Acute and Chronic Ethanol Administration Differentially Modulate Hepatic Autophagy and Transcription Factor EB.

BACKGROUND: Chronic ethanol (EtOH) consumption decelerates the catabolism of long-lived proteins, indicating that it slows hepatic macroautophagy (hereafter called autophagy) a crucial lysosomal catabolic pathway in most eukaryotic cells. Autophagy and lysosome biogenesis are linked. Both are regulated by the transcription factor EB (TFEB). Here, we tested whether TFEB can be used as a singular indicator of autophagic activity, by quantifying its nuclear content in livers of mice subjected to acute and chronic EtOH administration. We correlated nuclear TFEB to specific indices of autophagy. METHODS: In acute experiments, we gavaged GFP-LC3(tg) mice with a single dose of EtOH or with phosphate buffered saline (PBS). We fed mice chronically by feeding them control or EtOH liquid diets. RESULTS: Compared with PBS-gavaged controls, livers of EtOH-gavaged mice exhibited greater autophagosome (AV) numbers, a higher incidence of AV-lysosome co-localization, and elevated levels of free GFP, all indicating enhanced autophagy, which correlated with a higher nuclear content of TFEB. Compared with pair-fed controls, livers of EtOH-fed mice exhibited higher AV numbers, but had lower lysosome numbers, lower AV-lysosome co-localization, higher P62/SQSTM1 levels, and lower free GFP levels. The latter findings correlated with lower nuclear TFEB levels in EtOH-fed mice. Thus, enhanced autophagy after acute EtOH gavage correlated with a higher nuclear TFEB content. Conversely, chronic EtOH feeding inhibited hepatic autophagy, associated with a lower nuclear TFEB content. CONCLUSIONS: Our findings suggest that the effect of acute EtOH gavage on hepatic autophagy differs significantly from that after chronic EtOH feeding. Each regimen distinctly affects TFEB localization, which in turn, regulates hepatic autophagy and lysosome biogenesis.

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.

Proteasome activity and autophagosome content in liver are reciprocally regulated by ethanol treatment.

UNLABELLED: The proteasome and autophagy are two major intracellular protein degradation pathways and the regulation of each by ethanol metabolism affects cellular integrity. Using acute and chronic ethanol feeding to mice in vivo, and precision-cut rat liver slices (PCLS) ex vivo, we examined whether ethanol treatment altered these proteolytic pathways. In acute studies, we gave C57Bl/6 mice either ethanol or phosphate-buffered saline (PBS) by gastric intubation and sacrificed them 12h later. PCLS were exposed to 0 or 50mM ethanol for 12 and 24h with or without 4-methylpyrazole (4MP). In chronic studies we pair-fed control and ethanol liquid diets for 4-6 weeks to transgenic mice, expressing the green fluorescent protein (GFP) fused to the autophagic marker, microtubule associated protein-1 light chain 3 (LC3). Acute ethanol administration elevated autophagosomes (AVs), as judged by a 1.5-fold increase in LC3II content over PBS-gavaged control mice. Hepatic proteasome activity was unaffected by this treatment. Compared with controls, ethanol exposure for 12 and 24h to PCLS inhibited proteasome activity by 1.5- to 3-fold and simultaneously enhanced AVs by 2- to 5-fold. The decrease in proteasome activity and the rise in AVs both depended on ethanol oxidation as its inhibition by 4-methylpyrazole (4MP) blocked both proteasome inhibition and AV induction. Hepatocytes from mice chronically consuming ethanol exhibited a 1.6-fold decline in proteasome activity, and a 4-fold rise in GFP-LC3 puncta compared with pair-fed control mice. When we exposed hepatocytes from these animals to MG262, a proteasome inhibitor, LC3II puncta per cell further increased 2- to 5-fold over untreated cells. CONCLUSION: Our findings demonstrate that ethanol metabolism generates oxidants, the levels of which differentially influence the activities of the proteasome and autophagy.