and Metabolism, Roy J. and Lucille A. Carver College of Medicine, University of Iowa,

Transcription

1 1 Lipids, Lysosomes and Autophagy Bharat Jaishy 1 * and E. Dale Abel 1,2 1 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 52242, USA. 2 Corresponsdece to: E. Dale Abel, MB.BS., DPhil. Fraternal Order of Eagles Diabetes Research Center Division of Endocrinology and Metabolism Roy J. and Lucille A. Carver College of Medicine, University of Iowa 4312 PBDB, 169 Newton Road, Iowa City, IA Telephone: Fax: DRCadmin@uiowa.edu *Present Address Department of Molecular Genetics University of Texas Southwestern Medical Center Dallas, TX

2 2 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.

3 3 Overview of Dietary Lipid Metabolism: Fatty acids 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 fatty acid substrates to generate energy in the form of ATP. Fatty acids (FAs) enter the circulation from various sources (Fig. 1). In adipocytes, sequential hydrolysis of triglycerides (TGs) by three cytosolic lipases, adipose tissue triglyceride 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 free fatty acid (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 very low density lipoprotein (VLDL) particles. The lipoprotein lipase-mediated TG hydrolysis of circulating chylomicrons and VLDLs generates FAs and remnants of chylomicrons and VLDLs, respectively (1). The VLDL remnants are converted into low density lipoprotein particles and taken up by liver through lipoprotein receptor-mediated endocytosis along with chylomicron remnants. FA entry into the cell is facilitated by fatty acid transporters such as CD36 and fatty acid transport proteins (FATP) 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

4 4 adipose tissue (WAT) and liver and, to some extent, in other tissues. LDs also contain cholesterol esters and are surrounded by a phospholipid monolayer and several structural membrane proteins such as perilipins (PLIN), lipases, and the Rab family of small GTPases (3). Alternatively, FAs are transported into the mitochondria or peroxisomes as fatty acyl-coa and undergo β-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 fatty acids 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

5 5 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 non-oxidative pathways generating toxic lipid intermediates such as diacyl glycerol, 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, β-cell loss, hepatic steatosis and cardiovascular diseases (8-13). Furthermore, chronic lipid oversupply enhances FA oxidation rate and 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

6 6 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 FAinduced lysosomal dysfunction opens a new avenue for therapeutic approaches that may 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 chaperone mediated autophagy (CMA), microautophagy, organellar specific autophagy such as mitophagy and macroautophagy, all of which have been comprehensively reviewed (26,

7 7 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 activates 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 kd (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 (PI3K3 or VPS34) 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

8 8 phosphorylation by c-jun N-terminal kinase 1 (JNK1) (34). VPS34 catalyzes the formation of phospatidyl 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 lipid-conjugated microtubule-associated protein 1 light chain 3 (LC3-II). The ATG complex and LC3-II are required for the sequestration of autophagic substrates and maturation of autophagosomes. In addition, the adapter 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 autolysomes 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 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

9 9 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). β-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 peroxisome proliferator-activated receptor-α (PPARα) and PPAR-γ Coactivator 1 Alpha (PGC1α) 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 lysosomal acid lipase 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.

10 10 It is well established that FFAs generated from autophagy or lipolysis fuels mitochondrial β oxidation. Yet, how these two processes are coordinated and what is their relative contribution to the FFA pool used for mitochondrial β-oxidation is widely debated. Using mouse embryonic fibroblasts, Rambold et al. showed that when LDs were abundant, such as in the fed state, lipolysis of LDs by the cytosolic lipase ATGL supplied FFAs for mitochondrial oxidation, and autophagy was dispensable under these conditions (43). Only after prolonged starvation did bulk autophagy become essential to replenish the LD with TGs to maintain lipolysis mediated LD-to-mitochondria fatty acid transport. These finding are in line with previous findings which demonstrated that autophagy promotes LC3-mediated LD biogenesis in hepatocytes (18, 44). Since the Rambold study did not examine lipophagy, it is not known if lipophagy-derived FFAs follow the same fate as those of bulk-autophagy. Also, it remains to be seen if similar mechanisms exist in other cell types. Nevertheless, these studies support the notion that autophagy regulates lipid catabolism via selective and non-selective 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 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

11 11 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 high fat diet in mice or by FFA in hepatocytes (45). In contrast, autophagy deficiency sensitizes heptocytes to oxidative stress-induced apoptotic cell death (46). In addition, lipophagy may also regulate FFA supply required for VLDL production; since 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 WATs and, hence, their lipid-storing capacity. Protein levels of key mediators [PPAR-γ, CCAAT/enhancer binding protein-α (CEBP-α) and β (CEBP-β)] and markers [(fatty acid synthase, steroyl-coenzyme A 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-β is required to induce autophagy during adipocyte differentiation. CEBP-βinduced autophagy then degrades kruppel like factor 2 and 3 two negative regulators of adipogenesis to facilitate 3T3L1 adipocyte differentiation (49). In line with in vitro data, CEBP-β was shown bound to the Atg4b promoter in mouse WAT, and depletion of

12 12 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 (BAT)-like features in white adipose depots such as increased expression of PGC-1α, 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 lipid droplets (17, 48). Thus, knockout mice with enlarged BATlike mass exhibited high rates of β-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 and high-fat diets, which suggests an essential but undefined role for adipocyte-autophagy on organismal viability (48). Since 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. showed that palmitate-induced ER stress activates autophagy in mature 3T3L1 adipocytes (50). Pharmacological inhibition of autophagy increased expression of the pro-inflammatory cytokines monocyte chemoattractant protein-1and interleukin-6 (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

13 13 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 to non-obese controls (51). Furthermore, overexpression of death-associated kinase 2 (DAPK2) one of the most strongly repressed adipose tissue genes in obese humans and high fat diet-induced 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 post-surgery compared to the adipocytes from same patients prior to surgery (52). Despite the progress made in defining the role of autophagy in mature adipocytes, questions remains 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 of the 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 agouti-related peptide (AgRP) neurons of 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

14 14 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, if 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 important to note that muscles are not well suited to store lipids in bulk as liver or adipose tissue, it is possible that FFA 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 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

15 15 macrophages, CE hydrolysis occurs primarily via lipophagy to generate free cholesterols that are used in reverse cholesterol transport to apolipoprotein A1-rich high density lipoproteins (HDLs) (57). Activation of autophagy upon cholesterol loading is likely a protective mechanism since 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 if this could be a potential therapeutic target. Regulation of Autophagic Activity by Lipids With the role of autophagy in lipid catabolism well established, 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 tissue-specific 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 beta cells to palmitate induces autophagy in presence of hyperglycemia (61). Diabetic db/db mice or non-diabetic mice challenged with a high-fat diet exhibited marked induction of autophagosome formation in pancreatic beta cells (62). In these models of lipid overload, induction of autophagy played a protective role against lipotoxicity: inhibition of autophagy in INS-1E beta cells by depleting Atg5 or blocking autolysosome formation accentuated palmitate-induced cell death; likewise, beta cell specific

16 16 deletion of Atg7 in mice induced cell death and led to profound glucose intolerance, partly due to the lack of a compensatory increase in beta cell mass and insulin secretion following high-fat feeding. The loss of autophagy in Atg7-deficient beta cells significantly increased oxidative stress and toxic accumulation of ubiquitinated proteins and damaged organelles that might collectively contribute to beta cell degeneration (63). Inhibition of autophagic flux by chloroquine induced markers of ER stress (C/EBPhomologous protein and phosphorylated eukaryotic initiation factor-2α) and apotosis (cleaved caspse 3) in pancreatic beta cells of high-fat fed mice, which further supports the protective role of autophagy against beta cell lipotoxicity (63). While short-term high-fat feeding induces autophagy in pancreatic beta cells, chronic lipid overload impairs autophagic flux (64, 65). Prolonged exposure of INS1 beta 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 fatty acid also determines beta cell lipotoxicity. The saturated fatty acid (SFA) palmitate inhibits beta cell proliferation and induces apoptosis, whereas the monounsaturated fatty acid 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, long-term high-fat feeding or palmitate

17 17 exposure induces lipotoxicity by blocking this adaptive autophagic response. Other factors such as ceramide accumulation, increased ROS production may also induce lipotoxicity independent of autophagy (67). As in beta 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 inhibtion 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 high-fat diet 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 proess 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 high-fat diet-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 (71). Restoration of hepatic Atg7 expression in obese mice normalized autophagy, reduced ER stresss, 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,

18 18 apoptotic cell death and fibrosis (72-75). 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 (76, 77). 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, BECLIN1 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 high-fat diet-induced autophagy was absent in adiponectin-deficient mice and was restored upon adiponectin reexpression. 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 diet-induced autophagy, high fat feeding in skeletal muscle-specific histone deacetylase 1 and 2 (Hdac1 and Hdac2) null mice which normally exhibit autophagy deficiency restored autophagic flux and prevented myopathy in adult mice (78). However, under chronic lipid overload, such as in severely obese, hyperinsulnemic human subjects, autophagic flux was blunted in primary skeletal muscle myotubes, which could contribute to the insulin resistant phenotype (79). 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

19 19 nutrient excess is persistent, this adaptative response is lost and declining autophagy might then exacerbate insulin resistance. This model may need to be revised in the light of recent study, which showed that. blocking autophagy by deleting Atg7 in skeletal muscles protects mice from diet-induced systemic obesity and peripheral insulin resistance (80). Mitochondrial dysfunction, induced by lack of autophagy, led to increased expression of fibroblast growth factor 21 (Fgf21) in the muscle and protected mice from diet-induced obesity and insulin resistance. Interestingly, autophagy deficiency in liver also induced Fgf21 expression and provided similar protection from obesity and insulin resistance (80). 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 hyperactivates autophagy and accelerates protein degradation in the muscle (81). A recent in vitro study showed that the FOXO3 pathway of muscle atrophy was induced as a lipotoxic response to SFA overload (82). Coincubation with unsaturated fatty acid completely reversed lipotoxic

20 20 effects of saturated fatty acid, underscoring the fatty acid-specific regulation of autophagy and atrophy in skeletal muscles. In the heart, basal autophagy plays a prosurvial role (83). However, our understanding of the lipid-induced regulation of autophagy is still emerging. He et al. showed that 12 weeks of high fat feeding suppressed cardiac autophagy assessed by the increased level of p62 (84). Several other studies reported impairment in cardiac autophagy following long-term high-fat feeding (85-87). A recent study showed that impaired cardiac autophagy in high fat fed mouse induced 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 (88). 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 HFDinduced inhibition of autophagic flux (87). High-fat feeding suppressed the expression of Rab7 a small GTPase 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 heart (87). Besides AKT, AMPK-mediated regulation of protein tyrosine phosphatase -1B (PTP1B) also controls lipid-induced inhibition of autophagy (89). Mice fed with a 45% high fat diet 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 diet-induced cardiac hypertrophy and

21 21 contractile dysfunction. Pharmacological inhibition of AMPK, however, abrogated beneficial effects of PTP1B ablation on autophagy, cardiac structure and contractile function (89). In contrast to these studies, feeding a high fat diet 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 (90). 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 (90). 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 corrobrate 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 (88). 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 (91). Prolonged palmitate treatment for 12 h or longer suppressed autophagic flux with

22 22 concomitant increase in cellular lipotoxicity marked by an elevated ROS level, activation of the stress kinases JNK and p38mpak, and a profound increase in apoptotic markers such as cleaved caspase 3 and caspase 7 and their downstream effector poly ADP-ribose polymerase 1 (92). 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 medium-chain SFA myristate but not the long-chain SFA palmitate induced autophagic flux and promoted hypertrophy in isolated adult primary cardiomyocytes (90). This suggests that myristate or its derivatives induces autophgy 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 lipidinduced regulation of cardiac autophagy depending on composition of diet, type of fatty acids, duration of feeding, and type of cell culture and animal models used. Whether these pathways are linked, to provide a unifying mechanism or they 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. 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,

23 23 thereby stabilizing lysosomal membranes against acid hydrolases and other degradative agents (93). Like proteins, lipids are an intergral part of lysosomal membranes and play a key role in lysosomal biogenesis and function (reviewed in reference (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 low density lipoproteins (LDL) and remnants of very low density lipoproteins (VLDL) and chylomicrons (94, 95). Endocytosis of lipoproteins is facilitated by members of the LDL receptor family localized in either lipid rafts or clathrin-coated pits (96). Endocytosed LDL particles are transferred as such from late endosomes to the lysosomes where TGs and CEs are hydrolyzed by lysosomal acid lipase (LAL) to generate FFAs and free cholesterols, respectively (97). In contrast, for VLDL and chylomicrons remnants, their apolipoprotein E receptors are recycled back to the plasma membrane leaving remaining lipid particles to be degraded in the lysosome (96, 98). Endogenous lipids or LDs enter the lysosome primarily via autophagy and are hydrolyzed by LAL. Hydrolysis of LD TGs and CEs by LAL generate FFAs and free

24 24 cholesterols, respectively (16, 57). Normally, free cholesterols released from lysosomes are transferred to the ER for reesterification (99). However, as described earlier, in cholesterol-loaded macrophages, free cholesterols derived from lipophagy are incorporated into apolipoprotein A1-rich HDL (57). Biochemical studies on LAL isolated from rat-liver lysosomes suggest that the enzyme catalyzes both the hydrolysis and synthesis of CEs (100). Re-esterification of cholesterol was also observed in endosomes of cultured mouse macrophages exposed to LDL aggregates (101). 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 cholesterolreesterification 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 (102). 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. showed that cells sense amino acid content in the lysosomal lumen to determine their nutritional status (108). This information is relayed by lysosomal vacuolar ATPase (v-atpase) proton pump to the Ragulator 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 Ragulator complex (109). Ragulator charges small molecule GTPases RagA/B and

25 25 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 (108, 110). Active mtorc1 inhibits the transcription factor TFEB by direct phosphorylation and blocks PPARα transcriptional activity by promoting its interaction with nuclear receptor corepressor 1 (NCoR1) (111, 112). TFEB is the major transcription factor that globally activates several autophagic and lysosomal genes including LAL (113, 114). PPARα is the master transcriptional activator of several genes involved in fatty acid β-oxidation. Thus, by regulating mtorc1 activity in response to lumenal amino acid content, lysosomes control both lipophagy and mitochondrial fatty acid oxidation by sensing cellular nutrient status. It is interesting but 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 (115), excess lipid supply largely impair lysosomal function by multiple mechanisms (Fig. 5). Lysosomal lipid storage diseases (LSD), or lipidoses, are a constellation of inherited disorders characterized by the accumulation of lipids in late endosomes and lysosomes (116). Many of these diseases are caused by impaired function of lysosomal hydrolyses, 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 unesterifed cholesterol (117, 118). How lipid accumulation contributes to the pathogenesis of the disease is still unclear. A previous study linked cholesterol

26 26 accumulation to defects in trafficking between intralumenal vesicles and limiting membrane of the late endosomes (119). Such defects may impair trafficking or reccyling 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 lipid storage diseases ( ). Accumulation of lipids in lysosomes can also inhibit autophagosome turnover. Several studies in LSD, documented an impairment in fusion between autophagosomes and lipid-loaded lysosomes that leads to defective autophagic clearance ( ). Conversely, depletion of cholesterol or inhibition of cholesterol synthesis induces autophagy (127, 128). In vitro fusion assays showed that altered membrane composition of autophagosomes and lysosomes isolated from lipid-loaded fibroblasts or high fat 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 high fat diet 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 (129). Lipid-induced lysosomal dysfunction is also observed in diet-induced obesity and metabolic disorders (87, 130). Mice fed a high fat diet exhibited increased accumulation of phospholipids in lysosomes that was associated with reduced lysosomal enzyme activity, which was reversed by the chronic activation of AMPK (130). Likewise, high fat feeding in mice reduced the cardiac expression of Rab7 a small GTPase essential for lysosome maturation (131) which impaired autophagosome-lysosome fusion and

27 27 suppressed autophagic flux (87). Cardiac ablation of Atg2 restored Rab7 expression and normalized autophagic flux and prevented cardiac hypertrophy and contractile dysfunction induced by high fat feeding (87). Lysosomal membrane permeabilization (LMP) is a key mechanism by which chronic lipid overload promotes lysosome dysfunction and triggers apoptotic cell death (132, 133). Both high fat feeding and fatty acid exposure induced LMP in adipose tissue that led to the release and activation of the lysosomal protease cathepsin B (CTSB) in the cytosol following macrophage infiltration of adipose tissue and production proinflammatory cytokines (134). High fat feeding also induced LMP and lipotoxicity in the liver of a mouse model of diet-induced non-alcoholic steatohepatitis (NASH) although hepatic autophagy remained largely unchanged (135). In the heart, a high fat diet induced release of lysosomal cathepsin K into the cytosol a hallmark of LMP in parallel with cardiac hypertrophy, contractile dysfunction and the induction of apoptosis. Ablation of cardiac cathepsin K improved cardiac structure and function and suppressed apoptosis in high fat fed mice (136). In macrophages, exposure to atherogenic lipids such as oxidized LDL (oxldl) and cholesterol crystals precipitated lysosomal dysfunction characterized by increased lysosomal ph, reduced proteolytic activity, and increased LMP which led to impaired cholesterol efflux, potentially due to inhibition of autophagy (137). Induction of lysosomal biogenesis by TFEB overexpression attenuated lysosomal dysfunction and restored normal cholesterol efflux in lipid-loaded macrophages. Mechanistic studies on mouse hepatocytes revealed that SFA treatment initiates Bax translocation to lysosomes and lysosomal destabilization that releases cathepsins into the cytosol (138, 139). Cytosolic cathepsins induced mitochondrial

28 28 membrane permeabilization and caspase activation, triggering the intrinsic pathway of apoptosis and liver injury as observed in a dietary mouse model of non-alcoholic fatty liver disease (NAFLD). Genetic and pharmacological inactivation of CTSB was shown to be protective against hepatic steatosis and liver injury in dietary NAFLD mice (132). These studies indicate that LMP may be the key mediator of lipotoxicity. Besides lysosomal pathways, SFA-induced lipotoxicity can also activate non-lysosomal extrinsic pathways of apoptosis (140). Oxidative stress is another important regulator of lysosome function. The high iron content of lysosomes, derived from the degradation of metalloproteins such as ferritin, makes them susceptible to oxidative damage (141, 142). Ferric iron reacts with hydrogen peroxide (H 2 O 2 ) and superoxide anions (O2 - ) to generate the highly reactive hydroxyl radical (OH ) (143). Hydroxyl radicals can oxidize proteins, lipids, and other macromolecules that inhibit their degradation leading to the accumulation of indigestible wastes in lysosomes. Lysosomal wastes are known to impair lysosomal enzyme activity and promote LMP (144). Additionally, ROS and reactive nitrogen species (RNS) generated from various other cellular sources may also affect lysosome function. Previous studies on neutrophils showed that NADPH oxidase isoform 2 (NOX2) is recruited to the late endosomal membrane where it generates superoxides. Initially, superoxides can promote acidification of endosomes by trapping protons in the endosomal lumen. However, excessive superoxides can react with water to generate hydroxyl ions that can ultimately impair acidification of the endosomal vesicles by increasing ph (145). NOX2 activation and increased ROS production has been observed in animal models of diabetes and diet-induced obesity and in humans with

29 29 metabolic syndrome (15, ). Until recently, NOX2-derived ROS was known to regulate phagosomal ph only in inflammatory cells: for antigen presentation in dendritic cells, and for targeting phagosomes for antibacterial autophagy. Due to striking parallels between NOX activation and lysosomal dysfunction in the context of lipid oversupply, we examined if two processes are interconnected. Both high fat fed mouse hearts and palmitate exposed H9C2 cardiomyocytes markedly increased NOX2 activity. NOX2- derived superoxides impaired lysosomal acidification and hydrolase activity that blunted autophagosome degradation despite normal autophagosome-lysosome fusion (91). Genetic and pharmacological inhibition of NOX2 normalized superoxide production, and restored lysosomal function and autophagic flux. Mechanistically, palmitate treatment led to an accumulation of diacylglycerol, which activated protein kinase beta2, to increase NOX2 activity. NOX2-derived superoxide, increased oxidative S-nitrosylation of the Atp6v1a1 subunit of lysosomal vatpase at Cys-277, which masks the free thiol group potentially required for cleaving an inhibitory disulfide bond and activating vatpase (149, 150), leading to impaired lysosomal acidification and defective autophagic clearance. How NOX2-derived superoxides regulate S-nitrosylation of vatpase subunits is under further investigation. Consistent with the role of NOX2 in lysosomal function, Pal et al. found that NOX2-derived ROS impairs both lysosome biogenesis and autophagy in a mouse model of Duchenne muscular dystrophy. Genetic down regulation of NOX2 attenuated ROS production, restored normal autophagy, and improved muscle function in these mice (151). These studies suggest that NOX2 regulates lysosome biology by multiple mechanisms in diverse cells types in a stimulusdependent fashion.

30 30 It is important to note that in line with multiple studies examining FFA-induced lipotoxicity, we observed that only the saturated but not the monounsaturated FAs induced ROS-production and lysosomal dysfunction in cardiomyocytes (91). In fact, studies suggest that monounsaturated FAs may protect against saturated FFA-induced lipotoxicity, possibly by channeling toxic lipids to the relatively benign TG depot (82, 152, 153). Supporting these findings, we found that co-incubation with oleate increased TG accumulation and normalized autophagy in palmitate-treated cardiomyocytes (91). These studies underscore the need for defining the composition of lipids in an animal diet or in vitro experiments to better understand the mechanism of lipotoxicity. Such approaches may also reconcile discrepancies in various studies examining the effect of lipid-overload on lysosome function, autophagy, and other cellular pathways involving lysosome function. Conclusion: Lipids are central to cellular survival and viability by regulating fundamental processes that maintain cellular structure, organelle function and energy homeostasis. Oversupply of dietary lipids overwhelms the ability of adipocytes and peripheral tissues to store lipid, leading to cellular stress responses. Autophagy has emerged as an important process for maintaining cellular and whole body lipid homeostasis under normal conditions, upon nutrient depletion, and during the early phase of lipid challenge. This homeostatic mechanism falters upon chronic lipid overload by multiple mechanisms. Studies of autophagy and lipids over the last decade have greatly increased our understanding of their mutual relationship; however, several key questions still remain to be answered in the short-term and the long-term.

31 31 How are LDs recruited to lysosomes via lipophagy? How is the mechanism of lipophagy distinct from other types of autophagy? What are the molecular signals that govern selective targeting of LDs via lipophagy? What is the molecular switch between non-selective and selective degradation of LDs via autophagy? What are the differences in the utilization of FFAs generated from lipophagy, bulk autophagy and cytosolic lipolysis? Does reactivation of lipophagy protect cells from lipotoxicity when bulk autophagy is inhibited? Does lipophagy in mature adipose tissue the major organ for lipid metabolism play a major role in lipid mobilization as in hepatocytes, given the dogma that classical lipolysis in adipocytes is the major and most efficient lipolytic mechanism? Since lysosomes are the final destination for autophagy and other endocytotic pathways, endo/lysosomal dysfunction not only affects macroautophagy but also impairs CMA, recycling of membrane lipids, sorting of lipids and lipoproteins into appropriate intracellular compartments, and promotes lipotoxic cell death (23, 154, 155). Accordingly, the repertoire of lysosomal functions has greatly increased recently, transforming it from a recycling bag to a hub of nutrient signaling. However, the molecular mechanisms of many of these functions are incompletely understood. From the perspective of lipid metabolism, the following represent some outstanding questions that remain to be answered in future investigations. How do lysosomes process different types of lipids? Do lysosomal hydrolases have equal affinity for all types of lipids?

32 32 How are lysosomal lipid catabolites sorted into different cellular pathways? Are lysosomal membrane proteins and transporters also involved in lipid trafficking within and outside of cells? How do lysosomes coordinate lipophagy and other lipid mobilizing pathways? How do different fatty acid species modulate lysosomal function? Do lysosomes also sense nutrient signals other than amino acids, such as simple FFAs or sugars, and how do these signals crosstalk with amino acid-sensing signaling mechanisms? In conclusion, this review has discussed studies that broaden our understanding of how lysosomes import and process dietary lipids. Moreover, they have also provided additional insights into other cellular functions whereby the lysosome may act as a lipidregulated signaling hub in addition to its role as an organelle for degradation (156, 157). Acknowledgment Relevant studies in the Abel laboratory were supported by NIH grant R01HL108379

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42 42 triglycerides (TG) and cholesteryl esters (CE) and releases them into the circulation. (3) Adipose tissue TG hydrolysis generates FFA which are released into the blood stream bound to albumin. Hydrolysis of CM and VLDL by lipoprotein lipase (LPL) at the endothelial surface generates FFA that enter the peripheral tissue via fatty acid transporters CD36/FATP. The CM and VLDL remnants generated from LPL-mediated hydrolysis are taken up by liver for further processing. In the fed state, FFA are esterified into TG and stored as LDs in adipose tissue and liver or undergo β-oxidation to generate ATP in muscle and other peripheral tissues. In addition, de novo lipogenesis from glucose also generates FAA in the fed state. In the fasted state when lipid supply dwindles, TG hydrolysis generates FFA that are released into the circulation by adipose tissue or used to generate ketone bodies in liver. Albumin-bound FFA (Alb-FFA) and ketone bodies serve as the major source of energy during fasting. Fig. 2. Fatty acid overload-induced oxidative stress and lipotoxicity. Oversupply of FFA may overwhelm mitochondrial capacity to oxidize fatty acids for ATP production, which leads to accumulation of toxic lipid intermediates such as diacyl glycerol (DAG), ceramides and acyl carnitines. Toxic lipid intermediates impair insulin signaling through insulin receptor substrates and blunt insulin-stimulated glucose uptake. DAG also activates NADPH oxidase (NOX) via protein kinase C (PKC) to increase intracellular reactive oxygen species (ROS) production. In addition, dysregulation of mitochondrial electron transport chain (ETC) due to excessive FA β 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

43 43 autophagy, and activation of inflammatory responses in multiple tissues. FA-CoA, fatty acyl-coa; CPT I and CPT II, carnitine palmitoyltransferase I and II; NADH, reduced nicotinamide adenine dinucleotide; FADH 2, reduced flavin adenine dinucleotide. 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 (PI3KC3) 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 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 results 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 towards 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. Fig. 4. Degradation of lipid droplets (LDs) via autophagy. Lipid droplets, containing mostly triglycerides (TG) and cholesteryl esters (CE) at the core, undergo chaperone-

44 44 mediated autophagy (CMA) of their structural membrane proteins perilipin 2 and 3 (PLIN2 and PLIN3). PLIN removal activates RAB7 at the exposed LD surface. RAB7 interacts with 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 and B) or with other autophagic substrates via non-selective macroautophagy (C). Autophagosomes fuse with lysosomes to form autolysosomes wherein lysosomal hydrolases such as lysosomal acid lipases degrade LD TG and CE into free fatty acids (FFA) that are recycled back into the cytosol to support various energetic and structural requirements of the cell. Fig. 5. Lysosomal stress-response pathways induced by dietary lipid overload. Overload of free fatty acid (FFA) supplied exogenously or derived from triglyceride (TG) hydrolysis induces ROS production from mitochondrial electron transport chain complexes, the NADPH oxidase 2 (NOX2) complex, and other reactive oxygen species (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 lysosomal membrane permeabilization (LMP) and release of cathepsins into the cytosol. Cytosolic cathepsins induces apoptotic cell death either by direct activation caspases or through mitochondrial membrane permeabilization (MMP)- mediated caspase activation. ROS-induced protein oxidation impairs the function several lysosomal enzymes such as vacuolar ATPase (vatpase) proton pump and lysosomal hydrolases, which raises lysosomal ph and inhibits degradation of autophagic cargo, respectively. Lipid overload also induces lysosomal dysfunction by

45 45 ROS-independent 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). Free fatty acid (FFA)-induced translocation of mtor complex 1 (mtorc1) to the lysosomal membrane has been shown to promote endoplasmic reticulum (ER) stress-dependent apoptosis in podocytes.

46 46 Table 1. Lipid-overload induced regulation of autophagy in different metabolic tissues. (WT= wildtype) Study Model Pancreas Wildtype (WT) or db/db mice Diet or Treatment 60% HFD for 12 weeks Effect on autophagy Induced Observation (Reference) Autophagy maintained beta cell mass, insulin secretion, and normal glucose tolerance (44). INS1 or MIN6 beta cells Liver WT, ob/ob and liver-atg7 null mice 0.4 mm Palmitate and 20 mm Glucose for 14 h or longer 35% HFD for 22 weeks Suppressed Suppressed Impaired autophagic flux led to defective insulin secretion and apoptosis (46, 47). Autophagy suppression increased ER stress and insulin resistance (51). Skeletal muscle WT, Adiponectinnull and musle- HDAC 1/2 null mice WT and muscle- Atg7 null mice Heart WT and Akt2-null mice WT mice 60% HFD for 2 to 12 weeks 60% HFD for 13 weeks 45% HFD for 20 or 25 weeks 60% Milk fat-based diet for 18 weeks Induced Suppressed in Atg7 null mice Suppressed Induced Autophagy induction parallels upregulation of antioxidant genes and ameliorates insulin-resistance and myopathy (60,61) Suppression of autophagy protects from HFD-induced obesity and insulin-resistance (63). Impaired autophagic flux resulted in cardiac hypertrophy, contractile dysfunction, ER stress and apoptosis (70,71). De novo sphingolipid synthesis induced autophagy and cardiomyocytes hypertrophy and contractile dysfunction (73).

47 47 Table 2. Changes in lysosomal function associated with lipid overload. Study Model Effect on lysosomes Effect on autophagy Observation (Reference) Mouse models of Defective endocytic transport. Suppressed turnover due Accumulation of unesterified cholesterol, LSD (e.g., NPC1, MSD, and MPSIIIA). Reduced expression or activity of lysosomal hydrolases. to defective autophagosomelysosome fusion. phospholipids, glycolipids, sphingomyelin ( ), dysfunctional mitochondria and polyubiquitinated proteins (125). Abnormal lysosomal ph. Aberrant sequestration of SNARE complex and Enlarged lysosomal vesicles accumulation of autophagic cargo (123, 126). Mouse models of diet-induced obesity and metabolic syndrome Mouse models of diet-induced LMP Deficiency in Rab7 protein level. Enlarged lysosomal vesicles Bax-induced permeabilization of lysosomal membrane. Release of cathepsin B, D and K into cytosol. Suppressed autophagic flux linked to Rab7- or ER stress-dependent defects in autolysosome formation. Suppressed autophagy due to lack of intact, functional lysosomes Reduced Rab7 expression in the heart (87). Induced ER stress in liver (87, 158). Accumulation of phospholipids, ubiquitinin, and p62-positive protein aggregates in lysosomes in kidney and liver (130, 158). Cytosolic cathepsin-induced apoptotic cell death (132, 138). Cathepsin-induced mitochondrial membrane permeabilization and apoptotic cell death (138). Lipotoxic liver injury in NASH (138). Cardiac hypertrophy and contractile dysfunction (87, 88) Impaired cholesterol efflux in macrophages (137). Cell culture model of lipid-induced oxidative stress and lipotoxicity Increased lysosomal ph. Reduced lysomal cathepsin activity Oxidative post-translational modification of lysosomal Suppressed due to inhibition of autophagosome turnover Increased Nox2-derived superoxide production, s-nitrosylation of lysosomal vatpase, accumulation of defective autolysosomes (91). vatpase Decreased lysosomal Suppressed due to Accumulation of autophagosomes and acidification reduced lysosomal autophagic cargo, reduced mitochondrial Reduced cathepsin L activity acidification and oxidative phosphorylation and ATP hydrolase activity caused production, and β-cell dysfunction marked by by low cellular ATP defective insulin secretion and increase cell levels. death (64).

48 48 Bax-induced lysosomal membrane permeabilization Unknown Upregulation of ceramide and caspase dependent lysosomal membrane permeabilization and apoptotic cell death (132).

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