Nascent Lipid Droplets Research Articles

Changes in liver perilipin gene expression during an oral lipid load in obese OLETF rats

Non‐alcoholic fatty liver disease (NAFLD) is one of the most prevalent liver maladies in the US. NAFLD is associated with obesity, hyperlipidemia, hypertension, insulin resistance, and diabetes; all traits linked to metabolic syndrome, affecting 20% of the US population. Inappropriate hepatic lipid accumulation is associated with metabolic syndrome. Intracellular lipid storage is regulated by lipid droplets (LDs) that are active components of the cell. LDs have surface proteins of which the most studied belong to the PAT family (Perilipin1–5), which participate in storage and lipid metabolism. To evaluate the effect that a high‐lipid load on the expression and dynamics of hepatic perilipins during early onset T2D, an oral fat tolerance test (OFTT) was performed in 28 wk‐old rats (n=5–6/group): (1) healthy, strain control LETO, and (2) obese, diabetic OLETF. Liver and blood were collected before (T0) and after (T1, T2, T4, and T8 hours) the lipid challenge. An acute increase in serum TG was observed at T2 (p= < 0.0001) and was maintained over the next 6 hours in OLETF whereas in LETO it decreased after the T2 mark. The mRNA expression of Plin2 increased 2.5‐fold in OLETF at T2 compared to LETO, and increased 2.5‐fold at T8 in LETO compared to baseline. Plin3 remained higher (p= 0.0077) in LETO compared to OLETF at T8. Plin4 did not change significantly in response to the lipid challenge between groups. Plin5 increased 1.8‐fold in LETO at T8 compared to baseline. These changes help explain the contributions of LDs to the regulation of hepatic lipids during both normal and diabetic conditions in response to a high lipid challenge similar to the consumption of a high‐fat meal. As Plin2 and 3 have been described as structural LD‐associated proteins, being present mostly in nascent LD, suggesting that a high fat load induces an acute induction of hepatocyte lipid storage. The increase in Plin5 suggests that lipid metabolism pathways are being activated, as Plin5 has been related with mitochondrial FA metabolism. The differential expression patterns of perilipins in response to an acute lipid challenge illustrates the timing of the cellular changes during the development of hepatic steatosis, providing useful insights into future development of treatment by targeting the PAT family of proteins.Support or Funding InformationThis work was supported by NIH's MHRT program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cerebellar ataxia disease-associated Snx14 promotes lipid droplet growth at ER-droplet contacts.

Lipid droplets (LDs) are nutrient reservoirs used by cells to maintain homeostasis. Nascent droplets form on the endoplasmic reticulum (ER) and grow following an influx of exogenous fatty acids (FAs). The budding of LDs requires extensive ER-LD crosstalk, but how this is regulated remains poorly understood. Here, we show that sorting nexin protein Snx14, an ER-resident protein associated with the cerebellar ataxia SCAR20, localizes to ER-LD contacts following FA treatment, where it promotes LD maturation. Using proximity-based APEX technology and topological dissection, we show that Snx14 accumulates specifically at ER-LD contacts independently of Seipin, where it remains ER-anchored and binds LDs in trans. SNX14KO cells exhibit perturbed LD morphology, whereas Snx14 overexpression promotes LD biogenesis and extends ER-LD contacts. Multi-time point imaging reveals that Snx14 is recruited to ER microdomains containing the fatty acyl-CoA ligase ACSL3, where nascent LDs bud. We propose that Snx14 is a novel marker for ER-LD contacts and regulates FA-stimulated LD growth.

Lipid droplet and peroxisome biogenesis occur at the same ER subdomains

Nascent lipid droplet (LD) formation occurs in the endoplasmic reticulum (ER) membrane but it is not known how sites of biogenesis are determined. We previously identified ER domains in S. cerevisiae containing the reticulon homology domain (RHD) protein Pex30 that are regions where preperoxisomal vesicles (PPVs) form. Here, we show that Pex30 domains are also sites where most nascent LDs form. Mature LDs usually remain associated with Pex30 subdomains, and the same Pex30 subdomain can simultaneously associate with a LD and a PPV or peroxisome. We find that in higher eukaryotes multiple C2 domain containing transmembrane protein (MCTP2) is similar to Pex30: it contains an RHD and resides in ER domains where most nascent LD biogenesis occurs and that often associate with peroxisomes. Together, these findings indicate that most LDs and PPVs form and remain associated with conserved ER subdomains, and suggest a link between LD and peroxisome biogenesis.

Diacylglycerol acyltransferase-2 contains a c-terminal sequence that interacts with lipid droplets

Diacylglycerol acyltranferase-2 (DGAT2) is a resident protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerol. When lipid droplet formation is stimulated by incubating cells with fatty acids, DGAT2 becomes concentrated around the surface of cytosolic lipid droplets. Using confocal microscopy and directed mutagenesis, we have identified a 17-amino acid sequence in the C-terminal region of DGAT2 that is necessary and sufficient for targeting DGAT2 to lipid droplets. When this region was deleted, DGAT2 remained in the ER and did not target to lipid droplets. Fusing this sequence to mCherry directed the fluorescent reporter to lipid droplets. Similarly, when the corresponding region of monoacylglycerol acyltransferase-2 (MGAT2) was replaced with this sequence, MGAT2 was also targeted to lipid droplets. Lastly, we demonstrated that DGAT2 in ER membranes is continuous with lipid droplets. We propose a new model whereby DGAT2 remains in the ER during lipid droplet formation via it's transmembrane domains and interacts with nascent lipid droplets via its C-terminal lipid droplet interacting domain as they expand.

Syntaxin 17 promotes lipid droplet formation by regulating the distribution of acyl-CoA synthetase 3

Lipid droplets (LDs) are ubiquitous organelles that contain neutral lipids and are surrounded by a phospholipid monolayer. How proteins specifically localize to the phospholipid monolayer of the LD surface has been a matter of extensive investigations. In the present study, we show that syntaxin 17 (Stx17), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein whose expression in the liver is regulated by diet, participates in LD biogenesis by regulating the distribution of acyl-CoA synthetase (ACSL)3, a key enzyme for LD biogenesis that redistributes from the endoplasmic reticulum (ER) to LDs during LD formation. Stx17 interacts with ACSL3, but not with LD formation-unrelated ACSL1 or ACSL4, through its SNARE domain. The interaction occurs at the ER-mitochondria interface and depends on the active site occupancy of ACSL3. Depletion of Stx17 impairs ACSL3 redistribution to nascent LDs. The defect in LD maturation due to Stx17 knockdown can be compensated for by ACSL3 overexpression, suggesting that Stx17 increases the efficiency of ACSL3 redistribution to LDs. Moreover, we show that the interaction between Stx17 and ACSL3 during LD maturation may be regulated by synaptosomal-associated protein of 23 kDa.

The role of caveolin-1 in lipid droplets and their biogenesis

We address unresolved questions of the energetics and mechanism of lipid droplet (LD) biogenesis, and of the role of caveolins in the endoplasmic reticulum (ER) and in mature LDs. LDs are eukaryotic repositories of neutral lipids, which are believed to be synthesised in the ER. We investigate the effects of a curvature-inducing protein, caveolin-1, on the formation and structure of a spontaneously aggregated triolein (TO) lipid lens in a flat lipid bilayer using molecular dynamics (MD) simulations. A truncated form of caveolin-1 (Cav1) localises on the interface between the spontaneously formed TO aggregate and the bulk bilayer, and thins the bilayer at the edge of the aggregate, which may contribute to lowering the energy barrier for pinching off the aggregate from the host bilayer. Simulations of fully mature LDs do not conclusively establish the optimal localisation of Cav1 in LDs, but when Cav1 is in the LD core, the distribution of both neutral lipids in the LD core, and of phospholipids on the engulfing monolayer are altered significantly. Our simulations provide an unprecedented molecular description of the distribution and dynamics of various lipid species in both mature LDs and in the nascent LD inside the bilayer.

Seipin is required for converting nascent to mature lipid droplets.

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.

Lipid Droplets Form from Distinct Regions of the Cell in the Fission Yeast Schizosaccharomyces pombe.

Eukaryotic cells store cholesterol/sterol esters (SEs) and triacylglycerols (TAGs) in lipid droplets, which form from the contiguous endoplasmic reticulum (ER) network. However, it is not known if droplets preferentially form from certain regions of the ER over others. Here, we used fission yeast Schizosaccharomyces pombe cells where the nuclear and cortical/peripheral ER domains are distinguishable by light microscopy to show that SE-enriched lipid droplets form away from the nucleus at the cell tips, whereas TAG-enriched lipid droplets form around the nucleus. Sterols localize to the regions of the cells where droplets enriched in SEs are observed. TAG droplet formation around the nucleus appears to be a strong function of diacylglycerol (DAG) homeostasis with Cpt1p, which coverts DAG into phosphatidylcholine and phosphatidylethanolamine localized exclusively to the nuclear ER. Also, Dgk1p, which converts DAG into phosphatidic acid localized strongly to the nuclear ER over the cortical/peripheral ER. We also show that TAG more readily translocates from the ER to lipid droplets than do SEs. The results augment the standard lipid droplet formation model, which has SEs and TAGs flowing into the same nascent lipid droplet regardless of its biogenesis point in the cell.

A Conserved Family of Proteins Facilitates Nascent Lipid Droplet Budding from the ER

Lipid droplets (LDs) are found in almost all cells and play central roles in energy and lipid metabolism; it is also becoming clear that LDs have additional functions, including important roles in protein degradation, ER stress, and viral replication. Unlike all other membrane‐bound organelles, LDs are surrounded by a phospholipid monolayer rather than a bilayer. How this unique structure is formed has remained an important unsolved mystery for many years. Quite a number of models of LD biogenesis have been proposed but the simplest and arguably most popular is one that has sometimes been termed the “lens” model. Neutral lipids, triacylglycerols (TAG) and steryl esters (SE) which form the core of all LDs, are synthesized in the ER and it is thought that as these lipids accumulate in the ER bilayer they form a lens (or blister) in the ER that can grow and bud. We visualized nascent LD formation in yeast using electron microscopy (EM)‐tomography and found that the lens model is correct. Lens of neutral lipid approximately 30–60 nm wide were found between the two leaflets of the ER bilayer. This is the first time the beginnings of LD biogenesis have been visualized and resolves a long‐standing question in LD biogenesis. We next asked whether proteins are necessary to facilitate nascent LD budding from the ER and focused on a conserved family of proteins, called fat storage‐inducing transmembrane (FIT) proteins. Yeast has two FITs (called Fit2a and Fit2b) and we found that in cells missing both most LDs are wrapped by the ER membrane and that this is caused by LD budding into the ER lumen rather than the cytosol. The wrapping membrane is not derived from the phagophore or related to autophagy since we found that wrapping still occurred in mutants lacking the FIT proteins and various proteins required for autophagy. The role of FIT proteins in LD budding is conserved; knock down of FIT2 in 3T3 fibroblasts and in C.elegans caused LDs to become wrapped in the ER. Interestingly deletion of the single FIT protein in C. elegans is lethal, suggesting that LD budding is an essential process in this organism. Our findings indicate that nascent LDs form lenses in the ER and that FIT proteins are necessary to promote proper budding of LDs from the ER. The mechanism of how FIT proteins control the directionality of LD budding remains to be investigated.Support or Funding InformationThis work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases. Vineet Choudhary is supported by fellowship from Swiss National Science Foundation (Grant Nr; PA00P3_145358, P300P3_158454).

A conserved family of proteins facilitates nascent lipid droplet budding from the ER.

Lipid droplets (LDs) are found in all cells and play critical roles in lipid metabolism. De novo LD biogenesis occurs in the endoplasmic reticulum (ER) but is not well understood. We imaged early stages of LD biogenesis using electron microscopy and found that nascent LDs form lens-like structures that are in the ER membrane, raising the question of how these nascent LDs bud from the ER as they grow. We found that a conserved family of proteins, fat storage-inducing transmembrane (FIT) proteins, is required for proper budding of LDs from the ER. Elimination or reduction of FIT proteins in yeast and higher eukaryotes causes LDs to remain in the ER membrane. Deletion of the single FIT protein in Caenorhabditis elegans is lethal, suggesting that LD budding is an essential process in this organism. Our findings indicated that FIT proteins are necessary to promote budding of nascent LDs from the ER.

On the Formation of Lipid Droplets in Human Adipocytes: The Organization of the Perilipin–Vimentin Cortex

We report on the heterogeneity and diversity of lipid droplets (LDs) in early stages of adipogenesis by elucidating the cell and molecular biology of amphiphilic and cytoskeletal proteins regulating and stabilizing the generation of LDs in human adipose cells. A plethora of distinct and differently sized LDs was detected by a brief application of adipocyte differentiation medium and additional short treatment with oleic acid. Using these cells and highly specific antibodies for LD-binding proteins of the perilipin (PLIN) family, we could distinguish between endogenously derived LDs (endogenous LDs) positive for perilipin from exogenously induced LDs (exogenous LDs) positive for adipophilin, TIP47 and S3-12. Having optimized these stimulation conditions, we used early adipogenic differentiation stages to investigate small-sized LDs and concentrated on LD-protein associations with the intermediate-sized filament (IF) vimentin. This IF protein was described earlier to surround lipid globules, showing spherical, cage-like structures. Consequently - by biochemical methods, by immunofluorescence microscopy and by electron- and immunoelectron microscopy - various stages of emerging lipid globules were revealed with perilipin as linking protein between LDs and vimentin. For this LD-PLIN-Vimentin connection, a model is now proposed, suggesting an interaction of proteins via opposed charged amino acid domains respectively. In addition, multiple sheaths of smooth endoplasmic reticulum cisternae surrounding concentrically nascent LDs are shown. Based on our comprehensive localization studies we present and discuss a novel pathway for the LD formation.

Changes in the phospholipid fatty acid composition of the lipid droplet during the differentiation of 3T3-L1 adipocytes

Lipid droplets (LDs) are independent organelles in adipocytes that are composed of a lipid ester core surrounded by a phospholipid monolayer. The fatty acid composition of the phospholipid monolayer should determine the metabolism and dynamics of LDs. In this study, we examined the fatty acid composition of phospholipid monolayer in LDs during the differentiation of 3T3-L1 adipocytes. The levels of saturated fatty acids (SFAs), such as 16:0 and 18:0, in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of LDs decreased during differentiation. In contrast, the levels of monounsaturated fatty acids (MUFAs) such as 16:1n-7 and 18:1n-9 in PC and PE of LDs and the level of polyunsaturated fatty acids (PUFAs) such as 20:3n-6, 20:4n-6 and 22:6n-3 in PE of LDs increased during differentiation. These results suggest that the phospholipid monolayer in mature LDs is more fluid than that in nascent LDs. The fatty acid compositions of the LD monolayer were different from those of the microsome bilayer in the early stage of differentiation, but similar to those of the microsome bilayer in the late stage of differentiation. These data provide evidence that biophysical properties of the phospholipid monolayer in LDs change during adipocyte differentiation.

Influence of apolipoprotein A-V on the metabolic fate of triacylglycerol

Apolipoprotein (apo) A-V functions to modulate intracellular and extracellular triacylglycerol metabolism. The present review addresses molecular mechanisms underlying these effects. The relevance of apoA-V to human disease conditions is illustrated by the strong correlation between single nucleotide polymorphisms in APOA5, elevated plasma triacylglycerol and dyslipidemic disease. Despite undergoing processing for secretion from hepatocytes, a portion of apoA-V escapes this destiny and accumulates as a component of cytosolic lipid droplets. Expression of recombinant apoA-V in hepatocarcinoma cells results in increased lipid droplet size and number at the expense of triacylglycerol secretion.ApoA-V modulates atherosclerosis in hypercholesterolemic apoE null mice. ApoE null/human apoA-V transgenic mice had reduced levels of triacylglycerol and cholesterol in plasma along with decreased aortic lesion size. ApoA-V modulates triacylglycerol metabolic fate. Following its synthesis, apoA-V enters the endoplasmic reticulum and associates with membrane defects created by triacylglycerol accumulation. Association of apoA-V with endoplasmic reticulum membrane defects promotes nascent lipid droplets budding toward the cytosol. Despite its low concentration in plasma (∼150 ng/ml), apoA-V modulates lipoprotein metabolism by binding to glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1. This interaction effectively localizes triacylglycerol-rich lipoproteins in the vicinity of glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein1's other ligand, lipoprotein lipase.

The N-terminal region of acyl-CoA synthetase 3 is essential for both the localization on lipid droplets and the function in fatty acid uptake

Cytosolic lipid droplets (LDs) are storage organelles for neutral lipids derived from endogenous metabolism. Acyl-CoA synthetase family proteins are essential enzymes in this biosynthetic pathway, contributing activated fatty acids. Fluorescence microscopy showed that ACSL3 is localized to the endoplasmic reticulum (ER) and LDs, with the distribution dependent on the cell type and the supply of fatty acids. The N-terminus of ACSL3 was necessary and sufficient for targeting reporter proteins correctly, as demonstrated by subcellular fractionation and confocal microscopy. The N-terminal region of ACSL3 was also found to be functionally required for the enzyme activity. Selective permeabilization and in silico analysis suggest that ACSL3 assumes a hairpin membrane topology, with the N-terminal hydrophobic amino acids forming an amphipathic helix restricted to the cytosolic leaflet of the ER membrane. ACSL3 was effectively translocated from the ER to nascent LDs when neutral lipid synthesis was stimulated by the external addition of fatty acids. Cellular fatty acid uptake was increased by overexpression and reduced by RNA interference of ACSL3. In conclusion, the structural organization of ACSL3 allows the fast and efficient movement from the ER to emerging LDs. ACSL3 not only esterifies fatty acids with CoA but is also involved in the cellular uptake of fatty acids, presumably indirectly by metabolic trapping. The unique localization of the acyl-CoA synthetase ACSL3 on LDs suggests a function in the local synthesis of lipids.

A lipid-droplet-targeted O-GlcNAcase isoform is a key regulator of the proteasome

Protein-O-linked N-Acetyl-β-D-glucosaminidase (O-GlcNAcase, OGA; also known as hexosaminidase C) participates in a nutrient-sensing, hexosamine signaling pathway by removing O-linked N-acetylglucosamine (O-GlcNAc) from key target proteins. Perturbations in O-GlcNAc signaling have been linked to Alzheimer's disease, diabetes and cancer. Mammalian O-GlcNAcase exists as two major spliced isoforms differing only by the presence (OGA-L) or absence (OGA-S) of a histone-acetyltransferase domain. Here we demonstrate that OGA-S accumulates on the surface of nascent lipid droplets with perilipin-2; both of these proteins are stabilized by proteasome inhibition. We show that selective downregulation of OGA-S results in global proteasome inhibition and the striking accumulation of ubiquitinylated proteins. OGA-S knockdown increased levels of perilipin-2 and perilipin-3 suggesting that O-GlcNAc-dependent regulation of proteasomes might occur on the surface of lipid droplets. By locally activating proteasomes during maturation of the nascent lipid droplet, OGA-S could participate in an O-GlcNAc-dependent feedback loop regulating lipid droplet surface remodeling. Our findings therefore suggest a mechanistic link between hexosamine signaling and lipid droplet assembly and mobilization.

Molecular Mechanisms of Active Lipolysis in 3T3-L1 Adipocytes

Lipid droplets (LDs) of mammalian cells are coated with lipid-associated proteins (LAP) such as perilipin (PLIN), hormone sensitive lipase (HSL), and adipose triglyceride lipase (ATGL), which play important roles in regulating fat storage and mobilization, by interacting with each other. By using coherent anti-Stokes Raman Scattering (CARS) microscopy which visualizes LDs in living cells, we observed numerous micro-LDs (mLDs) emerging after lipolytic stimulation. However, the interplay of LAP and the physiological significance of mLDs are not fully understood. PURPOSE: In this study, we examined the localization, trafficking, and interactions of key proteins involved in lipolysis (i.e. LAP) as well as the physiological significance of mLDs during lipolysis in differentiated 3T3-L1 adipocytes. METHODS: The localization and trafficking of LAP during 2 hrs of cAMP-dependent PKA activation were examined by immunostaining. In addition, dynamic morphological changes of LDs and fatty acid (FA) turnover during lipolysis were obtained using multiplex CARS microspectroscopy, which is able to distinguish between deuterium-labeled FA and non-labeled FA. Lipolytic activity and the protein-protein interaction were biochemically analyzed. RESULTS: In the basal state, the localization of HSL was cytoplasmic, while that of PLIN was on large LDs. Upon lipolytic stimulation, HSL being redistributed to the surface of large LDs where being co-localized with PLIN. Furthermore, HSL and PLIN were co-localized at particulate structures, which are supposed to be mLDs. We found that these structures are derived from endoplasmic reticulum (ER) where nascent LDs are formed via triacylglycerol (TAG) synthesis, and perform active lipolysis. On the other hand, lipolytic stimulation did not alter the cytoplasmic distribution of ATGL. Following subcellular fractionation, we observed that ATGL and its co-activator CGI-58 were co-fractionated upon lipolytic stimulation in membrane fraction where organelles are enriched. Furthermore, immunoprecipitation analysis demonstrated that a small but significant amount of ATGL was co-precipitated with Myc-tagged CGI-58 in membrane fraction, in response to lipolytic stimulation. CONCLUSIONS: These results suggest that mLDs have important functions to enhance the efficiency of lipolysis, by increasing the total LD surface area where PLIN and HSL can interact. It is also possible that active lipolysis occurs by means of interplays of CGI-58 and ATGL, in organelles other than LDs.

Yolk nucleus – The complex assemblage of cytoskeleton and ER is a site of lipid droplet formation in spider oocytes

Oocytes (future egg cells) of various animal groups often contain complex organelle assemblages (Balbiani bodies, yolk nuclei). The molecular composition and function of Balbiani bodies, such as those found in the oocytes of Xenopus laevis, have been recently recognized. In contrast, the functional significance of more complex and highly ordered yolk nuclei has not been elucidated to date. In this report we describe the structure, cytochemical content and evolution of the yolk nucleus in the oocytes of a common spider, Clubiona sp. We show that the yolk nucleus is a spherical, rather compact and persistent cytoplasmic accumulation of several different organelles. It consists predominantly of a highly elaborate cytoskeletal scaffold of condensed filamentous actin and a dense meshwork of intermediate-sized filaments. The yolk nucleus also comprises cisterns of endoplasmic reticulum, mitochondria, lipid droplets and other organelles. Nascent lipid droplets are regularly found in the cortical regions of the yolk nucleus in association with the endoplasmic reticulum. Single lipid droplets become surrounded by filamentous cages formed by intermediate filaments. Coexistence of the forming lipid droplets with the endoplasmic reticulum in the cortical zone of the yolk nucleus and their later investment by intermediate-sized filamentous cages suggest that the yolk nucleus is the birthplace of lipid droplets.

Altered lipid droplet dynamics in hepatocytes lacking triacylglycerol hydrolase expression.

Lipid droplets (LDs) form from the endoplasmic reticulum (ER) and grow in size by obtaining triacylglycerols (TG). Triacylglycerol hydrolase (TGH), a lipase residing in the ER, is involved in the mobilization of TG stored in LDs for the secretion of very-low-density lipoproteins. In this study, we investigated TGH-mediated changes in cytosolic LD dynamics. We have found that TGH deficiency resulted in decreased size and increased number of LDs in hepatocytes. Using fluorescent fatty acid analogues to trace LD formation, we observed that TGH deficiency did not affect the formation of nascent LDs on the ER. However, the rate of lipid transfer into preformed LDs was significantly slower in the absence of TGH. Absence of TGH expression resulted in increased levels of membrane diacylglycerol and augmented phospholipid synthesis, which may be responsible for the delayed lipid transfer. Therefore, altered maturation (growth) rather than nascent formation (de novo synthesis) may be responsible for the observed morphological changes of LDs in TGH-deficient hepatocytes.

Targeting of acyl‐CoA synthetase 3 to lipid droplets

Lipid droplets emerge as dynamic organelles highly relevant for lipid homeostasis and the pathophysiology of metabolic diseases. We are using fatty acyl‐CoA synthetases (ACS), especially ACSL3 as a paradigm to investigate the formation and growth of lipid droplets. Out of eleven mammalian long chain ACS, ACSL3 is the only family member localizing specifically to lipid droplets and the ER. The distribution of ACSL3 between these two organelles depends on the cell type and whether exogenous fatty acids are being supplied. The N‐terminal region of ACSL3 is sufficient to direct reporter proteins to lipid droplets which we analysed by confocal microscopy. Mutational analysis demonstrated that only amino acids 12 to 51 are essential for targeting. ACSL3 is tightly membrane associated by an amphipathic helix contained within the N‐terminal sorting signal. During lipid droplet biogenesis, ACSL3 moves from the bilayer membrane of the ER to the phospholipid monolayer of nascent lipid droplets as we demonstrated by live microscopy. Overexpression of ACSL3 results not only in increased cellular enzyme activity but also in the enhanced uptake of exogenous fatty acids. This suggests that ACSL3 is functionally relevant for the regulation of fatty acid uptake. Moreover, we speculate that the specific localization of ACSL3 is important for the local biosynthesis of phospholipids at the surface of the lipid droplets.

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 beta-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 beta-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.