Fatty acid transport and metabolism in HepG2 cells

Abstract

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.

Cellular uptake of fatty acids driven by the ER-localized acyl-CoA synthetase FATP4

“…The enhanced oleate uptake was not accompanied by an accumulation of free oleate in the cells. Only 5-8% of oleate remained unesterified, which is in line with other reports (Guo et al, 2005;Pohl et al, 2002;Stremmel and Berk, 1986).…”

Fatty acids are rapidly delivered to and extracted from membranes by methyl-β-cyclodextrin

“…We demonstrated in both cell types that adsorption and transbilayer movement of OA delivered by MBCD are complete within seconds and that all of the OA that had been complexed with MBCD partitioned from MBCD into the cell membrane. We also showed that MBCD did not affect the recovery of BCECF fl uorescence in HepG2 cells, which closely follows FA metabolism (mainly FA esterifi cation) ( 25,37 ). Therefore, MBCD can deliver FA to cells without altering the plasma membrane structure and FA metabolism.…”

“…These cells metabolize FA faster than 3T3-L1 preadipocytes, which is detected as recovery in the BCECF fl uorescence ( 25 ). As shown in Fig.…”

“…Because the long-term goal of our studies of FA transport in membrane models is to apply our methods to cells, we tested the ability of MBCD to deliver OA to different HepG2 cells ( 25 ). Additionally, MBCD did not affect the BCECF fl uorescence recovery in HepG2 cells.…”

Colorectal cancer cell growth inhibition by linoleic acid is related to fatty acid composition changes

“…There are two main pathways by which LA may affect the cells: (1) etherification into phospholipids to influence the membrane fluidity, and (2) providing energy for cell cycle, while a part of LAs would be transferred into 9/13-hydroxyoctadecadienoic acid (9/13-HODE) by the action of 15-lipoxygenase (15-LOX) enzyme, and become elongated and desaturated into long chain PUFAs by the action of elongases and desaturases (Kelavkar et al, 2006a;2006b;Guo et al, 2006).…”