Effects of Ethanol in Uptake of Long Chain Fatty Acid in HepG2 Cells

Han, Nam Ik; Berk, Paul D

Abstract

Background/Airns: Plasma membrane fatty acid binding protein (FABPpm), a long chain fatty acid (FFA) transporter, is reported to be identical with mitochondrial (m) AST. Alcoholic liver disease (ALD) is characterized by increased plasma level of AST>ALT. In ALD, the elevated AST consists mainly of m-AST which are originated from the leakage of the morphologically abnormal mitochondria. However, increase in plasma AST is often found without mitochondrial injury. Moreover, since m-AST leaking from mitochondria first enters the cytoplasm, it is unclear why m-AST should preferentially escape from the cytoplasm into the circulation.
METHODS
We established four long term HepG2 cell cultures in 0, 20, 40 or 80 mM ethanol. In these cultures, plasma membrane expression of FABPpm was assessed by immunofluorescence, Western blotting of plasma membrane extracts: m-AST mRNA by slot blotting/scanning densitornetry; AST leakage into the medium enzymatically; and mitochondrial morphology by EM. The FFA uptake kinetics were quantified with [3H]-oleate.
RESULTS
HepG2 cells at all ethanol levels displayed FABPpm on the plasma membrane, with a progressive, ethanol related increase. This paralleled an increase in m-AST mRNA. Leakage of total AST into the culture medium per 24 hours was similar in cells cultured in 0 and 20 mM ethanol but the amount of leakage was increased 6 to 14 times in cells cultured at 40 and 80 mM ethanol. No gross alteration was studied. The Vmax for oleate uptake was closely correlated with m-AST mRNA level (p<0.01).
CONCLUSIONS
Ethanol upregulated with m-AST synthesis and the m-AST sorting to the plasma membrane. Increased plasma m-AST in ALD may not be derived from mitochondria, but from the plasma membrane FABPpm. Increased plasma membrane FABPpm mediates increased FFA uptake in hepatocyte, which may be a contributing factor to alcoholic fatty liver.