A new pathway regulating autophagy

Autophagy is required to remove damaged organelles and proteins in all healthy tissues. Within the heart, the turnover of intracellular damaged organelles and proteins might be especially important to respond to a variety of pathological conditions including ischemia and pressure overload-induced hypertrophy. Defective autophagy has been created in tissues by deletion of the autophagic mediators, most commonly autophagy proteins (ATG) 5 and ATG7 (1). These defects are invariably deleterious. Another role of autophagy is to provide energy during starvation. Many tissues store energy in lipid droplets and utilize these substrates primarily via hydrolysis of the esterified fatty acids by adipose triglyceride lipids and hormone sensitive lipase (2). In several tissues, including the liver (3) and heart (4), autophagy is an alternative pathway to acquire fatty acids. Autophagy is negatively regulated by mTOR signaling complex 1 (mTORC1) activation. When mTORC1 is inactivated in response to nutrient or growth factor deprivation, autophagy is induced by the formation of a multiprotein kinase complex primarily composed of ATGs leading to the nucleation of the autophagosome. Further processing results in the matured autophagosome, defined by the closed double membrane, which can then fuse with the lysosome membrane to form the autophagolysome. The autophagosome internal membrane and contents are digested by hydrolases within the lysosome. ATG regulation and autophagosome formation are not completely understood, so autophagic flux must be measured by a functional readout, i.e., examining autophagosome morphology and quantity as well as microtubule-associated protein 1A/1B-light chain 3 (LC3) lipidation (1, 5). LC3 linked to phosphotidylethanolamine resides on the autophagosome outer membrane and is commonly used as a signal for autophagic flux. The paper by Jaishy et al. (6) in this issue of the Journal of Lipid Research reports a novel pathway for regulation of the autophagy. The authors propose that changes in lysosomal pH due to fatty acids prevent the elimination of autophagosomes. Mice eating a high-fat diet had impaired cardiac autophagy, specifically at the level of lysosomal digestion of contents. This decrease in autophagic flux associated with increased cellular lipids had also been observed in insulin-resistant livers (3). However, these impairments in autophagic flux were independent of changes in mTORC1 because activation of mTORC1 was insufficient to restore autophagic flux. The surprising result was that this regulation of autophagy was independent of changes in protein kinase B/AKT and S6-kinase, which are thought to be major regulators of autophagy. Therefore, the autophagy defects occurred without defective insulin signaling, which one would expect to find with longer-term palmitate treatment. Furthermore, there was no cardiac dysfunction observed in this short-term high-fat diet feeding. The authors went on to define the specific mechanism whereby increased cardiac lipids reduced autophagy. In a series of elegant tissue culture experiments, Jaishy et al. (6) demonstrate that treatment of cultured cells with palmitate activated protein kinase Cbeta2, increased p47phox, and in turn elevated activity of NADPH-dependent oxidase (NOX)2, thereby increasing intracellular superoxide. Similar to the high-fat diet-fed mice, there was no observed cytotoxicity in these cultured cells, indicating that palmitate treatment was not increasing autophagy by initiation of cell death. Despite the increase in autophagosomes, the authors did not find increased autophagy of mitochondria. However, inhibiting mitochondrial fatty acid oxidation with etomoxir exacerbated the impairment of autophagy, indicating that mitochondrial reactive oxygen species (ROS) were not contributing to changes in autophagy. Next, they demonstrated that the lysosomal vacuolar-type H+-ATPase (V-ATPase), which is an ion channel that maintains low lysosomal pH, had decreased activity, which the authors suggest was likely due to elevated superoxide levels. Taken together, these surprising findings illustrate a new pathway whereby autophagic flux is modulated. Notably, these findings conflict with recent findings where increased superoxide levels actually elevated autophagic flux in glucose-deprived cardiac myocytes, where NOX4 in the endoplasmic reticulum was important and NOX2 was dispensable for regulation of autophagy (7). In some ways, these results parallel studies on the effects of lipids on lysosomal function in other situations. Two decades ago, Hoff and colleagues (8) showed that lysosomal processes such as those needed for recycling of the LDL receptor were inactivated when macrophages were overloaded with oxidized LDL. More recent data suggest that polyunsaturated fatty acid-derived ROS increases hepatic autophagy of nascent lipoproteins that are oxidized and aggregated (9). Thus, those data and those in the current study by Jaishy et al. (6) prove that intracellular lipid metabolism is central to regulation of autophagy. Future studies will confirm whether this regulation of autophagy is specific to cardiomyocytes or is more generalizable in other oxidative tissues, such as skeletal muscle, liver, and brown adipose tissue. It is difficult to assess the effects of altered autophagy in vivo, but knockout or transgenic animal studies altering NOX2 and lysosomal V-ATPase may facilitate further understanding of this pathway. Finally, it will be critical to assess whether clearance of defective autophagosomes has significance in living animals. The controversy regarding the role of ROS in the regulation of cardiac autophagy will also need to be resolved by future studies.