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
• ethanol
• GFP-LC3
• liver injury
• steatosis

Chronic ethanol exposure disrupts lysosome function, and animals with chronic ethanol exposure, as well as heavy drinkers, have a larger liver with increased hepatic protein accumulation. These early observations imply that chronic alcohol consumption may impair hepatic lysosomal degradation, but the underlying mechanisms are unclear. The lysosome is the terminal component of autophagy. TFEB (transcription factor EB) is a master regulator of lysosome biogenesis. In a recent study [1], we found that chronic feeding plus an acute binge of ethanol decreases hepatic TFEB and lysosomal biogenesis in mouse livers. To our surprise, no differences of lysosomal biogenesis, hepatic steatosis and liver injury are found between liver-specific tfeb knockout (KO) mice and the matched wild-type mice. In mammals, TFEB belongs to the MITF (melanogenesis associated transcription factor) family of proteins including MITF, TFEB, TFE3 and TFEC, which have redundant functions in regulating lysosomal biogenesis. It is likely that the presence of other MITF transcription factors in the liver may compensate for the chronic loss of TFEB. Indeed, more severe steatosis, liver injury and hepatic inflammation are found in acute TFEB knockdown mice and in tfe3 tfeb double-KO mice, compared with their matched wild-type mice. Overexpression of TFEB in mouse livers markedly protects against ethanol-induced steatosis and liver injury. Our study thus uncovers a novel protective mechanism against ethanol-induced steatosis and liver injury through TFEB-mediated lysosomal biogenesis. The negative results from the liver-specific tfeb or tfe3 single-KO mice after chronic ethanol also suggest that cautions on the compensatory and/or secondary effects should be emphasized when interpreting data from genetic KO mice.

In addition to lysosomal biogenesis, overexpression of TFEB improves hepatic mitochondria bioenergetics and mitochondrial fatty acid beta oxidation (FAO) gene expression likely via TFEB-mediated upregulation of PPARGC1A (peroxisome proliferative activated receptor, gamma, coactivator 1 alpha). It has been hypothesized that autophagic lysosomal removal of lipid droplets (LDs) may release free fatty acid from lysosomes, which are harmful to hepatocytes. Increased mitochondrial function and FAO may help to burn the free fatty acids and eliminate their potential lipotoxicity. Therefore, TFEB may be an ideal target for treating alcoholic and non-alcoholic fatty livers by coupling the catabolism of LDs via autophagy and lysosomal degradation and the burning of fatty acids via mitochondria.

Perhaps another intriguing finding was the discovery of a novel autophagic flux scenario, which we termed as insufficient autophagy. Autophagy is a highly dynamic process. Autophagic flux refers to the measure of autophagic degradation activity. Several autophagic flux assays have been elaborated and are mandatory in the autophagy guidelines. One of the most common autophagic flux assays that is accepted widely by many laboratories is to determine the changes of LC3-II levels or number of GFP-LC3 puncta in the presence or absence of a lysosome inhibitor. To determine the autophagic flux in mouse livers, we treated GFP-LC3 transgenic mice with chronic ethanol with or without the lysosomal inhibitor leupeptin. We found that the number of GFP-LC3 puncta, the levels of hepatic GFP-LC3-II and endogenous LC3-II in hepatocytes, were much higher in mice treated with ethanol together with leupetin than the mice treated with either ethanol or leupeptin alone. Based on the guidelines of autophagy research, we concluded that chronic ethanol increases autophagic flux in mouse livers. However, as discussed above, chronic ethanol impairs TFEB-mediated lysosomal biogenesis resulting in a decreased number of lysosomes. Therefore, chronic ethanol-increased autophagic flux in mouse livers may not be able to reach the full capacity of autophagic degradation, or chronic ethanol only induces insufficient autophagy. We found that the percentage of green-only GFP-LC3 puncta (which are not colocalized with lysosomes) was significantly higher in ethanol-treated mouse livers than that of control mice. These results suggest that there is a lack of a sufficient number of lysosomes to fuse with autophagosomes in hepatocytes after chronic ethanol, which ultimately leads to the accumulation of early autophagosomes and an incomplete autophagy process. Insufficient autophagy induced by chronic ethanol is also supported by the observation that chronic ethanol conditions resulted in a failure to degrade hepatic SQSTM1/p62.

How would decreased lysosome numbers still lead to increased autophagic flux? As illustrated in Figure 1, at basal conditions, a newly formed autophagosome (AV) travels and fuses with a lysosome (LY) to form an autolysosome (AL) to complete the autophagic process. In the absence of lysosomal inhibitors, the levels of LC3-II only reflect the newly synthesized numbers of AVs because LC3-II in an AL will either be degraded by lysosomal enzymes (inner membrane LC3-II) or will have been deconjugated from the surface of the AV by ATG4 (outer membrane LC3-II). Starvation induces a greater number of AVs and also activates TFEB to increase the number of lysosomes to ensure a sufficient number of lysosomes to fuse with the increased number of AVs, to meet the demand of autophagy. Most autophagic flux assays are performed at one single time point in a snapshot manner; therefore, in normal cells in the absence of lysosomal inhibitors, the levels of LC3-II could either be the same (same number of AVs, but more ALs), higher (more AVs, the same number of ALs) or lower (fewer AVs, and more ALs) compared with the basal conditions. However, in the presence of lysosomal inhibitors, the levels of LC3-II are much higher than the basal levels, thus indicating increased autophagic flux after starvation. In the chronic ethanol or insufficient autophagy scenario, despite there being a decreased number of lysosomes due to impaired TFEB, the existing lysosomes can still fuse with AVs to form functioning ALs. Accordingly, chronic ethanol-increased levels of hepatic LC3-II are further enhanced by leupetin that blocks degradation within the ALs (although AL numbers are insufficient). Thus, the traditional autophagic flux assay using the levels of LC3-II changes in the presence of lysosomal inhibitors may not be able to detect this insufficient autophagic flux scenario. To better assess autophagy status under certain conditions such as chronic ethanol, we therefore suggest that additional assays to include TFEB activity and determining the number of lysosomes should also be considered in conjunction with the traditional autophagic flux assay.

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

In summary, our study identified TFEB-mediated lysosomal biogenesis as a potential novel approach for treating and preventing ethanol-induced liver injury. Moreover, we also discovered a previously unappreciated insufficient autophagy scenario.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The research was supported in part by the NIAAA R01 AA020518, U01 AA024733, National Institute of General Medical Sciences P20GM103549 & P30GM118247.