Lipid droplet degradation through a novel and direct engulfment by endo‐lysosomes

Abstract

Lipid droplets (LDs) are fat‐storage organelles that play critical roles in energy homeostasis, but can excessively accumulate in metabolic diseases such as obesity and fatty liver where hepatocytes become grossly engorged with excess lipid. The catabolism of LDs aids in disease resolution and occurs in part by lipophagy, a form of selective autophagy, but the molecular and vesicle trafficking events that facilitate this process are not fully understood. During lipophagy, autophagosomes are thought to target LDs prior to fusion with acidic lysosomes, where neutral lipids are terminally degraded by lysosomal acid lipase. Ultrastructural viewing of hepatocytes showing LDs contained within electron‐dense acidic vesicles is consistent with this concept. In addition to canonical autophagy, we find that most LDs undergoing degradation are encased within single‐membrane endo‐lysosomes, and few LDs are observed within traditional double‐membrane autophagosomes. Further, studies of LD catabolism in yeast have shown that LDs can be directly internalized by the large acidic vacuole through a process known as microlipophagy. Thus, the GOAL of this study was to test if similar microlipophagy mechanisms occur in mammalian cells such as hepatocytes, and whether this process involves autophagy.METHODSTo test this, we used microscopic fatty acid tracing studies and time‐lapse confocal microscopy to determine the role of autophagosomes versus endo‐lysosomes in LD catabolism. To trace the fate of LDs into terminal degradative vesicles, cells were first loaded with the fluorescent lipid marker Bodipy C12, then treated for 24h with LAListat, an inhibitor of lysosomal acid lipase.RESULTSWe observed a ~5‐fold higher incidence of LDs within endo‐lysosomes (marked with the tetraspanin CD63), compared with autophagosomes (marked by the autophagy protein LC3). Furthermore, siRNA knockdown of macroautophagy proteins (Atg5, Atg7, and Fip200) did not inhibit LD internalization by endo‐lysosomes, suggesting that autophagy is not a prerequisite for this process. In addition, time‐lapse microscopy revealed a direct internalization of cytosolic LDs by endo‐lysosomes positive for lysotracker or CD63‐mCherry.CONCLUSIONThese findings suggest that hepatocytes utilize a distinct form of microautophagy that entails the direct envelopment of cytoplasmic LDs by endocytic vesicles such as multivesicular bodies and lysosomes/late endosomes. In addition to lipolysis and canonical macroautophagy, this process plays an important role in the catabolic utilization of LDs by the hepatocyte.Support or Funding InformationThis study was supported by NIH R01DK044650 (to M.A.M.), RO1AA020735 (to M.A.M.), and the K99AA020735 (to M.B.S.).