Effect of clofibrate on rat liver acylcarnitines

BIOCHEMICAL

MEDICINE

28, 204-209

( 1982)

Effect of Clofibrate on Rat Liver Acylcarnitines J. KERNER Department *Department

of Biochemistry, of Biochemistry,

AND

University Michigan

Received

L. L.

BIEBER*

Medical State 48823

School, University,

December

2, 1981

H-7624 Pees. Hungary. and East Lansing, Michigan

Previous studies showed that chronic clofibrate treatment of rats increases the activities of all known carnitine acyltransferases in liver. Short-chain carnitine acyltransferase activity was effected more than medium- and long-chain carnitine acyltransferases (I-4). Clofibrate treatment also increased hepatic camitine (5, 6). The role of camitine in shuttling long-chain fatty acids across the mitochondrial inner membrane is well documented (7-9). Although not firmly established, several other functions have been proposed for carnitine. These include shuttling acetyl groups between different cell compartments (8) and buffering the acetyl-CoA:CoASH ratio by forming acetylcamitine which can serve as a high-energy acetyl reservoir (IO, 11). Recently, several acid-soluble short-chain camitine esters, corresponding to the acyl-CoA intermediates of the catabolic pathways for branchedchain amino acid metabolism, as well as the presence of the carnitine acyltransferases necessary for their formation have been demonstrated in several tissues (12-15). These findings and the stimulatory effect of camitine on the oxidative breakdown of the keto acids derived from branched-chain amino acids (16-18) indicates carnitine can effect the metabolism of these amino acids. The regulated step for branched-chain amino acid catabolism is catalyzed by the mitochondrial branched-chain 2-oxoacid dehydrogenase. It is regulated by NADH and by branchedchain acyl-CoA esters (18-22) and possibly by phosphorylation and dephosphorylation (23, 24). Camitine can accept the acyl groups of branched-chain acyl-CoA esters, thus lowering the acyl-CoA:free CoASH ratio, which could effect flux through the pathway (18, 23). The above findings suggest that clofibrate might affect metabolism,

0006-2944/82/050204-06%02.00/O Copyright All rights

0 1982 by Academic Ress, Inc. of reproduction in any form reserved.

CLOFIBRATE

AND ACYLCARNITINES

205

especially metabolism of branched-chain amino acids by mechanisms which involve camitine and the short-chain camitine acyltransferases. In this study, we determined the amounts of total hepatic camitine, free and esterified camitine, and also the individual acid-soluble short-chain acylcamitines in control and clofibrate-treated rats. MATERIALS

AND METHODS

Materials

Camitine acetyltransferase, N-ethylmaleimide, and Dowex-50-8X (100-200 mesh) were purchased from Sigma; Dowex-l-X8 (100-200 mesh) and Bio-Gel P-2 (100-200 mesh) were purchased from Bio-Rad Laboratories. [l-‘4C]Acetyl-CoA, specific radioactivity 58 &i/~mol, was supplied by New England Nuclear. Acetyl-CoA was purchased from P-L Biochemicals. Column packing, 15% SP-1220 1% HsP04 on 100/200 Chromosorb WAW was purchased from Supelco. L-Camitine chloride was a generous gift from Sigma-Tau (Rome, Italy). n,L-Valerylcamitine was synthesized as described (25). Animals

Male 100-g Sprague-Dawley rats were divided into two groups and fed ad Zibitum on standard laboratory chow and water. One group received daily subcutaneous injections of clofibrate, 250 mg/kg body weight for 14 successive days, and the other group received a sham injection. On the 15th day the rats were sacrificed by decapitation, their livers quickly removed, frozen in dry ice/acetone, weighed, and stored at -70°C until processed. On Day 15, the body weight of the two groups was the same but the average liver weight was 15% greater for the clofibrate-treated group. Analytical

Procedures

Tissue extracts for determination of free and total camitine and longchain acylcamitines were prepared as referenced (26). Free camitine was quantitated by the radiochemical method of Cederblad and Lindstedt (27) with some modifications. The assay mixture contained the following in a final volume of 220 ~1: 50 mM potassium phosphate, pH 7.4, 2.0 mM iV-ethylmaleimide, 25 PM [l-‘4C]acetyl-CoA, specific radioactivity 3.3 nCi/p.mol, and the sample to be assayed. The reactions were initiated by adding 0.5 unit of camitine acetyltransferase. After incubation at room temperature for 30 min, 200-pl samples were applied to a 5 x 25-mm column of Dowex-l-X& Cl- (100-200 mesh), eluted with 1.0 ml of water directly into scintillation vials containing 10 ml of a scintillation cocktail, and counted. Acid-soluble total camitine

206

KERNER

AND BIEBER

and long-chain acylcarnitines were assayed as described above after alkaline hydrolysis. Esterified carnitine was obtained by substracting the amount of free carnitine from total acid-soluble carnitine. Acid-soluble short-chain acylcarnitines were separated from other acyl-containing compounds using gel filtration, anion- and cation-exchange chromatography; they were then saponified and the individual fatty acids were quantitated gas chromatographically (28). For quantitation valerylcamitine was used as the internal standard. The statistical significance of the results was calculated by Student’s t test. RESULTS AND DISCUSSION

Table 1 shows that livers from rats treated with clofibrate contained 2.4-fold more carnitine than the controls. This finding agrees with data of others (26). The greatest increase was in free carnitine. The elevation of total hepatic camitine and long-chain acylcarnitines together with the previously shown increase in carnitine palmitoyltransferase (l-4, 29) indicates a possible stimulation of mitochondrial fatty acid oxidation by clofibrate. The decrease in levels of hepatic malonyl-CoA (30), a potent inhibitor of palmitoyltransferase I (29) and the enhanced activity of acylcarnitine-carnitine translocase (6) are also consistent with increased p-oxidation. Quantitation of the individual short-chain camitine esters is given in Table 2. Acetylcarnitine and propionylcarnitine levels increased 1.8 and 3.0-fold, respectively, but the amounts of isobutyryl-, butyryl-, and isovalerylcarnitine did not change. The elevated levels of acetylcarnitine could be due to an accelerated mitochondrial P-oxidation. However, a TABLE AMOUNTS

1

OF FREE, ESTERIFIED, AND TOTAL CARNITINE IN LIVER OF CONTROL CLOFIBRATE-TREATED RATS“

Controls (nmoYg) Total camitineb Acid-soluble total camitine Free camitine Acid-soluble acylcamitines’ Long-chain acylcamitines

152 144 65 79 8.3

f 2 2 2 2

9.2 8.3 7.4 2.0 2.9

AND

Clofibrate-treated (nmolk) 359 346 177 169 12.9

2 It zt -re

14.0* 13.0* 7.4* 11.4* 4.1 (ns)

a The data are means it SE of five livers for each group. b Total camitine is the sum of total acid-soluble camitine plus long-chain acylcamitines. ’ Acid-soluble acylcamitine is obtained by subtracting the amount of free camitine from total acid-soluble carnitine. * P < 0.001 compared to the control group. ns, Not significant.

CLOFIBRATE

AND TABLE

THE EFFECT OF

CLOFIBRATE

ON THE

Controls (nmol/g) Acetylcamitine Propionylcamitine Isobutyrylcamitine Butyrylcamitine Isovalerylcamitine Total

52 23 3.0 4.3

t k 2 ‘-

2.3 2.5 0.7 0.78

1.1 f 0.25 83 2

1.83

207

ACYLCARNITINES 2

AMOUNTS OF RAT LIVER’I

SHORT-CHAIN

ACYLCARNITINES

IN

Acyl free

Clofibrate (nmol/ 8)

Acyl free

0.78 0.35 0.05 0.07 0.02 1.28

92 68 3.7 4.0 1.8

0.38 0.02 0.02

2 “r 2 r 169

7.2+ 9.7* 0.65 (ns) 0.94 (ns) 0.52 (ns) -r 14.3*

0.51

0.01 0.95

’ The results are means 2 SE for four rats in the controls and five for the clofibratetreated group. * P < 0.01 compared with values in the control group. ns, Not significant.

shift in the mitochondrial redox potential of the NAD’/NADH couple toward a more oxidized state and a significant decrease of succinyl-CoA content in liver (30) and the lack of elevated ketone bodies (31) in clofibrate-treated rats indicates accelerated citric acid cycle activity which compensates for the faster mitochondrial acetyl-CoA production. Alternatively, the elevated amounts of acetylcarnitine could indicate increased peroxisomal ‘fatty acid oxidation. Clofibrate treatment increases both peroxisomal carnitine acyltransferase activities (4) and the amount of peroxisomal CoA (32). Because the peroxisomal membrane is impermeable to CoA and its esters, carnitine via peroxisomal carnitine acetyltransferase could regenerate free CoASH needed for unimpaired poxidation. The increase in the hepatic levels of propionylcarnitine is puzzling (see Table 2). The ratio of propionylcarnitine :free carnitine was the only specific acylcarnitine : carnitine ratio that increased. If carnitine acetyltransferase approaches equilibrium with its reactants and products in vivo, the increase in propionylcarnitine indicates a corresponding increase in steady-state levels of propionyl-CoA. If so, clofibrate seems to affect reactions leading to production of and/or utilization of propionylCoA. The elevated levels of propionylcarnitine could be due to increased breakdown of fatty acids with both even and odd chain lengths and a concomittant differential effect of clofibrate on the metabolism of acetyland propionyl-CoA, respectively. Another potential source of propionyl-CoA is the branched-chain 2oxoacids derived from valine and isoleucine. The regulated step in their catabolism is catalyzed by the branched-chain 2-oxoacid dehydrogenase which is inhibited by products of the reaction (19-22). Because clofibratetreatment causes an approximate three fold increase in hepatic CoA

208

KERNER

AND BIEBER

levels (33) and also increases both cytoplasmic and mitochondrial NAD’ :NADH ratio (30), elevated flux through the pathway might be allowed. The major matabolic fate of propionyl-CoA is conversion to succinylCoA, which is either oxidized in the tricarboxylic acid cycle or converted to glucose. Clofibrate decreases the succinyl-CoA:free CoASH ratio (30). The elevation of propionylcarnitine and presumably propionyl-CoA and the decrease in succinyl-CoA levels indicates that in liver of clofibratetreated rats one of the enzymes in the pathway from propionyl-CoA to succinyl-CoA is rate limiting causing an increase in the steady-state level of propionylcarnitine. SUMMARY The effect of subcutaneous clofibrate injection on the amounts of longchain acylcarnitines and on individual short-chain acylcarnitines in rat liver was investigated. Total liver carnitine increased 2.4-fold. The largest increase was in free carnitine. The levels of isobutyryl-, butyryl- and isovalerylcarnitine did not change, whereas acetyl- and propionylcarnitine increased 1.8- and 3.0-fold, respectively. The ratios of the individual acylcarnitines to free carnitine all decreased, except propionylcarnitine which increased slightly. If the short-chain camitine acyltransferase(s) work near equilibirum in vivo, then the clofibrate-promoted increase in propionylcarnitine must reflect corresponding changes in propionyl-CoA levels, indicating that this lipid-lowering drug might specifically affect reactions producing or utilizing propionyl-CoA. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Solberg, H. E., Aas, M., and Daae, L. N. W., Biochim. Biophys. Acta 280,434 (1972). Daae, L. N. W., and Aas, M., Atherosclerosis 17, 389 (1973). Kahonen, M. T., and Ylikahri, R. H., Atherosclerosis 32, 47 (1979). Markwell, M. K., Bieber, L. L.. and Tolbert, N. E. Eiochem. Pharmacol. 26, 1697 (1977). Mannaerts, G. P., Debeer, L. J., Thomas, J., and DeSchepper, P. J., J. Biol. Chem. 254, 458.5 (1979). Pande, S. V., and Parvin, R., Biochim. Biophys. Acta 617, 363 (1980). Fritz, I. B., Adv. Lipid Res. 1, 285 (1963). Bremer, J., J. Biol. Chem. 237, 3628 (1962). Fritz, I. B., and Marquis, N. R., Proc. Natl. Acad. Sci. USA 72,, 4385 (1965). Childress, C. C.. Sacktor, B., and Traynor, D. R., J. Biol. Chem. 242, 754 (1966). Hutson, S. M., vanDop, C., and Lardy, H. A., J. Biol. Chem. 252, 1309 (1977). Choi, Y. R., Fogle, P. J., Clarke, P. R. H., and Bieber, L. L., J. Biol. Chem. 252, 7930 (1977). Bieber, L. L., and Choi, Y. R.. (1977) Proc. Natl. Acad. Sci. USA 74, 2795 (1977). Fogle, P. J., and Bieber, L. L., Biochem. Med. 22, 119 (1977). Choi, Y. R., Fogle, P. J., and Bieber, L. L., J. Nurr. 109, 155 (1979). Van Hinsberg, V. W., Veerkamp, J. H., Engelen, P. J. M., and Ghijsen, W. J.. Biochem. Med. 20, 115 (1978).

CLOFIBRATE

AND ACYLCARNITINES

209

17. Paul, H. S., and Adibi, S. A., Amer. .I. Physiol. 234, E494 (1978). 18. May, M. E., Aftring, R. P., and Buse, M. G., J. Biol. Chem. 255, 8394 (1980). 19. Pettit, F. H., Yeaman, S. J., and Reed, L. J., Proc. Natl. Acad. Sci. USA 75, 4881 (1978). 20. Parker, P. J., and Randle, P. J., &o&em. J. 171, 751 (1978). 21. Bremer, J., and Davis, E. J., Biochim. Biophys. Acta 528, 269 (1978). 22. Williamson, J. R., Walajtys-Rode. E., and Coil, K. E.. J. Biol. Chem. 254, 11511 (1979). 23. Odessey, R., Biochem. J. 192, 15.5 (1980). 24. Parker, P. J., and Randle, P. J., FEBS Let. 112, 186 (1980). 25. Bohmer, T., and Bremer, J. Biochim. Biophys. Acta 152, 559 (1%8). 26. Pearson, D. J., Tubbs, P. K., and Chase, J. F. A., in “Methods of Enzymatic Analysis” (H. U. Bergmeyer, Ed.), 2nd ed., Vol. 4, p. 1758. Verlag Chemie, Weinheim. 1974. 27. Cederblad, G., and Lindstedt, S. Clin. Chim. Acta 37, 235 (1972). 28. Bieber, L. L., and Lewin, L. Methods Enzymol. 72, 276 (1981). 29. McGarry, J. D., Leatherman. G. F.. and Foster. D. W., J. Biol. Chem. 253, 4128 (1978).

30. Bali, M. R., Gumaa, K. A., and McLean, P.. Biochem. Biophys. Res. Commun. 87, 489 (1979).

Burck, R. E., and Curran. G. L., F. Lipid Res. 10, 668 (1969). 32. VanBroekhoven, A., Peters, M.-C., Debeer. L. J., and Mannaerts, G. P., Biochem. Biophys. Res. Commun. 100, 30.5 (1981). 33. Voltti, H., Salvolainen, M. J., Jauhonen, V. P., and Hassinen, 1. E., Biochem. J. 182, 31.

9.5 (1979).