Chem. Res. Toxicol. 2005, 18, 277-282
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Monochloroacetic Acid Inhibits Liver Gluconeogenesis by Inactivating Glyceraldehyde-3-phosphate Dehydrogenase Akiko Sakai,*,† Hiroyasu Shimizu,‡ Koichi Kono,‡ and Eisuke Furuya† Departments of Chemistry and Hygiene and Public Health, Osaka Medical College, 2-7 Daigakumachi, Takatsuki, Osaka 569-8686, Japan Received August 17, 2004
We previously reported that a lethal dose of monochloroacetate (MCA) causes severe hypoglycemia and lactic acidosis. MCA has been thought to inhibit mitochondrial aconitase; however, the exact effect of MCA on hepatic glucose metabolism is not clear. In this study, we investigated the effects of MCA on liver gluconeogenesis using an isolated perfused rat liver system. Gluconeogenesis from 2.5 mM lactate was inhibited by 1 mM MCA and was completely abolished after 2 h of perfusion. Levels of citric acid cycle intermediates such as citrate, isocitrate, and 2-oxoglutarate (2-OG) were significantly reduced by MCA. The finding that the levels of citrate and 2-OG were similarly reduced (to 31 and 36% of control, respectively) indicates that aconitase was not inhibited by MCA. On the contrary, gluconeogenesis from glycerol, which can be converted to glucose without glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not inhibited by MCA. GAPDH was inactivated by MCA in vitro, but enolase, phosphoglycerate mutase, and phosphoglycerate kinase were not inactivated at the same or higher concentrations of MCA. Furthermore, GAPDH activity in the MCA-perfused liver decreased to 33-42% of control and that in the liver of rats exposed to MCA was reduced to 19% of control. We concluded that MCA inactivates GAPDH, and this is the cause of the inhibition of liver gluconeogenesis.
Introduction Monochloroacetic acid is a halogenated analogue of acetic acid and is used in chemical industries as an intermediate for the production of carboxymethylcellulose, phenoxy acetates, drugs, dyes, and other substances (1). Accidental occupational exposure has been reported for workers handling, transporting, and producing monochloroacetic acid (2, 3). It is strongly corrosive, and the small size and irritative nature of the compound allow extensive absorption via the skin. Burns from 80% monochloroacetic acid solution that cover only 25-30% of the body surface cause lethal systemic poisoning (2). We previously reported that a single lethal dose of monochloroacetate (MCA)1 causes severe hypoglycemia and lactic acidosis and that these symptoms can be prevented by continuous infusion of glucose solution (4). The metabolic fate of monofluoroacetate (MFA), another halogenated analogue of acetic acid, has been studied in detail. MFA is converted to fluoroacetyl-CoA by acetyl-CoA synthetase and then to fluorocitrate by citrate synthase. Fluorocitrate is a “suicide” substrate for aconitase, resulting in inhibition of citric acid cycle (5, * To whom correspondence should be addressed. Tel: 81-72-6831221 ext. 2962. Fax: 81-72-684-7086. E-mail:
[email protected]. † Department of Chemistry. ‡ Department of Hygiene and Public Health. 1 Abbreviations: DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); GAP, glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KHB, Krebs-Henseleit bicarbonate; MCA, monochloroacetate; MCT, monocarboxylate transporter; MFA, monofluoroacetate; MIA, monoiodoacetate; 2-OG, 2-oxoglutarate; PEG, poly(ethylene glycol); PEP, phosphoenolpyruvate; 3-PG, 3-phosphoglycerate; PGK, phosphoglycerate kinase; SH, thiol; TEA, triethanolamine.
6). Because of the structural similarity to MFA, it has been thought that MCA might also inhibit aconitase (7). However, it has been reported that MCA inhibits aconitase activity in the heart but not in the liver (8); therefore, the exact toxicological mechanism of action of MCA remains controversial. In the present study, we investigated the effects of MCA on liver gluconeogenesis, which plays a central role in maintaining blood glucose levels, using an isolated perfused rat liver system. The isolated perfused liver is useful for analyzing the effect of MCA on glucose metabolism in an intact organ because we can monitor and control the concentrations of drugs to which the liver is exposed and measure hepatic glucose production from lactate and other substrates.
Materials and Methods Materials. Sodium MCA and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Wako (Osaka, Japan). Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and phosphoglycerate kinase (PGK) were from Roche Molecular Biochemicals (Mannheim, Germany). dl-glyceraldehyde 3-phosphate (GAP) and 3-phosphoglycerate (3-PG) were from Sigma (St. Louis, MO). All chemicals were of the highest purity commercially available. Animals. Male Sprague-Dawley rats weighing 160-180 g were purchased from SLC (Hamamatsu, Japan) and housed under a 12 h light-dark cycle for at least 1 week with free access to rodent chow and water. Prior to liver perfusion experiments, animals were fasted for 40 h in order to deplete liver glycogen and maximize the ability of gluconeogenesis, as described in ref 9. The experimental procedures for animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Animal Research Laboratory, Osaka Medical College).
10.1021/tx0497705 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/15/2005
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Perfusion of Isolated Rat Liver. Rats were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg body wt), and the isolated perfused rat liver system was prepared according to the method reported by Sugano et al. (9). Briefly, after anesthesia, the abdomen was opened, and the liver, portal vein, and inferior vena cava were exposed. The portal vein was cannulated with a polyethylene drip tube, and the liver was perfused with the oxygenated perfusion medium described below. The flow rate of the perfusate was 3.5 mL/min per g of liver with a perfusion pump (Decarf-N, Taiyo Kagaku, Tokyo, Japan), and the perfusate temperature was maintained at 32 °C. The abdominal vena cava was incised immediately after cannulation, the diaphragm was then opened, and the thoracic vena cava was cannulated with a polyethylene tube. The abdominal vena cava was ligated, and the liver was excised. The whole operation took 5-8 min. Krebs-Henseleit bicarbonate (KHB) buffer (119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3) saturated with 95% oxygen/5% carbon dioxide was used. After a 30 min equilibration period, the substrates for gluconeogenesis (lactate or glycerol; final concentrations were 2.5 and 2 mM, respectively) with or without MCA (final concentration was 1 mM) were added to the perfusion fluid with a PHD 2000 syringe pump (Harvard Apparatus, Holliston, MA). The perfusate eluted from the liver was collected at 4-10 min intervals for an additional 120 min and used for determination of glucose concentration. At the end of perfusion, a small piece of the liver was removed, freeze-clamped with a pair of tongs precooled in liquid nitrogen, lyophilized, and stored at -80 °C. In separate experiments, the perfused whole liver was removed and homogenized for preparation of GAPDH as described below. Enzymatic Assays. To determine the rate of gluconeogenesis from a given substrate, the rate of hepatic glucose production was measured. The glucose concentration in the collected perfusate was assayed electrochemically after treatment with an immobilized glucose oxidase column (10). To determine the concentrations of metabolic intermediates in the perfused rat liver, lyophilized livers were sonicated using a Branson Sonifier 250 (Branson, Danbury, CT) in 10-16× volumes of 1 M perchloric acid. The homogenate was centrifuged for 5 min at 15000g, and the supernatant was neutralized with 5 M KOH. Supernatants were then used to measure concentrations of pyruvate, citrate, isocitrate, 2-oxoglutarate (2-OG), malate, and phosphoenolpyruvate (PEP) by the enzymatic methods described in ref 11. Adenine nucleotides were measured as described in ref 12. GAPDH Assay. GAPDH activity was measured according to the Boehringer procedure (Mannheim, Germany; assay instruction 5178). The reaction mixture (1.0 mL) contained 82.3 mM triethanolamine (TEA)-HCl buffer (pH 7.6), 0.5 mM EDTA, 0.2 mM NADH, 1 mM ATP, 2 mM MgSO4, 6 mM 3-PG, and 13 U/mL PGK. The reaction was started by the addition of GAPDH solution after a 5 min preincubation at 30 °C. For the inhibition assay, partially purified GAPDH from rat liver was treated with various concentrations of MCA in 50 mM TEA-HCl buffer (pH 7.6) at 30 °C for 30 min, and the remaining activity was measured with the above method. The enzyme activity was determined by measuring the initial rate of decrease in absorbance at 340 nm with a Shimadzu UV1600PC spectrophotometer. Assay of Other Enzymes. The 25% poly(ethylene glycol) (PEG) fraction of rat liver homogenate was used as the source of phosphoglycerate mutase and enolase in order to remove low molecular weight compounds that may interfere with the analysis. In brief, rat liver was homogenized in 50 mM TEA-HCl buffer (pH 7.6) containing 100 mM KCl, 0.5 mM EDTA, and 5 mM DTT with a Potter homogenizer. The homogenate was centrifuged at 100000g for 40 min, and the supernatant was added to an equal volume of 50% PEG 6000 solution. After centrifugation at 35000g for 30 min, the pellet was resuspended in the above buffer and used immediately. The 25% PEG fraction of rat liver homogenate was treated with 5 or 10 mM MCA in 50 mM TEA-HCl buffer (pH 7.6) containing 0.5
Sakai et al. mM EDTA at 30 °C for 30 min, and the remaining activity was measured. Isocitrate dehydrogenase, phosphoglycerate mutase, and enolase activities were measured as described in ref 11. The 0-75% saturation ammonium sulfate fraction (as described below) of rat liver homogenate, which contains no GAPDH activity, was used to study the effect of MCA on PGK activity. This fraction was incubated with 5 or 10 mM MCA at 30 °C for 30 min, and the remaining PGK activity was measured in the reaction mixture (1.0 mL) contained 82.3 mM TEA-HCl buffer (pH 7.6), 0.5 mM EDTA, 0.2 mM NADH, 1 mM ATP, 2 mM MgSO4, 6 mM 3-PG, and 20-fold excess activity of GAPDH. Exposure of Rats to MCA in Vivo. Overnight-fasted rats were injected subcutaneously with a lethal dose (80 mg/kg body wt) of MCA (4) or saline. In MCA-exposed rats, livers were excised immediately after death (approximately 3 h after MCA administration) and homogenized for preparation of GAPDH. Partial Purification of GAPDH by Ammonium Sulfate Fractionation. Livers from saline-treated rats (n ) 3), MCAexposed rats (n ) 3), or isolated perfused liver in the presence or absence of MCA (n ) 4-6) were homogenized in 3 × volumes of 10 mM TEA-HCl buffer (pH 7.6) containing 100 mM KCl and 0.5 mM EDTA with a Potter homogenizer. The homogenate was centrifuged at 16000g for 20 min, and the supernatant was further centrifuged at 125000g for 1 h. Then, 51.6 g of ammonium sulfate per 100 mL of supernatant was added to give 75% saturation, after 20 min the solution was centrifuged at 12000g for 20 min. The ammonium sulfate concentration in the supernatant was raised to 97% saturation by addition of 18 g of ammonium sulfate per 100 mL, after 30 min the precipitated proteins were collected by centrifugation at 125000g for 1 h (13, 14). The precipitate was resuspended in the above buffer, and aliquots were stored at -80 °C. The protein concentration was determined with the Bio-Rad protein assay using bovine serum albumin as a standard. Titration of Thiol (SH) Groups. Rat liver GAPDH was purified to homogeneity using ammonium sulfate fractionation, hydroxyapatite, and DEAE column chromatography. Briefly, ammonium sulfate fraction (75-97% saturation) prepared from 30 g of rat livers was desalted with a Sephadex G-50 column (2.5 cm × 21 cm) equilibrated with 10 mM potassium phosphate buffer (pH 6.9). The eluate was applied to a column of hydroxyapatite (2.5 cm × 7 cm) equilibrated with 10 mM potassium phosphate buffer (pH 6.9) and eluted with a 200 mL linear gradient of 10-400 mM potassium phosphate buffer (pH 6.9). The GAPDH fractions, as determined by SDS-PAGE, were concentrated and desalted with a Vivaspin 20 (Vivascience AG, Hannover, Germany) and then applied to a DE52 column (2.5 cm × 6 cm) equilibrated with 5 mM potassium phosphate buffer (pH 7.7) containing 1 mM DTT. The GAPDH fractions were pooled and concentrated. The purified preparation showed a single band on SDS-PAGE. The number of SH groups was measured by the method of Ellman (15). Briefly, purified rat liver GAPDH was incubated with 10 mM MCA in 50 mM TrisHCl buffer (pH 8.0) containing 1 mM EDTA at 30 °C for 0, 30, and 60 min. Desalted and concentrated GAPDH solutions (2.5-4 µM) in 50 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA and 6 M guanidine-HCl were incubated with excess DTNB in a 1 mL cuvette at 25 °C. We used a molar extinction coefficient of 13700 M-1 cm-1 at 412 nm for the thiolate dianion of DTNB in buffered 6 M guanidine-HCl (16). Values are means ( SD of three to five independent experiments. The protein concentration was determined with the Bio-Rad protein assay using rabbit skeletal muscle GAPDH as a standard. Statistical Analysis. Values are presented as means ( SD. The two population means were compared by unpaired Student’s t-test after testing for the equality of variances between the two groups using the F-test. Differences were considered statistically significant when the two-sided p values were
