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 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.

Early growth response-1 contributes to steatosis development after acute ethanol administration.

BACKGROUND: Previous work demonstrated that the transcription factor, early growth response-1 (Egr-1), participates in the development of steatosis (fatty liver) after chronic ethanol (EtOH) administration. Here, we determined the extent to which Egr-1 is involved in fatty liver development in mice subjected to acute EtOH administration. METHODS: In acute studies, we treated both wild-type and Egr-1 null mice with either EtOH or phosphate-buffered saline (PBS) by gastric intubation. At various times after treatment, we harvested sera and livers and quantified endotoxin, indices of liver injury, steatosis, and hepatic Egr-1 content. In chronic studies, groups of mice were fed liquid diets containing either EtOH or isocaloric maltose-dextrin for 7 to 8 weeks. RESULTS: Compared with controls, acute EtOH-treated mice showed a rapid, transient elevation in serum endotoxin beginning 30 minutes after treatment. One hour postgavage, livers from EtOH-treated mice exhibited a robust elevation of both Egr-1 mRNA and protein. By 3 hours postgavage, liver triglyceride increased in EtOH-treated mice as did lipid peroxidation. Acute EtOH treatment of Egr-1-null mice showed no Egr-1 expression, but these animals still developed elevated triglycerides, although significantly lower than EtOH-fed wild-type littermates. Despite showing decreased fatty liver, EtOH-treated Egr-1 null mice exhibited greater liver injury. After chronic EtOH feeding, steatosis and liver enlargement were clearly evident, but there was no indication of elevated endotoxin. Egr-1 levels in EtOH-fed mice were equal to those of pair-fed controls. CONCLUSIONS: Acute EtOH administration induced the synthesis of Egr-1 in mouse liver. However, despite its robust increase, the transcription factor had a smaller, albeit significant, function in steatosis development after acute EtOH treatment. We propose that the rise in Egr-1 after acute EtOH is an hepatoprotective adaptation to acute liver injury from binge drinking that is triggered by EtOH metabolism and elevated levels of endotoxin.