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Journal of Lipid Research, Vol. 43, 1390-1399, August 2002
Copyright © 2002 by Lipid Research, Inc.

Uptake of long-chain fatty acids in HepG2 cells involves caveolae

Jürgen Pohl¹, Axel Ring¹ and Wolfgang Stremmel²

Department of Internal Medicine IV, Ruprechts-Karls-University, Heidelberg, Germany

² To whom correspondence should be addressed. e-mail: wolfgang.stremmel{at}med.uni-heidelberg.de

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 [³H]oleic acid and [³H]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 [³H]oleic acid uptake (V₀) 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 [³H]oleic acid uptake by 54%, 45%, and 23%, respectively. Furthermore, filipin III inhibited the uptake of [³H]stearic acid and its fluorescent derivative 12-NBD stearate by 44% and 50%, respectively. Transfection studies with -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.

Abbreviations: CFP, cyano-fluorescent protein; FAT, fatty acid translocase; FABP, fatty acid binding protein; FATP, fatty acid transport protein; LCFA, long-chain fatty acids; LSM, laser scanning microscopy; 12-NBD, 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]

Supplementary key words vesicles • hepatocytes • intracellular trafficking • oleic acid

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