ARCHIVES

Vol.

OF BIOCHEMISTRY

289, No. 2, September,

AND

BIOPHYSICS

pp. 329-336,

1991

Peroxisomal

Fatty Acid ,&Oxidation

Paul A. Watkins,*,2

Edward

*Kennedy Baltimore,

Received

V. Ferrell,

Jr.,*

in HepG2 Cells’

Jan I. Pedersen,?

and Gerald

Hoefler*T3

Institute and Department of Neurology, Johns Hopkins University School of Medicine, Maryland 21205; and TZnstitute for Nutrition Research, University of Oslo, Oslo, Norway

February

25, 1991, and in revised

form

May

22, 1991

these fatty acids rather than from increased synthesis (35). Fatty acid P-oxidation pathways are present in two subcellular organelles, mitochondria and peroxisomes (6, 7). Since morphologically distinguishable peroxisomes are greatly reduced in number or are absent in tissues from Zellweger patients (8), it has been suggested that p-oxidation of VLCFA occurs primarily in this organelle (9). Although peroxisomes appear to be relatively normal in both size and number in the childhood (X-linked) form of adrenoleukodystrophy (10, ll), this disease has also been classified as a peroxisomal disorder because of defective @-oxidation of VLCFA (12, 13). The peroxisomal fatty acid P-oxidation pathway was initially described by Lazarow and deDuve (6). Although the reactions of both the peroxisomal and the mitochondrial pathways are similar, the individual enzymes of the two pathways are distinct (14). Hashimoto and co-workers (15-19) have purified and characterized the peroxisomal P-oxidation enzymes from rat liver. Antibodies raised against these proteins have been shown to cross-react with the peroxisomal P-oxidation enzyme proteins in human tissues (20-23). Since fresh human tissue, e.g., liver, is not readily available for study, it was desirable to develop a model The metabolism of very-long-chain fatty acids system for the investigation of the human peroxisomal (VLCFA)4 in human cells and tissues is of interest because fatty acid oxidation pathway. Cultured human cells such as skin fibroblasts are available, and techniques for the in certain diseases,such as X-linked adrenoleukodystroseparation of peroxisomes from other organelles have been phy and the Zellweger cerebra-hepato-renal syndrome, saturated VLCFA accumulate in tissues and body fluids reported (21, 24-28). Although peroxisomal &oxidation (l-3). In these disorders, elevated levels of VLCFA are enzymes are present in peroxisomes from fractionated thought to result primari1.y from defective P-oxidation of fibroblasts, the overall activity of this pathway in isolated organelles is low (25, 27). We therefore chose a human cell line of hepatic origin, HepG2, for study. HepG2 is i This work was supported in part by Grants HD10981 and HD24061 derived from a human hepatoblastoma and expresses from the National Institutes of Health. G.H. was supported by the Fonds many liver-specific functions, particularly with respect to zur Foerderung der Wissenschaftlichen Forschung, Project No. J0145M. ’ To whom correspondence slhould be addressed at the Kennedy Inlipid metabolism (29). HepG2 cells are capable of synstitute, 707 N. Broadway, Baltimore, MD 21205. thesizing and secreting cholesterol, apolipoproteins, and 3 Present address: Dept. of IMedical Biochemistry, Karl-Franzensbile acids (29); fatty acid oxidation by this cell line has Universitat, Graz, Austria. not yet been reported. In this paper, we present evidence ’ Abbreviations used: VLCFA., very long-chain fatty acid; TDGA, tetradecylglycidic acid, Bicine, N,.N-bis(2-hydroxyethyl)glycine. that peroxisomes from cultured human liver-derived cells

HepG2 cells, originally derived from a human hepatoblastoma, contain peroxisomes which could be separated from mitochondria and other subcellular organelles by density gradient centrifugation. To determine whether this cell line was a suitable model for human peroxisomal fatty acid &oxidation, we investigated the ability of these cells to catabolize very-long-chain fatty acids (VLCFA). HepG2 cell homogenates or digitonindisrupted cells oxidized both long chain fatty acids and VLCFA, although at somewhat lower rates than human liver homogenates. &Oxidation of VLCFA was observed in both peroxisomes and mitochondria of HepG2 cells. Peroxisomal B-oxidation ‘was independent of carnitine, insensitive to antimycin A and rotenone, and not blocked by an inhibitor of carnitine palmitoyl transferase I. HepG2 peroxisomes contained immunoreactive acyl-CoA oxidase, the first enzyme unique to the peroxisomal@oxidation pathway. In addition, HepG2 peroxisomes contained VLCFA-CoA synthetase activity. These results suggest that HepG2 may ble a useful model system for the study of human peroxisomal metabolic processes, including &oxidation of fatty acids. o 1991 Academic PRESS, IW.

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330

WATKINS

contain a P-oxidation pathway capable of degrading VLCFA. HepG2 cells should, therefore, prove useful in the investigation of other human peroxisomal metabolic processes. MATERIALS

AND

METHODS

Materials and general methods. Cell culture reagents were purchased from Mediatech or GIBCO Laboratories. [l-‘4C]Lignoceric acid (46 mCi/ mmol) was purchased from Research Products International and [l‘?]palmitic acid (58 mCi/mmol) was from New England Nuclear. Digitonin (approx. 80% pure) was obtained from Sigma Chemical Co. and was purified by the method of Janski and Cornell (30). Tetradecylglycidic acid (TDGA; McN-3802) was a gift from M. F. Ralston, McNeil Pharmaceutical (Spring House, PA). Nycodenz and Maxidens were from Accurate Chemical and Scientific. All other reagents were of analytical grade and were obtained from commercial sources. Catalase activity was assayed by the method of Peters et al. (31). Marker enzymes for subcellular fractions were assayed by established methods: succinate dehydrogenase (32), citrate synthase (33), lactate dehydrogenase (34), phosphoglucomutase (35), NADPH:cytochrome c reductase (36). Subcellular distribution of catalase in digitonin-treated cells was determined as previously described (37). Protein was determined by the method of Lowry et al. (38). Cells and tissues. HepG2 cells were obtained from the Wistar Institute (Philadelphia, PA). Cells were maintained in minimal essential medium supplemented with antibiotics and 10% fetal bovine serum. Samples of normal human liver (margins of resected tumors) were obtained from the Department of Surgical Pathology, Johns Hopkins University School of Medicine. Preparation of cell and tissue homogenates. Confluent monolayers of HepGL cells were harvested by gentle trypsinization, washed twice with phosphate-buffered saline and once with 0.25 M sucrose containing 1 mM Tris(Cll), pH 7.5, and 1 mM EDTA (homogenization buffer), and resuspended in this buffer. In some experiments, Hepes replaced Tris. Homogenization was carried out in a precision ball-bearing homogenizer of the type described by Balch and Rothman (39). Cell breakage without significant organelle damage, which was necessary for density gradient experiments (below), could not be adequately achieved when more conventional homogenizers such as Dounce or Potter-Elvehjem were used. Five passes of the cell suspension through the ball-bearing apparatus resulted in breakage of >90% of the cells, as determined by release of lactate dehydrogenase activity. In contrast, only 5-10% of the mitochondria and lo-15% of the peroxisomes were broken, as determined by the activities of citrate synthase and catalase, respectively. Liver was homogenized in 5 vol of homogenization buffer with one stroke of a loose fitting teflon pestle in a Potter-Elvehjem homogenizer. Homogenates were centrifuged at 3000g for 30 s to remove debris and nuclei (40). Fatty acid P-oxidation. Oxidation of [1-14C]fatty acids to water-soluble products was measured essentially as previously described (22). Fatty acids (5 nmol/assay; 20,000-35,000 dpm/nmol) were solubilized in 0.05 ml of a-cyclodextrin (10 mg/ml) in 10 mM Tris (Cl-), pH 8.0, at 37°C. The reaction mixture (final volume, 0.25 ml) contained 20 mM Tris (Cl-), pH 7.85, 30 mM KCl, 8.5 mM ATP, 8.5 mM MgClz, 0.16 mM coenzyme A, 1 mM dithiothreitol, 1 mM NAD, 0.17 mM FAD, 2.5 mM L-carnitine, 0.5 mM malate, 0.2 mM EDTA, and labeled fatty acid. The assay was initiated by the addition of cell or tissue preparation. After 60 min at 37’C, reactions were terminated by the addition of 50 ~1 of 1 N KOH and heating at 60°C for 60 min to hydrolyze fatty acyl-CoA’s. Perchloric acid was added to a final concentration of 6% and the samples were kept at 4°C for at least 1 h. The precipitate was removed by centrifugation and the supernatant was subjected to Folch partition (41). The aqueous phase was mixed with Liquiscint (National Diagnostics, Manville, NJ) and radioactivity was determined using a Beckman Model LS 3801 liquid scintillation spectrometer.

ET

AL.

Preliminary experiments indicated that P-oxidation rates could be overestimated by as much as 50% when the alkaline hydrolysis step was omitted. Significant amounts of [l-i4C]fatty acyl-CoA were found in the aqueous phase of Folch extractions under these conditions (data not shown). For HepG2 cells disrupted in isotonic buffer containing digitonin (40 wg/ml), P-oxidation of palmitic acid was linear for at least 60 min when O-75 pg of cell protein was present in the incubation (data not shown). For lignoceric acid P-oxidation, the assay was linear for at least 60 min when incubations contained O-350 pg of cell protein (data not shown). The pH optimum for oxidation of [l-‘4C]lignoceric acid was near 9.0 while that for [l-‘4C]palmitic acid was 7.5-8.0 (data not shown). Since P-oxidation of palmitic acid dropped off rapidly above pH 8, all assays were performed at pH 7.85-7.90. Fatty acyl-CoA synthetase. Activation of fatty acids to their coenzyme A derivatives was measured by a modification of the method of Nagamatsu et al. (42). [1-‘?]Fatty acids were solubilized with n-cyclodextrin as for B-oxidation experiments. Reaction mixtures contained, in a total volume of 0.25 ml, 50 mM Bicine, pH 8.1, 10 mM ATP, 1 mM MgC12, 0.2 mM CoA, 0.2 mM dithiothreitol, and gradient fractions. After 30 min at 37’C, 2.5 ml of isopropanol/heptane/l N H&SO, (40~10~1) was added, and extraction was carried out as described by Dole (43). Radioactivity in the aqueous phase was measured. Activation of TDGA. TDGA was activated to its CoA thioester prior to use in P-oxidation assays by incubation with rat liver microsomes. Normal rat liver was homogenized as described above for human liver; microsomes were prepared as described below for HepG2 cells. TDGA (0.1 pmol, in ethanol) was evaporated under N, and solubilized in 100 gl of a-cyclodextrin/Tris as described above. Solubilized TDGA was activated by incubation for 10 min at 37°C with liver microsomes (50100 pg protein) and fatty acyl-CoA synthetase reaction mixture (above) in a total volume of 0.5 ml. Microsomes were removed by ultrafiltration in a Centricon (Amicon Corp.); 25 ~1 of the filtrate (containing TDGACoA) was added to each P-oxidation assay. Control P-oxidation assays contained 25 ~1 of a similar filtrate prepared without TDGA. Differential centrifugation. All centrifugations were performed in a Sorvall RCPB centrifuge with an SS34 rotor unless otherwise indicated. HepGP cell homogenates were centrifuged for 5 min at 500g. After removing the supernatant, the pellet was resuspended in homogenization buffer and centrifuged again. The resuspended pellet from the second centrifugation is the crude nuclear fraction (N-fraction). The combined supernatants (E-fraction) were centrifuged for 10 min at 6000g. The resuspended 6000g pellet is the mitochondrial or M-fraction. The SOOOg supernatant was further centrifuged at 20,OOOg for 15 min to pellet the light mitochondrial or L-fraction. The 20,OOOg supernatant was centrifuged for 60 min at 100,OOOg in a Beckman L5-65 centrifuge with a Ti50 rotor to yield a microsomal pellet (P-fraction) and the cytosolic or Sfraction. Density gradient centrifugation. Homogenates (1.2 ml) were loaded directly onto linear gradients (10 ml) of increasing Nycodenz concentration (15-40%) and decreasing sucrose concentration (0.25-o M), similar to those described by Hart1 et al. (44) and Gould er al. (45); all gradient solutions contained 1 mM Tris(Cll), pH 7.5, and 0.1 mM EDTA. In some experiments, 0.25 M sucrose was present throughout the Nycodenz gradient, as described by Santos et al. (26) and Imanaka et al. (46). A cushion (1.5 ml) of Maxidens was at the bottom of the tube. Gradients were centrifuged in a vertical ultracentrifuge rotor (Beckman VTi 65.1) for 25 min at 74,3OOg,,,. Fractions of approximately 0.8 ml were collected from the bottom of the tube and assayed for marker enzyme activity. Zmmunoblot analysis. Ice-cold trichloroacetic acid was added to HepG2 subcellular fractions from Nycodenz gradients to a final concentration of 10%. Fractions were diluted fivefold with additional 10% trichloroacetic acid and were kept at 4°C overnight. Precipitates were collected by centrifugation, washed twice with diethyl ether, and solubilized for 5 min at 70°C in 80-160 ~1 of 67.5 mM Tris(Cl-), pH 6.8, containing 1% sodium dodecyl sulfate and 50 mM dithiothreitol. Sodium

PEROXISOMAL

@OXIDATION

dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis were then carried out as previously described (23), except that immunoreactive proteins were detected with alkaline phosphatase-conjugated second antibody rather than radiolabeled protein A.

IN

HepG2

TABLE

I

P-Oxidation of Long- and Very Long-Chain Fatty Acids in HepG2 Cells and Normal Human Liver C16:O oxidation

RESULTS

HepG2 cells contain perorisomes. When HepG2 cells were suspended in isotonic buffer containing digitonin and then centrifuged for 5 min in a Beckman microfuge, more than 80% of the catalase activity was in the pellet, whereas more than 80% of the lactate dehydrogenase activity was in the supernatant. This suggeststhat the peroxisomal matrix enzyme, c!atalase, was present in a particle-bound form under conditions where cytosolic enzymes were released. This finding was confirmed by subcellular fractionation of HepG2 homogenates (below). These results indicate that catalase-containing peroxisomes are present in HepG2 cells. P-Oxidation of VLCFA 15~HepG2 cells. HepG2 cells oxidized the VLCFA lignoceric acid (C24:0), as well as the long-chain fatty acid palmitic acid (C16:0), to watersoluble products (Table I). This suggested that HepG2 cells contain a peroxisom,al P-oxidation pathway, since VLCFA oxidation is thought to occur mainly in peroxisomes(9,25). Cells disrupted by digitonin had higher rates of oxidation than did intact cells or total cell homogenates when either [1-i4C]palmitic acid or [l-14C]lignoceric acid was the substrate (Table I). P-Oxidation of both longand very long-chain fatty acids by digitonin-disrupted HepG2 cells was linear with time and protein concentration (see Materials and Methods). The rate of palmitic acid P-oxidation in digitonin-disrupted HepG2 cells was 60% of that observed in fresh human liver (Table I). Oxidation of lignoceric acid in HepG2 cells was about 40% of the rate in liver, and the ratio of C24:O to C16:O oxidation in HepG2 cells was 65% of that in liver (Table I). These results suggest that the hepatoma cells have retained the capacity to oxidize VLCFA, although not as efficiently as normal human liver. P-Oxidation of VLCFA in HepG2 subcellular fractions prepared by differential centrifugation. To determine whether P-oxidation of lignoceric acid occurred in peroxisomes or mitochondria of HepG2 cells, homogenates were fractionated by differ~ential centrifugation techniques (Table II). The highest specific activity and the highest relative specific activity of C16:O P-oxidation was associated with the M-fractilon (heavy mitochondria); this fraction also contained the highest relative specific activity of the mitochondrial marker enzyme, succinate dehydrogenase. Although the highest specific activity and relative specific activity of C24:O oxidation was also associated with the M-fraction, there was significant @-oxidation activity observed i.n the light mitochondrial pellet (L-fraction). The L-fraction contained the highest relative specific activity of the peroxisomal marker enzyme, catalase. These results suggest that P-oxidation of C24:O oc-

331

CELLS

C24:O oxidation (nmol/h/mg

Experiment HepG2 HepG2 HepG2 Experiment HepG2 Human Experiment HepG2 Human

1 (Intact) (Digitonin) (Homogenate) 2 (Digitonin) liver 3 (Digitonin) liver

c24:o __ C16:O

protein)

1.78 8.28 3.31

0.88 1.02 0.48

0.108 0.116 0.136

6.67 10.31

1.06 2.73

0.159 0.265

4.83 8.61

0.92 2.30

0.191 0.267

Note. Oxidation of [l-i4C]palmitic acid (C16:O) and [1-Wjlignoceric acid (C24:O) to water-soluble products was assayed as described under Materials and Methods. In Experiment 1, washed HepG2 cells were resuspended in either homogenization buffer alone (intact, homogenate) or in this buffer containing digitonin (40 rig/ml). HepG2 homogenates were prepared using the ball-bearing apparatus described under Materials and Methods. Postnuclear supernatants of normal human liver homogenates were prepared as described under Materials and Methods. Each value is the mean of duplicate or triplicate determinations.

curs in both peroxisomes and mitochondria, whereas C16: 0 oxidation is primarily a mitochondrial process. P-Oxidation of VLCFA in HepG2 cells fractionated on Nycodenz gradients. To further investigate the role in VLCFA P-oxidation of the two organelles in which this pathway occurs, HepG2 cell homogenates were subjected to centrifugation on continuous Nycodenz-sucrose density gradients. Peroxisomes and mitochondria were well separated by this technique, as illustrated by the activities of catalase and succinate dehydrogenase (Fig. 1). Oxidation of palmitic acid in gradient fractions closely paralleled the succinate dehydrogenase activity, indicating that P-oxidation of this long-chain fatty acid is carried out primarily by mitochondria. P-Oxidation of lignoceric acid was observed in both the catalase-rich and the succinate dehydrogenase-rich gradient fractions. The proportion of lignocerate oxidation in catalase-rich vs succinate dehydrogenase-rich fractions was significantly greater than that of palmitate oxidation. These results confirm the data obtained from differential centrifugation (Table II), which indicated that VLCFA oxidation in HepG2 cells occurs, at least in part, in peroxisomes. Effect of antimycin A/rotenone, carnitine, and TDGA on fatty acid P-oxidation in gradient fractions. To verify that P-oxidation in catalase-rich (peroxisomal) fractions was not the result of mitochondrial contamination, we investigated the effects of several agents known to affect mitochondrial fatty acid oxidation on the catabolism of palmitic and lignoceric acids. The mitochondrial electron

332

WATKINS

ET

TABLE Separation

of Peroxisomes

and

Mitochondria

AL.

II of HepGX

Cells

by Differential

Centrifugation

(4 Catalase

Fraction

Protein

E+N N M L P S Recovery

(%)

Activity

(%)

Relative specific activity”

(%)

100 13 15 9 14 47

100 I 21 32 17 16

99

93

SDH

1.0 0.5 1.4 3.4 1.3 0.3

Activity

(%)

100 16 48 10 6 6

Relative specific activity” 1.0 1.2 3.2 1.0 0.6 0.1

86

(B) Cl&O Activity (%)

Fraction E+N N M L P S Recovery

100 8 48 12 4