Increased β-oxidation of erucic acid in perfused hearts from rats fed clofibrate

183

Biochimica et Biophysics Acta, 617 (1980) 183-191 0 El6evjer/Nort~-Holland Biomedical Press

BBA 57510

INCREASED ~-OXIDATION OF ERUCIC ACID IN PERFUSED HEARTS FROM RATS FED CLOFIBRATE

JON NORSETH Institute of Clinical Biochemistry,

Rikshospitalet,

University of Oslo, Oslo (Norway)

(Received July 3rd, 1979)

Key words: Clofibrate feeding; Erucic acid; &Oxidation;

(Perfused rat heart)

1. The metabolism of [14-14C]erucate and [U-‘4C]palmitate has been investigated in perfused heart from rats fed 0.3% clofibrate for 10 days and from control rats. 2. The total uptake of fatty acids in the heart increased in the clofibrate fed group. Clofibrate increased the oxidation of [14-‘4C]erucic acid by 100% and the oxidation of [ U-‘4C]palmitic acid by 30% compared to controls. 3. The chain-shortening of erucate to CZO:1 and C18:1 fatty acids in the perfused heart was stimulated at least two-fold by clofibrate feeding. 4. The activity of the peroxisomal marker enzyme catalase increased 60%, the activity of cytochrome oxidase increased approx. 16% and the content of total coenzyme A increased 30% in heart homogenates from rats fed clofibrate compared to controls. 5. The isolated mitochondrial fraction from clofibrate fed rats showed an increased capacity for oxidation of palmitoylcamitine and decanoylcarnitine, while the oxidation of erucoylcarnitine showed little change. 6. It is suggested that clofibrate increases the oxidation of [ 14-14C]erueic acid in the perfused heart by increasing the capacity for chain-shortening of [14“C]erucate in the peroxisomal P-oxidation system.

Introduction In feeding experiments, rapeseed oil, rich in erucic acid (22 : 1, n-9 cis), causes an accumulation of triacylglycerol in the heart of young rats [l]. We have earlier reported that addition of clofibrate (ethyl-cu-pchlorophenoxyisobutyrate) to the diet decreases this fatty infiltration significantly, probably because of an increased chin-sho~n~g of erucic acid in the liver and an

184

increased P-oxidation of fatty acids in the heart [2-41. Daae and Aas [5] have shown that clofibrate induces an increase in the activity of long chain acyl-CoA synthetase and camitine palmityltransferase, and a smaller increase in the glycerophosphate acylating enzymes in the liver. The drug is known to cause proliferation of mitochondria [6] and peroxisomes [7] in the liver. Recently, it has been shown that clofibrate also induces a peroxisomal proliferation in the heart [ 81. Lazarow [9] has found a P-oxidation system in the peroxisomes. The activity of the peroxisomal &oxidation system increases in liver of rats treated with clofibrate [9]. Christiansen [ 3,4] has found that clofibrate stimulated the oxidation, chain-shortening and to a smaller extent esterification of erucate in isolated hepatocytes. We have recently reported that erucic acid undergoes chain-shortening to shorter monoenes in perfused rat heart, probably by an adaptable peroxisomal P-oxidation [ lO,ll] . In the present work clofibrate feeding is shown to stimulate chain-shortening and oxidation of erucic acid also in the heart. Materials and Methods Chemicals [U-14C]Palmitic acid was obtained from the Radiochemical Centre, Amersham and [14-14C]erucic acid (22 : 1, n-9 cis) from Centre d’Etudes Nucleaires de Saclay, Gif-sur-Yvette, France. The [14-‘4C]erucic acid was purified as described [ lO,ll]. Essentially fatty acid free bovine serum albumin, erucic acid, palmitic acid, acetyl-CoA and acetylcamitine transferase were purchased from Sigma Chemical Co., St. Louis, MO., U.S.A. CoA was obtained from C.F. Boehringer, Mannheim, F.R.G. Other chemicals were commercial products of high purity. Animals Male Wistar rats (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 an? minerals. Clofibrate (a generous gift from Weiders Farmas@ytiske A/S, Oslo, Norway) was dissolved in acetone and mixed with the diet (0.3% (w/w)). The acetone was evaporated by air. Rats were fed this diet or a control diet without clofibrate for lo-12 days. There was no significant difference in weight gain between the two groups. Heart perfusion The rats were decapitated and their hearts perfused by a modified Langendorf preparation [ 121. The hearts were preperfused with 10 ml of buffer without fatty acid before perfusion in the recirculating system under a constant pressure of 70-80 mmHg for 30 min at 37°C. Coronary flow ranged from 6-10 ml/min, and the heart frequency was 200-240 beats/min. At the end of each perfusion the heart was quickly removed and rinsed 4 times in ice-cold 0.9% NaCl, and immediately homogenized in CH,OH. The 14C02 released during the perfusion was collected in phenylethylamine/ CH30H (1 : 2, v/v) as described before [ 111.

185

Analytical procedures Lipids were extracted from the hearts, and separated by thin-layer chromatography on silica gel as described [ll]. Fractions of free fatty acids and triacylglycerols were extracted from the gel with CHC1,/CH30H (2 : 1, v/v) and phospholipids with CHClJCH,OH/acetic acid/H,0 (65 : 25 : 2 : 2, v/v). The extracts of lipid classes were transmethylated [13], and the methyl esters of fatty acids were analyzed by radio-gas chromatography as described previously [ 111. The analyses of the fatty acid composition of total heart lipids were performed in lipid extracts of not-perfused hearts. Nonadecanoic acid was added as internal standard. Methyl esters of fatty acids were separated isothermically at 200°C in a Carl Erba gas-chromatograph model Fractovap 2101 AC equipped with a wall coated open tubular glass column SE-30 (Chrompack, The Netherlands). Phospholipids were estimated according to Zilversmith [ 141. Triacylglycerol analyses were performed with a Technicon Autoanalyzer after the phospholipids had been removed from the total lipid extracts [ 151. Catalase (EC 1.11.1.6) was assayed by the method of Baudhuin et al. [16]. Cytochrome oxidase (EC 1.9.3.1) was measured as described by Appelmans et al. [17]. Total CoA was measured in heart homogenates (10% w/v in 0.25 M sucrose and 2 mM EDTA pH 7.4) after alkaline hydrolysis (1 M KOH) at room temperature for 20 min. CoA was determined by an exchange assay with palmitoylcamitine transferase [ 181. The heart mitochondria were isolated according to Bremer and Davis [191. The measurements of the /?-oxidationdependent reduction of ferricyanide were performed as described by Osmundsen and Bremer [20]. Protein was determined by the method of Lowry et al. [21]. Student’s t-test was used for the statistical analyses of the results. Results The effect of clofibrate on fatty acid metabolism in perfused heart Table I shows that clofibrate-feeding doubled the rate of erucate oxidation while the palmitate oxidation increased approx. 25% in the perfused hearts. Clofibrate feeding decreased the relatively high amounts of “C-labelled free fatty acids in the heart after perfusion with [14-‘4C]erucic acid by approximately one third. The comparatively low level of labelled free fatty acids after perfusion with [U-‘4C]palmitate did not differ significantly between the hearts from rats fed clofibrate and the controls. The rate of esterification of erucate to triacylglycerol and phospholipids was increased by clofibrate feeding to some extent (Table I). In agreement with previous findings with perfused hearts from rats fed different fat diets [lO,ll], the fatty acid oxidation to 14C02 was markedly lower with [14-14C]erucate as substrate than with [U-‘4C]palmitate (Table I). The esterification of the two fatty acids to triacylglycerol and phospholipids was approximately equal in the heart. This is in contrast to the findings with liver were both the rate of esterification and rate of oxidation of erucate were only l/3 to l/4 of the rates with palmitate as substrate [ 3,4,22].

186 TABLE

I

THE METABOLISM OF HEART FROM CONTROL

[14-14C] ERUCIC ACID AND CLOFIBRATE-FED

AND RATS

[U-14C]PALMITIC

ACID

IN PERFUSED

The results are presented as mean ? SD. Number of animals is given in brackets. The oxidation is calculated from the sum of 14C02 released during perfusion and the 14C02 dissolved in the perfusion fluid after the perfusion. The initial erucic acid concn. in the perfusion fluid was 0.5 mM in a total volume of 50 ml. and the perfusion time 30 min. Clofibrate

Fatty acid

*

Control

nmol/g heart [14-14C]

[U-14C]

Erucic acid (n = 7)

Pahnitic

* Different

acid (n = 5)

from the control

Phosphohpid Diacylglycerol Triacylglycerol Free fatty acid Sum in tissue lipid Oxidized Total uptake

30t 6c 612f 55b 200 f 30a 1019 + 118 e 1030 f 167 a 2049 f 285 a

Phosphohpid Diacylglycerol Triacylglyceroi Free fatty acid Sum in tissue lipid Oxidized Total uptake

190+ 24c 192 3e 581 t 8le 33f 14e 823 f 122 e 2101 f 91 a 2924 f 213

with:

a P < 0.00i.b

P < 0.01, c P < 0.02,

%

nmol/g heart

%

8.6 1.5 29.8 9.8 49.1 50.3

137 f 29 23f 3 516 f 41 312+ 16 988 f 89 507 f 70 1495 + 159

9.2 1.5 34.5 20.9 66.1 33.9

6.5 0.1 19.9 1.1 28.2 71.8

1752 44 19+ 1 491? 41 39? 10 130 ? 102 1649 f 147 2379 ?: 249

7.4 0.8 20.9 1.6 30.7 69.3

d P < 0.05,

e P > 0.05 (N.s.).

After perfusion with [14-14C]erucate distinctly higher amounts of 14Clabelled CzO:1 (55% increase) and C,,: 1 (80% increase) fatty acids were present in the hearts from rats fed clofibrate than in the hearts from rats fed the control diet (Table II). “C-labelled C16:1 fatty acid was detectable in the hearts from animals fed clofibrate, but not in the control group. C,,:, fatty acid always constituted the major fraction of the chain-shortened fatty acids. The shortened fatty acids accounted for approx. 9% of the “C-labelled fatty

TABLE

II

THE EFFECT OF CLOFIBRATE ACID IN PERFUSED RAT HEART

FEEDING

ON THE

CHAIN-SHORTENING

OF

[14-14C3ERUCIC

The results are presented as nmol of 14C-labelled fatty acid per g heart (wet weight) as mean f S.D. Number of animals is given in brackets. Total heart weight was 0.98 ? 0.07 g in the clofibrate-fed group and 0.96 f 0.12 g in the control group. Diet

Lipid fraction

C16:l

Normal pellet (n = 7)

TriacylgIycerol Phospholipid

-

Clofibrate * (n = 14)

Triacylglycerol Phospholipid

2.4 t 1.3 0.7 f 0.3

* Different

from the control

with: a P < 0.001.

C18:l

7.9 + 2.5 a 3.7 f 0.4 a 14.6 + 3.7 6.0 + 1.9

C2O:l

C(16 : 1. 18: 1, 20 : 1)

C22:l

6.2 + 2.0 b 4.5 f 0.9 a

14.1 + 4.5 a 8.2 f 1.3 a

500 f 68 b 127 f 26c

9.8 f 3.6 6.8 f 1.3

26.8 + 7.3 13.5 f 3.5

583 f 45 158 f 19

b P < 0.01, c P < 0.02.

187

acids incorporated in the phospholipid fraction, and for 5% of the labelled fatty acids in the triacylglycerol fraction of the hearts from the animals fed clofibrate. It is interesting that erucic acid constituted a larger fraction in relation to chain-shortened fatty acids in triacylglycerol than in the phospholipids. Diacylglycerol with erucate is probably a relatively better substrate for diacylglycerol acyltransferases than for phospholipid synthesizing phosphotransferases as suggested by Christiansen [ 41. The free fatty acid fraction in the hearts (Table I) did not contain detectable amounts of labelled shortened fatty acids (not shown). The perfusion with [U-‘4C]palmitic acid did not lead to the accumulation of any detectable shorter or longer fatty acids, either in the clofibrate-fed group, or in the contro1.s (not shown). Fatty acid composition of total heart lipids The fatty acid composition of heart lipids of rats fed clofibrate differed from the control in that the content of C16:0, C16: 1, CZoZ4and CZZZ6was increased, while the content of C& was significantly decreased (Table III). The ratio C18:2/C20:4 decreased from 2.6 in the control group to 1.5 in the clofibrate fed group. Only small differences were observed in total triacylglycerol and phospholipid content (Table III). The effect of clofibrate on the activities of catalase, cytochrome oxidase and the con tent of total CoA in heart homogenates Table III shows that the activity of the peroxisomal marker enzyme catalase (EC 1.11.1.6) increased approx. 60% in heart homogenates from rats fed clo-

TABLE III THE EFFECT OF 0.3% HEART LIPIDS

CLOFIBRATE

IN THE DIET ON FATTY

ACID COMPOSITION

OF TOTAL

The results are presented as ~mol per g heart (wet weigbt)(mean 2 S.D. with R = 6 animals in each group). The fatty acids are indicated by number of carbon atoms: number of double bonds. Fatty acid

I.tmol/g heart Control

Clofibrate * 14 : 0 16 : 0 16 : 1 18 : 0 18: 1 18 : 2 20 : 1 20 : 4 22 : 5 22 : 6 Sum Triacylglycerol Phospholipid

0.36 10.44? 1.07 10.05 7.11 8.88 trace 7.12 2.03 4.28 51.94

+ 0.06 0.19 + 0.20 ?: 0.48 + 0.90 f 0.99

d b a c d a

-f 0.45 + 0.15 ?r 0.25 + 3.67

a a a d

7.71 + 1.27 d 31.60?

2.57 d

0.31 9.47 0.67 9.35 7.21 14.96 trace 5.70 1.48 2.77 51.92

+ + + + ? +

0.05 0.57 0.07 0.32 0.82 1.01

f * ? f

0.30 0.10 0.13 3.37

7.89 t 0.95 32.07

+ 1.52

* Diffirent from the control with: a P < 0.001. b P < 0.01. C P < 0.02,

d P > 0.05 (N.S.)

188 TABLE

IV

THE EFFECT OF CLOFIBRATE FEEDING ON THE ACTIVITY OF CATALASE. OXIDASE AND THE CONTENT OF TOTAL CoA IN RAT HEART HOMOGENATES The results are presented compared to control.

as mean + S.D. with n = 6 animals in each group.

Control Catalase (mU/mg

protein)

Cytochrome oxidase Total-CoA (nmol/mg * Different

47.0

(mU/mg protein)

from the control

protein)

with:

53.0 + 5.5 1.37 f 0.04

a P < 0.001.

% increase indicates

Clofibrate * 8.5

73.8

CYTOCHROME

*

% increase

% increase

? 10.4 a

57.0

61.5 f 4.5b 1.78 + 0.11 a

16.0 30.0

b P < 0.02.

fibrate compared to controls. This is in agreement with Latalski [8], who has found that clofibrate induces a peroxisomal proliferation in the rat heart. We have earlier suggested that chain-shortening of Czz:, fatty acids in the heart is localized extramitochondrially to the peroxisomes [ lO,ll]. Thus, the increased chain-shortening of [14-14C]erucate. in perfused heart from rats fed clofibrate (Table II) could probably be explained by an increased peroxisomal /3-oxidation. The content of total CoA in heart homogenates from rats fed clofibrate increased approx. 30% compared to the control hearts. The activity of cytochrome oxidase in hearts from rats fed clofibrate was also significantly increased, but to a smaller extent (Table IV). &Oxidation in isolated heart mitochondria The rates of @oxidation with acylcarnitines of different chain lengths in isolated heart mitochondria from clofibrate fed and control rats were also TABLE

V

THE EFFECT OF CLOFIBRATE TINES OF DIFFERENT CHAIN

FEEDING LENGTHS

ON THE RATES

OF P-OXIDATION

WITH ACYLCARNI-

The rates of P-oxidation were measured by spectrophotometric recordings of rates of acylcarnitinedependent reduction of ferricyanide in the presence of 10 mM oxaloacetate using heart mitochondria isolated from control rats and clofibrate-fed rats. The C22 acylcarnitine represents results obtained with erucoylcamitine. The values are arithmetical means of three experiments, range given in parenthesis. Fatty acid chain length

Ferricyanide (nmol/min/mg Control

reduction protein) Clofibrate

C6

14.7 (14.2-15.5)

17.9 (17.2-18.4)

Cl0

31.0 (29.1-32.6)

44.5 (39.0-49.1)

Cl6

23.3 (22.1-25.0)

34.5 (33.0-37.7)

C22

15.3 (14.4-16.5)

18.2 (17.8-18.7)

189

studied (Table V). The contaminating peroxisomes does not contribute to the reduction of ferricyanide in the assay used. The mitochondrial fraction from clofibrate fed rats showed a distinct relative increase in the capacity to oxidize C 10 and Cl6 fatty acids. The oxidation of decanoylcarnitine increased approx. 43% while the oxidation of palmitoylcarnitine increased approx. 48% per mg protein. The capacity to oxidize erucoylcarnitine and hexanoylcarnitine changed very little. Discussion The present results show that clofibrate feeding induces a 100% increase in the oxidation of [14-14C]erucate in the perfused rat heart. This can probably not be explained by an increased mitochondrial P-oxidation because the oxidation of erucoylcarnitine in isolated heart mitochondria is changed very little by clofibrate in the diet (Table V). The chain-shortening of [14-14C]erucate in perfused rate heart is increased nearly 2-fold in rats fed clofibrate compared to controls as judged from the pattern of labelled fatty acids in triacylglycerols and phosphpholipids after perfusion. An increased chain-shortening of [ 14-14C] erucic acid in the peroxisomes followed by an oxidation of the shortened products to 14C02, presumably in the mitochondria, may explain the increased oxidation of [14C]erucate in rats fed clofibrate. This is in accordance with the finding of an increased activity of the peroxisomal marker enzyme catalase (Table IV) and the proliferation of heart peroxisomes induced by clofibrate shown by others [ 81. The total amounts of chain-shortened fatty acids recovered in the triacylglycerol and phospholipid fraction at the end of perfusion were small compared to amounts of [14-14C]erucate esterified, also in the clofibrate fed group. Measurement of the shortened 14C-labelled fatty acids esterified (Table II) may, however, lead to an underestimation of the total chain-shortening capacity, especially in hearts from rats fed clofibrate, because of a further oxidation of chain-shortened products to COZ. Thus, erucoylcarnitine has been shown to be a poor substrate for P-oxidation in isolated heart mitochondria compared to shorter acylcarnitines, for example oleylcarnitine and palmitoylcamitine [23]. Fatty acids shorter than erucate are preferentially oxidized in the mitochondria. Clofibrate feeding also induces a marked increase in the ability of heart mitochondria to oxidize Cl0 and Cl6 acylcamitines compared to a relatively small increase in the oxidation of erucoylcarnitine (Table V). This effect of clofibrate has previously been demonstrated with liver mitochondria [3]. Fatty acids formed by peroxisomal chain-shortening of erucate may thus be canalized to mitochondrial P-oxidation so that a relatively small fraction of the shortened fatty acids may be esterified to triacylglycerol and phospholipid. The decrease in the content of radioactive labelled free fatty acid in the heart after perfusion seen in the clofibrate fed group may be due to an increase in the activity of acyl-CoA synthetase. In the liver clofibrate feeding has been shown to induce an increase in the activity of this enzyme. Experiments are presently in progress to clarify this point. The increased oxidation of [U-14C]palmitic acid in the perfused rat heart from rats fed clofibrate compared to controls can probably be explained by an

190

increased mitochondrial /3-oxidation (Table V). The increase in the content of total CoA and the activity of cytochrome oxidase in heart homogenates from rats fed clofibrate compared to controls (Table IV) also indicate an increased capacity for mitochondrial P-oxidation. Approx. 90% of total CoA in the heart from control rats is localized to the mitochondria [ 241. Thus, it seems that clofibrate principally has the same effect on fatty acid metabolism in heart and liver, although much more pronounced on the liver 13341. Clofibrate feeding induced a decrease in the content of C18:2 and an increase of CZoZ4 fatty acids in the heart. This observation suggest that clofibrate may induce an increased conversion (chain elongation) of linoleate to arachidonate, e.g. by activating the A6 desaturase which has been suggested to be the rate limiting enzyme in this conversion [ 251. Several reports [26,27] have shown that clofibrate given to man reduces coronar morbidity but not coronar mortality. The heart normally prefer fatty acids as substrate. However, fatty acid oxidation induces an increased oxygen uptake in the heart as compared to glucose oxidation [28,29]. Increased uptake and &oxidation of fatty acids in the heart caused by feeding clofibrate may then accelerate the ischemic necrosis when occlusion occurs. Acknowledgements Prof. B.O. Christophersen is acknowledged for his helpful advice, encouragement and great inspiration. Dr. H. Osmundsen is thanked for help in doing estimations by spectrophotometric measurement of &oxidation and making the acylcamitines available. This work was supported by a research grant from the royal Norwegian Council for Scientific and Industrial Research. The excellent technical assistance of Miss Live Horn is greatly appreciated. References 1 Vles. R.O. (1975) The role of Fats in Human Nutrition (Vergroessen. A.J.. ed.), PP. 434-468, Academic Press, London 2 Christiansen, R.Z.. Norseth, J. and Christiansen, E.N. (1979) Lipids 14, 614418 3 Christiansen. R.Z.. Osmundsen. H.. Borrebaek. B. and Bremer, J. (1978) Lipids, 13. 487-491 4 Christiansen, R.Z. (1978) Biochim. Biophys. Acta 530, 314-324 5 Daae. L.N.W. and Aas, M. (1973) Atherosclerosis 17, 389-400 T. (1970) Biochem. J. 116, 773-779 6 Kurup, C.K.R., Aithal, H.N. and Ramasarma, I Svoboda, P.J. and Azarhoff, D.L. (1966) J. Cell. Biol. 30, 442450 8 Latalski, M. and Obuchowska, D. (1977) Univ. Ma&e Curie-Sklodowska Ann. Sect. D 32,67~-70 9 Lazarow, P.B. (1978) J. Biol. Chem. 253, 1522-1528 B.O. (1979) FEBS Lett. 97, 163-165 10 Norseth, J.. Christiansen. E.N. and Christophersen, 11 Norseth, J. (1979) Biochim. Biophys. Acta 575. l+l 12 Morgan, H.E., Henderson. M.J.. Regan. D.M. and Park, CR. (1961) J. Biol. Chem. 236, 253-261 13 Metcalfe, L.D. and Schmitz, A.A. (1961) Anal. Biochem. 33, 363-364 14 Zilversmith, D.B. (1958) in Standard Methods of Clinical Chemistry Vol. II (Se&son. D. ed.,). P. 132, Academic Press. New York. NY 15 Kessler, G. and Lederer. H. (1966) in Automation in Analytical Chemistry, Technicon Symp. 1965 (Skeggs, L.T.. ed.), PP. 341-344. Mediad, New York, NY 16 Baudhuin, P., Beaufay, H.. Rahman-Li. Y., Sellinger, O.Z., Wattiaux. R., Jacques, P. and de Duve, Ch. (1964) Biochem. J. 92,179-184 17 Appelmans, F.. Wattiaux, R. and de Duve, Ch. (1955) Biochem. J. 59, 438-444 18 Skrede, S. and Bremer. J. (1970) Eur. J. Biochem. 14. 465472 19 Bremer, J. and Davis. J. (1973) Biochim. Biophys. Acta 326. 262-266

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Osmundsen, H. and Bremer. J. (1977) Biochem. J. 164, 621-633 Lowry, O.H.. Rosebrough, N.J.. Farr, A.L. and Randall, R.J. (1951)

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Rio de Janeiro,