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Mechanisms of liver steatosis in rats with systemic carnitine deficiency due to treatment with trimethylhydraziniumpropionate

2003; Elsevier BV; Volume: 44; Issue: 1 Linguagem: Inglês

10.1194/jlr.m200200-jlr200

1539-7262

Markus Spaniol, Priska Kaufmann, Konstantin Beier, Jenny Wüthrich, Michael Török, Hubert Scharnagl, Winfried März, Stephan Krähenbühl,

Diet and metabolism studies

Rats with systemic carnitine deficiency induced by treatment with trimethylhydraziniumpropionate (THP) develop liver steatosis. This study aims to investigate the mechanisms leading to steatosis in THP-induced carnitine deficiency. Rats were treated with THP (20 mg/100 g) for 3 or 6 weeks and were studied after starvation for 24 h. Rats treated with THP had reduced in vivo palmitate metabolism and developed mixed liver steatosis at both time points. The hepatic carnitine pool was reduced in THP-treated rats by 65% to 75% at both time points. Liver mitochondria from THP-treated rats had increased oxidative metabolism of various substrates and of β-oxidation at 3 weeks, but reduced activities at 6 weeks of THP treatment. Ketogenesis was not affected. The hepatic content of CoA was increased by 23% at 3 weeks and by 40% at 6 weeks in THP treated rats. The cytosolic content of long-chain acyl-CoAs was increased and the mitochondrial content decreased in hepatocytes of THP treated rats, compatible with decreased activity of carnitine palmitoyltransferase I in vivo. THP-treated rats showed hepatic peroxisomal proliferation and increased plasma VLDL triglyceride and phospholipid concentrations at both time points.A reduction in the hepatic carnitine pool is the principle mechanism leading to impaired hepatic fatty acid metabolism and liver steatosis in THP-treated rats. Cytosolic accumulation of long-chain acyl-CoAs is associated with increased plasma VLDL triglyceride, phospholipid concentrations, and peroxisomal proliferation. Rats with systemic carnitine deficiency induced by treatment with trimethylhydraziniumpropionate (THP) develop liver steatosis. This study aims to investigate the mechanisms leading to steatosis in THP-induced carnitine deficiency. Rats were treated with THP (20 mg/100 g) for 3 or 6 weeks and were studied after starvation for 24 h. Rats treated with THP had reduced in vivo palmitate metabolism and developed mixed liver steatosis at both time points. The hepatic carnitine pool was reduced in THP-treated rats by 65% to 75% at both time points. Liver mitochondria from THP-treated rats had increased oxidative metabolism of various substrates and of β-oxidation at 3 weeks, but reduced activities at 6 weeks of THP treatment. Ketogenesis was not affected. The hepatic content of CoA was increased by 23% at 3 weeks and by 40% at 6 weeks in THP treated rats. The cytosolic content of long-chain acyl-CoAs was increased and the mitochondrial content decreased in hepatocytes of THP treated rats, compatible with decreased activity of carnitine palmitoyltransferase I in vivo. THP-treated rats showed hepatic peroxisomal proliferation and increased plasma VLDL triglyceride and phospholipid concentrations at both time points. A reduction in the hepatic carnitine pool is the principle mechanism leading to impaired hepatic fatty acid metabolism and liver steatosis in THP-treated rats. Cytosolic accumulation of long-chain acyl-CoAs is associated with increased plasma VLDL triglyceride, phospholipid concentrations, and peroxisomal proliferation. Liver steatosis is a frequent finding in liver biopsies and a frequent cause of asymptomatic elevation of transaminases (1Skelly M.M. James P.D. Ryder S.D. Findings on liver biopsy to investigate abnormal liver function tests in the absence of diagnostic serology.J. Hepatol. 2001; 35: 195-199Google Scholar). Several risk factors have been identified, among them ingestion of certain drugs (2Lewis J.H. Ranard R.C. Caruso A. Jackson L.K. Mullick F. Ishak K.G. Seeff L.B. Zimmerman H.J. Amiodarone hepatotoxicity: prevalence and clinicopathologic correlations among 104 patients.Hepatology. 1989; 9: 679-685Google Scholar, 3Krähenbühl S. Mang G. Kupferschmidt H. Meier P.J. Krause M. Plasma and hepatic carnitine and coenzyme A pools in a patient with fatal, valproate induced hepatotoxicity.Gut. 1995; 37: 140-143Google Scholar, 4Oien K.A. Moffat D. Curry G.W. Dickson J. Habeshaw T. Mills P.R. MacSween R.N. Cirrhosis with steatohepatitis after adjuvant tamoxifen.Lancet. 1999; 353: 36-37Google Scholar, 5Fortgang I.S. Belitsos P.C. Chaisson R.E. Moore R.D. Hepatomegaly and steatosis in HIV-infected patients receiving nucleoside analog antiretroviral therapy.Am. J. Gastroenterol. 1995; 90: 1433-1436Google Scholar), alcohol abuse (6Lieber C.S. Alcohol and the liver: 1994.Gastroenterology. 1994; 106: 1085-1105Abstract Full Text PDF Google Scholar), viral hepatitis (7Goodman Z.D. Ishak K.G. Histopathology of hepatitis C virus infection.Semin. Liver Dis. 1995; 15: 70-81Google Scholar), diabetes (8Reid A.E. Nonalcoholic steatohepatitis.Gastroenterology. 2001; 121: 710-723Google Scholar), increased body weight (8Reid A.E. Nonalcoholic steatohepatitis.Gastroenterology. 2001; 121: 710-723Google Scholar, 9Wanless I.R. Lentz J.S. Fatty liver hepatitis (steatohepatitis) and obesity: an autopsy study with analysis of risk factors.Hepatology. 1990; 12: 1106-1110Google Scholar), and intoxications (10Mahler H. Pasi A. Kramer J.M. Schulte P. Scoging A.C. Bar W. Krähenbühl S. Fulminant liver failure in association with the emetic toxin of Bacillus cereus.N. Engl. J. Med. 1997; 336: 1173-1174Google Scholar). While impaired mitochondrial β-oxidation is considered to be the principle cause for microvesicular steatosis (11Fromenty B. Pessayre D. Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.Pharmacol. Ther. 1995; 67: 101-154Google Scholar), the mechanisms leading to macrovesicular steatosis have so far not been identified in detail. As shown in Fig. 1, important possibilities leading to this finding include a decrease in VLDL export and/or an increase in VLDL formation, which may result from impaired mitochondrial fatty acid metabolism or from other causes.We have recently developed and characterized a rat model with systemic carnitine deficiency (12Spaniol M. Brooks H. Auer L. Zimmermann A. Solioz M. Stieger B. Krähenbühl S. Development and characterization of an animal model of carnitine deficiency.Eur. J. Biochem. 2001; 268: 1876-1887Google Scholar). In this model, carnitine deficiency is induced within 3 weeks by feeding trimethylhydraziniumpropionate (THP), which inhibits carnitine biosynthesis and increases renal excretion of carnitine. Interestingly, rats treated with THP develop combined micro- and macrovesicular liver steatosis within 3 weeks but have no macro- or microscopic accumulation of lipids in skeletal muscle or heart (12Spaniol M. Brooks H. Auer L. Zimmermann A. Solioz M. Stieger B. Krähenbühl S. Development and characterization of an animal model of carnitine deficiency.Eur. J. Biochem. 2001; 268: 1876-1887Google Scholar, 13Hayashi Y. Murunaka Y. Kirimoto T. Asaka N. Miyake H. Matsuura N. Effects of MET-88, a buytrobetaine hydroxylase inhibitor, on tissue carnitine and lipid levels in rats.Biol. Pharm. Bull. 2000; 23: 770-773Google Scholar). Since carnitine is essential for transport of long-chain fatty acids into the mitochondrial matrix (14Bremer J. Carnitine–metabolism and functions.Physiol. Rev. 1983; 63: 1420-1480Google Scholar), it can be speculated that at least the microvesicular part of liver steatosis in THP-treated rats could be caused by hepatic carnitine deficiency. In support of this assumption, both in children with primary systemic carnitine deficiency and in mice with systemic carnitine deficiency (JVS mice), microvesicular liver steatosis has been reported (15Treem W.R. Stanley C.A. Finegold D.N. Hale D.E. Coates P.M. Primary carnitine deficiency due to a failure of carnitine transport in kidney, muscle, and fibroblasts.N. Engl. J. Med. 1988; 319: 1331-1336Google Scholar, 16Kuwajima M. Kono N. Horiuchi M. Imamura Y. Ono A. Inui Y. Kawata S. Koizumi T. Hayakawa J. Saheki T. Animal model of systemic carnitine deficiency: analysis in C3H-H-2 degrees strain of mouse associated with juvenile visceral steatosis.Biochem. Biophys. Res. Commun. 1991; 174: 1090-1094Google Scholar). However, at least the macrovesicular part of liver steatosis in rats treated with THP cannot be explained by an isolated defect in mitochondrial β-oxidation of fatty acids, suggesting additional mechanisms.Since the morphology of liver steatosis in THP-treated rats resembles that found in certain types of steatotic livers in humans, e.g., certain types of alcohol-induced liver steatosis (17Fromenty B. Grimbert S. Mansouri A. Beaugrand M. Erlinger S. Rotig A. Pessayre D. Hepatic mitochondrial DNA deletion in alcoholics: association with microvesicular steatosis.Gastroenterology. 1995; 108: 193-200Google Scholar) or steatosis observed during administration of amiodarone (18Lewis J.H. Mullick F. Ishak K.G. Ranard R.C. Ragsdale B. Perse R.M. Rusnock E.J. Wolke A. Benjamin S.B. Seeff L.B. Zimmerman H.J. Histopathologic analysis of suspected amiodarone hepatotoxicity.Hum. Pathol. 1990; 21: 59-67Google Scholar), knowledge about the mechanisms leading to liver steatosis in THP-treated rats may also be relevant for understanding this disease in humans. We therefore decided to study the mechanisms leading to liver steatosis in rats treated with THP for 3 or 6 weeks. Beside the mechanisms leading to liver steatosis, we also investigated adaptive changes secondary to a decrease in the hepatic carnitine pool and to impaired in vivo mitochondrial β-oxidation. Our studies demonstrate that hepatic carnitine deficiency is the most important cause for liver steatosis in THP-treated rats and suggest that reduced mitochondrial fatty acid oxidation may be partially compensated by increased peroxisomal fatty acid metabolism due to proliferation of peroxisomes.MATERIALS AND METHODSInduction of carnitine deficiency and in vivo palmitate metabolismThe experiments have been reviewed and accepted by the State Ethics Committee of animal research. Carnitine deficiency was induced in male Sprague-Dawley rats by feeding vegetarian food poor in carnitine (Kliba Futter 2435, Basel, Switzerland) and THP (20 mg/100 g/day) for 3 or 6 weeks (n = 6 rats for each time point) (12Spaniol M. Brooks H. Auer L. Zimmermann A. Solioz M. Stieger B. Krähenbühl S. Development and characterization of an animal model of carnitine deficiency.Eur. J. Biochem. 2001; 268: 1876-1887Google Scholar). Control rats were kept for the same periods of time with the same rat chow ad libitum (n = 12).In order to investigate a potential toxic effect on mitochondrial metabolism by THP itself in vivo, rats (n=3 in each group) were treated with THP (20 mg/100 g/day), with l-carnitine (50 mg/100 g/day), or with the combination of THP and l-carnitine (same dosages) for 3 weeks.Metabolism of palmitate was determined in vivo after 3 weeks of treatment with THP by intraperitoneal injection of [14C]palmitate and determination of exhaled 14CO2 as described previously (19Visarius T.M. Stucki J.W. Lauterburg B.H. Inhibition and stimulation of long chain fatty acid oxidation by chloroacetaldehyde and methylene blue in rats.J. Pharmacol. Exp. Ther. 1999; 289: 820-824Google Scholar).Isolation of rat liver mitochondriaRats were killed by decapitation and mitochondria were isolated from the liver by differential centrifugation according to a previously described method (20Hoppel C.L. DiMarco J.P. Tandler B. Riboflavin and rat hepatic cell structure and function. Mitochondrial oxidative metabolism in deficiency states.J. Biol. Chem. 1979; 254: 4164-4170Google Scholar). This method yields mitochondria of high purity with only minor contamination by peroxisomes or lysosomes (21Krähenbühl S. Talos C. Reichen J. Mechanisms of impaired hepatic fatty acid metabolism in rats with long-term bile duct ligation.Hepatology. 1994; 19: 1272-1281Google Scholar). The mitochondrial protein content was determined using the biuret method with BSA as a standard (22Gornall A.G. Bardawill G.J. David M.M. Determination of serum proteins by means of the biuret reaction.J. Biol. Chem. 1949; 177: 751-766Google Scholar). The content of mitochondrial protein/g liver was determined by correcting for the recovery of the mitochondria isolated using the activities of citrate synthase and succinate dehydrogenase (21Krähenbühl S. Talos C. Reichen J. Mechanisms of impaired hepatic fatty acid metabolism in rats with long-term bile duct ligation.Hepatology. 1994; 19: 1272-1281Google Scholar).Oxidative metabolism of intact mitochondriaOxygen consumption by freshly isolated liver mitochondria was measured in a chamber equipped with a Clark-type oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH) at 30°C as described previously (23Krähenbühl S. Chang M. Brass E.P. Hoppel C.L. Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria.J. Biol. Chem. 1991; 266: 20998-21003Google Scholar). The concentrations of the substrates used were 20 mmol/l for l-glutamate and succinate, 40 μmol/l for palmitoyl-l-carnitine, 20 μmol/l for palmitoyl-CoA, and 80 μmol/l for palmitate. All incubations with fatty acids contained 5 mmol/l l-malate, incubations with palmitoyl-CoA and palmitate contained in addition 2 mmol/l l-carnitine, and incubations with palmitate contained in addition 250 μmol/l ATP and 250 μmol/l CoASH.In vitro mitochondrial β-oxidation and formation of ketone bodiesThe β-oxidation of [1-14C] palmitic acid by liver mitochondria was assessed as described by Fréneaux et al. (24Fréneaux E. Labbe G. Letteron P. Le Dinh T. Degott C. Gèneve J. Larrey D. Pessayre D. Inhibition of the mitochondrial oxidation of fatty acids by tetracycline in mice and man: possible role in microvesicular steatosis induced by this antibiotics.Hepatology. 1988; 8: 1056-1062Google Scholar) with some modifications described previously (25Lang C. Schafer M. Serra D. Hegardt F. Krähenbühl L. Krähenbühl S. Impaired hepatic fatty acid oxidation in rats with short-term cholestasis: characterization and mechanism.J. Lipid Res. 2001; 42: 22-30Google Scholar). This assay measures the formation of acid-soluble products from mitochondrial palmitate metabolism, which equals production of ketone bodies and citric acid cycle intermediates (24Fréneaux E. Labbe G. Letteron P. Le Dinh T. Degott C. Gèneve J. Larrey D. Pessayre D. Inhibition of the mitochondrial oxidation of fatty acids by tetracycline in mice and man: possible role in microvesicular steatosis induced by this antibiotics.Hepatology. 1988; 8: 1056-1062Google Scholar).Ketone body formation was measured using freeze-thawed mitochondria according to Chapman et al. (26Chapman M.J. Miller L.R. Ontko J.A. Localization of the enzymes of ketogenesis in rat liver mitochondria.J. Cell Biol. 1973; 58: 284-306Google Scholar) with some modifications as described previously (25Lang C. Schafer M. Serra D. Hegardt F. Krähenbühl L. Krähenbühl S. Impaired hepatic fatty acid oxidation in rats with short-term cholestasis: characterization and mechanism.J. Lipid Res. 2001; 42: 22-30Google Scholar). The reactions were stopped by adding 100 μl of 30% perchloric acid (w/v). After having removed the precipitate by centrifugation, the supernatants were analyzed for acetoacetate according to Olsen (27Olsen C. An enzymatic fluorometric micromethod for the determination of aceto-acetate, β-hydroxybutyrate, pyruvate and lactate.Clin. Chim. Acta. 1971; 33: 293-300Google Scholar).Activities of the enzyme complexes of the respiratory chainComplex I (NADH:decylubiquinone-1 oxidoreductase) was determined spectrophotometrically as described by Veitch et al. (28Veitch K. Hombroeck A. Caucheteux D. Pouleur H. Hue L. Global ischaemia induces a biphasic response of the mitochondrial respiratory chain. Anoxic pre-perfusion protects against ischaemic damage.Biochem. J. 1992; 281: 709-715Google Scholar) with some modifications. Briefly, 0.1 mg mitochondria were preincubated in 35 mmol/l potassium phosphate buffer pH 7.4, 5 mmol/l magnesium chloride, 2 mmol/l potassium cyanide, and 60 μmol/l decylubiquinone at 30°C. The reaction was started by the addition of 0.13 mmol/l NADH and the decrease of absorption was recorded spectrophotometrically at 340 nm using rotenone as inhibitor.Complex II (succinate:dichloroindophenol oxidoreductase) was determined according to a previously described method (23Krähenbühl S. Chang M. Brass E.P. Hoppel C.L. Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria.J. Biol. Chem. 1991; 266: 20998-21003Google Scholar). This method is based on the reduction of dichloroindophenol by complex II using succinate as a substrate. The reaction is followed spectrophotometrically at 600 nm in the presence and absence of the inhibitor thenoyltrifluoroacetone.Complex III (ubiquinol:ferricytochrome c oxidoreductase) was determined spectrophotometrically at 550 nm by the conversion of ferricytochrome c to ferrocytochrome c using decylubiquinol as substrate (29Krähenbühl S. Talos C. Wiesmann U. Hoppel C.L. Development and evaluation of a spectrophotometric assay for complex III in isolated mitochondria, tissues and fibroblasts from rats and humans.Clin. Chim. Acta. 1994; 230: 177-187Google Scholar). The reaction velocity was assessed as the difference in absorption with and without antimycine as inhibitor.Complex IV (cytochrome c oxidase) activity was measured by following the oxidation of ferrocytochrome c at 550 nm as described by Wharton and Tzagaloff (30Wharton D.C. Tzagoloff A. Cytochrome oxidase from beef heart mitochondria.Methods Enzymol. 1967; 10: 245-250Google Scholar).Determination of CoA and carnitineLiver samples (about 50 mg/1 ml, prepared without thawing) were homogenized in 3% perchloric acid and centrifuged for 5 min at 10,000 g. Mitochondria (100 μl corresponding to about 10 mg protein) were mixed with 20 μl 0.2 mol/l dithiothreitol and precipitated with 1.88 ml 3.2% perchloric acid. The suspension was vortexed, kept on ice for 5 min, and then centrifuged for 10 min at 10,000 g. This perchloric acid treatment yields an acid soluble (supernatant) and an acid insoluble fraction (pellet). The acid soluble fraction is used to measure free and acetyl-CoA and, after alkaline hydrolysis, total acid soluble CoA. The difference between these values represents short chain acyl-CoA. Long chain CoA is determined in the pellet after alkaline hydrolysis. Total CoA refers to the sum of total acid soluble CoA and long-chain acyl-CoA.In these fractions, CoASH and acetyl-CoA, total acid soluble CoA (supernatant), and long-chain acyl-CoA (pellet) were determined using the CoA recycling assay of Allred and Guy (31Allred J.B. Guy D.G. Determination of Coenzyme A and acetyl-CoA in tissue extracts.Anal. Biochem. 1969; 29: 293-299Google Scholar), with some modifications in the work-up as described before (32Krähenbühl S. Brass E.P. Fuel Homeostasis and carnitine metabolism in rats with secondary biliary cirrhosis.Hepatology. 1991; 14: 927-934Google Scholar). Since the CoA recycling assay does not differentiate CoASH and acetyl-CoA, acetyl-CoA was determined specifically using a radioenzymatic assay as described previously (33Cederblad G. Carlin J.I. Constantin-Teodosiu D. Harper P. Hultman E. Radioisotopic assays of CoASH and carnitine and their acetylated forms in human skeletal muscle.Anal. Biochem. 1990; 185: 274-278Google Scholar).For the determination of carnitine, liver tissue was worked up as described above for CoA. Plasma was also treated with perchloric acid (final concentration 3%) to obtain a supernatant and a pellet. The determination of carnitine in these fractions was performed using the radioenzymatic assay described by Brass and Hoppel (34Brass E.P. Hoppel C.L. Carnitine metabolism in the fasting rat.J. Biol. Chem. 1978; 253: 2688-2693Google Scholar). Direct analysis of the supernatant yields free carnitine, and, after alkaline hydrolysis, total acid soluble carnitine. The difference between free and total acid soluble carnitine is the short-chain acylcarnitine fraction (up to a chain-length of the acyl-group of about 10). Long-chain acylcarnitines were determined in the pellet after alkaline hydrolysis. Addition of total acid soluble and long-chain acylcarnitine yields total carnitine.Lipid determination in liversLipids from rat livers were extracted according to the method described by Bligh and Dyer (35Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). Briefly, about 100 mg of liver tissue were homogenized in 2 ml 20 mmol/l potassium phosphate buffer, pH 7.4, and the lipids were extracted by the addition of 5.0 ml chloroform-methanol (1:1, v/v). The samples were vortexed and incubated for 60 min under periodical stirring. Then, 2.5 ml chloroform and 0.8 ml 0.74% potassium chloride were added and the mixture centrifuged for 5 min at 500 g. The lower phase was separated and washed with 1.5 ml of a mixture containing 0.74% potassium chloride-chloroform-methanol (94:96:6, v/v/v). The organic phase was then evaporated to dryness and the lipid extract stored at −20°C until analysis.For lipid determination, liver extracts were resuspended in 250 μl isopropanol. Total cholesterol, triacylglycerides, and free fatty acids were measured using enzymatic methods and reagents from Wako (Neuss, Germany). The measurements were calibrated using standards from Roche Diagnostics (Mannheim, Germany).Determination of plasma lipidsTotal cholesterol (TC), free cholesterol (FC), triacylglycerides (TG), phospholipids (PL), HDL cholesterol (HDL-C), and free fatty acids (FFA) were measured using enzymatic methods and reagents from Wako (Neuss, Germany). The measurements were performed on a Wako 30R automatic analyzer (Wako) and were calibrated using standards from Roche Diagnostics (Mannheim, Germany). Esterified cholesterol was calculated as the difference between TC and FC.The determination of the lipoprotein fractions was performed by a combined ultracentrifugation-precipitation method (36Bachorik P.S. Ross J.W. National Cholesterol Education Program recommendations for measurement of low-density lipoprotein cholesterol: executive summary. The National Cholesterol Education Program Working Group on Lipoprotein Measurement.Clin. Chem. 1995; 41: 1414-1420Google Scholar, 37Wanner C. Horl W.H. Luley C.H. Wieland H. Effects of HMG-CoA reductase inhibitors in hypercholesterolemic patients on hemodialysis.Kidney Int. 1991; 39: 754-760Google Scholar). VLDL were removed quantitatively by ultracentrifugation using a TFT 56.6 rotor (Kontron, Germany) with adapters for 0.8 ml polycarbonate tubes. Five hundred microliters of plasma was pipetted into tubes and 0.1 ml of 0.9% sodium chloride solution was layered on top of the plasma. After centrifugation (18 h at 30,000 rpm, 10°C), the floating VLDL fraction was aspired with a 2 ml syringe until the supernatant was completely clear. The volume was reconstituted to the original weight with 0.9% saline. LDL were precipitated in the infranatant using phosphotungstic acid/magnesium chloride (PTA, Roche, Mannheim, Germany), and HDL lipids were measured in the supernatant after LDL precipitation. Lipids in the LDL fraction were calculated as the difference between the concentrations in the density fraction d > 1.006 kg/l and the HDL fraction.Cytochemical localization of catalase in liver sectionsLivers from control rats and rats treated with THP for 3 weeks were fixed by perfusion through the portal vein with a fixative containing 0.25% glutaraldehyde and 2% sucrose in 0.1 M PIPES buffer, pH 7.4. The tissue was cut into 70 μm sections with a DSK-Microslicer (Dosaka EM Co., Kyoto, Japan). The sections were incubated in alkaline diaminobenzidine (DAB) medium for cytochemical visualization of catalase (38Fahimi H.D. Cytochemical localization of peroxidatic activity of catalase in rat hepatic microbodies (peroxisomes).J. Cell Biol. 1969; 43: 275-288Google Scholar), followed by osmication and embedding in Epon 812.SDS-PAGE and immunoblottingFor Western blotting, tissues were homogenized in a buffer containing 250 mM sucrose, 5 mM MOPS, 1 mM EDTA, 0.1% ethanol, pH 7.4, using an Ultra-Turrax (IKA Labortechnik, Staufen, Germany). Equal amounts of protein (20 μg per sample) were subjected to SDS-PAGE. After electrotransfer of the polypeptides onto nitrocellulose, the sheets were incubated overnight with the primary antibody at a concentration of 1 μg protein/ml. The polyclonal antibody against acyl-CoA oxidase (AOX) was a generous gift of A. Völkl, Institute of Anatomy and Cell Biology, University of Heidelberg. Its specificity was assessed as described previously (39Beier K. Völkl A. Hashimoto T. Fahimi H.D. Selective induction of peroxisomal enzymes by the hypolipidemic drug bezafibrate. Detection of modulations by automatic image analysis in conjunction with immunoelectron microscopy and immunoblotting.Eur. J. Cell Biol. 1988; 46: 383-393Google Scholar). After repeated washing, a peroxidase conjugated goat anti-rabbit antibody (Sigma, München, Germany) was added for 1 h at room temperature. The immunoreactive bands were visualized by enhanced chemoluminescence (ECL, Amersham International, Little Chalfont, England) according to the manufacturer's protocol.The blots for subunit IV of cytochrome c oxidase and apolipoprotein B (apoB) were performed according to the method described above. The polyclonal antibody against subunit IV of cytochrome c oxidase was obtained from Molecular Probes (Juro Supply, Lucerne, Switzerland) and used at a dilution of 0.5 μg/ml. The polyclonal antibody against apoB was obtained from LabForce AG (4208 Nunningen, Switzerland) and used at a dilution of 0.5 μg/ml.Determination of acyl-CoA oxidase activityAcyl-CoA oxidase activity was determined according to a previously described method (40Small G.M. Burdett K. Connock M.J. A sensitive spectrophotometric assay for peroxisomal acyl-CoA oxidase.Biochem. J. 1985; 227: 205-210Google Scholar). This method is based on the production of hydrogen peroxide by the action of acyl-CoA oxidase, which is measured spectrophotometrically at 502 nm using dichlorofluorescein diacetate as a chromophore. Briefly, 50 mg of frozen liver tissue were homogenized in 20 mmol/l potassium phosphate buffer, pH 7.4, and the volume made up to 2.0 ml. One hundred microliters of this homogenate were preincubated in 1.9 ml of 10 mmol/l potassium phosphate buffer pH 7.4 containing 0.05 mg 2′,7′dichlorfluorescein diacetate, 5.2 mg sodium azide, 74 mU horseradish peroxidase, and 0.01% TritonX at 37°C for 5 min. The reaction was started by the addition of 15 μmol/l palmitoyl-CoA and the increase in absorbance was recorded spectrophotometrically over 3 min at 502 nm. Activities were calculated using an extinction coefficient of 91,000 l × mol−1 × cm−1.StatisticsData are presented as mean ± SD. Groups were compared using the Student's t-test (comparison of two groups) or ANOVA followed by Tukey's protected Student's t-test (>2 groups). Since the values of control animals were generally not different between 3 and 6 weeks, they were pooled unless indicated otherwise.RESULTSThe studies were carried out to elucidate the principle mechanisms leading to liver steatosis in rats with systemic carnitine deficiency due to treatment with THP.Rats were treated with THP for 3 or 6 weeks and studied after starvation for 24 h. As shown in Fig. 2, and in agreement with previous studies (12Spaniol M. Brooks H. Auer L. Zimmermann A. Solioz M. Stieger B. Krähenbühl S. Development and characterization of an animal model of carnitine deficiency.Eur. J. Biochem. 2001; 268: 1876-1887Google Scholar, 13Hayashi Y. Murunaka Y. Kirimoto T. Asaka N. Miyake H. Matsuura N. Effects of MET-88, a buytrobetaine hydroxylase inhibitor, on tissue carnitine and lipid levels in rats.Biol. Pharm. Bull. 2000; 23: 770-773Google Scholar), rats treated with THP for 3 weeks developed micro- and macrovesicular liver steatosis predominantly in zone I and II of the liver lobules. Similar findings were obtained after 6 weeks of treatment with THP (results not shown).Fig. 2Liver steatosis in rats treated with THP for 3 weeks. Cryosections stained with Sudan Black for specific labeling of lipids. As shown in A (enlargement 64×), lipids accumulate predominantly in zones 1 and 2 of the liver lobules, while zone 3 is spared. A higher enlargement (Fig. 2B, enlargement 160×) shows that the size of the lipid droplets varies, small and larger droplets can be detected in most hepatocytes, in particular in the periportal regions. Similar findings were present after 6 weeks of treatment with THP. In comparison, livers from control animals contained no fat (not shown). P, portal vein; C, central vein.View Large Image Figure ViewerDownload (PPT)Palmitate metabolism was assessed in vivo in rats treated with THP for 3 weeks and corresponding control rats by injecting l-[14C]palmitate ip and measuring the amount of 14CO2 exhaled over the next 2 h. The percentage of radioactivity exhaled was decreased in THP treated rats (28.2 ± 6.3% vs. 54.8 ± 8.2% of the dose injected, P < 0.05), compatible with reduced hepatic metabolism of long-chain fatty acids. Accordingly, the hepatic lipid content was increased significantly (P < 0.05) in rats treated with THP for 6 weeks for all lipid classes investigated [cholesterol 0.77 ± 0.09 vs. 0.58 ± 0.09 mg/g liver (THP-treated vs. control), triglycerides 4.63 ± 2.78 vs. 1.54 ± 0.46 mg/g liver, and free fatty acids 0.80 ± 0.09 vs. 0.58 ± 0.02 μmol/g liver]. As can be seen in Fig. 1, reduced metabolism of long-chain fatty acids can be due to impaired activation of fatty a