Lipases in lysosomes, what for?

We have all learned in textbooks that “lysosomes contain hydrolases able to degrade all types of intracellular molecules which include proteases, glycosidases, nucleotidases and lipases.” To date, the only logical explanation for the presence of lipases inside of lysosomes was for the degradation of lipoproteins internalized by endocytosis, and for the breakdown of intralysosomal vesicles derived from fusion with autophagosomes or multivesicular bodies. However, in our recent work we found a novel role for lysosomal lipases in the basic cellular process that regulates intracellular lipid stores that we have named “macrolipophagy.”

Cells store lipids as cholesterol and triglycerides in the form of lipid droplets from which the lipids can be mobilized when nutrients are scarce, to provide cells with energy and essential elements to preserve biosynthesis. Lipolysis, or the breakdown of triglycerides into free fatty acids, has traditionally been attributed to the actions of both neutral and acidic lipases. However, very little has been known about how intracellular lipids could reach lysosomes, the only acidic compartment in the cell and presumably the preferential location for acid lipase(s). The similarities in the activation of lipolysis and macroautophagy—both are upregulated during nutrient deprivation and repressed by insulin—made us consider a potential link between these two catabolic processes and the possibility that macroautophagy delivers intracellular lipids to lysosomes for hydrolysis.

In our recent work, we show that functional macroautophagy is required for starvation-induced lipolysis in hepatocytes, the main repository for the increased lipids circulating in blood as the result of the mobilization of adipocyte fat stores under these conditions. Furthermore, macroautophagy continually contributes to the degradation of intracellular lipids because blockage of macroautophagy even under basal conditions results in abnormally high levels of triglycerides and cholesterol, and increased lipid droplet size and number. Basal macrolipophagy may thus downregulate the lipid content of hepatocytes, fibroblasts and presumably other cell types, except for the specialized lipid-storing cell, the adipocyte. Using both biochemical and morphological procedures, we confirmed the presence of integral components of lipid droplets (both lipids themselves and structural proteins associated with the lipid droplets) in autophagosomes and lysosomes. As predicted, conditions resulting in an upregulation of macroautophagy increased the association between lipid droplet components and autophagic compartments, both in cultured hepatocytes and in vivo in the livers of mice starved for increasing periods of time. We further confirmed this essential role of basal macroautophagy in the maintenance of hepatocyte lipid content using a mouse model with impaired autophagy in liver (conditional Atg7 knockout mice). The livers of these mice, even under basal conditions, displayed massive accumulation of lipid droplets and abnormally high levels of intracellular cholesterol and triglyceride.

Contrary to our preconceived idea that the limiting membrane of the autophagosome sequestered whole lipid droplets and delivered them to lysosomes for hydrolysis, we found that in most instances only a portion of the lipid droplet was engulfed at one time. In fact, conjugation of LC3 to form the limiting membrane occurred on the lipid droplet surface, resulting in a penetrating membrane that seals on itself, sequestering both lipids and lipid droplet structural proteins (Fig. 1). As with conventional macroautophagy, basal macrolipophagy does not seem to be a selective process, because portions of lipid droplets are engulfed along with other cytosolic cargo giving rise to autophagic vacuoles with heterogeneous content. However, as starvation persists, macroautophagy becomes somewhat selective as autophagosomes with only lipid droplet content in their lumens constitute the most abundant type of autophagic vacuole under this condition.

Figure 1. Schematic model of macrolipophagy. Lipid droplet contents are continuously delivered to lysosomes for breakdown by macrolipophagy, a specialized form of autophagy. Our findings support the concept that portions of the lipid droplets are sequestered by in situ-formed limiting membranes of autophagic nature (enriched in LC3) that then seal to form autophagosomes. Microtubule-dependent fusion of these lipid droplet-containing autophagosomes with lysosomes leads to hydrolysis of triglycerides into free fatty acids that can be used by mitochondria to generate energy for the cells. FFA: free fatty acids; LC3: light chain type 3 protein.

Macrolipophagy is not only upregulated with nutritional deprivation, but also to some extent when cells are exposed to challenges that increase cellular lipid levels (such as extracellular supplementation with free fatty acids or cholesterol). Activation of macrolipophagy in these conditions is likely required to prevent uncontrolled growth of the intracellular lipid stores which leads to the liver disease steatohepatitis. However, if autophagy is indeed one of the mechanisms responsible for maintaining lipid stores “in check,” what goes wrong in pathological situations such as the classic fatty liver in which hepatocytes accumulate massive amounts of lipids? Using both cultured hepatocytes and livers from mice maintained on a high-fat diet for months, we found that chronic exposure of hepatocytes to high levels of fatty acids exerts an inhibitory effect on macroautophagy. It is plausible that very fast growth of lipid droplets may make them no longer amenable to autophagic sequestration due to changes in the properties of their surface components (e.g., lower density of structural proteins, changes in the lipid/protein ratio, etc). However, the fact that the inhibitory effect on macroautophagy of a sustained lipid challenge affects not only lipid removal but also the degradation of other intracellular components by macroautophagy, makes us favor a primary defect in the macroautophagic machinery under these conditions.

This previously unknown role of macroautophagy in lipid metabolism expands the physiological relevance of this pathway and the possible pathological implications of a failure in macroautophagic activity. Functional macrolipophagy is important in maintenance of cellular homeostasis by assuring a regulated control of the volume of intracellular lipid stores. Furthermore, mobilization of lipid droplets by macrolipophagy could be considered part of the cellular response to stress as it may constitute the main source of energy for cells during nutritional deprivation. In light of our findings, alterations in cellular lipid storage and catabolism can now be added to the consequences of autophagic failure, such as the one described with aging, and in different disorders with a primary or secondary defect of the autophagic system. Conversely, alterations in autophagy may underlie the basis of metabolic disorders of as yet unknown etiology or could worsen the prognosis of common diseases with pre-existing defects in lipid metabolism.