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

Abstract

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-LC3tg 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. Confocal micrograph of mouse hepatocytes from pair-fed (control) and ethanol-fed GFP-LC3 transgenic mice. Autophagosomes (AVs): green puncta. Lysosomes: red puncta. AV-lysosome fusion events: yellow or orange puncta. Larger green structures are nuclei. Hepatocytes from the ethanol-fed mouse exhibit larger and more numerous AVs, lower lysosome numbers, and lower AV-lysosome fusion events than cells from the control mouse. Results indicate that chronic ethanol feeding causes AV accumulation, by reducing AV-lysosome fusion, thus slowing autophagy.