Multilevel regulation of autophagosome content by ethanol oxidation in HepG2 cells

Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental (nonmetabolizing) and recombinant (ethanol-metabolizing) Hep G2 cells to identify the ethanol metabolite that c...

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Published in:	Autophagy Vol. 9; no. 1; pp. 63 - 73
Main Authors:	Thomes, Paul G., Ehlers, Rebecca A., Trambly, Casey S., Clemens, Dahn L., Fox, Howard S., Tuma, Dean J., Donohue, Jr, Terrence M.
Format:	Journal Article
Language:	English
Published:	United States Taylor & Francis 01-01-2013 Landes Bioscience
Subjects:	acetaldehyde Acetaldehyde - pharmacology alcohol dehydrogenase autophagy Autophagy - drug effects Autophagy - physiology Basic Research Paper Binding Biology Bioscience Calcium Cancer Cell Cycle cytochrome P450 2E1 Ethanol - metabolism Ethanol - toxicity Fomepizole Glutathione - analogs & derivatives Glutathione - pharmacology Hep G2 Cells Hepatocytes - drug effects Hepatocytes - metabolism Humans Landes Lysosomal Membrane Proteins - metabolism lysosome Microtubule-Associated Proteins - genetics Microtubule-Associated Proteins - metabolism Models, Biological Organogenesis oxidant stress Oxidation-Reduction Phagosomes - drug effects Phagosomes - metabolism Proteins Pyrazoles - pharmacology RNA, Messenger - genetics RNA, Messenger - metabolism Vacuoles - drug effects Vacuoles - metabolism
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Summary:	Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental (nonmetabolizing) and recombinant (ethanol-metabolizing) Hep G2 cells to identify the ethanol metabolite that causes AV enhancement by quantifying AVs or their marker protein, microtubule-associated protein 1 l ight c hain 3-II (LC3-II). The ethanol-elicited rise in LC3-II was dependent on ethanol dose, was seen only in cells that expressed alcohol dehydrogenase (ADH) and was augmented in cells that coexpressed cytochrome CYP2E1 (P450 2E1). Furthermore, the rise in LC3-II was inversely related to a decline in proteasome activity. AV flux measurements and colocalization of AVs with lysosomes or their marker protein L ysosomal- A ssociated M embrane P rotein 1 (LAMP1) in ethanol-metabolizing VL-17A cells (ADH + /CYP2E1 + ) revealed that ethanol exposure not only enhanced LC3-II synthesis but also decreased its degradation. Ethanol-induced accumulation of LC3-II in these cells was similar to that induced by the microtubule inhibitor, nocodazole. After we treated cells with either 4-methylpyrazole to block ethanol oxidation or GSH-EE to scavenge reactive species, there was no enhancement of LC3-II by ethanol. Furthermore, regardless of their ethanol-metabolizing capacity, direct exposure of cells to acetaldehyde enhanced LC3-II content. We conclude that both ADH-generated acetaldehyde and CYP2E1-generated primary and secondary oxidants caused LC3-II accumulation, which rose not only from enhanced AV biogenesis, but also from decreased LC3 degradation by the proteasome and by lysosomes.
ISSN:	1554-8627 1554-8635
DOI:	10.4161/auto.22490