Skip to main content
SpringerLink
Log in

Dual action of neutral sphingomyelinase on rat hepatocytes: Activation of cholesteryl ester metabolism and biliary cholesterol secretion and inhibition of VLDL secretion

  • Articles
  • Published:
Lipids

Abstract

To address the role of cell membrane neutral sphingomyelinase (EC 3.1.4.12; SMase) in the regulation of cholesterol metabolism in the liver parenchymal cell, we examined the effect of exogenous neutral SMase on the metabolism of cholesteryl esters and the secretion of VLDL and biliary lipids in isolated rat hepatocytes. We show that treatment of hepatocytes with SMase (20 mU/mL) resulted in the intracellular buildup of cholesteryl esters, increased ACAT (EC 2.3.1.26) activity without affecting the ACAT2 mRNA level, and increased cytosolic and microsomal cholesteryl ester hydrolase (EC 3.1.1.13) activity. This was accompanied by increases in the secretion of biliary. bile acid, phospholipid, and cholesterol and in increased cholesterol 7α-hydroxylase (EC 1.14.13.17) activity and levels of mRNA, as well as decreased levels of apoB mRNA and a decreased secretion of VLDL apoB (apoB-48, ∼45%; apoB-100, ∼32%) and lipids (∼55%). Moreover, the VLDL particles secreted had an abnormal size and lipid composition; they were larger than controls, were relatively enriched in cholesteryl ester, and depleted in TG and cholesterol. Cell-permeable ceramides did not replicate any of the reported effects. These findings demonstrate that the increased cholesteryl ester turnover, oversecretion of biliary cholesterol and bile acids, and undersecretion of VLDL cholesterol and particles are concerted responses of the primary hepatocytes to exogenous neutral SMase brought about by regulation at several levels. We suggest that plasma membrane neutral SMase may have a specific, ceramide-independent effect in the regulation of cholesterol out-put pathways in hepatocytes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

ApoB:

apolipoprotein B

cCEH:

cytosolic cholesteryl oster hydrolase

CE:

cholesteryl ester

CEH:

cholesteryl ester hydrolase

C2-ceramide:

d-erythro-N-hexanoylsphingosine

C6-ceramide:

d-erythro-N-acetylsphingosine

CYP7A:

cholesterol 7α-hydroxylase

ER:

endoplasmic reticulum

erCEH:

endoplasmic reticulum cholesteryl ester hydrolase

FC:

ree cholesterol

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

MTP:

microsomal TG transfer protein

SCAP:

SREBP cleavage-activating protein

SMase:

sphingomyelinase

SREBP:

sterol regulatory element-binding protein

TNF-α:

tumor necrosis factor-α

References

  1. Chatterjee, S. (1999) Neutral Sphingomyelinase: Past, Present, and Future, Chem. Phys. Lipids 102, 79–96.

    Article  PubMed  CAS  Google Scholar 

  2. Liu, B., Obeid, L.M., and Hannun, Y.A. (1997) Sphingomyelinases in Cell Regulation, Semin. Cell Dev. Biol. 8, 311–322.

    Article  PubMed  CAS  Google Scholar 

  3. Ohvo-Rekilä, H., Ramstedt, B., Leppimäki, P., and Slotte, J.P. (2002) Cholesterol Interactions with Phospholipids in Membranes, Progr. Lipid Res. 41, 66–97.

    Article  Google Scholar 

  4. Slotte, J.P., and Bierman, E.L. (1988) Depletion of Plasma Membrane Sphingomyelin Rapidly Alters the Distribution of Cholesterol Between Plasma Membranes and Intracellular Cholesterol Pools in Cultured Fibroblasts, Biochem. J. 250, 653–658.

    PubMed  CAS  Google Scholar 

  5. Gupta, A.K., and Rudney, H. (1991) Plasma Membrane Sphingomyelin and the Regulation of HMG-CoA Reductase Activity and Cholesterol Biosynthesis in Cell Cultures, J. Lipid Res. 32, 125–136.

    PubMed  CAS  Google Scholar 

  6. Scheek, S., Brown, M.S., and Goldstein, J.L. (1997) Sphingomyelin Depletion in Cultured Cells Blocks Proteolysis of Stero Regulatory Element Binding Proteins at Site 1, Proc. Natl. Acad. Sci. USA 94, 11179–11183.

    Article  PubMed  CAS  Google Scholar 

  7. Lawler, J.F., Yin, M., Diehl, A.M., Roberts, E., and Chatterjee, S. (1998) Tumor Necrosis Factor-α Stimulates the Maturation of Sterol Regulatory Element Binding Protein-1 in Human Hepatocytes Through the Action of Neutral Sphingomyelinase, J. Biol. Chem. 273, 5053–5059.

    Article  PubMed  CAS  Google Scholar 

  8. Chang, T.Y., Chang, C.C.Y., and Cheng, D. (1997) Acyl-Coenzyme A: Cholesterol Acyltransferase, Annu. Rev. Biochem. 66, 613–638.

    Article  PubMed  CAS  Google Scholar 

  9. Chang, C.C.Y., Lee, C.Y.G., Chang, E.T., Cruz, J.C., Levesque, M.C., and Chang, T.Y. (1998) Recombinant Acyl-CoA: Cholesterol Acyltransferase-1 (ACAT-1) Purified to Essential Homogeneity Utilizes Cholesterol in Mixed Micelles or in Vesicles in a Highly Cooperative Manner, J. Biol. Chem. 273, 35132–35141.

    Article  PubMed  CAS  Google Scholar 

  10. Cases, S., Novak, S., Zheng, Y.W., Myers, T.A., Lear, S.R., Sande, E., Welch, C.B., Lusis, A.J., Spencer, T.A., Krause, B.R. et al. (1998) ACAT-2, a Second Mammalian Acyl-CoA: Cholesterol Acyltransferase. Its Cloning, Expression, and Characterization, J. Biol. Chem. 273, 26755–26764.

    Article  PubMed  CAS  Google Scholar 

  11. Buhman, K.K., Chen, H.C., and Farese, R.V. (2001) The Enzymes of Neutral Lipid Synthesis, J. Biol. Chem. 276, 40369–40372.

    Article  PubMed  CAS  Google Scholar 

  12. Carr, T.P., Hamilton, R.L., and Rudel, L.L. (1995) ACAT Inhibitors Decrease Secretion of Cholesteryl Esters and Apolipoprotein B by Perfused Livers of African Green Monkeys, J. Lipid Res. 36, 25–36.

    PubMed  CAS  Google Scholar 

  13. Ghosh, S., Mallonee, D.H., Hylemon, P.B., and Grogan, W.M. (1995) Molecular Cloning and Expression of Rat Hepatic Neutral Cholesteryl Ester Hydrolase, Biochim. Biophys. Acta 1259, 305–312.

    PubMed  Google Scholar 

  14. Fresnedo, O., López de Heredia, M., Martínez, M.J., Cristóbal, S., Rejas, M.T., Cuezva, J.M., and Ochoa, B. (2001) Immunolocalization of a Novel Cholesteryl Ester Hydrolase in the Endoplasmic Reticulum of Murine and Human Hepatocytes, Hepatology 33, 662–667.

    Article  PubMed  CAS  Google Scholar 

  15. Teng, B., Burant, C.F., and Davidson, N.O. (1993) Molecular Cloning of an Apolipoprotein B Messenger RNA Editing Protein, Science 260, 1816–1819.

    Article  PubMed  CAS  Google Scholar 

  16. Wang, S., McLeod, R.S., Gordon, D.A., and Yao, Z. (1996) The Microsomal Triacylglycerol Transfer Protein Facilitates Assembly and Secretion of Apolipoprotein B-Containing Lipoproteins and Decreases Cotranslational Degradation of Apolipoprotein B in Transfected COS-7 Cells, J. Biol. Chem. 271, 14124–14133.

    Article  PubMed  CAS  Google Scholar 

  17. Benoist, F., and Grand-Perret, T. (1997) Co-translational Degradation of Apolipoprotein B-100 by the Proteasome Is Prevented by Microsomal Triacylglycerol Transfer Protein, J. Biol. Chem. 272, 20435–20442.

    Article  PubMed  CAS  Google Scholar 

  18. Wu, X., Sakata, N., Dixon, J., and Ginsberg, H.N. (1994) Exogenous VLDL Stimulates Apolipoprotein B Secretion from HepG2 Cells by Both Pre- and Posttranslational Mechanisms, J. Lipid Res. 35, 1200–1210.

    PubMed  CAS  Google Scholar 

  19. Benoist, F., and Grand-Perret, T. (1996) ApoB-100 Secretion by HepG2 Cells Is Regulated by the Rate of Triacylglycerol Biosynthesis but Not by Intracellular Lipid Pools, Arterioscler. Thromb. Vasc. Biol. 16, 1229–1235.

    PubMed  CAS  Google Scholar 

  20. Khan, B., Wilcox, H.G., and Heimberg, M. (1989) Cholesterol Is Required for the Secretion of Very Low Density Lipoproteins by Rat Liver, Biochem. J. 258, 807–816.

    PubMed  CAS  Google Scholar 

  21. Cianflone, K.M., Yasruel, Z., Rodriguez, M.A., Vas, D., and Sniderman, A.D. (1990) Regulation of ApoB Secretion from HepG2 Cells: Evidence for a Critical Role for Cholesteryl Ester Synthesis in the Response to a Fatty Acid Challenge, J. Lipid Res. 31, 2045–2055.

    PubMed  CAS  Google Scholar 

  22. Cartwright, I.J., Higgins, J.A., Wilkinson, J., Bellavia, S., Kendrick, J.S., and Graham, J.M. (1997) Investigation of the Role of Lipids in the Assembly of Very Low Density Lipoproteins in Rabbit Hepatocytes, J. Lipid Res. 38, 531–545.

    PubMed  CAS  Google Scholar 

  23. Isusi, E., Aspichueta, P., Liza, M., Hernández, M.L., Díaz, C., Hernández, G., Martínez, M.J., and Ochoa, B. (2000) Short- and Long-Term Effects of Atorvastatin, Lovastatin and Simvastatin on the Cellular Metabolism of Cholesteryl Esters and VLDL Secretion in Rat Hepatocytes, Atherosclerosis 153, 283–294.

    Article  PubMed  CAS  Google Scholar 

  24. Funatsu, T., Suzuki, K., Goto, M., Arai, Y., Kakuta, H., Tanaka, H., Yasuda, S., Ida, M., Nishijima, S., and Miyata, K. (2001) Prolonged Inhibition of Cholesterol Synthesis by Atorvastatin Inhibits apoB-100 and Triacylglycerol Secretion from HepG2 Cells, Atherosclerosis 157, 107–115.

    Article  PubMed  CAS  Google Scholar 

  25. Yao, Z., and Vance, D.E. (1988) The Active Synthesis of Phosphatidylcholine Is Required for Very Low Density Lipoprotein Secretion from Rat Hepatocytes, J. Biol. Chem. 263, 2998–3004.

    PubMed  CAS  Google Scholar 

  26. Björkhem, I. (1985) Mechanism of Bile Acid Biosynthesis in Mammalian Liver, in Sterols and Bile Acids (Danielsson H., and Sjövall, J., eds.), pp. 231–278, Elsevier, Amsterdam.

    Google Scholar 

  27. Chawla, A., Saez, E., and Evans, R.N. (2000) Don’t Know Much Bile-ology, Cell 103, 1–4.

    Article  PubMed  CAS  Google Scholar 

  28. Brown, M.S., and Goldstein, J.L. (1997) The SREBP Pathway: Regulation of Cholesterol Metabolism by Proteolysis of a Membrane-Bound Transcription Factor, Cell 89, 331–340.

    Article  PubMed  CAS  Google Scholar 

  29. Hua, X., Nohturfft, A., Goldstein, J.L., and Brown, M.S. (1996) Sterol Resistance in CHO Cells Traced to Point Mutation in SREBP Cleavage-Activating Protein, Cell 87, 415–426.

    Article  PubMed  CAS  Google Scholar 

  30. Ruiz, M.B., Ochoa, B., and Lacort, M. (1990) Glucagon- and Dibutyryl Cyclic AMP-Produced Inhibition of Cholesterol Ester Hydrolase in Isolated Rat Hepatocytes: Role of Calcium, J. Biochem. 107, 476–479.

    PubMed  CAS  Google Scholar 

  31. Vance, D.E., Weinstein, D.B., and Steinberg, D. (1984) Isolation and Analysis of Lipoproteins Secreted by Rat Liver Hepatocytes, Biochim. Biophys. Acta 792, 39–47.

    PubMed  CAS  Google Scholar 

  32. Ruiz, J.I., and Ochoa, B. (1997) Quantification in the Subnanomolar Range of Phospholipids and Neutral Lipids by Monodimensional Thin-Layer Chromatography and Image Analysis, J. Lipid Res. 38, 1482–1489.

    PubMed  CAS  Google Scholar 

  33. Chico, Y., Fresnedo, O., Botham, K.M., Lacort, M., and Ochoa, B. (1996) Regulation of Bile Acid Synthesis by Estradiol and Progesterone in Primary Cultures of Rat Hepatocytes, Exp. Clin. Endocrinol. Diabetes 104, 137–144.

    Article  PubMed  CAS  Google Scholar 

  34. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory, New York.

    Google Scholar 

  35. Bradford, M.M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein Dye Binding, Anal. Biochem. 72, 248–254.

    Article  PubMed  CAS  Google Scholar 

  36. Liza, M., Romero, J.R., Chico, Y., Fresnedo, O., and Ochoa, B. (1996) Application of 2-Hydroxypropyl-β-Cyclodextrin in the Assay of Acyl-CoA: Cholesterol Acyltransferase and Neutral and Acid Cholesterol Ester Hydrolases, Lipids 31, 323–329.

    PubMed  CAS  Google Scholar 

  37. Martin, K.O., Budai, K., and Javitt, N.B. (1993) Cholesterol and 27-Hydroxycholesterol 7α-Hydroxylation: Evidence for Two Different Enzymes, J. Lipid Res. 34, 581–588.

    PubMed  CAS  Google Scholar 

  38. Yeagle, P.I., ed. (1988) Biology of Cholesterol, pp. 121–146, CRC Press, Boca Raton.

    Google Scholar 

  39. Eckhardt, E.R.M., van de Heijning, B.J.M., van Erpecum, K.J., Renooij, W., and vanBerge-Henegouwen, G.P. (1998) Quantitation of Cholesterol-Carrying Particles in Human Gallblader Bile, J. Lipid Res. 39, 594–603.

    PubMed  CAS  Google Scholar 

  40. Lange, Y., and Steck, T.L. (1997) Quantitation of the Pool of Cholesterol Associated with Acyl-CoA: Cholesterol Acyltransferase in Human Fibroblasts, J. Biol. Chem. 272, 13103–13108.

    Article  PubMed  CAS  Google Scholar 

  41. Ghosh, S., Natarajan, R., Pandak, W.M., Hylemon, P.B., and Grogan, W.M. (1998) Regulation of Hepatic Neutral Cholesteryl Ester Hydrolase by Hormones and Changes in Cholesterol Flux, Am. J. Physiol. 274, 662–668.

    Google Scholar 

  42. Natarajan, R., Ghosh, S., and Grogan, W.M. (1999) Regulation of the Rat Neutral Cytosolic Cholesteryl Ester Hydrolase Promoter by Hormones and Sterols: A Role for Nuclear Factor-Y in the Sterol-Mediated Response, J. Lipid. Res. 40, 2091–2098.

    PubMed  CAS  Google Scholar 

  43. Cristóbal, S., Ochoa, B., and Fresnedo, O. (1999) Purification and Properties of a Cholesteryl Ester Hydrolase from Rat Liver Microsomes, J. Lipid Res. 40, 715–725.

    PubMed  Google Scholar 

  44. Ochoa, B., Gee, A., Jackson, B., and Suckling, K.E. (1990) Regulation of Cholesterol Ester Metabolism in the Hamster Liver. Biochim. Biophys. Acta 1044, 133–138.

    PubMed  CAS  Google Scholar 

  45. Romero, J.R., Fresnedo, O., Isusi, E., Barrionuevo, J., and Ochoa, B. (1999) Hepatic Zonation of the Formation and Hydrolysis of Cholesteryl Esters in Periportal and Perivenous Parenchymal Cells, Lipids 34, 907–913.

    Article  PubMed  CAS  Google Scholar 

  46. Hannun, Y.A. (1996) Functions of Ceramide in Coordinating Cellular Responses to Stress, Science 274, 1855–1859.

    Article  PubMed  CAS  Google Scholar 

  47. Field, F.J., Chen, H., Born, E., Dixon, B., and Mathur, S. (1993) Release of Ceramide After Membrane Sphingomyelin Hydrolysis Decreases the Basolateral Secretion of Triacylglycerol and Apolipoprotein B in Cultured Human Intestinal Cells, J. Clin. Invest. 92, 2609–2619.

    Article  PubMed  CAS  Google Scholar 

  48. Davidson, N.O., and Shelness, G.S. (2000) Apolipoprotein B: mRNA Editing, Lipoprotein Assembly, and Presecretory Degradation, Annu. Rev. Nutr. 20, 169–193.

    Article  PubMed  CAS  Google Scholar 

  49. Elam, M.B., von Wronski, M.A., Cagen, L., Thorngate, F., Kummar, P., Heimberg, M., and Wilcox, H.G. (1999) Apolipoprotein B mRNA Editing and Apolipoprotein Gene Expression in the Liver of Hyperinsulinemic Fatty Zucker Rats: Relationship to Very Low Density Lipoprotein Composition, Lipids 34, 809–816.

    Article  PubMed  CAS  Google Scholar 

  50. Chan, L., Chang, B.H.J., Nakamuta, M., Li, W.H., and Smith, L.C. (1996) Apobec-1 and Apolipoprotein B mRNA Editing, Biochim. Biophys. Acta 1345, 11–26.

    Google Scholar 

  51. Cavallo, D., McLeod, R.S., Rudy, D., Aiton, A., Yau, Z., and Adeli, K. (1998) Intracellular Translocation and Stability of Apolipoprotein B Are Inversely Proportional to the Length of the Nascent Polypeptide, J. Biol. Chem. 273, 33397–33405.

    Article  PubMed  CAS  Google Scholar 

  52. Nilsson, I., Ohvo-Rekilä, H., Slotte, J.P., Johnson, A.E., and von Heijne, G. (2001) Inhibition of Protein Translocation Across the Endoplasmic Reticulum Membrane by Sterols, J. Biol. Chem. 276, 41748–41754.

    Article  PubMed  CAS  Google Scholar 

  53. Verkade, H.J., Fast, D.G., Rusiñol, A.E., Scraba, D.G., and Vance, D.E. (1993) Impaired Biosynthesis of Phosphatidylcholine Causes a Decrease in the Number of Very Low Density Lipoprotein Particles in the Golgi but Not in the Endoplasmic Reticulum of Rat Liver, J. Biol. Chem. 268, 24990–24996.

    PubMed  CAS  Google Scholar 

  54. Fast, D.G., and Vance, D.E. (1995) Nascent VLDL Phospholipid Composition Is Altered/when Phosphatidylcholine Biosynthesis Is Inhibited: Evidence for a Novel Mechanism That Regulates VLDL Secretion, Biochim. Biophys. Acta 1258, 159–168.

    PubMed  Google Scholar 

  55. Merrill, A.H., Lingrell, S., Wang, E., Nikolova-Karakashian, M., Vales, T.R., and Vance, D.E. (1995) Sphingolipid Biosynthesis de novo by Rat Hepatocytes in Culture, J. Biol. Chem. 270, 13834–13841.

    Article  PubMed  CAS  Google Scholar 

  56. Russell, D.W. (1999) Nuclear Orphan Receptors Control Cholesterol Metabolism, Cell 97, 539–542.

    Article  PubMed  CAS  Google Scholar 

  57. Post, S.M., Zoeteweij, J.P., Bos, M.H.A., de Wit, E.C.M., Havinga, R., Kuipers, F., and Princen, H.M.G. (1999) Acyl-Coenzyme A: Cholesterol Acyltransferase Inhibitor, Avasimibe, Stimulates Bile Acid Synthesis and Cholesterol 7α-Hydroxylase in Cultured Rat Hepatocytes and in vivo in the Rat, Hepatology 30, 491–500.

    Article  PubMed  CAS  Google Scholar 

  58. Slife, C.W., Wang, E., Hunter, R., Wang, S., Burgess, C., Liotta, D.C., and Merrill, A.H. (1989) Free Sphingosine Formation from Endogenous Substrates by a Liver Plasma Membrane System with a Divalent Cation Dependence and a Neutral pH Optimum, J. Biol. Chem. 264, 10371–10377.

    PubMed  CAS  Google Scholar 

  59. Chatterjee, S. (1993) Neutral Sphingomyelinase, Adv. Lipid Res. 26, 25–48.

    PubMed  CAS  Google Scholar 

  60. Tomiuk, S., Zunbansen, M., and Stoffel, W. (2000) Characterization and Subcellular Localization of Murine and Human, Magnesium-Dependent Neutral Sphingomyelinase, J. Biol. Chem. 275, 5710–5717.

    Article  PubMed  CAS  Google Scholar 

  61. Alessenko, A., and Chatterjee, S. (1995) Neutral Sphingomyelinase: Localization in Rat Liver Nuclei Acid Involvement in Regeneration/Proliferation, Mol. Cell. Biochem. 143, 169–174.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Physiology, University of the Basque Country Medical School, P.B. Box 699, 48080, Bilbao, Spain

    Mariana Liza, Yolanda Chico, Olatz Fresnedo & Begoña Ochoa

Authors
  1. Mariana Liza
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yolanda Chico
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Olatz Fresnedo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Begoña Ochoa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Begoña Ochoa.

About this article

Cite this article

Liza, M., Chico, Y., Fresnedo, O. et al. Dual action of neutral sphingomyelinase on rat hepatocytes: Activation of cholesteryl ester metabolism and biliary cholesterol secretion and inhibition of VLDL secretion. Lipids 38, 53–63 (2003). https://doi.org/10.1007/s11745-003-1031-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11745-003-1031-y

Keywords

Search

Navigation