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Reduction of β-oxidation capacity of rat liver mitochondria by feeding orotic acid
Btochimica er Biophysics Acta, I I I ( 1982) 494-502 Elsevier Biomedical Press
494
BBA 51109
REDUCTION OF j3-OXIDATION OROTIC ACID SHOKO
MIYAZAWA,
SHUICHI
FURUTA
CAPACITY
OF RAT LIVER MITOCHONDRIA
and TAKASHI
HASHIMOTO
BY FEEDING
*
Department of Biochemistry, Shinshu University School of Medicine, Matsumoto, Nagano-ken 390 (Japan) (Received
December
30th, 1981)
Key words: P-Oxidation; Orotic acid diet; (Rat liver mitochondria)
Rats were maintained on fat-free high carbohydrate diets either with or without erotic acid (l%, w/w), pantethine (I%, w/w), adenine (0.25%, w/w), and/or pchlorophenoxyisobutyrate (0.258, w/w). Oxidation of fatty acid by liver mitochondria was inhibited to less than half that of the control after administration of orotie acid. Activities of acyCCoA dehy~~ena~s were markedly decreased by erotic acid adminis~a~on, but the following enzyme activities were not, or only slightly, decreased: acyl-CoA synthetase, carnitine acyltransferases, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase. Simultaneous addition of pantethine in the erotic acid-containing diet prevented induction of fatty liver. It also prevented decreases in fatty acid oxidation capacity and aeyl-CoA dehydrogenase activity. Introduction of adenine or p-chlorophenoxyisobutyrate, which reverse erotic acid-induced fatty liver, reversed oxidation and acyl-CoA dehydrogenase activities to control levels. The oxidation capacity of the peroxisomal system remained unchanged after adminis~ation of erotic acid.
In~~uction
16 h after erotic acid was introduced into the diet at 1% (w/w), and a minimum plateau was reached in 4 days. The level was restored to that of normal plasma lipid within 16 h by addition of adenine [4]. Therefore, the basic mechanism underlying the pathogenesis of fatty liver induced by the compound has been considered to be a block in the release of triacylglycerol into plasma. Studies on the morphological analysis of fatty liver and on plasma lipoprotein in rats fed erotic acid further substantiate this hypothesis [5-S]. It has been reported that there is no change in the conversion of [14C ,]stearate to 14C02, using liver slices from rats which had been maintained on the experimental diets for 3 days [3]. Nevertheless, this study concerning the fatty acid oxidation of livers of rats which had received erotic acid was conducted because of the following reasons. Recently, the presence of the b-oxidation system in
Orotic, acid induces severely fatty liver in rats when added to purified diets for 7-14 days [I]. The fatty liver induced by erotic acid ingestion, unlike the fatty liver induced by a choline-deficient diet, does not seem to be accompanied by other serious pathological disturbances. The addition of adenine to such a diet prevents and also reverses fat accumulation in the liver [2]. Lipid accumulation in the liver could be seen 3 days after 1% (w/w) erotic acid had been added to the diet, reached a maximum by 14 days and persisted for at least 200 days if erotic acid remained in the diet [3]. Plasma lipid levels started to drop as early as
* To whom correspondence
should be addressed.
OOOS-2760/82/0000-OOOOooo/%O2.75 0 1982 Elsevier Biomedical
Press
495
hepatic peroxisomes has been reported [9] and the fatty acid oxidation capacity of this system is comparable to that of the mitochondrial system [lo]. The peroxisomal system is induced markedly by various peroxisome proliferators such as pchlorophenoxyisobutyrate [9]. A large part of the fatty acids oxidized in the liver is converted to ketone bodies. The conversion of 14C-labeled fatty acid into 14COZdoes not seem to be representative of the total ability to metabolize fatty acid. The present report is concerned with the fatty acid oxidation of livers of rats receiving erotic acid. Fatty acid oxidation via the ~tochondrial system, but not the peroxisomal one, was inhibited. The inhibition site seems to be the acylCoA dehydrogenase (EC 1.33.99.2 and 1.3.99.3) reaction. Materials and Methods Animals . Male rats of the Wistar strain, weighing 200-25Og, were fed with various diets. The control diet was a set-syn~etic fat-free, highcarbohydrate diet which contained 24% milk casein, 51% corn starch, 10% sucrose, 5% cellulose, 2% a-starch, 1% vitamin mixture and 7% salt mixture (Clea, Tokyo). The following compounds were added to the control diet: erotic acid (l%, w/w), pantethine (1% w/w), adenine (0.25%, w/w) and p-chlorophenoxyisobutyrate (0.25%, w/w). In experiment 1, rats were divided into seven groups (n = 4) and fed with seven kinds of diet for 1-2 weeks. Feeding duration of the erotic acid (1W) and adenine groups was 1 week and that of the erotic acid (2W) group was 2 weeks. The erotic acid + adenine group was maintained on the erotic acid diet for 1 week and then placed on the erotic acid + adenine diet for 1 week. The control, pantethine and erotic acid + pantethine groups were fed with the respective diets for 2 weeks. In experiment 2 (n = 3, each), the control group was maintained for 3 weeks. One of the erotic acid groups (erotic acid (2W)) was fed with the erotic acid diet for 2 weeks and the other (erotic acid (3W)) for 3 weeks. The erotic acid + pchlorophenoxyisobutyrate group was m~nt~ned on the erotic acid diet for 2 weeks and then placed on the erotic acid + p-chlorophenoxyisobutyrate diet for 1 week.
Fatty acid oxidation. The fatty acid o~dation capacities of mitochondria and peroxisomes were assayed with used of the mitochondrial fractions from rat liver as described previously [lo]. Recoveries of mitochondria and peroxisomes in the mitochondrial fractions were calculated using the rate of recovery of the marker enzymes, glutamate dehydrogenase (EC 1.4.1.3) and catalase (EC 1,11.1.6), respectively. The data are expressed as pmol 0, consumed/m& per g liver. Enzyme assay. Camitine acyltransferase (EC 2.3.1.7 and 2.3.1.21) activities were assayed in both directions: the formation of acylcarnitines with use of acyl-CoAs as substrates [ 111 and of the formation of palmitoyl-CoA from palmitoylcarnitine [ 121. The activities of acyl-CoA dehydrogenases and electron-transfer flavoprotein were determined after DEAE-cellulose column chromatography of the mitochondrial extract [ 131. The activity of acyl-CoA oxidase was assayed with pahnitoyl-CoA as described previously [14]. The following enzymes involved in p-oxidation were located in both ~t~hondria and peroxisomes: enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.l. 1.35), and 3-ketoacyl-CoA thiolase (EC 2.3.1.16). Mitochondrial and peroxisomal enzyme activities were distinguished with use of antibodies against the respective enzymes [ 151. Activities of glutamate dehydrogenase [16] and catalase [ 171 were assayed for the estimation of recovery of mitochondria and peroxisomes after subcellular fractionation. Adenylate kinase (EC 2.7.4.3) was also assayed to check the integrity of the isolated ~t~hond~a [18]. All enzyme activities, including the oxidation of fatty acids, were assayed under the specified conditions at 3O’C. Units of enzyme activities were expressed as pmol of substrates utilized or products formed/mm. The data are summarized as units per g liver. Materials. Saturated fatty acyl-CoAs were prepared by the mixed anhydride method [19]_ Enzymes and coenzymes were purchased from Boehringer. Fatty acid-free bovine serum albumin was from Sigma. L-Carnitine, ~tanoylcar~tine and palmitoylca~tine were gifts from Otsuka Pharmaceutical Co., Naruto, Japan. Pantethine was kindly donated from Daiichi Seiyaku Co., Ltd., Tokyo.
496
Results Fatty liver
On feeding the diet containing 1% (w/w) erotic acid, development of the characteristic fatty liver was confirmed. The contents of total lipids (mg/g liver, mean * S.D.) in experiment 1 were 57.6 -+ 13.8 for the control, 101.8 * 20.5 for the erotic acid (1 W), 163.7 2 36.6 for the erotic acid (2W). 62.9 2 5.0 for the pantethine, 53.6 * 13.0 for the erotic acid + pantethine, 54.2 r+ 12.6 for the adenine and 74.1 * 13. I for the erotic acid + adenine groups. The values in experiment 2 were 46.1 k 3.2 for the control, 196.0 t 27.1 for the erotic acid (2W), 229.3 2 63.2 for the erotic acid (3W), and 52.0* 8.3 for the erotic acid +pchlorophenoxyisobutyrate groups. Pantethine prevented induction of fatty liver, and adenine and p-chlorophenoxyisobutyrate reversed it, as previously reported [8]. When a powdered diet with the composition of the standard chow diet (Oriental M, Oriental Yeast Co., Tokyo) was used as the basal diet, the erotic acid feeding did not result in fatty liver. Fatty acid oxidation
As shown in TableI, palmitoyl-CoA oxidation by mitochondria was decreased markedly after TABLE
administration of erotic acid. Pantethine administration resulted in an increase in the capacity of the mitochondrial system. The simultaneous feeding of pantethine and erotic acid also resulted in a higher activity of this system than that of the control. Adminstration of adenine alone did not affect the oxidation. However, administration of adenine with erotic acid to rats previously given erotic acid reversed the capacity of the mitochondrial system to the control level. When ~-chlorophenoxyisobutyrate was administered simult~eously with erotic acid to rats previously given erotic acid, the rate of mitochondrial oxidation exceeded the control level. Palmitoylcarnitine is oxidized by mitochondria but not by peroxisomes. Oxidation of palmitoylcarnitine was studied with pooled samples of all groups. The oxidation rates were nearly the same as those of pamitoyl-CoA by mitochondria listed in Table I (data not shown). Administration of erotic acid decreased oxidation of fatty acids by ~tochondria without change of the substrate specificity (Fig. 1). Administration of adenine did not change the activities toward various substrates. The reversal effect of adenine did not affect the substrate specificity (data not shown). However, the effects of the pantethine, the erotic acid f pantethine, and the erotic acid fp-
I
EFFECTS ON PALMITOYL-CoA OXIDATION AND ~-CHLOROPHENOXYISO~U~~TE Experimental procedures are described mean* S.D. The levels of significances
OF ADMINISTRATION
OF OROTIC
in Materials and Methods. The oxidation values (gmol are determined by Student’s t-test. n.s., not significant.
ACID, O,/min
PANTETHINE,
per g liver) are
ADENINE
expressedas
Mitochondria
P
Peroxisomes
P
Expt. 1 (n=4) Control Orotic acid (1 W) Orotic acid (2W) Pantethlne Orotic acid+pantethine Adenine Orotic acid + adenine
0.424 * 0.050 0.308 f 0.048 0.172iO.028 1.298-t-0.378 0.934eO.278 0.456) 0.046 0.404) 0.038
CO.05
0.115~0.025 0.120~~.021 0.135 * 0.030 0.300~0.059 0.199~0.051 0.170* 0.053 0.124* 0.030
ns. n.s.
Expt. 2 (n =3f Control Orotic acid (2W) Orotic acid (3W) Erotic acid + p-chlorophenoxyisobutyrate
0.5~~0.1~ 0.092 * 0.020 0.058 kO.036 0.718r0.020
0.120~0.015 0.156~0.0~ 0.101~0.011 1.560~0.342
ns. ns. co.01
491
I.5
ti
.r IX 0, i ._
E \
0” 5
O.!
5.
0
6
8
IO
12
I4
I6
I8
Carbon chain length Fig. 1. Mitochondrial P-oxidation capacities. Saturated fatty acids with various carbon chain lengths were used: 6, hexanoate,; 8, octanoate; 10, decanoate; 12, dodecanoate; 14, tetradecanoate, 16, hexadecanoate; 18, octadecanoate. oxidation rates were determined with pooled mitochondrial fractions from four rats in Expt. 1 and that from three rats of the erotic acid+ p-chlorophenoxyisobutyrate group in Expt. 2. 0, Control; 0, erotic acid (2W); q, pantethine; n , erotic acid + pantethine; A, erotic acid + p-chlorophenoxyisobutyrate.
TABLE
chlorophenoxyisobutyrate groups were different from that of the control. In a previous study [lo], we observed that mitochondrial fatty acid oxidation was increased after administration of p-chlorophenoxyisobutyrate, and that the increase in the oxidation was more marked with long-chain fatty acids. The activities of marker enzymes for two organelles, glutamate dehydrogenase and catalase, are shown in Table II. Glutamate dehydrogenase activity remained unchanged by the erotic acid treatment in experiment 1. The enzyme activity was increased by pantethine adminstration. A marked reduction of the glutamate dehydrogenase activity was observed after administration of erotic acid in experiment 2. The result seems to be due to enlargement of cell volume, since the DNA contents (per g liver) were decreased. The ratios of the glutamate dehydrogenase activity to the DNA content (per g liver) of the erotic acid groups in experiment 2 were nearly the same as those in experiment 1. On feeding erotic acid + pchlorophenoxyisobutyrate, the enzyme activity was reversed to the control level. It was confirmed that adenylate kinase activity, which is confined to the intermembrane space of mitochondria, was re-
II
GLUTAMATE
DEHYDROGENASE
Data are expressed
as mean*S.D.
AND CATALASE The levels of significances
were determined
Glutamate dehydrogenase (units/g liver)
by Student’s P
r-test. n.s., not significant, Catalase (kunits/g
P liver)
Expt. 1 (n =4) Control Orotic acid (1 W) Orotic acid (2W) Pantethine Orotic acid+panthethine Adenine Orotic acid + adenine
217*40 215*34 202222 298-t32 266+52 208223 204* 19
n.s. n.s. < 0.05 n.s. n.s. ns.
84% 16 80f 18 672 13 136*24 102* 15 9O%20 93’18
n.s. n.s. CO.05 n.s. ns. n.s.
Expt. 2 (n = 3) Control Orotic acid (2W) Orotic acid (3W) Orotic acid + p-chlorophenoxyisobutyrate
186*22 133* 2 103% 12 188’13
CO.05 CO.05 n.s.
125~ 8 93*15 13-t 2 191c I
CO.05
498
dddd ti tl tt ii
499
covered in the mitochondrial fractions but was not detected in the soluble fraction for all experimental groups (data not shown). Recoveries of the glutamate dehydrogenase activities in the mitochondrial fractions by the subcellular fractionation were within the range of 38-41% suggesting that the integrity and number of mitochondria were not altered seriously by the treatments. These data, therefore, indicate that the reduction of fatty acid oxidation of the mitochondrial system after erotic acid administration and the preventive or reversal effect of feeding of pantethine, adenine or p-chlorophenoxyisobutyrate are not due to the structural alteration of mitochondria (Table II). Peroxisomal fatty acid oxidation remained unchanged after administration of erotic acid and/or adenine (Table I). Pantethine administration increased the capacity of this system. p-Chlorophenoxyisobutyrate administration induced the peroxisomal system enormously to nearly the same level as those observed previously [9]. Enzyme activities Camitine palmitoyltransferase activities were not affected by the erotic acid and/or adenine feeding in experiment 1, as shown in Table III. The enzyme activities in experiment 2 were de: creased after erotic acid administration. However, there is no difference between these groups when the activities are expressed per unit of glutamate dehydrogenase. Pantethine administration also has no effect on the enzyme level when the activities are experessed in relation to the glutamate dehydrogenase activity. It has been reported that peroxisome proliferators such as p-chlorophenoxyisobutyrate induce carnitine acyltransferases [2024]. Fig. 2 summarizes substrate specificities of carnitine acyltransferases, which are classified into carnitine acetyltransferase, carnitine octanoyltransferase and carnitine palmitoyltransferase. Administration of erotic acid and/or adenine did not change activity of carnitine the palmitoyltransferase (Table III) and other activities of carnitine acyltransferases (data not shown). Administration of pantethine or erotic acid plus pantethine increased the activities without changing the relative activities of the different trans-
1
27.3
13.5 A
12T
-I-
2-
O-
Carbon
chain
length
Fig. 2. Activities of carnitine acyltransferases. Enzyme activities were determined in the direction of acylcarnitine formation from saturated fatty acyl-CoAs with various carbon chain lengths. Homogenates of frozen livers were pooled and used for the enzyme assay. 0, Control; 0, erotic acid (2W); 0, pantethine; n , erotic acid+pantethine (Expt. 1). X, erotic acid (3W); A, erotic acid+ p-chlorophenoxyisobutyrate (Expt. 2).
ferases. Administration of p-chlorophenoxyisobutyrate, however, changed the relative amounts as described previously [20-241. Induction of carnitine acetyltransferase was the highest, and that of camitine palmitoyltransferase was the lowest. Acyl-CoA dehydrogenases in mammalian tissues are classified by their substrate specificities: short-chain, general and long-chain acyl-CoA dehydrogenase (cf. Ref. 25). There are three acylCoA dehydrogenases in rat liver [ 131. Table III shows that acyl-CoA dehydrogenase activity with palmitoyl-CoA, which represents long-chain acylCoA dehydrogenase activity, was decreased to less than half after administration of erotic acid. Administration of pantethine increased the dehydrogenase activity. Pantethine feeding with erotic acid did not change the dehydrogenase activity. Administration of adenine alone did not affect the
500
dehydrogenase activity. The effect of erotic acid on acyl-CoA dehydrogenase activity was reversed by administration of adenine or p-chlorophenoxyisobutyrate. Fig. 3 summarizes the enzyme activities of fatty acyl-CoAs with various carbon chain lengths. The pattern of activity dependent on chain length for the control group was slightly different from that previously reported [lo]. The difference seems to be due to a decrease in short-chain acyl-CoA dehydrogenase caused by the fat-free diet feeding. From the pattern of the erotic acid group, it appears possible that all three acyl-CoA dehydrogenases [ 131 are decreased. Acyl-CoA dehydrogenase activities were increased by pantethine administration, but the levels were the same as that of the control when rats were fed the diet containing erotic acid and pantethine. The reversal
4-
3-
effect of adenine or p-chlorophenoxyisobutyrate was marked for all three dehydrogenases. Activities of other individual enzymes involved in the mitochondrial P-oxidation system are listed in Table III. The enoyl-CoA hydratase activity remained unchanged by administration of erotic acid (except erotic acid (3W) in experiment II), pantethine, adenine, and/or p-chlorophenoxyisobutyrate. 3-Hydroxyacyl-CoA dehydrogenase activity was decreased by erotic acid administration. The activity was restored to the control level by administration of adenine or p-chlorophenoxyisobutyrate. The 3-ketoacyl-CoA thiolase was not affected by administration of erotic acid, adenine and/or pantethine, but was increased markedly by p-chlorophenoxyisobutyrate treatment, as previously described [15]. It has been reported that administration of peroxisome proliferations increases activities of 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase but not enoylCoA hydratase in mitochondria [ 151. The peroxisomal P-oxidation system consists of the specific enzymes: acyl-CoA oxidase, a bifunctional enzyme exhibiting enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities, and 3-ketoacyl-CoA thiolase. The levels of the peroxisomal enzymes remained unchanged after erotic acid administration in experiment 1, but decreased slightly in experiment 2 (data not shown). The levels of these enzyme activities of the erotic acid + p-chlorophenoxyisobutyrate groups were nearly the same as those of rats receiving a standard laboratory diet containing a peroxisome proliferator, di(2-ethylhexyl)phthalate [ 151 (data not shown). Discussion
0114
”
6
t ” ” ” 8 IO 12 14 16 I8 20 Carbon chain length
22
Fig. 3. Activities of aycl-CoA dehydrogenases. Enzyme activities of pooled samples were determined with saturated fatty acyl-CoAs with various carbon chain lengths. 0, Control; 0, erotic acid (2W); 0, pantethine; n , erotic acid+pantethine V, adenine (Expt. 1). X, erotic acid (3W); A, erotic acid+ pchlorophenoxyisobutyrate (Expt. 2).
The main mechanism of the pathogenesis of the erotic acid-induced fatty liver is a block in the serection of low density lipoproteins without any change of general liver protein synthesis [S-8]. In the present study, we report another aspect of the change in the lipid metabolism by erotic acid administration. Fatty acid oxidation by hepatic mitochondria was inhibited by the introduction of erotic acid into the diet. However, the peroxisomal P-oxidation was not affected by erotic acid administration. The activities of
501
adenylate kinase, carnitine palmitoyltransferase, glutamate dehydrogenase and the three enzymes involved in the mitochondrial P-oxidation system remained unchanged by the erotic acid feeding in experiment 1. The recoveries of these enzyme activities in the mitochondrial fractions after subcellular fractionation were also nearly the same for both control and erotic acid-fed groups. Therefore, the reduction of fatty acid oxidation seems not to be due to changes in the number and/or the integrity of the mitochondria. In experiment 2, the level of glutamate dehydrogenase was decreased after administration of erotic acid. However, the ratio of glutamate dehydrogenase activity to DNA content remained unchanged. The data suggest that the decreased fatty acid oxidation capacity of mitochondria in experiment 2 is not due to a decrease in the number of mitochondria in the cell, but to an enlargment of the cell volume. Oxidations of palmitate, palmitoyl-CoA, palmitoylcarnitine octanoate, octanoyl-CoA and octhe tanoylcarnitine were reduced. Therefore, responsible site for the reduction in the fatty acid oxidation cannot be the acyl-CoA synthetase (EC 6.2.1.3) reaction. The activity of long-chain acylCoA synthetase in the mitochondrial fraction, which was assayed by the conversion of CoA to palmitoyl-CoA [24] or by the formation of AMP from ATP in the presence of palmitate [26], was not changed by the erotic acid feeding in experiment 1 (data not shown). The activities of the carnitine acyltransferases, expressed per unit of the glutamate dehydrogenase, were also unchanged by erotic acid feeding (Table III and Fig. 2). Oxidation of octanoate is carnitine-independent, whereas that of octanoyl-CoA is carnitine-dependent. The oxidation rates of octanoate, octanoylCoA and octanoylcarnitine in the presence and absence of carnitine were determined using pooled samples of the control and the erotic acid groups in experiment 2. The oxygen consumptions of the erotic acid (2W) group with palmitate, palmitoylCoA in the presence of carnitine, and palmitoylcarnitine were 24, 18 and 21% of those of the control, respectively. Those with octanoate, octanoyl-CoA in the presence of carnitine, and octanoylcarnitine were 21, 20 and 24%, respectively. The involvement of carnitine acyltransferases, therefore, in the reduction of fatty acid oxidation is excluded.
For consideration of the physiological significance, however, the carnitine level in the cell should be taken into account, since the carnitine palmitoyltransferase step has been regulated by the carnitine content and is presumed to regulate fatty acid oxidation in intact cells [27-311. The total carnitine levels were 0.458 2 0.073 pmol/g liver in control, 0.867 2 0.008 Pmol/g liver in erotic acid (1W), 0.662 2 0.113 pmol/g liver in erotic acid (2W) groups in experiment 1. Therefore, in the early stage of development of fatty liver by erotic acid administration, deterioration of fatty acid oxidation seems to be compensated for by an increased level of carnitine. Adminstration of pantethine or adenine did not affect the carnitine level. Administration of p-chlorophenoxyisobutyrate increased the carnitine level to 1.50 * 0.19 pmol/g liver, as has been described previously [24]. The levels of total CoA were increased about 2-fold in the pantethine and adenine groups. The increase in the erotic acid + p-chlorophenoxyisobutyrate groups was about 5-fold. In the present study, we determined the mitochondrial fatty acid oxidation capacity under conditions fortified with an optimal concentration of carnitine. When the capacity of mitochondrial fatty acid oxidation is assayed in the presence of a sufficient amount of carnitine, the rate-limiting step is the acyl-CoA dehydrogenase reaction [lo]. Therefore, it appears likely that reduction of the fatty acid oxidation capacity by erotic acid feeding is due to a decrease in the acyl-CoA dehydrogenase activities. A marked increase in fatty acid oxidation was observed in the pantethine-fed group. The increase was more marked for the oxidation of long-chain fatty acids. The mechanism of the pantethine action is not clear. However, one effect of pantethine seems to be the increase of acyl-CoA dehydrogenase activity. Adenine administration reversed acyl-CoA dehydrogenase without affecting the levels of other enzyme. p-Chlorophenoxyisobutyrate feeding resulted in the increases in both the mitochondrial and peroxisomal fatty acid oxidation systems. Administration of peroxisome proliferators, such as p-chlorophenoxyisobutyrate or di(Zethylhexyl)phthalate, induces the capacity of mitochondrial fatty acid oxidation by increasing quantities of
502
acyl-CoA dehydrogenases, rate-limiting enzymes of the mitochondrial P-oxidation system [lo]. The data in the present study suggest that the reduction of mitochondrial fatty acid oxidation capacity by erotic acid feeding is prevented or reversed by prevention of a decrease in the acylCoA dehydrogenase activity or by restoration of the enzyme level. References 1 Standerfer, S.B. and Handler, P. (1955) Proc. Sot. Exp. Biol. Med. 90, 270-271 2 Handschumacher, R.E., Creasey, W.A., Jaffe. J.J., Pasternak, C.A. and Hankin, L. (1960) Proc. Natl. Acad. Sci. U.S.A. 46, 178- 186 3 Creasey, W.A., Hankin, L. and Handschumacher, R.E. (1961) J. Biol. Chem. 236, 2064-2070 4 Windmueller, H.G. (1963) B&hem. Biophys. Res. Commun. 11,496-500 5 Rajalakshmi, S., Adams, W.R. and Handschumacher, R.E. (1969) J. Cell Biol. 41, 625-636 6 Pottenger, L.A. and Getz, G.S. (1971) J. Lipid Res. 12, 450-459 7 Novilcoff, P.M., Roheim, P.S., Novikoff, A.B. and Edelstein, D. (1974) Lab. Invest. 30, 732-750 8 Novikoff, P.M. and Edelstein, D. (1977) Lab. Invest. 36, 215-231 Lazarow, P.B. and De Duve, C. (1976) Proc. Nat]. Acad. Sci. U.S.A. 73, 2043-2046 Furuta, S., Miyazawa, S. and Hashimoto, T. (1981) J. B&hem. 90, 1751-1756 Markwell, M.A.K., McGroarty, E.J.. Bieber, L.L. and Tolbert, N.E. (1973) J. Biol. Chem. 248, 3426-3432 Chase, J.F.A., Pearson, D.J. and Tubbs, P.K. (1965) Biochim. Biophys. Acta, 96, 162- 165
13 Furuta. S., Miyazawa, S.’ and Hashimoto. T. (1981) J. B&hem. 90, 1739- 1750 14 Hashimoto, T., Miyazawa, S., Gunarso, D. and Furuta. S. (1981) J. B&hem. 90, 415-421 15 Miyazawa, S., Furata, S., Osumi, T. and Hashimoto, T. (1980) Biochim. Biophys. Acta 630, 367-374 16 Schmidt, E. (1974) in Methods of Enzymatic Analysis. 2nd edn. (Bergmeyer, H.U., ed.), Vol. 2. pp. 650-656, Verlag Chemie, Weinheim 17 Aebi. H. (1974) in Methods of Enzymatic Analysis, 2nd edn. (Bergmeyer, H.U., Chemie, Weinheim
ed.), Vol. 2, pp. 673-678.
Verlag
18 Bergmeyer, H.U., Gawehn, K. and Grass]. M. (1974) in Methods of Enzymatic Analysis, 2nd edn. (Bergmeyer, H.U., ed.), Vol. 1.. pp. 486-487, Verlag Chemie, Weinheim 19 Wieland, T. and Rueff, L. (1953) Angew. Chem. 65, 186- 187 20 Solberg, H.E., Aas, M. and Daae, L.N.W. (1972) Biochim. Biophys. Acta 280, 434-439 21 Solberg. H.E. (1974) Biochim. Biophys. Acta 360. 101-112 22 Kahonen, M.T. (1976) Biochim. Biophys. Acta, 428, 690701 71 Markwell, *.z M.A.K., Bieber, L.L. and Tolbert. N.E. ( 1977) B&hem. Pharmacol. 26, 1697- 1702 24 Shindo, Y., Osumi, T. and Hashimoto, T. (1978) Biochem. Pharmacol. 27, 2683-2688 25 Hall, CL. (1978) Methods Enzymol. 53, 502-518 26 Tanaka, T., Hosaka, K., Hoshimaru. M. and Numa, S. (1979) Eur. J. B&hem. 98, 165-172 27 McGarry, J.D., Robles-Valdes, C. and Foster, D.W. (1975) Proc. Natl. Acad. Sci. U.S.A., 72, 4385-4388 28 Christiansen, R. Borrebaek, B. and Bremer, J. (1976) FEBS Lett. 62, 313-317 29 Christiansen, R.Z. (1977) B&him. Biophys. Acta 488, 249262 30 McGarry, J.D., Mannaerts, G.P. and Foster, D.W. ( 1977) J. Clin. Invest. 60, 265-270 31 McGarry, J.D., Mannaerts, G.P. and Foster, D.W. (1978) Biochim. Biophys. Acta, 530, 305-313
494
BBA 51109
REDUCTION OF j3-OXIDATION OROTIC ACID SHOKO
MIYAZAWA,
SHUICHI
FURUTA
CAPACITY
OF RAT LIVER MITOCHONDRIA
and TAKASHI
HASHIMOTO
BY FEEDING
*
Department of Biochemistry, Shinshu University School of Medicine, Matsumoto, Nagano-ken 390 (Japan) (Received
December
30th, 1981)
Key words: P-Oxidation; Orotic acid diet; (Rat liver mitochondria)
Rats were maintained on fat-free high carbohydrate diets either with or without erotic acid (l%, w/w), pantethine (I%, w/w), adenine (0.25%, w/w), and/or pchlorophenoxyisobutyrate (0.258, w/w). Oxidation of fatty acid by liver mitochondria was inhibited to less than half that of the control after administration of orotie acid. Activities of acyCCoA dehy~~ena~s were markedly decreased by erotic acid adminis~a~on, but the following enzyme activities were not, or only slightly, decreased: acyl-CoA synthetase, carnitine acyltransferases, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase. Simultaneous addition of pantethine in the erotic acid-containing diet prevented induction of fatty liver. It also prevented decreases in fatty acid oxidation capacity and aeyl-CoA dehydrogenase activity. Introduction of adenine or p-chlorophenoxyisobutyrate, which reverse erotic acid-induced fatty liver, reversed oxidation and acyl-CoA dehydrogenase activities to control levels. The oxidation capacity of the peroxisomal system remained unchanged after adminis~ation of erotic acid.
In~~uction
16 h after erotic acid was introduced into the diet at 1% (w/w), and a minimum plateau was reached in 4 days. The level was restored to that of normal plasma lipid within 16 h by addition of adenine [4]. Therefore, the basic mechanism underlying the pathogenesis of fatty liver induced by the compound has been considered to be a block in the release of triacylglycerol into plasma. Studies on the morphological analysis of fatty liver and on plasma lipoprotein in rats fed erotic acid further substantiate this hypothesis [5-S]. It has been reported that there is no change in the conversion of [14C ,]stearate to 14C02, using liver slices from rats which had been maintained on the experimental diets for 3 days [3]. Nevertheless, this study concerning the fatty acid oxidation of livers of rats which had received erotic acid was conducted because of the following reasons. Recently, the presence of the b-oxidation system in
Orotic, acid induces severely fatty liver in rats when added to purified diets for 7-14 days [I]. The fatty liver induced by erotic acid ingestion, unlike the fatty liver induced by a choline-deficient diet, does not seem to be accompanied by other serious pathological disturbances. The addition of adenine to such a diet prevents and also reverses fat accumulation in the liver [2]. Lipid accumulation in the liver could be seen 3 days after 1% (w/w) erotic acid had been added to the diet, reached a maximum by 14 days and persisted for at least 200 days if erotic acid remained in the diet [3]. Plasma lipid levels started to drop as early as
* To whom correspondence
should be addressed.
OOOS-2760/82/0000-OOOOooo/%O2.75 0 1982 Elsevier Biomedical
Press
495
hepatic peroxisomes has been reported [9] and the fatty acid oxidation capacity of this system is comparable to that of the mitochondrial system [lo]. The peroxisomal system is induced markedly by various peroxisome proliferators such as pchlorophenoxyisobutyrate [9]. A large part of the fatty acids oxidized in the liver is converted to ketone bodies. The conversion of 14C-labeled fatty acid into 14COZdoes not seem to be representative of the total ability to metabolize fatty acid. The present report is concerned with the fatty acid oxidation of livers of rats receiving erotic acid. Fatty acid oxidation via the ~tochondrial system, but not the peroxisomal one, was inhibited. The inhibition site seems to be the acylCoA dehydrogenase (EC 1.33.99.2 and 1.3.99.3) reaction. Materials and Methods Animals . Male rats of the Wistar strain, weighing 200-25Og, were fed with various diets. The control diet was a set-syn~etic fat-free, highcarbohydrate diet which contained 24% milk casein, 51% corn starch, 10% sucrose, 5% cellulose, 2% a-starch, 1% vitamin mixture and 7% salt mixture (Clea, Tokyo). The following compounds were added to the control diet: erotic acid (l%, w/w), pantethine (1% w/w), adenine (0.25%, w/w) and p-chlorophenoxyisobutyrate (0.25%, w/w). In experiment 1, rats were divided into seven groups (n = 4) and fed with seven kinds of diet for 1-2 weeks. Feeding duration of the erotic acid (1W) and adenine groups was 1 week and that of the erotic acid (2W) group was 2 weeks. The erotic acid + adenine group was maintained on the erotic acid diet for 1 week and then placed on the erotic acid + adenine diet for 1 week. The control, pantethine and erotic acid + pantethine groups were fed with the respective diets for 2 weeks. In experiment 2 (n = 3, each), the control group was maintained for 3 weeks. One of the erotic acid groups (erotic acid (2W)) was fed with the erotic acid diet for 2 weeks and the other (erotic acid (3W)) for 3 weeks. The erotic acid + pchlorophenoxyisobutyrate group was m~nt~ned on the erotic acid diet for 2 weeks and then placed on the erotic acid + p-chlorophenoxyisobutyrate diet for 1 week.
Fatty acid oxidation. The fatty acid o~dation capacities of mitochondria and peroxisomes were assayed with used of the mitochondrial fractions from rat liver as described previously [lo]. Recoveries of mitochondria and peroxisomes in the mitochondrial fractions were calculated using the rate of recovery of the marker enzymes, glutamate dehydrogenase (EC 1.4.1.3) and catalase (EC 1,11.1.6), respectively. The data are expressed as pmol 0, consumed/m& per g liver. Enzyme assay. Camitine acyltransferase (EC 2.3.1.7 and 2.3.1.21) activities were assayed in both directions: the formation of acylcarnitines with use of acyl-CoAs as substrates [ 111 and of the formation of palmitoyl-CoA from palmitoylcarnitine [ 121. The activities of acyl-CoA dehydrogenases and electron-transfer flavoprotein were determined after DEAE-cellulose column chromatography of the mitochondrial extract [ 131. The activity of acyl-CoA oxidase was assayed with pahnitoyl-CoA as described previously [14]. The following enzymes involved in p-oxidation were located in both ~t~hondria and peroxisomes: enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.l. 1.35), and 3-ketoacyl-CoA thiolase (EC 2.3.1.16). Mitochondrial and peroxisomal enzyme activities were distinguished with use of antibodies against the respective enzymes [ 151. Activities of glutamate dehydrogenase [16] and catalase [ 171 were assayed for the estimation of recovery of mitochondria and peroxisomes after subcellular fractionation. Adenylate kinase (EC 2.7.4.3) was also assayed to check the integrity of the isolated ~t~hond~a [18]. All enzyme activities, including the oxidation of fatty acids, were assayed under the specified conditions at 3O’C. Units of enzyme activities were expressed as pmol of substrates utilized or products formed/mm. The data are summarized as units per g liver. Materials. Saturated fatty acyl-CoAs were prepared by the mixed anhydride method [19]_ Enzymes and coenzymes were purchased from Boehringer. Fatty acid-free bovine serum albumin was from Sigma. L-Carnitine, ~tanoylcar~tine and palmitoylca~tine were gifts from Otsuka Pharmaceutical Co., Naruto, Japan. Pantethine was kindly donated from Daiichi Seiyaku Co., Ltd., Tokyo.
496
Results Fatty liver
On feeding the diet containing 1% (w/w) erotic acid, development of the characteristic fatty liver was confirmed. The contents of total lipids (mg/g liver, mean * S.D.) in experiment 1 were 57.6 -+ 13.8 for the control, 101.8 * 20.5 for the erotic acid (1 W), 163.7 2 36.6 for the erotic acid (2W). 62.9 2 5.0 for the pantethine, 53.6 * 13.0 for the erotic acid + pantethine, 54.2 r+ 12.6 for the adenine and 74.1 * 13. I for the erotic acid + adenine groups. The values in experiment 2 were 46.1 k 3.2 for the control, 196.0 t 27.1 for the erotic acid (2W), 229.3 2 63.2 for the erotic acid (3W), and 52.0* 8.3 for the erotic acid +pchlorophenoxyisobutyrate groups. Pantethine prevented induction of fatty liver, and adenine and p-chlorophenoxyisobutyrate reversed it, as previously reported [8]. When a powdered diet with the composition of the standard chow diet (Oriental M, Oriental Yeast Co., Tokyo) was used as the basal diet, the erotic acid feeding did not result in fatty liver. Fatty acid oxidation
As shown in TableI, palmitoyl-CoA oxidation by mitochondria was decreased markedly after TABLE
administration of erotic acid. Pantethine administration resulted in an increase in the capacity of the mitochondrial system. The simultaneous feeding of pantethine and erotic acid also resulted in a higher activity of this system than that of the control. Adminstration of adenine alone did not affect the oxidation. However, administration of adenine with erotic acid to rats previously given erotic acid reversed the capacity of the mitochondrial system to the control level. When ~-chlorophenoxyisobutyrate was administered simult~eously with erotic acid to rats previously given erotic acid, the rate of mitochondrial oxidation exceeded the control level. Palmitoylcarnitine is oxidized by mitochondria but not by peroxisomes. Oxidation of palmitoylcarnitine was studied with pooled samples of all groups. The oxidation rates were nearly the same as those of pamitoyl-CoA by mitochondria listed in Table I (data not shown). Administration of erotic acid decreased oxidation of fatty acids by ~tochondria without change of the substrate specificity (Fig. 1). Administration of adenine did not change the activities toward various substrates. The reversal effect of adenine did not affect the substrate specificity (data not shown). However, the effects of the pantethine, the erotic acid f pantethine, and the erotic acid fp-
I
EFFECTS ON PALMITOYL-CoA OXIDATION AND ~-CHLOROPHENOXYISO~U~~TE Experimental procedures are described mean* S.D. The levels of significances
OF ADMINISTRATION
OF OROTIC
in Materials and Methods. The oxidation values (gmol are determined by Student’s t-test. n.s., not significant.
ACID, O,/min
PANTETHINE,
per g liver) are
ADENINE
expressedas
Mitochondria
P
Peroxisomes
P
Expt. 1 (n=4) Control Orotic acid (1 W) Orotic acid (2W) Pantethlne Orotic acid+pantethine Adenine Orotic acid + adenine
0.424 * 0.050 0.308 f 0.048 0.172iO.028 1.298-t-0.378 0.934eO.278 0.456) 0.046 0.404) 0.038
CO.05
0.115~0.025 0.120~~.021 0.135 * 0.030 0.300~0.059 0.199~0.051 0.170* 0.053 0.124* 0.030
ns. n.s.
Expt. 2 (n =3f Control Orotic acid (2W) Orotic acid (3W) Erotic acid + p-chlorophenoxyisobutyrate
0.5~~0.1~ 0.092 * 0.020 0.058 kO.036 0.718r0.020
0.120~0.015 0.156~0.0~ 0.101~0.011 1.560~0.342
ns. ns. co.01
491
I.5
ti
.r IX 0, i ._
E \
0” 5
O.!
5.
0
6
8
IO
12
I4
I6
I8
Carbon chain length Fig. 1. Mitochondrial P-oxidation capacities. Saturated fatty acids with various carbon chain lengths were used: 6, hexanoate,; 8, octanoate; 10, decanoate; 12, dodecanoate; 14, tetradecanoate, 16, hexadecanoate; 18, octadecanoate. oxidation rates were determined with pooled mitochondrial fractions from four rats in Expt. 1 and that from three rats of the erotic acid+ p-chlorophenoxyisobutyrate group in Expt. 2. 0, Control; 0, erotic acid (2W); q, pantethine; n , erotic acid + pantethine; A, erotic acid + p-chlorophenoxyisobutyrate.
TABLE
chlorophenoxyisobutyrate groups were different from that of the control. In a previous study [lo], we observed that mitochondrial fatty acid oxidation was increased after administration of p-chlorophenoxyisobutyrate, and that the increase in the oxidation was more marked with long-chain fatty acids. The activities of marker enzymes for two organelles, glutamate dehydrogenase and catalase, are shown in Table II. Glutamate dehydrogenase activity remained unchanged by the erotic acid treatment in experiment 1. The enzyme activity was increased by pantethine adminstration. A marked reduction of the glutamate dehydrogenase activity was observed after administration of erotic acid in experiment 2. The result seems to be due to enlargement of cell volume, since the DNA contents (per g liver) were decreased. The ratios of the glutamate dehydrogenase activity to the DNA content (per g liver) of the erotic acid groups in experiment 2 were nearly the same as those in experiment 1. On feeding erotic acid + pchlorophenoxyisobutyrate, the enzyme activity was reversed to the control level. It was confirmed that adenylate kinase activity, which is confined to the intermembrane space of mitochondria, was re-
II
GLUTAMATE
DEHYDROGENASE
Data are expressed
as mean*S.D.
AND CATALASE The levels of significances
were determined
Glutamate dehydrogenase (units/g liver)
by Student’s P
r-test. n.s., not significant, Catalase (kunits/g
P liver)
Expt. 1 (n =4) Control Orotic acid (1 W) Orotic acid (2W) Pantethine Orotic acid+panthethine Adenine Orotic acid + adenine
217*40 215*34 202222 298-t32 266+52 208223 204* 19
n.s. n.s. < 0.05 n.s. n.s. ns.
84% 16 80f 18 672 13 136*24 102* 15 9O%20 93’18
n.s. n.s. CO.05 n.s. ns. n.s.
Expt. 2 (n = 3) Control Orotic acid (2W) Orotic acid (3W) Orotic acid + p-chlorophenoxyisobutyrate
186*22 133* 2 103% 12 188’13
CO.05 CO.05 n.s.
125~ 8 93*15 13-t 2 191c I
CO.05
498
dddd ti tl tt ii
499
covered in the mitochondrial fractions but was not detected in the soluble fraction for all experimental groups (data not shown). Recoveries of the glutamate dehydrogenase activities in the mitochondrial fractions by the subcellular fractionation were within the range of 38-41% suggesting that the integrity and number of mitochondria were not altered seriously by the treatments. These data, therefore, indicate that the reduction of fatty acid oxidation of the mitochondrial system after erotic acid administration and the preventive or reversal effect of feeding of pantethine, adenine or p-chlorophenoxyisobutyrate are not due to the structural alteration of mitochondria (Table II). Peroxisomal fatty acid oxidation remained unchanged after administration of erotic acid and/or adenine (Table I). Pantethine administration increased the capacity of this system. p-Chlorophenoxyisobutyrate administration induced the peroxisomal system enormously to nearly the same level as those observed previously [9]. Enzyme activities Camitine palmitoyltransferase activities were not affected by the erotic acid and/or adenine feeding in experiment 1, as shown in Table III. The enzyme activities in experiment 2 were de: creased after erotic acid administration. However, there is no difference between these groups when the activities are expressed per unit of glutamate dehydrogenase. Pantethine administration also has no effect on the enzyme level when the activities are experessed in relation to the glutamate dehydrogenase activity. It has been reported that peroxisome proliferators such as p-chlorophenoxyisobutyrate induce carnitine acyltransferases [2024]. Fig. 2 summarizes substrate specificities of carnitine acyltransferases, which are classified into carnitine acetyltransferase, carnitine octanoyltransferase and carnitine palmitoyltransferase. Administration of erotic acid and/or adenine did not change activity of carnitine the palmitoyltransferase (Table III) and other activities of carnitine acyltransferases (data not shown). Administration of pantethine or erotic acid plus pantethine increased the activities without changing the relative activities of the different trans-
1
27.3
13.5 A
12T
-I-
2-
O-
Carbon
chain
length
Fig. 2. Activities of carnitine acyltransferases. Enzyme activities were determined in the direction of acylcarnitine formation from saturated fatty acyl-CoAs with various carbon chain lengths. Homogenates of frozen livers were pooled and used for the enzyme assay. 0, Control; 0, erotic acid (2W); 0, pantethine; n , erotic acid+pantethine (Expt. 1). X, erotic acid (3W); A, erotic acid+ p-chlorophenoxyisobutyrate (Expt. 2).
ferases. Administration of p-chlorophenoxyisobutyrate, however, changed the relative amounts as described previously [20-241. Induction of carnitine acetyltransferase was the highest, and that of camitine palmitoyltransferase was the lowest. Acyl-CoA dehydrogenases in mammalian tissues are classified by their substrate specificities: short-chain, general and long-chain acyl-CoA dehydrogenase (cf. Ref. 25). There are three acylCoA dehydrogenases in rat liver [ 131. Table III shows that acyl-CoA dehydrogenase activity with palmitoyl-CoA, which represents long-chain acylCoA dehydrogenase activity, was decreased to less than half after administration of erotic acid. Administration of pantethine increased the dehydrogenase activity. Pantethine feeding with erotic acid did not change the dehydrogenase activity. Administration of adenine alone did not affect the
500
dehydrogenase activity. The effect of erotic acid on acyl-CoA dehydrogenase activity was reversed by administration of adenine or p-chlorophenoxyisobutyrate. Fig. 3 summarizes the enzyme activities of fatty acyl-CoAs with various carbon chain lengths. The pattern of activity dependent on chain length for the control group was slightly different from that previously reported [lo]. The difference seems to be due to a decrease in short-chain acyl-CoA dehydrogenase caused by the fat-free diet feeding. From the pattern of the erotic acid group, it appears possible that all three acyl-CoA dehydrogenases [ 131 are decreased. Acyl-CoA dehydrogenase activities were increased by pantethine administration, but the levels were the same as that of the control when rats were fed the diet containing erotic acid and pantethine. The reversal
4-
3-
effect of adenine or p-chlorophenoxyisobutyrate was marked for all three dehydrogenases. Activities of other individual enzymes involved in the mitochondrial P-oxidation system are listed in Table III. The enoyl-CoA hydratase activity remained unchanged by administration of erotic acid (except erotic acid (3W) in experiment II), pantethine, adenine, and/or p-chlorophenoxyisobutyrate. 3-Hydroxyacyl-CoA dehydrogenase activity was decreased by erotic acid administration. The activity was restored to the control level by administration of adenine or p-chlorophenoxyisobutyrate. The 3-ketoacyl-CoA thiolase was not affected by administration of erotic acid, adenine and/or pantethine, but was increased markedly by p-chlorophenoxyisobutyrate treatment, as previously described [15]. It has been reported that administration of peroxisome proliferations increases activities of 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase but not enoylCoA hydratase in mitochondria [ 151. The peroxisomal P-oxidation system consists of the specific enzymes: acyl-CoA oxidase, a bifunctional enzyme exhibiting enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities, and 3-ketoacyl-CoA thiolase. The levels of the peroxisomal enzymes remained unchanged after erotic acid administration in experiment 1, but decreased slightly in experiment 2 (data not shown). The levels of these enzyme activities of the erotic acid + p-chlorophenoxyisobutyrate groups were nearly the same as those of rats receiving a standard laboratory diet containing a peroxisome proliferator, di(2-ethylhexyl)phthalate [ 151 (data not shown). Discussion
0114
”
6
t ” ” ” 8 IO 12 14 16 I8 20 Carbon chain length
22
Fig. 3. Activities of aycl-CoA dehydrogenases. Enzyme activities of pooled samples were determined with saturated fatty acyl-CoAs with various carbon chain lengths. 0, Control; 0, erotic acid (2W); 0, pantethine; n , erotic acid+pantethine V, adenine (Expt. 1). X, erotic acid (3W); A, erotic acid+ pchlorophenoxyisobutyrate (Expt. 2).
The main mechanism of the pathogenesis of the erotic acid-induced fatty liver is a block in the serection of low density lipoproteins without any change of general liver protein synthesis [S-8]. In the present study, we report another aspect of the change in the lipid metabolism by erotic acid administration. Fatty acid oxidation by hepatic mitochondria was inhibited by the introduction of erotic acid into the diet. However, the peroxisomal P-oxidation was not affected by erotic acid administration. The activities of
501
adenylate kinase, carnitine palmitoyltransferase, glutamate dehydrogenase and the three enzymes involved in the mitochondrial P-oxidation system remained unchanged by the erotic acid feeding in experiment 1. The recoveries of these enzyme activities in the mitochondrial fractions after subcellular fractionation were also nearly the same for both control and erotic acid-fed groups. Therefore, the reduction of fatty acid oxidation seems not to be due to changes in the number and/or the integrity of the mitochondria. In experiment 2, the level of glutamate dehydrogenase was decreased after administration of erotic acid. However, the ratio of glutamate dehydrogenase activity to DNA content remained unchanged. The data suggest that the decreased fatty acid oxidation capacity of mitochondria in experiment 2 is not due to a decrease in the number of mitochondria in the cell, but to an enlargment of the cell volume. Oxidations of palmitate, palmitoyl-CoA, palmitoylcarnitine octanoate, octanoyl-CoA and octhe tanoylcarnitine were reduced. Therefore, responsible site for the reduction in the fatty acid oxidation cannot be the acyl-CoA synthetase (EC 6.2.1.3) reaction. The activity of long-chain acylCoA synthetase in the mitochondrial fraction, which was assayed by the conversion of CoA to palmitoyl-CoA [24] or by the formation of AMP from ATP in the presence of palmitate [26], was not changed by the erotic acid feeding in experiment 1 (data not shown). The activities of the carnitine acyltransferases, expressed per unit of the glutamate dehydrogenase, were also unchanged by erotic acid feeding (Table III and Fig. 2). Oxidation of octanoate is carnitine-independent, whereas that of octanoyl-CoA is carnitine-dependent. The oxidation rates of octanoate, octanoylCoA and octanoylcarnitine in the presence and absence of carnitine were determined using pooled samples of the control and the erotic acid groups in experiment 2. The oxygen consumptions of the erotic acid (2W) group with palmitate, palmitoylCoA in the presence of carnitine, and palmitoylcarnitine were 24, 18 and 21% of those of the control, respectively. Those with octanoate, octanoyl-CoA in the presence of carnitine, and octanoylcarnitine were 21, 20 and 24%, respectively. The involvement of carnitine acyltransferases, therefore, in the reduction of fatty acid oxidation is excluded.
For consideration of the physiological significance, however, the carnitine level in the cell should be taken into account, since the carnitine palmitoyltransferase step has been regulated by the carnitine content and is presumed to regulate fatty acid oxidation in intact cells [27-311. The total carnitine levels were 0.458 2 0.073 pmol/g liver in control, 0.867 2 0.008 Pmol/g liver in erotic acid (1W), 0.662 2 0.113 pmol/g liver in erotic acid (2W) groups in experiment 1. Therefore, in the early stage of development of fatty liver by erotic acid administration, deterioration of fatty acid oxidation seems to be compensated for by an increased level of carnitine. Adminstration of pantethine or adenine did not affect the carnitine level. Administration of p-chlorophenoxyisobutyrate increased the carnitine level to 1.50 * 0.19 pmol/g liver, as has been described previously [24]. The levels of total CoA were increased about 2-fold in the pantethine and adenine groups. The increase in the erotic acid + p-chlorophenoxyisobutyrate groups was about 5-fold. In the present study, we determined the mitochondrial fatty acid oxidation capacity under conditions fortified with an optimal concentration of carnitine. When the capacity of mitochondrial fatty acid oxidation is assayed in the presence of a sufficient amount of carnitine, the rate-limiting step is the acyl-CoA dehydrogenase reaction [lo]. Therefore, it appears likely that reduction of the fatty acid oxidation capacity by erotic acid feeding is due to a decrease in the acyl-CoA dehydrogenase activities. A marked increase in fatty acid oxidation was observed in the pantethine-fed group. The increase was more marked for the oxidation of long-chain fatty acids. The mechanism of the pantethine action is not clear. However, one effect of pantethine seems to be the increase of acyl-CoA dehydrogenase activity. Adenine administration reversed acyl-CoA dehydrogenase without affecting the levels of other enzyme. p-Chlorophenoxyisobutyrate feeding resulted in the increases in both the mitochondrial and peroxisomal fatty acid oxidation systems. Administration of peroxisome proliferators, such as p-chlorophenoxyisobutyrate or di(Zethylhexyl)phthalate, induces the capacity of mitochondrial fatty acid oxidation by increasing quantities of
502
acyl-CoA dehydrogenases, rate-limiting enzymes of the mitochondrial P-oxidation system [lo]. The data in the present study suggest that the reduction of mitochondrial fatty acid oxidation capacity by erotic acid feeding is prevented or reversed by prevention of a decrease in the acylCoA dehydrogenase activity or by restoration of the enzyme level. References 1 Standerfer, S.B. and Handler, P. (1955) Proc. Sot. Exp. Biol. Med. 90, 270-271 2 Handschumacher, R.E., Creasey, W.A., Jaffe. J.J., Pasternak, C.A. and Hankin, L. (1960) Proc. Natl. Acad. Sci. U.S.A. 46, 178- 186 3 Creasey, W.A., Hankin, L. and Handschumacher, R.E. (1961) J. Biol. Chem. 236, 2064-2070 4 Windmueller, H.G. (1963) B&hem. Biophys. Res. Commun. 11,496-500 5 Rajalakshmi, S., Adams, W.R. and Handschumacher, R.E. (1969) J. Cell Biol. 41, 625-636 6 Pottenger, L.A. and Getz, G.S. (1971) J. Lipid Res. 12, 450-459 7 Novilcoff, P.M., Roheim, P.S., Novikoff, A.B. and Edelstein, D. (1974) Lab. Invest. 30, 732-750 8 Novikoff, P.M. and Edelstein, D. (1977) Lab. Invest. 36, 215-231 Lazarow, P.B. and De Duve, C. (1976) Proc. Nat]. Acad. Sci. U.S.A. 73, 2043-2046 Furuta, S., Miyazawa, S. and Hashimoto, T. (1981) J. B&hem. 90, 1751-1756 Markwell, M.A.K., McGroarty, E.J.. Bieber, L.L. and Tolbert, N.E. (1973) J. Biol. Chem. 248, 3426-3432 Chase, J.F.A., Pearson, D.J. and Tubbs, P.K. (1965) Biochim. Biophys. Acta, 96, 162- 165
13 Furuta. S., Miyazawa, S.’ and Hashimoto. T. (1981) J. B&hem. 90, 1739- 1750 14 Hashimoto, T., Miyazawa, S., Gunarso, D. and Furuta. S. (1981) J. B&hem. 90, 415-421 15 Miyazawa, S., Furata, S., Osumi, T. and Hashimoto, T. (1980) Biochim. Biophys. Acta 630, 367-374 16 Schmidt, E. (1974) in Methods of Enzymatic Analysis. 2nd edn. (Bergmeyer, H.U., ed.), Vol. 2. pp. 650-656, Verlag Chemie, Weinheim 17 Aebi. H. (1974) in Methods of Enzymatic Analysis, 2nd edn. (Bergmeyer, H.U., Chemie, Weinheim
ed.), Vol. 2, pp. 673-678.
Verlag
18 Bergmeyer, H.U., Gawehn, K. and Grass]. M. (1974) in Methods of Enzymatic Analysis, 2nd edn. (Bergmeyer, H.U., ed.), Vol. 1.. pp. 486-487, Verlag Chemie, Weinheim 19 Wieland, T. and Rueff, L. (1953) Angew. Chem. 65, 186- 187 20 Solberg, H.E., Aas, M. and Daae, L.N.W. (1972) Biochim. Biophys. Acta 280, 434-439 21 Solberg. H.E. (1974) Biochim. Biophys. Acta 360. 101-112 22 Kahonen, M.T. (1976) Biochim. Biophys. Acta, 428, 690701 71 Markwell, *.z M.A.K., Bieber, L.L. and Tolbert. N.E. ( 1977) B&hem. Pharmacol. 26, 1697- 1702 24 Shindo, Y., Osumi, T. and Hashimoto, T. (1978) Biochem. Pharmacol. 27, 2683-2688 25 Hall, CL. (1978) Methods Enzymol. 53, 502-518 26 Tanaka, T., Hosaka, K., Hoshimaru. M. and Numa, S. (1979) Eur. J. B&hem. 98, 165-172 27 McGarry, J.D., Robles-Valdes, C. and Foster, D.W. (1975) Proc. Natl. Acad. Sci. U.S.A., 72, 4385-4388 28 Christiansen, R. Borrebaek, B. and Bremer, J. (1976) FEBS Lett. 62, 313-317 29 Christiansen, R.Z. (1977) B&him. Biophys. Acta 488, 249262 30 McGarry, J.D., Mannaerts, G.P. and Foster, D.W. ( 1977) J. Clin. Invest. 60, 265-270 31 McGarry, J.D., Mannaerts, G.P. and Foster, D.W. (1978) Biochim. Biophys. Acta, 530, 305-313