Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1979 Nov 15;184(2):291–301. doi: 10.1042/bj1840291

The metabolism of 4-methyl-2-oxopentanoate in rat pancreatic islets.

J C Hutton, A Sener, W J Malaisse
PMCID: PMC1161764  PMID: 43143

Abstract

1. Radioactively labelled 4-methyl-2-oxopentanoate was taken up by isolated pancreatic islets in a concentration- and pH-dependent manner and led to the intracellular accumulation of labelled amino acid and to a decrease in the intracellular pH. Uptake of 4-methyl-2-oxopentanoate did not appear to be either electrogenic or Na+-dependent. The islet content of 2-oxo acid radioactivity was not affected by either 2-cyano-3-hydroxy-cinnamate (10mM) or pyruvate (10mM), although both these substances inhibited the oxidation of [U-14C]4-methyl-2-oxopentanoate by islet tissue. 2. 4-Methyl-2-oxopentanoate markedly stimulated islet-cell respiration, ketone-body formation and biosynthetic activity. The metabolism of endogenous nutrients by islets appeared to be little affected by the compound. 3. Studies with the 3H- and 14C-labelled substrate revealed that 4-methyl-2-oxopentanoate was incorporated by islets into CO2, water, acetoacetate, L-leucine and to a lesser extent into islet protein and lipid. Carbon atoms C-2, C-3 and C-4 of the acetoacetate produced were derived from the carbon skeleton of the 4-methyl-2-oxopentanoate, but the acetoacetate carboxy group was derived from the incorporation of CO2. These results, and consideration of the relative rates of 14CO2 and acetoacetate formation from 1-14C-labelled as opposed to U-14C-labelled 4-methyl-2-oxopentanoate, led to the conclusion that the pathway of catabolism of this 2-oxo acid in pancreatic islets is identical with that described in other tissues. The amination of 4-methyl-2-oxopentanoate by islets was attributed to the presence of a branched-chain amino acid aminotransferase (EC 2.6.1.42) activity in the tissue. Although glutamate dehydrogenase activity was demonstrated in islet tissue, the reductive amination of 2-oxoacids did not seem to be of importance in the formation of leucine from 4-methyl-2-oxopentanoate. 4. The results of experiments with respiratory inhibitors and uncouplers, and the finding that 14CO2 production and islet respiration were linked in a 1:1 stoicheiometry suggested that 4-methyl-2-oxopentanoate catabolism was coupled to mitochondrial oxidative phosphorylation. The catabolism of 4-methyl-2-oxopentanoate in islet tissue appeared to be regulated at the level of the initial 2-oxo acid dehydrogenase (EC 1.2.1.25) reaction.

Full text

PDF
291

Selected References

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

  1. Atwater I., Ribalet B., Rojas E. Cyclic changes in potential and resistance of the beta-cell membrane induced by glucose in islets of Langerhans from mouse. J Physiol. 1978 May;278:117–139. doi: 10.1113/jphysiol.1978.sp012296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berne C. Determination of D-3-hydroxybutyrate dehydrogenase in mouse pancreatic islets with a photokinetic technique using bacterial luciferase. Enzyme. 1976;21(2):127–136. doi: 10.1159/000458851. [DOI] [PubMed] [Google Scholar]
  3. COON M. J. The metabolic fate of the isopropyl group of leucine. J Biol Chem. 1950 Nov;187(1):71–82. [PubMed] [Google Scholar]
  4. Chaykin S. Assay of nicotinamide deamidase. Determination of ammonia by the indophenol reaction. Anal Biochem. 1969 Oct 1;31(1):375–382. doi: 10.1016/0003-2697(69)90278-4. [DOI] [PubMed] [Google Scholar]
  5. Donatsch P., Lowe D. A., Richardson B. P., Taylor P. The functional significance of sodium channels in pancreatic beta-cell membranes. J Physiol. 1977 May;267(2):357–376. doi: 10.1113/jphysiol.1977.sp011817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  7. Gylfe E. Amino acid content as indicator of membrane permeability in pancreatic beta-cells. Horm Res. 1974;5(6):339–343. doi: 10.1159/000178648. [DOI] [PubMed] [Google Scholar]
  8. Halestrap A. P., Brand M. D., Denton R. M. Inhibition of mitochondrial pyruvate transport by phenylpyruvate and alpha-ketoisocaproate. Biochim Biophys Acta. 1974 Oct 10;367(1):102–108. doi: 10.1016/0005-2736(74)90140-0. [DOI] [PubMed] [Google Scholar]
  9. Halestrap A. P. The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors. Biochem J. 1975 Apr;148(1):85–96. doi: 10.1042/bj1480085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Halestrap A. P. Transport of pyruvate nad lactate into human erythrocytes. Evidence for the involvement of the chloride carrier and a chloride-independent carrier. Biochem J. 1976 May 15;156(2):193–207. doi: 10.1042/bj1560193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hellerström C. Effects of carbohydrates on the oxygen consumption of isolated pancreatic islets of mice. Endocrinology. 1967 Jul;81(1):105–112. doi: 10.1210/endo-81-1-105. [DOI] [PubMed] [Google Scholar]
  12. Hellman B., Sehlin J., Täljedal I. B. The intracellular pH of mammalian pancreatic -cells. Endocrinology. 1972 Jan;90(1):335–337. doi: 10.1210/endo-90-1-335. [DOI] [PubMed] [Google Scholar]
  13. Hutton J. C., Sener A., Malaisse W. J. The stimulus--secretion coupling 4-methyl-2-oxopentanoate-induced insulin release. Biochem J. 1979 Nov 15;184(2):303–311. doi: 10.1042/bj1840303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ichihara A., Koyama E. Transaminase of branched chain amino acids. I. Branched chain amino acids-alpha-ketoglutarate transaminase. J Biochem. 1966 Feb;59(2):160–169. doi: 10.1093/oxfordjournals.jbchem.a128277. [DOI] [PubMed] [Google Scholar]
  15. Johnson W. A., Connelly J. L. Cellular localization and characterization of bovine liver branched-chain -keto acid dehydrogenases. Biochemistry. 1972 May 9;11(10):1967–1973. doi: 10.1021/bi00760a036. [DOI] [PubMed] [Google Scholar]
  16. Jungas R. L. Fatty acid synthesis in adipose tissue incubated in tritiated water. Biochemistry. 1968 Oct;7(10):3708–3717. doi: 10.1021/bi00850a050. [DOI] [PubMed] [Google Scholar]
  17. Krebs H. A., Eggleston L. V. Metabolism of acetoacetate in animal tissues. 1. Biochem J. 1945;39(5):408–419. [PMC free article] [PubMed] [Google Scholar]
  18. Krebs H. A., Lund P. Aspects of the regulation of the metabolism of branched-chain amino acids. Adv Enzyme Regul. 1976;15:375–394. doi: 10.1016/0065-2571(77)90026-7. [DOI] [PubMed] [Google Scholar]
  19. Lacy P. E., Kostianovsky M. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes. 1967 Jan;16(1):35–39. doi: 10.2337/diab.16.1.35. [DOI] [PubMed] [Google Scholar]
  20. Lenzen S. Effects of alpha-ketocarboxylic acids and 4-pentenoic acid on insulin secretion from the perfused rat pancreas. Biochem Pharmacol. 1978 May 1;27(9):1321–1324. doi: 10.1016/0006-2952(78)90114-4. [DOI] [PubMed] [Google Scholar]
  21. MEISTER A. Enzymatic preparation of alpha-keto acids. J Biol Chem. 1952 May;197(1):309–317. [PubMed] [Google Scholar]
  22. Malaisse W. J., Boschero A. C., Kawazu S., Hutton J. C. The stimulus secretion coupling of glucose-induced insulin release. XXVII. Effect of glucose on K+ fluxes in isolated islets. Pflugers Arch. 1978 Mar 20;373(3):237–242. doi: 10.1007/BF00580830. [DOI] [PubMed] [Google Scholar]
  23. Malaisse W. J., Brisson G., Malaisse-Lagae F. The stimulus-secretion coupling of glucose-induced insulin release. I. Interaction of epinephrine and alkaline earth cations. J Lab Clin Med. 1970 Dec;76(6):895–902. [PubMed] [Google Scholar]
  24. Malaisse W. J., Malaisse-Lagae F., Walker M. O., Lacy P. E. The stimulus-secretion coupling of glucose-induced insulin release. V. The participation of a microtubular-microfilamentous system. Diabetes. 1971 May;20(5):257–265. doi: 10.2337/diab.20.5.257. [DOI] [PubMed] [Google Scholar]
  25. Malaisse W. J., Sener A., Mahy M. The stimulus-secretion coupling of glucose-induced insulin release. Sorbitol metabolism in isolated islets. Eur J Biochem. 1974 Sep 1;47(2):365–370. doi: 10.1111/j.1432-1033.1974.tb03701.x. [DOI] [PubMed] [Google Scholar]
  26. Noda C., Ichihara A. Control of ketogenesis from amino acids. II. Ketone bodies formation from alpha-ketoisocaproate, the keto-analogue of leucine, by rat liver mitochondria. J Biochem. 1974 Nov;76(5):1123–1130. [PubMed] [Google Scholar]
  27. Panten U. Effects of alpha-ketomonocarboxylic acids upon insulin secretion and metabolism of isolated pancreatic islets. Naunyn Schmiedebergs Arch Pharmacol. 1975;291(4):405–420. doi: 10.1007/BF00501798. [DOI] [PubMed] [Google Scholar]
  28. Panten U., Kriegstein E. v., Poser W., Schönborn J., Hasselblatt A. Effects of L-leucine and alpha-ketoisocaproic acid upon insulin secretion and metabolism of isolated pancreatic islets. FEBS Lett. 1972 Feb 1;20(2):225–228. doi: 10.1016/0014-5793(72)80801-9. [DOI] [PubMed] [Google Scholar]
  29. Sener A., Kawazu S., Hutton J. C., Boschero A. C., Devis G., Somers G., Herchuelz A., Malaisse W. J. The stimulus-secretion coupling of glucose-induced insulin release. Effect of exogenous pyruvate on islet function. Biochem J. 1978 Oct 15;176(1):217–232. doi: 10.1042/bj1760217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sener A., Malaisse W. J. Measurement of lactic acid in nanomolar amounts. Reliability of such a method as an index of glycolysis in pancreatic islets. Biochem Med. 1976 Feb;15(1):34–41. doi: 10.1016/0006-2944(76)90072-7. [DOI] [PubMed] [Google Scholar]
  31. WADDELL W. J., BUTLER T. C. Calculation of intracellular pH from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO); application to skeletal muscle of the dog. J Clin Invest. 1959 May;38(5):720–729. doi: 10.1172/JCI103852. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES