Lipid Droplet Biology Research Articles

Autophagic Clearance of Lipid Droplets Alters Metabolic Phenotypes in a Genetic Obesity–Diabetes Mouse Model

Lipid droplets (LDs) are intracellular organelles that store neutral lipids, and their aberrant accumulation is associated with many diseases including metabolic disorders such as obesity and diabetes. Meanwhile, the potential pathological contributions of LDs in these diseases are unclear, likely due to a lack of chemical biology tools to clear LDs. We recently developed LD-clearance small molecule compounds, Lipid Droplets·AuTophagy TEthering Compounds (LD·ATTECs), that are able to induce autophagic clearance of LDs in cells and in the liver of db/db (C57BL/6J Leprdb/Leprdb) mouse model, which is a widely used genetic model for obesity–diabetes. Meanwhile, the potential effects on the metabolic phenotype remain to be elucidated. Here, using the metabolic cage assay and the blood glucose assay, we performed phenotypic characterization of the effects of the autophagic degradation of LDs by LD·ATTECs in the db/db mouse model. The study reveals that LD·ATTECs increased the oxygen uptake of mice and the release of carbon dioxide, enhanced the heat production of animals, partially enhanced the exercise during the dark phase, decreased the blood glucose level and improved insulin sensitivity. Collectively, the study characterized the metabolic phenotypes induced by LD·ATTECs in an obesity–diabetes mouse model, revealing novel functional impacts of autophagic clearance of LDs and providing insights into LD biology and obesity–diabetes pathogenesis from the phenotypic perspective.

Mitoguardin-2-mediated lipid transfer preserves mitochondrial morphology and lipid droplet formation.

Lipid transport proteins at membrane contacts, where organelles are closely apposed, are critical in redistributing lipids from the endoplasmic reticulum (ER), where they are made, to other cellular membranes. Such protein-mediated transfer is especially important for maintaining organelles disconnected from secretory pathways, like mitochondria. We identify mitoguardin-2, a mitochondrial protein at contacts with the ER and/or lipid droplets (LDs), as a lipid transporter. An x-ray structure shows that the C-terminal domain of mitoguardin-2 has a hydrophobic cavity that binds lipids. Mass spectrometry analysis reveals that both glycerophospholipids and free-fatty acids co-purify with mitoguardin-2 from cells, and that each mitoguardin-2 can accommodate up to two lipids. Mitoguardin-2 transfers glycerophospholipids between membranes in vitro, and this transport ability is required for roles both in mitochondrial and LD biology. While it is not established that protein-mediated transfer at contacts plays a role in LD metabolism, our findings raise the possibility that mitoguardin-2 functions in transporting fatty acids and glycerophospholipids at mitochondria-LD contacts.

RIPK3 dampens mitochondrial bioenergetics and lipid droplet dynamics in metabolic liver disease.

Receptor-interacting protein kinase 3 (RIPK3) mediates NAFLD progression, but its metabolic function is unclear. Here, we aimed to investigate the role of RIPK3 in modulating mitochondria function, coupled with lipid droplet (LD) architecture in NAFLD. Functional studies evaluating mitochondria and LD biology were performed in wild-type (WT) and Ripk3-/- mice fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks and in CRISPR-Cas9 Ripk3 -null fat-loaded immortalized hepatocytes. The association between hepatic perilipin (PLIN) 1 and 5, RIPK3, and disease severity was also addressed in a cohort of patients with NAFLD and in PLIN1 -associated familial partial lipodystrophy. Ripk3 deficiency rescued impairment in mitochondrial biogenesis, bioenergetics, and function in CDAA diet-fed mice and fat-loaded hepatocytes. Ripk3 deficiency was accompanied by a strong upregulation of antioxidant systems, leading to diminished oxidative stress upon fat loading both in vivo and in vitro. Strikingly, Ripk3-/- hepatocytes displayed smaller size LD in higher numbers than WT cells after incubation with free fatty acids. Ripk3 deficiency upregulated adipocyte and hepatic levels of LD-associated proteins PLIN1 and PLIN5. PLIN1 upregulation controlled LD structure and diminished mitochondrial stress upon free fatty acid overload in Ripk3-/- hepatocytes and was associated with diminished human NAFLD severity. Conversely, a pathogenic PLIN1 frameshift variant was associated with NAFLD and fibrosis, as well as with increased hepatic RIPK3 levels in familial partial lipodystrophy. Ripk3 deficiency restores mitochondria bioenergetics and impacts LD dynamics. RIPK3 inhibition is promising in ameliorating NAFLD.

Lipid droplets and perilipins in canine osteosarcoma. Investigations on tumor tissue, 2D and 3D cell culture models

Lipid droplets were identified as important players in biological processes of various tumor types. With emphasis on lipid droplet-coating proteins (perilipins, PLINs), this study intended to shed light on the presence and formation of lipid droplets in canine osteosarcoma. For this purpose, canine osteosarcoma tissue samples (n = 11) were analyzed via immunohistochemistry and electron microscopy for lipid droplets and lipid droplet-coating proteins (PLINs). Additionally, we used the canine osteosarcoma cell lines D-17 and COS4288 in 2D monolayer and 3D spheroid (cultivated for 7, 14, and 21 days) in vitro models, and further analyzed the samples by means of histochemistry, immunofluorescence, molecular biological techniques (RT-qPCR, Western Blot) and electron microscopical imaging. Lipid droplets, PLIN2, and PLIN3 were detected in osteosarcoma tissue samples as well as in 2D and 3D cultivated D-17 and COS4288 cells. In spheroids, specific distribution patterns of lipid droplets and perilipins were identified, taking into consideration cell line specific zonal apportionment. Upon external lipid supplementation (oleic acid), a rise of lipid droplet amount accompanied with an increase of PLIN2 expression was observed. Detailed electron microscopical analyzes revealed that lipid droplet sizes in tumor tissue were comparable to that of 3D spheroid models. Moreover, the biggest lipid droplets were found in the central zone of the spheroids at all sampling time-points, reaching their maximum size at 21 days. Thus, the 3D spheroids can be considered as a relevant in vitro model for further studies focusing on lipid droplets biology and function in osteosarcoma.

A 3,4-dimethoxy-1,8-naphthalimide for lipid droplet imaging in live and fixed cells

Lipid droplets (LDs) are found in most eukaryotic cells and in addition to serving as lipid reservoirs, these highly dynamic organelles play fundamental roles in cell metabolism and growth. The study of LDs and their interactions with other cellular compartments provides new insights into normal cell biology and disease processes such as virus packaging, metabolic disorders, and cancer progression. Here we report the synthesis and comprehensive evaluation of two readily prepared, low molecular weight 3,4-dimethoxy-1,8-naphthalimides, as selective LD stains. The newly synthesised 1,8-naphthalimide derivative (DMN-LD), demonstrated impressive versatility compared to currently available options, by staining LDs in both live and fixed cells, as well as a three-dimensional spheroid, using both single and two-photon excitation. This new imaging agent has the potential to further unravel the complex biology of LDs, which are essential to cell survival, and when altered, underpin many disease states.

Adaptive and maladaptive roles of lipid droplets in health and disease.

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.

Homeostatic regulation of lipid droplet content in mammalian oocytes and embryos.

Lipid droplets (LDs) consist of a core of neutral lipids such as triacylglycerols and cholesteryl esters covered by a phospholipid monolayer. Recent studies have shown that LDs not only store neutral lipids but are also associated with various physiological functions. LDs are found in most eukaryotic cells and vary in size and quantity. It has long been known that mammalian oocytes contain LDs. Porcine and bovine oocytes contain substantial amounts of LDs, which cause their cytoplasm to darken, whereas mouse and human oocytes are translucent due to their low LD content. A sufficient amount of LDs in mammalian oocytes has been thought to be associated with oocyte maturation and early embryonic development, but the necessity of LDs has been questioned because embryonic development proceeds normally even when LDs are removed. However, recent studies have revealed that LDs play a crucial role during implantation and that maintaining an appropriate amount of LDs is important for early embryonic development, even in mammalian species with low amounts of LDs in their oocytes. This suggests that a fine-tuned balance of LD content is essential for successful mammalian embryonic development. In this review, we discuss the physiological importance of LDs in mammalian oocytes and preimplantation embryos based on recent findings on LD biology.

The disassembly of lipid droplets in Chlamydomonas.

Lipid droplets (LDs) are ubiquitous and specialized organelles in eukaryotic cells. Consisting of a triacylglycerol core surrounded by a monolayer of membrane lipids, LDs are decorated with proteins and have myriad functions, from carbon/energy storage to membrane lipid remodeling and signal transduction. The biogenesis and turnover of LDs are therefore tightly coordinated with cellular metabolic needs in a fluctuating environment. Lipid droplet turnover requires remodeling of the protein coat, lipolysis, autophagy and fatty acid β-oxidation. Several key components of these processes have been identified in Chlamydomonas (Chlamydomonas reinhardtii), including the major lipid droplet protein, a CXC-domain containing regulatory protein, the phosphatidylethanolamine-binding DTH1 (DELAYED IN TAG HYDROLYSIS1), two lipases and two enzymes involved in fatty acid β-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil.

Lipidomics profiling of goose granulosa cell model of stearoyl-CoA desaturase function identifies a pattern of lipid droplets associated with follicle development

BackgroundDespite their important functions and nearly ubiquitous presence in cells, an understanding of the biology of intracellular lipid droplets (LDs) in goose follicle development remains limited. An integrated study of lipidomic and transcriptomic analyses was performed in a cellular model of stearoyl-CoA desaturase (SCD) function, to determine the effects of intracellular LDs on follicle development in geese.ResultsNumerous internalized LDs, which were generally spherical in shape, were dispersed throughout the cytoplasm of granulosa cells (GCs), as determined using confocal microscopy analysis, with altered SCD expression affecting LD content. GC lipidomic profiling showed that the majority of the differentially abundant lipid classes were glycerophospholipids, including PA, PC, PE, PG, PI, and PS, and glycerolipids, including DG and TG, which enriched glycerophospholipid, sphingolipid, and glycerolipid metabolisms. Furthermore, transcriptomics identified differentially expressed genes (DEGs), some of which were assigned to lipid-related Gene Ontology slim terms. More DEGs were assigned in the SCD-knockdown group than in the SCD-overexpression group. Integration of the significant differentially expressed genes and lipids based on pathway enrichment analysis identified potentially targetable pathways related to glycerolipid/glycerophospholipid metabolism.ConclusionsThis study demonstrated the importance of lipids in understanding follicle development, thus providing a potential foundation to decipher the underlying mechanisms of lipid-mediated follicle development.

Dissecting lipid droplet biology with coherent Raman scattering microscopy.

Lipid droplets (LDs) are lipid-rich organelles universally found in most cells. They serve as a key energy reservoir, actively participate in signal transduction and dynamically communicate with other organelles. LD dysfunction has been associated with a variety of diseases. The content level, composition and mobility of LDs are crucial for their physiological and pathological functions, and these different parameters of LDs are subject to regulation by genetic factors and environmental inputs. Coherent Raman scattering (CRS) microscopy utilizes optical nonlinear processes to probe the intrinsic chemical bond vibration, offering label-free, quantitative imaging of lipids in vivo with high chemical specificity and spatiotemporal resolution. In this Review, we provide an overview over the principle of CRS microscopy and its application in tracking different parameters of LDs in live cells and organisms. We also discuss the use of CRS microscopy in genetic screens to discover lipid regulatory mechanisms and in understanding disease-related lipid pathology.

Inhibition of Elongation Factor 1A1 Activity Decreases Lipid Droplet Accumulation

Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatocyte lipid accumulation, which can cause hepatocyte injury through lipotoxicity. Eukaryotic elongation factor 1A1 (EEF1A1), which is a component of the protein synthetic machinery and regulates actin cytoskeleton turnover, is induced in human hepatocyte-like HepG2 cells undergoing lipotoxicity, in association with ER stress. Moreover, EEF1A1 has been identified in the lipid droplet (LD) proteome in various cell and tissue types, suggesting that it may have a role in LD biology. Partial inhibition of EEF1A1 peptide elongation activity with the marine compound didemnin B (DB) decreases lipotoxic HepG2 cell death. However, the effects of EEF1A1 inhibition on cellular lipid accumulation are unclear. Based on the potential association of EEF1A1 with LDs, and its apparent roles in responding to ER stress and in regulating cytoskeleton dynamics, we hypothesized that inhibition of EEF1A1 with DB would alter hepatocyte LD accumulation. Male 129S6 mice were fed a western diet for 26 weeks to induce NAFLD, followed by intervention with DB (50 μg/kg, i.p.) once every 3 days for 14 days. Treatment with DB decreased liver lipid accumulation, as determined by biochemical and histological analyses, and upregulated expression of genes involved in fatty acid oxidation (Cpt1a, Fgf21). Assessments of LD size in mouse liver revealed a significant decrease in median size upon treatment with DB, suggesting that DB promotes the formation of smaller LDs. To explore whether this effect was conserved in human hepatocyte-like cells, HepG2 cells were treated for 6 h with DB (80 nM) under lipotoxic conditions (1 mM palmitate plus oleate) and LDs were visualized by confocal microscopy. Cells exposed to lipotoxic conditions exhibited increased LD area, and treatment with DB appeared to decrease the formation of large LDs (-63%, p=0.11). This occurred alongside upregulation of CPT1A mRNA, which was consistent with the upregulation of genes involved in fatty acid oxidation observed in mouse liver. Finally, a Chinese hamster ovary cell line that harbors a mutation leading to reduced EEF1A1 expression accumulated less triglyceride under high fatty acid conditions compared to wild-type cells. Together, these findings suggest that that disruption of EEF1A1 activity may diminish LD accumulation and channel excess fatty acids toward oxidation, which could have implications for the treatment of NAFLD.

CG32803 is the fly homolog of LDAF1 and influences lipid storage in vivo

The Seipin protein is a conserved key component in the biogenesis of lipid droplets (LDs). Recently, a cooperation between human Seipin and the Lipid droplet assembly factor 1 (LDAF1) was described. LDAF1 physically interacts with Seipin and the holocomplex safeguards regular LD biogenesis. The function of LDAF1 proteins outside mammals is less clear. In yeast, the lipid droplet organization (LDO) proteins, which also cooperate with Seipin, are the putative homologs of LDAF1. While certain functional aspects are shared between the LDO and mammalian LDAF1 proteins, the relationship between the proteins is under debate. Here, we identify the Drosophila melanogaster protein CG32803, which we re-named to dmLDAF1, as an insect member of this protein family. dmLDAF1 decorates LDs in cultured cells and in vivo and the protein is linked to the fly and mouse Seipin proteins. Altering the dmLDAF1 abundance affects LD size, number and overall lipid storage amounts. Our results suggest that the LDAF1 proteins thus fulfill an evolutionarily conserved function in the biogenesis and biology of LDs.

Novel Mechanism of Conventional BODIPY Conjugates for Live-cell SMLM Reveals Lipid Droplet Biology at Super-resolution

Novel Mechanism of Conventional BODIPY Conjugates for Live-cell SMLM Reveals Lipid Droplet Biology at Super-resolution

Lipid Droplets in Neurodegenerative Disorders.

Knowledge of lipid droplets (LDs) has evolved from simple depots of lipid storage to dynamic and functionally active organelles involved in a variety of cellular functions. Studies have now informed significant roles for LDs in cellular signaling, metabolic disease, and inflammation. While lipid droplet biology has been well explored in peripheral organs such as the liver and heart, LDs within the brain are relatively understudied. The presence and function of these dynamic organelles in the central nervous system has recently gained attention, especially in the context of neurodegeneration. In this review, we summarize the current understanding of LDs within the brain, with an emphasis on their relevance in neurodegenerative diseases.

Grease on—Perspectives in lipid droplet biology

Lipid droplets (LDs) are now recognized as omnipresent and dynamic subcellular organelles of amazing morphological and functional diversity. Beyond the obvious benefit of having molecules full of chemical energy stored in a dedicated structural entity, LDs may also be viewed as a safe harbor for potentially damaging metabolites. This protective function might in many cases even supersede the relevance of lipid storage for eventual energy gain and membrane biogenesis. Furthermore, the LD surface constitutes a unique membrane environment, creating a platform for hosting specific proteins and thus enabling their interactions. These metabolic hotspots would contribute decisively to compartmentalized metabolism in the cytosol. LDs are also communicating extensively with other subcellular organelles in directing and regulating lipid metabolism. Deciphering the relevance of LD storage and regulation at the organismic level will be essential for the understanding of widespread and serious metabolic complications in humans. Increasing attention is also devoted to pathogens appropriating LDs for their own benefit. LD biology is still considered an emerging research area in rapid and vibrant development, attracting scientists from all disciplines of the life sciences and beyond, which is mirrored by the accompanying review collection. Here, we present our personal views on areas we believe are especially exciting and hold great potential for future developments. Particularly, we address issues relating to LD biogenesis and heterogeneity, required technological advances, and the complexity of human physiology.

Faster and More Specific: Excited-State Intramolecular Proton Transfer-Based Dyes for High-Fidelity Dynamic Imaging of Lipid Droplets within Cells and Tissues.

Lipid droplets (LDs), a type of dynamic organelle residing at the center of cellular lipid storage, have been identified to play important roles in multiple biological processes, metabolic disorders, and diseases. The highly dynamic characters of LDs were found to correspond to their physiological and pathological functions. Hence, the fluorescent probes which enable dynamic tracking of LDs should be very helpful for better understanding the mechanisms of LDs involved biological processes and diseases. Herein we present, to the best of our knowledge, the first class of excited-state intramolecular proton transfer (ESIPT) fluorescence dyes (Flp-(11-13, 19)) for dynamic imaging of LDs based on 3-hydroxyflavone (3HF) derivatives. Flp-(11-13, 19) display strong fluorescence from yellow to NIR in lipid but exhibit almost nonfluorescence in aqueous solution. Besides, they also show large Stokes shifts (>150 nm), narrow absorption and emission peaks, and good oil-water separation efficiency, which makes them specifically target and stain LDs with very low background noisy in both living cells and fixed cells. They stain intracellular LDs quite quickly (within 30 s) with very low dosage (as low as 500 nM). Benefitting from these advantages, Flp-(11-13, 19) are applied successfully in tracking the dynamic nature of LDs and accumulation of LDs in both aqueous solution and living cells, 3D imaging of LDs for visualization of their repartition within the cells, and visualizing LDs in tissues of diseases mice models including adipose, skeletal muscle, and fatty liver tissues, underscoring the potential utility of these dyes in both LDs biology research and medical diagnosis of LDs involved diseases.

A Review of Diatom Lipid Droplets.

The dynamic nutrient availability and photon flux density of diatom habitats necessitate buffering capabilities in order to maintain metabolic homeostasis. This is accomplished by the biosynthesis and turnover of storage lipids, which are sequestered in lipid droplets (LDs). LDs are an organelle conserved among eukaryotes, composed of a neutral lipid core surrounded by a polar lipid monolayer. LDs shield the intracellular environment from the accumulation of hydrophobic compounds and function as a carbon and electron sink. These functions are implemented by interconnections with other intracellular systems, including photosynthesis and autophagy. Since diatom lipid production may be a promising objective for biotechnological exploitation, a deeper understanding of LDs may offer targets for metabolic engineering. In this review, we provide an overview of diatom LD biology and biotechnological potential.

Dynamic cyclic behaviors of lipid droplets monitored by two-photon fluorescence probe with high photostability.

Lipid droplets (LDs) are organelles featuring in intracellular storage of neutral lipids, which are involved with many biological processes. Monitoring the dynamical cyclic behaviors of cellular LDs poses great importance for early disease diagnosis. Herein, two coumarin-based LDs-specific fluorescence probes exhibited "turn-on" and two-photon fluorescence triggered by breaking their aggregation states. By virtue of establishing oil/water emulsions model to simulate LDs, the behaviors of "turn-on" fluorescence were elucidated, which benefited for the enhancement of selectivity of the probes to LDs. Then, we highlight a LDs-specific coumarin-based two-photon probe (L1) with high photo-stability to monitor the dynamic cyclic behaviors of LDs in cells, unraveling the changes of LDs quantity during lipophagy and the reproduction of LDs to prevent lipotoxicity. We believe the probe offers a convenient way to investigate the biology of LDs.

Proteomics-Based Monitoring of Pathway Activity Reveals that Blocking Diacylglycerol Biosynthesis Rescues from Alpha-Synuclein Toxicity

SummaryProteinaceous inclusions containing alpha-synuclein (α-Syn) have been implicated in neuronal toxicity in Parkinson’s disease, but the pathways that modulate toxicity remain enigmatic. Here, we used a targeted proteomic assay to simultaneously measure 269 pathway activation markers and proteins deregulated by α-Syn expression across a panel of 33 Saccharomyces cerevisiae strains that genetically modulate α-Syn toxicity. Applying multidimensional linear regression analysis to these data predicted Pah1, a phosphatase that catalyzes conversion of phosphatidic acid to diacylglycerol at the endoplasmic reticulum membrane, as an effector of rescue. Follow-up studies demonstrated that inhibition of Pah1 activity ameliorates the toxic effects of α-Syn, indicate that the diacylglycerol branch of lipid metabolism could enhance α-Syn neuronal cytotoxicity, and suggest a link between α-Syn toxicity and the biology of lipid droplets.

Three-dimensional label-free imaging throughout adipocyte differentiation by stimulated Raman microscopy.

Lipid droplets are lipid-storage organelles with a key role in lipid accumulation pathologies such as diabetes, obesity and atherosclerosis. Despite their important functions many aspects of lipid droplets biology are still unknown. This is partially due to the current use of exogenous labels to monitor their formation and remodelling by invasive imaging methods. Here, we apply stimulated Raman scattering microscopy to acquire images with high spatial resolution along with resolving capabilities of lipids and proteins and three-dimensional sectioning. Our images and data analysis demonstrate an increase in the number of large (>15μm2) lipid droplets in human adipocyte cells during differentiation process. In addition, spatially-resolved maps of lipids and proteins inside cells and three dimensional reconstructions of lipids at the initial and final steps of adipocyte differentiation are reported, too.