Departments of 1Urology and 2Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
Submitted 12 May 2004 ; accepted in final form 6 July 2004
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ABSTRACT |
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liver; peroxisomes; hepatocytes; hyperoxaluria; alanine:glyoxylate aminotransferase; glyoxylate reductase
Several factors contribute to the limitations of our knowledge of oxalate synthesis, including a low rate of synthesis, technical difficulties in measuring the low concentration of oxalate in cells, and the lack of suitable cellular or animal models to study synthetic steps. Isolated rat hepatocytes or primary cultures of rat hepatocytes have been used as cellular models (3, 29, 30), but these cells have important metabolic differences from human hepatocytes. These differences include the activity of AGT1, which is not only much lower in rat liver compared with human liver but is also compartmentalized in mitochondria as well as in peroxisomes (8, 32).
HepG2 cells were derived from a human hepatoma, and they retain many important hepatocyte functions (20, 36). They have been shown to retain AGT and GO activities and have been proposed to be a useful model to investigate oxalate synthesis (36). We therefore studied oxalate synthesis in these cells, focusing on the metabolism of the potential immediate precursors of oxalate, glycolate and glyoxylate, and compared the enzymatic properties of these cells with human liver tissue.
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MATERIALS AND METHODS |
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Cells. HepG2 cells were obtained from the American Type Culture Collection (Rockville, MD) at passage 76 and were used only until passage 120. They were routinely grown at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS in a humidified atmosphere containing 5% CO2 and used for experiments when confluent.
Preparation of mitochondria. Mitochondria were prepared as described previously, with modifications (2, 31). Confluent monolayers of cells were washed twice with PBS, scraped from dishes, suspended in mitochondria isolation buffer (1:10 wt/vol; 0.25 M sucrose, 1.0 mM EDTA, and 5 mM Tris·HCl pH 7.5), and homogenized with a microfuge tube homogenizer for 15 s. A portion of the homogenate was centrifuged at 1,500 g (5 min) to remove unbroken cells and cellular debris. The postnuclear supernatant was subsequently centrifuged at 16,000 g (20 min) to form a crude mitochondrial pellet. The pellet was resuspended in 2 ml of isolation buffer containing 15% Percoll, which was layered over a 40%, 23% Percoll discontinuous gradient. Gradients were centrifuged at 30,700 g for 10 min, and the mitochondria were collected at the interface between the 23% and 40% Percoll layers.
Oxalate analysis. To determine oxalate levels within cells, washed cell monolayers (3 x PBS) were extracted with ice-cold 10% trichloroacetic acid (TCA) for 30 min. The TCA was removed from extracts by vigorously vortexing with an equal volume of 1,1,2-trichlorotrifluoroethane (Freon)-trioctylamine (3:1, vol/vol; Aldrich, Milwaukee, WI), centrifuging at 4°C to promote phase separation, and collecting the upper aqueous layer for analysis (26). After treatment of samples with the Ag-treated resin as described by Hagen et al. (13), oxalate was measured by ion chromatography following their procedure. The HPLC equipment consisted of a Waters 510 pump, a Rheodyne 7125 injector, a Waters 431 conductivity detector, a Dionex AS 10 4 x 250-mm ion exchange column, and a Dionex ASRS-ULTRA 4-mm suppressor. A mobile phase of 30 mM sodium tetraborate at a flow rate of 1 ml/min was used.
Glycolate, lactate, and amino acid analyses. Glycolate and lactate in extracts were separated from other anions by ion exclusion chromatography on a Bio-Rad Aminex HPX-87H column, where they coeluted, with a running solvent of 0.7 mM H2SO4 and a flow rate of 0.4 ml/min. The glycolate in the peak was converted to glyoxylate by spinach GO, and lactate was converted to pyruvate by Pediococcus lactate oxidase with a 15-min incubation in 50 mM Tris buffer (pH 8.5) containing 10 mM phenylhydrazine. The phenylhydrazone derivatives of glyoxylate and pyruvate were measured by reverse-phase HPLC as previously described (17). Amino acids were estimated by phenyl isothiocyanate (PITC) derivatization and HPLC analysis (9).
Enzymatic assays.
Cell extracts were prepared by incubating monolayers with hypotonic detergent lysis buffer (25 mM HEPES pH 7.0, 0.1% Triton X-100) for 20 min on ice. Liver homogenates (10% wt/vol) were prepared in the same hypotonic detergent lysis buffer. The activities of GO and AGT were measured by HPLC techniques as previously described (17). GR activity was measured spectrophotometrically with the assay conditions of Giafi and Rumsby (12), except for the reduction of the NADPH concentration to 0.2 mM to accommodate absorbance limitations of the Beckman DU 640i spectrophotometer. LDH activity was also measured spectrophotometrically at 37°C by following the formation of NADH at 340 nm in an assay mixture containing (in mM) 100 Tris·HCl (pH 9.0), 2 NAD+, and 20 lithium lactate. Catalase activity was measured spectrophotometrically by following the direct oxidation of hydrogen peroxide (1). The -alanine-pyruvate aminotransferase (
-AlaAT) II activity of AGT2 was measured as described by Tamaki et al. (33) by measuring the formation of malonate semialdehyde from
-alanine.
Protein analysis. The protein content of cell monolayers was measured after dissolution of the cells with 0.1 M NaOH. A bicinchoninic acid assay was used with a kit supplied by Pierce (Rockford, IL) with bovine serum albumin as the standard. The absorbance at 562 nm (A562) of samples was compared with a standard curve to determine protein concentrations.
RNA isolation and RT-PCR. Total RNA was isolated from HepG2 cells by the guanidinium-phenol-chloroform extraction method of Chomczynski and Sacchi (5). One microgram of total RNA was used to carry out reverse transcription using oligo(dT) primers and the GeneAmp RNA PCR kit (Perkin-Elmer, Branchburg, NJ) according to the manufacturer's instructions. The reverse transcription reaction was used as template in subsequent nested PCR reactions to amplify AGT2 message in a 100-µl PCR reaction volume. The first reaction contained 1x thermophilic DNA polymerase buffer (Promega, Madison, WI), 2.5 mM MgCl2, each dNTP at 100 µM (Promega), each primer at 150 nM, and 5 U of Taq DNA polymerase (Promega). The sequence of the 5' primer was 5'-AAATGACTCTAATCTGGAGACATTTGC-3'. The sequence of the 3' primer was 5'-GGCTTTCTCTCTGGATACCAGTGG-3'. In the nested set of PCR reactions, 20 µl of the first reaction was used as template with 5' primer 5'-AAATGACTCTAATCTGGAGACATTTGC-3' and 3' primer 5'-GTGGCAAATTCTTGAGACTTGTGG- 3'. In the first set of reactions, the samples were heated to 94°C for 5 min and then 80°C for 5 min. After 1 min at 80°C DNA polymerase was added. Subsequent to this, 30 amplification reactions were carried out as follows: 94°C, 1 min; 57°C, 1 min; 72°C, 1 min. A final extension was performed at 72°C for 7 min. All reaction conditions were identical in the nested amplification except that after the 5-min incubation at 80°C 30 cycles of 94°C, 1 min; 52°C, 1 min; and 72°C, 1 min were performed, followed by a final extension at 72°C for 7 min. Samples were stored at 4°C until they were used. For a positive control, GAPDH mRNA was amplified with human GAPDH primers purchased from Stratagene (La Jolla, CA) according to the manufacturer's instructions (not shown). Water in place of RNA was included in RT-PCR as well as PCR reactions as a negative control (not shown). PCR products were electrophoresed on 1% agarose gels and visualized by staining with ethidium bromide.
Cell volume measurements. The volume of HepG2 cells under our experimental conditions was measured by radioisotopic methods previously described, which used [14C]urea and [14C]sucrose as markers of the total and extracellular spaces, respectively (4, 23).
Data analysis. The results, unless otherwise indicated, are presented as means ± SE. The data shown are representative of two or three independent experiments that showed similar results.
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RESULTS |
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Oxalate and glycolate in growth medium. The concentration of glycolate and oxalate in the growth medium of confluent monolayers of HepG2 cells increased over time, compatible with an ongoing synthesis of these metabolites in cells (Fig. 2). The rate of oxalate synthesis in serum-containing medium at 72 h, as measured by the change in oxalate concentration in the medium, was 0.26 ± 0.03 pmol·min1·mg protein1. Substantially more glycolate than oxalate was excreted into the medium (4.80 ± 0.35 pmol·min1·mg protein1). The ratio of glycolate to oxalate excretion over this time period was 32.8 ± 5.7. Glyoxylate was not detected in the growth medium under any experimental conditions. The synthesis of oxalate was 3.0-fold higher and that of glycolate 2.4-fold higher in media containing 10% FBS compared with growth in media lacking serum for 72 h (Fig. 2). This stimulation suggests that either hormones or growth factors in serum increase the metabolism associated with oxalate synthesis.
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Uptake and metabolism of glycolate. The uptake of glycolate by HepG2 cells, as measured by its intracellular content, was rapid and was near equilibrium by the first time point of 5 min (Fig. 4A). This rapid equilibration across the plasma membrane is consistent with the properties of the monocarboxylate transporter that has been identified in hepatocyte plasma membranes and has been shown to transport glycolate as effectively as lactate (19). If the glycolate is equally distributed throughout the cell, the intracellular glycolate concentration would be 6.8 mM, compared with the extracellular concentration of 10 mM. There was a time- and concentration-dependent increase in the oxalate concentration of the medium (Fig. 4B), with 10-fold higher concentrations than those observed with basal medium (Fig. 2). The intracellular concentration of glycolate varied linearly with the glycolate content of the medium (Fig. 4C). Despite this large change in glycolate concentration, the intracellular oxalate concentration remained unchanged at 423 ± 34 nmol/mg protein. A hyperbolic relationship was observed between the glycolate content of the medium and the amount of oxalate excreted into the medium (Fig. 4D). Oxalate production did not plateau, even with 50 mM glycolate in the medium (results not shown). The increased osmolality in the media with high glycolate concentrations did not influence oxalate synthesis, because the addition of 20 mM acetate had no effect. Metabolism in the cell also did not appear to be affected by the high glycolate concentration, because the lactate concentration in cells was unaffected: 44.7 ± 5.0 µmol/mg protein in control cells and 41.4 ± 3.3 µmol/mg protein in cells grown with 20 mM glycolate.
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DISCUSSION |
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In HepG2 cells and liver tissue, the concentrations were oxalate > glycolate > glyoxylate. The concentration of oxalate was similar in liver tissue and HepG2 cells (60 µM) and at this level may influence metabolism. At micromolar concentrations, oxalate is a potent inhibitor of both pyruvate kinase (28) and pyruvate carboxylase (10, 37) activities. Whereas the oxalate concentration was similar in cells and tissue, glycolate and glyoxylate concentrations were higher in liver than in HepG2 cells. Such differences may reflect differences in metabolism or may result from anoxia that occurred in the human tissue before freezing. They may also reflect changes in metabolism with the adaptation of cells to growth in culture or changes associated with malignancy. The amount of glycolate detected in human liver is similar to that reported in guinea pig liver (17). We have detected glycolate at concentrations similar to that observed in HepG2 cells in a wide range of cells, and this appears to be associated with the presence of GR activity (RP Holmes, M Kennedy, and SD Cramer, unpublished observations). These observations suggest that GR contributes to the synthesis of glycolate in most cells, disposing of endogenously produced glyoxylate and limiting oxalate production. Glyoxylate concentrations are low in HepG2 cells (Table 1), consistent with the low values we observed in guinea pig liver tissue (17) and other reports of a low concentration in rodent liver tissue (11). The source of the glyoxylate for oxalate and glycolate synthesis in HepG2 cells is not known, if as proposed below, GO plays a very limited role in glyoxylate production in these cells. We have identified pyruvate as a possible direct or indirect source of glyoxylate in HepG2 cells, but a more extensive evaluation is required to confirm its role and to identify other precursors (14).
Glycolate is considered to be a major precursor of oxalate in hepatocytes (7). In HepG2 cells, high concentrations of glycolate were required to stimulate oxalate synthesis. This requirement was not due to a limited glycolate uptake, because uptake was rapid and glycolate equilibrated within the cell at a concentration that was 70% of that in the extracellular medium (Fig. 4). No response was detected with 500 µM glycolate in the medium where the intracellular glycolate concentration reached
350 µM. HepG2 cells have a low activity of GO compared with liver tissue (Table 1), and we have argued previously (18) that this activity is too low to account for the oxalate synthesis observed, particularly when the AGT activity is over 100-fold higher. The failure to detect the synthesis of glycine from glycolate in these cells is also consistent with GO activity not being involved, because the substantial AGT activity in these cells should have converted GO-derived glyoxylate primarily to glycine. Experiments with purified LDH indicated that it was able to catalyze the conversion of glycolate to glyoxylate at high substrate concentrations. Thus the conversion of glycolate to oxalate in HepG2 cells is compatible with an LDH-catalyzed sequential conversion of glycolate to glyoxylate and the glyoxylate formed to oxalate.
The failure to observe the conversion of [14C]glycolate to [14C]glycine in HepG2 cells unless the detergent Triton X-100 was present suggests that peroxisomes in these cells are not readily permeable to glycolate. This impermeability is in contrast to a report that the peroxisomal membrane is freely permeable to glycolate in rat hepatocytes (35). One potential problem with the experimental approach in the studies of Verleur and Wanders (35) is that digitonin was used to permeabilize the plasma membrane. It is possible that the digitonin permeabilized both the plasma and peroxisomal membranes. The disruptive effect of digitonin on the permeability of isolated peroxisomes has been described by Pahan and Singh (25). In experiments with HepG2 cells, and presumably isolated hepatocytes, digitonin is not required to permeabilize cells because the monocarboxylate transporter facilitates a rapid equilibration of glycolate across the plasma membrane as shown in Fig. 4. Preliminary experiments in our laboratory with human hepatocytes indicate that their peroxisomes are more permeable to glycolate. However, the permeability is enhanced approximately threefold by Triton X-100, illustrating that their permeability to glycolate is also restricted.
Our investigations revealed that human hepatocyte mitochondria possess AGT2 activity. This aminotransferase is quite distinct from, and has no homology with, AGT1 (22). The nucleotide sequence coding for the human enzyme has been deposited in GenBank. Previous reports that this activity was not present in human liver were based on the use of antibodies to the porcine enzyme (32). It seems possible that this antibody did not recognize the human enzyme. This aminotransferase has several activities, including D-3-aminobutyrate:pyruvate aminotransferase, -alanine:pyruvate aminotransferase, and dimethylarginine:pyruvate aminotransferase activities, as well as AGT activity (21, 24). Wanders et al. (36) previously reported that AGT activity was apparent in the mitochondria of HepG2 cells. We observed that HepG2 mitochondria also contained glyoxylate reductase with an activity similar to the activity detected in cell homogenates, suggesting that the cytoplasm and mitochondria have equivalent activities. An analysis of the 5' terminus reveals a sequence of 17 amino acids from the second methionine that has a 98.3% probability of targeting the enzyme to mitochondria (6). Furthermore, this enzyme was recently identified as a component of human heart mitochondria (34). These two enzyme activities, GR and AGT2, could function in mitochondria to convert the glyoxylate produced from hydroxyproline metabolism to glycolate and glycine. The reactions associated with these two enzymes, their cellular compartmentation, and their association with other steps involved in oxalate synthesis are shown in Fig. 6.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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