Adaptive and maladaptive roles of lipid droplets in health and disease

Abstract

Advances in the understanding of lipid droplet biology have revealed essential roles for these organelles in mediating proper cellular homeostasis and stress response. Lipid droplets were initially thought to play a passive role in energy storage. However, recent studies demonstrate that they have substantially broader functions, including protection from reactive oxygen species, endoplasmic reticulum stress, and lipotoxicity. Dysregulation of lipid droplet homeostasis is associated with various pathologies spanning neurological, metabolic, cardiovascular, oncological, and renal diseases. This review provides an overview of the current understanding of lipid droplet biology in both health and disease.

PHYSIOLOGY OF LDs

Lipid droplets (LDs), initially considered mere subcellular cargo, are now recognized as cellular organelles with pivotal physiological functions. This shift was driven by the observation that LDs play a critical role in mediating normal cellular homeostasis and that dysregulation of this process is associated with various diseases (1). LDs store a variety of lipids, including fats, oils, and hormones. Lipids serve as energy-rich metabolic substrates, structural components of membranes, cell signal mediators, and posttranslational modifiers of proteins (2). Cells use multiple mechanisms to internalize, synthesize, store, and mobilize lipids within LDs to guarantee proper cellular homeostasis (3).

Structure, Biogenesis, and Composition of LDs

LDs are composed of a hydrophobic core consisting of primarily neutral lipids, such as triglycerides (TG) and cholesterol esters (CE), surrounded by an amphipathic phospholipid monolayer (4, 5). Phospholipids are synthesized at the endoplasmic reticulum (ER), and their presence in the phospholipid monolayer of LDs confers stability and regulates their size and numbers within cells (6). Other lipids, such as waxes, free cholesterol, and long-chain alcohols are also found in LDs but in significantly lesser quantities (7, 8). The amount and molecular composition of neutral lipids (NL) in LDs varies substantially among different cell types and may reflect cell-type specialization (9–11). LDs in adipocytes mostly contain TG (9), whereas LDs of nonadipocytes usually have a higher content of CE (10). Similarly, the CE content of LDs is higher in macrophages than in hepatocytes, whereas LDs of stellate cells are particularly enriched with retinoic acids (11). The NL composition of LDs also varies in response to pathological stressors. The NL, leukotriene, and prostaglandin content of LDs are elevated in neutrophils from patients with infectious and inflammatory diseases (12). The arrangement of these phospholipids in the phospholipid monolayer can vary in composition between different cell types and physiological states.

LDs originate from the ER; however, the exact mechanism of LD biogenesis is still a subject of active research. The current model of LD biogenesis involves resident ER proteins that synthesize NLs at the ER membrane (13–15). NLs aggregates develop between the membrane leaflets, leading to crescent or curved areas in the membrane, which ultimately bud into the cytoplasm with the help of specialized ER proteins to form the LD (16–20). In yeast, Pah1, which is involved in diacylglycerol (DAG) synthesis, and phospholipid:diacylglycerol acyltransferase (LRO1), a triacylglycerol (TAG) biosynthesis enzyme, were shown to localize to the ER-LD sites (20–22), suggesting DAG and TAG synthases are involved in LD formation. In mammalian cells, acyl-CoA synthetase long-chain family member 3 (ACSL3) and seipin coordinate the synthesis of LDs at the ER, with the recruitment of ACSL3 being regulated by seipin (23, 24). High-resolution structural data demonstrate that both human and Drosophila seipin form a helical ring-like structure with β-sheets oriented parallel to the membrane and α-helices situated perpendicular to the membrane (25, 26). These α-helices have been shown to interact with TAG, possibly ensuring efficient packaging of NLs into the LD.

Following the initiation of LD formation at the ER-LD biogenesis sites, LD expansion is the predominant process that determines LD size based on the number of NLs produced or sequestered (27–29). This process occurs upon relocation of ER-resident proteins to the LD via the ER-LD contact sites. Acetyltransferases, including glycerol-3-phosphate acyltransferase 3 (GPAT3), 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3), and diacylglycerol O-acyltransferase 2 (DGAT2), move from the ER to the LD and induce LD expansion (27). As the LD matures, LD-associated proteins are trafficked to the LD, affecting its size, shape, and behavior. Approximately 200 different LD-associated proteins with essential functions in regulating LD lipid content and stability have been described (30). These proteins are critical for LD transport, intracellular distribution, and association with filaments and other organelles (2). LD-associated lipases, such as adipose triglyceride lipase (ATGL), degrade NLs, facilitate fatty acid synthesis, and contribute to the transport of sterols to other organelles (31, 32). Induction of lipase activity leads to a reduction in the size of LDs (33), whereas the activation of lipid synthases leads to the generation of LDs with more NLs or enlarged LDs (34). Perilipins (PLINs) are a group of proteins that influence LD catabolism by modulating the access of lipases to the LD surface (35) and have been implicated in chaperone-mediated autophagy (CMA) (36). Similarly, the loss of lysosomal-associated membrane protein 2 (LAMP2A) and PLIN3 activity leads to LD accumulation due to a decrease in CMA levels. Further evidence suggests that PLIN2, and possibly PLIN3, are needed on the surface of LDs to allow ATGL and other autophagic proteins access to LD contents (37). Within the cell, TG-rich LDs are separated from CE-rich LDs. Such separation of the two distinct LD populations was also observed using cell lines. For example, in rat McARH777 hepatocytes CE-rich LDs were found clustered in the cytoplasm centered around the nucleus whereas TG-rich LDs were localized in the periphery (38). More studies are needed to reveal the physiological significance and functional consequences of this spatial segregation.

After their initial formation, LDs undergo various fusion processes, ripening, or coalescence (39). Ripening defines the process by which one LD transfers its contents to another LD (39). A crucial component of ripening is cell death-inducing DNA fragmentation factor-α (DFFA)-like effector C/fat specific protein 27 (CIDEC/FSP27), a member of the cell death-inducing DNA fragmentation factor-α (DFFA)-like effector (CIDE) family of proteins, which potentially play a pivotal role in the formation of giant LDs (40–42). Conversely, coalescence is a much more rapid process by which LDs fuse. Conditions that favor coalescence include decreases in the phosphatidylcholine:phosphatidylethanolamine ratio of the LD membrane, whereby phosphatidylcholine prevents coalescence by acting as a surfactant (43, 44). Thus, adequate levels of phosphatidylcholine are necessary to prevent large LDs from accumulating through coalescence (45).

The Role of LDs in Lipid Metabolism

Metabolic responsiveness is critical for the ability of cells to cope with changing energetic demands. Cells store energy in the form of fatty acids (FAs) and NLs when the nutrient load is high, which can be released in instances of stress or low nutrient levels. The most abundant form of these NLs are TAG and sterol esters. LDs function as a site for the storage of these NLs and thus play a central role in energy and lipid metabolism, and the contact sites associated with LDs and organelles are instrumental for FA synthesis and breakdown (46, 47). FA metabolism utilizes multiple intracellular organelles in concert with LDs (46). Lipid transfer proteins and FA-modifying enzymes reside and function at LD-organelle contact sites and compartmentalize FA metabolism. FAs must be broken down into acyl-CoA via β oxidation, acyl-CoA can then enter the citric acid cycle. This process occurs in mitochondria and peroxisomes, the primary sites of lipid metabolism in the cell.

Lipolysis and Lipophagy

Lipolysis is the breakdown of LDs stored as TGs to free fatty acids (FFAs) to produce energy through the action of cytosolic lipases, including hormone-sensitive lipase (HSL), monoacylglycerol lipase, and ATGL (48–50). Traditionally, this was the predominant model of LD degradation until other regulatory mechanisms between lipolysis and autophagy were identified. Although insulin inhibits lipolysis and autophagy, glucagon acts in a stimulatory manner. A seminal paper by Singh et al. described the discovery of a novel mechanism of LD degradation by autophagy in hepatocytes. This observation led to the discovery of lipophagy, a process by which LDs are catabolized through autophagic pathways leading to lysosomal degradation of stored lipids into FFAs. This mechanism, termed lipophagy, was confirmed by others (51–53).

Lipophagy begins with the interaction of LDs with the autophagosome membrane through binding to microtubule-associated protein 1 light chain 3 (MAP1LC3) (54). This binding involves the cooperation between one or more proteins that function as cargo adapters (i.e., p62 and optineurin) that connect the LD and LC3 (55). The exact regulators of lipophagy are largely unknown. However, some Rab family proteins are thought to be involved in the induction or cessation of lipophagy (56–59). Rab proteins are readily detected on LDs in various disease states (56), and Rab7 (58), Rab10 (57), and Rab25 (59) are regulators of lipophagy. The cytosolic lipolysis-associated lipase ATGL, acting through the deacetylase SIRT1, functions as an initiator of lipophagy in mouse liver cells (60). Although there is tight coordination and connection between these two pathways, ATGL cannot initiate LD catabolism without lipophagy. LDs, depending on the size, can undergo macroautophagy or microautophagy. In macroautophagy, an entire small LD is incorporated into the autophagosome and processed. In microautophagy, small parts of larger LDs accumulate LC3 and bud off to be incorporated into the autophagosome for gradual processing (53). LDs are catabolized by special lipases known as lysosomal acid lipases (LALs) that have a broad range of lipid substrates (61–64). These lipases differ from their cytosolic counterparts in that they function in the acidic environment of the autophagosome (65).

Lipophagy is regulated by the nutrient status of the cell and proteins that detect changes in nutrient stores. Examples of these regulatory receptors include the nuclear receptors farnesoid X receptor (FXR), the transcriptional activator cAMP response element-binding protein (CREB), peroxisome proliferator-activated receptor alpha (PPARα) (66, 67), mammalian target of rapamycin (mTOR) (68–70), and AMP-activated protein kinase (AMPK) (71, 72). Increased intracellular nutrient stores inhibit lipophagy, whereas cellular starvation promotes it (73). Lipophagy also acts as a defense mechanism against lipotoxicity and is increased in a genetic mouse model of SOCE (store-operated calcium entry) deficiency (54). In this model there are reduced levels of cytosolic lipolysis, further demonstrating the importance of lipophagy as a mechanism for neutral lipid catabolism and in maintaining cellular homeostasis (54).

Although the primary function of each of these pathways lies in the catabolism of LD content, lipolysis functions mainly to release energy stores in the form of FFA β-oxidation. Lipophagy can also alleviate lipotoxic stressors through acidic degradation in the lysosome. Initially, it was thought that these two processes, lipolysis and lipophagy were independent. Recent studies propose that these two pathways may regulate each other directly (74). Evidence suggests that levels of autophagy are regulated by LDs and lipases (74). However, although both processes regulate FFA levels in the cell, lipolysis also functions in lipid signaling, which has implications in multiple downstream processes integral to homeostasis. Lipophagy has broader substrate specificity than lipolysis and can export hydrolyzed lipids out of the cell through a process called exophagy (63). Aberrant lipophagy may lead to a decreased ability of the cells to process a broad scope of lipid substrates and export excess FFA out of the cell, leading to persistent increases in LDs. Although lipolysis and lipophagy pathways and their interconnected regulation have been reviewed in detail elsewhere (62), this review will include a brief discussion on the lipophagy signaling cascades that may contribute to the accumulation of LDs in diseases including cancer, metabolic, and liver disease.

Lipid Export from LDs

The transport of lipids into LDs and subsequently outside of the cell counteracts the accumulation of lipids inside the cell and is necessary for proper protection from lipotoxicity. LDs can export toxic or excess lipid species through active and passive transport mechanisms to acceptor particles. One example of such a transport mechanism is reverse cholesterol transport (RCT), where cholesterol transport proteins such as ATP binding cassette subfamily A member 1 (ABCA1) and ATP binding cassette subfamily G member 1 (ABCG1) efflux cholesterol to the extracellular acceptor high-density lipid (HDL) particle in an apoprotein A1 (APOA1)-dependent mechanism (75, 76). Free cholesterol and phospholipids transported to HDL by ABCA1 are immediately esterified by lecithin-cholesterol acyltransferase (LCAT) (77). The interaction of ABCG1 and HDL further increases cholesterol efflux and the total capacity of HDL to uptake cholesterol (78). Although there are also passive mechanisms of lipid efflux, they account only for a minor fraction of the total efflux capacity of a cell (79). Therefore, the dysfunction of active efflux mechanisms mainly contributes to the accumulation of LDs and the pathophysiology of LD accumulation/lipotoxicity in disease. Reduced HDL levels and activity has been associated with many diseases resulting in LD accumulation and cell injury (80, 81). Although much is known about ABCA1- and ABCG1-mediated lipid efflux mechanisms, more studies are necessary to investigate the existence of other mechanisms that could be utilized as therapeutic targets for the treatment of disorders involving LD accumulation.

LDs IN RESPONSE TO CELLULAR STRESS

The essential role of LDs in maintaining homeostasis includes functions pertaining to lipid storage and trafficking, lipid metabolism, innate immunity, and cellular signaling, which have been extensively reviewed elsewhere (2, 13, 82–89). In addition to these functions, LDs mediate cellular stress responses common to multiple disease states. Recent studies have demonstrated that LDs may mediate ER stress, reactive oxygen species (ROS), and lipotoxicity, and maladaptive processing of LDs can have deleterious effects (Fig. 1).

Figure 1.

Recent studies demonstrating lipid droplets (LDs) may mediate endoplasmic reticulum (ER) stress, reactive oxygen species (ROS), and lipotoxicity, and maladaptive processing of LDs can have deleterious effects. Figure created with Biorender.com.

ER Stress

ER stress and unfolded protein response (UPR) signaling are central in the pathogenesis of many diseases, including metabolic diseases, neurodegenerative diseases, and cancer (90–98). ER stress is a general term that refers to aberrant calcium uptake and lipid composition, which leads to dysfunction of ER protein folding capacity (99). In response to ER stress, cells initiate UPR, which re-establishes proper ER homeostasis (99). Accumulation of unfolded proteins initiates the activity of three independent UPR transducers, inositol-requiring protein 1 (IRE1), protein kinase RNA (PKR)-like ER kinase (PERK; also known as EIF2AK3), and activating transcription factor 6 (ATF6) (2). This initiates signaling cascades to slow protein translation and to increase the expression of genes involved in protein folding, protein degradation, and lipid biosynthesis (2). In yeast, any perturbation in LD biogenesis results in the upregulation of UPR (100). However, the mechanisms connecting UPR activation to LD biogenesis remain unclear. It is possible that alterations in the ER composition directly activate the UPR, independent of complications in protein folding (97, 101–103).

Polyubiquitylated proteins marked for degradation are observed in LD-enriched cellular fractions during ER stress, suggesting that LDs aid in the removal of defective proteins (100). However, the mechanism of these faulty proteins localizing to LDs is unclear and warrants further investigation. Research suggests that lipid export or trafficking of ubiquitinated proteins leads to the removal of proteins and decreased ER stress. In yeast and hepatocytes, impaired ER protein degradation and UPR inducers (e.g., tunicamycin and dithiothreitol) lead to LD accumulation, indicating that LDs are induced in response to ER stress to help restore cellular homeostasis (104–106). LD accumulation may also counteract ER stress through the direct redistribution of specific phospholipids and the restoration of proper function to the ER membrane. Increased UPR activation is observed in yeast strains that lack the enzymes required for the biosynthesis of LDs (107–109). LDs accumulate near ER aggregates in yeast models of ER stress (100). The absence of LDs leads to an increase in phosphatidylinositol, resulting in atypical ER morphology (109, 110). In mammalian adipocytes, LDs also play a role in preventing ER stress and UPR activation. Recent studies demonstrate that noradrenergic stimulation of lipolysis results in the release of fatty acids and the generation of LDs, which inhibitors of acyl-CoA synthetases and DGAT1 can block, indicating a role for fatty acid re-esterification and packaging of lipids into DGAT1-dependent LDs (111–113). These data strongly suggest an essential role for LDs in ER homeostasis and as a critical organelle that mediates intracellular lipid trafficking in response to ER stress.

Reactive Oxygen Species

LDs play an essential role in protecting cells from ROS-induced injury (114). High levels of ROS are produced during mitochondrial dysfunction, hypoxia, and aberrant lipid catabolism. Studies demonstrate a positive correlation between ROS levels and LD numbers and indicate an ability for LDs to ameliorate ROS. Increases in ROS lead to the expression of LD-associated proteins that may increase LDs (108, 109) directly. LDs appear to provide a buffer against damage caused by ROS by sequestering fatty acids that would otherwise become oxidative species (115, 116). LDs function to export neutral and peroxidized lipids that are not substrates for cytoplasmic lipases, and which would otherwise contribute to cell damage and activate cell death processes. Although nuclear and mitochondrial DNA, proteins, lipids, mitochondrial enzymes, calcium signaling, receptor trafficking, and metabolic enzymes are all cell components affected by ROS, LDs play a crucial role in reducing ROS by preventing the further oxidation of lipids, which could lead to mitochondrial dysfunction.

Lipotoxic Stress

In models of cancer, LDs can function as a buffer to prevent toxic lipid buildup and lipotoxicity by redistributing lipids within and outside the cell (117). This protective mechanism mediated by LDs inside the cell involves lipolysis via autophagy pathways (59, 62, 118–124). LDs can also accumulate inert TGs, CEs, and acyl ceramides and mitigate their potentially harmful impact on cell structures by processing, neutralizing, and exporting lipid species (125–129). Free fatty acids at high levels can be incorporated into cytotoxic lipid species or act as detergents that disrupt cellular membranes. In cancer cell models, LDs limit peroxidation by actively accumulating polyunsaturated FAs, a lipid species prone to oxidation, in their lipid core (130). This sequestration of oxidation-prone lipids maintains cellular membrane composition and cellular homeostasis during periods of lipotoxic stress (115, 125, 131). LDs also protect against increases in intracellular lipids, a hallmark of many metabolic diseases (132, 133).

LDs IN DISEASE

LDs decrease the amount of toxic lipids, accumulated proteins, and oxidative stressors freely available within the cell, limiting the damage from exogenous and endogenous stressors. Once these toxic species are incorporated into LDs, there is a need to further process and ultimately export or neutralize the contents. Dysfunction of export mechanisms associated with LD trafficking can lead to cellular insult and inability to recover from cellular stress. The emerging evidence indicates that in various diseases, LD accumulation can initially act to remedy lipotoxicity mediated by ER stress and ROS, whereby LDs aid in the sequestration, trafficking, and ultimate export of cytotoxic components. However, disruptions in these dynamic processes that control LD distribution, composition, and transport may overwhelm and destabilize this system, resulting in a failed stress response and contributing to disease pathology.

The roles of LDs in pathologies including cancer, atherosclerosis, obesity, nonalcoholic fatty liver disease, renal disease, and neurological disorders provide the option for the discovery of new therapeutic targets with a potential for novel clinical interventions.

Metabolic Disease

Obesity and diabetes.

Adipose tissue functions as the body’s energy reservoir by storing TGs during energy surplus and converting these to FFA during energy deficit. There are two types of adipose tissue, white adipose tissue (WAT) and brown adipose tissue (BAT). In vertebrates, around 90% of TGs are stored in WAT. WAT can expand its capacity to buffer energy stores by fat cell hypertrophy and function predominantly as an energy storage mechanism. On the other hand, BAT is a highly oxidative tissue with high mitochondrial density to generate heat. These two types of adipose tissue function very differently, and the role of LDs predominantly involves the storage and release of lipids in WAT. These cells are capable of de novo synthesis of lipids from intermediates of carbohydrate metabolism, storing dietary lipids, and regulating their distribution. Excessive lipids in adipocytes induce enlargement of the adipose tissue and eventually cause the release of stress signals that induce macrophage migration and infiltration. Macrophage migration and inflammation within adipose tissue can modulate the endocrine response in this tissue. Oversecretion of cytokines by adipocytes, such as PAI-1, tumor necrosis factor-α (TNF), or interleukin 6 (IL-6) in conjunction with hyposecretion of adipocytokines, such as adiponectin, may contribute to the pathogenesis of lifestyle-related diseases including diabetes mellitus, hyperlipidemia, hypertension, and atherosclerosis, comprising metabolic syndrome (134).

Elevated levels of circulating FAs and altered endocrine function results in ectopic lipid accumulation in peripheral tissues, including the liver and skeletal muscle. Accumulation of intramyocellular lipids is associated with the loss of insulin sensitivity and altered systemic metabolism (30). The number of LDs in the subsarcolemmal space of patients with type 2 diabetes was larger than in athletes and nondiabetic patients with obesity. Exercise intervention in these patients with type 2 diabetes reduced the number of LDs to almost the same as a control group of nondiabetic and nonathlete subjects (135). Therefore, exercise may play a role in cycling LDs and initiating LD clearance from muscle cells and potentially other cells in the body.

Atherosclerosis.

In atherosclerosis, arterial injury causes endothelial dysfunction and the retention and oxidation of low-density lipoproteins (LDL) and other apolipoprotein B-containing lipoproteins rich in cholesterol in the arteries. Oxidized LDL promotes the infiltration of macrophages in the subendothelial space. Scavenger receptors expressed in macrophages recognize and internalize oxidized LDL. Internalization of oxidized LDL causes remarkable lipid accumulation and enlargement of LDs in macrophages, earning them the name “foam cells.” Foam cells comprise the majority of cells found in the earliest lesions during atherogenesis, called the “fatty streak” (136). The actual chemical composition of oxidized LDL varies and may depend on the extent of oxidation of the LDL molecule. Oxidation may affect the phospholipid monolayer, the apolipoprotein, or the lipids in the internal core. Thus, oxidized LDL can contain various oxidation products like lysophosphatidic choline in the phospholipid monolayer, peroxidized FAs, aldehydes, and cross-linked proteins (137). LDL oxidation is catalyzed by transition metal ions, several free radicals, and some oxidizing enzymes. Exacerbated LD accumulation in foamy macrophages leads to perturbations in lipid signaling, resulting in an inflammatory phenotype.

Macrophage inflammation results in increased oxidative stress and cytokine/chemokine secretion, causing a vicious cycle of additional endothelial cell recruitment, LDL oxidation, macrophage infiltration, and foam cell formation. Macrophage inflammatory chemokines promote infiltration of other cell types, including lymphocytes and dendritic cells, which also participate in the modulation of cytokine production within the lesion and smooth muscle cells. Smooth muscle cells become activated, increase, and overexpress extracellular matrix components, forming a fibrous cap over the core of foam cells. Smooth muscle cells in the vicinity of foamy macrophages also accumulate CEs in LDs and become foamy (138). Foamy macrophages and smooth muscle cells show dysfunctional lysosomal activity and cholesterol efflux. The resulting atherosclerotic plaque can remain stable until exacerbated inflammation, and oxidation triggers the formation of a vulnerable plaque, with a more necrotic macrophage core, thinning of the fibrous cap, and enrichment in 18:0 lysophosphatidylcholine, that is more prone to rupture. Rupture of atherosclerotic plaque releases prothrombotic factors and results in thrombus formation at the site of plaque rupture (138). Foamy macrophages have also been found in other pathologies, including multiple sclerosis and spinal cord injury (139). Therefore, it is possible that strategies initially designed to alleviate foamy macrophage formation in atherosclerosis could have utility in the treatment of other neurological diseases as well (140).

Neurological Disease

Nervous tissue contains the highest relative amount of lipids and the greatest variety in lipid species, suggesting a complex role of lipid trafficking and signaling involved in neuronal homeostasis and function compared with other tissue types (141). LD accumulation occurs in both neurons and glial cells (141–143), with the latter being the primary target of LD accumulation in Parkinson’s disease and Alzheimer’s disease (AD), multiple sclerosis (139), and aging of the brain (144). Lipids and nutrients are transferred between glial cells and neurons, which results in preferential accumulation of LDs in glial cells (145). This may reflect a critical interplay between these two cell types whereby glial cells protect from neuronal dysfunction by preventing an excessive LD accumulation in neurons. In support of this concept, in the Drosophila larvae model of neuronal damage, mutations in mitochondrial genes introduced in neurons lead to neuronal damage via ROS accumulation, accompanied by a transient accumulation of LDs in glial cells but not in neurons. In contrast, when the same mutations were introduced in glial cells, they did not cause an increase in LDs (146). These data support the concept that glial cells assist in the export of LDs from neuronal cells. Moreover, it was found that glial cells supply lactate to neurons, which neurons utilize to synthesize lipids (147). Neuronal damage via ROS accumulation induces the synthesis of lipids, which are then transferred to glial cells via apolipoproteins, ApoD, or ApoE (116). In the presence of neuronal stressors, including ROS and ER stress, LD accumulation worsens neurodegeneration (146, 148). Reduction of LDs via lipase overexpression or downregulation of TG synthesis in the absence of neuronal damage induced neurodegeneration (149), indicating that dysregulation of LD composition and dynamics, not the mere presence of LDs, is detrimental to the cell. Additional studies are required to elucidate the role of LDs in normal and neurological disease states and identify therapeutic targets of pathological LD accumulation in neurological diseases.

Alzheimer’s disease.

Alois Alzheimer first reported lipid inclusions in glial cells in AD. Increases in lipid species, such as lysophosphatidic acid, sphingomyelin, ganglioside GM3, and CEs, are present in the entorhinal cortex of patients with AD, suggesting a pathogenic mechanism associated with endo-lysosomal storage disorders (150). In patients, APOE4, an isoform of the APOE apolipoprotein, is the strongest genetic risk factor for the development of late-onset AD. APOE has a multifaceted function in cholesterol efflux and the clearance of amyloid-β plaques (151). Fibroblasts treated with cultured media from APOE4 expressing astrocytes show an increased number of LDs and increased phospholipid synthesis and CE accumulation compared with those exposed to the media from APOE3 expressing astrocytes (116, 146). Liu et al. (116) demonstrated that APOE and APOD mediate the transport of lipids from neurons to glial cells in a Drosophila larvae model of AD. When APOE4 Drosophila larvae were exposed to rotenone, which increases ROS production, APOE4 harboring neurons failed to transport lipids to glia, leading to neurodegeneration (116). Astrocytes harboring APOE4 contained an increased number of LDs of reduced size than those harboring APOE3, possibly indicating that LD fission occurs and suggesting incomplete fatty acid oxidation (152). These findings support a role for LD accumulation in the pathogenesis of AD, caused by increases in ROS or ER stress, hallmarks of the disease (145).

Age-related macular degeneration.

Age-related macular (AMD) degeneration is one of the significant causes of blindness worldwide. Genome-wide association studies have identified lipid metabolism and inflammation as AMD-associated pathogenic pathways. Cultured retinal epithelial cells from AMD donors exhibit accumulation of LDs, impaired autophagy, increased ROS, susceptibility to oxidative stress, and decreased mitochondrial activity compared with regular donors (153). The role of lipid efflux in the homeostasis of retinal epithelial cells was recently demonstrated in a study showing that mice with retinal pigment epithelium-specific ABCA1 and ABCG1 deletions were characterized by increased accumulation of LDs, reduced retinal function, retinal inflammation, and retinal pigment epithelium/photoreceptor degeneration (154). LDs in the retinal pigment epithelium were enriched with CEs (154), which have been shown to increase under conditions of ROS stress (155). LD accumulation in macular degeneration may be a response to increased ROS levels, and failure to export lipids, such as cholesterol, via ABCA1 and ABCG1 transporters could promote the disease phenotype.

Parkinson’s disease.

Parkinson’s disease is characterized by the formation of fibrillary lesions containing α-synuclein (αS) aggregates that are neurotoxic. Changes in membrane lipids drive the formation of toxic pathological S aggregates. Under normal conditions, αS undergoes transient cyclic conformations, binds to membranes, is assembled in homo-tetramers, and then disassembles into the soluble form. Under certain pathological conditions where the membrane composition is altered, αS self-aggregates into toxic intermediates resulting in progressive neurotoxicity (156, 157). Overexpression of A53T αS, a mutant that preferentially adopts the toxic conformation, induces accumulation of LDs and TGs in neurons (158). In vitro, αS binds to LDs with low affinity to phospholipids, likely due to a more exposed hydrophobic core (39). The presence of free monounsaturated fatty acid, particularly oleic acid, favors the formation of toxic αS aggregates in neurons, and the addition of oleic acid worsens the neurotoxicity of αS (159). Reduction of stearoyl-CoA, a key enzyme in the synthesis of oleic acid, via pharmacological inhibition or gene deletion, reduces the formation of toxic aggregates, thus preventing the neurotoxic effect (159). Interestingly, the accumulation of LDs in response to αS is associated with a beneficial effect, whereas the neurons with lower LD numbers had worsened neurotoxicity (157). This effect highlights the need for LDs in preventing the damaging effects associated with the accumulation of toxic lipid species. The accumulation of LD in Parkinson’s disease that coincides with the increase in TGs may therefore sequester the toxic αS aggregates and embed them in the core of high TG-containing LDs.

Liver Disease

Steatosis occurs when hepatocytes accumulate TGs due to aberrant lipid metabolism (160). Excess TG is stored in LDs, and the formation of very large LDs is a hallmark of liver steatosis (161). Phosphatidylcholine is required for very low-density lipoprotein (VLDL) production and secretion in hepatocytes, which lysophosphatidylcholine acyltransferase 3 (LPCAT3) contributes to a critical step in VLDL production (162, 163). Mice lacking LPCAT3 accumulate LDs in the liver when fed a high-sucrose diet and show altered TG secretion, which are hallmarks of steatosis (163).

Increased LD accumulation in hepatic steatosis can act to remedy lipotoxicity, ER stress, and ROS stress (160). In mouse models of obesity, hepatocytes demonstrate high levels of ER stress and concurrent accumulation of LDs (92, 164). ER stress increases the expression of lipogenic genes in hepatocytes and initiates inflammatory cascades that further promote insulin resistance and lipogenesis (165). These events can contribute to the progression of other diseases, including atherosclerosis, diabetes, obesity, and renal disease, all of which are characterized by additional local tissue increases in LDs (164, 166). Lipotoxicity-induced LD accumulation in hepatic steatosis is also driven by dysregulation of lipophagy (103, 104). Studies have shown decreased levels of transcription factor EB (TFEB), a transcription regulator involved in lipophagy, and glycine N-methyltransferase (GNMT), an enzyme involved in autophagy initiation, are due to LD accumulation in hepatic steatosis (103, 104). People with naturally occurring mutations in immunity-related GTPase family M (IRGM), a protein involved in autophagic clearance of bacteria, demonstrate increased levels of hepatic lipid accumulation, which is also observed in IRGM knockout mice (106–108). This indicates that dysregulation of lipophagy-related pathways contributes to the progression of hepatic steatosis. Several therapeutics that upregulate autophagy are currently under investigation as potential treatments for liver disease (86, 109, 110). Future studies are needed to investigate the contribution of LD lipophagy, including exophagy-mediated clearance of LD contents, and lipolysis in liver diseases to better understand the role of LD accumulation and lipotoxicity, and to enable the development of novel therapeutics.

Cancer

The solid tumor microenvironment is characterized by abnormal vascularization and decreased blood supply (167). Hypoxia, the condition of low oxygen levels in tissue, occurs in most solid and more significant tumor types (168). Cancer cells have increased levels of ROS due to internal oncogenic signaling and immune cell infiltration into the tumor microenvironment, both of which lead to cytokine release, inflammation, and activation of cell proliferation pathways in human cancer cells (169). High levels of ROS lead to the oxidation of other molecules in the cell, which is typically associated with cell damage and death. However, cancer cells can upregulate redox pathways and activate mechanisms to counteract prolonged exposure to elevated ROS. NADH production in cancer cells is essential in protecting these cells from ROS damage (170). Increased FA accumulation in response to impaired lipolysis occurs in cancer cells, which induces the formation of LDs (171, 172). LD accumulation in response to hypoxia has been observed in various cancer types, including brain, breast, renal, and prostate cancers. It may protect cancer cells by embedding potentially toxic lipid species into the core of the LD (117). Studies in a rat glioma model demonstrate that LDs accumulate mainly in the necrotic areas of the tumor core and function to decrease the ROS build-up induced by the hypoxic conditions associated with necrotic tissue (173, 174). Hypoxia-inducing factors, HIF-1α and HIF-2α, mediate the production of DAG and stimulate LD accumulation in cancer cells through Lipin 1 and PLIN2, respectively (175, 176). In addition, hypoxia leads to decreases in endogenous production of FAs through the inhibition of stearoyl-coenzyme A desaturase 1, SCD1, thus increasing FA uptake to compensate for hypoxia-induced ER stress (171, 177, 178). LD accumulation in breast cancer and glioblastoma cells exposed to hypoxia promoted cell survival (172). Knockdown of FABP3, FABP7, or PLIN2 inhibited LD formation during hypoxia resulting in reduced NADPH levels and increased ROS accumulation (172). LDs aggregate lipotoxic species generated by increased ROS levels, which need to be processed or exported for the survival of cancer cells in the hypoxic tumor microenvironment. Lipophagy is a significant contributor to the catalytic breakdown of LDs, and recent studies indicate a role for this pathway in cancer progression (179–184). Different experimental models of cancer and cancer cell types exhibit different modulations on lipophilic signaling proteins. Future work to clarify the role of LD accumulation and catabolism, lipophagy, and the specialized export of autophagosomal contents by exophagy in various cancer types and tumor microenvironments may unveil new therapeutic targets for the treatment of cancer.

Renal Disease

LD accumulation has been strongly implicated in the progression and pathology of renal disease. Here, we will describe mechanisms in which LDs accumulate, the responses of different renal cells to LD accumulation, and the contribution of LD accumulation to renal disease.

Tubular epithelial disease.

Renal lipid accumulation has been extensively documented in acute and chronic kidney injury (185). In acute kidney injury, most damage is typically to kidney tubular epithelial cells. These cells consume and store fatty acids to accommodate the high energy demand required for the active transport of ions and molecules and are highly susceptible to hypoxia. Proximal tubules, which account for ∼ 70% of kidney cortex mass, are rich in TGs compared with glomeruli, accounting for most of the TGs present in the kidney cortex (186). Acute kidney injury in the left kidney is induced within 15 min after ischemia in angiotensin II-infused mice with nephrectomy of the right kidney and persists for hours after reperfusion (187) or starvation (188). In mice, CEs and TGs accumulated in proximal tubules after acute kidney injury is induced by different insults, including LPS injection, glycerol-induced rhabdomyolysis, and ischemia/reperfusion (187, 189). This phenomenon occurs independently from cholesterol and TG blood levels (187, 189), suggesting an important role for cellular rather than systemic lipids in acute kidney injury. LD accumulation in tubular cells may be mediated by increased TG synthesis, increased fatty acid uptake, or decreased fatty acid oxidation. Increased phospholipase A2 (PLA2) activity releases FFAs from phospholipids of the cell membrane, supplying FFA for LD biogenesis. Decreased expression of genes involved in cholesterol efflux, such as ABCA1 and ABCG1, can also account for LD accumulation (190). Whether lipid accumulation in tubular cells is beneficial or detrimental in renal disease depends on the duration of the injury and/or the extent of the damage. For example, LD accumulation after LPS injection protects tubular cells by limiting further exposure to hypoxia or oxidative stress (187). On the other hand, oleate-loaded tubular cells are more sensitive to oxidants and ATP depletion insults (187). Moreover, genetic or drug activation of PPARα receptors, which induces FA oxidation and consumption of LD cargo, protected mice from tubulointerstitial fibrosis (191, 192). LD accumulation forms part of a cytoprotective response but can become part of a maladaptive response that worsens the disease if left unresolved.

Glomerular disease.

Glomerular LD accumulation has been documented in many renal diseases of metabolic and nonmetabolic origin by us and others (190, 193–202). LD accumulation in podocytes, endothelial cells, and mesangial cells has been described in kidney biopsies from patients with diabetic kidney disease (203). Decreased expression of genes participating in cholesterol efflux and fatty acid oxidation along with upregulation of LDL and oxidized LDL receptors was found in kidney biopsies of these patients (203). Studies from our research group demonstrated that cultured human podocytes exposed to sera from patients with diabetic kidney disease are characterized by an increased number of LDs and downregulation of ABCA1 (190). In this context, renal lipid accumulation occurs in a manner independent of serum lipid levels (194), suggesting that this is an active cytopathological process and not the mere passive accumulation of circulating lipids in podocytes.

Glomerular diseases, such as focal segmental glomerulosclerosis (FSGS) and Alport syndrome (195), are other notable examples in which glomerular lipid accumulation was described (197–200). We previously described the accumulation of LDs in glomeruli of Col4A3 knockout (KO) mice, a mouse model of renal disease associated with Alport syndrome. These mice present with proteinuria and exhibit substantial glomerular damage and renal failure (195). Similarly, glomerular LD accumulation can be observed in mice with adriamycin-induced nephropathy, a mouse model for FSGS. More importantly, in both mouse models, compounds that induce ABCA1 expression, and ABCA1-mediated cholesterol efflux, significantly reduce the number of LDs and proteinuria. This suggests that promoting the resolution of the LD stress response in podocytes by preventing or remedying excessive LD accumulation and lipotoxicity improves renal function in glomerular disease.

TRANSLATIONAL APPLICATIONS

One strategy to accelerate moving drugs that target lipid metabolism forward into the clinical setting is repurposing existing FDA-approved drugs. For example, Verapamil, a calcium channel blocker commonly used to treat high blood pressure, was also shown to reduce hepatic lipid droplet accumulation, insulin resistance, and steatohepatitis, indicating a potential therapeutic advantage of calcium channel blockers that could be utilized in the treatment of general nonalcoholic fatty liver disease pathologies (204). Furthermore, several small molecules that target lipid metabolic pathways are currently being investigated as potential cancer therapeutics, which have been reviewed extensively elsewhere (205). The role of LDs in multiple disease states represents a significant opportunity for therapeutic intervention. LDs are ubiquitous among all cellular life forms, and thus the mechanisms that regulate their biosynthesis and activity are highly conserved. This allows for translational study of LDs in various organisms, including Drosophila, Caenorhabditis elegans, yeast, mammals, and fish. Efforts are underway to identify small molecule modulators of LD accumulation using phenotypic drug screens, which will provide further insight into the mechanisms involved in regulating LD formation and function. Phenotypic drug screens using these model organisms that investigate the potential to remedy LD accumulation will provide further insight into the mechanisms involved in regulating LD formation and function. These phenotypic screens could identify new compounds that can promote the resolution of the LD stress response by preventing excessive lipid buildup and broaden the pharmacological tools available to treat diseases characterized by uncontrolled LD dysregulation.

CONCLUSION

LD dysregulation occurs in multiple diseases including metabolic disease, neurological disease, liver disease, renal disease, and cancer. Targeting dysregulated cellular lipid metabolism may represent a potential therapeutic strategy for treating many of these diseases. The emerging evidence summarized in this review indicates that in various diseases, LD accumulation can initially act to remedy lipotoxicity mediated by ER stress and ROS, whereby LDs aid in the sequestration, trafficking, and ultimately the export of cytotoxic components. Disruptions in these dynamic processes that control LD distribution, composition, and transport may overwhelm and destabilize this system, resulting in a failed stress response and contributing to disease pathology. Depending on the pathophysiology and progression of known diseases involving LD accumulation, drugs that modulate cellular lipid metabolism may prove to represent a robust tool in the clinical setting.

In future studies, insights into the time scale of lipid accumulation in disease development and progression will provide important mechanistic data to enhance our understanding of LD dysfunction. Although LDs exercise an initial beneficial role in maintaining proper cellular homeostasis, the excessive accumulation of LDs is pathological and therefore represents an attractive new target for drug discovery. Live imaging of LDs in disease will provide valuable information on the behavior of LDs in disease states and is currently gaining momentum with the increase in lipid-specific dyes and advanced techniques. Determining the signaling cascades that enable the switch between various LD behaviors, size, and composition will be instrumental in treating metabolic, liver, renal, and neurological diseases. Molecular interventions that switch the behavior of LDs may provide novel clinical tools for the treatment of many diseases, including cancer.

This review underscores the critical need for further research into mechanisms of intercellular lipid trafficking, lipid metabolism, and the behavior of LDs. The knowledge obtained from further research will benefit the development of future therapeutic strategies aimed at adjusting maladaptive lipid accumulation, which can have a substantial clinical impact on an extensive range of diseases.

GRANTS

This work was supported by Boeheringer Ingelheim (to A.F. and S. M.), NIH Grants R01CA227493, R01DK117599, and R01DK104753 (to J.D.P., M.Z.G, and J.T.V.); Grants UL1TR000460, U54DK083912, UM1DK100846, and U01DK116101 (to A.F.); Grants DK076169 and DK115255 (to H.A-A.); and US Department of Defense Grant W81XWH2110940 (to H.A-A.).

DISCLOSURES

A. Fornoni and S. Merscher are inventors on pending (PCT/US2019/032215; PCT/US2019/041730; PCT/US2013/036484; US17/057,247; US17/259,883; Japan No. 501309/2021; Europe No. 19834217.2; China No. 201980060078.3; Canada Nos. 2,852,904, 2,930,119, 3,012,773) and issued patents (US10,183,038; US10,052,345) aimed at preventing and treating renal disease. They stand to gain royalties from their future commercialization. A. Fornoni and S. Merscher hold indirect equity interest in and potential royalty from ZyVersa Therapeutics Inc. by virtue of assignment and licensure of a patent estate. H. Al-Ali is an inventor on pending patent application PCT/US2018/058411 (China CN111801322A, Europe EP3704103A1, South Korea KR20200080273A, Japan JP2021503442A, Australia AU2018359413A1, and Israel IL274067D0). He is also a cofounder of Truvitech LLC, a university of Miami-based startup focused on developing and commercializing idTRAX, a biotechnology platform for drug target identification. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

J.D.P. prepared figures; J.D.P., M.Z.G., and J.T.V.S. drafted manuscript; J.D.P., M.Z.G., J.T.V.S., A.F., S.M., and H.A-A. edited and revised manuscript; J.D.P., J.T.V.S., A.F., S.M., and H.A-A. approved final version of manuscript.