Perilipin overexpression in white adipose tissue induces a brown fat-like phenotype

Background

Perilipin A (PeriA) exclusively locates on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Previously, we reported that adipocyte specific overexpression of PeriA caused resistance to diet-induced obesity and resulted in improved insulin sensitivity. In order to better understand the biological basis for this observed phenotype, we performed additional studies in this transgenic mouse model.

Methodology and Principal Findings

When compared to control animals, whole body energy expenditure was increased in the transgenic mice. Subsequently, we performed DNA microarray analysis and real-time PCR on white adipose tissue. Consistent with the metabolic chamber data, we observed increased expression of genes associated with fatty acid β-oxidation and heat production, and a decrease in the genes associated with lipid synthesis. Gene expression of Pgc1a, a regulator of fatty acid oxidation and Ucp1, a brown adipocyte specific protein, was increased in the white adipose tissue of the transgenic mice. This observation was subsequently verified by both Western blotting and histological examination. Expression of RIP140, a regulator of white adipocyte differentiation, and the lipid droplet protein FSP27 was decreased in the transgenic mice. Importantly, FSP27 has been shown to control gene expression of these crucial metabolic regulators. Overexpression of PeriA in 3T3-L1 adipocytes also reduced FSP27 expression and diminished lipid droplet size.

Conclusions

These findings demonstrate that overexpression of PeriA in white adipocytes reduces lipid droplet size by decreasing FSP27 expression and thereby inducing a brown adipose tissue-like phenotype. Our data suggest that modulation of lipid droplet proteins in white adipocytes is a potential therapeutic strategy for the treatment of obesity and its related disorders.     

The Studies of Phase Equilibria and Efficiency Assessment for Self-Emulsifying Lipid-Based Formulations

The study was designed to build up a database for the evaluation of the self-emulsifying lipid formulations performance. A standard assessment method was constructed to evaluate the self-emulsifying efficiency of the formulations based on five parameters including excipients miscibility, spontaneity, dispersibility, homogeneity, and physical appearance. Equilibrium phase studies were conducted to investigate the phase changes of the anhydrous formulation in response to aqueous dilution. Droplet size studies were carried out to assess the influence of lipid and surfactant portions on the resulted droplet size upon aqueous dilution. Formulations containing mixed glycerides showed enhanced self-emulsification with both lipophilic and hydrophilic surfactants. Increasing the polarity of the lipid portion in the formulation leaded to progressive water solubilization capacity. In addition, formulations containing medium chain mixed glycerides and hydrophilic surfactants showed lower droplet size compared with their long chain and lipophilic counterparts. The inclusion of mixed glycerides in the lipid formulations enormously enhances the formulation efficiency.     

Adhesion of intermediate filaments and lipid droplets in adrenal cells studied by field emission scanning electron microscopy

High-resolution field emission scanning electron microscopy was used to study the organisation of intermediate filaments around lipid droplets and their binding to these droplets, in primary culture of bovine adrenal cells. Whole-mount preparations of intermediate filaments and bound lipid droplets were prepared from cells grown on Formvar-coated grids and processed by freeze-drying. Intermediate filaments were seen as an interconnected network enveloping the entire droplet. The bound filaments appear to be directly adherent to the surface of the droplet and hence take on its curved contour. The binding of the filaments to the droplets was determined by means of tilting. This study provides a new approach to investigate the cytoskeleton and its associated structures with high-resolution three-dimensional images.     

The protein and neutral lipid composition of lipid droplets isolated from the fission yeast, <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Lipid droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer with bound proteins. Much of the information on lipid droplet function comes from proteomic and lipodomic studies that identify the components of droplets isolated from organisms throughout the phylogenetic tree. Here, we add to that important inventory by reporting lipid droplet factors from the fission yeast, Schizosaccharomyces pombe. Unique to this study was the fact that cells were cultured in three different environments: 1) late log growth phase in glucose-based media, 2) stationary phase in glucosebased media, and 3) late log growth phase in media containing oleic acid. We confirmed colocalization of major factors with lipid droplets using live-cell fluorescent microscopy. We also analyzed droplets from each of the three conditions for sterol ester (SE) and triacylglycerol (TAG) content, along with their respective fatty acid compositions. We identified a previously undiscovered lipid droplet protein, Vip1p, which affects droplet size distribution. The results provide further insight into the workings of these ubiquitous organelles.     

Proteomic profiling of lipid droplet-associated proteins in primary adipocytes of normal and obese mouse

Lipid droplets in adipocytes serve as the principal long-term energy storage depot of animals. There is increasing recognition that lipid droplets are not merely a static neutral lipid storage site, but in fact dynamic and multi-functional organelles. Structurally, lipid droplet consists of a neutral lipid core surrounded by a phospholipid monolayer and proteins embedded in or bound to the phospholipid layer. Proteins on the surface of lipid droplets are crucial to droplet structure and dynamics. To understand the lipid droplet-associated proteome of primary adipocyte with a large central lipid droplet, lipid droplets of white adipose tissue from C57BL/6 mice were isolated. And the proteins were extracted and analyzed by liquid chromatography coupled with tandem mass spectrometry. A total of 193 proteins including 73 previously unreported proteins were identified. Furthermore, the isotope-coded affinity tags (ICAT) was used to compare the difference of lipid droplet-associated proteomes between the normal lean and the high-fat diet-induced obese C57BL/6 mice. Of 23 proteins quantified by ICAT analysis, 3 proteins were up-regulated and 4 proteins were down-regulated in the lipid droplets of adipose tissue from the obese mice. Importantly, two structural proteins of lipid droplets, perilipin A and vimentin, were greatly reduced in the lipid droplets of the adipose tissue from the obese mice, implicating reduced protein machinery for lipid droplet stability.     

Fat-specific protein 27, a novel lipid droplet protein that enhances triglyceride storage

Fat-specific protein (FSP)27/Cidec is most highly expressed in white and brown adipose tissues and increases in abundance by over 50-fold during adipogenesis. However, its function in adipocytes has remained elusive since its discovery over 15 years ago. Here we demonstrate that FSP27/Cidec localizes to lipid droplets in cultured adipocytes and functions to promote lipid accumulation. Ectopically expressed FSP27-GFP surrounds lipid droplets in 3T3-L1 adipocytes and colocalizes with the known lipid droplet protein perilipin. Immunostaining of endogenous FSP27 in 3T3-L1 adipocytes also confirmed its presence on lipid droplets. FSP27-GFP expression also markedly increases lipid droplet size and enhances accumulation of total neutral lipids in 3T3-L1 preadipocytes as well as other cell types such as COS cells. Conversely, RNA interference-based FSP27/Cidec depletion in mature adipocytes significantly stimulates lipolysis and reduces the size of lipid droplets. These data reveal FSP27/Cidec as a novel adipocyte lipid droplet protein that negatively regulates lipolysis and promotes triglyceride accumulation.     

Development of swine adipose tissue: morphology and chemical composition.

Differentiation and growth of swine subcutaneous adipose tissue was assessed by chemical analysis of tissue components, cell size measurements of isolated adipocytes, and light and electron microscopic observations. At birth all adipocytes were multilocular (contained multiple small lipid droplets), but by day 3 postpartum, many were already differentiated to the unilocular state (one major, central lipid droplet). Microscopic observations of fixed tissue, cell size determinations on isolated adipocytes, and chemical analysis of tissue composition indicated a marked increase in adipocyte size accompanied by an increase in the size of the central lipid droplet with age. Small cells were observed at all ages (in both fixed tissue and isolated cell preparations), yielding biphasic size distributions. Although the adipocyte stem cell was not discerned, an early stage in differentiation, designated an adipoblast, was observed.     

Sterol carrier protein-2 expression modulates protein and lipid composition of lipid droplets

Despite the critical role lipid droplets play in maintaining energy reserves and lipid stores for the cell, little is known about the regulation of the lipid or protein components within the lipid droplet. Although immunofluorescence of intact cells as well as Western analysis of isolated lipid droplets revealed that sterol carrier protein-2 (SCP-2) was not associated with lipid droplets, SCP-2 expression significantly altered the structure of the lipid droplet. First, the targeting of fatty acid and cholesterol to the lipid droplets was significantly decreased. Second, the content of several proteins important for lipid droplet function was differentially increased (perilipin A), reduced severalfold (adipose differentiation-related protein (ADRP), vimentin), or almost completely eliminated (hormone-sensitive lipase and proteins >93 kDa) in the isolated lipid droplet. Third, the distribution of lipids within the lipid droplets was significantly altered. Double labeling of cells with 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-octadecanoic acid (NBD-stearic acid) and antisera to ADRP showed that 70, 24, and 13% of lipid droplets contained ADRP, NBD-stearic acid, or both, respectively. SCP-2 expression decreased the level of ADRP in the lipid droplet but increased the proportion wherein ADRP and NBD-stearic acid colocalized by 3-fold. SCP-2 expression also decreased the lipid droplet fatty acid and cholesterol mass (nmol/mg protein) by 5.2- and 6.6-fold, respectively. Finally, SCP-2 expression selectively altered the pattern of esterified fatty acids in favor of polyunsaturated fatty acids within the lipid droplet. Displacement studies showed differential binding affinity of ADRP for cholesterol and fatty acids. These data suggested that SCP-2 and ADRP play a significant role in regulating fatty acid and cholesterol targeting to lipid droplets as well as in determining their lipid and protein components.     

Immunocytochemical demonstration of a lipid droplet-specific capsule in cultured Leydig cells of the golden hamsters

In this report, we provide direct evidence for the presence of a lipid droplet-associated capsule in hamster steroidogenic Leydig cells by using a monoclonal antibody A2. Leydig cells are characterized by containing many lipid droplets and having 3β-hydroxysteroid dehydrogenase activity. Immunofluorescence staining with this antibody demonstrated a rim or capsule surrounding the lipid droplets in Leydig cells, a pattern not seen with anti-vimentin antibody. Immunogold labelling confirmed ultrastructurally that antibody binding was distributed on the lipid droplet surface. In order to investigate the possible function of the capsule, we examined the morphological changes induced in the capsule following stimulation with LH or dibutyryl cAMP; the fluorescent intensity of the capsule was seen to gradually decrease, accompanied by a decrease in number and size of lipid droplets, and the response to both reagents was time- and concentration-dependent. We thus conclude that hormonal stimulation resulting in the detachment of certain capsular proteins from the surface of lipid droplets is mediated via the cAMP signaling pathway and may allow cholesterol ester hydrolytic enzyme direct access to its substrate in the lipid droplet. © 1996 Wiley-Liss, Inc.     

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.     

Identification of the lipid droplet targeting domain of the Cidea protein

Cidea, the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) domain-containing protein, is targeted to lipid droplets in mouse adipocytes, where it inhibits triglyceride hydrolysis and promotes lipid storage. In mice, Cidea may prevent lipolysis by binding and shielding lipid droplets from lipase association. Here we demonstrate that human Cidea localizes with lipid droplets in both adipocyte and nonadipocyte cell lines, and we ascribe specific functions to its protein domains. Expression of full-length Cidea in undifferentiated 3T3-L1 cells or COS-1 cells increases total cellular triglyceride and strikingly alters the morphology of lipid droplets by enhancing their size and reducing their number. Remarkably, both lipid droplet binding and increased triglyceride accumulation are also elicited by expression of only the carboxy-terminal 104 amino acids, indicating this small domain directs lipid droplet targeting and triglyceride shielding. However, unlike the full-length protein, expression of the carboxy-terminus causes clustering of small lipid droplets but not the formation of large droplets, identifying a novel function of the N terminus. Furthermore, human Cidea promotes lipid storage via lipolysis inhibition, as the expression of human Cidea in fully differentiated 3T3-L1 adipocytes causes a significant decrease in basal glycerol release. Taken together, these data indicate that the carboxy-terminal domain of Cidea directs lipid droplet targeting, lipid droplet clustering, and triglyceride accumulation, whereas the amino terminal domain is required for Cidea-mediated development of enlarged lipid droplets.     

ARAP2 promotes GLUT1-mediated basal glucose uptake through regulation of sphingolipid metabolism

Lipid droplet formation, which is driven by triglyceride synthesis, requires several droplet-associated proteins. We identified ARAP2 (an ADP-ribosylation factor 6 GTPase-activating protein) in the lipid droplet proteome of NIH-3T3 cells and showed that knockdown of ARAP2 resulted in decreased lipid droplet formation and triglyceride synthesis. We also showed that ARAP2 knockdown did not affect fatty acid uptake but reduced basal glucose uptake, total levels of the glucose transporter GLUT1, and GLUT1 levels in the plasma membrane and the lipid micro-domain fraction (a specialized plasma membrane domain enriched in sphingolipids). Microarray analysis showed that ARAP2 knockdown altered expression of genes involved in sphingolipid metabolism. Because sphingolipids are known to play a key role in cell signaling, we performed lipidomics to further investigate the relationship between ARAP2 and sphingolipids and potentially identify a link with glucose uptake. We found that ARAP2 knockdown increased glucosylceramide and lactosylceramide levels without affecting ceramide levels, and thus speculated that the rate-limiting enzyme in glycosphingolipid synthesis, namely glucosylceramide synthase (GCS), could be modified by ARAP2. In agreement with our hypothesis, we showed that the activity of GCS was increased by ARAP2 knockdown and reduced by ARAP2 overexpression. Furthermore, pharmacological inhibition of GCS resulted in increases in basal glucose uptake, total GLUT1 levels, triglyceride biosynthesis from glucose, and lipid droplet formation, indicating that the effects of GCS inhibition are the opposite to those resulting from ARAP2 knockdown. Taken together, our data suggest that ARAP2 promotes lipid droplet formation by modifying sphingolipid metabolism through GCS.     

Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7

Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species. In mammalian cells, there exist proteins specifically localize in lipid droplets. However, the protein profile in the droplet remains yet to be clarified. In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques. Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction. The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11). Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins. When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction. In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP. These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction.     

An RDH‐Plin2 axis modulates lipid droplet size by antagonizing Bmm lipase

The size of lipid droplets varies greatly in vivo and is determined by both intrinsic and extrinsic factors. From an RNAi screen in Drosophila, we found that knocking down subunits of COP9 signalosome (CSN) results in enlarged lipid droplets under high‐fat, but not normal, conditions. We identified CG2064, a retinol dehydrogenase (RDH) homolog, as the proteasomal degradation target of CSN in regulating lipid droplet size. RDH/CG2064 interacts with the lipid droplet‐resident protein Plin2 and the RDH/CG2064‐Plin2 axis acts to reduce the overall level and lipid droplet localization of Bmm/ATGL lipase. This axis is important for larval survival under prolonged starvation. Thus, we discovered an RDH‐Plin2 axis modulates lipid droplet size.     

Retinyl esters form lipid droplets independently of triacylglycerol and seipin

Lipid droplets store neutral lipids, primarily triacylglycerol and steryl esters. Seipin plays a role in lipid droplet biogenesis and is thought to determine the site of lipid droplet biogenesis and the size of newly formed lipid droplets. Here we show a seipin-independent pathway of lipid droplet biogenesis. In silico and in vitro experiments reveal that retinyl esters have the intrinsic propensity to sequester and nucleate in lipid bilayers. Production of retinyl esters in mammalian and yeast cells that do not normally produce retinyl esters causes the formation of lipid droplets, even in a yeast strain that produces only retinyl esters and no other neutral lipids. Seipin does not determine the size or biogenesis site of lipid droplets composed of only retinyl esters or steryl esters. These findings indicate that the role of seipin in lipid droplet biogenesis depends on the type of neutral lipid stored in forming droplets.     

Label-free imaging of lipid-droplet intracellular motion in early Drosophila embryos using femtosecond-stimulated Raman loss microscopy

Lipid droplets are complex organelles that exhibit highly dynamic behavior in early Drosophila embryo development. Imaging lipid droplet motion provides a robust platform for the investigation of shuttling by kinesin and dynein motors, but methods for imaging are either destructive or deficient in resolution and penetration to study large populations of droplets in an individual embryo. Here we report real-time imaging and quantification of droplet motion in live embryos using a recently developed technique termed "femtosecond-stimulated Raman loss" microscopy. We captured long-duration time-lapse images of the developing embryo, tracked single droplet motion within large populations of droplets, and measured the velocity and turning frequency of each particle at different apical-to-basal depths and stages of development. To determine whether the quantities for speed and turning rate measured for individual droplets are sufficient to predict the population distributions of droplet density, we simulated droplet motion using a velocity-jump model. This model yielded droplet density distributions that agreed well with experimental observations without any model optimization or unknown parameter estimation, demonstrating the sufficiency of a velocity-jump process for droplet trafficking dynamics in blastoderm embryos.     

Stability of the oil-in-water type triacylglycerol emulsions

Lipid emulsions with saturated triacylglycerols (TAGs) with 4 to 10 carbons in each acyl chain were prepared to study how the oil component alters the stability of the lipid emulsions when phosphatidylcholines were used as emulsifiers. The average droplet size of the emulsions became smaller as the chain length of the TAG increased. For a given oil, emulsion with smaller droplets was formed with an emulsifier having higher HLB value. The influence of HLB values on the droplet size was biggest for the tributyrin (C4) emulsions. For the tricaprylin (C8) emulsions, droplet size was identical at given emulsifier concentrations regardless of HLB values. The HLB value and the concentration of the emulsifiers also affect the droplet size of the emulsions. The emulsions with smaller average droplet size were more stable than with bigger size for 20 days. The oil and water (o/w) interfacial tension is inversely proportional to the initial droplet size of the emulsion.     

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.