title: Multilevel regulation of autophagosome content by ethanol oxidation in HepG2 cells.
abstract: 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 light chain 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 Lysosomal-Associated Membrane Protein 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.
authors.affiliation: Liver Study Unit, Department of Veterans Affairs, VA Nebraska-Western Iowa Health Care System (NWIHCS), Omaha, NE, USA. pthomes@unmc.edu
authors.forename: Paul G
authors.initials: PG
authors.lastname: Thomes
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Ehlers
authors.forename: Casey S
authors.initials: CS
authors.lastname: Trambly
authors.forename: Dahn L
authors.initials: DL
authors.lastname: Clemens
authors.forename: Howard S
authors.initials: HS
authors.lastname: Fox
authors.forename: Dean J
authors.initials: DJ
authors.lastname: Tuma
authors.forename: Terrence M
authors.initials: TM
authors.lastname: Donohue
mesh_terms: D000079:Acetaldehyde / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D000431:Ethanol / Q000378:metabolism* / Q000633:toxicity; D000077604:Fomepizole; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D010084:Oxidation-Reduction; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D000079:Acetaldehyde; D000431:Ethanol; D000077604:Fomepizole; D005978:Glutathione; D051907:Lysosomal Membrane Proteins; D008869:Microtubule-Associated Proteins; D011720:Pyrazoles; D012333:RNA, Messenger; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C477012:MAP1LC3A protein, human; C042431:S-ethyl glutathione