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. 1980 Jul;66(1):88–93. doi: 10.1172/JCI109839

Branched Chain Amino Acid Oxidation in Cultured Rat Skeletal Muscle Cells

SELECTIVE INHIBITION BY CLOFIBRIC ACID

William M Pardridge 1,2, Delia Casanello-Ertl 1,2, Luiza Duducgian-Vartavarian 1,2
PMCID: PMC371509  PMID: 7400311

Abstract

Leucine metabolism in skeletal muscle is linked to protein turnover. Since clofibrate is known both to cause myopathy and to decrease muscle protein content, the present investigations were designed to examine the effects of acute clofibrate treatment on leucine oxidation. Rat skeletal muscle cells in tissue culture were used in these studies because cultivated skeletal muscle cells, like muscle in vivo, have been shown to actively utilize branched chain amino acids and to produce alanine. The conversion of [1-14C]leucine to 14CO2 or to the [1-14C]keto-acid of leucine (α-keto-isocaproate) was linear for at least 2 h of incubation; the production of 14CO2 from [1-14C]leucine was saturable with a Km = 6.3 mM and a maximum oxidation rate (Vmax) = 31 nmol/mg protein per 120 min. Clofibric acid selectively inhibited the oxidation of [1-14C]leucine (Ki = 0.85 mM) and [U-14C]isoleucine, but had no effect on the oxidation of [U-14C]glutamate, -alanine, -lactate, or -palmitate. The inhibition of [1-14C]leucine oxidation by clofibrate was also observed in the rat quarter-diaphragm preparation. Clofibrate primarily inhibited the production of 14CO2 and had relatively little effect on the production of [1-14C]keto-acid of leucine. A physiological concentration—3.0 g/100 ml—of albumin, which actively binds clofibric acid, inhibited but did not abolish the effects of a 2-mM concentration of clofibric acid on leucine oxidation. Clofibrate treatment stimulated the net consumption of pyruvate, and inhibited the net production of alanine. The drug also increased the cytosolic NADH/NAD+ ratio as reflected by an increase in the lactate/pyruvate ratio, in association with a decrease in cell aspartate levels. The changes in pyruvate metabolism and cell redox state induced by the drug were delayed compared with the nearly immediate inhibition of leucine oxidation. These studies suggest that clofibric acid, in concentrations that approximate high therapeutic levels of the drug, selectively inhibits branched chain amino acid oxidation, possibly at the level of the branched chain keto-acid dehydrogenase.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barrett A. M., Thorp J. M. Studies on the mode of action of clofibrate: effects on hormone-induced changes in plasma free fatty acids, cholesterol, phospholipids and total esterified fatty acids in rats and dogs. Br J Pharmacol Chemother. 1968 Feb;32(2):381–391. doi: 10.1111/j.1476-5381.1968.tb00980.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Buse M. G., Reid S. S. Leucine. A possible regulator of protein turnover in muscle. J Clin Invest. 1975 Nov;56(5):1250–1261. doi: 10.1172/JCI108201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen T. R. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res. 1977 Feb;104(2):255–262. doi: 10.1016/0014-4827(77)90089-1. [DOI] [PubMed] [Google Scholar]
  4. Dusheiko G., Kew M. C., Joffe B. I., Lewin J. R., Mantagos S., Tanaka K. Recurrent hypoglycemia associated with glutaric aciduria type II in an adult. N Engl J Med. 1979 Dec 27;301(26):1405–1409. doi: 10.1056/NEJM197912273012601. [DOI] [PubMed] [Google Scholar]
  5. Felig P. Amino acid metabolism in man. Annu Rev Biochem. 1975;44:933–955. doi: 10.1146/annurev.bi.44.070175.004441. [DOI] [PubMed] [Google Scholar]
  6. Garber A. J., Karl I. E., Kipnis D. M. Alanine and glutamine synthesis and release from skeletal muscle. I. Glycolysis and amino acid release. J Biol Chem. 1976 Feb 10;251(3):826–835. [PubMed] [Google Scholar]
  7. Goldberg A. L., Odessey R. Oxidation of amino acids by diaphragms from fed and fasted rats. Am J Physiol. 1972 Dec;223(6):1384–1391. doi: 10.1152/ajplegacy.1972.223.6.1384. [DOI] [PubMed] [Google Scholar]
  8. Houin G., Thébault J. J., D'Athis P., Tillement J. P., Beaumont J. L. A GLC method for estimation of chlorophenoxyisobutyric acid in plasma. Pharmacokinetics of a single oral dose of clofibrate in man. Eur J Clin Pharmacol. 1975 Aug 14;8(6):433–437. doi: 10.1007/BF00562318. [DOI] [PubMed] [Google Scholar]
  9. Hutson S. M., Cree T. C., Harper A. E. Regulation of leucine and alpha-ketoisocaproate metabolism in skeletal muscle. J Biol Chem. 1978 Nov 25;253(22):8126–8133. [PubMed] [Google Scholar]
  10. Khatra B. S., Chawla R. K., Sewell C. W., Rudman D. Distribution of branched-chain alpha-keto acid dehydrogenases in primate tissues. J Clin Invest. 1977 Mar;59(3):558–564. doi: 10.1172/JCI108671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Langer T., Levy R. I. Acute muscular syndrome associated with administration of clofibrate. N Engl J Med. 1968 Oct 17;279(16):856–858. doi: 10.1056/NEJM196810172791604. [DOI] [PubMed] [Google Scholar]
  12. Lee S. H., Davis E. J. Carboxylation and decarboxylation reactions. Anaplerotic flux and removal of citrate cycle intermediates in skeletal muscle. J Biol Chem. 1979 Jan 25;254(2):420–430. [PubMed] [Google Scholar]
  13. Odessey R., Goldberg A. L. Leucine degradation in cell-free extracts of skeletal muscle. Biochem J. 1979 Feb 15;178(2):475–489. doi: 10.1042/bj1780475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Odessey R., Khairallah E. A., Goldberg A. L. Origin and possible significance of alanine production by skeletal muscle. J Biol Chem. 1974 Dec 10;249(23):7623–7629. [PubMed] [Google Scholar]
  15. Pardridge W. M., Casanello-Ertl D. Effects of glutamine deprivation on glucose and amino acid metabolism in tissue culture. Am J Physiol. 1979 Mar;236(3):E234–E238. doi: 10.1152/ajpendo.1979.236.3.E234. [DOI] [PubMed] [Google Scholar]
  16. Pardridge W. M., Davidson M. B. Alanine metabolism in skeletal muscle in tissue culture. Biochim Biophys Acta. 1979 Jun 1;585(1):34–42. doi: 10.1016/0304-4165(79)90322-2. [DOI] [PubMed] [Google Scholar]
  17. Pardridge W. M., Davidson M. B., Casanello-Ertl D. Glucose and amino acid metabolism in an established line of skeletal muscle cells. J Cell Physiol. 1978 Sep;96(3):309–318. doi: 10.1002/jcp.1040960306. [DOI] [PubMed] [Google Scholar]
  18. Pardridge W. M., Duducgian-Vartavarian L., Casanello-Ertl D., Jones M. R., Kopple J. D. Glucose and amino acid metabolism in neonatal rat skeletal muscle in tissue culture. J Cell Physiol. 1980 Jan;102(1):91–98. doi: 10.1002/jcp.1041020113. [DOI] [PubMed] [Google Scholar]
  19. Paul H. S., Adibi S. A. Paradoxical effects of clofibrate on liver and muscle metabolism in rats. Induction of myotonia and alteration of fatty acid and glucose oxidation. J Clin Invest. 1979 Aug;64(2):405–412. doi: 10.1172/JCI109476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ruderman N. B., Berger M. The formation of glutamine and alanine in skeletal muscle. J Biol Chem. 1974 Sep 10;249(17):5500–5506. [PubMed] [Google Scholar]
  21. Schneider E. L., Stanbridge E. J., Epstein C. J. Incorporation of 3H-uridine and 3H-uracil into RNA: a simple technique for the detection of mycoplasma contamination of cultured cells. Exp Cell Res. 1974 Mar 15;84(1):311–318. doi: 10.1016/0014-4827(74)90411-x. [DOI] [PubMed] [Google Scholar]
  22. Spector A. A., Santos E. C., Ashbrook J. D., Fletcher J. E. Influence of free fatty acid concentration on drug binding to plasma albumin. Ann N Y Acad Sci. 1973 Nov 26;226:247–258. doi: 10.1111/j.1749-6632.1973.tb20486.x. [DOI] [PubMed] [Google Scholar]
  23. Teräväinen H., Larsen A., Hillbom M. Clofibrate-induced myopathy in the rat. Acta Neuropathol. 1977 Aug 16;39(2):135–138. doi: 10.1007/BF00703319. [DOI] [PubMed] [Google Scholar]
  24. Wahren J., Felig P., Hagenfeldt L. Effect of protein ingestion on splanchnic and leg metabolism in normal man and in patients with diabetes mellitus. J Clin Invest. 1976 Apr;57(4):987–999. doi: 10.1172/JCI108375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wilkening J., Wolpert F., Schwandt P. Glycolytic metabolites and adenosine triphosphate in skeletal and cardiac muscle of rats after clofibrate feeding. Biochem Pharmacol. 1978 Jan 15;27(2):244–245. doi: 10.1016/0006-2952(78)90308-8. [DOI] [PubMed] [Google Scholar]
  26. Wolfe B. M., Kane J. P., Havel R. J., Brewster H. P. Mechanism of the hypolipemic effect of clofibrate in postabsorptive man. J Clin Invest. 1973 Sep;52(9):2146–2159. doi: 10.1172/JCI107399. [DOI] [PMC free article] [PubMed] [Google Scholar]

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