Nascent Lipid Droplets Research Articles

Phospholipid demixing and the birth of a lipid droplet

The biogenesis of lipid droplets (LD) in the yeast Saccharomyces cerevisiae was theoretically investigated on basis of a biophysical model. In accordance with the prevailing model of LD formation, we assumed that neutral lipids oil-out between the membrane leaflets of the endoplasmic reticulum (ER), resulting in LD that bud-off when a critical size is reached. Mathematically, LD were modeled as spherical protuberances in an otherwise planar ER membrane. We estimated the local phospholipid composition, and calculated the change in elastic free energy of the membrane caused by nascent LD. Based on this model calculation, we found a gradual demixing of lipids in the membrane leaflet that goes along with an increase in surface curvature at the site of LD formation. During demixing, the phospholipid monolayer was able to gain energy during LD growth, which suggested that the formation of curved interfaces was supported by or even driven by lipid demixing. In addition, we show that demixing is thermodynamically necessary as LD cannot bud-off otherwise. In the case of Saccharomyces cerevisiae our model predicts a LD bud-off diameter of about 12 nm. This diameter is far below the experimentally determined size of typical yeast LD. Thus, we concluded that if the standard model of LD formation is valid, LD biogenesis is a two step process. Small LD are produced from the ER, which subsequently ripe within the cytosol through a series of fusions.

High-Speed Vibrational Imaging and Spectral Analysis of Lipid Bodies by Compound Raman Microscopy

Cells store excess energy in the form of cytoplasmic lipid droplets. At present, it is unclear how different types of fatty acids contribute to the formation of lipid droplets. We describe a compound Raman microscope capable of both high-speed chemical imaging and quantitative spectral analysis on the same platform. We used a picosecond laser source to perform coherent Raman scattering imaging of a biological sample and confocal Raman spectral analysis at points of interest. The potential of the compound Raman microscope was evaluated on lipid bodies of cultured cells and live animals. Our data indicate that the in vivo fat contains much more unsaturated fatty acids (FAs) than the fat formed via de novo synthesis in 3T3-L1 cells. Furthermore, in vivo analysis of subcutaneous adipocytes and glands revealed a dramatic difference not only in the unsaturation level but also in the thermodynamic state of FAs inside their lipid bodies. Additionally, the compound Raman microscope allows tracking of the cellular uptake of a specific fatty acid and its abundance in nascent cytoplasmic lipid droplets. The high-speed vibrational imaging and spectral analysis capability renders compound Raman microscopy an indispensible analytical tool for the study of lipid-droplet biology.

Group IVA Phospholipase A2 Is Necessary for the Biogenesis of Lipid Droplets

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A(2) inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A(2) (cPLA(2)alpha). Knocking down cPLA(2)alpha expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA(2)alpha fusion protein (EGFP-cPLA(2)) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA(2)alpha at Ser(505). Transfection of a S505A mutant cPLA(2)alpha showed that phosphorylation at Ser(505) is key for enzyme activity and LD formation. cPLA(2)alpha contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA(2)alpha inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA(2)alpha in the formation of nascent LD from the endoplasmic reticulum.

Intracellular lipid droplet targeting by apolipoprotein A-V requires the carboxyl-terminal segment

The expression of apolipoprotein A-V (apoA-V) in hepatoma cells results in homing of this protein to intracellular lipid droplets. When hepatoma cells transfected with a full-length apoA-V-green fluorescent protein fusion protein were cultured in medium that was not supplemented with oleic acid (OA), intracellular lipid droplet size and number were reduced compared with those of cells supplemented with OA. Confocal microscopy studies revealed that apoA-V associates with lipid droplets under both conditions. To define the structural requirements for apoA-V lipid droplet association, hepatoma cells were transfected with a series of C-terminal truncated apoA-V variants. Confocal microscopy analysis revealed that, in a manner similar to mature full-length apoA-V (343 amino acids), truncation variants apoA-V(1-292), apoA-V(1-237), and apoA-V(1-191) associated with lipid droplets, while apoA-V(1-146) did not. Western blot analysis of the relative abundance of apoA-V in cell lysates versus conditioned medium indicated that apoA-V variants associated with lipid droplets were poorly secreted while apoA-V(1-146) was efficiently secreted. Ultracentrifugation of conditioned medium revealed that, unlike full-length apoA-V, which associates with lipoproteins, apoA-V(1-146) was present solely in the lipoprotein-deficient fraction. Deletion of the N-terminal signal peptide from apoA-V resulted in an inability of the protein to be secreted into the medium, although it associated with lipid droplets. Taken together, these data suggest that the C terminus of apoA-V is essential for lipid droplet association in transfected hepatoma cells and lipoprotein association in conditioned medium while the signal peptide is required for extracellular trafficking of this protein.

Requirement of Vimentin Filament Assembly for β3-Adrenergic Receptor Activation of ERK MAP Kinase and Lipolysis

Catecholamine stimulation of beta-adrenergic receptors (betaAR) in adipocytes activates the cAMP-dependent protein kinase to promote liberation of fatty acids as a fuel source. The adipocyte beta3AR also activates extracellular signal-regulated kinases (ERK)-1 and -2 through direct recruitment and activation of Src kinase. This pathway together with cAMP-dependent protein kinase contributes to maximal beta3AR-stimulated lipolysis. In a search for other molecules that might associate with beta3AR upon agonist stimulation, we identified vimentin using a proteomics approach. Immunoprecipitation of beta3AR from adipocytes in the absence or presence of the beta3AR agonist CL316,243, followed by Western blotting for vimentin confirmed this specific interaction. Since vimentin has also been identified on lipid droplets, the functional consequences of blocking the expression or structural integrity of vimentin intermediate filaments on beta3AR regulation of ERK activation and lipolysis was assessed. Following disruption of intermediate filaments with beta,beta'-iminodipropionitrile, as confirmed by confocal microscopy, beta3AR-stimulated ERK activation was blocked, and lipolysis was reduced by more than 40%. Independently, depletion of vimentin by small hairpin RNA (shRNA) completely inhibited beta3AR-mediated ERK activation and significantly reduced lipolysis. By contrast, disruption of actin-containing microfilaments by cytochalasin D or microtubules by nocodazole had no effect on either lipolysis or ERK activation. These results indicate that vimentin plays an essential role in the signal transduction pathway from beta3AR to the activation ERK and its contribution to lipolysis.

Temporal and spatial variations of lipid droplets during adipocyte division and differentiation

By capturing time-lapse images of primary stromal-vascular cells (SVCs) derived from rat mesenteric adipose tissue, we revealed temporal and spatial variations of lipid droplets (LDs) in individual SVCs during adipocyte differentiation. Numerous small LDs (a few micrometers in diameter) appeared in the perinuclear region at an early stage of differentiation; subsequently, several LDs grew to more than 10 microm in diameter and occupied the cytoplasm. We have developed a method for the fluorescence staining of LDs in living adipocytes. Time-lapse observation of the stained cells at higher magnification showed that nascent LDs (several 100 nm in diameter) grew into small LDs while moving from lamellipodia to the perinuclear region. We also found that adipocytes are capable of division and that they evenly distribute the LDs between two daughter cells. Immunofluorescence observations of LD-associated proteins revealed that such cell divisions of SVCs occurred even after LDs were coated with perilipin, suggesting that the "final" cell division during adipocyte differentiation occurs considerably later than that characterized in 3T3-L1 cells. Our time-lapse observations have provided a detailed account of the morphological changes that SVCs undergo during adipocyte division and differentiation.

A proposed model of fat packaging by exchangeable lipid droplet proteins

Humans have evolved mechanisms of efficient fat storage to survive famine, but these mechanisms contribute to obesity in our current environment of plentiful food and reduced activity. Little is known about how animals package fat within cells. Five related structural proteins serve roles in packaging fat into lipid droplets. The proteins TIP47, S3–12, and OXPAT/MLDP/PAT-1 move from the cytosol to coat nascent lipid droplets during rapid fat storage. In contrast, perilipin and adipophilin constitutively associate with lipid droplets and play roles in sustained fat storage and regulation of lipolysis. Different tissues express different complements of these lipid droplet proteins. Thus, the tissue-specific complement of these proteins determines how tissues manage lipid stores.

Identification and Characterization of Associated with Lipid Droplet Protein 1: A Novel Membrane‐Associated Protein That Resides on Hepatic Lipid Droplets

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

S3-12, Adipophilin, and TIP47 Package Lipid in Adipocytes

Animals have evolved mechanisms to maintain circulating nutrient levels when energy demands exceed feeding opportunities. Mammals store most of their energy as triacylglycerol in the perilipin-coated lipid droplets of adipocytes. How newly synthesized triacylglycerol is delivered to perilipin-coated lipid droplets is poorly understood. Perilipin is a member of the evolutionarily related family of PAT proteins (Perilipin, Adipophilin, TIP47), which is defined by sequence similarity and association with lipid droplets. We previously showed that S3-12, which is also a member of this family, associates with a separate pool of lipid droplets that emerge when triacylglycerol storage is driven by adding oleate to the culture medium of adipocytes. Our current data extend these findings to demonstrate that nascent lipid droplets emerge with a coat composed of S3-12, TIP47, and adipophilin. After 100 min of oleate treatment, the nascent lipid droplets are more heterogeneous: S3-12 and TIP47 coat smaller, peripheral droplets and adipophilin coats a more medial population of droplets. Fractionation of untreated and oleate-treated adipocytes shows oleate-dependent redistribution of TIP47 and adipophilin from cytosolic fractions to the lipid droplet fraction. Inhibition of protein synthesis with cycloheximide does not block the oleate-induced formation of the nascent lipid droplets, nor does it prevent TAG accumulation. We suggest that the non-lipid droplet pools of S3-12, adipophilin, and TIP47 constitute a ready reservoir of coat proteins to permit rapid packaging of newly synthesized triacylglycerol and to maximize energy storage during nutrient excess.

Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets.

Although caveolins normally reside in caveolae, they can accumulate on the surface of cytoplasmic lipid droplets (LDs). Here, we first provided support for our model that overaccumulation of caveolins in the endoplasmic reticulum (ER) diverts the proteins to nascent LDs budding from the ER. Next, we found that a mutant H-Ras, present on the cytoplasmic surface of the ER but lacking a hydrophobic peptide domain, did not accumulate on LDs. We used the fact that wild-type caveolin-1 accumulates in LDs after brefeldin A treatment or when linked to an ER retrieval motif to search for mutants defective in LD targeting. The hydrophobic domain, but no specific sequence therein, was required for LD targeting of caveolin-1. Certain Leu insertions blocked LD targeting, independently of hydrophobic domain length, but dependent on their position in the domain. We propose that proper packing of putative hydrophobic helices may be required for LD targeting of caveolin-1.

Temporal and spatial assembly of lipid droplet-associated proteins in 3T3-L1 preadipocytes.

This study aimed to investigate the relationship between newly formed lipid droplets and lipid droplet surface proteins, including perilipin, adipose differentiation related protein (ADRP), and p200 kDa protein (p200) in 3T3-L1 preadipocytes, during lipogenesis. Sterol ester was used to induce nascent lipid droplets in 3T3-L1 preadipocytes and the sequence of lipids and lipid droplet surface proteins was studied using a combination of immunohistochemistry and Nile red staining/Oil red O. We demonstrated that, although most growing lipid droplets appeared to have a lipid core surrounded by a fluorescent rim of ADRP, perilipin, and p200, tiny protein aggregates of ADRP, perilipin, or p200 could also be found to occur in the absence of lipid accumulation. In addition, ADRP associated with nascent lipid droplets prior to that of perilipin or p200. We provide evidence that lipid droplet surface proteins, especially ADRP and perilipin, are important in serving as a nucleation center for the assembly of lipid to form nascent lipid droplets.

TIP47 Associates with Lipid Droplets

Most mammalian cells package neutral lipids into droplets that are surrounded by a monolayer of phospholipids and a specific set of proteins including the adipose differentiation-related protein (ADRP; also called adipophilin), which is found in a wide array of cell types, and the perilipins, which are restricted to adipocytes and steroidogenic cells. TIP47 was initially identified in a yeast two-hybrid screen for proteins that interact with the cytoplasmic tail of the mannose 6-phosphate receptor, yet its sequence is highly similar to the lipid droplet protein, ADRP, and more distantly related to perilipins. Hence, we hypothesized that TIP47 might be associated with lipid droplets. In HeLa cells grown in standard low lipid-containing culture media, immunofluorescence microscopy revealed that the cells had few lipid droplets; however, TIP47 and ADRP were found on the surfaces of the small lipid droplets present. When the cells were grown in media supplemented with physiological levels of fatty acids, the amount of neutral lipid stored in lipid droplets increased dramatically, as did the staining of TIP47 and ADRP surrounding these droplets. TIP47 was found primarily in the cytosolic fractions of HeLa cells and murine MA10 Leydig cells grown in low lipid-containing culture medium, while ADRP was undetectable in these fractionated cell homogenates. When HeLa and MA10 Leydig cells were lipid-loaded, significant levels of ADRP were found in the floating lipid droplet fractions and TIP47 levels remained constant, but the distribution of a significant portion of TIP47 shifted from the cytosolic fractions to the lipid droplet fractions. Thus, we conclude that TIP47 associates with nascent lipid droplets and can be classified as a lipid droplet-associated protein.

Reorganization of a novel vimentin-associated protein in 3T3-L1 cells during adipose conversion

We have found that the antibody A2, a marker for the capsule of steroidogenic lipid droplets, reacts with an intermediate filament-associated protein, P200, in 3T3-L1 preadipocytes. Supporting evidence came from the colocalization pattern of P200 with vimentin in double label experiments. The association of P200 with vimentin was further confirmed by its copurification with vimentin after high salt extraction and colocalization of these two proteins in high salt-extracted and vinblastine-treated cells. In preadipocytes this protein was distributed on the vimentin filament network. At the early stage of adipose conversion, this protein was found to encircle nascent lipid droplets ranging from 0.1 to 0.2 micron, accompanied with a decreased distribution on the vimentin filament system. This infers a possible translocation of P200 from the vimentin filaments to the droplet surface. Meanwhile, the vimentin filaments remained in a normal distribution in the cytoplasm and were apparently not associated with the nascent droplet. The association of vimentin filaments to droplet surfaces became prominent in lipid droplets larger than 0.2 micron, forming a typical vimentin cage. Immunogold staining also confirmed the translocation of P200 immunoreactivity from the droplet surface to the vimentin cage. The relocation of P200 from the cytoplasmic vimentin filaments to the droplet surface prior to the formation of the vimentin cage, as well as the reorganization of this protein in the vimentin cage, suggests a stabilizing role in the lipid droplet formation and an inducing function of this protein in the formation of the vimentin cage.

Reorganization of a novel vimentin‐associated protein in 3T3‐L1 cells during adipose conversion

We have found that the antibody A2, a marker for the capsule of steroidogenic lipid droplets, reacts with an intermediate filament-associated protein, P200, in 3T3-L1 preadipocytes. Supporting evidence came from the colocalization pattern of P200 with vimentin in double label experiments. The association of P200 with vimentin was further confirmed by its copurification with vimentin after high salt extraction and colocalization of these two proteins in high salt-extracted and vinblastine-treated cells. In preadipocytes this protein was distributed on the vimentin filament network. At the early stage of adipose conversion, this protein was found to encircle nascent lipid droplets ranging from 0.1 to 0.2 μm, accompanied with a decreased distribution on the vimentin filament system. This infers a possible translocation of P200 from the vimentin filaments to the droplet surface. Meanwhile, the vimentin filaments remained in a normal distribution in the cytoplasm and were apparently not associated with the nascent droplet. The association of vimentin filaments to droplet surfaces became prominent in lipid droplets larger than 0.2 μm, forming a typical vimentin cage. Immunogold staining also confirmed the translocation of P200 immunoreactivity from the droplet surface to the vimentin cage. The relocation of P200 from the cytoplasmic vimentin filaments to the droplet surface prior to the formation of the vimentin cage, as well as the reorganization of this protein in the vimentin cage, suggests a stabilizing role in the lipid droplet formation and an inducing function of this protein in the formation of the vimentin cage. J. Cell. Biochem. 67:84–91, 1997. © 1997 Wiley-Liss, Inc.

Disruption of the vimentin intermediate filament system during adipose conversion of 3T3-L1 cells inhibits lipid droplet accumulation.

During the differentiation of 3T3-L1 pre-adipocytes, vimentin intermediate filaments are reorganized to form cage-like structures around the nascent lipid droplets. Initial studies with 3T3-L1 cells indicated that aggregation of vimentin filaments by nocodazole treatment during or shortly after induction of adipose conversion dramatically reduced the lipid droplet content of 3T3-L1 cells 96-120 hours after induction. Specific but transient disruption of vimentin following anti-IFA antibody injection also resulted in a decrease in lipid droplet formation in differentiating cells. To specifically and stably affect filament organization, 3T3-L1 cells lines were established by transfection with a glucocorticoid-regulatable, dominant negative mutant vimentin cDNA expression plasmid. Treatment of these cells (83 delta C) with dexamethasone resulted in expression of vimentin with a carboxyl-terminal deletion, which led to the disruption of the endogenous filament network. Induction of adipose conversion in 83 delta C cells lead to the formation of lipid droplets comparable to those seen in untransfected 3T3-L1 cells. Addition of dexamethasone during the adipose conversion of 83 delta C cells did not affect the induction of the marker enzyme glycerol-3-phosphate dehydrogenase or the incorporation of [14C]palmitate into triglycerides during a 10 minute pulse label. There was, however, a failure to form prominent lipid droplets and to accumulate [14C]palmitate-labeled triglycerides. Pulse-chase experiments indicated that the failure of these cells to accumulate triglyceride was associated with an increased rate of turnover. These studies indicate that vimentin filaments provide a function that influences lipid stability during adipose conversion of 3T3-L1 cells.

Formation of oleosomes (storage lipid bodies) during embryogenesis and their breakdown during seedling development in cotyledons of Sinapis alba L.

Electron microscopic and biochemical investigations of developing embryonic mustard cotyledons provided no evidence for the widely accepted hypothesis that oleosomes of fat-storing tissues originate from the endoplasmic reticulum and are surrounded by a unit- or half-unit membrane. In contrast, it was found that the first lipid droplets appear (about 12-14 d after pollination) in the ground cytoplasm near the surface of plastids. Subsequently these nascent lipid droplets, which lack any detectable boundary structure at this stage, become encircled by a cisterna of rough endoplasmic reticulum. At the same time an osmiophilic coat of about 3 nm thickness becomes detectable at the lipid/water interface. In the cotyledon cells of germinating seedlings a centrifugally moving front of fat degradation moves from the central vacuoles(s) towards the cell periphery, leaving behind collapsed coats of oleosomes which are depleted of their lipid contents (saccules). Although saccules appear tripartite in cross section, they are structurally different from endoplasmic reticulum membranes. The oleosome coats can be isolated from oleosome preparations by extracting lipids with organic solvents. The coat material is insoluble in detergents like Triton X-100 or deoxycholate and shows a tripartite, lamellar structure (similar to collapsed saccules) under the electron microscope. Upon dissolution with dodecylsulfate, polyacrylamide gel electrophoresis revealed a polypeptide composition (9 major bands) which is qualitatively different from that of the endoplasmic reticulum membrane. Also the buoyant densities of defatted oleosome coats and defatted endoplasmic reticulum membranes are very different. It is concluded that oleosome lipids accumulate in the ground cytoplasm and are bounded by a lamellar structure originating de novo from proteinaceous elements synthesized by specific regions of the endoplasmic reticulum.