Cidea controls lipid droplet fusion and lipid storage in brown and white adipose tissue

Excess lipid storage in adipose tissue results in the development of obesity and other metabolic disorders including diabetes,fatty liver and cardiovascular diseases.The lipid droplet(LD)is an important subcellular organelle responsible for lipid storage.We previously observed that Fsp27,a member of the CIDE family proteins,is localized to LD-contact sites and promotes atypical LD fusion and growth.Cidea,a close homolog of Fsp27,is expressed at high levels in brown adipose tissue.However,the exact role of Cidea in promoting LD fusion and lipid storage in adipose tissue remains unknown.Here,we expressed Cidea in Fsp27-knockdown adipocytes and observed that Cidea has similar activity to Fsp27 in promoting lipid storage and LD fusion and growth.Next,we generated Cidea and Fsp27 double-deficient mice and observed that these animals had drastically reduced adipose tissue mass and a strong lean phenotype.In addition,Cidea/Fsp27 double-deficient mice had improved insulin sensitivity and were intolerant to cold.Furthermore,we observed that the brown and white adipose tissues of Cidea/Fsp27double-deficient mice had significantly reduced lipid storage and contained smaller LDs compared to those of Cidea or Fsp27single deficient mice.Overall,these data reveal an important role of Cidea in controlling lipid droplet fusion,lipid storage in brown and white adipose tissue,and the development of obesity.     

Fat-specific Protein 27 Undergoes Ubiquitin-dependent Degradation Regulated by Triacylglycerol Synthesis and Lipid Droplet Formation

The fat-specific protein 27 (Fsp27), a protein localized to lipid droplets (LDs), plays an important role in controlling lipid storage and mitochondrial activity in adipocytes. Fsp27-null mice display increased energy expenditure and are resistant to high fat diet-induced obesity and diabetes. However, little is known about how the Fsp27 protein is regulated. Here, we show that Fsp27 stability is controlled by the ubiquitin-dependent proteasomal degradation pathway in adipocytes. The ubiquitination of Fsp27 is regulated by three lysine residues located in the C-terminal region. Substitution of these lysine residues with alanines greatly increased Fsp27 stability and enhanced lipid storage in adipocytes. Furthermore, Fsp27 was stabilized and rapidly accumulated following treatment with β-agonists that induce lipolysis and fatty acid re-esterification in adipocytes. More importantly, Fsp27 stabilization was dependent on triacylglycerol synthesis and LD formation, because knockdown of diacylglycerol acyltransferase in adipocytes significantly reduced Fsp27 accumulation in adipocytes. Finally, we observed that increased Fsp27 during β-agonist treatment preferentially associated with LDs. Taken together, our data revealed that Fsp27 can be stabilized by free fatty acid availability, triacylglycerol synthesis, and LD formation. The stabilization of Fsp27 when free fatty acids are abundant further enhances lipid storage, providing positive feedback to regulate lipid storage in adipocytes.     

新型荧光探针mMaple3与mEos3.2应用于Fsp27介导脂滴融合的功能研究

目的:Fsp27已经被证明定位在脂滴上并且介导脂滴融合与增大。为研究Fsp27介导脂滴融合的动态分子机制,我们构建了Fsp27-mMaple3和Fsp27-mEos3.2两种新型荧光探针的融合蛋白并研究其对脂滴融合的功能影响,进而为研发Fsp27相关生理功能的光学显像技术奠定基础。方法:对照传统绿色荧光的融合蛋白Fsp27-EGFP,在共聚焦显微镜下观察Fsp27-mMaple3和Fsp27-mEos3.2两种新型融合蛋白的亚细胞定位和介导脂滴融合的功能,并利用荧光漂白恢复术(fluorescence recovery after photo-bleaching,FRAP)以判断脂滴与脂滴之间是否存在脂的交换。结果:表达Fsp27-mMaple3和Fsp27-mEos3.2两种新型融合蛋白的细胞中脂滴显著增大;同时,融合蛋白皆集中在脂滴与脂滴的接触位点上,且中性脂的交换实验显示脂滴与脂滴之间可以相互连通。结论:我们建构的两种新型荧光探针融合蛋白Fsp27-mMaple3和Fsp27-mEos3.2保持了Fsp27介导脂滴融合的功能,并为我们进一步研发新型的超分辨光学显像技术提供功能基础。     

FSP27 promotes lipid droplet clustering and then fusion to regulate triglyceride accumulation

Fat Specific Protein 27 (FSP27), a lipid droplet (LD) associated protein in adipocytes, regulates triglyceride (TG) storage. In the present study we demonstrate that FSP27 plays a key role in LD morphology to accumulate TGs. We show here that FSP27 promotes clustering of the LDs which is followed by their fusion into fewer and enlarged droplets. To map the domains of FSP27 responsible for these events, we generated GFP-fusion constructs of deletion mutants of FSP27. Microscopic analysis revealed that amino acids 173-220 of FSP27 are necessary and sufficient for both the targeting of FSP27 to LDs and the initial clustering of the droplets. Amino acids 120-140 are essential but not sufficient for LD enlargement, whereas amino acids 120-210 are necessary and sufficient for both clustering and fusion of LDs to form enlarged droplets. In addition, we found that FSP27-mediated enlargement of LDs, but not their clustering, is associated with triglyceride accumulation. These results suggest a model in which FSP27 facilitates LD clustering and then promotes their fusion to form enlarged droplets in two discrete, sequential steps, and a subsequent triglyceride accumulation.     

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.     

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.     

Visualization of lipid droplet composition by direct organelle mass spectrometry

An expanding appreciation for the varied functions of neutral lipids in cellular organisms relies on a more detailed understanding of the mechanisms of lipid production and packaging into cytosolic lipid droplets (LDs). Conventional lipid profiling procedures involve the analysis of tissue extracts and consequently lack cellular or subcellular resolution. Here, we report an approach that combines the visualization of individual LDs, microphase extraction of lipid components from droplets, and the direct identification of lipid composition by nanospray mass spectrometry, even to the level of a single LD. The triacylglycerol (TAG) composition of LDs from several plant sources (mature cotton (Gossypium hirsutum) embryos, roots of cotton seedlings, and Arabidopsis thaliana seeds and leaves) were examined by direct organelle mass spectrometry and revealed the heterogeneity of LDs derived from different plant tissue sources. The analysis of individual LDs makes possible organellar resolution of molecular compositions and will facilitate new studies of LD biogenesis and functions, especially in combination with analysis of morphological and metabolic mutants. Furthermore, direct organelle mass spectrometry could be applied to the molecular analysis of other subcellular compartments and macromolecules.     

The size matters:regulation of lipid storage by lipid droplet dynamics

Adequate energy storage is essential for sustaining healthy life.Lipid droplet(LD) is the subcellular organelle that stores energy in the form of neutral lipids and releases fatty acids under energy deficient conditions.Energy storage capacity of LDs is primarily dependent on the sizes of LDs.Enlargement and growth of LDs is controlled by two molecular pathways:neutral lipid synthesis and atypical LD fusion.Shrinkage of LDs is mediated by the degradation of neutral lipids under energy demanding conditions and is controlled by neutral cytosolic Upases and lysosomal acidic Upases.In this review,we summarize recent progress regarding the regulatory pathways and molecular mechanisms that control the sizes and the energy storage capacity of LDs.     

Quantitative electron microscopy shows uniform incorporation of triglycerides into existing lipid droplets

The lipid droplet (LD) is an organelle with a lipid ester core and a surface phospholipid monolayer. The mechanism of LD biogenesis is not well understood. The present study aimed to elucidate the LD growth process, for which we developed a new electron microscopic method that quantifies the proportion of existing and newly synthesized triglycerides in individual LDs. Our method takes advantage of the reactivity of unsaturated fatty acids and osmium tetroxide, which imparts LDs an electron density that reflects fatty acid composition. With this method, existing triglyceride-rich LDs in 3Y1 fibroblasts were observed to incorporate newly synthesized triglycerides at a highly uniform rate. This uniformity and its persistence even after microtubules were depolymerized suggest that triglycerides in fibroblasts are synthesized in the local vicinity of individual LDs and then incorporated. In contrast, LDs in 3T3-L1 adipocytes showed heterogeneity in the rate at which lipid esters were incorporated, indicating different mechanisms of LD growth in fibroblasts and adipocytes.     

The FATP1-DGAT2 complex facilitates lipid droplet expansion at the ER-lipid droplet interface

At the subcellular level, fat storage is confined to the evolutionarily conserved compartments termed lipid droplets (LDs), which are closely associated with the endoplasmic reticulum (ER). However, the molecular mechanisms that enable ER-LD interaction and facilitate neutral lipid loading into LDs are poorly understood. In this paper, we present evidence that FATP1/acyl-CoA synthetase and DGAT2/diacylglycerol acyltransferase are components of a triglyceride synthesis complex that facilitates LD expansion. A loss of FATP1 or DGAT2 function blocked LD expansion in Caenorhabditis elegans. FATP1 preferentially associated with DGAT2, and they acted synergistically to promote LD expansion in mammalian cells. Live imaging indicated that FATP1 and DGAT2 are ER and LD resident proteins, respectively, and electron microscopy revealed FATP1 and DGAT2 foci close to the LD surface. Furthermore, DGAT2 that was retained in the ER failed to support LD expansion. We propose that the evolutionarily conserved FATP1-DGAT2 complex acts at the ER-LD interface and couples the synthesis and deposition of triglycerides into LDs both physically and functionally.     

Long-term live cell microscopy studies of lipid droplet fusion dynamics in adipocytes

During the adipogenic differentiation process of mesenchymal stem cells, lipid droplets (LDs) grow slowly by transferring lipids between each other. Recent findings hint at the possibility that a fusion pore is involved. In this study, we analyze lipid transfer data obtained in long-term label-free microscopy studies in the framework of a Hagen-Poiseuille model. The data obtained show a LD fusion process in which the lipid transfer directionality depends on the size difference between LDs, whereas the respective rates depend on the size difference and additionally on the diameter of the smaller LDs. For the data analysis, the viscosity of the transferred material has to be known. We demonstrate that a viscosity-dependent molecular rotor dye can be used to measure LD viscosities in live cells. On this basis, we calculate the diameter of a putative lipid transfer channel which appears to have a direct dependence on the diameter of the smaller of the two participating LDs.     

Regulated localization of Rab18 to lipid droplets: effects of lipolytic stimulation and inhibition of lipid droplet catabolism

Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3DGV, a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3DGV labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by beta-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs.     

Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes

Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions.     

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.     

Remodeling of lipid droplets during lipolysis and growth in adipocytes

Synthesis, storage, and turnover of triacylglycerols (TAGs) in adipocytes are critical cellular processes to maintain lipid and energy homeostasis in mammals. TAGs are stored in metabolically highly dynamic lipid droplets (LDs), which are believed to undergo fragmentation and fusion under lipolytic and lipogenic conditions, respectively. Time lapse fluorescence microscopy showed that stimulation of lipolysis in 3T3-L1 adipocytes causes progressive shrinkage and almost complete degradation of all cellular LDs but without any detectable fragmentation into micro-LDs (mLDs). However, mLDs were rapidly formed after induction of lipolysis in the absence of BSA in the culture medium that acts as a fatty acid scavenger. Moreover, mLD formation was blocked by the acyl-CoA synthetase inhibitor triacsin C, implicating that mLDs are synthesized de novo in response to cellular fatty acid overload. Using label-free coherent anti-Stokes Raman scattering microscopy, we demonstrate that LDs grow by transfer of lipids from one organelle to another. Notably, this lipid transfer between closely associated LDs is not a rapid and spontaneous process but rather occurs over several h and does not appear to require physical interaction over large LD surface areas. These data indicate that LD growth is a highly regulated process leading to the heterogeneous LD size distribution within and between individual cells. Our findings suggest that lipolysis and lipogenesis occur in parallel in a cell to prevent cellular fatty acid overflow. Furthermore, we propose that formation of large LDs requires a yet uncharacterized protein machinery mediating LD interaction and lipid transfer.     

Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii

In plants, neutral lipids are frequently synthesized and stored in seed tissues, where the assembly of lipid droplets (LDs) coincides with the accumulation of triacylglycerols (TAGs). In addition, photosynthetic, vegetative cells can form cytosolic LDs and much less information is known about the makeup and biogenesis of these LDs. Here we focus on Chlamydomonas reinhardtii as a reference model for LDs in a photosynthetic cell, because in this unicellular green alga LD dynamics can be readily manipulated by nitrogen availability. Nitrogen deprivation leads to cellular quiescence during which cell divisions cease and TAGs accumulate. The major lipid droplet protein (MLDP) forms a proteinaceous coat surrounding mature LDs. Reducing the amount of MLDP affects LD size and number, TAG breakdown and timely progression out of cellular quiescence following nitrogen resupply. Depending on nitrogen availability, MLDP recruits different proteins to LDs, tubulins in particular. Conversely, depolymerization of microtubules drastically alters the association of MLDP with LDs. LDs also contain select chloroplast envelope membrane proteins hinting at an origin of LDs, at least in part, from chloroplast membranes. Moreover, LD surface lipids are rich in de novo synthesized fatty acids, and are mainly composed of galactolipids which are typical components of chloroplast membranes. The composition of the LD membrane is altered in the absence of MLDP. Collectively, our results suggest a mechanism for LD formation in C. reinhardtii involving chloroplast envelope membranes by which specific proteins are recruited to LDs and a specialized polar lipid monolayer surrounding the LD is formed.     

Seipin regulates ER–lipid droplet contacts and cargo delivery

Seipin is an endoplasmic reticulum (ER) membrane protein implicated in lipid droplet (LD) biogenesis and mutated in severe congenital lipodystrophy (BSCL2). Here, we show that seipin is stably associated with nascent ER–LD contacts in human cells, typically via one mobile focal point per LD. Seipin appears critical for such contacts since ER–LD contacts were completely missing or morphologically aberrant in seipin knockout and BSCL2 patient cells. In parallel, LD mobility was increased and protein delivery from the ER to LDs to promote LD growth was decreased. Moreover, while growing LDs normally acquire lipid and protein constituents from the ER, this process was compromised in seipin‐deficient cells. In the absence of seipin, the initial synthesis of neutral lipids from exogenous fatty acid was normal, but fatty acid incorporation into neutral lipids in cells with pre‐existing LDs was impaired. Together, our data suggest that seipin helps to connect newly formed LDs to the ER and that by stabilizing ER–LD contacts seipin facilitates the incorporation of protein and lipid cargo into growing LDs in human cells.     

Quantitative analysis of lipid droplet fusion: inefficient steady state fusion but rapid stimulation by chemical fusogens

Lipid droplets (LDs) are dynamic cytoplasmic organelles containing neutral lipids and bounded by a phospholipid monolayer. Previous studies have suggested that LDs can undergo constitutive homotypic fusion, a process linked to the inhibitory effects of fatty acids on glucose transporter trafficking. Using strict quantitative criteria for LD fusion together with refined light microscopic methods and real-time analysis, we now show that LDs in diverse cell types show low constitutive fusogenic activity under normal growth conditions. To investigate the possible modulation of LD fusion, we screened for agents that can trigger fusion. A number of pharmacological agents caused homotypic fusion of lipid droplets in a variety of cell types. This provided a novel cell system to study rapid regulated fusion between homotypic phospholipid monolayers. LD fusion involved an initial step in which the two adjacent membranes became continuous (<10 s), followed by the slower merging (100 s) of the neutral lipid cores to produce a single spherical LD. These fusion events were accompanied by changes to the LD surface organization. Measurements of LDs undergoing homotypic fusion showed that fused LDs maintained their initial volume, with a corresponding decrease in surface area suggesting rapid removal of membrane from the fused LD. This study provides estimates for the level of constitutive LD fusion in cells and questions the role of LD fusion in vivo. In addition, it highlights the extent of LD restructuring which occurs when homotypic LD fusion is triggered in a variety of cell types.     

Adipose triglyceride lipase regulates basal lipolysis and lipid droplet size in adipocytes

In adipocytes, lipid droplet (LD) size reflects a balance of triglyceride synthesis (lipogenesis) and hydrolysis (lipolysis). Perilipin A (Peri A) is the most abundant phosphoprotein on the surface of adipocyte LDs and has a crucial role in lipid storage and lipolysis. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major rate-determining enzymes for lipolysis in adipocytes. Each of these proteins (Peri A, ATGL, and HSL) has been demonstrated to regulate lipid storage and release in the adipocyte. However, in the absence of protein kinase A (PKA) stimulation (basal state), the lipases (ATGL and HSL) are located mainly in the cytoplasm, and their contribution to basal rates of lipolysis and influence on LD size are poorly understood. In this study, we utilize an adenoviral system to knockdown or overexpress ATGL and HSL in an engineered model system of adipocytes in the presence or absence of Peri A. We are able to demonstrate in our experimental model system that in the basal state, LD size, triglyceride storage, and fatty acid release are mainly influenced by the expression of ATGL. These results demonstrate for the first time the relative contributions of ATGL, HSL, and Peri A on determination of LD size in the absence of PKA stimulation.     

The MAP1-LC3 conjugation system is involved in lipid droplet formation

Lipid droplets (LDs) are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. We show here that the LC3 lipidation system essential for macroautophagy is involved in LD formation. LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin (LD marker)-positive lipid fraction from the starved liver. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.