Skip to main content
SpringerLink
Log in

Niemann-Pick C2 Protein Expression Regulates Lithogenic Diet-Induced Gallstone Formation and Dietary Cholesterol Metabolism in Mice

  • Original Article
  • Published:
Lipids

Abstract

Niemann-Pick C2 protein (NPC2) is a lysosomal soluble protein that is highly expressed in the liver; it binds to cholesterol and is involved in intracellular cholesterol trafficking, allowing the exit of lysosomal cholesterol obtained via the lipoprotein endocytic pathway. Thus, this protein may play an important role in controlling hepatic cholesterol transport and metabolism. The aim of this work was to study the relevance of NPC2 protein expression in hepatic cholesterol metabolism, biliary lipid secretion and gallstone formation by comparing NPC2 hypomorph [NPC2 (h/h)] and wild-type mice fed control, 2% cholesterol, and lithogenic diets. NPC2 (h/h) mice exhibited resistance to a diet-induced increase in plasma cholesterol levels. When consuming the chow diet, we observed increased biliary cholesterol and phospholipid secretions in NPC2 (h/h) mice. When fed the 2% cholesterol diet, NPC2 (h/h) mice exhibited low and high gallbladder bile cholesterol and phospholipid concentrations, respectively. NPC2 (h/h) mice fed with the lithogenic diet showed reduced biliary cholesterol secretion, gallbladder bile cholesterol saturation, and cholesterol crystal and gallstone formation. This work indicates that hepatic NPC2 expression is an important factor in the regulation of diet-derived cholesterol metabolism and disposal as well as in diet-induced cholesterol gallstone formation in mice.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

ABC:

ATP-binding cassette

HDL:

High density lipoproteins

LDL:

Low density lipoproteins

LDLR:

LDL receptor

NPC:

Niemann-Pick type C

NPC1:

Niemann-Pick C1 protein

NPC2:

Niemann-Pick C2 protein

NPC2 (h/h) mice:

NPC2 hypomorph BALB/c mice

SR-BI:

Scavenger receptor class B, type I

VLDL:

Very low-density lipoproteins

Wild-type mice:

Control wild-type BALB/c mice

References

  1. Patterson MC, Vanier MT, Suzuki K et al (2001) The metabolic and molecular basis of inherited disease: Niemann-Pick disease type C. A lipid trafficking disorder. In: Valle D, Beaudet AL, Vogelstein B, Kinzler KW, Antonarakis SE, Ballabio A, Scriver CR, Childs B, Sly WS (eds) The online metabolic & molecular bases of inherited disease. McGraw-Hill, New York

    Google Scholar 

  2. Liscum L, Sturley SL (2004) Intracellular trafficking of Niemann-Pick C proteins 1 and 2: obligate components of subcellular lipid transport. Biochim Biophys Acta 1685:22–27

    PubMed  CAS  Google Scholar 

  3. Loftus SK, Morris JA, Carstea ED, Gu JZ, Cummings C, Brown A, Ellison J, Ohno K, Rosenfeld MA, Tagle DA, Pentchev PG, Pavan WJ (1997) Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science 277:232–235

    Article  PubMed  CAS  Google Scholar 

  4. Naureckiene S, Sleat DE, Lackland H, Fensom A, Vanier MT, Wattiaux R, Jadot M, Lobel P (2000) Identification of HE1 as the second gene of Niemann-Pick C disease. Science 290:2298–2301

    Article  PubMed  CAS  Google Scholar 

  5. Sleat DE, Wiseman JA, El-Banna M, Price SM, Verot L, Shen MM, Tint GS, Vanier MT, Walkley SU, Lobel P (2004) Genetic evidence for nonredundant functional cooperativity between NPC1 and NPC2 in lipid transport. Proc Natl Acad Sci USA 101:5886–5891

    Article  PubMed  CAS  Google Scholar 

  6. Okamura N, Kiuchi S, Tamba M, Kashima T, Hiramoto S, Baba T, Dacheux F, Dacheux JL, Sugita Y, Jin YZ (1999) A porcine homolog of the major secretory protein of human epididymis, HE1, specifically binds cholesterol. Biochim Biophys Acta 1438:377–387

    PubMed  CAS  Google Scholar 

  7. Friedland N, Liou HL, Lobel P, Stock AM (2003) Structure of a cholesterol-binding protein deficient in Niemann-Pick type C2 disease. Proc Natl Acad Sci USA 100:2512–2517

    Article  PubMed  CAS  Google Scholar 

  8. Xu S, Benoff B, Liou HL, Lobel P, Stock AM (2007) Structural basis of sterol binding by NPC2, a lysosomal protein deficient in Niemann-Pick type C2 disease. J Biol Chem 282:23525–23531

    Article  PubMed  CAS  Google Scholar 

  9. Infante RE, Radhakrishnan A, Abi-Mosleh L, Kinch LN, Wang ML, Grishin NV, Goldstein JL, Brown MS (2008) Purified NPC1 protein: II. Localization of sterol binding to a 240-amino acid soluble luminal loop. J Biol Chem 283:1064–1075

    Article  PubMed  CAS  Google Scholar 

  10. Infante RE, Wang ML, Radhakrishnan A, Kwon HJ, Brown MS, Goldstein JL (2008) NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes. Proc Natl Acad Sci USA 105:15287–15292

    Article  PubMed  CAS  Google Scholar 

  11. Kwon HJ, Abi-Mosleh L, Wang ML, Deisenhofer J, Goldstein JL, Brown MS, Infante RE (2009) Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol. Cell 137:1213–1224

    Article  PubMed  Google Scholar 

  12. Chikh K, Vey S, Simonot C, Vanier MT, Millat G (2004) Niemann-Pick type C disease: importance of N-glycosylation sites for function and cellular location of the NPC2 protein. Mol Genet Metab 83:220–230

    Article  PubMed  CAS  Google Scholar 

  13. Mutka AL, Lusa S, Linder MD, Jokitalo E, Kopra O, Jauhiainen M, Ikonen E (2004) Secretion of sterols and the NPC2 protein from primary astrocytes. J Biol Chem 279:48654–48662

    Article  PubMed  CAS  Google Scholar 

  14. Ong WY, Sundaram RK, Huang E, Ghoshal S, Kumar U, Pentchev PG, Patel SC (2004) Neuronal localization and association of Niemann Pick C2 protein (HE1/NPC2) with the postsynaptic density. Neuroscience 128:561–570

    Article  PubMed  CAS  Google Scholar 

  15. Klein A, Amigo L, Retamal MJ, Morales MG, Miquel JF, Rigotti A, Zanlungo S (2006) NPC2 is expressed in human and murine liver and secreted into bile: potential implications for body cholesterol homeostasis. Hepatology 43:126–133

    Article  PubMed  CAS  Google Scholar 

  16. Turley SD, Dietschy JM (1988) The metabolism and excretion of cholesterol by the liver. In: Arias IM, Jakoby WB, Popper H, Schachter D, Shafritz DA (eds) The liver: biology and pathology. Raven Press, New York

    Google Scholar 

  17. Dietschy JM, Turley SD, Spady DK (1993) Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans. J Lipid Res 34:1637–1659

    PubMed  CAS  Google Scholar 

  18. Zanlungo S, Rigotti A (2009) Determinants of transhepatic cholesterol flux and their relevance for gallstone formation. Liver Int 29:323–330

    Article  PubMed  CAS  Google Scholar 

  19. Miquel JF, Covarrubias C, Villaroel L, Mingrone G, Greco AV, Puglielli L, Carvallo P, Marshall G, Del Pino G, Nervi F (1998) Genetic epidemiology of cholesterol cholelithiasis among Chilean Hispanics, Amerindians, and Maoris. Gastroenterology 115:937–946

    Article  PubMed  CAS  Google Scholar 

  20. Lammert F, Miquel JF (2008) Gallstone disease: from genes to evidence-based therapy. J Hepatol 48(Suppl 1):S124–S135

    Article  PubMed  CAS  Google Scholar 

  21. Amigo L, Mendoza H, Castro J, Quinones V, Miquel JF, Zanlungo S (2002) Relevance of Niemann-Pick type C1 protein expression in controlling plasma cholesterol and biliary lipid secretion in mice. Hepatology 36:819–828

    PubMed  CAS  Google Scholar 

  22. Morales MG, Amigo L, Balboa E, Acuna M, Castro J, Molina H, Miquel JF, Nervi F, Rigotti A, Zanlungo S (2010) Deficiency of Niemann-Pick C1 protein protects against diet-induced gallstone formation in mice. Liver Int 30:887–897

    Article  PubMed  CAS  Google Scholar 

  23. Amigo L, Quinones V, Mardones P, Zanlungo S, Miquel JF, Nervi F, Rigotti A (2000) Impaired biliary cholesterol secretion and decreased gallstone formation in apolipoprotein E-deficient mice fed a high-cholesterol diet. Gastroenterology 118:772–779

    Article  PubMed  CAS  Google Scholar 

  24. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC (1974) Enzymatic determination of total serum cholesterol. Clin Chem 20:470–475

    PubMed  CAS  Google Scholar 

  25. Baginski ES, Foa PP, Zak B (1967) Microdetermination of inorganic phosphate, phospholipids, and total phosphate in biologic materials. Clin Chem 13:326–332

    PubMed  CAS  Google Scholar 

  26. Talalay P (1960) Enzymic analysis of steroid hormones. Methods Biochem Anal 8:119–143

    Article  PubMed  CAS  Google Scholar 

  27. Carey MC (1978) Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 19:945–955

    PubMed  CAS  Google Scholar 

  28. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36

    Article  PubMed  Google Scholar 

  29. Wang DQ, Paigen B, Carey MC (1997) Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: physical–chemistry of gallbladder bile. J Lipid Res 38:1395–1411

    PubMed  CAS  Google Scholar 

  30. Lee FY, Lee H, Hubbert ML, Edwards PA, Zhang Y (2006) FXR, a multipurpose nuclear receptor. Trends Biochem Sci 31:572–580

    Article  PubMed  CAS  Google Scholar 

  31. Repa JJ, Berge KE, Pomajzl C, Richardson JA, Hobbs H, Mangelsdorf DJ (2002) Regulation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 by the liver × receptors alpha and beta. J Biol Chem 277:18793–18800

    Article  PubMed  CAS  Google Scholar 

  32. Rege RV, Prystowsky JB (1998) Inflammation and a thickened mucus layer in mice with cholesterol gallstones. J Surg Res 74:81–85

    Article  PubMed  CAS  Google Scholar 

  33. Dixit SS, Sleat DE, Stock AM, Lobel P (2007) Do mammalian NPC1 and NPC2 play a role in intestinal cholesterol absorption? Biochem J 408:1–5

    Article  PubMed  CAS  Google Scholar 

  34. Uronen RL, Lundmark P, Orho-Melander M, Jauhiainen M, Larsson K, Siegbahn A, Wallentin L, Zethelius B, Melander O, Syvanen AC, Ikonen E (2010) Niemann-Pick C1 modulates hepatic triglyceride metabolism and its genetic variation contributes to serum triglyceride levels. Arterioscler Thromb Vasc Biol 30:1614–1620

    Article  PubMed  CAS  Google Scholar 

  35. Xie C, Turley SD, Pentchev PG, Dietschy JM (1999) Cholesterol balance and metabolism in mice with loss of function of Niemann-Pick C protein. Am J Physiol 276:E336–E344

    PubMed  CAS  Google Scholar 

  36. Goldman SD, Krise JP (2010) Niemann-Pick C1 functions independently of Niemann-Pick C2 in the initial stage of retrograde transport of membrane-impermeable lysosomal cargo. J Biol Chem 285:4983–4994

    Article  PubMed  CAS  Google Scholar 

  37. van Helvoort A, de Brouwer A, Ottenhoff R, Brouwers JF, Wijnholds J, Beijnen JH, Rijneveld A, van der Poll T, van der Valk MA, Majoor D, Voorhout W, Wirtz KW, Elferink RP, Borst P (1999) Mice without phosphatidylcholine transfer protein have no defects in the secretion of phosphatidylcholine into bile or into lung airspaces. Proc Natl Acad Sci USA 96:11501–11506

    Article  PubMed  Google Scholar 

  38. Wu MK, Hyogo H, Yadav SK, Novikoff PM, Cohen DE (2005) Impaired response of biliary lipid secretion to a lithogenic diet in phosphatidylcholine transfer protein-deficient mice. J Lipid Res 46:422–431

    Article  PubMed  CAS  Google Scholar 

  39. Kanno K, Wu MK, Scapa EF, Roderick SL, Cohen DE (2007) Structure and function of phosphatidylcholine transfer protein (PC-TP)/StarD2. Biochim Biophys Acta 1771:654–662

    PubMed  CAS  Google Scholar 

  40. Scapa EF, Pocai A, Wu MK, Gutierrez-Juarez R, Glenz L, Kanno K, Li H, Biddinger S, Jelicks LA, Rossetti L, Cohen DE (2008) Regulation of energy substrate utilization and hepatic insulin sensitivity by phosphatidylcholine transfer protein/StarD2. FASEB J 22:2579–2590

    Article  PubMed  CAS  Google Scholar 

  41. Kang HW, Wei J, Cohen DE (2010) PC-TP/StARD2: of membranes and metabolism. Trends Endocrinol Metab 21:449–456

    Article  PubMed  CAS  Google Scholar 

  42. Ross PE, Butt AN, Gallacher C (1990) Cholesterol absorption by the gall bladder. J Clin Pathol 43:572–575

    Article  PubMed  CAS  Google Scholar 

  43. Frolov A, Zielinski SE, Crowley JR, Dudley-Rucker N, Schaffer JE, Ory DS (2003) NPC1 and NPC2 regulate cellular cholesterol homeostasis through generation of low density lipoprotein cholesterol-derived oxysterols. J Biol Chem 278:25517–25525

    Article  PubMed  CAS  Google Scholar 

  44. Zhang JR, Coleman T, Langmade SJ, Scherrer DE, Lane L, Lanier MH, Feng C, Sands MS, Schaffer JE, Semenkovich CF, Ory DS (2008) Niemann-Pick C1 protects against atherosclerosis in mice via regulation of macrophage intracellular cholesterol trafficking. J Clin Invest 118:2281–2290

    PubMed  CAS  Google Scholar 

  45. Wang MD, Franklin V, Sundaram M, Kiss RS, Ho K, Gallant M, Marcel YL (2007) Differential regulation of ATP binding cassette protein A1 expression and ApoA-I lipidation by Niemann-Pick type C1 in murine hepatocytes and macrophages. J Biol Chem 282:22525–22533

    Article  PubMed  CAS  Google Scholar 

  46. Yamanashi Y, Takada T, Yoshikado T, Shoda J, Suzuki H (2011) NPC2 regulates biliary cholesterol secretion via stimulation of ABCG5/G8-mediated cholesterol transport. Gastroenterology 140:1664–1674

    Article  PubMed  CAS  Google Scholar 

  47. Wang ML, Motamed M, Infante RE, Abi-Mosleh L, Kwon HJ, Brown MS, Goldstein JL (2010) Identification of surface residues on Niemann-pick C2 essential for hydrophobic Handoff of cholesterol to NPC1 in lysosomes. Cell Metab 12:166–173

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank Dr. Juan Francisco Miquel for helpful discussions. We also acknowledge the Humboldt Foundation (Germany) for donating the ABI 7500 sequence detection system used for gene expression analysis. Finally, Dr. David Sleat and Dr. Peter Lobel generously provided NPC2 (h/h) mice used in this study. This work was supported by the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT Grant # 1070622), FONDAP 15090007 and Dirección de Investigación, Facultad de Medicina, UC (Programa Inmersión de Verano, Grant to PA).

Author information

Authors and Affiliations

  1. Departmento de Gastroenterología, Escuela de Medicina, Facultad de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 367, Casilla 114-D, Santiago, Chile

    Elisa Balboa, Gabriela Morales, Paula Aylwin, Ludwig Amigo, Juan Castro, Attilio Rigotti & Silvana Zanlungo

  2. Fundación Hospital Parroquial de San Bernardo, Santiago, Chile

    Gonzalo Carrasco

  3. Departamento de Nutrición, Diabetes y Metabolismo, Pontificia Universidad Católica de Chile, Santiago, Chile

    Attilio Rigotti

  4. Fondap-Center of Genome Regulation, Santiago, Chile

    Silvana Zanlungo

Authors
  1. Elisa Balboa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gabriela Morales
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paula Aylwin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gonzalo Carrasco
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ludwig Amigo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Juan Castro
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Attilio Rigotti
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Silvana Zanlungo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Silvana Zanlungo.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 27 kb)

About this article

Cite this article

Balboa, E., Morales, G., Aylwin, P. et al. Niemann-Pick C2 Protein Expression Regulates Lithogenic Diet-Induced Gallstone Formation and Dietary Cholesterol Metabolism in Mice. Lipids 47, 13–25 (2012). https://doi.org/10.1007/s11745-011-3625-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11745-011-3625-2

Keywords

Search

Navigation