Ethanol up-regulates fatty acid uptake and plasma membrane expression and export of mitochondrial aspartate aminotransferase 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 (pHin), indicating diffusion of fatty acids across the plasma membrane. pHin 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 pHin drop after the addition of oleate but greatly suppressed pHin recovery. Using radiolabeled oleate, we showed that its esterification was almost completely inhibited by phloretin or triacsin C, supporting the correlation between pHin 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; pHin decreased rapidly and simultaneously but did not recover within 5 min. Phloretin had no effect on the PA-mediated pHin 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.

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Effect of surface and intracellular pH on hepatocellular fatty acid uptake

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1996.271.6.g1067 ◽

1996 ◽

Vol 271 (6) ◽

pp. G1067-G1073

Author(s):

C. Elsing ◽

A. Kassner ◽

W. Stremmel

Keyword(s):

Fatty Acids ◽

Plasma Membrane ◽

Fatty Acid ◽

Intracellular Ph ◽

Outer Surface ◽

Fatty Acid Uptake ◽

Proton Gradient ◽

Fatty Acid Transport ◽

Acid Uptake ◽

Acid Transport

Fatty acids enter hepatocytes, at least in part, by a carrier-mediated uptake mechanism. The importance of driving forces for fatty acid uptake is still controversial. To evaluate possible driving mechanisms for fatty acid transport across plasma membranes, we examined the role of transmembrane proton gradients on fatty acid influx in primary cultured rat hepatocytes. After hepatocytes were loaded with SNARF-1 acetoxymethyl ester, changes in intracellular pH (pHi) under different experimental conditions were measured and recorded by confocal laser scanning microscopy. Fatty acid transport was increased by 45% during cellular alkalosis, achieved by adding 20 mM NH4Cl to the medium, and a concomitant paracellular acidification was observed. Fatty acid uptake was decreased by 30% during cellular acidosis after withdrawal of NH4Cl from the medium. Cellular acidosis activates the Na+/H+ antiporter to export excessive protons to the outer cell surface. Inhibition of Na+/H+ antiporter activity by amiloride diminishes pHi recovery and thereby accumulation of protons at the outer surface of the plasma membrane. Under these conditions, fatty acid uptake was further inhibited by 57% of control conditions. This suggests stimulation of fatty acid influx by an inwardly directed proton gradient. The accelerating effect of protons at the outer surface of the plasma membrane was confirmed by studies in which pH of the medium was varied at constant pHi. Significantly higher fatty acid influx rates were observed at low buffer pH. Recorded differences in fatty acid uptake appeared to be independent of changes in membrane potential, because BaCl2 did not influence initial uptake velocity during cellular alkalosis and paracellular acidosis. Moreover, addition of oleate-albumin mixtures to the NH4Cl incubation buffer did not change the observed intracellular alkalinization. In contrast, after cells were acid loaded, addition of oleate-albumin solutions to the recovery buffer increased pHi recovery rates from 0.21 +/- 0.02 to 0.36 +/- 0.05 pH units/min (P < 0.05), indicating that fatty acids further stimulate Na+/H+ antiporter activity during pHi recovery from an acid load. It is concluded that carrier-mediated uptake of fatty acids in hepatocytes follows an inwardly directed transmembrane proton gradient and is stimulated by the presence of H+ at the outer surface of the plasma membrane.

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Fatty acid uptake in Escherichia coli: regulation by recruitment of fatty acyl-CoA synthetase to the plasma membrane

Biochemistry and Cell Biology ◽

10.1139/o93-008 ◽

1993 ◽

Vol 71 (1-2) ◽

pp. 51-56 ◽

Cited By ~ 25

Author(s):

Dev Mangroo ◽

Gerhard E. Gerber

Keyword(s):

Escherichia Coli ◽

Plasma Membrane ◽

Fatty Acid ◽

Fatty Acid Uptake ◽

Fatty Acyl ◽

Acid Uptake ◽

Bacterial Membranes ◽

Rate Limiting Step ◽

Triton X ◽

Rate Limiting

Fatty acid uptake in Escherichia coli has been shown to be inhibited by starvation and to be reversed by a short preincubation of the starved cells with D- or L-lactate, succinate, and acetate; these effects on oleate uptake were due to regulation of the rate-limiting step which involves fatty acyl-CoA synthetase. Investigation into the mechanism of regulation of fatty acyl-CoA synthetase showed that D-lactate did not affect the activity of the enzyme directly. Fatty acyl-CoA synthetase was found to be activated by about 20-fold by Triton X-100 and by another 4-fold by the addition of bacterial membranes. D-Lactate treatment was shown to result in coisolation of fatty acyl-CoA synthetase with the plasma membrane; these results are consistent with the interpretation that recruitment of the enzyme to the plasma membrane by D-lactate results in its activation and consequently in the increased level of fatty acid uptake.Key words: fatty acid, uptake, regulation, recruitment, fatty acyl-CoA synthetase, Escherichia coli, plasma membrane.

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3T3 fibroblasts transfected with an aspartate aminotransferase cDNA express both plasma mebrane fatty acid binding frotein and saturable fatty acid uptake . Depts. of Medicine & Biochemistry, Mt. Sinai School of Medicine, New York, NY 10029

Hepatology ◽

10.1016/0270-9139(93)92075-b ◽

1993 ◽

Vol 18 (4) ◽

pp. A137

Keyword(s):

New York ◽

Fatty Acid ◽

Aspartate Aminotransferase ◽

Fatty Acid Uptake ◽

Acid Uptake ◽

Fatty Acid Binding ◽

3T3 Fibroblasts ◽

School Of Medicine

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DHHC4 and DHHC5 Facilitate Fatty Acid Uptake by Palmitoylating and Targeting CD36 to the Plasma Membrane

Cell Reports ◽

10.1016/j.celrep.2018.12.022 ◽

2019 ◽

Vol 26 (1) ◽

pp. 209-221.e5 ◽

Cited By ~ 17

Author(s):

Juan Wang ◽

Jian-Wei Hao ◽

Xu Wang ◽

Huiling Guo ◽

Hui-Hui Sun ◽

...

Keyword(s):

Plasma Membrane ◽

Fatty Acid ◽

Fatty Acid Uptake ◽

Acid Uptake

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Sex differences in hepatic fatty acid uptake reflect a greater affinity of the transport system in females

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1992.263.3.g380 ◽

1992 ◽

Vol 263 (3) ◽

pp. G380-G385 ◽

Cited By ~ 2

Author(s):

D. Sorrentino ◽

S. L. Zhou ◽

E. Kokkotou ◽

P. D. Berk

Keyword(s):

Plasma Membrane ◽

Fatty Acid ◽

Transport System ◽

Fatty Acid Uptake ◽

Surface Expression ◽

Female Rats ◽

Acid Uptake ◽

Fatty Acid Binding ◽

Male And Female Rats

In this study, we examined the hypothesis that the reported sex difference in hepatic free fatty acid (FFA) uptake involves the putative FFA transport system, the plasma membrane fatty acid binding protein (FABPpm). In hepatocytes isolated from both male and female rats, initial [3H]oleate uptake velocity reflected transmembrane influx and not subsequent metabolism and was a saturable function of the unbound oleate concentration. Although Vmax values were similar (61 +/- 2 vs. 65 +/- 5 pmol.min-1.5 x 10(4) cells-1 for females and males, respectively), the apparent Km was significantly smaller in females (40 +/- 4 vs. 90 +/- 11 nM; P less than 0.05), reflecting faster influx velocities in female cells over a range of unbound oleate concentrations. The oleate efflux rate constant was also greater in females (0.280 +/- 0.014 vs. 0.198 +/- 0.020 min-1; P less than 0.05) despite their greater hepatic content of cytosolic FABP. Finally, despite the greater rates of transmembrane FFA flux in female hepatocytes, the surface expression of FABPpm was virtually identical in the two sexes (2.5 +/- 0.5 vs. 2.4 +/- 0.4 microgram/10(6) cells). Collectively, these data indicate that at FFA-to-albumin ratios occurring in vivo the plasma membrane of female hepatocytes transports oleate bidirectionally at a greater rate than that of male hepatocytes. A sex-related difference in the functional affinity of FABPpm for FFA appears the most likely explanation for the greater oleate uptake in females.

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