as determined by the stable isotope dilution method with 1-[13C1]serine, there is a significantly greater serine production compared with utilization that accounts for the net increase in medium serine. Under these culture conditions, 50% of serine produced is derived from glycine. With high serine medium, there is a shift to net serine utilization due to an increase in serine utilization without a significant change in serine production(Table 2).

Table 2 Serine and glycine production and utilization in cultured mid-gestation fetal ovine hepatocytes
Full size table

To determine the relative contribution of the coupled action of SHMT and GCS to serine biosynthesis, both 1-[13C1]glycine and 2-[13C1]glycine were used as tracers in parallel experiments. With each tracer, there was a net increase in the MPE of M + 1 serine over 24 h with a greater enrichment in serine from 2-[13C1]glycine (3.07± 0.26%) compared with 1-[13C1]glycine (2.34 ± 0.12%). Any increase in serine enrichment from 2-[13C1]glycine compared with 1-[13C1]glycine is due to the action of GCS and labeling of serine via the number 2 carbon of glycine labeling the methylene tetrahydrofolate pool (Fig. 2). The ratio of enrichment of M + 1 serine from 2-[13C1]glycine/1-[13C1]glycine in standard medium is 1.31 ± 0.09, indicating some coupling of GCS and SHMT. However, this coupling ratio is lower than the value of 1.7 we found in late gestation fetal hepatocytes cultured in similar conditions(7).

Glycine metabolism. The rates of glycine production and utilization in the two media are shown in Table 2. In standard medium, glycine utilization exceeds glycine production with a resultant net decline in medium glycine concentration over 24 h. Of the glycine produced, 18% was derived from serine. In high serine medium, there was a significant increase in glycine utilization with no effect on the percent of glycine produced derived from serine or on glycine production. Thus, despite culturing in medium with a serine concentration higher than that of glycine (where glycine formation from serine via SHMT should be favored), there is not an increase in synthesis of glycine from serine.

Transamination studies. As noted above, the contribution of nitrogen from glutamine via transamination reactions to serine synthesis can be estimated from the enrichment in serine by 2,5-[15N2]glutamine. This can be used to estimate the relative contribution to serine biosynthesis from phosphoserine aminotransferase and serine pyruvate aminotransferase (Fig. 1). Initial experiments with 1,2-[13C2]glutamine demonstrated a significant increase in the enrichment in M + 2 glutamate at 24 h, but not in serine or glycine. Thus, as expected, glutamine carbon does not contribute to serine biosynthesis. Using 2-[15N1]-glutamine as the tracer, there was a slight increase in M + 1 serine at 24 h (M + 1 MPE at 24 h: serine 0.8, glutamate 1.0, alanine 0.8 n = 2). In contrast, with 2,5-[15N2]glutamine as the tracer, there was a much more significant increase in the enrichment in M + 1 serine (M + 1 MPE at 24 h: serine 1.8 ± 0.6, glutamate 2.2 ± 0.3, alanine 3.2 ± 0.6n = 4). Thus, a substantial percentage of the serine nitrogen is derived directly or indirectly from the 5 position nitrogen of glutamine. The production and utilization rates for glutamine nitrogen, the estimated aminotransferase flux to serine, glutamate, alanine, and glycine, and the percent contribution to serine biosynthesis from 2,5-[15N2]glutamine are shown in Table 3. There is a significant utilization of glutamine over 24 h of culture. There is a high estimated flux rate to glutamate, alanine, and serine. The number 5 nitrogen of glutamine is used in 18 ± 7% of new serine biosynthesis.

Table 3 Glutamine production and utilization and relative amino acid flux from glutamine in mid-gestation fetal ovine hepatocytes
Full size table

SHMT activity. To validate that SHMT activity was stable for 24 h in the culture conditions chosen for the stable isotope studies, the specific activity of hepatocyte mitochondrial and cytosolic SHMT at isolation and after 24 h of culture in the presence of 10 nM dexamethasone compared with 10 nM dexamethasone and 100 nM insulin was determined and is shown inTable 4. There were no significant changes in SHMT isoform activity over the period of time involved in the stable isotope studies. Similarly, there were no changes in citrate synthase activity over the duration of the cultures.

Table 4 Activity of SHMT in primary culture of mid-gestation fetal ovine hepatocytes
Full size table

DISCUSSION

Several in vivo studies have suggested that serine and glycine occupy a unique metabolic position in the fetus. Fetal serine production has been demonstrated by the fetal ovine liver at both mid and late gestation(1, 2, 21). In addition, poor transport of serine and glycine from the mother to the fetus across the placenta has been demonstrated in sheep(2, 3). In humans, poor transport of glycine across the placenta has been recently documented(4). In growth-retarded human fetuses, plasma serine and glycine concentrations are significantly reduced compared with other amino acids(5, 6). Thus, the fetus must rely on endogenous production of serine and glycine, and changes in the regulation of fetal serine and glycine biosynthesis and utilization appear to be the only mechanisms available to the fetus to affect the supply of these two amino acids. This study has shown that, similar to the findings in vivo across the fetal liver, serine is produced in excess of its utilization by hepatocytes isolated from mid-gestation fetal lambs. The finding of a net increase in medium serine in cultured hepatocytes is in contrast to data fromin vivo studies in adult animals and in vitro studies in hepatocytes from adult rodents, where a net uptake of serine across the liver and a net decrease in medium serine concentration over 24 h has been documented(22, 23).

This is the first study to measure serine and glycine production rates using stable isotope dilution in hepatocyte culture. In this study, the serine and glycine production and utilization rates are consistent with the changes in the medium concentration of serine and glycine over 24 h. In standard medium, serine production exceeds utilization with a net result of an increase in medium serine concentration over 24 h. In contrast, glycine production is less than utilization with a net result of a decrease in medium glycine over 24 h. The site of regulation of this fetal hepatic serine production is unknown. We have demonstrated that one half of the fetal hepatic serine production is derived from glycine via the actions of SHMT and GCS. The SHMT isoform most associated with serine production is uncertain; however, studies in yeast and Chinese hamster ovary cells have suggested that cSHMT activity is important for serine biosynthesis(24, 25). This suggests that alterations in the specific activity of cSHMT might have a significant impact on serine production in the fetal liver. Both Pittset al.(8) and Rowsell et al.(9) have suggested that, in the kidney, SHMT and GCS operate in a coupled manner in the production of serine from glycine. In late gestation fetal ovine hepatocytes, we found a coupling ratio of 1.7, whereas the ratio in this study in mid-gestation hepatocytes is only 1.3. These data suggest that there is a developmental increase in the degree of coupling of GCS and SHMT from mid to late gestation in the fetal ovine liver, although the mechanism is still uncertain.

Some authors have suggested that serine is a semiessential amino acid in the fetus. In these experiments, increasing the available serine supply by increasing medium serine concentration resulted in a 2-fold increase in serine utilization (Table 2) and a change in the medium amino acid pattern at 24 h with a sparing of the decline in the concentration of most other amino acids at 24 h. These data could be interpreted to suggest that serine may be a semiessential amino acid in the fetus, particularly in terms of growth. This may partially explain the association between low plasma serine levels and growth retardation in human fetuses(5, 6).

Although 50% of serine biosynthesis is via the GCS and SHMT pathways, the source of the other half of serine biosynthesis is uncertain. Studies by Bismut et al.(26) have suggested that, in the rat fetus, glucose may be an important precursor for serine biosynthesis. However, studies in the sheep have not confirmed this(13). Cheung et al.(10) have shown that the flux of serine biosynthesis through the phosphorylated pathway of serine biosynthesis is high in fetal liver, whereas it is the nonphosphorylated pathway that is high in adult liver. However, the relative contribution of these pathways to serine biosynthesis is unknown. Using the labeling of serine via transamination reactions, we have made the first attempt to estimate the combined contribution of these two pathways to serine biosynthesis. Our data suggest that approximately one fifth of serine biosynthesis is derived from transamination reactions involving glutamine, glutamate, and alanine. This methodology is only an estimation of the contribution of various transamination reactions to the rate of serine biosynthesis as alanine and glutamate can also serve as nitrogen sources for transamination reactions. Both alanine and glutamate are labeled with the 2,5-[15N2]glutamine (MPE at 24 h of M + 1 glutamate = 2.2± 0.3, alanine = 3.2 ± 0.6, serine = 1.8 ± 0.6). Therefore, some of the incorporation of 15N into serine may have come from alanine or glutamate which would underestimate the total contribution from nitrogen donors to serine biosynthesis. Further studies are required to identify the exact precursors and metabolic pathways for these transamination reactions.

The measurement of serine production clearly includes the release of serine from protein degradation in the hepatocytes. Thus, it is possible that the entire de novo synthesis of serine is accounted for by the combined action of SHMT, GCS, and the transamination reactions.

In conclusion, serine production is high in the mid-gestation fetal hepatocyte. Fifty percent of serine production is derived from glycine via the combined actions of SHMT and GCS. Transamination contributes at least 20% of serine production. Serine may function as a semiessential amino acid in the fetal liver and thus be important in the regulation of fetal growth.