Fatty acid transport and metabolism in HepG2 cells.
The mechanism(s) of fatty acid uptake by liver cells is not fully understood. We applied new approaches to address long-standing controversies of fatty acid uptake and to distinguish diffusion and protein-based mechanisms. Using HepG2 cells containing an entrapped pH-sensing fluorescence dye, we showed that the addition of oleate (unbound or bound to cyclodextrin) to the external buffer caused a rapid (seconds) and dose-dependent decrease in intracellular pH (pH(in)), indicating diffusion of fatty acids across the plasma membrane. pH(in) returned to its initial value with a time course (in min) that paralleled the metabolism of radiolabeled oleate. Preincubation of cells with the inhibitors phloretin or triacsin C had no effect on the rapid pH(in) drop after the addition of oleate but greatly suppressed pH(in) recovery. Using radiolabeled oleate, we showed that its esterification was almost completely inhibited by phloretin or triacsin C, supporting the correlation between pH(in) recovery and metabolism. We then used a dual-fluorescence assay to study the interaction between HepG2 cells and cis-parinaric acid (PA), a naturally fluorescent but slowly metabolized fatty acid. The fluorescence of PA increased rapidly upon its addition to cells, indicating rapid binding to the plasma membrane; pH(in) decreased rapidly and simultaneously but did not recover within 5 min. Phloretin had no effect on the PA-mediated pH(in) drop or its slow recovery but decreased the absolute fluorescence of membrane-bound PA. Our results show that natural fatty acids rapidly bind to, and diffuse through, the plasma membrane without hindrance by metabolic inhibitors or by an inhibitor of putative membrane-bound fatty acid transporters.
Guo
Wen
W
Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118, USA.
Huang
Nasi
N
Cai
Jun
J
Xie
Weisheng
W
Hamilton
James A
JA
D001693:Biological Transport, Active / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors; D004058:Diffusion; D005231:Fatty Acids, Unsaturated / Q000493:pharmacokinetics; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019301:Oleic Acid / Q000378:metabolism / Q000493:pharmacokinetics*; D010693:Phloretin / Q000494:pharmacology; D014226:Triazenes / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000378:metabolism
D005231:Fatty Acids, Unsaturated; D005452:Fluoresceins; D005456:Fluorescent Dyes; D014226:Triazenes; D047392:beta-Cyclodextrins; D019301:Oleic Acid; C034613:triacsin C; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D003066:Coenzyme A Ligases; C031215:betadex; C013641:parinaric acid; D010693:Phloretin