Apolipoprotein A-V association with intracellular lipid droplets

Apolipoprotein A-V (apoA-V) plays a key role in the regulation of triglyceride (TG) metabolism. Given the very low concentration of apoA-V in plasma, we hypothesized that apoA-V may influence plasma TG levels by affecting the assembly and/or secretion of apoB-containing lipoproteins. When apoA-V was overexpressed in cultured Hep3B cells, neither the amount of apoB secreted nor the density distribution of apoB-containing lipoproteins was affected. Fluorescence microscopy and cell lysate immunoprecipitation studies revealed that apoA-V is not associated with apoB intracellularly, yet immunoprecipitation of apoA-V from the cell culture medium resulted in coprecipitation of apoB. These data suggest that the apoA-V association with apoB-containing lipoproteins is a postsecretory event. Confocal fluorescence microscopy revealed the presence of apoA-V in distinct cellular structures. Based on Nile Red staining, we identified these structures to be intracellular lipid droplets. These data suggest that apoA-V has a unique association with cellular lipids and, therefore, may be involved in the storage or mobilization of intracellular lipids.     

The obligate intracellular pathogen Chlamydia trachomatis targets host lipid droplets

Lipid droplets (LDs) are ubiquitous but poorly understood neutral-lipid-rich eukaryotic organelles that may participate in functions as diverse as lipid homeostasis, membrane traffic, and signaling . We report that infection with the obligate intracellular pathogen Chlamydia trachomatis, the causative agent of trachoma and many sexually transmitted diseases , leads to the accumulation of neutral-lipid-rich structures with features of LDs at the cytoplasmic surface of the bacteria-containing vacuole. To identify bacterial factors that target these organelles, we screened a collection of yeast strains expressing GFP-tagged chlamydial ORFs and identified several proteins with tropism for eukaryotic LDs. We determined that three of these LD-associated (Lda) proteins are translocated into the mammalian host and associate with neutral-lipid-rich structures. Furthermore, the stability of one Lda protein is dependent on binding to LDs, and pharmacological inhibition of LD formation negatively impacted chlamydial replication. These results suggest that C. trachomatis targets LDs to enhance its survival and replication in infected cells. The co-option of mammalian LD function by a pathogenic bacterium represents a novel mechanism of eukaryotic organelle subversion and provides unique research opportunities to explore the function of these understudied organelles.     

The assembly of lipid droplets and their roles in challenged cells

Cytoplasmic lipid droplets are important organelles in nearly every eukaryotic and some prokaryotic cells. Storing and providing energy is their main function, but they do not work in isolation. They respond to stimuli initiated either on the cell surface or in the cytoplasm as conditions change. Cellular stresses such as starvation and invasion are internal insults that evoke changes in droplet metabolism and dynamics. This review will first outline lipid droplet assembly and then discuss how droplets respond to stress and in particular nutrient starvation. Finally, the role of droplets in viral and microbial invasion will be presented, where an unresolved issue is whether changes in droplet abundance promote the invader, defend the host, to try to do both. The challenges of stress and infection are often accompanied by changes in physical contacts between droplets and other organelles. How these changes may result in improving cellular physiology, an ongoing focus in the field, is discussed.     

Nile red: a selective fluorescent stain for intracellular lipid droplets

We report that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red-stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.     

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.     

The life of lipid droplets

Lipid droplets are the least characterized of cellular organelles. Long considered simple lipid storage depots, these dynamic and remarkable organelles have recently been implicated in many biological processes, and we are only now beginning to gain insights into their fascinating lives in cells. Here we examine what we know of the life of lipid droplets. We review emerging data concerning their cellular biology and present our thoughts on some of the most salient questions for investigation.     

Redistribution of macrophage cholesteryl ester hydrolase from cytoplasm to lipid droplets upon lipid loading

Hydrolysis of intracellular cholesteryl esters (CEs) represents the first step in the removal of cholesterol from lipid-laden foam cells associated with atherosclerotic lesions. Neutral cholesteryl ester hydrolase (CEH) catalyzes this reaction, and we recently cloned the cDNA for the human macrophage CEH and demonstrated increased mobilization of intracellular CE droplets by CEH overexpression. The present study was undertaken to test the hypothesis that for CE hydrolysis, CEH must become associated with the surface of the cytoplasmic lipid droplets. Our data show the redistribution of CEH from cytosol to lipid droplets upon lipid loading of human THP-1 macrophages. Depletion of triacylglycerol (TG) by incubation with the acyl-CoA synthetase inhibitor Triacsin D had no effect on CEH association with the lipid droplets, suggesting that CEH associates with mixed (CE + TG) as well as TG-depleted CE droplets. However, CEH had 2.5-fold higher activity when mixed droplets were used as substrate in an in vitro assay, consistent with the reported higher cholesterol efflux from cells containing mixed isotropic droplets. Perilipin as well as adipophilin, two lipid droplet-associated proteins, were also present on the lipid droplets in THP-1 macrophages. In conclusion, CEH associates with its intracellular substrate (lipid droplets) and hydrolyzes CE more efficiently from mixed droplets.     

Biogenesis of cytoplasmic lipid droplets: from the lipid ester globule in the membrane to the visible structure

The cytoplasmic lipid droplet (CLD) and very low-density lipoprotein are generated from the lipid ester synthesized in the endoplasmic reticulum. The lipid ester deposited between the two membrane leaflets is supposed to bulge toward the cytoplasm to make a nascent CLD, but its size must be below the resolution limit of conventional techniques and the detectable CLD should only form after acquisition of additional lipid esters. The CLD is different from vesicular organelles in that the internal content is highly hydrophobic and the shape is invariably spherical. Due to its unique characteristics, quantitative discordance between the surface and the volume may occur in the growth and/or involution processes of the CLD. The possibility that these processes may give rise to the structural and functional diversities of the CLD is discussed.     

The birth and life of lipid droplets: learning from the hepatitis C virus

LDs (lipid droplets) are probably the least well-characterized cellular organelles. Having long been considered simple lipid storage depots, they are now considered to be dynamic organelles involved in many biological processes. However, most of the mechanisms driving LDs biogenesis, growth and intracellular movement remain largely unknown. As for other cellular mechanisms deciphered through the study of viral models, HCV (hepatitis C virus) is an original and relevant model for investigations of the birth and life of these organelles. Recent studies in this model have raised the hypothesis that the HCV core protein induces the redistribution of LDs through the regression and regeneration of these organelles in specific intracellular domains.     

Chk1 in the DNA damage response: conserved roles from yeasts to mammals

Chk1 is an evolutionarily conserved protein kinase that functions to ensure genomic integrity upon genotoxic stress. Studies to date have revealed striking similarities among Chk1 pathways of different organisms. In this review we discuss what is known about Chk1 activation and what downstream factors are regulated by Chk1 to counter replication blocks and DNA damage induced by UV, IR, and other genotoxic agents. Where applicable, we also compare the role of Chk1 with that of the Chk2 protein kinase in the checkpoint responses.     

Biomodulator-mediated susceptibility of endogenous lipid droplets from rat adipocytes to hormone-sensitive lipase

The amount of fatty acid release by a fat cell homogenate without pretreatment with epinephrine was found to be slightly more than that released from fat cells by epinephrine, suggesting that fat cells contain high lipolytic activity even in the absence of lipolytic agents. Fat cells contain high hormone-sensitive lipase activity (1383 mumole free fatty acids/g/hr) in the absence of epinephrine, and addition of epinephrine to the cells did not increase the activity, significantly. Like epinephrine, DBcAMP and/or theophylline also elicited marked release of glycerol from fat cells without activating the hormone-sensitive lipase activity. However, although fat cells contain a large amount of hormone-sensitive lipase, lipolysis was negligible in the absence of these lipolytic agents. These results suggest that lipolytic agents such as epinephrine, DBcAMP, and theophylline induce lipolysis in fat cells through some mechanism other than activation of hormone-sensitive lipase and that in the absence of lipolytic agents, some system in fat cells inhibits lipolysis of endogenous lipid droplets by hormone-sensitive lipase. The lipid droplets in fat cells consist mainly of triglyceride with phospholipids, cholesterol, carbohydrate, and protein as minor constituents. The phospholipid fraction was found to consist of 75% phosphatidylcholine and 25% phosphatidylethanolamine. Of the minor constituents of endogenous lipid droplets, only phosphatidylcholine strongly inhibited hormone-sensitive lipase activity in a [3H]triolein emulsion. These results suggest that phosphatidylcholine in endogenous lipid droplets may be responsible for inhibition of hormone-sensitive lipase. Then, a cell-free system was established in which epinephrine, DBcAMP, and theophylline stimulated lipolysis of endogenous lipid droplets from fat cells by lipase solution. In this system, these lipolytic agents did not induce lipolysis in the absence of added lipase. Lipolysis in the mixture of the endogenous lipid droplets and lipase solution was accelerated by phospholipase C with concomitant loss of epinephrine-induced lipolysis. After pretreatment of the endogenous lipid droplets with phospholipase C, these lipolytic agents no longer induced lipolysis. Pretreatment of the endogenous lipid droplets with phospholipase C reduced their phospholipid content with the formation of phosphorylcholine, but did not affect their triglyceride and cholesterol contents. Treatment of the endogenous lipid droplets with phospholipase D did not affect lipolysis in the cell-free system. These results suggest that phosphatidylcholine in the endogenous lipid droplets may inhibit their lipolysis by hormone-sensitive lipase in fat cells and also be involved in the mechanisms of the stimulatory effects of epinephrine, DBcAMP, and theophylline on lipolysis.     

Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets

The biosynthesis and storage of triglyceride (TG) is an important cellular process conserved from yeast to man. Most mammalian cells accumulate TG in lipid droplets, most prominent in adipocytes, which are specialized to store large amounts of the TG over long periods. In this study, we followed TG biosynthesis and targeting by fluorescence imaging in living 3T3-L1 adipocytes and COS7 fibroblasts. Key findings were (i) not only TG but also its direct metabolic precursor diacylglycerol, DG, accumulates on lipid droplets; (ii) the essential enzyme diacylglycerol acyltransferase 2 associates specifically with lipid droplets where it catalyzes the conversion of DG to TG and (iii) individual lipid droplets within one cell acquire TG at very different rates, suggesting unequal access to the biosynthetic machinery. We conclude that at least part of TG biosynthesis takes place in the immediate vicinity of lipid droplets. In vitro assays on purified lipid droplets show that this fraction of the biosynthetic TG is directly inserted into the growing droplet.     

Cytosolic lipid droplets: From mechanisms of fat storage to disease

Abstract

The lipid droplet (LD) is a phylogenetically conserved organelle. In eukaryotes, it is born from the endoplasmic reticulum, but unlike its parent organelle, LDs are the only known cytosolic organelles that are micellar in structure. LDs are implicated in numerous physiological and pathophysiological functions. Many aspects of the LD has captured the attention of diverse scientists alike and has recently led to an explosion in information on the LD biogenesis, expansion and fusion, identification of LD proteomes and diseases associated with LD biology. This review will provide a brief history of this fascinating organelle and provide some contemporary views of unanswered questions in LD biogenesis.     

The architecture of Cidec-mediated interfaces between lipid droplets

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Controlling the size of lipid droplets: lipid and protein factors

Recent advances have transformed our understanding of lipid droplets (LDs). Once regarded as inert lipid storage granules, LDs are now recognized as multi-functional organelles that affect many aspects of cell biology and metabolism. However, fundamental questions concerning the biogenesis and growth of LDs remain unanswered. Recent studies have uncovered novel modes of LD growth (including rapid/homotypic as well as slow/atypical LD fusion), and identified key proteins (e.g. Fsp27, seipin, FITM2 and perilipin 1) and lipids (e.g. phosphatidylcholine and phosphatidic acid) that regulate the size of LDs. Phospholipids appear to have an evolutionarily conserved role in LD growth. Protein factors may regulate LD expansion directly and/or indirectly through modulating the level and composition of phospholipids on LD surface.     

Propranolol-sensitive and phenoxybenzamine-insensitive binding of norepinephrine to endogenous lipid droplets from rat adipocytes

Norepinephrine, epinephrine, and isoproterenol at concentrations of 5.5 x 10(-8) M were found to elicit lipolysis in a cell-free system containing lipid droplets from fat cells and lipase solution. In the cell-free system, the beta-blockers propranolol and dichloroisoproterenol at concentrations of 1 microM inhibited lipolysis induced by norepinephrine, whereas similar concentrations of the alpha-blockers phenoxybenzamine and yohimbine did not inhibit lipolysis. The binding of norepinephrine to endogenous lipid droplets was inhibited by propranolol, but not by phenoxybenzamine. We concluded that the propranolol-sensitive, phenoxybenzamine-insensitive binding of norepinephrine to endogenous lipid droplets is involved in lipolysis in fat cells. Treatment of endogenous lipid droplets with phospholipase C, but not phospholipase D, trypsin, chymotrypsin, or neuraminidase, inhibited the propranolol-sensitive binding of norepinephrine to the droplets. These results suggest that the phosphate group of phospholipid in endogenous lipid droplets may be the site of propranolol-sensitive binding of norepinephrine. The physiological significance of the propranolol-sensitive binding is discussed.     

Targeting the motor regulator Klar to lipid droplets

Background  

In Drosophila, the transport regulator Klar displays tissue-specific localization: In photoreceptors, it is abundant on the nuclear envelope; in early embryos, it is absent from nuclei, but instead present on lipid droplets. Differential targeting of Klar appears to be due to isoform variation. Droplet targeting, in particular, has been suggested to occur via a variant C-terminal region, the LD domain. Although the LD domain is necessary and sufficient for droplet targeting in cultured cells, lack of specific reagents had made it previously impossible to analyze its role in vivo.     

Cell biology of lipid droplets

Lipid storage has attracted much attention in the past years, both by the broader public and the biomedical scientific community. Driven by concerns about the obesity epidemic that affects most industrialized countries and even substantial parts of the population in less and least developed countries, work from researchers of many disciplines has shed light on the genetics, the physiology, and the cellular mechanisms of fat accumulation. This review focuses on the actual organelle of fat deposition, the lipid droplet (LD), and on the recent progress in mechanistic understanding of processes like LD biogenesis, LD growth and degradation, protein targeting to LDs and LD fusion.     

The proteomics of lipid droplets: structure, dynamics, and functions of the organelle conserved from bacteria to humans

Lipid droplets are cellular organelles that consists of a neutral lipid core covered by a monolayer of phospholipids and many proteins. They are thought to function in the storage, transport, and metabolism of lipids, in signaling, and as a specialized microenvironment for metabolism in most types of cells from prokaryotic to eukaryotic organisms. Lipid droplets have received a lot of attention in the last 10 years as they are linked to the progression of many metabolic diseases and hold great potential for the development of neutral lipid-derived products, such as biofuels, food supplements, hormones, and medicines. Proteomic analysis of lipid droplets has yielded a comprehensive catalog of lipid droplet proteins, shedding light on the function of this organelle and providing evidence that its function is conserved from bacteria to man. This review summarizes many of the proteomic studies on lipid droplets from a wide range of organisms, providing an evolutionary perspective on this organelle.