Lipid Droplet Biology Research Articles

Lipid droplets in activated mast cells – a significant source of triglyceride-derived arachidonic acid for eicosanoid production

Mast cells are potent effectors of immune reactions and key players in various inflammatory diseases such as atherosclerosis, asthma, and rheumatoid arthritis. The cellular defense response of mast cells represents a unique and powerful system, where external signals can trigger cell activation resulting in a stimulus-specific and highly coordinated release of a plethora of bioactive mediators. The arsenal of mediators encompasses preformed molecules stored in cytoplasmic secretory granules, as well as newly synthesized proteinaceous and lipid mediators. The release of mediators occurs in strict chronological order and requires proper coordination between the endomembrane system and various enzymatic machineries. For the generation of lipid mediators, cytoplasmic lipid droplets have been shown to function as a major intracellular pool of arachidonic acid, the precursor for eicosanoid biosynthesis. Recent studies have revealed that not only phospholipids in mast cell membranes, but also triglycerides in mast cell lipid droplets are a substrate source for eicosanoid formation. The present review summarizes current knowledge about mast cell lipid droplet biology, and discusses expansions and challenges of traditional mechanistic models for eicosanoid production.

Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis.

Chaperone-mediated autophagy (CMA) selectively degrades a subset of cytosolic proteins in lysosomes. A potent physiological activator of CMA is nutrient deprivation, a condition in which intracellular triglyceride stores or lipid droplets (LD) also undergo hydrolysis (lipolysis) to generate free fatty acids for energetic purposes. Here we report that LD-associated proteins perilipin 2 (PLIN2) and perilipin 3 (PLIN3) are CMA substrates and their degradation via CMA precedes lipolysis. In vivo studies revealed that CMA degradation of PLIN2 and PLIN3 was enhanced during starvation, concurrent with elevated levels of cytosolic adipose triglyceride lipase (ATGL) and macroautophagy proteins on LD. CMA blockage both in cultured cells and mouse liver or expression of CMA-resistant PLINs lead to reduced association of ATGL and macrolipophagy-related proteins with LD and the subsequent decrease in lipid oxidation and accumulation of LD. We propose a role of CMA in LD biology and in the maintenance of lipid homeostasis.

Hepatic lipid droplet biology: Getting to the root of fatty liver.

Hepatic steatosis is defined by the accumulation of lipid droplets (LDs). Once thought to be only inert energy storage depots, LDs are increasingly recognized as organelles that have important functions in hepatocytes beyond lipid storage. The lipid and protein composition of LDs is highly dynamic and influences their intrinsic metabolism and signaling properties, which ultimately links them to the changes in hepatic function. This concise review highlights recent discoveries in LD biology and unique aspects of hepatic LDs and their role in liver disease.

Characterization of a new class of blue-fluorescent lipid droplet markers for live-cell imaging in plants.

The present work demonstrates the use and advantages of novel, live cell permeable, lipid droplet localizing, non toxic, blue fluorochromes for use in live plant cells. Lipid droplets (LDs) are ubiquitous components of both animal and plant cells. They consist of a core of neutral lipids surrounded by a monolayer of phospholipids, glycolipids and/or sterols with embedded amphipathic proteins. Although initially considered to be simple energy depots, they have recently emerged as organelles that serve important regulatory functions. Here we report three new fluorochromes as markers for LDs in plants. These bright blue fluorochromes with their unique spectral properties can easily be combined with other green and red fluorescent reporters for multicolor fluorescence imaging. The fluorochromes are non-toxic and photo-stable. All in all, they represent a reliable tool to use, for the investigation of dynamic LD biology within living plant cells using fluorescence microscopy.

Microscopic and spectroscopic techniques to investigate lipid droplet formation and turnover in yeast.

In spite of some progress in understanding the molecular basis of lipid-associated disorders, major questions about the regulation of synthesis and degradation of lipids and the interaction of these processes with other aspects of cellular physiology are still unresolved. Studies in reference organisms such as various yeast species, the fruit fly Drosophila melanogaster, or the nematode Caenorhabditis elegans complement efforts in mouse models as well as clinical studies in humans to address these questions. Imaging techniques play a pivotal role in understanding lipid droplet biology, and the implementation of imaging-based high-content screens of mutant collections has led to the identification of novel molecular players. This study focuses on novel fluorescent probes as well as spectroscopic imaging techniques to investigate lipid droplet formation and turnover in yeast. The application and limitations of such techniques in understanding lipid storage and turnover are discussed.

New automated single-cell technique for segmentation and quantitation of lipid droplets.

Lipid droplets are the major organelle for intracellular storage of triglycerides and cholesterol esters. Various methods have been attempted for automated quantitation of fluorescently stained lipid droplets using either thresholding or watershed methods. We find that thresholding methods deal poorly with clusters of lipid droplets, whereas watershed methods require a smoothing step that must be optimized to remove image noise. We describe here a novel three-stage hybrid method for automated segmentation and quantitation of lipid droplets. In this method, objects are initially identified by thresholding. They are then tested for circularity to distinguish single lipid droplets from clusters. Clusters are subjected to a secondary watershed segmentation. We provide a characterization of this method in simulated images. Additionally, we apply this method to images of fixed cells containing stained lipid droplets and GFP-tagged proteins to provide a proof-of-principle that this method can be used for colocalization studies. The circularity measure can additionally prove useful for the identification of inappropriate segmentation in an automated way; for example, of non-cellular material. We will make the programs and source code available to the community under the Gnu Public License. We believe this technique will be of interest to cell biologists for light microscopic studies of lipid droplet biology.

Modifications of the C terminus Affect Functionality and Stability of Yeast Triacylglycerol Lipase Tgl3p

Lipid droplets are specific organelles for the storage of triacylglycerols and steryl esters. They are surrounded by a phospholipid monolayer with a small but specific set of proteins embedded. Assembly and insertion of proteins into this surface membrane is an intriguing question of lipid droplet biology. To address this question we studied the topology of Tgl3p, the major triacylglycerol lipase of the yeast Saccharomyces cerevisiae, on lipid droplets. Employing the method of limited proteolysis of lipid droplet surface proteins, we found that the C terminus of Tgl3p faces the inside of the organelle, whereas the N terminus is exposed at the cytosolic side of lipid droplets. Detailed analysis of the C terminus revealed a stretch of seven amino acids that are critical for protein stability and functionality. The negative charge of two aspartate residues within this stretch is crucial for lipase activity of Tgl3p. A portion of Tgl3p, which is located to the endoplasmic reticulum, exhibits a different topology. In the phospholipid bilayer of the endoplasmic reticulum the C terminus faces the cytosol, which results in instability of the protein. Thus, the topology of Tgl3p is important for its function and strongly dependent on the membrane environment.

Integrated physiology and systems biology of PPARα.

The Peroxisome Proliferator Activated Receptor alpha (PPARα) is a transcription factor that plays a major role in metabolic regulation. This review addresses the functional role of PPARα in intermediary metabolism and provides a detailed overview of metabolic genes targeted by PPARα, with a focus on liver. A distinction is made between the impact of PPARα on metabolism upon physiological, pharmacological, and nutritional activation. Low and high throughput gene expression analyses have allowed the creation of a comprehensive map illustrating the role of PPARα as master regulator of lipid metabolism via regulation of numerous genes. The map puts PPARα at the center of a regulatory hub impacting fatty acid uptake, fatty acid activation, intracellular fatty acid binding, mitochondrial and peroxisomal fatty acid oxidation, ketogenesis, triglyceride turnover, lipid droplet biology, gluconeogenesis, and bile synthesis/secretion. In addition, PPARα governs the expression of several secreted proteins that exert local and endocrine functions.

Functional Mutation Analysis Provides Evidence for a Role of REEP1 in Lipid Droplet Biology

Hereditary axonopathies are frequently caused by mutations in proteins that reside in the endoplasmic reticulum (ER). Which of the many ER functions are pathologically relevant, however, remains to be determined. REEP1 is an ER protein mutated in hereditary spastic paraplegia (HSP) and hereditary motor neuropathy (HMN). We found that HSP-associated missense variants at the N-terminus of REEP1 abolish ER targeting, whereas two more central variants are either rare benign SNPs or confer pathogenicity via a different mechanism. The mis-targeted variants accumulate at lipid droplets (LDs). N-terminal tagging, deletion of the N-terminus, and expression of a minor REEP1 isoform had the same effect. We also confirmed an increase in LD size upon cooverexpression of atlastins and REEP1. Neither wild-type REEP1, LD-targeted HSP variants, nor a non-LD-targeted HMN variant reproduced this effect when expressed alone. We conclude that the N-terminus of REEP1 is necessary for proper targeting to and/or retention in the ER. The protein's potential to also associate with LDs corroborates a synergistic effect with atlastins on LD size. Interestingly, LD size is also altered upon knockdown of seipin, mutations of which also cause HSP and HMN. Regulation of LDs may thus be an ER function critical for long-term axonal maintenance.

The biophysics and cell biology of lipid droplets

Lipid droplets are intracellular organelles that are found in most cells, where they have fundamental roles in metabolism. They function prominently in storing oil-based reserves of metabolic energy and components of membrane lipids. Lipid droplets are the dispersed phase of an oil-in-water emulsion in the aqueous cytosol of cells, and the importance of basic biophysical principles of emulsions for lipid droplet biology is now being appreciated. Because of their unique architecture, with an interface between the dispersed oil phase and the aqueous cytosol, specific mechanisms underlie their formation, growth and shrinkage. Such mechanisms enable cells to use emulsified oil when the demands for metabolic energy or membrane synthesis change. The regulation of the composition of the phospholipid surfactants at the surface of lipid droplets is crucial for lipid droplet homeostasis and protein targeting to their surfaces.

The Lipid Droplet as a Potential Therapeutic Target in NAFLD

Nonalcoholic fatty liver disease (NAFLD) is a growing problem worldwide. Nonalcoholic fatty liver disease is characterized by an abnormal accumulation of triglyceride-rich lipid droplets (LDs) in the liver, which can lead to liver inflammation and metabolic disturbances. Lipid droplets are dynamic organelles that have recently gained considerable scientific interest. Their formation and growth are regulated processes requiring the participation of many endoplasmic reticulum- (ER-) and LD-associated proteins, which may serve as potential therapeutic targets for NAFLD. Protein families such as fat-inducing transmembrane proteins 1 and 2 (FITM1/FIT1 and FITM2/FIT2), the CIDE family of proteins, and the perilipin family, play important roles in LD biology. In this review, the authors discuss current views on LD formation and growth, and how various proteins may affect LD metabolism and lipoprotein assembly in the pathogenesis of NAFLD.

Lipid droplet metabolism

With the realization that lipid droplets are not merely inert fat storage organelles, but highly dynamic and actively involved in cellular lipid homeostasis, there has been an increased interest in lipid droplet biology. Recent studies have begun to unravel the roles that lipid dropletss play in cellular physiology and provide insights into the mechanisms by which lipid droplets contribute to cellular homeostasis. This review provides a summary of these recent publications on lipid droplet metabolism. Perilipins have different preferences for associating with triacylglycerol (TAG) or cholesteryl esters, different tissue distributions, and each contributes to lipid metabolism in its unique way. Cell death-inducing DFF45-like effector proteins are not only involved in lipid droplet expansion, but also in the cellular response to stress and lipid secretion. Lipid droplets undergo an active cycle of lipolysis and re-esterification to form microlipid droplets. TAG synthesis for lipid droplet formation and expansion occurs in the endoplasmic reticulum and on lipid droplets, and TAG transfers between lipid droplets during lipid droplet fusion. Lipid droplets interact with the endoplasmic reticulum and mitochondria to facilitate lipid transfer, lipid droplet expansion, and metabolism. Lipid droplets are dynamically active, responding to changes in cellular physiology, as well as interacting with cytosolic proteins and other organelles to control lipid homeostasis.

Rab GTPases Associate with Isolated Lipid Droplets (LDs) and Show Altered Content After Ethanol Administration: Potential Role in Alcohol-Impaired LD Metabolism

Alcoholic liver disease is manifested by the presence of fatty liver, primarily due to accumulation of hepatocellular lipid droplets (LDs). The presence of membrane-trafficking proteins (e.g., Rab GTPases) with LDs indicates that LDs may be involved in trafficking pathways known to be altered in ethanol (EtOH) damaged hepatocytes. As these Rab GTPases are crucial regulators of protein trafficking, we examined the effect EtOH administration has on hepatic Rab protein content and association with LDs. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8weeks. Whole liver and isolated LD fractions were analyzed. Identification of LDs and associated Rab proteins was performed in frozen liver or paraffin-embedded sections followed by immunohistochemical analysis. Lipid accumulation was characterized by larger LD vacuoles and increased total triglyceride content in EtOH-fed rats. Rabs 1, 2, 3d, 5, 7, and 18 were analyzed in postnuclear supernatant (PNS) as well as LDs. All of the Rabs were found in the PNS, and Rabs 1, 2, 5, and 7 did not show alcohol-altered content, while Rab 3d content was reduced by over 80%, and Rab 18 also showed EtOH-induced reduction in content. Rab 3d was not found to associate with LDs, while all other Rabs were found in the LD fractions, and several showed an EtOH-related decrease (Rabs 2, 5, 7, 18). Immunohistochemical analysis revealed the enhanced content of a LD-associated protein, perilipin 2 (PLIN2) that was paralleled with an associated decrease of Rab 18 in EtOH-fed rat sections. Chronic EtOH feeding was associated with increased PLIN2 and altered Rab GTPase content in enriched LD fractions. Although mechanisms driving these changes are not established, further studies on intracellular protein trafficking and LD biology after alcohol administration will likely contribute to our understanding of fatty liver disease.

Balancing the fat: lipid droplets and human disease.

Lipid droplets (LDs) are dynamic, cytosolic lipid-storage organelles found in nearly all cell types. Too many or too few LDs during excess or deficient fat storage lead to many different human diseases. Recent insights into LD biology and LD protein functions shed new light on mechanisms underlying those metabolic pathologies. These findings will likely provide opportunities for treatment of diseases associated with too much or too little fat.

Overexpression of Sir2 in the adult fat body is sufficient to extend lifespan of male and female Drosophila

Sir2 is the most intensively discussed longevity gene in current aging research. Although, the gene encoding a NAD(+)-dependent histone deacetylase initially was found to extend lifespan of various organisms ranging from yeast to mammals, serious doubts regarding its role in longevity have been expressed recently. In this study, we tested whether tissue-specific overexpression of Sir2 in the adult fat body can extend lifespan when compared to genetically identical controls. We also wanted to elucidate the mechanisms by which fat body Sir2 promotes longevity by studying the phenotypic and transcriptional changes in the fat body. We found that moderate (3-fold) Sir2 overexpression in the fat body during adulthood only can promote longevity in both sexes by roughly 13 %. In addition, we obtained transcriptional profiles elicited by this overexpression and propose a role for Sir2 in lipid droplet biology especially under conditions of starvation. Furthermore, our data do not support the idea of Sir2 mediating the response to dietary restriction (DR) because transcriptional profiles of fat bodies after DR or Sir2 overexpression do not match. This study provides additional independent evidence for the concept of Sir2 as a longevity gene and as a promising pharmacological target to cure age-related diseases.

Abstract 1893: Spectroscopic imaging unveils altered cholesterol metabolism in prostate cancer .

Abstract Metabolic reprogramming allows cancer cells to sustain high proliferative rates and resist cell death. Whereas alterations to glucose and amino acid metabolism have been extensively studied, altered lipid metabolism in cancer is increasingly recognized based on the findings of imbalanced lipid signaling network and dysregulated lipogenic enzymes. Storage of neutral lipids in lipid droplets (LDs), an essential aspect of lipid metabolism, is however long underappreciated until recent advances in LD biology. In human prostate cancer cell lines, neutral lipid accumulation was commonly seen and partially attributed to the up-regulation of fatty acid synthase, a key lipogenic enzyme catalyzing fatty acid biosynthesis and implicated in many human malignancies including prostate cancer. However, because the composition of LDs is not accessible with fluorescence microscopy, the exact role of lipid accumulation in prostate cancer progression remains elusive. By vibration-based spectroscopic imaging of 65 specimens collected from 61 prostate cancer patients and healthy donors, we found for the first time a marked shift from lipofuscin-like autofluorescent granules in normal, benign, and prostatic intraepithelial neoplasia lesions to cholesteryl ester (CE)-rich LDs in high-grade and metastatic prostate cancers. Quantitatively, CE amount was found ∼5 folds higher in high-grade relative to low-grade prostate cancers. Cellular studies demonstrate that the activated PI3K/Akt/mTOR/SREBP pathway accounts for accumulation of CEs via elevated LDL uptake and cholesterol esterification. Blockage of cholesterol esterification with an acetyl-coA cholesterol acyltransferase (ACAT) inhibitor significantly slows the growth and induces apoptosis of prostate cancer both in vitro and in vivo. These findings suggest that CE can be a quantifiable molecular marker for diagnosis of prostate cancer, especially for differentiation between low-grade and high-grade prostate cancers which currently has up to 40% inter-observer discordance. Moreover, targeting the altered cholesterol metabolism opens new opportunities for treating advanced prostate cancer in human patients. Citation Format: Shuhua Yue, Junjie Li, Seung Young Lee, Tian Shao, Bing Song, Liang Cheng, Chang-Deng Hu, Xiaoqi Liu, Timothy L. Ratliff, Ji-Xin Cheng. Spectroscopic imaging unveils altered cholesterol metabolism in prostate cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1893. doi:10.1158/1538-7445.AM2013-1893 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

PPARγ coactivator-1α contributes to exercise-induced regulation of intramuscular lipid droplet programming in mice and humans

Intramuscular accumulation of triacylglycerol, in the form of lipid droplets (LD), has gained widespread attention as a hallmark of metabolic disease and insulin resistance. Paradoxically, LDs also amass in muscles of highly trained endurance athletes who are exquisitely insulin sensitive. Understanding the molecular mechanisms that mediate the expansion and appropriate metabolic control of LDs in the context of habitual physical activity could lead to new therapeutic opportunities. Herein, we show that acute exercise elicits robust upregulation of a broad program of genes involved in regulating LD assembly, morphology, localization, and mobilization. Prominent among these was perilipin-5, a scaffolding protein that affects the spatial and metabolic interactions between LD and their surrounding mitochondrial reticulum. Studies in transgenic mice and primary human skeletal myocytes established a key role for the exercise-responsive transcriptional coactivator PGC-1α in coordinating intramuscular LD programming with mitochondrial remodeling. Moreover, translational studies comparing physically active versus inactive humans identified a remarkably strong association between expression of intramuscular LD genes and enhanced insulin action in exercise-trained subjects. These results reveal an intimate molecular connection between intramuscular LD biology and mitochondrial metabolism that could prove relevant to the etiology and treatment of insulin resistance and other disorders of lipid imbalance.

The evolutionary conserved protein CG9186 is associated with lipid droplets, required for their positioning and for fat storage

Lipid droplets (LDs) are specialized cell organelles for the storage of energy-rich lipids. Although lipid storage is a conserved feature of all cells and organisms, little is known about fundamental aspects of the cell biology of LDs, including their biogenesis, structural assembly and subcellular positioning, and the regulation of organismic energy homeostasis. We identified a novel LD-associated protein family, represented by the Drosophila protein CG9186 and its murine homolog MGI:1916082. In the absence of LDs, both proteins localize at the endoplasmic reticulum (ER). Upon lipid storage induction, they translocate to LDs using an evolutionarily conserved targeting mechanism that acts through a 60-amino-acid targeting motif in the center of the CG9186 protein. Overexpression of CG9186, and MGI:1916082, causes clustering of LDs in both tissue culture and salivary gland cells, whereas RNAi knockdown of CG9186 results in a reduction of LDs. Organismal RNAi knockdown of CG9186 results in a reduction in lipid storage levels of the fly. The results indicate that we identified the first members of a novel and evolutionarily conserved family of lipid storage regulators, which are also required to properly position LDs within cells.

Caveolin-1 Deficiency Leads to Increased Susceptibility to Cell Death and Fibrosis in White Adipose Tissue: Characterization of a Lipodystrophic Model

Caveolin-1 (CAV1) is an important regulator of adipose tissue homeostasis. In the present study we examined the impact of CAV1 deficiency on the properties of mouse adipose tissue both in vivo and in explant cultures during conditions of metabolic stress. In CAV1−/− mice fasting caused loss of adipose tissue mass despite a lack of hormone-sensitive lipase (HSL) phosphorylation. In addition, fasting resulted in increased macrophage infiltration, enhanced deposition of collagen, and a reduction in the level of the lipid droplet protein perilipin A (PLIN1a). Explant cultures of CAV1−/− adipose tissue also showed a loss of PLIN1a during culture, enhanced secretion of IL-6, increased release of lactate dehydrogenase, and demonstrated increased susceptibility to cell death upon collagenase treatment. Attenuated PKA-mediated signaling to HSL, loss of PLIN1a and increased secretion of IL-6 were also observed in adipose tissue explants of CAV1+/+ mice with diet-induced obesity. Together these results suggest that while alterations in adipocyte lipid droplet biology support adipose tissue metabolism in the absence of PKA-mediated pro-lipolytic signaling in CAV1−/− mice, the tissue is intrinsically unstable resulting in increased susceptibility to cell death, which we suggest underlies the development of fibrosis and inflammation during periods of metabolic stress.

Revisiting the connection between intramyocellular lipids and insulin resistance: a long and winding road

In the mid-1990s, researchers began to re-examine type 2 diabetes from a more 'lipocentric' perspective; giving strong consideration to the idea that systemic lipid imbalances give rise to glucose dysregulation, rather than vice versa. At the forefront of this paradigm shift was a report by Krssak and colleagues (Diabetologia 1999; 42:113-116) showing that intramyocellular lipid content, measured via the (then) novel application of proton nuclear magnetic resonance spectroscopy, served as a robust indicator of muscle insulin sensitivity in healthy individuals. A subsequent wave of investigations produced compelling correlative evidence linking ectopic lipid deposition within skeletal myocytes to the development of obesity-associated insulin resistance. But this relationship has proven much more complex than originally imagined, and scientists today are still left wondering if and how the intramyocellular accumulation of lipid droplets has a direct bearing on insulin action. Originally viewed as a simple storage depot, the lipid droplet is now recognised as an essential and sophisticated organelle that actively participates in numerous cellular processes. This edition of 'Then and now' revisits the connection between intramuscular lipids and insulin resistance and looks to future research aimed at understanding the dynamic interplay between lipid droplet biology and metabolic health.