Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Apr 1;323(Pt 1):17–21. doi: 10.1042/bj3230017

Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion.

M R Owen 1, C C Corstorphine 1, V A Zammit 1
PMCID: PMC1218291  PMID: 9173878

Abstract

The possibility that triacylglycerol (TAG) synthesis occurs on both aspects of the endoplasmic-reticular membrane during the process of incorporation of TAG into secreted very-low-density lipoprotein (VLDL) [Zammit (1996) Biochem. J. 314, 1-14] was investigated by measuring the latency of diacylglycerol acyltransferase (DGAT) in microsomal fractions obtained from rat liver homogenates. Permeabilization of microsomes with taurocholate resulted in the doubling of the activity, indicating that DGAT activities of approximately equal magnitude occur on either aspect of the microsomal membrane. The taurocholate concentrations required for exposure of the latent activity of DGAT were identical with those that resulted in the exposure of marker enzymes for the lumen of the endoplasmic reticulum. Fractionation of the microsomes into smooth and rough populations indicated that the distribution of overt and latent DGAT activities was the same throughout. The possibility that taurocholate effects may result from non-specific activation of the overt enzyme was excluded by employing the channel-forming peptide alamethicin to effect permeabilization, and by varying the mode of delivery of diacylglycerol substrate to the microsomal membranes. Permeabilization using alamethicin gave a slightly higher latent/overt ratio for DGAT. The possible roles of overt and latent DGAT activities in the synthesis and secretion of TAG by the liver are discussed.

Full Text

The Full Text of this article is available as a PDF (365.0 KB).

Selected References

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

  1. Alexander C. A., Hamilton R. L., Havel R. J. Subcellular localization of B apoprotein of plasma lipoproteins in rat liver. J Cell Biol. 1976 May;69(2):241–263. doi: 10.1083/jcb.69.2.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allan D., Thomas P., Michell R. H. Rapid transbilayer diffusion of 1,2-diacylglycerol and its relevance to control of membrane curvature. Nature. 1978 Nov 16;276(5685):289–290. doi: 10.1038/276289a0. [DOI] [PubMed] [Google Scholar]
  3. Bates E. J., Saggerson E. D. A study of the glycerol phosphate acyltransferase and dihydroxyacetone phosphate acyltransferase activities in rat liver mitochondrial and microsomal fractions. Relative distribution in parenchymal and non-parenchymal cells, effects of N-ethylmaleimide, palmitoyl-coenzyme A concentration, starvation, adrenalectomy and anti-insulin serum treatment. Biochem J. 1979 Sep 15;182(3):751–762. doi: 10.1042/bj1820751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bell R. M., Ballas L. M., Coleman R. A. Lipid topogenesis. J Lipid Res. 1981 Mar;22(3):391–403. [PubMed] [Google Scholar]
  5. Borén J., Rustaeus S., Olofsson S. O. Studies on the assembly of apolipoprotein B-100- and B-48-containing very low density lipoproteins in McA-RH7777 cells. J Biol Chem. 1994 Oct 14;269(41):25879–25888. [PubMed] [Google Scholar]
  6. Borén J., Wettesten M., Sjöberg A., Thorlin T., Bondjers G., Wiklund O., Olofsson S. O. The assembly and secretion of apoB 100 containing lipoproteins in Hep G2 cells. Evidence for different sites for protein synthesis and lipoprotein assembly. J Biol Chem. 1990 Jun 25;265(18):10556–10564. [PubMed] [Google Scholar]
  7. Broadway N. M., Saggerson E. D. Microsomal carnitine acyltransferases. Biochem Soc Trans. 1995 Aug;23(3):490–494. doi: 10.1042/bst0230490. [DOI] [PubMed] [Google Scholar]
  8. Broadway N. M., Saggerson E. D. Solubilization and separation of two distinct carnitine acyltransferases from hepatic microsomes: characterization of the malonyl-CoA-sensitive enzyme. Biochem J. 1995 Sep 15;310(Pt 3):989–995. doi: 10.1042/bj3100989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brown A. E., Lok M. P., Elovson J. Improved method for the isolation of rat liver plasma membrane. Biochim Biophys Acta. 1976 Mar 19;426(3):418–432. doi: 10.1016/0005-2736(76)90387-4. [DOI] [PubMed] [Google Scholar]
  10. Cartwright I. J., Higgins J. A. Intracellular events in the assembly of very-low-density-lipoprotein lipids with apolipoprotein B in isolated rabbit hepatocytes. Biochem J. 1995 Sep 15;310(Pt 3):897–907. doi: 10.1042/bj3100897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Coleman R., Bell R. M. Evidence that biosynthesis of phosphatidylethanolamine, phosphatidylcholine, and triacylglycerol occurs on the cytoplasmic side of microsomal vesicles. J Cell Biol. 1978 Jan;76(1):245–253. doi: 10.1083/jcb.76.1.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fukuda N., Azain M. J., Ontko J. A. Altered hepatic metabolism of free fatty acids underlying hypersecretion of very low density lipoproteins in the genetically obese Zucker rats. J Biol Chem. 1982 Dec 10;257(23):14066–14072. [PubMed] [Google Scholar]
  13. Gibbons G. F., Khurana R., Odwell A., Seelaender M. C. Lipid balance in HepG2 cells: active synthesis and impaired mobilization. J Lipid Res. 1994 Oct;35(10):1801–1808. [PubMed] [Google Scholar]
  14. Gibbons G. F., Wiggins D. Intracellular triacylglycerol lipase: its role in the assembly of hepatic very-low-density lipoprotein (VLDL). Adv Enzyme Regul. 1995;35:179–198. doi: 10.1016/0065-2571(94)00006-o. [DOI] [PubMed] [Google Scholar]
  15. Glaumann H., Bergstrand A., Ericsson J. L. Studies on the synthesis and intracellular transport of lipoprotein particles in rat liver. J Cell Biol. 1975 Feb;64(2):356–377. doi: 10.1083/jcb.64.2.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gordon D. A., Jamil H., Gregg R. E., Olofsson S. O., Borén J. Inhibition of the microsomal triglyceride transfer protein blocks the first step of apolipoprotein B lipoprotein assembly but not the addition of bulk core lipids in the second step. J Biol Chem. 1996 Dec 20;271(51):33047–33053. doi: 10.1074/jbc.271.51.33047. [DOI] [PubMed] [Google Scholar]
  17. Haude W., Wagner H., Theil S., Haase H., Hünicke G., Goetze E. Bestimmung der Umsätze und Flussraten von Fettsäuren und Cholesterin in Serum und Leber der Ratte mit Hilfe der mathematischen Simulierung von Isotopenverdünnungskurven am Analogrechner. Acta Biol Med Ger. 1972;28(6):963–975. [PubMed] [Google Scholar]
  18. Higgins J. A., Fieldsend J. K. Phosphatidylcholine synthesis for incorporation into membranes or for secretion as plasma lipoproteins by Golgi membranes of rat liver. J Lipid Res. 1987 Mar;28(3):268–278. [PubMed] [Google Scholar]
  19. Innerarity T. L., Borén J., Yamanaka S., Olofsson S. O. Biosynthesis of apolipoprotein B48-containing lipoproteins. Regulation by novel post-transcriptional mechanisms. J Biol Chem. 1996 Feb 2;271(5):2353–2356. doi: 10.1074/jbc.271.5.2353. [DOI] [PubMed] [Google Scholar]
  20. Jones L. R., Maddock S. W., Besch H. R., Jr Unmasking effect of alamethicin on the (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles. J Biol Chem. 1980 Oct 25;255(20):9971–9980. [PubMed] [Google Scholar]
  21. Kondrup J., Damgaard S. E., Fleron P. Metabolism of palmitate in perfused rat liver. Computer models of subcellular triacylglycerol metabolism. Biochem J. 1979 Oct 15;184(1):73–81. doi: 10.1042/bj1840073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lad P. J., White A. A. Effect of alamethicin, gramicidin S and melittin upon the particulate guanylate cyclase from rat lung. Biochim Biophys Acta. 1979 Sep 12;570(1):198–209. doi: 10.1016/0005-2744(79)90214-6. [DOI] [PubMed] [Google Scholar]
  23. Miller J. C., Weinhold P. A. Cholinephosphotransferase in rat lung. The in vitro synthesis of dipalmitoylphosphatidylcholine from dipalmitoylglycerol. J Biol Chem. 1981 Dec 25;256(24):12662–12665. [PubMed] [Google Scholar]
  24. Moir A. M., Park B. S., Zammit V. A. Quantification in vivo of the effects of different types of dietary fat on the loci of control involved in hepatic triacylglycerol secretion. Biochem J. 1995 Jun 1;308(Pt 2):537–542. doi: 10.1042/bj3080537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Munro H. N., Fleck A. Recent developments in the measurement of nucleic acids in biological materials. A supplementary review. Analyst. 1966 Feb;91(79):78–88. doi: 10.1039/an9669100078. [DOI] [PubMed] [Google Scholar]
  26. Murthy M. S., Pande S. V. Malonyl-CoA-sensitive and -insensitive carnitine palmitoyltransferase activities of microsomes are due to different proteins. J Biol Chem. 1994 Jul 15;269(28):18283–18286. [PubMed] [Google Scholar]
  27. Polokoff M. A., Bell R. M. Limited palmitoyl-CoA penetration into microsomal vesicles as evidenced by a highly latent ethanol acyltransferase activity. J Biol Chem. 1978 Oct 25;253(20):7173–7178. [PubMed] [Google Scholar]
  28. Swift L. L. Assembly of very low density lipoproteins in rat liver: a study of nascent particles recovered from the rough endoplasmic reticulum. J Lipid Res. 1995 Mar;36(3):395–406. [PubMed] [Google Scholar]
  29. Wiggins D., Gibbons G. F. Origin of hepatic very-low-density lipoprotein triacylglycerol: the contribution of cellular phospholipid. Biochem J. 1996 Dec 1;320(Pt 2):673–679. doi: 10.1042/bj3200673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wiggins D., Gibbons G. F. The lipolysis/esterification cycle of hepatic triacylglycerol. Its role in the secretion of very-low-density lipoprotein and its response to hormones and sulphonylureas. Biochem J. 1992 Jun 1;284(Pt 2):457–462. doi: 10.1042/bj2840457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Woolley G. A., Wallace B. A. Temperature dependence of the interaction of alamethicin helices in membranes. Biochemistry. 1993 Sep 21;32(37):9819–9825. doi: 10.1021/bi00088a037. [DOI] [PubMed] [Google Scholar]
  32. Yang L. Y., Kuksis A., Myher J. J., Steiner G. Contribution of de novo fatty acid synthesis to very low density lipoprotein triacylglycerols: evidence from mass isotopomer distribution analysis of fatty acids synthesized from [2H6]ethanol. J Lipid Res. 1996 Feb;37(2):262–274. [PubMed] [Google Scholar]
  33. Yang L. Y., Kuksis A., Myher J. J., Steiner G. Origin of triacylglycerol moiety of plasma very low density lipoproteins in the rat: structural studies. J Lipid Res. 1995 Jan;36(1):125–136. [PubMed] [Google Scholar]
  34. Zammit V. A. Role of insulin in hepatic fatty acid partitioning: emerging concepts. Biochem J. 1996 Feb 15;314(Pt 1):1–14. doi: 10.1042/bj3140001. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES