Insufficient autophagy: a novel autophagic flux scenario uncovered by impaired liver TFEB-mediated lysosomal biogenesis from chronic alcohol-drinking mice

Abstract

Macroautophagy/autophagy is a dynamic process, and newly synthesized autophagosomes need to fuse with lysosomes to complete the full process, which is referred to as autophagic degradation or autophagic flux. Therefore, the proper number and function of lysosomes are critical for accomplishing autophagic flux. In a recent study, we found that chronic ethanol consumption impairs TFEB (transcription factor EB) function, which leads to decreased lysosomal biogenesis resulting in hepatic steatosis and liver injury in mice. Interestingly, using the autophagic flux assay recommended by the autophagy guidelines, we discovered a novel autophagic flux scenario, which we termed insufficient autophagy. Insufficient autophagy is a scenario in which cells have a decreased number of lysosomes resulting in the accumulation of early autophagosomes. Insufficient autophagy is marked by a partially increased autophagic flux, but the process cannot reach its full degradative capacity due to the lack of a sufficient number of lysosomes. Our work demonstrated that pharmacological or genetic activation of TFEB-mediated lysosomal biogenesis enhances autophagic flux coupled with mitochondrial biogenesis in protecting against ethanol-induced liver injury. Overall, these findings not only identified the steps in which chronic ethanol impairs autophagic flux, but also discovered insufficient autophagy as a novel previously unappreciated autophagic flux scenario.

Keywords: Autophagosome; GFP-LC3; ethanol; liver injury; steatosis.

Figure 1.

Scheme of insufficient autophagy. Under basal conditions, among 3 synthesized AVs, 2 of them already fused with a LY to form an AL (thus eliminating their LC3-II) and there is 1 free AV (with LC3-II). If, for simplicity, we assume that 1 AV has 3 LC3-II proteins, then, in the presence of lysosomal inhibitors (leupeptin or chloroquine [CQ]), the level of basal autophagy (i.e., the number of LC3-II detected) would be 9. Under starvation conditions, both the numbers of AV and LY increase resulting in an increased number of AL. In the presence of lysosomal inhibitors, the level of starvation-induced autophagic flux (again, the number of LC3-II) would be 12. Chronic ethanol increases the number of AVs but also decreases the number of LYs, resulting in fewer ALs. In the presence of lysosomal inhibitors, the level of ethanol-induced autophagic flux would still be 12. However, in this scenario the degradative capacity is only 1/3 that of starvation-induced autophagy because the majority of the LC3-II levels are from AVs but not ALs. Thus, we have termed this situation insufficient autophagy. Although it is technically possible to differentiate among these scenarios based on the absolute changes in the level of LC3-II in the absence and presence of lysosomal inhibitors, this may be technically challenging. Accordingly, we propose that it is easier to monitor TFEB and/or lysosome numbers as part of the flux analysis.