Uptake of long-chain fatty acids in HepG2 cells involves caveolae: analysis of a novel pathway.
We investigated the role of caveolae in uptake and intracellular trafficking of long chain fatty acids (LCFA) in HepG2 human hepatoma cells. The uptake of [(3)H]oleic acid and [(3)H]stearic acid into HepG2 cells was measured by radioactive assays and internalization of the non-metabolizable fluorescent fatty acid 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] (12-NBD) stearate into single HepG2 cells was semi-quantitatively assessed by laser scanning microscopy. The initial rate of [(3)H]oleic acid uptake (V(0)) in HepG2 cells exhibited saturable transport kinetics with increasing concentrations of free oleic acid (V(max) 854 +/- 46 pmol mg protein(-1) min(-1), K(m) 100 +/- 14 nmol/l). While inhibition of clathrin coated pits did not influence LCFA uptake in HepG2, inhibition of caveolae formation by filipin III, cyclodextrin, and caveolin-1 antisense oligonucleotides resulted in reduction of [(3)H]oleic acid uptake by 54%, 45%, and 23%, respectively. Furthermore, filipin III inhibited the uptake of [(3)H]stearic acid and its fluorescent derivative 12-NBD stearate by 44% and 50%, respectively. Transfection studies with alpha-caveolin-1/cyanofluorescent protein chimeras showed significant colocalization of caveolae and internalized 12-NBD stearate. In conclusion, these data suggest a significant role for caveolae mediated uptake and intracellular trafficking of LCFA in HepG2 cells.
Pohl
Jürgen
J
Department of Internal Medicine IV, Ruprechts-Karls-University, Heidelberg, Germany.
Ring
Axel
A
Stremmel
Wolfgang
W
D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics / Q000378:metabolism; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D003505:Cyclodextrins / Q000494:pharmacology; D004705:Endocytosis; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism*; D005372:Filipin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D019301:Oleic Acid / Q000378:metabolism; D013229:Stearic Acids / Q000378:metabolism; D014407:Tumor Cells, Cultured
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D002966:Clathrin; D003505:Cyclodextrins; D005227:Fatty Acids; D013229:Stearic Acids; D019301:Oleic Acid; C031183:stearic acid; D005372:Filipin