Lipid droplets lighting up: Insights from live microscopy

Lipid droplets emerge as important intracellular organelles relevant for lipid homeostasis and the pathophysiology of metabolic diseases. Here, we present a personal view on the current knowledge about the biogenesis of mammalian cytoplasmic lipid droplets, with a focus on microscopy and especially live imaging. We also discuss difficulties related to the lipid droplet proteome, contentious views on lipid droplet growth, and last but not least the evidence for the heterogeneity of lipid droplets within a single cell. We conclude with an outline of the most important future challenges.     

脂滴——细胞脂类代谢的细胞器

脂滴是细胞内中性脂贮存的主要场所,由极性单磷脂层包裹疏水核心组成。近年来的蛋白质组学研究表明,脂滴表面还存在着许多功能蛋白,进一步揭示了脂滴可能参与细胞内物质的代谢和转运,以及细胞信号传导等过程,是一个活动旺盛的多功能细胞器。实验结果还证明,脂滴不但是甘油三酯贮存和分解、花生四烯酸代谢和前列腺素合成的主要场所,脂滴还具有合成甘油三酯和磷酯的功能。由此可见,脂滴可能是细胞内参与脂类合成代谢的细胞器。     

Bodipy-C12在癌细胞过氧化物酶体中富集

目的:研究并比较Bodipy标记的月桂酸(Bodipy-C12)在癌细胞与正常细胞中的亚细胞定位。方法:在多种癌细胞和正常细胞的培养基中加入Bodipy-C12(1μg/m L),利用共聚焦显微镜活细胞定时间隔拍摄,结合不同细胞器的分子标记蛋白,观察Bodipy-C12五分钟内在不同细胞器中的定位。结果:在人肝癌细胞系HepG2细胞中,Bodpy-C12信号不仅仅存在于线粒体和脂滴,同时富集于过氧化物酶体中。我们分别采用Pex3-GFP、Pex14-GFP、GFP-Pex16、GFP-SKL和GFP-Pmp34等特异性定位的过氧化物酶体蛋白,确认Bodpy-C12信号富集于过氧化物酶体。此外,过氧化物酶体中Bodpy-C12信号的富集发生在更多的癌细胞系中,例如结肠癌细胞HCT116和乳腺癌细胞MCF7。不同的是,在正常细胞系如3T3-L1,NRK和COS7中,Bodipy-C12信号存在于线粒体和脂滴中,但未在过氧化物酶体中检测到。结论:Bodipy-C12信号存在于正常细胞和癌细胞的脂滴和线粒体中,且其在癌细胞过氧化物酶体中富集,而不存在于正常细胞的过氧化物酶体中,预示癌细胞中过氧化物酶体脂代谢的差异。     

The Retinol Dehydrogenase Rdh10 Localizes to Lipid Droplets during Acyl Ester Biosynthesis

Rdh10 catalyzes the first step of all-trans-retinoic acid biogenesis physiologically, conversion of retinol into retinal. We show that Rdh10 associates predominantly with mitochondria/mitochondrial-associated membrane (MAM) in the absence of lipid droplet biosynthesis, but also locates with lipid droplets during acyl ester biosynthesis. Targeting to lipid droplets requires the 32 N-terminal residues, which include a hydrophobic region followed by a net positive charge. Targeting to mitochondria/MAM and/or the stability of Rdh10 require both the N-terminal and the 48 C-terminal hydrophobic residues. Rdh10 behaves similarly to cellular retinol-binding protein, type 1, which also localizes to mitochondria/MAM before lipid droplet synthesis, and associates with lipid droplets during acyl ester synthesis (Jiang, W., and Napoli, J. L. (2012) Biochem. Biophys. Acta 1820, 859–8692). LRAT, an ER protein, also associates with lipid droplets upon acyl ester biosynthesis. Colocalization of Rdh10, Crbp1, and LRAT on lipid droplets suggests a metabolon that mediates retinol homeostasis.     

Interactions of perilipin-5 (Plin5) with adipose triglyceride lipase

Members of the perilipin family of lipid droplet scaffold proteins are thought to play important roles in tissue-specific regulation of triglyceride metabolism, but the mechanisms involved are not fully understood. Present results indicate that adipose triglyceride lipase (Atgl) interacts with perilipin-5 (Plin5) but not perilipin-1 (Plin1). Protein interaction assays in live cells and in situ binding experiments showed that Atgl and its protein activator, α-β-hydrolase domain-containing 5 (Abhd5), each bind Plin5. Surprisingly, competition experiments indicated that individual Plin5 molecules bind Atgl or Abhd5 but not both simultaneously. Thus, the ability of Plin5 to concentrate these proteins at droplet surfaces involves binding to different Plin5 molecules, possibly in an oligomeric complex. The association of Plin5-Abhd5 complexes on lipid droplet surfaces was more stable than Plin5-Atgl complexes, and oleic acid treatment selectively promoted the interaction of Plin5 and Abhd5. Analysis of chimeric and mutant perilipin proteins demonstrated that amino acids 200-463 are necessary and sufficient to bind both Atgl and Abhd5 and that the C-terminal 64 amino acids of Plin5 are critical for the differential binding of Atgl to Plin5 and Plin1. Mutant Plin5 that binds Abhd5 but not Atgl was defective in preventing neutral lipid accumulation compared with wild type Plin5, indicating that the ability of Plin5 to concentrate these proteins on lipid droplets is critical to functional Atgl activity in cells.     

Lipid droplet formation is dispensable for endoplasmic reticulum-associated degradation

Proteins that fail to fold or assemble in the endoplasmic reticulum (ER) are destroyed by cytoplasmic proteasomes through a process known as ER-associated degradation. Substrates of this pathway are initially sequestered within the ER lumen and must therefore be dislocated across the ER membrane to be degraded. It has been proposed that generation of bicellar structures during lipid droplet formation may provide an "escape hatch" through which misfolded proteins, toxins, and viruses can exit the ER. We have directly tested this hypothesis by exploiting yeast strains defective in lipid droplet formation. Our data demonstrate that lipid droplet formation is dispensable for the dislocation of a plant toxin and the degradation of both soluble and integral membrane glycoproteins.     

脂滴与线粒体相互作用的研究进展

肥胖和多种代谢类疾病的发生有着密切的关系,而导致肥胖的脂肪多以中性脂的形式储存于细胞的一种细胞器——脂滴中。越来越多的研究表明,脂滴能够和其它细胞器发生相互作用,而它和线粒体的相互作用可能与Ⅱ型糖尿病的形成密切相关:非正常的脂滴和线粒体的相互作用有可能是导致细胞胰岛素抵抗的重要原因。我们通过对脂滴表面蛋白质组学、脂滴与线粒体的空间位置,以及相关蛋白等研究的总结,结合本实验室的研究结果,对脂滴与线粒体相互作用的物质基础及可能方式、受骨骼肌有氧运动的影响,及其与骨骼肌胰岛素抵抗发生的关系等,进行了讨论。     

PAT proteins,an ancient family of lipid droplet proteins that regulate cellular lipid stores

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3–12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.     

Nitric oxide-independent lipid metabolism in RAW 264.7 macrophages loaded with oleic acid

The role of nitric oxide (NO) in the regulation of lipogenesis and lipolysis in RAW 264.7 macrophages loaded with oleic acid (OA) was investigated in this paper. Magnolol stimulated full lipolysis without affecting NO levels. Both inhibition and elevation of NO production in OA-loaded macrophages did not induce lipolysis. Besides, lipopolysaccharide (LPS)-induced increased accumulation of lipid droplets was not reduced by down-regulation of NO levels. Moreover, incubation of macrophages with sodium nitroprusside (SNP), an NO donor, stimulated significant NO production without altering the lipid droplet accumulation. All these results clearly demonstrate that NO is not involved in the modulation of lipid metabolism in macrophages loaded with OA.     

PAT蛋白抑制饥饿诱导的脂滴快速融合

目的:脂滴快速融合是增大脂滴直径的方式之一,但其研究相对少。本研究旨在建立脂滴快速融合的细胞模型,以便对其进行深入的生物学研究。方法:本研究使用大鼠肾成纤维细胞系NRK和小鼠前脂肪细胞系3T3-L1两种细胞系,先用油酸诱导细胞内产生大量脂滴,再使用饥饿缓冲液培养细胞,利用显微镜实时观测技术跟踪脂滴动态变化,建立脂滴快速融合的模型。而后在此模型中,加入自噬抑制剂或者以过表达CCT为阳性对照,过表达PAT蛋白(PLIN1、ADRP和TIP47),来探究它们在调控脂滴快速融合方面的功能。结果:饥饿缓冲液处理约3小时可诱导细胞发生脂滴快速融合,其融合速率很快,从脂滴接触到融合完成可发生在20秒内,显然不同于CIDE蛋白调控的缓慢脂滴融合过程。自噬抑制剂可以抑制自噬,但是并没有显著影响脂滴快速融合,说明饥饿诱导的脂滴快速融合不依赖于自噬。另发现,与过表达GFP相比,过表达定位于脂滴的GFP-CCT、GFP-PLIN1、GFP-ADRP或GFP-TIP47均能显著性抑制快速融合导致的脂滴变大的现象。结论:本研究建立了饥饿缓冲液诱导脂滴发生快速融合的细胞模型,并证明PAT蛋白(PLIN1、ADRP、TIP47)能抑制脂滴快速融合。     

Human frame shift mutations affecting the carboxyl terminus of perilipin increase lipolysis by failing to sequester the adipose triglyceride lipase (ATGL) coactivator AB-hydrolase-containing 5 (ABHD5)

Perilipin (PLIN1) is a constitutive adipocyte lipid droplet coat protein. N-terminal amphipathic helices and central hydrophobic stretches are thought to anchor it on the lipid droplet, where it appears to function as a scaffold protein regulating lipase activity. We recently identified two different C-terminal PLIN1 frame shift mutations (Leu-404fs and Val-398fs) in patients with a novel subtype of partial lipodystrophy, hypertriglyceridemia, severe insulin resistance, and type 2 diabetes (Gandotra, S., Le Dour, C., Bottomley, W., Cervera, P., Giral, P., Reznik, Y., Charpentier, G., Auclair, M., Delépine, M., Barroso, I., Semple, R. K., Lathrop, M., Lascols, O., Capeau, J., O'Rahilly, S., Magré, J., Savage, D. B., and Vigouroux, C. (2011) N. Engl. J. Med. 364, 740-748.) When overexpressed in preadipocytes, both mutants fail to inhibit basal lipolysis. Here we used bimolecular fluorescence complementation assays to show that the mutants fail to bind ABHD5, permitting its constitutive coactivation of ATGL, resulting in increased basal lipolysis. siRNA-mediated knockdown of either ABHD5 or ATGL expression in the stably transfected cells expressing mutant PLIN1 reduced basal lipolysis. These insights from naturally occurring human variants suggest that the C terminus sequesters ABHD5 and thus inhibits basal ATGL activity. The data also suggest that pharmacological inhibition of ATGL could have therapeutic potential in patients with this rare but metabolically serious disorder.     

Dynamic modification of sphingomyelin in lipid microdomains controls development of obesity, fatty liver, and type 2 diabetes

Lipid microdomains or caveolae, small invaginations of plasma membrane, have emerged as important elements for lipid uptake and glucose homeostasis. Sphingomyelin (SM) is one of the major phospholipids of the lipid microdomains. In this study, we investigated the physiological function of sphingomyelin synthase 2 (SMS2) using SMS2 knock-out mice, and we found that SMS2 deficiency prevents high fat diet-induced obesity and insulin resistance. Interestingly, in the liver of SMS2 knock-out mice, large and mature lipid droplets were scarcely observed. Treatment with siRNA for SMS2 also decreased the large lipid droplets in HepG2 cells. Additionally, the siRNA of SMS2 decreased the accumulation of triglyceride in liver of leptin-deficient (ob/ob) mice, strongly suggesting that SMS2 is involved in lipid droplet formation. Furthermore, we found that SMS2 exists in lipid microdomains and partially associates with the fatty acid transporter CD36/FAT and with caveolin 1, a scaffolding protein of caveolae. Because CD36/FAT and caveolin 1 exist in lipid microdomains and are coordinately involved in lipid droplet formation, SMS2 is implicated in the modulation of the SM in lipid microdomains, resulting in the regulation of CD36/FAT and caveolae. Here, we established new cell lines, in which we can completely distinguish SMS2 activity from SMS1 activity, and we demonstrated that SMS2 could convert ceramide produced in the outer leaflet of the plasma membrane into SM. Our findings demonstrate the novel and dynamic regulation of lipid microdomains via conformational changes in lipids on the plasma membrane by SMS2, which is responsible for obesity and type 2 diabetes.     

Endolysosomal phospholipidosis and cytosolic lipid droplet storage and release in macrophages

This review summarizes the current knowledge of endolysosomal and cytoplasmic lipid storage in macrophages induced by oxidized LDL (Ox-LDL), enzymatically degraded LDL (E-LDL) and other atherogenic lipoprotein modifications, and their relation to the adapter protein 3 (AP-3) dependent ABCA1 and ABCG1 cellular lipid efflux pathways. We compare endolysosomal lipid storage caused either through drug induced phospholipidosis, inheritable endolysosomal and cytosolic lipid storage disorders and Ox-LDL or E-LDL induced phagosomal uptake and cytosolic lipid droplet storage in macrophages. Ox-LDL is resistant to rapid endolysosomal hydrolysis and is trapped within the endolysosomal compartment generating lamellar bodies which resemble the characteristics of phospholipidosis. Various inherited lysosomal storage diseases including sphingolipidosis, glycosphingolipidosis and cholesterylester storage diseases also present a phospholipidosis phenotype. In contrast E-LDL resembling coreless unesterified cholesterol enriched LDL-particles, with a multilamellar, liposome-like structure, lead to rapid phagosomal degradation and cytosolic lipid droplet accumulation. As a consequence the uptake of E-LDL through type I and type II phagocytosis leads to increased lipid droplet formation and moderate upregulation of ABCA1 and ABCG1 while uptake of Ox-LDL leads to a rapid expansion of the lysosomal compartment and a pronounced upregulation of the ABCA1/ABCG1/AP-3 lipid efflux pathway.     

Filling up adipocytes with lipids. Lessons from caveolin-1 deficiency

Caveolins are primarily known as the main constituents of the protein coat of caveolae invaginations at the plasma membrane. They have also been found at the surface of intracellular lipid droplets but their function in this lipid storage organelle remains poorly understood. This paper reviews recent studies in adipocytes, the specialized cell type for fatty acid storage, which suggest a role for caveolins in the formation, maintenance or mobilization of lipid droplet stores. These new functions emerged from studies of fat cells in which caveolin expression was invalidated, highlighting the metabolic phenotype of caveolin-deficient mice or human patients who develop progressive lipoatrophy.     

The stoichiometry of protein phosphorylation in adipocyte lipid droplets: Analysis by N‐terminal isotope tagging and enzymatic dephosphorylation

Most phosphoproteomic studies to date have been limited to the identification of phosphoproteins and their phosphorylation sites, and have not assessed the stoichiometry of protein phosphorylation, a critical parameter reflecting the dynamic equilibrium between phosphorylated and non‐phosphorylated pools of proteins. Here, we used a method for measuring phosphorylation stoichiometry through isotope tagging and enzymatic dephosphorylation of tryptic peptides. Using this method, protein digests are divided into two equal aliquots that are modified with either light or heavy isotope tags. One aliquot is dephosphorylated by alkaline phosphatase. Finally, the peptide mixtures are recombined and LC‐MS/MS analysis is performed. With this method, we studied adipocytes of mice stimulated with CL316,243, a β‐3 adrenergic agonist known to induce lipolysis and marked phosphorylation changes in proteins of the lipid droplet surface. In lipid droplet preparations, CL316,243 administration increased phosphorylation of proteins related to regulation of signaling, metabolism and intracellular trafficking in white adipose tissue, including hormone‐sensitive lipase which was 80% phosphorylated at the previously reported site, Ser‐559, and the lipid surface protein perilipin, which was phosphorylated by ～60 and ～40% at previously unreported sites, Ser‐410 and Ser‐460.     

Immunocytochemical studies on lipid droplet-surface proteins in adrenal cells

Perilipin and ADRP, located on the surface of intracellular lipid droplets, are proposed to be involved in adipocyte lipid metabolism. The aim of the present study was to investigate the effect of PKA and PKC activities on the distribution of perilipin and ADRP in primary cultured adrenal cells, and the role of ERK in PMA- and calphostin C-induced steroidogenesis. Immunofluorescence staining indicated that in addition to p160, a capsular protein of steroidogenic lipid droplets, perilipin and ADRP were localized on the lipid droplet surface. Stimuli such as activation of PKA by db cAMP or inhibition of PKC by calphostin C, which increase corticosterone synthesis in various magnitudes, caused detachment of p160 and perilipin, but not ADRP, from the lipid droplet surface. Activation of PKC by PMA induced increase in corticosterone synthesis, however, it did not affect the distribution of perilipin, p160, or ADRP on the lipid droplet surface, suggesting the presence of mechanisms for promoting sterodiogensis other than causing detachment of lipid droplet surface proteins. We further demonstrated that ERK pathway was involved in PMA-induced steroidogenesis, since PD98059, specific inhibitor of MEK, blocked the increases in steroidogenesis and phosphorylation of ERK caused by PMA, but not by cAMP-PKA. These data indicate that p160, perilipin, and ADRP were all located on the lipid droplet surface in rat adrenal cells. On the basis of its non-responsiveness to lipolytic stimulation, ADRP may be a structural protein of the lipid droplet surface, whereas their immediate response to lipolytic stimuli suggest that perilipin and p160 are functional proteins. PKC regulates adrenal steroidogenesis through ERK cascade, whereas PKA pathway does not involve ERK.     

Modifications of the C terminus Affect Functionality and Stability of Yeast Triacylglycerol Lipase Tgl3p

Lipid droplets are specific organelles for the storage of triacylglycerols and steryl esters. They are surrounded by a phospholipid monolayer with a small but specific set of proteins embedded. Assembly and insertion of proteins into this surface membrane is an intriguing question of lipid droplet biology. To address this question we studied the topology of Tgl3p, the major triacylglycerol lipase of the yeast Saccharomyces cerevisiae, on lipid droplets. Employing the method of limited proteolysis of lipid droplet surface proteins, we found that the C terminus of Tgl3p faces the inside of the organelle, whereas the N terminus is exposed at the cytosolic side of lipid droplets. Detailed analysis of the C terminus revealed a stretch of seven amino acids that are critical for protein stability and functionality. The negative charge of two aspartate residues within this stretch is crucial for lipase activity of Tgl3p. A portion of Tgl3p, which is located to the endoplasmic reticulum, exhibits a different topology. In the phospholipid bilayer of the endoplasmic reticulum the C terminus faces the cytosol, which results in instability of the protein. Thus, the topology of Tgl3p is important for its function and strongly dependent on the membrane environment.     

S-Adenosyl methionine synthetase 1 limits fat storage in Caenorhabditis elegans

Cytosolic lipid droplets are versatile, evolutionarily conserved organelles that are important for the storage and utilization of lipids in almost all cell types. To obtain insight into the physiological importance of lipid droplet size, we isolated and characterized a new S-adenosyl methionine synthetase 1 (SAMS-1)-deficient Caenorhabditis elegans mutant, which have enlarged lipid droplets throughout its life cycle. We found that the sams-1 mutant showed a markedly reduced body size and progeny number; impaired synthesis of phosphatidylcholine, a major membrane phospholipid; and elevated expression of key lipogenic genes, such as dgat-2, resulting in the accumulation of triacylglyceride in fewer, but larger, lipid droplets. The sams-1 mutant store more than 50 % (wild type: 10 %) of its intestinal fat in large lipid droplets, ≥10 μm³ in size. In response to starvation, SAMS-1 deficiency causes reduced depletion of a subset of lipid droplets located in the anterior intestine. Given the importance of liberation of fatty acids from lipid droplets, we propose that the physiological function of SAMS-1, a highly conserved enzyme involved in one-carbon metabolism, is the limitation of fat storage to ensure proper growth and reproduction.

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