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Lipid droplet changes in proliferating and quiescent 3T3 fibroblasts

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Abstract

Lipid droplets (LDs) are fat-storing organelles present in virtually all eukaryotic cells and involved in many aspects of cell biology related to lipid metabolism and cholesterol homeostasis. In this study, we investigated the presence of LDs in proliferating and quiescent (contact-inhibited) 3T3 fibroblasts to verify a correlation with cell growth. LDs were characterized by Nile red staining, positivity to adipophilin and negativity to perilipin. LDs were numerous in proliferating cells, but very few in quiescent cells. However, the fraction of quiescent cells, which resumed proliferation after scratch-wound assay, also resumed the formation of LDs. In proliferating cells, the number of LDs correlated with the DNA content, suggesting a continuous accumulation of LDs during cell growth. These findings were supported by biochemical data showing much higher rates of cholesterol esterification and triglyceride synthesis in proliferating cells. Both filipin staining and the fluorescent cholesterol analog dehydroergosterol revealed the presence of an intense traffic of free cholesterol, mediated by acidic vesicles, in proliferating cells. Nile red ratiometric measurements revealed a different lipid composition of LDs in proliferating and quiescent cells. Changes in the number and composition of LDs were also found in growing cells treated with inhibitors of cholesterol esterification (Sandoz 58-035), endosomal cholesterol efflux (U18666A) and V-ATPase (bafilomycin-A1).

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References

  • Adelman SJ, Glick JM, Phillips MC, Rothblat GH (1984) Lipid composition and physical state effects on cellular cholesteryl ester clearance. J Biol Chem 259:13844–13850

    PubMed  CAS  Google Scholar 

  • Bartz R, Li WH, Venables B, Zehmer JK, Roth MR, Welti R, Anderson RG, Liu P, Chapman KD (2007) Lipidomics reveals that adiposomes store ether lipids and mediate phospholipid traffic. J Lipid Res 48:837–847

    Article  PubMed  CAS  Google Scholar 

  • Brown MS, Goldstein JL, Krieger M, Ho YK, Anderson RG (1979) Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins. J Cell Biol 82:597–613

    Article  PubMed  CAS  Google Scholar 

  • Brown WJ, Sullivan TR, Greenspan P (1992) Nile red staining of lysosomal phospholipid inclusions. Histochemistry 97:349–354

    Article  PubMed  CAS  Google Scholar 

  • Coller HA, Sang L, Roberts JM (2006) A new description of cellular quiescence. PLoS Biol 4:e83

    Article  PubMed  CAS  Google Scholar 

  • Delahunty TJ, Rubinstein D (1970) Accumulation and release of triglycerides by rat liver following partial hepatectomy. J Lipid Res 11:536–543

    PubMed  CAS  Google Scholar 

  • Dessì S, Batetta B (2004) Cholesterol esters and cell growth: coregulation in animal models. In: Pani A, Dessì S (eds) Cell growth and cholesterol esters. Kluwer Academic/Plenum Publishers, New York, pp 25–34 (also available at http://www.eurekah.com; published by Landes Bioscience, Georgetown)

    Google Scholar 

  • Fernandez MA, Albor C, Ingelmo-Torres M, Nixon SJ, Ferguson C, Kurzchalia T, Tebar F, Enrich C, Parton RG, Pol A (2006) Caveolin-1 is essential for liver regeneration. Science 313:1628–1632

    Article  PubMed  CAS  Google Scholar 

  • Furuchi T, Aikawa K, Arai H, Inoue K (1993) Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, blocks lysosomal cholesterol trafficking in macrophages. J Biol Chem 268:27345–27348

    PubMed  CAS  Google Scholar 

  • Glende EA Jr, Morgan WS (1968) Alteration in liver lipid and lipid fatty acid composition after partial hepatectomy in the rat. Exp Mol Pathol 8:190–200

    Article  PubMed  CAS  Google Scholar 

  • Glick JM, Adelman SJ, Rothblat GH (1987) Cholesteryl ester cycle in cultured hepatoma cells. Atherosclerosis 64:223–230

    Article  PubMed  CAS  Google Scholar 

  • Goldstein JL, Ho YK, Basu SK, Brown MS (1979) Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Natl Acad Sci USA 76:333–337

    Article  PubMed  CAS  Google Scholar 

  • Greenspan P, Fowler SD (1985) Spectrofluorometric studies of the lipid probe, nile red. J Lipid Res 26:781–789

    PubMed  CAS  Google Scholar 

  • Greenspan P, Mayer EP, Fowler SD (1985) Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100:965–973

    Article  PubMed  CAS  Google Scholar 

  • Kamada H, Sato K, Iwai M, Zhang WR, Nagano I, Manabe Y, Shoji M, Abe K (2003) Temporal and spatial changes of free cholesterol and neutral lipids in rat brain after transient middle cerebral artery occlusion. Neurosci Res 45:91–100

    Article  PubMed  CAS  Google Scholar 

  • Kellner-Weibel G, McHendry-Rinde B, Haynes MP, Adelman S (2001) Evidence that newly synthesized esterified cholesterol is deposited in existing cytoplasmic lipid inclusions. J Lipid Res 42:768–777

    PubMed  CAS  Google Scholar 

  • Lada AT, Willingham MC, St Clair RW (2002) Triglyceride depletion in THP-1 cells alters cholesteryl ester physical state and cholesterol efflux. J Lipid Res 43:618–628

    PubMed  CAS  Google Scholar 

  • Lange Y, Ye J, Rigney M, Steck TL (2002) Dynamics of lysosomal cholesterol in Niemann–Pick type C and normal human fibroblasts. J Lipid Res 43:198–204

    PubMed  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr JL, Randall RJ (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  • Martin S, Parton RG (2006) Lipid droplets: a unified view of a dynamic organelle. Nat Rev Mol Cell Biol 7:373–378

    Article  PubMed  CAS  Google Scholar 

  • McGookey DJ, Anderson RG (1983) Morphological characterization of the cholesteryl ester cycle in cultured mouse macrophage foam cells. J Cell Biol 97:1156–1168

    Article  PubMed  CAS  Google Scholar 

  • Mukherjee S, Zha X, Tabas I, Maxfield FR (1998) Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog. Biophys J 75:1915–1925

    Article  PubMed  CAS  Google Scholar 

  • Murphy DJ, Vance J (1999) Mechanisms of lipid-body formation. Trends Biochem Sci 24:109–115

    Article  PubMed  CAS  Google Scholar 

  • Pol A, Martin S, Fernandez MA, Ferguson C, Carozzi A, Luetterforst R, Enrich C, Parton RG (2004) Dynamic and regulated association of caveolin with lipid bodies: modulation of lipid body motility and function by a dominant negative mutant. Mol Biol Cell 15:99–110

    Article  PubMed  CAS  Google Scholar 

  • Ross AC, Go KJ, Heider JG, Rothblat GH (1984) Selective inhibition of acyl coenzyme A:cholesterol acyltransferase by compound 58-035. J Biol Chem 259:815–819

    PubMed  CAS  Google Scholar 

  • Sanna F, Bonatesta RR, Frongia B, Uda S, Banni S, Melis MP, Collu M, Madeddu C, Serpe R, Puddu S, Porcu G, Dessì S, Batetta B (2007) Production of inflammatory molecules in peripheral blood mononuclear cells from severely glucose-6-phosphate dehydrogenase-deficient subjects. J Vasc Res 44:253–263

    Article  PubMed  CAS  Google Scholar 

  • Shteyer E, Liao Y, Muglia LJ, Hruz PW, Rudnick DA (2004) Disruption of hepatic adipogenesis is associated with impaired liver regeneration in mice. Hepatology 40:1322–1332

    Article  PubMed  CAS  Google Scholar 

  • Smyth MJ, Wharton W (1992) Differentiation of A31T6 proadipocytes to adipocytes: a flow cytometric analysis. Exp Cell Res 199:29–38

    Article  PubMed  CAS  Google Scholar 

  • Vejux A, Kahn E, Dumas D, Bessede G, Menetrier F, Athias A, Riedinger JM, Frouin F, Stoltz JF, Ogier-Denis E, Todd-Pokropek A, Lizard G (2005) 7-Ketocholesterol favors lipid accumulation and colocalizes with Nile red positive cytoplasmic structures formed during 7-ketocholesterol-induced apoptosis: analysis by flow cytometry, FRET biphoton spectral imaging microscopy, and subcellular fractionation. Cytometry A 64:87–100

    PubMed  Google Scholar 

  • Watari H, Blanchette-Mackie EJ, Dwyer NK, Sun G, Glick JM, Patel S, Neufeld EB, Pentchev PG, Strauss JF 3rd (2000) NPC1-containing compartment of human granulosa-lutein cells: a role in the intracellular trafficking of cholesterol supporting steroidogenesis. Exp Cell Res 255:56–66

    Article  PubMed  CAS  Google Scholar 

  • Wüstner D (2007) Fluorescent sterols as tools in membrane biophysics and cell biology. Chem Phys Lipids 146:1–25

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Giacomo Diaz.

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Diaz, G., Batetta, B., Sanna, F. et al. Lipid droplet changes in proliferating and quiescent 3T3 fibroblasts. Histochem Cell Biol 129, 611–621 (2008). https://doi.org/10.1007/s00418-008-0402-2

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