The effect of clofibrate-feeding on hepatic fatty acid metabolism

The effect of clofibrate-feeding on hepatic fatty acid metabolism

314 Biochimica et Bdophysica 0 Elsevier~Nol~th-Holland Rcta, 530 (1978) Biomedical Press 314-324 BBA 57241 THE EFFECT OF CLOFIBRATE-FEEDING ~ETA3...

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314

Biochimica et Bdophysica 0 Elsevier~Nol~th-Holland

Rcta, 530 (1978) Biomedical Press

314-324

BBA 57241

THE EFFECT OF CLOFIBRATE-FEEDING ~ETA3OLIS~

RENATA Institute (Received

ON HEPATIC FATTY ACID

Z. CHRISTIANSEN of Medical Biochemistry, February

Sognsvamzsveien

9, University

of Oslo, Oslo 3 (Norway)

ZOth, 1978)

Summary 1. The hepatocytes isolated from clofibrate-fed rats oxidized ~almi~te to ketone bodies and CO2 more rapidly than did hepatocytes from control rats. Glucagon stimulated the oxidation of palmitate further. The extent of stimulation was approximately the same in cells from control and clofibrate-fed animals. The esterification of palmitate was decreased by clofibrate-feeding. 2. Clofibrate stimulated the oxidation, chain shortening and esterification of erucate in isolated hepatocytes. The oxidation of erucate was not stim~ated by glucagon. The increase in the esterification seemed to depend on the availability of the chain-shortened fatty acids derived from [ 14-14C]erucic acid. 3. The pattern of chain-shortened fatty acids changed towards longer fatty acids (C,,) with increasing concentration of erucic acid in the medium, suggesting a competition between erucate and shorter fatty acids for the limited capacity of the cllain-sho~ening system. 4. The chain-shortened fatty acids derived from [14-‘4C]erucate were found mainly in phospholipids and triacylglycerol. Relatively more unchanged erucate was found in the cellular lipids at higher concentrations of erucate in the medium. 5. (~)-Dec~oylc~itine had a much smaller inhibitory effect on the oxidation of palmitate and erucate in cells from clofibrate-fed rats. The inhibitor had a small stimulatory effect on the chain-shortening of erucate. 6. It is concluded that both oxidation and esterification of very long chain fatty acids are limited by the capacity of the chain-shortening system which is localized extramitochondrially, most probably in peroxisomes. The peroxisomal oxidation system may also contribute to the oxidation of palmitate especially when carnitine p~mitoyltr~sferase is rate-limiting. ._ ___-. ~

315

Introduction Clofibrate (ethyl a-(p-chlorophenoxy) isobutyrate) acts as an inducer of several enzymatic activities. Clofibrate administration to animals increases the activity of carnitine palmitoyltransferase [1,2] and especially that of carnitine acetyltransferase [ 21. Clofibrate induces also the proliferation of peroxisomes [ 31 and mitochondria [ 41 in the liver. Therefore it was of interest to investigate whether an increased rate of fatty acid oxidation may be a primary effect of clofibrate. It has been recently reported, that peroxisomes contain a P-oxidation system different from that of mitochondria [ 51. The activity of the peroxisomal system increases approximately one order of magnitude in livers of rats treated with clofibrate. Thus,, the liver contains two systems for P-oxidation of fatty acids which may have different properties, e.g. different chain-length specificity. Erucic acid (CZz: 1) given in small doses in vivo is oxidized as fast as oleic acid [6]. However, all the studies performed on subcellular fractions of liver and heart showed, that this long-chain fatty acid is a very poor substrate for a number of enzymes. Its activation, esterification and oxidation proceeds with a much lower rate than that of palmitate [7-lo]. The isolated mitochondria oxidize erucoylcarnitine with a rate which is only about 40% of that of palmitoylcamitine. Erucic acid undergoes chain-shortening both when administered in vivo [ 111 and when added to the heart cells in culture [ 121. These reports prompted us to investigate the effects of clofibrate-feeding on the overall metabolism of palmitate and erucate in isolated liver cells. Special attention was payed to a possibility that an extramitochondrial system of poxidation may take part in the metabolism of very long chain fatty acids. Materials and Methods Chemicals. [1-‘4C]- and [16-14C]palmitic acid was obtained from N.E.M. Chemicals GmbH., G.F.R. [14-14C]Erucic acid was from Centre d’Etudes Nucleaires, Gif-sur-Yvette, France. [ 14-14C] Erucic acid was routinely purified by thin-layer chromatography using hexane/diethyl ether/acetic acid (80 : 20 : 1, v/v/v). The band corresponding to free fatty acid was scraped off and extracted with CHCIJCHJOH (2 : 1, v/v). Clofibrate was obtained from ICI Industrial Company, Macclesfield, U.K.; glucagon was fom Ely Lilly Pharmaceutical Comp., Indianapolis, U.S.A. Essentially fatty acid-free bovine serum albumin, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) and ,&hydroxybutyrate dehydrogenase were purchased from Sigma Chemical Co., St. Louis, MO., U.S.A. Animals. Male Wistar rats weighing 160-180 g were fed a laboratory diet containing 55% digestible carbohydrates, 25% protein and 2.1% fat (by weight) together with all necessary vitamins and minerals. The clofibrate (0.3 g/100 g chow) was dissolved in acetone and mixed with the diet. The solvent was blown off by air during shaking. The rats were fed the normal diet or the clofibrate-containing diet for 8 days. Fed rats were used throughout the experiments. At least four animals in each group were used for the liver cell preparations.

316

Incubation conditions. Isolated hepatocytes were prepared and purified according to Seglen [13] except that Ca*‘-free Krebs-Henseleit bicarbonate buffer was used as the suspension and incubation medium. The hepatocytes were routinely preincubated at 37°C with 1 mM carnitine to secure optimal concentration of intracellular carnitine [ 141. The incubation conditions were as described previously [15] except that 1 ml cell suspension (5-7 mg protein) was mixed with 1 ml incubation medium containing fatty acids (800 cpm/ nmol) and other additions. The incubation time was 30 mm. Analytical procedures. The measurements of 14C02 release was performed in 25-ml Erlenmeyer flasks stoppered with a rubber cup and provided with a suspended plastic cup. The reaction was stopped by the injection of HC104 through the cup. Phenylethylamine/CH,OH (1 : 1, v/v) was injected into the suspended cup 15 s before the addition of HC104 and the flasks were incubated with shaking for another 45 min to collect 14C02. The measurements of radioactive acid-soluble products, the extraction of lipids and the separation of lipid classes were performed as described previously [16]. Total lipid extracts of phospholipids, free fatty acids and triacylglycerol were transmethylated according to MetcaIfe and Schmitz [17]. The methyl esters of fatty acids were analyzed by radio-gas chromatography as described previously [ 181. Results The effect of clofibrate on fatty acids oxidation and ketogenesis The oxidation rate of palmitate in isolated hepatocytes was significantly stimulated by clofibrate-feeding througout the whole concentration range of this fatty acid (Fig. 1A). The stimulation was most pronounced at high concentrations of palmitate (a-fold at 1.5 mM palmitate). Feeding clofibrate did not convert fed cells to the status of fasted cells since the rate of oxidation was

(b)

(a)

72 c 50 .o +G 0 -I c" s m z iT

/@+g?50k~;

1.0

1.5

Concentration

0.5

1.0

1.5

of fatty acids imM)

Fig. 1. Total oxidation of [l- “Clpalmitate and [14-‘“Clerucate in hepatocytes isolated from control and clofibrate-fed rats. The incubation conditions were as described in Materials and Methods. The results arc presented as the sum of radioactive acid-soluble products and 14C02 in nmol fatty acid per mg pmkin. (a), palmitate; (b), erucate. cl. cells from control rats; l, cells from clofibarte-fed rats. The results obtained with 0.5 and 1.5 mM fatty acids are presented as mean f S.D. from hepatocytes isolated from 4 animals in each group.

317

stimulated further by glucagon (see later) and did not show saturation up to 1.5 mM palmitate in the medium (see also ref. 15). The oxidation of erucate was stimulated much more than that of palmitate (Fig. lB), but the maximal rate of oxidation was reached already at 0.75 mM erucate in the medium. The increase in the total oxidation of radioactive fatty acids, in hepatocytes isolated from clofibrate-fed rats, was also accompanied by the increase in total ketogenesis (Table I). The increase of palmitate concentration from 0.5 to 1.5 mM resulted in an approx. 2-fold rise in total ketogenesis. The same increase in erucate concentration resulted only in a small or non-significant stimulation of total ketogenesis (P < 0.005 for the cells from control rats, P < 0.25 for the cells from clofibrate-fed rats). The radioactive ketone bodies produced from [l14C]palmitate (acid-soluble products, X4) constituted approximately the same percentage of total ketone bodies in both types of cells; 64.1 * 7.4% and 66.7 * 4.9% (P < 0.25), respectively, in the cells of clofibrate-fed rats and the cells of control rats with 0.5 mM palmitate and 90.8 f 2.9% and 97.0 * 3.3% (P < O.Ol), respectively with 1.5 mM palmitate in the medium (mean f SD. from six determinations). The percentage of radioactive ketone bodies synthesized from [14-‘4C]erucate (acid-soluble products, X5.5) in relation to total ketogenesis increased from 40.3 f 5.8% in the cells of control rats to 72.5 * 7.1% (P < 0.001) in the cells of clofibrate rats with 0.5 mM erucate in the medium, and from 59.1 ? 6.4 to 83.2 + 4.8% (P < 0.001) with 1.5 mM erucate in the medium (mean + S.D. from six determinations). This shows a specific stimulation of erucic acid oxidation in the hepatocytes isolated from clofibrate-fed rats. The factor of 5.5 used in the above calculations of the radioactive ketone bodies synthesized from erucate (22 carbons) is probably the minimum factor since the formation of the non-radioactive ketone bodies in the process of chain-shortening of erucate (see later) was not taken into consideration. The separation of acid-soluble products on a Dowex-1 formate column according to La Noue et al. [ 191 revealed that 90--95% of the radioactivity was associated with acetoacetate and /3-hydroxybutyrate. TABLE THE

I TOTAL

KETOGENESIS

BRATE-FED The

results

from

IN

are presented

4 animals

in each

as nmol group).

None

ketone

Glucagon

bodies

per

was added

ISOLATED

FROM

CONTROL

AND

CLOFI-

mg

protein

to the

(mean

concentration

? S.D.

from

of

. 1O-8

2.5

hepatocytes

isolated

M.

Clofibrate

+

-

+ Glucagon

Glucagon 46.0

mM

HEPATOCYTES

Control

Additions

0.5

THE

RATS

6.5

90.0

7.0

a

paImitate

128.3

+ 13.9

250.0

+

+ 14.1

a

Glucagon 70.0

?

+ Glucagon 6.0

115.0

*

208.4

r 19.0

408.0

t 18.3

a

7.5

a

1.5

mM

paImitate

228.8

? 22.7

283.0

i 21.0

c

522.0

+ 31.7

613.0

+ 29.0

b

0.5

mM

erucate

107.8

t

9.1

121.0

k

7.5

d

313.3

I21.0

357.5

* 23.0

d

1.5

mM

erucate

134.9

+

6.0

148.0

f

8.0

d

333.8

? 20.0

378.0

? 18.0

c _

Different

from

aP


bP


c P

< 0.02;

d P

< 0.05.

the samples

without

glucagon

with:

318

Both endogenous ketogenesis and ketogenesis from added palmitate were stimulated by glucagon in cells of control and clofibrate-fed rats (Table I). The extent of stimulation was similar in both types of cells when palmitate was present in the incubation medium. The total ketogenesis in the presence of erucate was only slightly stimulated by glucagon. This is probably due to the stimulation of ketogenesis from endogenous fatty acids since the oxidation of [14-‘4C]erucate to acid-soluble products and CO;? was not significantly stimulated (not shown). This indicates that the rate-limiting step in the oxidation of erucate is not stimulated by glucagon. Since palmitate and erucate used in previous experiments (Fig. 1) were labelled in different positions, the oxidation of [ 1-‘4C]- and [ 16-i4C]paImitate and [14-‘4C]erucate to acid-soluble products and 14C0, were compared (Table II). A big difference in the labelling of CO;, from [1;14C]- and [16-‘4C]palmitate was observed. The decrease in CO2 radioactivity formed from [16-‘4C]palmitate was matched by the increase in acid-soluble radioactivity. The oxidation rate expressed as the sum of nmol fatty acid converted to acid-soluble products and CO* was the same for [l-14C]- and [16-‘4C]palmitate. The higher labelling of ketone bodies synthesized from [ 16-‘4C]palmitate observed here is in accordance with previous reports, that the w-terminal &-unit of a fatty acid is preferentially incorporated into ketone bodies [ 203. This does not explain low labelling of CO2 formed from [14-‘4C]erucic acid since in this case the label is located in the middle of the molecule. One possible explanation could be the suggested inhibition of the oxidation of the Krebs cycle intermediates by erucoylcarnitine [ 211. The effect of clofibrate on the total metabolism of fatty acids The results presented in Table III show that the relatively small stimulation of 0.5 mM palmitate oxidation was matched primarily by a decrease in triacylglycerol synthesis. With 1.5 mM palmitate in the medium, the increase in the oxidation resulted in an increase of palmitate uptake since the relatively small decrease in triacylglycerol synthesis did not compensate for the increased oxi-

TABLE THE

II OXIDATION

BLE

PRODUCTS

The

results

4 animals Fatty (0.5

are

OF

14C02

expressed

in each

acids

DIFFERENTLY

AND

LABELLED

IN ISOLATED

as nmol

fatty

acid

PALMITATE

AND

ERUCATE

per mg

protein

(mean

? S.D.

from

hepatowtes

isolated

from

Acid-soluble

products

14co2

Totally

oxidired

mM) Clofibrate

Control

Clofibrate

[l-14ClPalmitate

16.5

r 1.1

26.6

i 2.8

a

4.9

? 0.8

6.8

2 I.od

[lF-14ClPalmitate

21.4

!

1.8

33.1

i 2.4

b

0.6

i 0.2

1.2

+0.2

c

I 0.9

39.9 _____.

f 3.5

a

0.6

i 0.1

1.4

i 0.3

b

[14-‘4ClErucate Different

from

\ 0.001;

b f’ ._o.005; c I’ \ 0.01; drJ

ACID-SOLU-

group).

Control

a I’

TO

HEPATOCYTES

< 0.05.

7.3 the control

with:

Control

Clofibrate

21.4

t 2.0

33.4

? 3.4

22.0

I 1.8

34.3

? 2.9

a

k 0.9

41.3

i- 3.9

a

7.9

a

from

f P

e

d

c

b

0.025;

0.02;

0.01;

0.005;

0.001:

erucate

< 0.05.

< < < < <

Different

P P P P P

mM

1.5

a

erucate

mM

0.5

palmitate

mM

1.5

palmitate

mM

acid

0.5

Fatty

[l-‘4ClpaJmitate

OF

[14-14C]ERUCIC

of

minus

fatty

(+)

-

with:

DC

(+> DC

-

DC

(0.9)

(0.5)

(1.5)

(0.5)

2.1

14.4

3.9

(1.6)

(+)

7.9

(4.1)

55.6

13.9

-

(4.1) (1.8)

50.5

(1.5)

32.8

(3.9)

24.5

(5.1)

94.8 41.3

(6.0)

118.5

(2.1)

(3.4)

(1.1)

27.1

33.4

(2.0)

7.5

DC

21.4

(+)

-

Clofibrate

products

protein

(mean

ACID

=

a

=

a

=

a

a

a

acid

t

IN

given ((+)-DC)

at the end

S.D..

(1.0) (0.8) (1.2) (1.1)

4.0 7.4 7.4

(2.3)

(2.8)

4.2

23.0

23.9

(1.0) (1.1)

13.0

(0.5)

12.3

(0.8)

(2.3)

20.0

7.5

(2.6)

22.0

7.7

(0.5)

13.0 f

a

a

a

b

f

15.5

12.6

9.3

7.0

82.0

ti9.8

21.0

(1.2)

(1.3)

(0.9)

(1.2)

(6.2)

(6.6)

(2.7)

(2.6)

21.5

(0.8)

f

14.0

(0.9)

12.7 12.7

(0.7)

Control

TriacyJgJycerol

incubation

Clofibrate e

the 30-min

159.0

a

a

27.5

35.0

(2.8) (2.6)

32.3 37.5

19.1 15.2

d

(0.9) b

124.0 (2.0)

a

a

56.1 47.2

13.0

(4.0)

a

a

(2.1)

(2.1)

(2.5)

(1.8)

(2.0)

(5.0)

(5.6)

The

RATS.

85.5

98.0

45.0

60.5

175.0

192.5

54.4

60.5

(4.3)

(4.6)

(2.4)

(4.6)

(6.0)

(7.7)

(2.5)

(0.9)

Clofibrate

a

a

a

a

=

a

b

c

total

oxidation equals

uptake

uptake

acid

(2.0)

Control

Fatty

11.0

56.0

(4.8)

(2.0)

40.3

(1.9)

10.6 13.6

Clofibrate

period.

acid

4 animals).

CLOFIBRATE-FED

Fatty

from

AND

of 2 mM.

isolated

CONTROL

hepetocytes

FROM

to the concentration

Control

of

added

from

ISOLATED

brackets,

was

in

HEPATOCYTES

Phosphoiipids -

recovered

(+)-Decanoyl-carnitine

____

mg

14C-free

’ 4C02.

Control

Oxidation

added

and

per

[1-‘4C]PALMITIC

acids

AND

fatty

14C]erucate

of

products

as nmoles

acid-soluble

Additions

or [14-

sum

presented

(+)-DECANOYLCARNITINE

the control

are the

are

results

The

products

OF

METABOLISM

EFFECT

THE

III

THE

TABLE

320

dation. We have previously shown [ 181 that clofibrate induces a a-fold increase in the activity of camitine palmitoyltransferase while that of glycerophosphate acyltr~sfer~e increases insi~ificantly. This may explain the observed decrease in trialcylglycerol formation and increase in oxidation of palmitate in cells from clofibrate-fed animals, since it is probably the relationship between the activities of these two key enzymes which is responsible for the partition of fatty acids between oxidation and esterification. Another contributing cause of the observed fall in triacylglycerol formation might be the pronounced decrease in glycerol 3-phosphate concentration in the liver caused by clofibrate administration [22]. Table III shows that in the case of erucate both the oxidation and esterification were stimulated and the erucate uptake increased significantly. of f+)-decanoylcarnitine Lazarow and de Duve [5] suggested that one of the main reasons for the hypolipidemic action of clofibrate might be the increase of the peroxisomal fatty acid oxidation. Therefore we have investigated the effect of (+)-decanoylcamitine, the inhibitor of carnitine acyltransferase, i.e. the mitochondrial fatty acids oxidation, in hepatocytes isolated from control and clofibrate-fed rats. Table III shows that (+)-dec~oylc~itine inhibited very strongly the oxidation of both palmitate and erucate (65-75% inhibition) in the cells from control rats. However, the extent of inhibition was suprisingly much smaller in cells from clofibrate-fed rats; it decreased from 65-75% inhibition in the cells from control animals to 19-20% inhibition in the cells from clofibrate-fed rats when palmitate was used as a substrate. The inhibition of erucate oxidation was also diminished by clofibrate, but the difference was smaller in this case. The extent of inhibition decreased from 73% in the cells from control rats to 35-41% in the cells from clofibrate-fed rats. The effect

Chain-shortening of erucic acid The results presented in Table III show that feeding clofibrate to rats increased the esterification of erucic acid in contrast to that of palmitate which decreased. Presumably erucic acid becomes more available as a substrate for glycerol a-phosphate acylating enzymes due to its chain-shortening. Fig. 2 shows that the chain-shortening proceeds both in cells from control and clofibrate-fed rats. The main products were: CzO:,, Cz0:2, CXEzland Ci6:t (Fig. 2). Oleic acid always constituted the major fraction of the chain-shortened fatty acids. The pattern of fatty acids derived from [14-‘4C]erucic acid changed depending on the concentration of erucic acid in the medium. At low concentrations of erucate significant amounts of C16:, were found, but this fatty acid disappeared almost completely at higher concentrations of erucate. On the other hand both C,,:, and C& increased continously, while the accumulation of C,,:, reached the plateau above 0.75 mM erucate m the medium. The pattern of chain-shortened fatty acids changed in a similar way in cells from control and clofibrate-fed rats and presumably illustrates the competition between the excess of erucic acid and shorter fatty acids for the limited capacity of the chain-shortening system. Feeding clofibrate to the rats increased significantly (2.1-2.6 fold} the total capacity of the chain-shortening system

321

15 (a)

Erucate

r

(b)

concentration

(mM)

Fig. 2. The pattern of chain-shortened fatty acids derived from [14-14C]erucate centration of erucate in the medium. (a), cells from control rats; (b), cells o-, 18 : 1: l -m, 20: 1;00.20: 2;aA, 16 : 1.

(Fig. 2). shortening bra&fed strongly of erucic Synthesis

(+)-Decanoylcamitine caused a of erucic acid in hepatocytes rats (Table IV). Glucagon had support the hypothesis that the acid proceeds extramitochondrially. of cellular lipid from

depending on the confrom clofibrate-fed rats.

slight apparent increase of the chainisolated from both control and clofino effect (not shown). These results clofibrate-stimulated chain-shortening

[14-‘4C]erucic

acid

The maximal rate of erucate oxidation was observed at 0.75 mM erucate in the medium (Fig. 1B). The rate of chain-shortening also reached saturation at 0.75 mM erucate when the results presented in Fig. 2 were calculated as nmol TABLE

IV

THE EFFECT OF (+)-DECANOYLCARNITINE LATED HEPATOCYTES

ON THE CHAIN-SHORTENING

OF ERUCATE

IN ISO-

The results are presented as the sum of chain-shortened fatty acids derived from [14-‘4Clerucate. in nmol per mg protein (mean * S.D. from hepatocytes isolated from 4 rats in each group). (+)-Decanoylcarnitine ((+)-DC) was added to the concentration of 2 mM. Erucate

Additions

Control

Clofibrate

(mM) 0.5 (+)-DC 1.5 (+)-DC Different +P < ** P < ***p <

7.2 ? 1.1 * 9.5 f 0.8 13.4 16.6

from the samples with inhibitor 0.02; 0.025; 0.3.

r 1.6 * ? 1.2 with:

17.5 19.9

+ 1.2 ** * 1.0

28.0 30.5

k 3.2 *** ? 2.9

322

TABLE THE

V DISTRIBUTION

FATTY The

ACIDS

presented

OF results

OF

RADIOACTIVITY

DIFFERENT (mean

DERIVING

CHOPIN-LENGTH

from

3 experiments)

FROM

were

obtained

ACID

[14-14ClERUCIC

IN PHOSPHOLIFIDS with

AND

hepatocytes

BETWEEN

TRIACYLGLYCEROL isolated

from

clofibrate-

fed rats. Fraction

of lipids

Phosph~lipids

Triacylglycerol

Percentage ~.~_

of total

of erucate

(mM>

16

: 1

18

Concentration

--

____~

: 1

-

20

: 1

___.-

:2 -_

20

22

0.5

9.0

59.7

11.8

trace

19.5

1.5

3.0

45.9

13.5

4.0

33.6

0.5

13.2

32.6

21.2

trace

33.0

1.5

2.8

28.1

4.4

4.4

60.3

:1

of &-units theoretic~ly split off erucic acid [(C16:1 , X3) + (C18:1, X2) + CZD:1 + C&1. However, incorporation into complex lipids continued to increase with higher concentrations of erucate (Table III). Table V shows that concomitantly relatively more unchanged erucate was incorporated in the complex lipids. The ratio between shorter fatty acids and unchanged erucate decreased from 4.3 to 2.0 in phospholipids and from 2.0 to 0.66 in triacylglycerol when the concentration of erucate in the medium was increased from 0.5 to 1.5 mAL Simultaneously, the synthesis of phospholipids increased 1%fold and that of triacylglycerol 3-fold. It is interesting that erucic acid constituted a much smaller fraction in relation to chain-shortened fatty acids, in phospholipids than in triacylglycerol at both concentrations of erucate. Thus, the erucate-containing dia~ylglycerol is probably a relatively better substrate for diacylglycerol acyltransferases than for phospholipid-synthesizing phosphotransferases. The chainshortened fatty acids derived from [14-14C]erucate were recovered mainly in phospholipids and triacylglycerol. Only an insignificant amount was found in the free fatty acid fraction. Discussion The increase in the activity of carnitine palmitoyltransferase [18] may probably account for the observed stimulation of palmitate oxidation in hepatocytes isolated from clofibrate-fed rats (Fig. 1 and Table III). The much stronger effect of clofibrate on the oxidation of erucate requires an additional explanation which might be the increase in the capacity of the chain-shortening system. The chain-shortening capacity of the isolated hepatocytes is, most likely, much higher than it is apparent from Fig. 2 since fatty acids shorter than erucic acid will be preferentially oxidized [lo]. (+)-Decanoylcarnitine apparently stimulated the chain-shortening of erucate (Table IV) while glucagon did not have any effect. Further, the inhibitor effect of (~)-dec~oylc~itine on the oxidation of both erucate and palmitate was much smaller in cells from clofibrate-fed rats than in cells from control rats (Table III). These results suggest that, in the presence of the inhibitor of mitochondrial P-oxidation, fatty acids are oxidized by an extramitochondrial system, the capacity of which is enhanced by clofibrate treatment.

323

It seems likely that the increased esterification of erucate in hepatocytes from clofibrate-fed rats is, at least in part, explained by the increased conversion of erucate to shorter fatty acids. However, a 3-fold increase in triacylglycerol formation between 0.5 and 1.5 mM erucate is associated with only a 1.7-fold increase in chain-shortened fatty acids (Tables III and V). This discrepancy cannot be explained with certainty. The oxidation rate of erucate reaciles the maximum at 0.75 mM erucate. Then more erucoyl-CoA will be available for esterification at higher concentrations of erucate (1.5 mM), since the uptake of erucate is increased by clofibrate-feeding. It is striking though, that, even with 1.5 mM erucate in the medium, only 60% of the radioactive fatty acids incorporated into triacylglycerol is erucic acid (Table V). This is also the case in cells from control rats (not shown). This suggests that trierucoylglycerol can not be formed and that the availability of shorter fatty acids may limit the esterification of erucic acid. In the hepatocytes isolated from clofibrate-fed rats the ratio between radioactive and total ketone bodies was approximately the same as in hepatocytes from control rats both in the presence and in the absence of (+)-decanoylcarnitine when palmitate was used as the substrate. This means that the oxidation products of palmitate were chanelled to the ketone body formation pathway no matter which way the P-oxidation proceeded. Besides, we have not found any intermediate fatty acids shorter than C,6 when [16-14C]palmitate was incubated with isolated liver cells. This may be due to their very rapid oxidation in mitochondria. It is likely that carnitine acetyltransferase, the activity of which increases lo-fold in mitochondria and 2-fold in peroxisomes in the liver of clofibrate-fed rats [2], facilitates the transfer of acetyl-CoA formed extramitochondrially to the mitochondria. The capacity for the oxidation of acetylcarnitine and other short-chain acylcarnitines is increased several fold in the mitochondria isolated from clofibrate-fed rats [ 181. In the previous studies we have shown that the oxidation rate of palmitoyland erucoylcarnitine in the mitochondrial fraction isolated from livers of clofibrate-fed rats was only slightly stimulated (10% on a mitochondrial protein basis) [18]. Thus, the reported 50-100% increase in the content of mitochondria in livers from clofibrate-fed rats [4] cannot account for the 3.5---5fold increase in erucate oxidation reported here. The presented results strongly support the idea that the extramitochondrial chain-shortening of erucic acid is rate-limiting for the further metabolism of this fatty acid. First, clofibrate increases both the oxidation and esterification of erucate and at the same time the capacity for chain-shortening of erucate is enhanced. Second, both the rate of oxidation and the rate of chain-shortening reach saturation at 0.75 mM erucate in the medium. Third, glucagon does not have any significant effect on erucate oxidation while palmitate oxidation is stimulated. These results suggest that the extramitochondrial system of fatty acid oxidation primarily handles very long-chain fatty acids. In addition, it probably plays an important role in the oxidation of more usual dietary fatty acids (palmitate) when the mito chondrial long-chain carnitine acyltransferase and/or long-chain acyl-CoA dehydrogenase becomes rate-limiting. The extramitochondrial system, mentioned above, is, most probably, the /3oxidation system of peroxisomes. It has been reported that clofibrate treat-

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ment causes the proliferation of peroxisomes [3] and the capacity of liver to oxidize palmitoyl-CoA in the presence of cyanide increases several fold [ 51. So far, nothing is known about the peroxisomal oxidation of C22 fatty acids. In this context it is interesting that the capacity of the isolated liver cells to chain-shorten and oxidize erucic acid is increased in rats fed a diet containing high cal.% of rape-seed oil or marine oil for 3 weeks (Christiansen, R.Z., and Christiansen, E.N., unpublished). It is, however, still not established if tile presence of very long chain fatty acids in the diet can induce a proliferation of peroxisomes or an increase in the activity of the peroxisomal P-oxidation system. Acknowledgements Professor Jon Bremer is gratefully acknowledged for helpful suggestions and discussions, Dr. Harald Osmundsen for help in doing estimations by radio-gas chromatography. The technical assistance of Mr. Tore Gjelvold and Miss June Taje is greatly appreciated. This work was supported by a grant from the Norwegian Research Council for Science and the Humanities. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

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