Lipid droplet changes in proliferating and quiescent 3T3 fibroblasts.
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).
Diaz
Giacomo
G
Department of Cytomorphology, University of Cagliari, Cittadella Universitaria Monserrato, 09042 Monserrato (CA), Italy. gdiaz@unica.it
Batetta
Barbara
B
Sanna
Francesca
F
Uda
Sabrina
S
Reali
Camilla
C
Angius
Fabrizio
F
Melis
Marta
M
Falchi
Angela Maria
AM
D016475:3T3 Cells; D000577:Amides / Q000494:pharmacology; D000736:Androstenes / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins; D049109:Cell Proliferation; D002784:Cholesterol / Q000378:metabolism; D003260:Contact Inhibition; D003593:Cytoplasm / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004875:Ergosterol / Q000031:analogs & derivatives / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005372:Filipin / Q000378:metabolism; D007150:Immunohistochemistry; D002479:Inclusion Bodies / Q000187:drug effects / Q000378:metabolism*; D050356:Lipid Metabolism / Q000187:drug effects; D008055:Lipids / Q000032:analysis / Q000737:chemistry; D018942:Macrolides / Q000494:pharmacology; D008565:Membrane Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D017646:Organosilicon Compounds / Q000494:pharmacology; D010078:Oxazines / Q000737:chemistry; D010455:Peptides / Q000378:metabolism; D000070778:Perilipin-1; D000070780:Perilipin-2; D010750:Phosphoproteins / Q000378:metabolism
D000577:Amides; D000736:Androstenes; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008055:Lipids; D018942:Macrolides; D008565:Membrane Proteins; D017646:Organosilicon Compounds; D010078:Oxazines; D010455:Peptides; D000070778:Perilipin-1; D000070780:Perilipin-2; D010750:Phosphoproteins; C000602983:Plin2 protein, mouse; C010905:dehydroergosterol; C006261:3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one; C040256:SAN 58035; D005372:Filipin; C040929:bafilomycin A1; D002784:Cholesterol; C044808:nile red; D004875:Ergosterol