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1 thematic review series Thematic Review Series: Lipotoxicity: Many Roads to Cell Dysfunction and Cell Death Lipids, lysosomes, and autophagy Bharat Jaishy 1 and E. Dale Abel 2 Fraternal Order of Eagles Diabetes Research Center and Division of Endocrinology and Metabolism, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA ORCID IDs: (E.D.A.); (B.J.) Abstract Lipids are essential components of a cell providing energy substrates for cellular processes, signaling intermediates, and building blocks for biological membranes. Lipids are constantly recycled and redistributed within a cell. Lysosomes play an important role in this recycling process that involves the recruitment of lipids to lysosomes via autophagy or endocytosis for their degradation by lysosomal hydrolases. The catabolites produced are redistributed to various cellular compartments to support basic cellular function. Several studies demonstrated a bidirectional relationship between lipids and lysosomes that regulate autophagy. While lysosomal degradation pathways regulate cellular lipid metabolism, lipids also regulate lysosome function and autophagy. In this review, we focus on this bidirectional relationship in the context of dietary lipids and provide an overview of recent evidence of how lipid-overload lipotoxicity, as observed in obesity and metabolic syndrome, impairs lysosomal function and autophagy that may eventually lead to cellular dysfunction or cell death. Jaishy, B., and E. D. Abel. Lipids, lysosomes, and autophagy. J. Lipid Res : Supplementary key words lipid metabolism lipotoxicity lysosomal dysfunction lipophagy oxidative stress reactive oxygen species lipases, adipose tissue TG lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MGL), releases FAs that circulate bound to albumin. In the intestine, dietary lipids are hydrolyzed by pancreatic lipase into FFA and glycerol, which are reesterified into TGs and packaged into lipoprotein particles, called chylomicrons, in the enterocytes. In liver, TGs and cholesterol are assembled into VLDL particles. The LPL-mediated TG hydrolysis of circulating chylomicrons and VLDLs generates FAs and remnants of chylomicrons and VLDLs, respectively (1). The VLDL remnants are converted into LDL particles and taken up by the liver through lipoprotein receptor-mediated endocytosis along with chylomicron remnants. FA entry into the cell is facilitated by FA transporters, such as CD36 and FA transport proteins (FATPs), at the cell surface (2). FAs can also be synthesized from carbohydrate precursors by de novo lipogenesis when there is a surplus of carbohydrates. FAs serve multiple functions. As a metabolic fuel, they are either stored or oxidized. FAs are stored as TGs in specialized organelles called lipid droplets (LDs) primarily in white adipose tissue (WAT) and liver and, to some extent, OVERVIEW OF DIETARY LIPID METABOLISM FAs are an essential component of cellular structure and function. They provide backbones for biological membranes, act as potent signaling molecules, coordinate whole body homeostasis as lipid hormones and, importantly, provide FA substrates to generate energy in the form of ATP. FAs enter the circulation from various sources (Fig. 1). In adipocytes, sequential hydrolysis of TGs by three cytosolic This work was supported by Office of Extramural Research, National Institutes of Health Grant R01HL The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Manuscript received 28 February 2016 and in revised form 13 June Published, JLR Papers in Press, June 21, 2016 DOI /jlr.R Abbreviations: AgRP, agouti-related peptide; AMPK, AMP-activated protein kinase; ATGL, adipose tissue triglyceride lipase; BCL2, B-cell lymphoma 2; CE, cholesteryl ester; CEBP, CCAAT/enhancer binding protein; CMA, chaperone-mediated autophagy; DAPK2, death-associated kinase 2; ER, endoplasmic reticulum; FATP, fatty acid transport protein; FIP200, family interacting protein of 200 kda; Hdac, histone deacetylase; HFD, high-fat diet; JNK1, c-jun N-terminal kinase 1; IL, interleukin; LAL, lysosomal acid lipase; LC3-II, lipid-conjugated microtubuleassociated protein 1 light chain 3; LD, lipid droplet; LMP, lysosomal membrane permeabilization; LSD, lipid storage disease; mtorc1, mechanistic target of rapamycin complex 1; NOX2, NADPH oxidase isoform 2; NPC, Niemann-Pick type C; p62, sequestosome 1; PLIN, perilipin; POMC, proopiomelanocortin; PTP1B, protein tyrosine phosphatase 1B; Rag, Ragulator; ROS, reactive oxygen species; SFA, saturated FA; TFEB, transcription factor EB; ULK1/2, unc-51-like autophagy activating kinase 1 and 2; v-atpase, vacuolar ATPase; VPS34, class III phosphatidylinositol-3-kinase; WAT, white adipose tissue. 1 Present address of B. Jaishy: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX To whom correspondence should be addressed. DRCadmin@uiowa.edu Copyright 2016 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at Journal of Lipid Research Volume 57,

2 Fig. 1. Overview of dietary TG metabolism. Dietary lipids enter the circulation by three major pathways. 1) Pancreatic lipases (PLs) digest fats to generate FFAs and glycerol, which are reesterified and released into the circulation packaged into chylomicrons (CM). 2) The liver synthesizes VLDL from TGs and CEs and releases them into the circulation. 3) Adipose tissue TG hydrolysis generates FFAs, which are released into the blood stream bound to albumin. Hydrolysis of CM and VLDL by LPL at the endothelial surface generates FFAs that enter the peripheral tissue via FA transporters CD36/FATP. The CM and VLDL remnants generated from LPLmediated hydrolysis are taken up by liver for further processing. In the fed state, FFAs are esterified into TG and stored as LDs in adipose tissue and liver or undergo -oxidation to generate ATP in muscle and other peripheral tissues. In addition, de novo lipogenesis from glucose also generates FFA in the fed state. In the fasted state when lipid supply dwindles, TG hydrolysis generates FFAs that are released into the circulation by adipose tissue or used to generate ketone bodies in liver. Albumin-bound FFA (Alb-FA) and ketone bodies serve as the major source of energy during fasting. DGAT, diacylglycerol acyltransferase; GPAT; glycerol-3-phosphate acyltransferase; HSL, hormone-sensitive lipase; LPIN, lipin; MGL, monoacylglycerol lipase. in other tissues. LDs also contain cholesterol esters and are surrounded by a phospholipid monolayer and several structural membrane proteins, such as perilipins (PLINs), lipases, and the Rab family of small GTPases (3). Alternatively, FAs are transported into the mitochondria or peroxisomes as fatty acyl-coa and undergo -oxidation, which sequentially removes two carbon units from their chain by a series of cyclic reactions catalyzed by acyl-coa dehydrogenases, enoyl-coa hydratase, and 3-ketoacyl-CoA thiolase to generate acetyl-coa. Acetyl-CoA enters the TCA-cycle generating reducing equivalents, nicotinamide adenine dinucleotide and flavin adenine dinucleotide, which drive mitochondrial oxidative phosphorylation to generate ATP (4). FA oxidation is more prominent in tissues with high energy demands, such as skeletal muscles and the heart. Synthesis of FAs or their oxidation is tightly regulated in response to organismal nutritional status. In the fed state, glucose is the primary source of energy and FAs are stored as TGs in LDs. Upon fasting, although hepatic glucose production is increased, rates of TG hydrolysis also increase to supply FFAs, which are utilized as such or converted to ketone bodies by the liver to meet energy requirements of the peripheral tissues during starvation. The intimate regulation of FA metabolism and nutrient availability are essential for organismal lipid homeostasis and are regulated at multiple levels by a complex network of autocrine, paracrine, and endocrine signaling. However, chronic feeding of energy-rich diets combined with a sedentary life style and genetic predisposition may disrupt these homeostatic networks leading to lipid overload, which contributes, in part, to insulin resistance in obesity and the metabolic syndrome. Lipid overload eventually overwhelms adipose tissue s capacity to store TG that leads to rerouting of lipids in tissues with minimal lipid storage capacity, such as skeletal muscle, heart, liver, vascular endothelial cells, and pancreas (5 7). Ectopically stored lipids are mostly shunted into nonoxidative pathways generating toxic lipid intermediates, such as diacylglycerol, ceramides, acyl carnitines, and long-chain fatty acyl-coa, which may cause several deleterious effects, collectively known as lipotoxicity, that contribute to impaired cellular signaling, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and cell death (Fig. 2). Consequences of these maladaptations include insulin resistance, -cell loss, hepatic steatosis, and cardiovascular diseases (8 13). Furthermore, chronic lipid oversupply enhances FA oxidation rate and 1620 Journal of Lipid Research Volume 57, 2016

3 Fig. 2. FA overload-induced oxidative stress and lipotoxicity. Oversupply of FFA may overwhelm mitochondrial capacity to oxidize FAs for ATP production, which leads to accumulation of toxic lipid intermediates, such as diacylglycerol (DAG), ceramides, and acyl carnitines. Toxic lipid intermediates impair insulin signaling through insulin receptor substrates and blunt insulin-stimulated glucose uptake. DAG also activates NOX via protein kinase C (PKC) to increase intracellular ROS production. In addition, dysregulation of mitochondrial electron transport chain (ETC) due to excessive FA -oxidation promotes mitochondrial ROS production. ROS-induced oxidative stress coupled with reduced expression or activity of antioxidant enzymes eventually leads to cellular lipotoxicity manifested by lipid peroxidation, DNA damage, mitochondrial and lysosomal dysfunction, defective autophagy, and activation of inflammatory responses in multiple tissues. FA-CoA, fatty acyl-coa; CPT I and CPT II, carnitine palmitoyltransferase I and II; FADH 2, reduced flavin adenine dinucleotide. imposes constant pressure on the mitochondrial electron transport chain to increase electron flux, which increases reactive oxygen species (ROS) production from electron transport chain complexes I and III (14). Lipid overload also leads to ROS production from extramitochondrial sources, such as NADPH oxidases and other pro-oxidant enzymes. In concert with decreased expression of antioxidative enzymes, these changes precipitate oxidative stress and lead to lipid peroxidation, DNA damage, lysosomal dysfunction, defective autophagy, and activation of inflammatory responses in multiple tissues (15). Emerging evidence suggests that the lysosome-mediated catabolic process, called autophagy, plays a pivotal role in maintaining cellular lipid homeostasis in multiple tissues (16 18). Accordingly, systemic dysregulation of autophagy is observed in animal models of diet-induced obesity, oxidative stress, and metabolic syndrome (19 22), suggesting a role for defective autophagy in lipid overload-induced metabolic dysfunction. For lysosome-mediated recycling of lipids in general, readers are referred to excellent reviews published previously (23, 24). This review focuses on recent advances in our understanding of the bidirectional relationship between autophagy and the metabolism of dietary lipids, such as FAs, TGs, and cholesterol, and highlights recent findings on how lysosomal dysfunction may impair autophagy and exacerbate lipotoxicity in the setting of dietary lipid overload. Understanding the mechanisms of FA-induced lysosomal dysfunction opens a new avenue for therapeutic approaches that may be aimed at mitigating lipotoxicity and improving clinical outcomes of obesity. OVERVIEW OF AUTOPHAGY Adaptation to changes in nutrient status is fundamental to the survival of all organisms. Autophagy is an evolutionarily conserved process that is central for cellular survival during periods of nutrient deprivation. Dysregulation of autophagy is associated with diverse diseases, including cancer, cardiovascular, neurodegenerative, muscular, and metabolic diseases (25). Autophagy is a catabolic process wherein cells degrade their own cytoplasmic materials in lysosomes to supply precursor molecules for processes critical for cell survival. In addition to providing alternative energy sources during starvation, autophagy is also involved in growth and differentiation, metabolic regulation, and macromolecule and organelle quality control. As such, the repertoire of autophagic substrates targeted for degradation is highly heterogeneous and includes proteins, lipids, carbohydrates, and damaged organelles, such as mitochondria, ER, and peroxisomes. Distinct forms of autophagy exist, based on the mode of substrate delivery to lysosomes and distinct molecular mediators. These include Lipids, lysosomes, and autophagy 1621

4 chaperone-mediated autophagy (CMA), microautophagy, and organelle-specific autophagy, such as mitophagy and macroautophagy, all of which have been comprehensively reviewed (26, 27). Macroautophagy (hereafter autophagy ) is the major form of autophagy that plays a key role in lipid metabolism and will be the focus of this review. REGULATION OF AUTOPHAGY Autophagy is a multistep process that involves several core autophagy proteins (ATG) and an increasing number of upstream signaling molecules that respond to various intracellular and extracellular stimuli, such as nutrient deprivation (28). Initial mechanistic insight into autophagy came from the genetic screening of autophagy-deficient yeast mutants, which formed the basis for the study of more complex mammalian autophagy (29). The mechanistic target of rapamycin complex 1 (mtorc1), a nutrient-sensing kinase, is a central regulator of autophagy that integrates signals from multiple upstream pathways to modulate autophagy (Fig. 3). In the fed state, growth factor and nutrient signaling activate mtorc1. Active mtorc1 directly interacts unc-51-like autophagy activating kinase 1 and 2 (ULK1/2)-ATG13-focal adhesion kinase family interacting protein of 200 kda (FIP200) complex and phosphorylates ULK1/2 and ATG13 to suppress autophagy to a low basal level (30, 31). However, nutrient depletion or mtorc1 inhibition relieves this inhibitory phosphorylation to initiate activation of autophagy. Additionally, prolonged nutrient depletion activates the AMP-activated protein kinase (AMPK), which simultaneously inhibits mtorc1 and induces ULK1 (32). ULK1/2 phosphorylates ATG13 and FIP200 to activate the ULK1/2-ATG13-FIP200 complex, which in turn activates the Beclin1-class III phosphatidylinositol-3-kinase (VPS34 or PI3K3) complex to initiate autophagosome formation (33). Prior to its activation, Beclin1 dissociates from its inhibitory interaction with B-cell lymphoma 2 (BCL2) and interacts with VPS34, a process mediated by nutrient depletion and BCL2 phosphorylation by c-jun N-terminal kinase 1 (JNK1) (34). VPS34 catalyzes the formation of phosphatidyl inositol-3 phosphate (PI3P), which is essential for the recruitment of several regulatory ATG proteins to the phagophore for autophagosome expansion. Initiation of autophagy is followed by two ubiquitin-like conjugation events: one is mediated by ubiquitin-like E1 and E2 ligases, ATG7 and ATG10, to form an ATG5-ATG12-ATG16 complex; and the other is mediated by the cysteine protease, ATG4, and ubiquitin-like ligases, ATG7 and ATG3, resulting in formation of lipidconjugated microtubule-associated protein 1 light chain 3 (LC3-II). The ATG complex and LC3-II are required Fig. 3. Molecular mechanism of mammalian autophagy. Under nutrient-rich conditions, mtorc1 binds and inactivates Ulk1/2 complex by inhibitory phosphorylation to suppress autophagy. An autophagy stimulus such as starvation relieves inhibition of Ulk1/2 complex by mtorc1. Activation of AMPK upon starvation inhibits mtorc1 and further activates the Ulk1/2 complex to initiate autophagy. The Ulk1/2 complex, in turn, activates Beclin1-Vps34 (PI3K3) complex either by phosphorylating Beclin1 or through Ambra1 phosphorylation. The kinase activity of Vps34 is regulated by Beclin1, which in turn is controlled by its inhibitory interaction with Bcl-2. The catalytic activity of Vps34 results in the formation of phosphatidyl inositol-3 phosphate (PI3P): a key lipid molecule that recruits specific membrane and protein components to the growing isolation membrane (phagophore). In addition, autophagosome expansion and maturation requires two ubiquitin-like conjugation systems that result in ATG16-ATG5-ATG12 complex and lipidation of LC3-I to LC3-II. LC3-II is required for the selection of autophagic substrate, determining autophagosome size and membrane curvature, and for the bidirectional movement of autophagosomes toward lysosomes for fusion. Mature autophagosomes fuse with lysosomes to form autolysosomes. The autophagic substrates are degraded by lysosomal hydrolases into simple substrates which are utilized for energy production and biomolecule synthesis to maintain cellular homeostasis Journal of Lipid Research Volume 57, 2016

5 for the sequestration of autophagic substrates and maturation of autophagosomes. In addition, the adaptor protein sequestosome 1 (p62 or SQSTM1) also interacts with autophagic substrates and targets them to the growing autophagosome vesicle (35). Matured autophagosomes fuse with lysosomes to form autolysosomes where autophagic substrates are degraded by lysosomal hydrolases into simple molecules and released into the cytosol to complete the process of autophagy. ROLE OF AUTOPHAGY IN LIPID METABOLISM The remarkable similarities between the events regulating autophagy and classical cytosolic lipolysis prompted several studies to examine the link between the two processes. The role of autophagy in lipid metabolism has also garnered considerable interest due to its potential implication in obesity and metabolic syndrome (36). Studies in liver and cultured hepatocytes showed that LDs can be selectively sequestered in autophagosomes and delivered to lysosomes for degradation by lysosomal acid lipases (LALs), a process known as lipophagy (Fig. 4), which is distinct from classical lipolysis wherein lipids in LDs are directly hydrolyzed by cytosolic lipases without being sequestered by autophagosomes for degradation (16). Although, like lipolysis, lipophagy is activated in response to fasting or sustained lipid challenge, our understanding of the molecular mechanism of lipophagy is incomplete. Removal of the LD surface proteins, PLIN2 and PLIN3, by CMA represents the preliminary step that opens up the lipid components of the LD core for degradation by lipophagy or cytosolic lipases (37). -Adrenergic receptor agonist-induced activation of lipophagy suggests that PLIN removal allows activation of the small GTPase, RAB7, at the exposed LD surface to initiate lysosomal degradation (38). RAB7 directs both autophagosomes and lysosomes to the LD surface to coordinate lipophagy following nutrient deprivation (39). At the transcriptional level, the transcription factor EB (TFEB) activates PPAR- and PPAR- coactivator 1 (PGC1 ) target genes involved in FA catabolism, including lipophagy, to accommodate increased hepatic lipid load following starvation (40). Further study revealed that transcriptional activation of TFEB by camp response element-binding protein (CREB) promotes fasting-induced lipophagy; whereas, activation of farnesoid X receptor upon refeeding inhibits TFEB activity (41). Like TFEB, activation of transcription factor forkhead box O1 (FOXO1) by nutrient depletion was shown to induce LAL and lipophagy in adipocytes (42). Together, these studies indicate that mobilization of lipid depots by lipophagy is regulated by a complex network of transcriptional and posttranslational signaling in response to starvation. However, the mechanism of lipophagy in response to lipid challenge was not explored in these studies and requires further investigation, especially given the role of lipid overload in diet-induced metabolic diseases. It is well-established that FFAs generated from autophagy or lipolysis fuel mitochondrial -oxidation. Yet, how these two processes are coordinated and what is their relative contribution to the FFA pool used for mitochondrial -oxidation is widely debated. Using mouse embryonic fibroblasts, Rambold, Cohen, and Lippincott-Schwartz (43) showed that when LDs were abundant, such as in the fed Fig. 4. Degradation of LDs via autophagy. LDs, containing mostly TGs and CEs at the core, undergo CMA of their structural membrane proteins, PLIN2 and PLIN3. PLIN removal activates RAB7 at the exposed LD surface. RAB7 interacts with autophagy proteins, such as p62 and LC3-II, to facilitate the engulfment of the LD by the growing autophagosome. LDs are sequestered by autophagosomes either selectively via lipophagy (A, B) or with other autophagic substrates via nonselective macroautophagy (C). Autophagosomes fuse with lysosomes to form autolysosomes wherein lysosomal hydrolases, such as LALs, degrade LD TGs and CEs into FFAs that are recycled back into the cytosol to support various energetic and structural requirements of the cell. Lipids, lysosomes, and autophagy 1623

6 state, lipolysis of LDs by the cytosolic lipase, ATGL, supplied FFAs for mitochondrial oxidation, and autophagy was dispensable under these conditions. Only after prolonged starvation did bulk autophagy become essential to replenish the LDs with TGs to maintain lipolysis-mediated LD-to-mitochondria FA transport. These findings are in line with previous findings, which demonstrated that autophagy promotes LC3-mediated LD biogenesis in hepatocytes (18, 44). Because the Rambold study did not examine lipophagy, it is not known whether lipophagy-derived FFAs follow the same fate as those of bulk autophagy. Also, it remains to be seen whether similar mechanisms exist in other cell types. Nevertheless, these studies support the notion that autophagy regulates lipid catabolism via selective and nonselective pathways based on nutritional cues, which may serve diverse physiological roles in diverse tissues as described below. In contrast to mouse embryonic fibroblasts, inhibition of autophagy by ablating the autophagy gene, Atg7, in mouse liver increased hepatic lipid accumulation following starvation or high-fat diet (HFD) feeding (16). In hepatocytes, inhibition of autophagy by silencing Atg5 or by inhibiting VPS34 significantly increased cellular TG content following oleate exposure. The increased lipid storage was characterized by an increase in LD size and number in autophagy-deficient hepatocytes (16). Lipid accumulation was due to reduced lysosomal lipolysis resulting from the decreased delivery of lipid substrates into lysosomes and not due to increased de novo lipogenesis. This observation suggests that lipophagy may act as a major lipolytic pathway in liver where cytoplasmic lipolysis has been considered significantly lower than that in adipocytes. As such, lipophagy may play a crucial role in maintaining lipid homeostasis and protecting cells against hepatolipotoxicity. Supporting this notion, hormonal activation of autophagy protects against hepatic lipotoxicity induced by HFD in mice or by FFA in hepatocytes (45). In contrast, autophagy deficiency sensitizes hepatocytes to oxidative stress-induced apoptotic cell death (46). In addition, lipophagy may also regulate the FFA supply required for VLDL production because inhibition of autophagy suppresses VLDL secretion, whereas induction of autophagy upregulates VLDL secretion (47). Unlike in hepatocytes, autophagy promotes adipogenesis and lipid storage in adipocytes. Inhibition of autophagy in 3T3L1 preadipocytes, pharmacologically or by genetic knockdown of Atg5 or Atg7, blocked their differentiation into mature WAT and, hence, their lipid-storing capacity. Protein levels of key mediators [PPAR-, CCAAT/enhancer binding protein (CEBP)-, and CEBP- ] and markers (FA synthase, steroyl-coa desaturase, and glucose transporter 4) of adipocyte differentiation were reduced in autophagy-deficient adipocytes, suggesting that the impaired lipid storage is secondary to a defect in adipocyte differentiation (17, 48). Studies on 3T3L1 preadipocytes showed that transcriptional activation of Atg4b by CEBP- is required to induce autophagy during adipocyte differentiation. CEBP- -induced autophagy then degrades Kruppel-like factors 2 and 3, two negative regulators of adipogenesis, to facilitate 3T3L1 adipocyte differentiation (49). In line with in vitro data, CEBP- was shown bound to the Atg4b promoter in mouse WAT, and depletion of Atg4b increased Kruppel-like factor 2/3 levels and reduced WAT mass in mice (49). The adipogenic role of autophagy was further validated in adipocyte-specific Atg7-null mice, which showed significantly reduced WAT mass, resulting in a lean insulin-sensitive phenotype. Loss of WAT mass in these mice was accompanied by increased brown adipose tissue-like features in white adipose depots, such as increased expression of PGC-1, a key regulator of mitochondrial biogenesis, and uncoupling protein 1 (UCP1); increased levels of mitochondrial enzymes cytochrome oxidase and cytochrome C; increased mitochondrial number; and the presence of rounded nuclei and small multilocular LDs (17, 48). Thus, knockout mice with enlarged brown adipose tissue-like mass exhibited high rates of - oxidation that might have prevented TG accumulation in skeletal muscles and liver that increased insulin sensitivity. Despite improved FA oxidation and insulin sensitivity, adipose-specific Atg7 knockout mice died prematurely after 6 weeks of age on both regular diets and HFDs, which suggests an essential but undefined role for adipocyte-autophagy on organismal viability (48). Because the studies described above were performed in undifferentiated adipocytes or in mice with germline knockout manipulations, it was not possible to distinguish the effects of autophagy on adipocyte differentiation from effects on mature adipocyte physiology. Recently, Yin et al. (50) showed that palmitateinduced ER stress activates autophagy in mature 3T3L1 adipocytes. Pharmacological inhibition of autophagy increased expression of the pro-inflammatory cytokines, monocyte chemoattractant protein-1 and interleukin (IL)- 6, and exacerbated palmitate-induced ER stress and cell death (50). These findings imply that autophagy in mature adipocytes may play an adaptive role in mitigating ER stress and low-grade inflammation associated with obesity and insulin resistance. In fact, autophagy was induced in adipose tissue from obese individuals, and inhibition of autophagy increased expression of the pro-inflammatory genes, IL-1, IL-6, and IL-8, both in adipocytes and adipose tissue explants of obese individuals compared with nonobese controls (51). Furthermore, overexpression of deathassociated kinase 2 (DAPK2), one of the most strongly repressed adipose tissue genes in obese humans and HFDinduced obese mice, increased autophagic clearance in 3T3L1 adipocytes in nutrient-rich conditions (52). Consistent with these observations, in adipocytes isolated from obese patients recovering from bariatric surgery-induced weight loss, both DAPK2 expression and autophagic clearance were markedly increased and adipocyte size was proportionally reduced postsurgery compared with the adipocytes from the same patients prior to surgery (52). Despite the progress made in defining the role of autophagy in mature adipocytes, questions remain as to whether bulk autophagy or selective lipophagy is involved in mediating the changes observed in these studies and what the molecular mechanisms of autophagic regulation are. A conditional deletion of core autophagic genes or genes specifically involved in lipophagy may ultimately clarify the 1624 Journal of Lipid Research Volume 57, 2016

7 role of autophagy in mature adipocytes and in adipocyte lipid metabolism. Lipophagy controls whole body energy homeostasis by regulating the activity of hypothalamic neurons. The agoutirelated peptide (AgRP) neurons in the hypothalamus express AgRP and neuropeptide Y that stimulate food intake and reduce energy expenditure. A recent study showed that autophagy of neuronal lipids in AgRP neurons provides FFAs to induce AgRP expression in response to starvation (53). Inhibition of autophagy by ablating Atg7 in these neurons lowered AgRP expression and resulted in leaner mice with higher activity and reduced food intake following starvation, possibly due to increased levels of the catabolic neuropeptide, proopiomelanocortin (POMC), in POMC neurons. Consistent with these findings, mice lacking Atg7 in both AgRP and POMC neurons exhibited increased body weight, total fat mass, and lower energy expenditure (54). In skeletal muscles and the heart, autophagy plays important roles in maintaining tissue and energy homeostasis; yet, a comprehensive study on how autophagy regulates lipid metabolism in these tissues is lacking (55, 56). Based on existing evidence, one can assume that TGs or other dietary lipids are degraded via bulk autophagy to provide FFA substrates. However, whether lipids are selectively degraded via lipophagy is unknown. Therefore, studies in skeletal muscles and the heart examining the role of autophagy in mobilizing lipids accumulated in obese, insulin resistant, and type II diabetic models carries great therapeutic implications. At the same time, it is important to note that muscles are not as well suited to store lipids in bulk as liver or adipose tissue; it is possible that FFAs entering muscles are oxidized for energy production or utilized for other physiological functions rather than stored as lipids under normal physiological conditions. Under such conditions, lipid mobilization through autophagy may be too subtle to be detected. Therefore, an obese model of muscle lipid accumulation may provide a good starting point to study lipid catabolism in response to starvation or other autophagic stimuli. In addition to TGs, lipophagy is also involved in the hydrolysis of cholesteryl esters (CEs). In normal macrophages, CEs in LDs are hydrolyzed by cytosolic lipases to release free cholesterols, which are effluxed from the cell. However, in cholesterol-loaded macrophages, CE hydrolysis occurs primarily via lipophagy to generate free cholesterols that are used in reverse cholesterol transport to apolipoprotein A1- rich HDLs (57). Activation of autophagy upon cholesterol loading is likely a protective mechanism because the inhibition of autophagy in cholesterol-loaded smooth muscle cells significantly induced cell death. In contrast, rapamycin-induced autophagy protected cells from free cholesterol overload-induced cell death (58). These findings imply that inhibition of autophagy may be a key mediator of cholesterol overload-induced vascular cell death in the progression of atherosclerosis. This is an important hypothesis which requires further study to establish the role of autophagy in atherosclerosis and to determine whether this could be a potential therapeutic target. REGULATION OF AUTOPHAGIC ACTIVITY BY LIPIDS With the role of autophagy in lipid catabolism wellestablished, several studies show that an inverse relationship exists where dietary lipid overload changes cellular lipid content or composition to alter autophagic activity, which may have beneficial or detrimental effects in a tissuespecific manner (Table 1). Such lipid-induced changes are distinct from the fundamental role lipids and LDs play in autophagosome biogenesis and in the overall process of autophagy (59, 60). For example, exposure of INS-1E cells to palmitate induces autophagy in the presence of hyperglycemia (61). Diabetic db/db mice or nondiabetic mice challenged with a HFD exhibited marked induction of autophagosome formation in pancreatic cells (62). In these models of lipid overload, induction of autophagy TABLE 1. Lipid overload-induced regulation of autophagy in different metabolic tissues Study Model Diet or Treatment Effect on Autophagy Observation (Reference) Pancreas WT or db/db mice 60% HFD for 12 weeks Induced Autophagy maintained cell mass, insulin secretion, and normal glucose tolerance (62) INS1 or MIN6 cells Liver WT, ob/ob, and liver-atg7-null mice Skeletal muscle WT, adiponectin-null, and muscle-hdac 1/2-null mice 0.4 mm palmitate and 20 mm glucose for 14 h or longer Suppressed Impaired autophagic flux led to defective insulin secretion and apoptosis (64, 65) 35% HFD for 22 weeks Suppressed Autophagy suppression increased ER stress and insulin resistance (19) 60% HFD for 2 12 weeks Induced Autophagy induction parallels upregulation of antioxidant genes and ameliorates insulin resistance and myopathy (22, 77) WT and muscle-atg7-null mice 60% HFD for 13 weeks Suppressed in Atg7-null mice Suppression of autophagy protects from HFDinduced obesity and insulin resistance (20) Heart WT and Akt2-null mice 45% HFD for 20 or 25 weeks Suppressed Impaired autophagic flux resulted in cardiac hypertrophy, contractile dysfunction, ER stress, and apoptosis (85, 86) WT mice 60% milk fat-based diet for 18 weeks Induced De novo sphingolipid synthesis induced autophagy and cardiomyocyte hypertrophy and contractile dysfunction (88) Lipids, lysosomes, and autophagy 1625

8 played a protective role against lipotoxicity: inhibition of autophagy in INS-1E cells, by depleting Atg5 or blocking autolysosome formation, accentuated palmitate-induced cell death; likewise, cell-specific deletion of Atg7 in mice induced cell death and led to profound glucose intolerance, partly due to the lack of a compensatory increase in cell mass and insulin secretion following high-fat feeding. The loss of autophagy in Atg7-deficient cells significantly increased oxidative stress and toxic accumulation of ubiquitinated proteins and damaged organelles that might collectively contribute to cell degeneration (63). Inhibition of autophagic flux by chloroquine induced markers of ER stress (C/EBP-homologous protein and phosphorylated eukaryotic initiation factor-2 ) and apoptosis (cleaved caspse 3) in pancreatic cells of high-fat-fed mice, which further supports the protective role of autophagy against cell lipotoxicity (63). While short-term high-fat feeding induces autophagy in pancreatic cells, chronic lipid overload impairs autophagic flux (64, 65). Prolonged exposure of INS1 cells and human pancreatic islets to high levels of palmitate and glucose increased the accumulation of autophagosomes and autophagic protein markers, and suppressed autophagic degradation of long-lived proteins. Impairment in autophagic flux led to defective insulin secretion and increased apoptotic cell death. Inhibition of mtorc1 by rapamycin partially restored autophagic flux, insulin secretion, and cell viability by mechanisms that are incompletely understood (64, 65). In addition to the chronicity of lipid overload, the type of FA also determines cell lipotoxicity. The saturated FA (SFA), palmitate, inhibits cell proliferation and induces apoptosis, whereas the monounsaturated FA, palmitoleate, with identical chain length, counteracts the toxic effect of palmitate and promotes cell proliferation (66). Taken together, these in vivo and in vitro data support the notion that lipid overload-induced autophagy plays a protective role against lipotoxicity; however, longterm high-fat feeding or palmitate exposure induces lipotoxicity by blocking this adaptive autophagic response. Other factors, such as ceramide accumulation and increased ROS production, may also induce lipotoxicity independent of autophagy (67). As in cells, chronic lipid accumulation inhibits hepatic autophagy. Hepatocytes acutely loaded with oleate failed to mobilize LDs through autophagic pathways (16). Mutliple mechanisms appear to drive lipid-induced inhibition of hepatic autophagy. One potential mechanism is the sustained activation of mtorc1 in high-fat-fed mouse liver that may inhibit autophagic initiation (68). Consistent with this notion, mice on a HFD failed to move LDs to autophagic vesicles in response to starvation (16). In vitro studies showed that high-fat feeding also impairs autophagosome-lysosome fusion (the distal step in the autophagic process) by disrupting the normal membrane composition of autophagosomes and lysosomes isolated from mouse liver (69). Suppression of hepatic autophagy may also occur by transcriptional mechanisms. In a mouse model of HFD-induced hyperinsulinemia and insulin resistance, hepatic autophagy was suppressed partly due to transcriptional repression of FOXO1-dependent autophagic genes, Vps34, Atg12, and GABA(A) receptor-associated protein like 1(Gabarapl1), which may contribute to autophagy inhibition (70). A separate study in genetic and dietary models of obesity reported suppression of autophagy in terms of Atg7 expression in liver that paralleled increased ER stress and insulin resistance (19). Restoration of hepatic Atg7 expression in obese mice normalized autophagy, reduced ER stress, and improved hepatic insulin action and systemic glucose tolerance. Lipid overload in skeletal muscles and the heart is associated with multiple metabolic abnormalities, such as oxidative stress, mitochondrial dysfunction, insulin resistance, apoptotic cell death, and fibrosis (71 74). The role autophagy plays in these cellular stress responses is complex and incompletely understood. Some studies showed that basal autophagy remained intact in skeletal muscles of high-fat-fed animals, although fat accumulation and oxidative stress was markedly elevated and insulin signaling pathways impaired (75, 76). In contrast, most studies showed altered autophagic activity in response to lipid overload. High-fat feeding was shown to induce autophagic flux in skeletal muscles (marked by an increase in LC3-II, BE- CLIN1, and ULK1, and a decrease in p62 level) in a mouse model of diet-induced obesity and insulin resistance (22). Autophagic induction was correlated with increased oxidative stress, manifested by induction of superoxide dismutase activity, lipid peroxidation, and mrna levels of key antioxidant enzymes. This increase in HFD-induced autophagy was absent in adiponectin-deficient mice and was restored upon adiponectin re-expression. The insulin-resistant phenotype was recapitulated in an autophagy-deficient cell culture model expressing an Atg5 inactive mutant (22). These data suggest that HFD-induced skeletal muscle autophagy and antioxidant capacity ameliorates HFD-induced insulin resistance. Consistent with the adaptive role of dietinduced autophagy, high-fat feeding in skeletal musclespecific histone deacetylase (Hdac)1- and Hdac2-null mice, which normally exhibit autophagy deficiency, restored autophagic flux and prevented myopathy in adult mice (77). However, under chronic lipid overload, such as in severely obese hyperinsulinemic human subjects, autophagic flux was blunted in primary skeletal muscle myotubes, which could contribute to the insulin resistant phenotype (78). Thus in the face of nutrient overload, the skeletal muscle adaptation to autophagy might be biphasic, with an initial increase that might limit insulin resistance. However, if nutrient excess is persistent, this adaptive response is lost and declining autophagy might then exacerbate insulin resistance. This model may need to be revised in the light of a recent study, which showed that blocking autophagy by deleting Atg7 in skeletal muscles protects mice from diet-induced systemic obesity and peripheral insulin resistance (20). Mitochondrial dysfunction, induced by lack of autophagy, led to increased expression of fibroblast growth factor 21 (Fgf21) in the muscle and protected mice from dietinduced obesity and insulin resistance. Interestingly, autophagy deficiency in liver also induced Fgf21 expression and 1626 Journal of Lipid Research Volume 57, 2016

9 provided similar protection from obesity and insulin resistance (20). Together with previous findings, these data portray a complex role of autophagy in lipid-loaded skeletal muscle and warrants further study to reconcile these seemingly contradictory findings, or to more clearly delineate those changes that represent additional mechanisms that might be independent of autophagy per se, but which may impact metabolic homeostasis. The presence of these alternative pathways underscores the pleiotropic adaptations that may defend against diet-induced obesity and insulin resistance. While modest increases in autophagy may improve muscle function, excessive autophagy was linked to muscle atrophy. For example, induction of several key autophagy genes by the transcription factor, FOXO3, hyper-activates autophagy and accelerates protein degradation in the muscle (79). A recent in vitro study showed that the FOXO3 pathway of muscle atrophy was induced as a lipotoxic response to SFA overload (80). Coincubation with unsaturated FA completely reversed the lipotoxic effects of SFA, underscoring the FA-specific regulation of autophagy and atrophy in skeletal muscles. In the heart, basal autophagy plays a prosurvival role (81). However, our understanding of the lipid-induced regulation of autophagy is still emerging. He et al. (82) showed that 12 weeks of high-fat feeding suppressed cardiac autophagy, assessed by the increased level of p62. Several other studies reported impairment in cardiac autophagy following long-term high-fat feeding (83 85). A recent study showed that impaired cardiac autophagy in high-fat-fed mouseinduced lipotoxicity was marked by an increase in ER stress and cardiomyocyte apoptosis, suggesting that autophagy might play a protective role against diet-induced cardiac lipotoxicity (86). However, high-fat feeding also induces hyperglycemia, hyperinsulinemia, and cardiac hypertrophy in these mice, which could independently impair autophagy. The mechanism of lipid-induced inhibition of autophagy in the heart is complex and multifactorial. A recent study showed that AKT2 is an important component of HFD-induced inhibition of autophagic flux (85). High-fat feeding suppressed the expression of Rab7 (a small GT- Pase involved in lysosomal biogenesis) and, thus, reduced autophagosome turnover. Knockdown of Akt2 prevented the suppression of Rab7 by an unknown mechanism and restored normal autophagic flux in high-fat-fed mouse hearts (85). Besides AKT, AMPK-mediated regulation of protein tyrosine phosphatase 1B (PTP1B) also controls lipid-induced inhibition of autophagy (87). Mice fed with a 45% HFD for 5 months exhibited impaired autophagosome turnover in the heart in parallel with reduced AMPK phosphorylation. Deletion of PTP1B gene restored normal autophagic flux in high-fat-fed mice and reversed dietinduced cardiac hypertrophy and contractile dysfunction. Pharmacological inhibition of AMPK, however, abrogated beneficial effects of PTP1B ablation on autophagy, cardiac structure, and contractile function (87). In contrast to these studies, feeding a HFD rich in medium-chain SFAs was found to activate autophagy and promote subsequent development of cardiac hypertrophy and left ventricular contractile dysfunction in the murine heart (88). In this model, induction of autophagy required de novo synthesis of the specific sphingolipid, C 14 -ceramide, by ceramide synthase 5 (CERS5). Depletion of CerS5 mrna blocked autophagy induction and hypertrophy in isolated adult cardiomyocytes treated with myristate, a precursor for C 14 - ceramide (88). This study showed that the specificity of the lipid species is a key determinant of the effect of diet on autophagy, lipotoxicity, and cardiac hypertrophy. These studies underscore the importance of defining in even greater detail the composition of diets used in various metabolic studies, which may help reconcile some of the contradictory data in the literature. In vitro studies in H9C2 cardiomyocytes corroborate some of the in vivo findings on lipid overload-induced cardiac autophagy. Treatment of these cells with palmitate induced apoptosis and increased the accumulation of autophagosomes and lysosomes. Induction of autophagy by rapamycin treatment ameliorated palmitate-induced apoptosis, whereas inhibition of autophagy by 3-MA exacerbated apoptosis (86). Data from multiple studies suggest that lipid overload induces dynamic autophagic response in these cells in a time-dependent manner. For instance, acute palmitate exposure may induce autophagy without inflicting cytotoxicity. Extending palmitate treatment to 4 h, however, inhibited autophagic flux by impairing lysosomal function with no cytotoxicity (89). Prolonged palmitate treatment for 12 h or longer suppressed autophagic flux with concomitant increase in cellular lipotoxicity, marked by an elevated ROS level, activation of the stress kinases, JNK and p38mapk, and a profound increase in apoptotic markers, such as cleaved caspase 3 and caspase 7 and their downstream effector, poly ADP-ribose polymerase 1 (90). Thus, the initial activation of autophagy may protect cardiomyocytes by preventing accumulation of toxic lipids. The subsequent impairment in autophagic flux offsets this protective adaptation, which leads to cellular lipotoxicity. However, a recent study showed that prolonged treatment with the mediumchain SFA, myristate, but not the long-chain SFA, palmitate, induced autophagic flux and promoted hypertrophy in isolated adult primary cardiomyocytes (88). This suggests that myristate, or its derivatives, induces autophagy and hypertrophy by specific mechanisms that are not shared by other toxic FAs known to inhibit autophagy. Together, in vivo and in vitro studies indicate that multiple pathways mediate lipid-induced regulation of cardiac autophagy depending on composition of diet, type of FAs, duration of feeding, and type of cell culture and animal models used. Whether these pathways are linked, to provide a unifying mechanism, or represent independent mechanisms, is unknown and requires a more comprehensive study. LYSOSOMES AND LIPIDS Lysosomes are acidic organelles containing at least 50 different hydrolases that degrade a wide variety of macromolecules, including DNA, proteins, carbohydrates, and lipids delivered via autophagy and other endocytic pathways. Lipids, lysosomes, and autophagy 1627

10 Lysosomes contain membrane-bound vacuolar ATPases that maintain the acidic ph of the lysosomal lumen for efficient functioning of the hydrolases. Lysosomal membrane proteins undergo various modifications, including glycosylation, that protect them against proteolysis, thereby stabilizing lysosomal membranes against acid hydrolases and other degradative agents (91). Like proteins, lipids are an integral part of lysosomal membranes and play a key role in lysosomal biogenesis and function [reviewed in (23)]. Reciprocally, lysosomes play a major role in lipid metabolism and the maintenance of cellular lipid homeostasis. A role for lysosomes in the recycling of membrane lipids has been described (24). In this part of the review, we primarily focus on the mutual interrelationship between dietary lipids and lysosomal function. LYSOSOMES AS REGULATORS OF LIPID METABOLISM Both exogenous and endogenous lipids are delivered to lysosomes. Exogenous lipids enter the lysosomes via endocytosis. These lipids primarily include lipoproteins and CEs derived from LDLs and remnants of VLDLs and chylomicrons (92, 93). Endocytosis of lipoproteins is facilitated by members of the LDL receptor family localized in either lipid rafts or clathrin-coated pits (94). Endocytosed LDL particles are transferred, as such, from late endosomes to the lysosomes where TGs and CEs are hydrolyzed by LAL to generate FFAs and free cholesterols, respectively (95). In contrast, for VLDLs and chylomicron remnants, their apolipoprotein E receptors are recycled back to the plasma membrane leaving the remaining lipid particles to be degraded in the lysosome (94, 96). Endogenous lipids or LDs enter the lysosome primarily via autophagy and are hydrolyzed by LAL. Hydrolysis of LD TGs and CEs by LAL generates FFAs and free cholesterols, respectively (16, 57). Normally, free cholesterols released from lysosomes are transferred to the ER for reesterification (97). However, as described earlier, in cholesterolloaded macrophages, free cholesterols derived from lipophagy are incorporated into apolipoprotein A1-rich HDL (57). Biochemical studies on LAL isolated from ratliver lysosomes suggest that the enzyme catalyzes both the hydrolysis and synthesis of CEs (98). Reesterification of cholesterol was also observed in endosomes of cultured mouse macrophages exposed to LDL aggregates (99). Whether LAL catalyzes CE formation in these macrophages is not clear. These findings may have an implication in CE storage disease where cholesterol concentration in the membrane was found near saturation, a condition under which cholesterol-reesterification was upregulated in mouse macrophage endosomes. Deficiency of LAL results in two major diseases: CE storage disease and the more severe Wolman disease. The latter is characterized by the accumulation of both CEs and TGs (100). Besides TGs and CEs, a variety of glycerophospholipids and glycosphingolipids are also hydrolyzed in the lysosomes by specific phospholipases and water-soluble acid hydrolases ( ). Besides hydrolyzing and recycling various lipids, lysosomes also act as a nutrient sensor to regulate lipophagy. Zoncu et al. (106) showed that cells sense amino acid content in the lysosomal lumen to determine their nutritional status. This information is relayed by the lysosomal vacuolar ATPase (v-atpase) proton pump to the Ragulator (Rag) complex outside the lysosome via human member 9 of the solute carrier family 38, a recently identified lysosomal membrane resident-protein, which forms a part of the Rag complex (107). Rag charges small molecule GT- Pases (RagA/B and RagC/D) with GTP to activate them. Active Rags recruit mtorc1 to the lysosomal surface where the GTP binding protein, ras homolog enriched in brain (RHEB), activates mtorc1 (106, 108). Active mtorc1 inhibits the transcription factor, TFEB, by direct phosphorylation and blocks PPAR- transcriptional activity by promoting its interaction with nuclear receptor corepressor 1 (NCoR1) (109, 110). TFEB is the major transcription factor that globally activates several autophagic and lysosomal genes, including LAL (111, 112). PPAR- is the master transcriptional activator of several genes involved in FA - oxidation. Thus, by regulating mtorc1 activity in response to lumenal amino acid content, lysosomes control both lipophagy and mitochondrial FA oxidation by sensing cellular nutrient status. It is interesting, but as yet unknown, why lysosomes use amino acids to relay cellular nutrient status to control lipid metabolism. REGULATION OF LYSOSOMAL FUNCTION BY LIPIDS As in autophagy, lipids play an important role in regulating lysosomal function. While some lipid-induced protein modifications facilitate the transport of lysosomal proteins and enzymes to the lysosome (113), excess lipid supply largely impairs lysosomal function by multiple mechanisms (Fig. 5). Lysosomal lipid storage diseases (LSDs), or lipidoses, are a constellation of inherited disorders characterized by the accumulation of lipids in late endosomes and lysosomes (114). A majority of LSDs are associated with impaired function of lysosomal hydrolases, membrane transporters, and other accessory proteins (Table 2). For instance, in Niemann-Pick disease type C (NPC), defects in the transporter proteins, NPC1 and NPC2, impair cholesterol efflux and fill the late endosomes with unesterified cholesterol (115, 116). How lipid accumulation contributes to the pathogenesis of the disease is still unclear. A previous study linked cholesterol accumulation to defects in trafficking between intralumenal vesicles and limiting membrane of the late endosomes (117). Such defects may impair trafficking or recycling of proteins, lipids, and other membrane components between endocytic vesicles and other organelles, such as Golgi, ER, and plasma membrane, which is often observed in several lysosomal LSDs ( ). Accumulation of lipids in lysosomes can also inhibit autophagosome turnover. Several studies in LSD documented an impairment in fusion between autophagosomes 1628 Journal of Lipid Research Volume 57, 2016

11 Fig. 5. Lysosomal stress-response pathways induced by dietary lipid overload. Overload of FFA supplied exogenously or derived from TG hydrolysis induces ROS production from mitochondrial electron transport chain complexes, the NADPH oxidase 2 (NOX2) complex, and other ROS producing enzymes. The ROS molecules hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2 ) undergo Fenton s reaction in lysosomes to generate hydroxyl radical (OH ), a highly reactive ROS. ROS-induced peroxidation of lysosomal membrane lipids leads to LMP and release of cathepsins into the cytosol. Cytosolic cathepsins induce apoptotic cell death either by direct activation of caspases or through mitochondrial membrane permeabilization (MMP)- mediated caspase activation. ROS-induced protein oxidation impairs the function of several lysosomal enzymes, such as v-atpase proton pump and lysosomal hydrolases, which raises lysosomal ph and inhibits degradation of autophagic cargo, respectively. Lipid overload also induces lysosomal dysfunction by ROSindependent pathways. Incubation with excess FFAs alters the membrane composition of lysosomes and autophagosomes that impairs heterotypic fusion between the two organelles. Macrophages loaded with atherogenic lipids exhibited an increase in lysosomal ph that contributed to a defect in cholesterol efflux that also inhibits chaperone mediated autophagy (CMA) by promoting selective degradation of the lysosome-associated membrane protein 2A (LAMP2A). FFA-induced translocation of mtorc1 to the lysosomal membrane has been shown to promote ER stress-dependent apoptosis in podocytes. and lipid-loaded lysosomes that leads to defective autophagic clearance ( ). Conversely, depletion of cholesterol or inhibition of cholesterol synthesis induces autophagy (125, 126). In vitro fusion assays showed that altered membrane composition of autophagosomes and lysosomes isolated from lipid-loaded fibroblasts or highfat-fed mouse liver inhibits the fusion between these organelles, which could account for the impaired autophagy in many lipid-induced lysosomal disorders (69). In addition to macroautophagy, dietary lipid overload also affects CMA. Mice fed with HFD showed selective degradation of lysosome-associated membrane protein 2A (LAMP2A) in hepatocytes that led to inhibition of CMA in high-fat-fed mice, but not in chow-fed controls (127). Lipid-induced lysosomal dysfunction is also observed in diet-induced obesity and metabolic disorders (85, 128). Mice fed a HFD exhibited increased accumulation of phospholipids in lysosomes that was associated with reduced lysosomal enzyme activity, which was reversed by the chronic activation of AMPK (128). Likewise, high-fat feeding in mice reduced the cardiac expression of Rab7, a small GTPase essential for lysosome maturation (129), which impaired autophagosomelysosome fusion and suppressed autophagic flux (85). Cardiac ablation of Atg2 restored Rab7 expression and normalized autophagic flux and prevented cardiac hypertrophy and contractile dysfunction induced by high-fat feeding (85). Lysosomal membrane permeabilization (LMP) is a key mechanism by which chronic lipid overload promotes lysosome dysfunction and triggers apoptotic cell death (130, 131). Both high-fat feeding and FA exposure induced LMP in adipose tissue that led to the release and activation of the lysosomal protease, cathepsin B, in the cytosol following macrophage infiltration of adipose tissue and production of pro-inflammatory cytokines (132). High-fat feeding also induced LMP and lipotoxicity in the liver of a mouse model of diet-induced nonalcoholic steatohepatitis, although hepatic autophagy remained largely unchanged (133). In the heart, a HFD induced release of lysosomal Lipids, lysosomes, and autophagy 1629