Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues

Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues

211 Biochimica et Biophysics Acta, 1044 (1990) 211-221 Elsevier BBALIP 53409 Fatty acid metabolism in liver of rats treated with hypolipidemic sul...

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211

Biochimica et Biophysics Acta, 1044 (1990) 211-221 Elsevier

BBALIP

53409

Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues Daniel Asiedu ‘, Asle Aarsland ‘, Jon Skorve ‘, Asbjmn and Rolf K. Berge ’

M. Svardal

*

’ University of Bergen, Laboratov of Clinical Biochemistry and 2 Department of Pharmacology and Toxicology, Haukeland Sykehur, Bergen (Norway)

(Revised

Key words:

Hypolipidemic

(Received manuscript

fatty acid analog;

20 October 1989) received 12 February

Fatty acid metabolism;

1990)

Long chain fatty acid; (Rat liver peroxisome)

The purpose of this study was to investigate early biochemical changes and possible mechanisms via which alkyl(C12)thioacetic acid (CM’ITD, blocked for /l-oxidation), alkyl(C12)thiopropionic acid (CETI’D, undergo one cycle of /%oxididation) and a 34hiadicarboxylic acid (BCMTD, blocked for both w- (and /l-oxidation) influence the peroxisomal &oxidation in liver of rats. Treatment of rats with CMTTD caused a stimulation of the palmitoyl-CoA synthetase activity accompanied with increased concentration of hepatic acid-insoluble CoA. This effect was already established during 12-24 h of feeding. From 2 days of feeding, the cellular level of acid-insoluble CoA began to decrease, whereas free CoASH content increased. Stimulation of [l-t4C]palmitoyl-CoA oxidation in the presence of KCN, palmitoyl-CoA-dependent dehydrogenase (termed peroxisomal /l-oxidation) and pahnitoyl-CoA hydrolase activities were revealed after 36-48 h of CMTTD-feeding. Administration of BCMTD affected the enzymatic activities and altered the distribution of CoA between acid-insoluble and free forms comparable to what was observed in CMTTDtreated rats. It is evident that treatment of peroxisome proliferators (BCMTD and CMTTD), the level of acyl-CoA esters and the enzyme activity involved in their formation precede the increase in peroxisomal and palmitoyl-CoA hydrolase activities. In CMTTD-fed animals the activity of cyanide-insensitive fatty acid oxidation remained unchanged when the mitochondriai j%oxidation and camitine palmitoyltransferase operated at maximum rates. The sequence and redistribution of CoA and enzyme changes were interpreted as support for the hypothesis that substrate supply is an important factor in the regulation of peroxisomal fatty acid metabolism, i.e., the fatty acyl-CoA species appear to be catabolized by peroxisomes at high rates only when uptake into mitochondria is saturated. Administration of CETID led to an inhibition of mitochondrial fatty acid oxidation accompanied with a rise in the concentration of &yl-CoA esters in the liver. Consequently, fatty liver developed. The peroxisomal &oxidation was marginally affected. \IYh&er inhibition of mitochondriai /&oxidation may be involved in regulation of peroxisomal fatty acid metabolism and in development of fatty liver should be considered.

Introduction Alkylthioacetic acid (CMTTD) and 3-thiadicarboxylic acid (BCMTD), represent non-b-oxidizable fatty acid analogues in which one sulphur atom and two sulphur atoms, respectively, substitute the P-methylene group (3-position) in the chains. It has recently been described that when these fatty acid analogues are fed to rats they induce a drop in plasma triacylglycerol and cholesterol together with a reduction in the hepatic

Correspondence: R.K. Berge, University of Bergen, Laboratory of Clinical Biochemistry, Hankeland Sykehus, N-5021 Bergen, Norway. 0005-2760/90/$03.50

0 1990 Elsevier Science Publishers

B.V. (Biomedical

triacylglycerol level [1,2]. A considerable dose-dependent proliferation of peroxisomes and mitochondria are observed concomitant with increased peroxisomal /3oxidation [3]. Among the changes observed in the liver are a dose-dependent increase in the activities of palmitoyl-CoA synthetase, glycerophosphate acyltransferase and redistribution of palmitoyl-CoA hydrolase activity

L31. Our previous studies have established that the effects of feeding BCMTD and CMTTD mimic most of the hepatic responses seen on administration of other hypolipidemic peroxisome proliferating drugs, clofibrate, tiadenol [4-71 and high-fat diets [8]. When alkylthiopropionic acid (CETTD), which may Division)

212 undergo one cycle of /3-oxidation, was fed to rats, the most striking effect was accumulation of triacylglycerols and increased numbers of lipid droplets in the liver [1,3]. Whether increased intrahepatic direction of fatty acids towards esterification as opposed to mitochondrial and peroxisomal /I-oxidation, may effect the development of fatty metamorphosis in the liver, induced by CETTD, are to be considered. Various mechanisms have been proposed to explain the induction of peroxisomal P-oxidation. Enzyme induction may be regulated by substrate supply or the hypolipidemic drugs themselves are converted into unmetabolizable CoA thioesters [7,9]. Several peroxisome proliferators are known to inhibit mitochondrial fatty acid oxidation in rat liver, suggesting that the inhibition of the mitochondria may be involved in the induction of peroxisome proliferation [lo-131. Notably, it has been established that the coenzyme A esters of the sulphur substituted fatty acid analogues interfere with acyl-CoA dehydrogenases and act as inhibitors of these enzymes 1141. To better understand the sequence of events leading to peroxisome proliferation we have studied the rapid responses (as a function of time) which occur in the liver of rats fed CMTTD and CETTD, especially focusing on ‘long-chain acyl-CoA’ (CoA-thioesters of native long-chain fatty acid and/or the CoA-thioesters of the concerned drugs) formation as a regulatory component in peroxisomal fatty acid metabolism. l-o-dicarboxylic acid are predominantly oxidized in peroxisomes and by trapping of coenzyme A, the CoA-esters of dicarboxylic acid may represent a trigger for peroxisome proliferation. Therefore, we also wanted to examine whether or not the 3-thiadicarboxylic acid (BCMTD) could change the distribution of CoA between acylated and free forms. We also have in the cellular fractions measured the activity of key enzymes involved in the formation and breakdown of long-chain acyl-CoA in order to establish whether or not the increase in palmitoyl-CoA synmitochondrial P-oxidation and carnitine thetase, palmitoyltransferase activities preceded the rise in peroxisomal P-oxidation and palmitoyl-CoA hydrolase activity. Materials and Methods Chemicals and drugs [l-‘4C]Palmitic acid, [1-t4C]palmitoyl-CoA, L-[~~C] carnitine, L-[14C]palmitoyl-~-carnitine and [1-t4C]glycerol 3-phosphate, purchased from New England Nuclear (Boston, MA, U.S.A.), were mixed with unlabelled palmitic acid, palmitoyl-CoA, L-carnitine and glycerol 3-phosphate (Sigma, MO, U.S.A.) to give a specific radioactivity of 1500-1800 cpm/nmol. l.lO-Bis (carboxymethylthio)decane (BCMTD, thiadicarboxylic acid), 1-(carboxymethylthio)tetradecane (CMTTD,

alkylthioacetic acid) and l-(carboxyethylthio)tetradecane (CETTD, alkylthio propionic acid) were prepared as earlier described [15,16]. All other chemicals were obtained from common commercial sources and were of reagent grade. Animals and treatments Male Wistar rats from Mollegaard Breeding Laboratory (Ejby, Denmark) weighing 170-180 g, were housed individually in metal wire cages in a room maintained at 12 h light-dark cycles and at a constant temperature of 20 + 3” C. The animals were acclimatized for at least 1 week under these conditions before the start of the experiment. BCMTD, CMTTD and CETTD were suspended in 0.5% sodium carboxymethyl cellulose (CMS). The individual agents were administered by gastric incubation in a volume of 1 ml once a day and the animals were killed after 12 h of starvation. The animals were separately treated with the fatty acid analogues and the doses were the same for all of them: BCMTD, 150 mg/day per kg body wt; CMTTD, 150 mg/day per kg body wt; CETTD, 150 mg/day per kg body wt. The control animal groups received only CMS. All animals had free access to water and food. The body weights were measured daily. At the end of the experiments, the fasted rats were weighed. Under light halothane anesthesia, cardiac puncture was performed to obtain blood samples and the livers were removed and immediately chilled on ice and weighed. Preparation of total homogenate and the different subcellular fractions The livers from individual rats were homogenized in ice-cold sucrose-medium (0.25 M sucrose in 10 mM Hepes buffer (pH 7.4) and 2 mM EDTA), centrifuged and the resulting nuclear plus postnuclear fractions were used as the total homogenate. For further analytical differential centrifugation experiments, postnuclear fractions from three animals were pooled and a mitochondrial-enriched fraction (M), peroxisome-enriched fraction (L), microsomal fraction (P) and cytosolic fraction (S) were isolated [17]. Purified peroxisomes were prepared as earlier described [ 171. The variation in the response from animal to animal was estimated separately for selected enzymes in the group of control animals. Assay of enzymes and other analytical methods The subcellular marker enzymes were determined as earlier described [18]. Protein was assayed by Bio-Rad protein assay kit (Bio-Rad, Richmond, CA). The enzymatic activity of palmitoyl-CoA synthetase (EC 6.2.1.3), camitine palmitoyltransferase (EC 2.3.1. 21), palmitoyl-CoA hydrolase (EC 3.1.2.2) and palmitoyl-CoA-dependent dehydrogenase (usually termed

213 peroxisomal P-oxidation) were determined as earlier described 181. Free CoASH and acid-insoluble CoA, ‘long-chain acyl-CoA’ (the CoA-thioesters of native fatty acids and/or the CoA-thioesters of the three drugs) were measured in liver extracts processed as described previously [19,20].

The palmitoyl-CoA and palmitoyl-L-carnitine oxidation was measured as acid-soluble products. The assay medium (0.3 ml) contained 10 mM Hepes buffer (pH 7.3), 60 mM KC1 11.0 mM MgCl,, 11.1 mM dithiothreitol, 5.6 mM ADP, 0.2 mM NAD+, 0.6 mM EDTA and IO-l.2 mg protein from the isolated mitochondria or postnuclear fractions. The palmitoyl-t-carnitine oxidation was measured with 80 PM ]l-14C]palmitoylL-carnitine and the pal~toyl-CoA oxidation was measured with 100 FM palmitoyl-CoA supplemented with 2 mM L-carnitine. The cyanide-sensitive fatty acid oxidation was measured with the same assay medium in the presence of 3.5 mM KCN. After incubation at 30 o C for 90-120 s, the rate of oxidation was stopped by addition of 25 ~1 fatty acid-free bovine serum albumin (100 mg/ml) followed by 150 ~11.5 M KOH and 500 ~14 M HClO,. After centrifugation, a sample of the proteinand fatty-and-fry supernat~t was assayed for radioactivity, Presentation of the results The variation in the response from animal to animal

was estimated separately for selected enzymes in total liver homogenates in control and treated animals. In all tables and figures, the observed differences are compared to 0,7 and 14 days, tabulated as means 2 SD. to day 0 (n = 12 in control group and n = 3 is in the experimental groups) as there was no significant changes in the control animals of the 14 day period (varied between 5 and 10%). Data on enzyme activities in total liver homogenates and on the concentration of CoASH and acid-insoluble CoA in treated rats are presented as means f SD. (n = 3). For isolation of cellular fractions the postnuclear fractions from three animals were pooled. The tabulated values on enzymatic activities in cellular fraction of treated rats are given as means (n = 3) and in control rats, means + SD. (days 0, 7 and 14). P > 0.05 was taken to be statistically insignificant. Results Liuer weight and protein context

Diet intakes of all animal groups were comparable and the increase in body weights were similar for the treated groups (data not shown). Ad~~stratio~ of the a~ylt~oacetic acid (CMTTD), characterized as a non-&oxidizable fatty acid, at a dose of 150 mg/day per kg body weight caused a moderate

effect on liver weights within 7 days of feeding (Table I). No hepatomegaly was demonstrated with alkylthiopropionic acid (CETTD) feeding, a fatty acid which may undergo one cycle of ~-o~dation (Table I), The @and w-blocked sulphur substituted dicarboxylic acid (BCMTD), however, significantly increased the liver weight within 2 days of feeding. With BCMTD feeding the protein content in the mitochond~aland peroxisomal-en~ched fractions tended to increase, which was already revealed during the first day of treatment. CMTTD tended to increase the protein mass in the peroxisomal-enriched fraction after 2 days of feeding, whereas no increases in protein content in the cellular fractions was observed in the CETTD-administered rats (Table I). Liver triacylglyceroI

and CoA

In CMTTD- and BCMTD-treated rats the hepatic triacylglycerol content tended to decrease within hours of feeding and after 3 days of treatment a reduction at about 50% was revealed (Table I). Notably, in CETTDtreated animals, the hepatic t~acyl~ycerol content was greatly increased. Treatment of rats with CMTI’D and BCMTD resulted in an acute enhancement of the hepatic CoA level which was already established in 12-48 h of treatment and amounted to over 3-fold increase in total liver CoA (Table I). In CETTD-treated rats the hepatic CoA content was marginally affected (Table I). Fig. 1A shows that in the CMTTD-fed animals the hepatic acid-insoluble CoA levels was increased already after 12 h and continued to increase for up to 36 h of feeding amounting a 8-fold increase. Subsequently, the level of acid-insoluble CoA tended to decrease. In the liver of CETTD-treated animals, the acid-insoluble CoA content was mar~nally changed during the first 3 days of feeding. From 3 days until 14 days, the long-chain acyl-CoA content increased, however, resulting in a 2.5-fold elevation compared to normal values (Fig. 1B). The time-course pattern of the acid-insoluble CoA in the liver of BCMTD-treated rats (Fig. 1C) was comparable with those revealed after CMTTD feeding (Fig. 1A). BCMTD increased the concentration of hepatic CoA-esters within 24 h which continued to rise up to 2 days when a &fold increase was observed. Subsequently, from 3 days until 14 days, the acid-insoluble CoA content returned to approximately normal values (Fig. 1C). The free CoASH content in liver of CMTTD-fed rats remained unchanged after hours of feeding, but from 3 days until 10 days it gradually increased resulting to over 2-fold the normal values (Fig. 1A). The free CoA content in liver of CETTD-treated animals was marginally effected during the feeding period (Fig. 1B).

214 TABLE

I

Effect of sulphur-substituted

fatty acid analagues

5n

reiatiue liver weight and hepatic protein, lipid and CoA contents

CMITD, the alkylthioacetic acid; CETTD, the alkylthio propionic acid: BCMTD, the thiadicarboxylic acid. The tabulated values represent means 1 SD. of twelve control animals ’ and three rats in each treatment group 2 (150 mg/day per kg body wt), 3 mean values obtained from three animals

(see Materials

and Methods).

* P < 0.01, * * P < 0.05. n.d., not determined.

Parameters

Compounds

Days of treatment o 1

Relative liver

CMTTD

’ 3.55 * 0.45

I

1

1;

2

3

7

10

14

‘! 3.60 kO.04

3.82 +0.15

3.84 f 0.30

3.41 * 0.25

3.89 kO.14

4.20 f 0.26

4.16 *to.20

4.64 f 0.66

3.56 f 0.29

3.30 f 0.26

3.63 10.15

*0.15

3.75 f 0.10

3.89 i 0.20

3.86 *0.25

3.55 io.14

3.74 *IO.30

3.91 f 0.06

4.11 f 0.23

fO.10

4.41 * * f 0.24

5.13 * * 0.20

5.18 * + 0.25

4.87 * +0.32

2 152.7 & 5.6

153.4 *5.3

144.1 k9.2

150.0 f 2.2

146.8 k9.6

148.9 f 5.2

150.4 i-4.8

146.6 +4.2

150.8 * 7.6 160.7 k3.6

164.6 f 4.2 171.8 k2.3

165.6 f 9.6 169.6 * 5.2

166.4 -f 4.2 160.0 f 1.4

158.4 & 8.2 162.6 + 2.4

148.3 f 5.6 162.8 f 5.2

153.4 + 2.4 162.4 *1.4

133.4 * + 2.4 162.4 +4.7

3 25.3

25.0

25.9

24.5

25.6

23.7

26.2

25.8

weight (%) (g/g body wt)

CETTD

BCMTD

Protein

CMTTD

tmg/g liver)

’ 156.7 57.0

CETT’D BCMTD

CMTTD

a 26.3 * 1.1

2.89 * *

4.13 * *

Protein in mite chondrial fraction

CETTD

26.0

26.3

24.8

27.2

26.3

23.7

24.9

28.7

(mg/g liver)

BCMTD

28.0

30.2 * *

25.4

25.7

25.3

25.3

25.6

26.6

Protein in peroxisomalenriched fraction (mg/g liver) Protien in microsomal fraction (mg/g liver)

Triacylglycerol

36.6

7.3

6.7

8.6 **

8.1

8.6 *+

8.0

9.0 * *

CETT’D

6.7

6.7

6.4

7.6

6.3

6.8

6.4

6.8

BCMTD

7.9

CMTTD

27.4 *0.7

10.4 *

10.9 *

10.2 *

11.4 *

12.1 *

15.0 *

14.3 *

3 11.9

11.0

11.7

13.0

13.0

11.6

12.9

11.6

CETTD

12.4

12.3

11.6

12.5

12.7

11.8

11.9

12.0

BCMTD

14.4

13.6

13.8

14.4

14.9

11.8

12.7

12.8

CM’M’D

CMTTD

* 12.6 *0.9

t 4.5 i-O.5

2 3.7 * * *0.2

3.4 * +0.3

2.6 * *0.5

4.5 * ztO.6

+1.0

2.7 *

4.0 *1.2

3.9 *0.8

3.5 *0.5

5.1 *0.3 4.6 *0.6

5.2 20.5 3.8 5 0.6

3.8 + 0.6 3.7 f 0.4

8.5 * 10.5 5.1 f 0.4

5.2 f0.6 2.6 * *OS

15.1 * f 1.6 2.5 * +0.4

17.8 * f1.3 2.6 * jrO.6

22.6 * i2.1 2.4 * *0.3

’ 146.8 * * k4.3

192.4 * *6.2

270.6 * f 8.4

152.1 * f 6.7

180.1 * *11.4

195.2 * f 18.6

300.4 * f 40.6

n.d.

110.4 * * f 4.2 100.6 * * *4.2

84.6 * 4.8 162.6 * *2

74.6 * 5.2 193.6 * + 10.1

93.4 +6.1 238.6 * + 10.8

92.8 *5.2 160.1 * *5.8

120.1 ** f 15.6 250.6 * f 20.1

104.6 jz6.2 200.6 * k15.8

115.1 * 7.3 215.6 *20.6

(PmoVg liver) CETTD BCMTD

CoA (aeidsoluble + acid-insoluble)

CMT’TD

(nmoVg liver)

CE’ITD BCMTD

’ 82.3 *9.f3

215 TABLE II Subcellular distribution (BCMTD)-fed rats

of marker

enzymes of normal, alkylthioacetic

acid (CMTTD);

alkylthiopropionic

acid (CETTD)-

and thiadicarboxylic

acid

(M), microsomal (P) and peroxisomal (L) fractions are expressed as percent of whole homogenates, whereas purified peroxisomes are expressed as percent of the prepared L-fraction (see Materials and Methods). The tabulated values represent the means of three animals of the experimental groups at day 1 t and day 5 ’ and the means & S.D. for twelve control animals 3. The total activity was measured in the combined nuclear + postnuclear fractions. PMS, phenazine methosulphate.

The sums of the enzyme activities in the ~t~hond~al

Mitochondrial fraction (M) (a)

Enzymes

Succinate : PMS oxidoreductase

normal CMTTD CETTD BCMTD

Rotenone-insensitive NADPHcytochrome c oxidoreductase

normal CMTID CETTD BCMTD

8*8 8-8 9-8 7-9

Urate oxidase

normal CMTTD CETTD BCMTD

10*4 9-8 7-8 8-7

98k3 3 861-932 87-88 90-94

Fig. 1C shows that in the liver of BCMTD-fed animals, the free CoA content was not significantly changed during the first day of treatment, but increased in a time-dependent manner from 3 to 7 days of feeding. At this time, the hepatic free CoA concentration was increased more than 4-f&d. Cellular fractionation and distribution

The distribution of marker enzymes for mitochondria, pero~somes and ~crosomes was essentially similar for normal and treated animals (l- and 5 days) (Table II). High purity of the cellular fractions, especially the purified peroxisomes, was found, as judged by the distribution of marker enzymes. Recovery of enzyme activities was for all animal groups in the range [email protected]% (data not shown). Effect on fatty acid activation

Since the sulFhur-substituted fatty acid analogues increased acid-insoluble CoA and promoted alteration in the distribution of CoA between acylated and free forms during feeding, we wanted to examine whether or not this increase was due to stimulated palmitoyl-CoA and/or xenobiotic acyl-CoA synthetase activities. In homogenates of rat liver the palmitoyl-CoA synthetase activity was enriched in the mitochondrial- and microsomal fractions (Fig. 2). Administration of CMTTD increased the ~t~hon~al enzyme activity which was already revealed during the 12 h of treatment (Fig. 2A). The mitochondrial palmitoyl-CoA synthetase

Microsoma1 fraction (P) (%)

Peroxisoma1 fraction (L) (W

Purified peroxisomes (W

2*1’ 2’-3’ 2-2 2-2

0.4+33.2 3 0.3 ‘-0.2 2 0.4-0.4 0.3-0.2

65*5 67-65 68-66 63-67

5+3 7-8 6-9 7-6

3+1 2-l 2-1 l-2

2+1 2-3 3-2 2-4

74*4 76-11 71-77 76-72

88+4 87-84 85-87 86-82

3fl 3 3’-2’ 3-4 3-3

activity continued to rise for up to 3 days, when a 1.5-fold stimulation over the basal value was observed. The enzyme activity then fell and returned to normal values within 14 days of feeding. Treatment of rats with CMTTD resulted also in an acute stimulation of the microsomal palmitoyl-CoA synthetase activity, but in contrast to the mitochondrial enzyme activity, the microsomal palmitoyl-CoA synthetase activity increased gradually as a function of feeding time (Fig 2A). In CUD-treated animals, the peroxisomal p~~toylCoA synthetase activity remained unaffected after 24 h of feeding, but was found elevated for the rest of the feeding period. A similar time-course pattern of the synthetase activity was observed in the purified peroxisomes (data not shown). When the rats were fed alkylthiopropionic acid (CETTD), the mitochondrial enzyme activity was unaffected, whereas the microsomal palmitoyl-CoA synthetase tended to decrease (Fig. 2B). The peroxisomal palmitoyl-CoA synthetase activity, however, significantly increased during 24 h of feeding when a 2-fold stimulation over the basal value was observed. The activity then fell and returned to normal values within 7 days of feeding (Fig. 3B). Of the cellular palmitoyl-CoA synthetase activities, only the peroxisomal enzyme activity began to increase within hours of feeding BCMTD (Fig. 2C). After longer feeding periods, however, both the microsomal- and mitochondrial palmitoyl-CoA synthetasae activities increased (Fig. 2C).

216

;:

decrease in microsomal palmitoyl-CoA hydrolase activity was observed from 3 to 14 days. In contrast, the cytosolic palmitoyl-CoA hydrolase activity was increased within this feeding period (data not shown). Administration of CETTD, however, inhibited total palmitoyl-CoA hydrolase activity and the peroxisomaland microsomal enzyme activities, whereas both the cytosolic- and mitochondrial enzyme activities remained unaffected (Fig. 3B). As in CMTTD-treated rats, decreased peroxisomaland microsomal palmitoyl-CoA hydrolase activities and increased mitochondrial palmitoyl-CoA hydrolase activity was observed in BCMTD-treated rats (Fig. 3C), but in this experimental group, the changes were observed at earlier feeding times than in the CMTTD-treated animals (Fig. 3A). A similar phenomenon was observed concerning the cytosolic palmitoyl-CoA hydrolase activity, which in BCMTD-adapted animals, significantly

150 -

: .~

loo-

'0 E C

. I

4

50-

0 90-

8 u n z

60 -

z E 0

30-

2. 200

4

8 I

-2 m

150

400

c 2 0 1

100

z s

50

300 200 150

I

0

I

2

4

Time

6

of exposure

8

10

12

14

t '5 s h P

12a 80

(days)

Fig. 1. Changes of acid-insoluble (0) and acid-soluble (free CoASH) (0) products during the course of CMTTD (A), CETTD (B) and BCMTD (C) exposure. Data are expressed as means f S.D. of twelve control animals (from 0, 7 and 14 days) and means f S.D. of three animals in each experimental group. For more details, see Materials and Methods.

It is worth noting that in the three, treated animal groups, it was the peroxisomal palmitoyl-CoA synthetase activity which was most enhanced (Fig. 2). Recently we have shown that BCMTD, CM’ITD and CETTD can be activated to their respective CoA-thioesters [15].

YE4a E 2

0

E C

160

P .$

120

:

80

:: 2

40

r 5

40:

4, ': 300 F .E

2oa

z

150 120

Effect on long-chain acyl-CoA hydrolase activities The next step in the investigation was to determine whether long-chain acyl-CoA and/or xenobiotic acylCoA degradation was increased, since the ratio of acidinsoluble CoA to CoASH decreased in CMTTDand BCMTD-treated rats after 3 days feeding (Fig. 1). The cellular palmitoyl-CoA hydrolase activities remained unchanged within 24 h of CMTTD-feeding, except for the mitochondrial palmitoyl-CoA hydrolase activity which tended to increase (Fig. 3A). The peroxisomal palmitoyl-CoA hydrolase activity showed a maximum at 2 days of feeding, then this enzyme activity was decreased under the normal value. A similar

80 40 0

i

i

i Time

i of exposure

i

lb

ii

li

(days)

Fig. 2. Changes of CMTTD (A), CETTD (B) and BCMTD (C) exposure on palmitoyl CoA-synthetase activities. The enzyme activity in the postnuclear (O), mitochondrial (A), peroxisomal (C) (0) and microsomal (A) fraction. Data are expressed as the means f S.D. (see legend to Fig. I), the data in the cellular fractions of the experimental groups represent the means of three animals (see Materials and Methods).

217 tions of CMTTD-fed animals after 1 day of feeding. The level of this fatty acid oxidation system reached a peak after 36 h of feeding and continued to increase during the rest of the feeding period. A similar effect was obtained in the peroxisomal fraction and purified peroxisomes (data not shown) and notably, in the microsomal fraction, resulting in a 6-fold stimulation. Peroxisomal P-oxidation of CETTD-treated animals (Fig. 4B) was moderately stimulated compared with CMTTD-treated animals (Fig. 4A). Compared to controls, however, a 1.8-2-fold increase in stimulation was observed, but no increased activity was observed in the microsomal fraction (Fig. 4B). In contrast to CMTTDfeeding, the peroxisomal P-oxidation in BCMTD-treated animals began to increase in liver homogenates and cellular fractions after only 12 h and continued to increase within 10 days of feeding (Fig. 4C). It is also worth noting that the stimulating effect of BCMTD was much more pronounced in the microsomal fraction (> 1Cfold) than in the other cellular fractions, including the purified peroxisomes (> 6-8-fold) (Fig. 4C).

OL

a

s8

6 .= E

% 1

0

2

4

lima

6

of rxporure

0

10

12

14

(days)

Fig. 3. Effect of CMlTD (A), CETTD (B) and BCMTD (C) exposure on palmitoyl-CoA hydrolase activities as a function of time. The enzyme activity in the postnuclear (O), mitochondrial (A),peroxisomal (o), microsomal (A) and cytosolic (0) fractions. Data are expressed as means f S.D. and only means (see legend to Fig. 1 and Fig. 2).

increased within hours of feeding, and continued to rise up to 14 days. At that time, the cytosolic palmitoyl-CoA hydrolase was increased about &fold (Fig. 3C). CoA-thioester of the sulphur substituted fatty acid analogues are good substrates for an inducible xenobiotic acyl-CoA hydrolase (Berge, unpublished results). Peroxisomal /FLoxidation BCMTD and CMTTD are reported to be peroxisome proliferators where the newly formed peroxisomes are smaller than those observed in control rats [3]. Thus, it would seem possible that some of the peroxisomes in BCMTD- and CMTTD-treated rats will be sedimented in microsomal fraction during cellular fractionation [3]. As peroxisomal P-oxidation uses activated fatty acids as substrates, we wanted to examine peroxisomal /?-oxidation not only in liver homogenates and peroxisomal-enriched fractions, but also in the microsomal fraction. Fig. 4A shows that peroxisomal p-oxidation remains unaffected in liver homogenates and microsomal frac-

0

2

4

6

a

Time of exposure

10

12

11

(days)

Fig. 4. Effect of CMTTD (A), CETTD (B) and BCMTD (C) exposure on peroxisomal B-oxidation as a function of time. Enzyme activity in the postnuclear fraciton (0) (data are expressed as the means* SD. of three and twelve individual animals); in the peroxisomal fraction (0); in the purified peroxisomes (m) and in the microsomal fraction (A). The data in the cellular fractions of the experimental groups represent the means of three animals.

218

‘2

6A). The oxidation of [l-‘4C]palmitoyl-L-carnitine was marginally affected in liver homogenates and mitochondrial fractions from rats fed BCMTD (Fig. 6B). Fig. 7 shows that the carnitine palmitoyltransferase activity in the CMTTD-fed animals was increased in a time-dependent manner up to 7 days of feeding, resulting in a 1.7-fold stimulation over the basal values (control rats). In CETTD-treated rats, however, the carnitine palmitoyltransferase activity decreased within 2

600

T .," 500 .E E 2 400 E 5 yJ 300 s -o g

200

'u D

3 100 =: 0 2 50 0.

I A I

0

2

4

6

6

Time of CMTTD exposure

10

12

I

14

(days)

Fig. 5. Effect of CMTTD exposure on [l-‘4C]palmitoyl-L-carnitine oxidation (0) and of [l-‘4C]palmitoyl-CoA oxidation in the absence (0) and presence (m) of KCN in total liver homogenates. Data are expressed as means 5 S.D.

Effect on cyanide-sensitive and -insensitive fatty acid oxidation and carnitine palmitoyltransferase activity Administration of CMTTD rapidly enhanced the concentration of activated fatty acid (Fig. 1) and stimulation of the mitochondrial palmitoyl-CoA synthetase activity occurred simultaneously (Fig. 2). Since activated fatty acids are substrates for the mitochondrial /3-oxidation, we set out to investigate whether or not the mitochondrial fatty acid oxidation precedes the palmitoylCoA hydrolase activity and/or peroxisomal /3-oxidation. Fig. 5 shows that the total oxidation of [l-‘4C]palmitoyl-L-carnitine and especially of [l-‘4C]palmitoyl-CoA in the absence of KCN was rapidly increased to almost its maximum value after only 12 h of feeding CMTTD. At that time, the palmitoyl-CoA oxidation in the total liver homogenates was increased about 2-fold. Subsequently, the oxidation of both substrates decreased and showed a new maximum within 3 days of feeding. From 3 to 14 days, the total fatty acid oxidation decreased and returned to normal values (Fig. 5). Notably, in the presence of KCN, the oxidation of [1-i4C]palmitoylCoA remained unaffected within 2 days of feeding. In the 3-day-fed animals, however, an increased cyanideinsensitive oxidation of palmitoyl-CoA was observed (Fig. 5) resulting in a 7-fold stimulation. Similar results were observed with isolated rnitochondrial fractions (data to be published). In CETTD-fed animals, the oxidation of palmitoylCoA and palmitoyl-L-carnitine in the absence of KCN decreased as a function of time and amounted 70-75s inhibition of mitochondrial fatty acid oxidation (Fig.

I

2

0

4

6

Time of CETTD T 0) 600 ._)

6

exposure

10

12

1

14

(days.1

I 6

i

i

k

Time of BCMTD

i exposure

lb

li

1;

(days)

Fig. 6. Effect of CETTD exposure on [l-‘4C]pahnitoyl-L-camitine oxidation (0) and on [l-‘4C]pahnitoyl-CoA oxidation (0) in liver homogenates (A). Effect of BCMTD exposure on [l-‘4C]palmitoyl-Lcamitine oxidation in liver homogenates (0) and in mitochondrial fractions (0) (B). Data are expressed as means f SD. and only means (see legends to Fig. 1 and Fig. 2).

219

7

0

2

&

6

0

Time of exposure

10

12

lr,

(days]

Fig. 7. Effect of CMTTD (*), CmD (A) and BCMTD (0) exposure on carnitine palmitoyltransferase activity in mitochondrial fractions. The tabulated values represent the means of three animals of the experimental groups and the means* S.D. for twelve control animals from 0.7 and 14 days.

days of feeding but after larger feeding periods, the enzyme activity returned to normal values. BCMTD administration increased the camitine palmitoyltransferase activity within hours of feeding and almost reached maximum within 36 h. Notably, the activity then decreased and from day 10 until 14 days, the enzyme activity was reduced to 40-50% compared to normal values (control animals).

3-thiadicarboxylic acid as hypolipidemic peroxisome proliferating drug When 3-thiadicarboxylic acid (BCMTD) was fed to rats it reduced the triacylglycerol and cholesterol contents in plasma [2] and lowered the hepatic triacylglycerol concentration (Table I). The present study has further confirmed previous findings [3] that BCMTD exposure induced increased peroxisomal P-oxidation activity (Fig. 4), increased activities of palmitoyl-CoA synthetase (Fig. 2), palmitoyl-CoA hydrolase (Fig. 3) and camitine pal~toyltr~sferasc (Fig. 7). Stimulation of all these enzyme activities were already established during the first day of treatment. From the measured increase in the cellular content of CoA (Table I), and alteration of the CoA distribution between free and acyIated forms (Fig. I), it is evident that the 3-thiadicarboxylic acid has similar effects as clofibrate, tiadenol, niadenate [4,7] and other hypolipidemic drugs [21,22]. w-Dicarboxylic acids are predominantly peroxisomal substrates 1231 and they are oxidized mainly in

peroxisomes 124,251, although some authors have suggested dicarboxylic acid oxidation in ~tochond~a (261. BCMTD may behave essentially like ‘normal’ w-dicarboxylic acids, except for the block in their &oxidation due to the S-atom in the position of the carbon chain. BCMTD is reported to be a potent peroxisome pro~ferator f15,27] accompanied with stimulation of the peroxisomal P-oxidation. Thus, the w-dicarboxylic acids or their CoA-thioesters, formed by the P-450 w-oxidation pathway and thereby trapping of coenzyme A, may represent a trigger signal for peroxisome proliferation [28]. This appears to be unlikely in BCMTDand CM~D-treaty rats, as no rapid consumption of coenzyme A was observed (Fig. 1) and induction of P-450 IVAI does not precede increase in the peroxisomal enzymes (unpublished data). Furthermore, it is conceivable that the potency of the selected sulphur substituted fatty acid analogues (representing a monocarboxylic acid and a dicarboxylic acid) as proliferators of peroxisomes and inducers of the associated enzymes depends on their accessibility for P-oxidation [1,3]. Indeed, CoA-ester accumulation has been proposed to be a major factor in stimulation of peroxisome proliferation [5]. Alkylthioacetic acid A hypolipidemic fatty acid. When alkylthioacetic acid (CMTTD) was fed to rats it induced a drop in plasma t~acylgly~rol and cholesterol [2] and hepatic triacylglycerol (Table I). In addition, CMTTD induced a rapid increase in the activity of the palmitoyl-CoA synthetase (Fig. 2) and tnitochondrial /?-oxidation (Fig. 5) whereas the cytosolic- and peroxisomal palmitoyl-CoA hydrolase activities (Fig. 3) and the peroxisomal &oxidation system (Fig. 4) were induced later within the feeding period. From the measured increase in total CoA content (Table I), changes in distribution of CoA between free and acylated forms (Fig. 1) and formation of enlarged mitochondria [1,3], CMTTD feeding in this respect is more like high-fat diets. Thus, the long-chain alkylt~oacetic acid behave essentially like normal fatty acids except for the impaired &oxidation due to the S-atom in the 3-position of the carbon chain. Factors influencing peroxisomal P-oxidation. Longchain acyl-CoA correlates well with induction of peroxisomal &oxidation and pahnitoyl-CoA hydrolase activity under starvation, diabetes, high-fat diets and different hypolipidemic drugs (tiadenol, clofibrate, niadenate, nicotinic acid) [7], conditions which cause an increased fatty acid influx to the liver. Assuming that acyl-CoA and/or the CoA-thioesters of drugs play a causable role for induction of peroxisomal ~-o~dation, their formation are expected to increase in response to inducers. Treatment of rats with 150 mg/day per kg body weight CMTTD caused a stimulation in activities of pahnitoyl-CoA synthetase (Fig. 2) and xenobiotic

220 acyl-CoA synthetase [15] accompanied with increased concentration of hepatic acid-insoluble CoA (Fig. 1). This effect was already established during the 12 h of treatment. Stimulation of peroxisomal p-oxidation (Fig. 4) and palmitoyl-CoA hydrolase activity (Fig. 3) and xenobiotic acyl-CoA hydrolase activity (data to be published) resulted in an increase after 36 h. Thus, it is evident that the level of acyl-CoA esters and the enzymatic activity involved in their formation preceded the increase in peroxisomal and p~~toyl-CoA/ xenobiotic acyl-CoA hydrolase activities, enzyme systems involved in the breakdown of long-chain acyl-CoA and CoA-thioesters of the drugs. This sequence of responses is further strengthened when the ratio between the specific content/ activity (’ acyl-CoA’ : /3-oxidation and ‘acyl-CoA” : hydrolase) (see Materials and Methods) is plotted as a function of time, the ratios (data not shown) actually increase sharply the 12-24 h of feeding and thereafter decreased within longer feeding periods (based on data given in Fig. 1, Fig. 2 and Fig. 4). The by~olipid~~c drugs themselves are converted into unmetabollizable CoA esters [9,15] which may represent a trigger signal for peroxisomal /&oxidation and xenobiotic acyl-CoA hydrolase activity [29]. Whether the g-fold increase in the cellular level of long-chain acyl-CoA after 36 h of CMTTD treatment (Fig. 1) involves a rise in the content of long-chain acyl-CoA and/or 3-thia acyl-CoA is a question that requires further investigation. The present data show that BCMTD and CMTTD administration alter the distribution of CoA between acid-insoluble and free forms as a function of time (Fig. 1). From 2 days of feeding, the cellular levels of acid-insoluble CoA began to decrease, whereas the free CoA content increased. The increased activities in peroxisomal &oxidation (Fig. 4) and palmitoyl-CoA hydrolase (Fig. 3) probably limit the rise in the concentration of acyl-CoA esters in the liver (Fig. IA, C), which might otherwise become toxic (Fig. 1B). This can often result in the formation of a fatty liver, i.e., CETTD feeding. The present study has demonstrated a close relationship between the increase in specific activity of peroxisomal P-oxidation and changes of ~t~hond~al @oxidation and specific activity of carnitine palmitoyltransferase. In CMTTD-treated animals the increased cyanide-sensitive fatty acid oxidation clearly preceded the rise in peroxisomal P-oxidation (Fig. 5, Fig. 4) and the H~O~~generating acyl=CoA oxidase activity (data not shown). The ratio between specific activities of mitochondrial /3-oxidation/ peroxisomal P-oxidation was increased within the first day of feeding. In addition, a slight increase in the mitochondrial palmitoylCoA synthetase activity was observed within hours of feeding (Fig. 2) and the ratio between specific activities of palmitoyl-CoA synthetase/pal~toyl-CoA hydrolase

tended to increase up to 36 h (based on data given in Fig. 2 and Fig. 3). Altogether, it is conceivable that the mitochondrial fatty acid oxidation preceded the increase in peroxisomal &oxidation activity. Several factors may regulate peroxisomal @oxidation. Chain-length specificity for fatty acids of the acylCoA oxidase influences rates of peroxisomal P-oxidation. The supply of fatty acid is a major determinant both for peroxisomal [30] and mitochondrial ,&oxidation in the liver (311, The fact that in CLAD-fed animals the cyanide-insensitive activity remained unchanged when the mitochondrial P-oxidation (Fig. 5) and carnitine palmitoyltransferase operate at almost maximum rates (Fig. 7) is consistent with the view that fatty acyl-CoA compounds are transported preferentially into mitochondria within hours. The data are interpreted as support for the hypothesis that when transport of acyl-CoA into mitochondria is saturated, acyl-CoA compounds are transported into peroxisomes for P-oxidation. This is in accordance with the fact that increased ~tochond~al fatty acid oxidation in rat hepatocytes are observed when the cells are supplemented with low concentrations of fatty acids and that fairly high concentrations of fatty acids [ll] and high-fat diets [32] are required to increase the peroxisomal /3oxidation. Whether proliferation of peroxisomes and stimulated peroxisomal ~-o~dation may represent a protective mechanism against accumulation of longchain acyl-CoA/3-thia acyl-CoA generated by the palmitoyl-CoA synthetase activities awaits consideration. Alkylt~~~pr~~~~nic acid treatment

in i~d~~ti~n of fatty

Ii&T

Alkylthiopropionic acid represents a P-oxidizable fatty acid analogue as it may undergo one cycle of /?-oxidation. When CETTD was fed to rats it reduced the plasma triacylglycerol and cholesterol contents at low doses, but elevated the serum lipids at high doses [2,3]. Furthermore, it induced a moderate increase of peroxisomal /?-oxidation (Fig. 4) and marginally changed the activities of palmitoyl-CoA synthetase (Fig. 2) and palmitoyl-CoA hydrolase (Fig. 3). The most striking effect of CETTD-treated rats was inhibited ~tochond~al fatty acid oxidation and an increased hepatic triacylglycerol concentration (Table I) and accumulation of numerous lipid droplets [l]. In this respect, CETTD is apparently like choline-deficient diets [33,343 and ethionine treatment [35,36] and different from hypohpidemic peroxisome proliferators and high fat diets. Recently, we have found that the concentra~on in free fatty acid in plasma was increased in CETTD-fed animals {unpublished data), indicating an effect on adipose tissue lipolysis. As a result, the influx of fatty acids into the pathway of triacylglycerol biosynthesis exceeded the capacity of the liver to transport triacylglycerol. Consequently, fatty liver developed. These re-

221 sults emphasize the importance of the availability of the substrate, i.e., fatty acids as a major determinant of the rate of triacylglycerol biosynthesis. Indeed, in CETTDfed animals a progressive increase of hepatic long-chain acyl-CoA content was observed (Fig. lB), concomitant with increased hepatic triacylglycerol concentration (Table I). Acknowledgements The authors are grateful to Mr. Svein Kruger for excellent technical assistance. The work was supported by the Norwegian Research Council for Science and Humanities and the Norwegian Cancer Society. References 1 Berge, R.K., Aarsland, A., Kryvi, H., Bremer, J. and Aarsaether, N. (1989) Biochim. Biophys. Acta 1004, 345-356. 2 Aarsland, A., Aarsaether, N., Bremer, J. and Berge, R.K. (1989) J. Lipid Res. 30, 1711-1719. 3 Berge, R.K., Aarsland, A., Kryvi, H., Bremer, J. and Aarsaether, Biochem. Pharmacol. 38, 3969-3979. 4 Berge, R.K. and Bakke, O.M. (1981) B&hem. Pharmacol. 30, 2251-2254. 5 Berge, R.K. and Aarsland, A. (1985) Biochim. Biophys. Acta 837, 141-151. 6 Thomassen, MS., Christiansen, E.N. and Norum, K.R. (1982) Biochem. J. 206, 195-202. 7 Berge, R.K., Aarsland, A.,Osmundsen, H., Aarsaether, N. and Male, R. (1987) in Peroxisomes in Biolgoy and Medicine (Fahimi, H.D. and Sies, H., eds.), pp. 273-278, Springer, Berlin. 8 Berge, R.K., Nilsson, A. and Husey, A.M. (1988) B&him. Biophys. Acta 960, 417-426. 9 Lygre, T., Aarsaether, N., Stensland, E., Aarsland, A. and Berge, R.K. (1986) J. Chromatogr. 381, 95-105. 10 Hertz, R. and Bar-Tana, J. (1987) B&hem. J. 245, 387-392. 11 Hertz, R., Arnon, J. and Bar-Tana, J. (1985) B&him. Biophys. Acta 836, 192-200. 12 Gerondaes, P., George, K., Alberti, M.M. and Agius, L. (1988) B&hem. J. 253, 169-173. 13 Foxworthy, P.S. and Eacho, P.I. (1988) B&hem. J. 252, 409-414. 14 Lau, S.M., Brantley, R.K. and Thorpe, C. (1988) Biochemistry 27, 5089-5095.

15 Aarsland, A., Aarsaether, N., Bremer, J. and Berge, R.K. (1989) Biochim. Biophys. Acta 1004, 345-356. 16 Spydevold, 0. and Bremer, J. (1989) B&him. Biophys. Acta 1003, 72-79. 17 Berge, R.K.., Flatmark, T. and Osmundsen, H. (1984) Eur. J. B&hem. 141, 637-644. 18 Berge, R.K., Flatmark, T. and Christiansen, E.N. (1987) Arch. Biochem. Biophys. 252, 269-276. 19 Ingebretsen, O.C. and Farstad, M. (1980) J. Chromatgr. 202, 439-445. 20 Berge, R.K., Aarsland, A., Bakke, O.M. and Farstad, M. (1983) Int. J. Biochem. 15, 191-204. 21 Reddy, J.K. and Krishnakanta, T.P. (1975) Science 190, 787-789. 22 Eacho, PI., Foxworthy, P.S., Johnson, W.D., Hoover, D.M. and White, S.L. (1986) Toxicol. Appl. Pharmacol. 83, 430-437. 23 Cerdan, S., Ktinnecke, B., Dolle, A. and Seelig, I. (1988) J. Biol. Chem. 263,11664-11674. 24 Mortensen, P.B., Kelvraa, S., Gregersen, N. and Rasmussen, K. (1982) Biochim. Biophys. Acta 876, 515-525. 25 Leighton, F., Bergseth, S., Rortveit, T., Christiansen, E.N. and Bremer, J. (1989) J. Biol. Chem. 264, 10347-10350. 26 Draye, J.P., Weitch, K., Vamecq, J. and Van Hoof, F. (1988) Eur. J. Biochem. 178, 183-189. 27 Aarsland, A., Berge, R.K., Bremer, J., Stensland, E. and Aarsaether, N. (1984) Arch. Toxicol. Suppl. 12, 260-264. 28 Sharma, R., Lake, B.G., Foster, J. and Gibson, G.G. (1988) Biochem. Pharmacol. 37, 1193-1201. 29 Berge, R.K., Stensland, E., Aarsland, A., Tsegai, G., Osmundsen, A., Aarsaether, N. and Gjellesvik, D.R. (1987) B&him. Biophys. Acta 918, 60-66. 30 Handler, J.A. and Thurman, R.G. (1988) Eur. J. Biochem. 176, 477-484. 31 Williamson, J.R., Scholz, R., Browning, E.T., Thurman, R.G. and Fukami, M.H. (1969) J. Biol. Chem. 244, 5044-5054. 32 Berge, R.K. and Thomassen, M.S. (1984) Lipids 20, 49-52. 33 Berge, R.K., Aarsaether, N., Aarsland, A., Svardal, A. and Ueland, P.M. (1988) Carcinogenesis 9, 619-624. 34 Aarsaether, N., Berge, R.K., Aarsland, A., Svardal, A. and Ueland, P.M. (1988a) B&him. Biophys. Acta 958, 70-80. 35 Aarsaether, N., Berge, R.K., Husey, A.M., Aarsland, A., Kryvi, H., SvardaI, A., Ueland, P.M. and Farstad, M. (1988) B&him. Biophys. Acta 963, 349-358. 36 Aarsaether, N., Aarsland, A., Kryvi, H., Nilsson, A., Svardal, A., Ueland, P.M. and Berge, R.K. (1989) Carcinogenesis 10, 987-994.