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Inhibitors of fatty acid oxidation
Life Sciences, Vol. 40, pp. 1443-1449 Printed in the U.S.A.
Pergamon Journals
MINIREVIEW INHIBITORS OF FATTY ACID OXIDATION Horst Schulz Department of Chemistry, City College of the City University of New York, New York, N.Y. 10031
Summar y
This review discusses inhibitors of f a t t y acid oxidation for which sites and mechanisms of inhibition are reasonably well understood. Included in this review are hypoglycin, an i n h i b i t o r of butyryl-CoA dehydrogenase (EC 1.3.99.2), 4-pentenoic acid, 2-bromooctanoic acid, and 4-bromocrotonic acid a l l of which i n h i b i t mitochondrial thiolases (EC 2.3.1.9 and 2.3.1.16) as well as several inhibitors of carnitine palmitoyltransferase I (EC 2.3.1.21) as for example 2-tetradecylglycidic acid, 2-bromopalmitic acid and aminocarnitine. Most of these inhibitors of f a t t y acid oxidation have been shown to cause hypoglycemia in animals and some also cause hypoketonemia. The advantages and limitations of using these inhibitors in metabolic studies are discussed. The ~-oxidation of f a t t y acids is an important metabolic pathway in many animals. The significance of this pathway in humans is emphasized by an increasing number of reports about diseases due to an impairment of f a t t y acid oxidation. For example Jamaican vomiting disease is caused by the plant toxin hypoglycin which inhibits B-oxidation (1). More recently other inhibitors of f a t t y acid oxidation have been developed either as potential hypoglycemic drugs or as tools for studying the regulation of f a t t y acid oxidation. I t is the intent of this report to review those inhibitors which i n h i b i t f a t t y acid oxidation by s p e c i f i c a l l y inhibiting one reaction of the pathway. Listed in Table I are seven inhibitors for which sites and mechanisms of i n h i b i t i o n are d e f i n i t e l y or reasonably well established. I t is noteworthy that these inhibitors affect only three reactions of the pathway of f a t t y acid oxidation; those catalyzed by acyl-CoA dehydrogenases, 3-ketoacyl-CoA thiolases and carnitine palmitoyltransferase I (CPT I ) . All inhibitors with the exception of acylaminocarnitine are carboxylic acids with reactive or potentially reactive functional groups. They must be converted to their coenzyme A thioesters and in most cases further metabolized in order to bind reversibly or i r r e v e r s i b l y to the active site of the target enzyme thereby inhibiting i t . The general principle underlying the design of these inhibitors is their structural s i m i l a r i t y to f a t t y acids which permits t h e i r c e | l u l a r and/or mitochondrial uptake and the presence of a functional group which, as a consequence of metabolic conversions, w i l l yield a highly reactive and active site-directed i n h i b i t o r .
Copyright
0024-3205/87 $3.00 + .00 (c) 1987 Pergamon Journals Ltd.
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TABLE I Inhibitors of Fatty Acid Oxidation Compound
Inhibitory metabolite
Inhibited enzyme
Hypoglycin
Methylenecyclopropylacetyl-CoA
Butyryl-CoA dehydrogenase
4-Pentenoic acid 2-Bromooctanoic acid 4-Bromocrotonic acid
3-Keto-4-pentenoyl-CoA 2-Bromo-3-ketooctanoyl-CoA 3-Keto-4-bromobutyryl-CoA
3- Ketoa cyl -CoA thiolase
2-Bromopalmitic acid 2-Tetradecylglycidic acid Acylaminocarnitine
2- BromopaI mi toyl- CoA 2-Tetradecy} glycidyl- CoA Acyl ami nocarni t i ne
Carni ti ne palmi toyl tra nstransferase I
3-Mercaptopropanoic acid
S-Acyl-3-mercaptopropanoyl-CoA
Acyl-CoA dehydrogenase
Hypoglycin y C C 0 the toxic p r i n c i p l e of the unrip i b i t i o n of f a t t y acid oxidation and is believed to cause secondarily an impairment of gluconeogenesis which results in hypoglycemia ( I - 3 ) . Hypoglycin poisoning also induces ketosis (4). The hypoglycemic e f f e c t of hypoglycin was f i r s t observed in man but studied in d e t a i l with rats and mice. In the cytosol of animal c e l l s , hypoglycin is converted by transamination to methyl enecyclopropylpyruvic acid which, in mitochondria, is o x i d a t i v e l y decarboxylated to y i e l d methylenecyclopropylacetyl-CoA (i). The l a t t e r compound i n h i b i t s acyl-CoA dehydrogenases i r r e v e r s i b l y , being most e f f e c t i v e against butyryl-CoA dehydrogenase (3,5), by forming a covalent adduct with the f l a v i n adenine dinucleotide cofactor of the enzyme (6). Methylenecyclopropylacetic acid causes the i n h i b i t i o n of butyryl-CoA dehydrogenase and thereby of B-oxidation in isolated l i v e r mitochondria (3). Rats treated with hypoglycin (80-100 mg/kg of body weight) become severely hypoglycemic within a few hours and the ~-oxidation capacity of mitochondria isolated from those animals is i n h i b i t e d by more than 50% (2). This i n h i b i t i o n is a consequence of a more than 80% i n h i b i t i o n of butyryl-CoA dehydrogenase. Isovaleryl-CoA dehydrogenase is also i n h i b i t e d but palmitoyl-CoA dehydrogenase is not (2). Inhibitors of Thiolase The search for structurally similar but simpler analogs of hypoglycin led to the identification of 4-pentenoic acid as a hypoglycemic agent. Like hypoglycin, 4-pentenoic acid causes the inhibition of fatty acid oxidation and is believed to cause, secondarily, an impairment of gluconeogenesis which results in hypoglycemia (1-3). 4-Pentenoic acid also induces ketosis (7). The inhibition of fatty acid oxidation has been demonstrated in a number of systems including rats (2), the perfused rat l i v e r (8), myocytesl, rat l i v e r mitochondria (3) and rat heart mitochondria (9). The reported inhibition of pyruvate oxidation (8) appears to be nonspecific because i t is also observed with 1
S. Abd EI-Aleem and H. Schulz, unpublished results.
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non-hypoglycemic compounds like pentanoic acid (9). 4-Pentenoic acid was found to be metabolized to acetyl-CoA and acryloyl-CoA (10). In perfused rat hearts 4-pentenoic acid is e f f i c i e n t l y converted to tricarboxylic acid cycle intermediates which presumably are formed from propionyl-CoA (11). Studies of 4-pentenoate degradation proved the intramitochondrial metabolism of this compound to proceed via two pathways. Co,non to both pathways is the activation of 4-pentenoate to 4-pentenoyl-CoA followed by its dehydrogenation to 2,4-pentadienoylCoA. The l a t t e r compound is converted by the NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34) of the major pathway to 3-pentenoyl-CoA which is isomerized to 2-pentenoyl-CoA and then B-oxidized to propionyl-CoA and acetyl-CoA (12,13). The minor pathway involves the direct ~-oxidation of 2,4-pentadienoylCoA to acryloyl-CoA and acetyl-CoA (13). The minor pathway but not the major pathway yields 3-keto-4-pentenoyl-CoA which is a reversible as well as an irreversible inhibitor of 3-ketoacyl-CoA thiolase (EC 2.3.1.16) and acetoacetyl-CoA thiolase (EC 2.3.1.9) (13). Consequentlyany condition, as for example c l o f i brate feeding (14), which results in an increased a c t i v i t y of 2,4-dienoyl-CoA reductase, will reduce the toxic effect of 4-pentenoate. 3-Keto-4-pentenoyl-CoA also inactivates carnitine acetyltransferase (EC 2.3.1.7) although at a slower rate than i t inhibits either of the two thiolases (9,15). Another compound which inhibits 3-ketoacyl-CoA thiolase and thereby fatty acid oxidation is 4-bromocrotonic acid (16). This inhibitor is effective at low micromolar concentrations in isolated rat heart mitochondria (16) as well as in the perfused rat heart (17). Since 4-bromocrotonic acid must f i r s t be converted to its CoA derivative, i t is not inhibitory in uncoupled mitochondria. 4-Bromocrotonyl-CoA is converted intramitochondrially byE-oxidation to 3-keto-4-bromobutyryl-CoA which inactivates irreversibly both 3-ketoacyl-CoA thiolase and acetoacetyl-CoA thiolase. Consequently, fatty acid oxidation as well as ketone body degradation are inhibited. Since the oxidation of pyruvate in heart mitochondria is not inhibited by 4-bromocrotonic acid, this compound does not seem to affect the tricarboxylic acid cycle or oxidative phosphorylation. A third inhibitor of thiolases and thus of fatty acid oxidation is 2-bromooctanoic acid. This compound inhibits fatty acid oxidation and ketogenesis in the perfused rat l i v e r (18). I t also causes a decrease in hepatic gluconeogenesis. When rat l i v e r mitochondria are incubated with 10 ~M 2-bromooctanoic acid, 3-ketoacyl-CoA thiolase is completely inactivated and consequently fatty acid oxidation is inhibited (19). Under identical conditions acetoacetyl-CoA thiolase is only partially inactivated (19). Experiments with isolated rat l i v e r mitochondria suggest that 2-bromooctanoic acid is activated intramitochondrially to 2-bromooctanoyl-CoA and then converted via ~-oxidation to 2-bromo-3-ketooctanoyl-CoA which causes the irreversible inactivation of both thiolases. When [1-14C]-labeled 2-bromooctanoic acid was used, 3-ketoacyl-CoA was found to be radioactively labeled thereby proving the covalent modification of the enzyme (19). Although 2-bromooctanoic is an effective inhibitor in perfused rat l i v e r and rat l i v e r mitochondria, i t is v i r t u a l l y without effect in rat heart mitochondria (16) and myocytesI. In addition to its effect on E-oxidation, 2-bromooctanoic acid also inhibits triacylglycerol synthesis in hepatocytes (20). This inhibition is probably a consequence of its conversion to 2-bromooctanoyl-CoA which is a competitive inhibitor of diacylglycerol acyltransferase (EC 2.3.1.20) (20). Inhibitors of Carnitine Palmitoyltransferase I Of the three listed i nhibitors of carnitine palmitoyltransferase I (see Table I), 2-tetradecylglycidic acid has been studied most extensively. 2-Tetradecylglycidic acid is an effective hypoglycemic agent in fasted or diabetic animals; i t also lowers the plasma levels of ketone bodies (21). This compound
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at micromolar concentrations strongly inhibits the oxidation of long-chain, but not of short-chain f a t t y acids in rat hepatocytes, l i v e r mitochondria, the perfused heart, as well as in other systems (21). An inhibition of gluconeogenesis appears to be secondary to the inhibition of f a t t y acid oxidation (22). Since 2-tetradecylglycidic acid inhibits the oxidation of palmitate, but not of palmitoylcarnitine or octanoate (22), the site of the inhibition was assumed to be the reaction catalyzed by carnitine palmitoyltransferase I or a preceding reaction. The inhibitory compound was believed to be the CoA thioester of 2-tetradecylglycidic acid. This hypothesis was confirmed by demonstrating with isolated mitochondria that 2-tetradecylglycidic acid is a substrate of acyl-CoA synthetase (21) and that the resultant 2-tetradecylglycidyl-CoA is a powerful inhibitor of carnitine palmitoyltransferase I (23). Since the inactivation of carnitine palmitoyltransferase I by 2-tetradecylglycidyl-CoA is a time dependent process, not reversible by prolonged dialysis and p a r t i a l l y prevented by palmitoyl-CoA or CoASH, the inactivation of the carnitine palmitoyltransferase I seems to be a consequence of the inhibitory compound binding to the active site of the enzyme and forming a covalent adduct (23). A compound structurally and mechanistically related to 2-tetradecylglycidic acid is 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylicacid. This compound is a hypoglycemic and hypoketonemic agent in fasted and diabetic animals (24). These metabolic effects seem to be a consequence of an inhibition of f a t t y acid oxidation at the level of carnitine palmitoyltransferase I which is strongly inhibited by the CoA derivative of this compound (25). 2-Bromopalmitic acid inhibits the oxidation of palmitate, but not of palmitoylcarnitine in rat hepatocytes (26). T h i s inhibition was attributed to an impaired uptake of f a t t y acids caused by 2-bromopalmitate (26). Experiments with isolated rat l i v e r mitochondria revealed that the inhibition of f a t t y acid oxidation required the conversion of 2-bromopalmitate to 2-bromopalmitoyl-CoA. The l a t t e r compound in the presence of L-carnitine is a powerful inhibitor of the oxidation of palmitoyl-CoA, but not of palmitoylcarnitine, pyruvate, or hexanoate by rat l i v e r mitochondria (27). These observations point to carnitine palmitoyltransferase I as the site of the inhibition. Since the inhibiton is carnitine dependent, i t may be mechanistically similar to the inactivation of carnitine acetyltransferase (EC 2.3.1.7) by bromoacetyl-CoA in the presence of L-carnitine (28). The inactivation of carnitine acetyltransferase involves the enzyme-catalyzed transfer of the bromoacetyl group from CoA to carnitine f o l lowed by alkylation of CoASH by bromoacetylcarnitine at the active site of the enzyme. The resultant compound, which remains very t i g h t l y , but noncovalently bound to the enzyme, consists of CoA and carnitine bridged by an acetate residue. 2-Bromopalmitoylcarnitine in contrast to 2-bromopalmitoyl-CoA i n h i b i t s the oxidation of short-chain f a t t y acids and pyruvate in addition to i n h i b i t i n g the oxidation of long-chain f a t t y acids, The mechanism by which 2-bromopalmitoyl~ c a r n i t i n e i n h i b i t s f a t t y acid oxidation is unknown, whereas the i n h i b i t i o n of pyruvate oxidation is most l i k e l y due to depletion of CoASH because i t can be reversed by the addition of c a r n i t i n e (27). More recently aminocarnitine and acylaminocarnitines, as for example palmitoylaminocarnitine, have been i d e n t i f i e d as r e v e r s i b l e i n h i b i t o r s of c a r n i t i n e palmitoyltransferase I and thus of f a t t y acid oxidation (29,30). These compounds have hypoglycemic and hypoketonemic effects in diabetic mice (30). Aminocarnitine and i t s acyl d e r i v a t i v e s are more potent i n h i b i t o r s of c a r n i t i n e palmitoyltransferase than are a c y l - D - c a r n i t i n e s , the acyl d e r i v a t i v e s of the unnatural c a r n i t i n e enantiomer, which have previously been used to i n h i b i t the oxidation of long-chain f a t t y acids in r a t l i v e r mitochondria (31).
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Oxfenicine (S-4-hydroxyphenylglycine) inhibits f a t t y acid oxidation in heart and stimulates glucose u t i l i z a t i o n , thereby stimulating cardiac performance without an increase in oxygen consumption (32). The active principle causing these effects seems to be 4-hydroxyphenylglyoxylate which most l i k e l y i n h i b i t s carnitine palmitoyltransferase I (33). 3-Mercaptopropanoic Acid The convulsant 3-mercaptopropanoic acid was found to be an effective inhibi t o r of f a t t y acid oxidation in isolated rat heart mitochondria (34). The oxidation of pyruvate was hardly affected under conditions which resulted in an almost complete i n h i b i t i o n of the oxidation of palmitoylcarnitine. Tests with rat heart myocytes confirmed the results obtained with rat heart mitochondria and also proved the i n h i b i t i o n to be reversible . This situation prompted a study of the mitochondrial metabolism of 3-mercaptopropanoic acid which revealed that the compound is activated intramitochondrially to 3-mercaptopropanoyl-CoA (35). The l a t t e r compound is an e f f i c i e n t analog of CoASH in reactions catalyzed by t h i o l ases thereby giving rise to S-acyl-3-mercaptopropanoyl-CoA thioesters within the mitochondrial matrix (35). When various mitochondrial metabolites of 3-mercaptopropanoic acid were tested for their effects on the a c t i v i t i e s of the #-oxidation enzymes, long-chain and medium-chain acyl-CoA dehydrogenases were found to be severely inhibited by long-chain S-acyl-3-mercaptopropanoyl-CoA thioesters in a manner which suggested that this inhibition may be the cause of the inhibition of ~-oxidation (34). 2-Mercaptoacetic acid is another mercapto acid which inhibits f a t t y acid oxidation. Its administration to rats results in an inhibition of hepatic f a t t y acid oxidation possibly via an i n h i b i t i o n of acyl-CoA dehydrogenase (36,37). However, the potency of 2-mercaptoacetic acid as an i n h i b i t o r of #-oxidation is s i g n i f i c a n t l y lower than that of 3-mercaptopropanoic acid (34). Conclusion Hypoglycin and a l l inhibitors of f a t t y acid oxidation that have been tested in vivo cause hypoglycemia in animals. The hypoglycemia is a consequence of gluconeogenesis being inhibited and glucose u t i l i z a t i o n being increased. Although the mechanism of gluconeogenesis inhibition has not been f u l l y elucidated, i t seems to be at least p a r t i a l l y due to a lowered a c t i v i t y of pyruvate carboxylase possibly because the concentration of i t s a l l o s t e r i c activator acetyl-CoA is decreased and the concentration of inhibitory acyl-CoA thioesters is increased. Inhibitors of carnitine palmitoyltransferase I also induce hypoketonemia as a direct consequence of inhibiting hepatic f a t t y acid oxidation. In contrast, hypoglycin and 4-pentenoic acid induce ketosis possibly by inhibiting peripheral ketone body u t i l i z a t i o n more than hepatic ketone body formation. The inhibitors of f a t t y acid oxidation listed in Table I can be used to i n h i b i t the pathway at specific steps and to study the metabolic consequences of such inhibitions. The question as to which i n h i b i t o r should be used in a given study depends on the step at which f a t t y acid oxidation should be affected as well as on the s e l e c t i v i t y and effectiveness of the available inhibitors. Since 2-tetradecylglycidic acid and 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxy l i c acid are very selective and effective inhibitors, they are the compounds of choice when carnitine palmitoyltransferase I should be inhibited i r r e v e r s i b l y . I f a reversible inhibition of the same enzjnne is desired, aminocarnitine or one of i t s acyl derivatives seems to be an excellent choice. 2-Bromopalmiticacid, which has not yet been studied thoroughly, may affect more than one reaction and is therefore not recommended as an i n h i b i t o r for metabolic studies. However,
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when carnitine palmitoyltransferase I should be inhibited in isolated mitochondria, 2-bromopalmitoyl-CoA is an excellent inhibitor because of its s e l e c t i v i t y and effectiveness. Of the thiolase inhibitors 2-bromooctanoic acid is effective in l i v e r but not in heart. Thus, i t may be a good agent for in vivo experiments aimed at distinguishing between the contributions of l i v e r and muscle to the oxidation of fatty acids in animals. 4-Pentenoic acid, because of its complicated metabolism and multitude of effects, may yield results that are d i f f i c u l t to interpret. However, 4-bromocrotonic acid, although so far tested in heart only, seems to cause the inhibition of fatty acid oxidation via a specific and effective inactivation of the mitochondrial thiolases. Since hypoglycin is not readily available and 3-mercaptopropionic acid causes convulsions in animals, a need exists for developing other inhibitors of acyl-CoA dehydrogenases. However, most important is the design and testing of inhibitors for those enzymes for which no inhibitors are known so far. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
R. BRESSLER, C. CORREDOR,and K. BRENDEL, Pharmacol. Rev. ~1, 105-130 (1969). D. BILLINGTON, H. OSMUNDSEN,and H. S. A. SHERRATT, Biochem. Pharmacol. 27, 2891-2900 (1978). D. BILLINGTON, H. OSMUNDSEN,and H. S. A. SHERRATT, Biochem. Pharmac~l. 27, 2879-2890 (1978). D. H. WILLIAMS, and M. B. WILSON, Biochem. J. 94, 19C-20C (1965). E. A. KEAN, Biochim. Biophys. Acta 422, 8-14 (~76). A. WENZ, C. THORPE, and S. GHISLA, J. Biol. Chem. 256, 9809-9812 (1981). A. E. SENIOR, and H. S. A. SHERRATT, J. Pharm. Pharmac. 21, 85-92 (1969). J. R. WILLIAMSON, S. G. ROSTAND,and M. J. PETERSON, J. Biol. Chem. 245, 3242-3251 (1970). J. C. FONG, and H. SCHULZ, J. Biol. Chem. 253, 6917-6922 (1978). K. BRENDEL, C. F. CORREDOR,and R. BRESSLER,~Biochem. Biophys. Res. Commun. 34, 340-347 (1969). J-~- K. HILTUNEN, Biochem. J. 170, 241-247 (1978). J. K. HILTUNEN, and E. J. D A ~ , Biochem. J. 194, 427-432 (1981). H. SCHULZ, Biochemistry 22, 1827-1832 (1983). B. BORREBACK,H. OSMUNDS~, and J. BREMER, Biochem. Biophys. Res. Commun. 93, 1173-1180 (1980). J. ZHONG, J. C. FONG, and H. SCHULZ, Arch. Biochem. Biophys. 240, 524-529 (1985). Y. OLOWE, and H. SCHULZ, J. Biol. Chem. 257, 5408-5413 (1982). J. F. HUTTER, C. SCHWEICKHARDT, H. M. PIP--~-R, and P. G. SPIECKERMANN, J. Mol. Cell. Cardiol. 16, 105-108 (1984). B. 11. RAAKA, and J. M. LOWENSTEIN, J. Biol. Chem. 254, 3303-3310 (1979). B. M. RAAKA, and J. M. LOWENSTEIN, J. Biol. Chem. 554, 6755-6762 (1979). N. MAYOREK, and J. BAR-TANA, J. Biol. Chem. 260, 6528-6532 (1985). G. F. TUTWILER, W. HO, and R. J. MOHRBACHER,Methods in Enzymology (J. M. Lowenstein, ed.) Vol. 7__2, pp. 533-551, Academic Press, New York. (1985). G. F. TUTWILERand P. DELLEVIGNE, J. Biol. Chem. 254, 2935-2941 (1979). T. C. KIORPES, D. HOERR, W. HO, L. E. WEANER, M. G. INMAN, and G. F. TUTWILER, J. Biol. Chem. 259, 9750-9755 (1984). H. P. O. WOLF, K. EISTETTER, and G. LUDWIG, Diabetologia 22, 456-463 (1982). D. M. TURNBULL, K. BARTLETT, S. I. M. YOUNAN, and H. S. A. SHERRATT, Biochem. Pharmacol. 33, 475-481 (1984). S. MAHADEVAN,and F. SAUER, J. Biol. Chem. 246, 5862-5867 (1971). J. F. A. CHASE, and P. K. TUBBS, Biochem. J ~ 2 9 , 55-65 (1972).
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28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
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J. F. A. CHASE, and P. K. TUBBS, Biochem. J. 111, 225-235 (1969). D. L. JENKINS, and O. W. GRIFFITH, J. Biol. Chem. 260, 14748-14755 (1985). D. L. JENKINS, and O. W. GRIFFITH, Proc. Natl. Acad. Sci. U.S.A. 83, 290-294 (1986). I. B. FRITZ, and N. R. MARQUIS, Proc. Natl. Acad. Sci. U.S.A. 54, 1226-1233 (1965). A. J. HIGGINS, ~I. MORVILLE, R. A. BURGES, D. G. GARDINER, M. G. PAGE, and K. J. BLACKBURN, Life Sci. 2~, 963-970 (1980). A. J. HIGGINS, M. MORVILLE, R. A. BURGES, and K. J. BLACKBURN, Biochem. Biophys. Res. Commun. 100, 291-296 (1981). E. SABBAGH, D. CUEBAS, and H. SCHULZ, J. Biol. Chem. 260, 7337-7342 (1985). D. CUEBAS, J. D. BECKMANN, F. E. FRERMAN, and H. SCHUL-~r~,J. Biol. Chem. 260, 7330-7336 (1985). F. BAUCHE, D. SABOURAULT, Y. GIUDICELLI, J. NORDMANN,and R. NORDMANN, Biochem. J. 196, 803-809 (1981). F. BAUCHE, D. SABOURAULT, Y. GIUDICELLI, J. NORDMANN,and R. NORDMANN, Biochem. J. 215, 457-464 (1983).
Pergamon Journals
MINIREVIEW INHIBITORS OF FATTY ACID OXIDATION Horst Schulz Department of Chemistry, City College of the City University of New York, New York, N.Y. 10031
Summar y
This review discusses inhibitors of f a t t y acid oxidation for which sites and mechanisms of inhibition are reasonably well understood. Included in this review are hypoglycin, an i n h i b i t o r of butyryl-CoA dehydrogenase (EC 1.3.99.2), 4-pentenoic acid, 2-bromooctanoic acid, and 4-bromocrotonic acid a l l of which i n h i b i t mitochondrial thiolases (EC 2.3.1.9 and 2.3.1.16) as well as several inhibitors of carnitine palmitoyltransferase I (EC 2.3.1.21) as for example 2-tetradecylglycidic acid, 2-bromopalmitic acid and aminocarnitine. Most of these inhibitors of f a t t y acid oxidation have been shown to cause hypoglycemia in animals and some also cause hypoketonemia. The advantages and limitations of using these inhibitors in metabolic studies are discussed. The ~-oxidation of f a t t y acids is an important metabolic pathway in many animals. The significance of this pathway in humans is emphasized by an increasing number of reports about diseases due to an impairment of f a t t y acid oxidation. For example Jamaican vomiting disease is caused by the plant toxin hypoglycin which inhibits B-oxidation (1). More recently other inhibitors of f a t t y acid oxidation have been developed either as potential hypoglycemic drugs or as tools for studying the regulation of f a t t y acid oxidation. I t is the intent of this report to review those inhibitors which i n h i b i t f a t t y acid oxidation by s p e c i f i c a l l y inhibiting one reaction of the pathway. Listed in Table I are seven inhibitors for which sites and mechanisms of i n h i b i t i o n are d e f i n i t e l y or reasonably well established. I t is noteworthy that these inhibitors affect only three reactions of the pathway of f a t t y acid oxidation; those catalyzed by acyl-CoA dehydrogenases, 3-ketoacyl-CoA thiolases and carnitine palmitoyltransferase I (CPT I ) . All inhibitors with the exception of acylaminocarnitine are carboxylic acids with reactive or potentially reactive functional groups. They must be converted to their coenzyme A thioesters and in most cases further metabolized in order to bind reversibly or i r r e v e r s i b l y to the active site of the target enzyme thereby inhibiting i t . The general principle underlying the design of these inhibitors is their structural s i m i l a r i t y to f a t t y acids which permits t h e i r c e | l u l a r and/or mitochondrial uptake and the presence of a functional group which, as a consequence of metabolic conversions, w i l l yield a highly reactive and active site-directed i n h i b i t o r .
Copyright
0024-3205/87 $3.00 + .00 (c) 1987 Pergamon Journals Ltd.
1444
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Vol. 40, No. 15, 1987
TABLE I Inhibitors of Fatty Acid Oxidation Compound
Inhibitory metabolite
Inhibited enzyme
Hypoglycin
Methylenecyclopropylacetyl-CoA
Butyryl-CoA dehydrogenase
4-Pentenoic acid 2-Bromooctanoic acid 4-Bromocrotonic acid
3-Keto-4-pentenoyl-CoA 2-Bromo-3-ketooctanoyl-CoA 3-Keto-4-bromobutyryl-CoA
3- Ketoa cyl -CoA thiolase
2-Bromopalmitic acid 2-Tetradecylglycidic acid Acylaminocarnitine
2- BromopaI mi toyl- CoA 2-Tetradecy} glycidyl- CoA Acyl ami nocarni t i ne
Carni ti ne palmi toyl tra nstransferase I
3-Mercaptopropanoic acid
S-Acyl-3-mercaptopropanoyl-CoA
Acyl-CoA dehydrogenase
Hypoglycin y C C 0 the toxic p r i n c i p l e of the unrip i b i t i o n of f a t t y acid oxidation and is believed to cause secondarily an impairment of gluconeogenesis which results in hypoglycemia ( I - 3 ) . Hypoglycin poisoning also induces ketosis (4). The hypoglycemic e f f e c t of hypoglycin was f i r s t observed in man but studied in d e t a i l with rats and mice. In the cytosol of animal c e l l s , hypoglycin is converted by transamination to methyl enecyclopropylpyruvic acid which, in mitochondria, is o x i d a t i v e l y decarboxylated to y i e l d methylenecyclopropylacetyl-CoA (i). The l a t t e r compound i n h i b i t s acyl-CoA dehydrogenases i r r e v e r s i b l y , being most e f f e c t i v e against butyryl-CoA dehydrogenase (3,5), by forming a covalent adduct with the f l a v i n adenine dinucleotide cofactor of the enzyme (6). Methylenecyclopropylacetic acid causes the i n h i b i t i o n of butyryl-CoA dehydrogenase and thereby of B-oxidation in isolated l i v e r mitochondria (3). Rats treated with hypoglycin (80-100 mg/kg of body weight) become severely hypoglycemic within a few hours and the ~-oxidation capacity of mitochondria isolated from those animals is i n h i b i t e d by more than 50% (2). This i n h i b i t i o n is a consequence of a more than 80% i n h i b i t i o n of butyryl-CoA dehydrogenase. Isovaleryl-CoA dehydrogenase is also i n h i b i t e d but palmitoyl-CoA dehydrogenase is not (2). Inhibitors of Thiolase The search for structurally similar but simpler analogs of hypoglycin led to the identification of 4-pentenoic acid as a hypoglycemic agent. Like hypoglycin, 4-pentenoic acid causes the inhibition of fatty acid oxidation and is believed to cause, secondarily, an impairment of gluconeogenesis which results in hypoglycemia (1-3). 4-Pentenoic acid also induces ketosis (7). The inhibition of fatty acid oxidation has been demonstrated in a number of systems including rats (2), the perfused rat l i v e r (8), myocytesl, rat l i v e r mitochondria (3) and rat heart mitochondria (9). The reported inhibition of pyruvate oxidation (8) appears to be nonspecific because i t is also observed with 1
S. Abd EI-Aleem and H. Schulz, unpublished results.
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non-hypoglycemic compounds like pentanoic acid (9). 4-Pentenoic acid was found to be metabolized to acetyl-CoA and acryloyl-CoA (10). In perfused rat hearts 4-pentenoic acid is e f f i c i e n t l y converted to tricarboxylic acid cycle intermediates which presumably are formed from propionyl-CoA (11). Studies of 4-pentenoate degradation proved the intramitochondrial metabolism of this compound to proceed via two pathways. Co,non to both pathways is the activation of 4-pentenoate to 4-pentenoyl-CoA followed by its dehydrogenation to 2,4-pentadienoylCoA. The l a t t e r compound is converted by the NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34) of the major pathway to 3-pentenoyl-CoA which is isomerized to 2-pentenoyl-CoA and then B-oxidized to propionyl-CoA and acetyl-CoA (12,13). The minor pathway involves the direct ~-oxidation of 2,4-pentadienoylCoA to acryloyl-CoA and acetyl-CoA (13). The minor pathway but not the major pathway yields 3-keto-4-pentenoyl-CoA which is a reversible as well as an irreversible inhibitor of 3-ketoacyl-CoA thiolase (EC 2.3.1.16) and acetoacetyl-CoA thiolase (EC 2.3.1.9) (13). Consequentlyany condition, as for example c l o f i brate feeding (14), which results in an increased a c t i v i t y of 2,4-dienoyl-CoA reductase, will reduce the toxic effect of 4-pentenoate. 3-Keto-4-pentenoyl-CoA also inactivates carnitine acetyltransferase (EC 2.3.1.7) although at a slower rate than i t inhibits either of the two thiolases (9,15). Another compound which inhibits 3-ketoacyl-CoA thiolase and thereby fatty acid oxidation is 4-bromocrotonic acid (16). This inhibitor is effective at low micromolar concentrations in isolated rat heart mitochondria (16) as well as in the perfused rat heart (17). Since 4-bromocrotonic acid must f i r s t be converted to its CoA derivative, i t is not inhibitory in uncoupled mitochondria. 4-Bromocrotonyl-CoA is converted intramitochondrially byE-oxidation to 3-keto-4-bromobutyryl-CoA which inactivates irreversibly both 3-ketoacyl-CoA thiolase and acetoacetyl-CoA thiolase. Consequently, fatty acid oxidation as well as ketone body degradation are inhibited. Since the oxidation of pyruvate in heart mitochondria is not inhibited by 4-bromocrotonic acid, this compound does not seem to affect the tricarboxylic acid cycle or oxidative phosphorylation. A third inhibitor of thiolases and thus of fatty acid oxidation is 2-bromooctanoic acid. This compound inhibits fatty acid oxidation and ketogenesis in the perfused rat l i v e r (18). I t also causes a decrease in hepatic gluconeogenesis. When rat l i v e r mitochondria are incubated with 10 ~M 2-bromooctanoic acid, 3-ketoacyl-CoA thiolase is completely inactivated and consequently fatty acid oxidation is inhibited (19). Under identical conditions acetoacetyl-CoA thiolase is only partially inactivated (19). Experiments with isolated rat l i v e r mitochondria suggest that 2-bromooctanoic acid is activated intramitochondrially to 2-bromooctanoyl-CoA and then converted via ~-oxidation to 2-bromo-3-ketooctanoyl-CoA which causes the irreversible inactivation of both thiolases. When [1-14C]-labeled 2-bromooctanoic acid was used, 3-ketoacyl-CoA was found to be radioactively labeled thereby proving the covalent modification of the enzyme (19). Although 2-bromooctanoic is an effective inhibitor in perfused rat l i v e r and rat l i v e r mitochondria, i t is v i r t u a l l y without effect in rat heart mitochondria (16) and myocytesI. In addition to its effect on E-oxidation, 2-bromooctanoic acid also inhibits triacylglycerol synthesis in hepatocytes (20). This inhibition is probably a consequence of its conversion to 2-bromooctanoyl-CoA which is a competitive inhibitor of diacylglycerol acyltransferase (EC 2.3.1.20) (20). Inhibitors of Carnitine Palmitoyltransferase I Of the three listed i nhibitors of carnitine palmitoyltransferase I (see Table I), 2-tetradecylglycidic acid has been studied most extensively. 2-Tetradecylglycidic acid is an effective hypoglycemic agent in fasted or diabetic animals; i t also lowers the plasma levels of ketone bodies (21). This compound
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at micromolar concentrations strongly inhibits the oxidation of long-chain, but not of short-chain f a t t y acids in rat hepatocytes, l i v e r mitochondria, the perfused heart, as well as in other systems (21). An inhibition of gluconeogenesis appears to be secondary to the inhibition of f a t t y acid oxidation (22). Since 2-tetradecylglycidic acid inhibits the oxidation of palmitate, but not of palmitoylcarnitine or octanoate (22), the site of the inhibition was assumed to be the reaction catalyzed by carnitine palmitoyltransferase I or a preceding reaction. The inhibitory compound was believed to be the CoA thioester of 2-tetradecylglycidic acid. This hypothesis was confirmed by demonstrating with isolated mitochondria that 2-tetradecylglycidic acid is a substrate of acyl-CoA synthetase (21) and that the resultant 2-tetradecylglycidyl-CoA is a powerful inhibitor of carnitine palmitoyltransferase I (23). Since the inactivation of carnitine palmitoyltransferase I by 2-tetradecylglycidyl-CoA is a time dependent process, not reversible by prolonged dialysis and p a r t i a l l y prevented by palmitoyl-CoA or CoASH, the inactivation of the carnitine palmitoyltransferase I seems to be a consequence of the inhibitory compound binding to the active site of the enzyme and forming a covalent adduct (23). A compound structurally and mechanistically related to 2-tetradecylglycidic acid is 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylicacid. This compound is a hypoglycemic and hypoketonemic agent in fasted and diabetic animals (24). These metabolic effects seem to be a consequence of an inhibition of f a t t y acid oxidation at the level of carnitine palmitoyltransferase I which is strongly inhibited by the CoA derivative of this compound (25). 2-Bromopalmitic acid inhibits the oxidation of palmitate, but not of palmitoylcarnitine in rat hepatocytes (26). T h i s inhibition was attributed to an impaired uptake of f a t t y acids caused by 2-bromopalmitate (26). Experiments with isolated rat l i v e r mitochondria revealed that the inhibition of f a t t y acid oxidation required the conversion of 2-bromopalmitate to 2-bromopalmitoyl-CoA. The l a t t e r compound in the presence of L-carnitine is a powerful inhibitor of the oxidation of palmitoyl-CoA, but not of palmitoylcarnitine, pyruvate, or hexanoate by rat l i v e r mitochondria (27). These observations point to carnitine palmitoyltransferase I as the site of the inhibition. Since the inhibiton is carnitine dependent, i t may be mechanistically similar to the inactivation of carnitine acetyltransferase (EC 2.3.1.7) by bromoacetyl-CoA in the presence of L-carnitine (28). The inactivation of carnitine acetyltransferase involves the enzyme-catalyzed transfer of the bromoacetyl group from CoA to carnitine f o l lowed by alkylation of CoASH by bromoacetylcarnitine at the active site of the enzyme. The resultant compound, which remains very t i g h t l y , but noncovalently bound to the enzyme, consists of CoA and carnitine bridged by an acetate residue. 2-Bromopalmitoylcarnitine in contrast to 2-bromopalmitoyl-CoA i n h i b i t s the oxidation of short-chain f a t t y acids and pyruvate in addition to i n h i b i t i n g the oxidation of long-chain f a t t y acids, The mechanism by which 2-bromopalmitoyl~ c a r n i t i n e i n h i b i t s f a t t y acid oxidation is unknown, whereas the i n h i b i t i o n of pyruvate oxidation is most l i k e l y due to depletion of CoASH because i t can be reversed by the addition of c a r n i t i n e (27). More recently aminocarnitine and acylaminocarnitines, as for example palmitoylaminocarnitine, have been i d e n t i f i e d as r e v e r s i b l e i n h i b i t o r s of c a r n i t i n e palmitoyltransferase I and thus of f a t t y acid oxidation (29,30). These compounds have hypoglycemic and hypoketonemic effects in diabetic mice (30). Aminocarnitine and i t s acyl d e r i v a t i v e s are more potent i n h i b i t o r s of c a r n i t i n e palmitoyltransferase than are a c y l - D - c a r n i t i n e s , the acyl d e r i v a t i v e s of the unnatural c a r n i t i n e enantiomer, which have previously been used to i n h i b i t the oxidation of long-chain f a t t y acids in r a t l i v e r mitochondria (31).
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Oxfenicine (S-4-hydroxyphenylglycine) inhibits f a t t y acid oxidation in heart and stimulates glucose u t i l i z a t i o n , thereby stimulating cardiac performance without an increase in oxygen consumption (32). The active principle causing these effects seems to be 4-hydroxyphenylglyoxylate which most l i k e l y i n h i b i t s carnitine palmitoyltransferase I (33). 3-Mercaptopropanoic Acid The convulsant 3-mercaptopropanoic acid was found to be an effective inhibi t o r of f a t t y acid oxidation in isolated rat heart mitochondria (34). The oxidation of pyruvate was hardly affected under conditions which resulted in an almost complete i n h i b i t i o n of the oxidation of palmitoylcarnitine. Tests with rat heart myocytes confirmed the results obtained with rat heart mitochondria and also proved the i n h i b i t i o n to be reversible . This situation prompted a study of the mitochondrial metabolism of 3-mercaptopropanoic acid which revealed that the compound is activated intramitochondrially to 3-mercaptopropanoyl-CoA (35). The l a t t e r compound is an e f f i c i e n t analog of CoASH in reactions catalyzed by t h i o l ases thereby giving rise to S-acyl-3-mercaptopropanoyl-CoA thioesters within the mitochondrial matrix (35). When various mitochondrial metabolites of 3-mercaptopropanoic acid were tested for their effects on the a c t i v i t i e s of the #-oxidation enzymes, long-chain and medium-chain acyl-CoA dehydrogenases were found to be severely inhibited by long-chain S-acyl-3-mercaptopropanoyl-CoA thioesters in a manner which suggested that this inhibition may be the cause of the inhibition of ~-oxidation (34). 2-Mercaptoacetic acid is another mercapto acid which inhibits f a t t y acid oxidation. Its administration to rats results in an inhibition of hepatic f a t t y acid oxidation possibly via an i n h i b i t i o n of acyl-CoA dehydrogenase (36,37). However, the potency of 2-mercaptoacetic acid as an i n h i b i t o r of #-oxidation is s i g n i f i c a n t l y lower than that of 3-mercaptopropanoic acid (34). Conclusion Hypoglycin and a l l inhibitors of f a t t y acid oxidation that have been tested in vivo cause hypoglycemia in animals. The hypoglycemia is a consequence of gluconeogenesis being inhibited and glucose u t i l i z a t i o n being increased. Although the mechanism of gluconeogenesis inhibition has not been f u l l y elucidated, i t seems to be at least p a r t i a l l y due to a lowered a c t i v i t y of pyruvate carboxylase possibly because the concentration of i t s a l l o s t e r i c activator acetyl-CoA is decreased and the concentration of inhibitory acyl-CoA thioesters is increased. Inhibitors of carnitine palmitoyltransferase I also induce hypoketonemia as a direct consequence of inhibiting hepatic f a t t y acid oxidation. In contrast, hypoglycin and 4-pentenoic acid induce ketosis possibly by inhibiting peripheral ketone body u t i l i z a t i o n more than hepatic ketone body formation. The inhibitors of f a t t y acid oxidation listed in Table I can be used to i n h i b i t the pathway at specific steps and to study the metabolic consequences of such inhibitions. The question as to which i n h i b i t o r should be used in a given study depends on the step at which f a t t y acid oxidation should be affected as well as on the s e l e c t i v i t y and effectiveness of the available inhibitors. Since 2-tetradecylglycidic acid and 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxy l i c acid are very selective and effective inhibitors, they are the compounds of choice when carnitine palmitoyltransferase I should be inhibited i r r e v e r s i b l y . I f a reversible inhibition of the same enzjnne is desired, aminocarnitine or one of i t s acyl derivatives seems to be an excellent choice. 2-Bromopalmiticacid, which has not yet been studied thoroughly, may affect more than one reaction and is therefore not recommended as an i n h i b i t o r for metabolic studies. However,
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when carnitine palmitoyltransferase I should be inhibited in isolated mitochondria, 2-bromopalmitoyl-CoA is an excellent inhibitor because of its s e l e c t i v i t y and effectiveness. Of the thiolase inhibitors 2-bromooctanoic acid is effective in l i v e r but not in heart. Thus, i t may be a good agent for in vivo experiments aimed at distinguishing between the contributions of l i v e r and muscle to the oxidation of fatty acids in animals. 4-Pentenoic acid, because of its complicated metabolism and multitude of effects, may yield results that are d i f f i c u l t to interpret. However, 4-bromocrotonic acid, although so far tested in heart only, seems to cause the inhibition of fatty acid oxidation via a specific and effective inactivation of the mitochondrial thiolases. Since hypoglycin is not readily available and 3-mercaptopropionic acid causes convulsions in animals, a need exists for developing other inhibitors of acyl-CoA dehydrogenases. However, most important is the design and testing of inhibitors for those enzymes for which no inhibitors are known so far. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
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J. F. A. CHASE, and P. K. TUBBS, Biochem. J. 111, 225-235 (1969). D. L. JENKINS, and O. W. GRIFFITH, J. Biol. Chem. 260, 14748-14755 (1985). D. L. JENKINS, and O. W. GRIFFITH, Proc. Natl. Acad. Sci. U.S.A. 83, 290-294 (1986). I. B. FRITZ, and N. R. MARQUIS, Proc. Natl. Acad. Sci. U.S.A. 54, 1226-1233 (1965). A. J. HIGGINS, ~I. MORVILLE, R. A. BURGES, D. G. GARDINER, M. G. PAGE, and K. J. BLACKBURN, Life Sci. 2~, 963-970 (1980). A. J. HIGGINS, M. MORVILLE, R. A. BURGES, and K. J. BLACKBURN, Biochem. Biophys. Res. Commun. 100, 291-296 (1981). E. SABBAGH, D. CUEBAS, and H. SCHULZ, J. Biol. Chem. 260, 7337-7342 (1985). D. CUEBAS, J. D. BECKMANN, F. E. FRERMAN, and H. SCHUL-~r~,J. Biol. Chem. 260, 7330-7336 (1985). F. BAUCHE, D. SABOURAULT, Y. GIUDICELLI, J. NORDMANN,and R. NORDMANN, Biochem. J. 196, 803-809 (1981). F. BAUCHE, D. SABOURAULT, Y. GIUDICELLI, J. NORDMANN,and R. NORDMANN, Biochem. J. 215, 457-464 (1983).