Mammalian lipid droplets: structural,pathological, immunological and anti-toxicological roles

Mammalian lipid droplets (LDs) are specialized cytosolic organelles consisting of a neutral lipid core surrounded by a membrane made up of a phospholipid monolayer and a specific population of proteins that varies according to the location and function of each LD. Over the past decade, there have been significant advances in the understanding of LD biogenesis and functions. LDs are now recognized as dynamic organelles that participate in many aspects of cellular homeostasis plus other vital functions. LD biogenesis is a complex, highly-regulated process with assembly occurring on the endoplasmic reticulum although aspects of the underpinning molecular mechanisms remain elusive. For example, it is unclear how many enzymes participate in the biosynthesis of the neutral lipid components of LDs and how this process is coordinated in response to different metabolic cues to promote or suppress LD formation and turnover. In addition to enzymes involved in the biosynthesis of neutral lipids, various scaffolding proteins play roles in coordinating LD formation. Despite their lack of ultrastructural diversity, LDs in different mammalian cell types are involved in a wide range of biological functions. These include roles in membrane homeostasis, regulation of hypoxia, neoplastic inflammatory responses, cellular oxidative status, lipid peroxidation, and protection against potentially toxic intracellular fatty acids and lipophilic xenobiotics. Herein, the roles of mammalian LDs and their associated proteins are reviewed with a particular focus on their roles in pathological, immunological and anti-toxicological processes.     

Spatial compartmentalization of lipid droplet biogenesis

Lipid droplets (LDs) are ubiquitous organelles that store metabolic energy in the form of neutral lipids (typically triacylglycerols and steryl esters). Beyond being inert energy storage compartments, LDs are dynamic organelles that participate in numerous essential metabolic functions. Cells generate LDs de novo from distinct sub-regions at the endoplasmic reticulum (ER), but what determines sites of LD formation remains a key unanswered question. Here, we review the factors that determine LD formation at the ER, and discuss how they work together to spatially and temporally coordinate LD biogenesis. These factors include lipid synthesis enzymes, assembly proteins, and membrane structural requirements. LDs also make contact with other organelles, and these inter-organelle contacts contribute to defining sites of LD production. Finally, we highlight emerging non-canonical roles for LDs in maintaining cellular homeostasis during stress.     

Emerging role of lipid droplets in host/pathogen interactions

Lipid droplets (LDs) are highly dynamic cell organelles involved in energy homeostasis and membrane trafficking. Here, we review how select pathogens interact with LDs. Several RNA viruses use host LDs at different steps of their life cycle. Some intracellular bacteria and parasites usurp host LDs or encode their own lipid biosynthesis machinery, thus allowing production of LDs independently of their host. Although many mechanistic details of host/pathogen LD interactions are unknown, a picture emerges in which the unique cellular architecture and energy stored in LDs are important in the replication of diverse pathogens.     

Lipid droplet-organelle interactions; sharing the fats

Lipid droplets (LDs) are key cellular organelles involved in lipid storage and mobilisation. While the major signalling cascades and many of the regulators of lipolysis have been identified, the cellular interactions involved in lipid mobilisation and release remain largely undefined. In non-adipocytes, LDs are small, mobile and interact with other cellular compartments. In contrast, adipocytes primarily contain very large, immotile LDs. The striking morphological differences between LDs in adipocytes and non-adipocytes suggest that key differences must exist in the manner in which LDs in different cell types interact with other organelles. Recent studies have highlighted the complexity of LD interactions, which can be both homotypic, with each other, and heterotypic, with other organelles. The molecules involved in these interactions are also now emerging, including Rab proteins, key regulators of membrane traffic, and caveolin, an integral membrane protein providing a functional link between the cell surface and LDs. Here we summarise recent insights into the cell biology of the LD particularly focussing on the homotypic and heterotypic interactions in both adipocytes and non-adipocytes. We speculate that these interactions may involve inter-organelle membrane contact sites or a hemi-fusion type mechanism to facilitate lipid transfer.     

Lipid droplets in skeletal muscle during grass snake (Natrix natrix L.) development

Lipid droplets (LDs) are common organelles observed in Eucaryota. They are multifunctional organelles (involved in lipid storage, metabolism, and trafficking) that originate from endoplasmic reticulum (ER). LDs consist of a neutral lipid core, made up of diacyl- and triacylglycerols (DAGs and TAGs) and cholesterol esters (CEs), surrounded by a phospholipid monolayer and proteins, which are necessary for their structure and dynamics.Here, we report the protein and lipid composition as well as characterization and dynamics of grass snake (Natrix natrix) skeletal muscle LDs at different developmental stages. In the present study, we used detailed morphometric, LC-MS, quantitative lipidomic analyses of LDs isolated from the skeletal muscles of the snake embryos, immunofluorescence, and TEM.Our study also provides a valuable insight concerning the LDs' multifunctionality and ability to interact with a variety of organelles. These LD features are reflected in their proteome composition, which contains scaffold proteins, metabolic enzymes signalling polypeptides, proteins necessary for the formation of docking sites, and many others. We also provide insights into the biogenesis and growth of muscle LDs goes beyond the conventional mechanism based on the synthesis and incorporation of TAGs and LD fusion. We assume that the formation and functioning of grass snake muscle LDs are based on additional mechanisms that have not yet been identified, which could be related to the unique features of reptiles that are manifested in the after-hatching period of life, such as a reptile-specific strategy for energy saving during hibernation.     

LC3, a microtubule-associated protein1A/B light chain3, is involved in cytoplasmic lipid droplet formation

The cytoplasmic lipid droplet (LD) is one of organelles that has a neutral lipid core with a single phospholipid layer. LDs are believed to be generated between the two leaflets of the endoplasmic reticulum (ER) membrane and to play various roles, such as high effective energy storage. However, it remains largely unknown how LDs are generated and grow in the cytoplasm. We have previously shown that the Atg conjugation system that is essential for autophagosome formation is involved in LD formation in hepatocytes and cardiac myocytes. We show here that LC3 itself is involved in LD formation by using RNA interference (RNAi). All cultured cell lines examined, in which the expression of LC3 was suppressed by RNAi, showed reduced LD formation. Triacylglycerol, a major component of LDs, was synthesized and degraded in LC3 mRNA-knockdown cells as well as in control cells. Interestingly, potential of the bulk protein degradation in the knockdown-cells was also evident in the control cells. These findings indicate that LC3 is involved in the LD formation regardless of the bulk degradation, and that LC3 has two pivotal roles in cellular homeostasis mediated by autophagy and lipid metabolism.     

Lipid Droplets and Peroxisomes: Key Players in Cellular Lipid Homeostasis or A Matter of Fat—Store ’em Up or Burn ’em Down

Lipid droplets (LDs) and peroxisomes are central players in cellular lipid homeostasis: some of their main functions are to control the metabolic flux and availability of fatty acids (LDs and peroxisomes) as well as of sterols (LDs). Both fatty acids and sterols serve multiple functions in the cell—as membrane stabilizers affecting membrane fluidity, as crucial structural elements of membrane-forming phospholipids and sphingolipids, as protein modifiers and signaling molecules, and last but not least, as a rich carbon and energy source. In addition, peroxisomes harbor enzymes of the malic acid shunt, which is indispensable to regenerate oxaloacetate for gluconeogenesis, thus allowing yeast cells to generate sugars from fatty acids or nonfermentable carbon sources. Therefore, failure of LD and peroxisome biogenesis and function are likely to lead to deregulated lipid fluxes and disrupted energy homeostasis with detrimental consequences for the cell. These pathological consequences of LD and peroxisome failure have indeed sparked great biomedical interest in understanding the biogenesis of these organelles, their functional roles in lipid homeostasis, interaction with cellular metabolism and other organelles, as well as their regulation, turnover, and inheritance. These questions are particularly burning in view of the pandemic development of lipid-associated disorders worldwide.WORK for the past five decades on the yeast Saccharomyces cerevisiae has contributed fundamental insight into peroxisome biogenesis and function that is also relevant for mammalian cells. While LD research in yeast is still in its infancy and looks back to a much shorter history—the previous edition of YeastBook did not even mention LDs as an “organelle”—combined biochemical, cell biological, lipidomic, and proteomic studies in recent years have already contributed significant insight into LD biogenesis and function.     

Lipid droplet proteins and metabolic diseases

Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage which are composed of a neutral lipid core bounded by a protein decorated phospholipid monolayer. Although lipid storage is their most obvious function, LDs are far from inert as they participate in maintaining lipid homeostasis through lipid synthesis, metabolism, and transportation. Furthermore, they are involved in cell signaling and other molecular events closely associated with human disease such as dyslipidemia, obesity, lipodystrophy, diabetes, fatty liver, atherosclerosis, and others. The last decade has seen a great increase in the attention paid to LD biology. Regardless, many fundamental features of LD biology remain obscure. In this review, we will discuss key aspects of LD biology including their biogenesis, growth and regression. We will also summarize the current knowledge about the role LDs play in human disease, especially from the perspective of the dynamics of the associated proteins. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

Identification and characterization of associated with lipid droplet protein 1: A novel membrane-associated protein that resides on hepatic lipid droplets

Alcoholic and nonalcoholic liver steatosis and steatohepatitis are characterized by the massive accumulation of lipid droplets (LDs) in the cytosol of hepatocytes. Although LDs are ubiquitous and dynamic organelles found in the cells of a wide range of organisms, little is known about the mechanisms and sites of LD biogenesis. To examine the participation of these organelles in the pathophysiological disorders of steatotic livers, we used a combination of mass spectrometry (matrix-assisted laser desorption ionization-time of flight and LC-MS electrospray) and Western blot analysis to study the composition of LDs purified from rat liver after a partial hepatectomy. Fifty proteins were identified. Adipose differentiation-related protein was the most abundant, but other proteins such as calreticulin, TIP47, Sar1, Rab GTPases, Rho and actin were also found. In addition, we identified protein associated with lipid droplets I ALDI (tentatively named Associated with LD protein 1), a novel protein widely expressed in liver and kidney corresponding to the product of 0610006F02Rik (GI:27229118). Our results show that, upon lipid loading of the cells, ALDI translocates from the endoplasmic reticulum into nascent LDs and indicate that ALDI may be targeted to the initial lipid deposits that eventually form these droplets. Moreover, we used ALDI expression studies to view other processes related to these droplets, such as LD biogenesis, and to analyze LD dynamics. In conclusion, here we report the composition of hepatic LDs and describe a novel bona fide LD-associated protein that may provide new insights into the mechanisms and sites of LD biogenesis.     

Born this way – Biogenesis of lipid droplets from specialized ER subdomains

Both the endoplasmic reticulum (ER) and lipid droplets (LDs) are key players in lipid handling. In addition to this functional connection, the two organelles are also tightly linked due to the fact that the ER is the birthplace of LDs. LDs have an atypical architecture, consisting of a neutral lipid core that is covered by a phospholipid monolayer. LD biogenesis starts with neutral lipid synthesis in the ER membrane and formation of small neutral lipid lenses between its leaflets, followed by budding of mature LDs toward the cytosol.Several ER proteins have been identified that are required for efficient LD formation, among them seipin, Pex30, and FIT2. Recent evidence indicates that these LD biogenesis factors might cooperate with specific lipids, thus generating ER subdomains optimized for LD assembly. Intriguingly, LD biogenesis reacts dynamically to nutrient stress, resulting in a spatial reorganization of LD formation in the ER.     

CIDE family proteins control lipid homeostasis and the development of metabolic diseases

Dysregulation of lipid homeostasis leads to the development of metabolic disorders including obesity, diabetes, cardiovascular disease and cancer. Lipid droplets (LDs) are subcellular organelles vital in the maintenance of lipid homeostasis by coordinating lipid synthesis, lipid storage, lipid secretion and lipolysis. Under fed condition, free fatty acids (FFAs) are remodeled and esterified into neutral lipids by lipogenesis and stored in the LDs. The lipid storage capacity of LDs is controlled by its growth via local lipid synthesis or by LD fusion. During fasting, neutral lipids are hydrolyzed by lipolysis, released as FFAs and secreted to meet energy demand. C ell death‐i nducing D NA fragmentation factor alpha (DFFA)‐like e ffector (CIDE) family proteins composed of Cidea, Cideb and Cidec/Fsp27 are ER‐ and LD‐associated proteins and have emerged as important regulators of lipid homeostasis. Notably, when localized on the LDs, CIDE proteins enrich at the LD‐LD contact sites (LDCSs) and control LD fusion and growth. Here, we summarize these recent advances made on the role of CIDE proteins in the regulation of lipid metabolism with a particular focus on the molecular mechanisms underlying CIDE‐mediated LD fusion and growth.     

Lipid droplets and lipotoxicity during autophagy

Lipid droplets (LDs) are neutral lipid storage organelles that provide a rapidly accessible source of fatty acids (FAs) for energy during periods of nutrient deprivation. Surprisingly, lipids released by the macroautophagic/autophagic breakdown of membranous organelles are packaged and stored in new LDs during periods of prolonged starvation. Why cells would store FAs during an energy crisis was unknown. In our recent study, we demonstrated that FAs released during MTORC1-regulated autophagy are selectively channeled by DGAT1 (diacylglycerol O-acyltransferase 1) into triacylglycerol (TAG)-rich LDs. These DGAT1-dependent LDs sequester FAs and prevent the accumulation of acylcarnitines, which otherwise directly disrupt mitochondrial integrity. Our findings establish LD biogenesis as a general cellular response to periods of high autophagic flux that provide a lipid buffering system to mitigate lipotoxic cellular damage.     

The lipid droplet is an important organelle for hepatitis C virus production

The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER). These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases. HCV capsid protein (Core) associates with the LD, envelope proteins E1 and E2 reside in the ER lumen, and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions.     

High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation

Accurate protein inventories are essential for understanding an organelle’s functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae. Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism.     

Bidirectional Lipid Droplet Velocities Are Controlled by Differential Binding Strengths of HCV Core DII Protein

Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV.     

Packaging of fat: an evolving model of lipid droplet assembly and expansion

Lipid droplets (LDs) are organelles found in most types of cells in the tissues of vertebrates, invertebrates, and plants, as well as in bacteria and yeast. They differ from other organelles in binding a unique complement of proteins and lacking an aqueous core but share aspects of protein trafficking with secretory membrane compartments. In this minireview, we focus on recent evidence supporting an endoplasmic reticulum origin for LD formation and discuss recent findings regarding LD maturation and fusion.     

Peroxisomes,lipid droplets,and endoplasmic reticulum “hitchhike” on motile early endosomes

Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3– and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell.     

The dynamic roles of intracellular lipid droplets: from archaea to mammals

During the past decade, there has been a paradigm shift in our understanding of the roles of intracellular lipid droplets (LDs). New genetic, biochemical and imaging technologies have underpinned these advances, which are revealing much new information about these dynamic organelles. This review takes a comparative approach by examining recent work on LDs across the whole range of biological organisms from archaea and bacteria, through yeast and Drosophila to mammals, including humans. LDs probably evolved originally in microorganisms as temporary stores of excess dietary lipid that was surplus to the immediate requirements of membrane formation/turnover. LDs then acquired roles as long-term carbon stores that enabled organisms to survive episodic lack of nutrients. In multicellular organisms, LDs went on to acquire numerous additional roles including cell- and organism-level lipid homeostasis, protein sequestration, membrane trafficking and signalling. Many pathogens of plants and animals subvert their host LD metabolism as part of their infection process. Finally, malfunctions in LDs and associated proteins are implicated in several degenerative diseases of modern humans, among the most serious of which is the increasingly prevalent constellation of pathologies, such as obesity and insulin resistance, which is associated with metabolic syndrome.     

The seipin complex Fld1/Ldb16 stabilizes ER–lipid droplet contact sites

Lipid droplets (LDs) are storage organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer and a set of LD-specific proteins. Most LD components are synthesized in the endoplasmic reticulum (ER), an organelle that is often physically connected with LDs. How LD identity is established while maintaining biochemical and physical connections with the ER is not known. Here, we show that the yeast seipin Fld1, in complex with the ER membrane protein Ldb16, prevents equilibration of ER and LD surface components by stabilizing the contact sites between the two organelles. In the absence of the Fld1/Ldb16 complex, assembly of LDs results in phospholipid packing defects leading to aberrant distribution of lipid-binding proteins and abnormal LDs. We propose that the Fld1/Ldb16 complex facilitates the establishment of LD identity by acting as a diffusion barrier at the ER–LD contact sites.