Introduction

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Based on sensitivity to the alcohol dehydrogenase (ADH) inhibitor 4-methylpyrazole, it was reported previously that ADH was involved in the swift increase in alcohol metabolism (SIAM) (Yuki and Thurman, 1980). This phenomenon is characterized by increased basal oxygen uptake, which provides NAD⁺ for ADH-dependent alcohol metabolism (Yuki and Thurman, 1980). As glycogen reserves are depleted, glycolysis is slowed and ADP is shuttled into the mitochondria, where respiration is increased. More recently, however, it has been shown that 4-methylpyrazole also inhibits acyl CoA synthetase (Bradford et al., 1993a), a pivotal enzyme in the synthesis of acyl CoA compounds required for the generation of H₂O₂ via peroxisomal -oxidation. Catalase is localized in the peroxisome, and the catalase pathway requires H₂O₂, which is provided largely by metabolism of fatty acids via peroxisomal -oxidation (Lazarow and de Duve, 1976; Handler and Thurman, 1988). Methanol is a known selective substrate for catalase in rodents and is an excellent tool for the evaluation of catalase-dependent alcohol metabolism without the use of inhibitors (Feytmans et al., 1974; Bradford et al., 1993a). It is well known that ethanol stimulates peripheral lipolysis and increases circulating triglycerides (Khanna et al., 1974); however, whether catalase participates in the mechanism of SIAM is not known.

It has been demonstrated that Kupffer cells, the resident hepatic macrophages, participate in the pathophysiology of ethanol-induced liver damage (Adachi et al., 1995). When Kupffer cells are activated, potent cytokines such as platelet-activating factor and tumor necrosis factor- are released. In addition, Kupffer cells release prostaglandin E₂ (PGE₂), which stimulates oxygen uptake by parenchymal cells via increases in cAMP (Qu et al., 1996). GdCl₃ specifically destroys large Kupffer cells without causing other morphological changes in liver (Hardonk et al., 1992). Moreover, GdCl₃ diminishes inflammation and necrosis due to ethanol (Adachi et al., 1994) and fibrosis using model compounds (Sullivan et al., 1995; Wall et al., 1995). Furthermore, GdCl₃ treatment blocked the increase in hypoxia and production of -hydroxyethyl radicals associated with chronic ethanol exposure (Adachi et al., 1994; Knecht et al., 1995). Therefore, this study will test the hypothesis that Kupffer cells and catalase are involved in the hypermetabolic state and the swift increase in alcohol metabolism (SIAM). Preliminary accounts of this work have appeared elsewhere (Bradford et al., 1994).

	Materials and Methods

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Treatment of Rats. Fed, female Sprague-Dawley rats (100-120 g) were used in this study. GdCl₃ (10 mg/kg) dissolved in acidic saline (pH 3.0) was injected into the tail vein 24 h before perfusion in some rats. This dose of GdCl₃ has been shown to remove large Kupffer cells without causing other morphological changes in the liver (Hardonk et al., 1992). Moreover, it eliminates about 80% of Kupffer cells based on mRNA for a specific Kupffer cell lectin (Koop et al., 1997). Ethanol (5.0 g/kg), methanol (5.0 g/kg), and olive oil (2 ml/100 g b.wt.), a good source of oleate, were administered intragastrically 2.5 h before liver perfusion. In some experiments, indomethacin (3.0 mg/kg, in dimethylsulfoxide) was administered intragastrically 1 h before ethanol.

Liver Perfusion and Alcohol Metabolism. Livers were perfused using hemoglobin-free Krebs-Henseleit buffer under conditions that were established more than 30 years ago (Scholz, 1968) and have been used for studies of hepatic metabolism, oxidation of xenobiotics, and metabolism of alcohols (reviewed in Brouwer and Thurman, 1996). The oxygen concentration in the effluent perfusate was monitored using a Teflon-shielded, Clark-type oxygen electrode. After oxygen uptake reached steady state values in about 15 min, the perfusion system was converted to a closed system with a 50-ml volume containing either 25 mM ethanol or methanol. Perfusate was reoxygenated using a Silastic tube oxygenator (Handler et al., 1986). Samples of perfusate (0.5 ml) were collected every 10 to 15 min, and the decrease in alcohol concentration over time was measured with head-space gas chromatography as described in detail elsewhere (Bradford et al., 1993a). Rates of alcohol metabolism were calculated based on changes in concentration over time and were expressed per gram of liver per hour. After perfusion, the liver was fixed with a solution of 5% buffered formalin and was perfused with alcohol for 15 min. Alcohol elimination was monitored during this period to correct for vaporization of alcohol from the organ surface, which was minimal.

Enzyme Measurements. Liver homogenates (1:10) were prepared in 0.25 M sucrose, and activities of catalase, acyl CoA synthetase, and acyl CoA oxidase were determined as described previously (Bradford et al., 1993a). Lipoprotein lipase (LPL) activity was determined in homogenized retroperitoneal fat pads as described elsewhere (Borensztajn et al., 1970). Measurement of LPL activity required chylomicrons that were harvested from fasted rats treated for 4 h with olive oil (2 ml/100 g b.wt. i.g.). Rats were anesthetized with pentobarbital (50 mg/kg) and chylomicrons (100-150 µEq of triglyceride fatty acid/ml) were collected from the thoracic duct for 2 h and frozen (20°C) for subsequent use (Borensztajn et al., 1970). Rats were anesthetized, and retroperitoneal fat pads were harvested and homogenized in saline (1:40). Homogenates (56 µl) were incubated at 37°C for 2 h in 0.1 ml of a cocktail containing 2 volumes of albumin (20% w/v in water, pH 8.1), 1 volume of 0.7 M Tris·HCl (pH 8.1), and 0.5 volume each of serum, heparin (14 IU/ml), and chylomicrons to provide appropriate substrates for LPL. The reaction was stopped by adding 50 µl of incubation mixture to 250 µl of Dole's extraction mixture, and free fatty acids were extracted. Free fatty acids were washed with heptane, isolated, and determined colorimetrically (Novak, 1965). Triglycerides were determined in plasma using a spectrophotometric assay after hydrolysis to glycerol and free fatty acids (Bucolo and David, 1973).

PGE₂ from Kupffer Cells. Rats were treated with saline or ethanol (5.0 g/kg) and were killed 2 h later for isolation of Kupffer cells. Isolation was performed using collagenase digestion and differential centrifugation with Percoll as described previously (Pertoft and Smedsrod, 1987). Primary cultures of Kupffer cells from control or ethanol treated rats were incubated for 4 h. Supernatants were assayed for PGE₂ by competitive radioimmunoassay using ¹²⁵I-labeled PGE₂ (Advanced Magnetics, Cambridge, MA).

Statistics. Statistical comparisons were made using analysis of variance (ANOVA) with Bonferroni's post hoc comparisons, Student's t test, or two-way ANOVA with Tukey's post hoc comparisons on ranks as appropriate. p < .05 was selected before the study as the level of significance.

	Results

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Oxygen Uptake and Alcohol Metabolism by Perfused Liver after Acute Exposure to Alcohol In Vivo (SIAM). Figure 1 (top) depicts typical liver perfusion experiments from a control animal and a rat 2.5 h after treatment with 5.0 g/kg methanol in vivo. In this experiment, basal rates of oxygen uptake, which were monitored continuously, were around 100 µmol/g/h and were increased to 200 µmol/g/h after methanol treatment in vivo. Values increased about 20% after the addition of 25 mM methanol to the perfusate, most likely due to an increase in peroxisomal oxygen demand (see also Fig. 3, top). The methanol concentration in the perfusate decreased over time at a rate of 23 µmol/g/h in the control and 67 µmol/g/h after methanol treatment (Fig. 1, bottom). Figure 3 summarizes the effect of methanol treatment in vivo on rates of oxygen and methanol uptake by the perfused liver. Average rates of oxygen uptake by the perfused liver were increased significantly from 114 ± 12 to 192 ± 5 µmol/g/h by methanol treatment in vivo. Methanol uptake was likewise increased from 22 ± 6 to 83 ± 18 µmol/g/h. Thus, like ethanol, methanol, which is not a substrate for ADH in rodents, can produce a SIAM phenomenon.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effect of methanol treatment in vivo on rates of oxygen and methanol uptake in the perfused liver. Top, oxygen uptake by an isolated perfused liver from a control rat or from a rat treated 2.5 h earlier with methanol in vivo (5.0 g/kg i.p.). The liver was perfused for 10 min using a nonrecirculating system with Krebs-Henseleit buffer (pH 7.4, 37°C), followed by 35 min with buffer containing 25 mM methanol in a recirculating system. Rates of oxygen uptake were determined with a Clark-type electrode. Bottom, methanol metabolism by the perfused liver. Samples of perfusate were collected and analyzed for methanol as described in Materials and Methods. Rates of methanol uptake were 23 µmol/g/h in the control and 67 µmol/g/h after methanol treatment determined by measuring the decrease in concentration and values calculated based on liver weight, volume of the perfusate, and time. Results are from a typical experiment repeated eight times.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effect of ethanol treatment in vivo on rates of oxygen and methanol uptake by the perfused liver. Rats were treated with GdCl3 and/or ethanol in vivo as described in Materials and Methods, and livers were perfused as described in Fig. 1. Statistical comparisons were made with the control group using ANOVA with Bonferroni's post hoc comparisons. Values are mean ± S.E.M. from five to nine livers per group. *p < .05, for comparisons with control. +p < .05 for comparisons with ethanol group.

Effect of Acute Alcohol Treatment on Ketogenesis. Because free fatty acids could provide substrate for catalase-dependent alcohol metabolism, rates of ketogenesis were calculated from ketone body release into the effluent perfusate. Basal rates of ketogenesis were low and not different in those livers from control or GdCl₃-treated rats (Table 1); however, there was a significant, nearly 2-fold increase after acute ethanol treatment in this study, confirming earlier work (Yuki and Thurman, 1980). Moreover, this increase in ketone body production due to ethanol was blocked by GdCl₃ treatment (Table 1). Because peroxisomal fatty acid metabolism generates H₂O₂ for metabolism of alcohols via catalase, it is possible that GdCl₃ blocks the production of H₂O₂ when excess fatty acid is present. To test this hypothesis, rats were given olive oil, a good source of oleate, to provide exogenous fatty acids in vivo for H₂O₂ production 2.5 h before perfusion (Fig. 3). Under these conditions, rates of oxygen uptake were elevated significantly from 114 ± 13 to 156 ± 14 µmol/g/h, a phenomenon that was unaffected by GdCl₃. Methanol metabolism was also increased about 3-fold by the addition of exogenous fatty acids, a phenomenon that was also not affected by GdCl₃.

View this table: [in this window] [in a new window]   TABLE 1 Effect of acute ethanol treatment and GdCl3 in vivo on ketogenesis in the perfused liver Ethanol or saline was given i.g. 2.5 h before perfusion to fed rats after treatment in vivo with 10 mg/kg GdCl3 or saline i.v. 24 h before perfusion as described in the text. Livers were perfused for 20 min, and acetoacetate (A) and -hydroxybutryrate (B) were determined enyzmatically in samples of effluent perfusate (Bergmeyer, 1988). Results are expressed as mean ± S.E.M., n = 5-7. Statistical comparisons were made using two-way ANOVA on ranks and Tukey's post hoc test.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of methanol and oleate treatment in vivo on oxygen and methanol uptake by the perfused liver. Rats were treated with methanol or olive oil in vivo, and livers were perfused as described in Materials and Methods. GdCl3 was administered to some rats as described in Materials and Methods. Statistical comparisons were made using ANOVA with Bonferroni's post hoc comparisons. Values are mean ± S.E.M. from five to eight livers per group, *p < .05 for comparison with the control group.

Fatty Acid Supply. Because peroxisomal -oxidation requires fatty acids, plasma triglycerides and several key enzymes involved in lipid metabolism (e.g., LPL and acyl CoA synthetase) were measured. LPL activity in retroperitoneal fat pads was decreased significantly by about 60% after alcohol treatment, an effect blocked by GdCl₃ (Table 2). Plasma triglycerides were increased nearly 2-fold by ethanol treatment as expected, an effect also blocked by treatment with GdCl₃ (Table 2). Hepatic acyl CoA synthetase activity was increased slightly but significantly 2.5 h after ethanol treatment (Table 2), and this effect was also blocked by GdCl₃. On the other hand, catalase was unaltered by GdCl₃ (control, 2891 ± 367 U/g liver; GdCl₃, 2781 ± 180 U/g liver), and acyl CoA oxidase remained unchanged (control, 0.8 ± 0.3 nmol/mg protein/min; GdCl₃, 1.1 ± 0.6 nmol/mg protein/min).

	Discussion

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The Catalase Pathway Is Activated Rapidly by Both Ethanol and Methanol. When ethanol or methanol was given in vivo, rates of methanol metabolism increased significantly in only a few hours, leading to the conclusion that the catalase pathway is stimulated by alcohols (Figs. 2 and 3).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Working hypothesis for the involvement of peroxisomal fatty acids in SIAM. Alcohol increases plasma endotoxin, which activates Kupffer cells to produce mediators such as PGE2 that inhibit LPL and elevate free fatty acids. PGE2 elevates cAMP, which is involved in the hypermetabolic state caused by ethanol. Adrenergic stimulation by alcohol also increases peripheral lipolysis and increases triglycerides in blood. Fatty acids are metabolized, and fatty acyl CoA oxidase generates H2O2 for catalase-dependent alcohol metabolism in the peroxisomes. Importantly, these biochemical events are blocked if Kupffer cells are destroyed by GdCl3.

PGE₂ Participates in SIAM by Providing Lipid. SIAM requires activation of oxygen uptake and cofactor supply for alcohol metabolism. Is there evidence to support the hypothesis that PGE₂ plays a role in SIAM? A link between Kupffer cells and regulation of oxygen uptake was made recently. Qu et al. (1996) demonstrated that Kupffer cells produce PGE₂ in sufficient quantities to stimulate respiration of isolated parenchymal cells. Media from cultured Kupffer cells isolated from rats fed ethanol chronically stimulated respiration about 30% in parenchymal cells. In this study, PGE₂ was significantly higher in media from Kupffer cells isolated from rats after acute ethanol treatment (see Results). Furthermore, the stimulation of oxygen uptake was blocked by the cyclooxygenase inhibitor indomethacin (see Results) (Qu et al., 1996). This study demonstrated that activation of oxygen uptake in the perfused liver by alcohol is dependent on mediators such as PGE₂ from Kupffer cells.