Lipid storage and lipophagy regulates ferroptosis

The synthesis, storage, and degradation of lipids are highly regulated processes. Impaired lipid metabolism is implicated in inflammation and cell death. Although ferroptosis is a recently described form of regulated cell death driven by lipid peroxidation, the impact of lipid droplets on ferroptosis remains unidentified. Here, we demonstrate that lipophagy, the autophagic degradation of intracellular lipid droplets, promotes RSL3-induced ferroptotic cell death in hepatocytes. Lipid droplet accumulation is increased at the early stage but decreased at the late stage of ferroptosis in mouse or human hepatocytes. Importantly, either genetically enhancing TPD52-dependent lipid storage or blocking ATG5-and RAB7A-dependent lipid degradation prevents RSL3-induced lipid peroxidation and subsequent ferroptosis in vitro and in vivo. These studies support an antioxidant role for lipid droplets in cell death and suggest novel strategies for the inhibition of ferroptosis by targeting the lipophagy pathway.     

Lipid storage in cultured articular chondrocytes due to prostanoid precursors and a prostanoid synthesis inhibitor

Lapine articular chondrocytes were subcultured in the presence or absence of the prostanoid precursors, arachidonic acid or dihomo-gamma-linolenic acid, and the cyclooxygenase inhibitor indomethacin. Lipid storage was studied microscopically using the Sudan black staining method. Control chondrocyte cultures showed a weakly positive staining reaction until confluence was reached, at which point the intra-cytoplasmic lipid content decreased. Both arachidonic acid and dihomo-gamma-linolenic acid at 100 mumol/l caused a marked increase in lipid storage which continued even after confluence was achieved. 1 mumol/l concentrations were indistinguishable from controls, whereas 10 mumol/l concentrations elicited a slight increase in lipid storage compared with controls. The prostaglandin cyclooxygenase inhibitor indomethacin did not affect chondrocyte lipid storage. However, administration of a prostanoid precursor in the presence of indomethacin caused a massive increase in intra-cytoplasmic storage of lipid, eventually leading to cell death. A possible explanation is that indomethacin may alter chondrocyte lipid metabolism in the presence of substrate molecules by rechanneling lipid synthesis away from the prostaglandin pathway to other lipid synthetic pathways.     

The role of membrane lipid in the platelet storage lesion.

Because of their hemostatic and structural importance and their chemical and physical lability, membrane lipids are likely to be involved in the development of the platelet storage lesion. Chemical analysis using the new method of high-performance liquid chromatography with laser light scattering detection (HPLC-LLS) reveals platelet lipid to be composed of more than 22 individual components, the most abundant of which are phosphatidylcholine (PC), phosphatidylethanolamine (PE), cholesterol (C), sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylinositol (PI). Surprisingly, an asymmetric distribution of these lipids is maintained in the resting platelet with PS concentrated in the inner leaflet of the plasma membrane. The exposure of PS may be important in platelet activation because of its powerful procoagulant effect. Studies of the effect of blood bank storage on platelet lipid composition have repeatedly shown a steady loss of all components, which may be temperature dependent. Studies of platelet factor 3 activity and flow cytometry of stored platelets have revealed the lipid is lost through the process of microvesiculation. Coupled to this storage induced depletion of platelet lipid is a loss of more than half of the potential capacity of lipid-dependent platelet functions by day 5. The most likely underlying mechanism for this loss of lipid mass and functional capacity is lipid peroxidation, a process that could be blocked with antioxidants. Lipid peroxidation may also interfere with other membrane constituents such as glycoprotein IIb/IIIa and the aminophospholipid-specific translocase. Thus, lipid peroxidation should be a major focus in studies aimed at preventing or reversing the platelet storage lesion.     

中华绒螯蟹肝胰腺R和F细胞及其脂类储存的电镜研究

电镜下研究了中华绒螯蟹不同生长发育时期（９月份卵巢处于快速发育阶段，１２月成熟阶段，胚胎发育４－１４天和卵孵出以后）肝胰腺Ｒ和Ｆ细胞的结构及其脂类储存。肝胰腺的脂类主苛以脂肪滴（Ｌ）的形式存在于Ｒ细胞中。Ｒ细胞在不同阶段Ｌ的存在形式和脂类的储存量不同，细胞数量也有所不同，显示Ｒ细胞对不同生长时期的敏感性。Ｆ细胞的结构特征是胞质中的发达的粗面内质网系统。细胞中几乎没有脂肪滴的储存。     

Lipid droplets as fat storage organelles in Caenorhabditis elegans: Thematic Review Series: Lipid Droplet Synthesis and Metabolism: from Yeast to Man

Lipid droplets are evolutionarily conserved organelles where cellular fat storage and mobilization are exquisitely regulated. Recent studies have defined lipid droplets in C. elegans and explored how they are regulated by genetic and dietary factors. C. elegans offers unique opportunities to visualize lipid droplets at single-cell resolution in live animals. The development of novel microscopy techniques and protein markers for lipid droplets will accelerate studies on how nutritional states and subcellular organization are linked in vivo. Together with powerful tools for genetic and biochemical analysis of metabolic pathways, alteration in lipid droplet abundance, size, and distribution in C. elegans can be readily connected to whole-animal energy homeostasis, behavior, and life span. Therefore, further studies on lipid droplets in C. elegans promise to yield valuable insights that complement our knowledge gained from yeast, Drosophila, and mammalian systems on cellular and organismal fat storage.     

Clinical and genetic characterization of Chanarin-Dorfman syndrome

We describe the clinical features, muscle pathology features, and molecular studies of seven patients with Chanarin-Dorfman syndrome (CDS) or neutral lipid storage disease and ichthyosis (NLSDI), a multisystem triglyceride storage disease with massive accumulation of lipid droplets in muscle fibers.All patients presented with congenital ichthyosiform erythroderma, cytoplasmic lipid droplets in blood cells, mild to severe hepatomegaly, and increased serum CK levels and liver enzymes. Three patients showed muscle symptoms and three had steathorrea. Molecular analysis identified five mutations, three of which are novel.These findings expand the clinical and mutational spectrum and underline the genetic heterogeneity of this disease.     

Lipid oversupply,selective insulin resistance,and lipotoxicity: Molecular mechanisms

The accumulation of fat in tissues not suited for lipid storage has deleterious consequences on organ function, leading to cellular damage that underlies diabetes, heart disease, and hypertension. To combat these lipotoxic events, several therapeutics improve insulin sensitivity and/or ameliorate features of metabolic disease by limiting the inappropriate deposition of fat in peripheral tissues (i.e. thiazolidinediones, metformin, and statins). Recent advances in genomics and lipidomics have accelerated progress towards understanding the pathogenic events associated with the excessive production, underutilization, or inefficient storage of fat. Herein we review studies applying pharmacological or genetic strategies to manipulate the expression or activity of enzymes controlling lipid deposition, in order to gain a clearer understanding of the molecular mechanisms by which fatty acids contribute to metabolic disease.     

Perilipin A and the control of triacylglycerol metabolism

Perilipin A is the most abundant protein associated with the lipid droplets of adipocytes and functions to control both basal and stimulated lipolysis. Under basal or fed conditions, perilipin A shields stored triacylglycerols from cytosolic lipases, thus promoting triacylglycerol storage. When catecholamines bind to cell surface receptors to initiate signals that activate cAMP-dependent protein kinase (PKA), phosphorylated perilipin A facilitates maximal lipolysis. Mutagenesis studies have revealed that central sequences of moderately hydrophobic amino acids are required to target nascent perilipin A to lipid droplets and provide an anchor into the hydrophobic environment of lipid droplets. Sequences of amino acids in the unique carboxyl terminus of perilipin A and those in amino terminal sequences flanking the first hydrophobic stretch are required for the barrier function of perilipin A in promoting triacylglycerol storage. Site-directed mutagenesis studies of serine residues within six PKA consensus sites of perilipin A reveal functions for phosphorylation of at least three of the sites. Phosphorylation of one or more of the serines within three amino terminal PKA sites is required to facilitate hormone-sensitive lipase access to lipid substrates. Phosphorylation of serines within two carboxyl terminal sites is also required for maximal lipolysis. Phosphorylation of serine 492 (site 5) triggers a massive remodeling of lipid droplets, whereby large peri-nuclear lipid droplets fragment into myriad lipid micro-droplets that scatter throughout the cytoplasm. We hypothesize that perilipin A binds accessory proteins to provide assistance in carrying out these functions.     

Alteration in biological properties of human albumin during storage

The effects of human albumin preparations on oxidative energy metabolism and lipid svnthesis were investigated in rat liver slices incubated with sodium [1-¹⁴C]acetate as precursor. Labeled CO₂ production and incorporation of precursor into the major lipid classes was increased 2 to 3-fold by fresh preparations of albumin (fraction V), and by defatted fraction V, whereas highly purified cystalline albumin was less active. Albumin preparations from various commercial suppliers varied widely in activity. Activity of fraction V was preserved during storage at ?20°C, and gradually lost at +3°C in the course of 1 year. In contrast, defatted fractions rapidly lost activity in storage at both temperatures. After 1 year in storage at +3°C, albumin preparations became inhibitory to CO₂ production and lipid synthesis. The results suggest that commercial albumin used in metabolic studies, and in clinical situations may have unpredictable or undesirable effects related to state of purity and storage conditions of the protein.     

Functional conservation for lipid storage droplet association among Perilipin,ADRP, and TIP47 (PAT)-related proteins in mammals,Drosophila, and Dictyostelium

Intracellular neutral lipid storage droplets are essential organelles of eukaryotic cells, yet little is known about the proteins at their surfaces or about the amino acid sequences that target proteins to these storage droplets. The mammalian proteins Perilipin, ADRP, and TIP47 share extensive amino acid sequence similarity, suggesting a common function. However, while Perilipin and ADRP localize exclusively to neutral lipid storage droplets, an association of TIP47 with intracellular lipid droplets has been controversial. We now show that GFP-tagged TIP47 co-localizes with isolated intracellular lipid droplets. We have also detected a close juxtaposition of TIP47 with the surfaces of lipid storage droplets using antibodies that specifically recognize TIP47, further indicating that TIP47 associates with intracellular lipid storage droplets. Finally, we show that related proteins from species as diverse as Drosophila and Dictyostelium can also target mammalian or Drosophila lipid droplet surfaces in vivo. Thus, sequence and/or structural elements within this evolutionarily ancient protein family are necessary and sufficient to direct association to heterologous intracellular lipid droplet surfaces, strongly indicating that they have a common function for lipid deposition and/or mobilization.     

Membrane traffic in sphingolipid storage diseases

In this review, we summarize our studies of membrane lipid transport in sphingolipid storage disease (SLSD) fibroblasts. We recently showed that several fluorescent SL analogs were internalized from the plasma membrane predominantly to the Golgi complex of normal cells, while in ten different SLSD cell types, these lipids accumulated in endosomes and lysosomes (The Lancet 1999;354: 901-905). Additional studies showed that cholesterol homeostasis is perturbed in multiple SLSDs secondary to SL accumulation and that mistargeting of SL analogs was regulated by cholesterol (Nature Cell Biol 1999;1: 386-388). Based on these findings, we hypothesize that endogenous sphingolipids, which accumulate in SLSD cells due to primary defects in lipid catabolism, result in an altered intracellular distribution of cholesterol, and that this alteration in membrane composition then results in defective sorting and transport of SLs. The importance of SL/cholesterol interactions and potential mechanisms underlying the regulation of lipid transport and targeting are also discussed. These studies suggest a new paradigm for regulation of membrane lipid traffic along the endocytic pathway and could have important implications for future studies of protein trafficking as well as lipid transport. This work may also lead to important future clinical developments (e.g. screening tests for SLSD, new methodology for screening drugs which abrogate lipid storage, and possible therapeutic approaches to SLSD).     

Connecting lipid droplet biology and the metabolic syndrome

In the recent years, new advances in the biology of lipid droplets led these structures specialized for lipid storage to be considered as new universal intracellular organelles playing active roles in cell physiology. Concomitantly, studies on the pathogenesis of metabolic diseases such as type 2 diabetes or atherosclerosis, associated with ongoing epidemic obesity, have pointed out the importance of lipotoxic effects in metabolic dysfunction, generated by ectopic lipid storage in non-adipose tissues. The purpose of this paper is to establish connections between recent discoveries in lipid droplet biology and novel views in the pathology of the metabolic syndrome. Bringing together the new concepts produced in these two separated fields might show the way towards the definition of innovative strategies to treat metabolic diseases. Particular attention is given to the role of adipocyte-specific proteins that interact with lipid droplets and confer unique functions to adipocyte lipid storage by limiting the spill-over of fatty acids and their lipotoxic effects.     

Drosophila Perilipin/ADRP homologue Lsd2 regulates lipid metabolism

Many cells store neutral lipids, as triacylglycerol and sterol esters, in droplets. PAT-domain proteins form a conserved family of proteins that are localized at the surface of neutral lipid droplets. Two mammalian members of this family, Perilipin and adipose differentiation-related protein, are involved in lipid storage and regulate lipolysis. Here, we describe the Drosophila PAT-family member Lsd2. We showed that Lsd2 is predominantly expressed in tissues engaged in high levels of lipid metabolism, the fat body and the germ line of females. Ultrastructural analysis in the germ line showed that Lsd2 localizes to the surface of lipid droplets. We have generated an Lsd2 mutant and described its phenotype. Mutant adults have a reduced level of neutral lipid content compared to wild type, showing that Lsd2 is required for normal lipid storage. In addition, ovaries from Lsd2 mutant females exhibit an abnormal pattern of accumulation of neutral lipids from mid-oogenesis, which results in reduced deposition of lipids in the egg. Consistent with its expression in the female germ line, we showed that Lsd2 is a maternal effect gene that is required for normal embryogenesis. This work demonstrates that Lsd2 has an evolutionarily conserved function in lipid metabolism and establishes Drosophila melanogaster as a new in vivo model for studies on the PAT-family of proteins.     

Appearance of smaller lipid bodies and protein kinase activation in the lipid body fraction are induced by an increase in the nitrogen source in the Mortierella fungus

We studied the regulation of lipid body biogenesis in the oleaginous fungus Mortierella ramanniana var. angulispora by investigating culture conditions to modulate lipid body size, which we found was affected by the carbon-to-nitrogen ratio (C/N ratio) in the culture medium. Increasing the nitrogen source or decreasing the C/N ratio from 38 to 9 induced the appearance of lipid bodies with diameters less than 2-3 micro m, which are usually found at a C/N ratio of 38 in this fungus. To determine factors regulating lipid body size, we compared lipid body fractions from fungal cells cultured at different C/N ratios. We found some differences in polypeptide profiles between lipid body fractions from fungal cells cultured at different C/N ratios for 2 days when the lipid bodies were enlarged at a C/N ratio of 38. We then compared the phosphorylation of lipid body proteins, since protein phosphorylation plays a pivotal role in various aspects of signal transduction. In vitro phosphorylation in the lipid body fraction indicated that protein kinase activity toward endogenous and exogenous substrates such as histone IIIS, VIIS, and myelin basic protein increased in the lipid body fraction at a C/N ratio of 9. Further analysis by in-gel protein kinase assay indicated the presence of at least three activated protein kinases with molecular masses of 75, 72, and 42 kDa, which were also autophosphorylated. These results indicate the presence of nutrient-regulated protein kinases and increased phosphorylation in lipid bodies, which correlate with the appearance of smaller lipid bodies in this fungus. Further studies to characterize these protein kinases at the molecular level should provide new insights into the link between nutrient sensing and lipid storage.     

Characterization of the Drosophila lipid droplet subproteome

Lipid storage droplets are universal organelles essential for the cellular and organismal lipometabolism including energy homeostasis. Despite their apparently simple design they are proposed to participate in a growing number of cellular processes, raising the question to what extent the functional multifariousness is reflected by a complex organellar proteome composition. Here we present 248 proteins identified in a subproteome analysis using lipid storage droplets of Drosophila melanogaster fat body tissue. In addition to previously known lipid droplet-associated PAT (Perilipin, ADRP, and TIP47) domain proteins and homologues of several mammalian lipid droplet proteins, this study identified a number of proteins of diverse biological function, including intracellular trafficking supportive of the dynamic and multifaceted character of these organelles. We performed intracellular localization studies on selected newly identified subproteome members both in tissue culture cells and in fat body cells directly. The results suggest that the lipid droplets of fat body cells are of combinatorial protein composition. We propose that subsets of lipid droplets within single cells are characterized by a protein "zip code," which reflects functional differences or specific metabolic states.     

Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets

The lipid fraction ("fat cake") of rat epididymal adipocytes contains a prominent phosphoprotein (62 kDaapp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) that is multiply phosphorylated by cAMP-dependent protein kinase in vivo, at which point it migrates as a 65/67-kDaapp doublet by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is by far the most heavily radiolabeled protein in the cell. Western blot analysis of various tissues with immunopurified antibodies purified from antisera raised against the 62-kDa species suggests that the protein is specific for adipocytes. This protein, which we term perilipin, is found in differentiated cultured 3T3-L1 adipocytes, but not in their precursor 3T3-L1 fibroblasts. Immunocytochemical studies with specific antiserum shows that the perilipin is closely associated with the periphery of lipid storage droplets in cultured adipocytes. Given its adipocyte specificity, acute regulation by hormones, and subcellular location, we speculate that perilipin plays a role in the specialized lipid storage function of adipocytes.     

Studies on the compartmentation of lipid in adipose cells. I. Subcellular distribution, composition, and transport of newly synthesized lipid: liposomes

The subcellular distribution and composition of endogenously synthesized lipid in isolated white adipose cells were studied to determine the nature and extent of lipid compartmentation. After brief incubation of cells with labeled glucose, acetate, or palmitic acid, over 90% of newly synthesized triglyceride was localized in the bulk-lipid phase, indicating rapid intracellular transport and storage. From 13 to 20% of the newly formed lipid was diglyceride, and over 95% of it was localized in the central lipid-storage vacuole rather than in organelle systems concerned with esterification, thus indicating intracellular segregation of newly synthesized partial glycerides. Most of the newly synthesized phosphatides partitioned with membranous organelles. Synthesis of cholesterol or cholesteryl ester was negligible. After brief incubation of cells with labeled glucose, the relative specific activity of organelle triglyceride was mitochondria > microsomes > liposomes > soluble supernatant > bulk lipid. In pulse-chase studies the specific activity of organelle triglyceride decreased and that of the bulk fraction increased reflecting intracellular lipid transport. The data suggest that a significant proportion of newly formed lipid is transferred from mitochondrial membranes into the storage vacuole by direct lipid-lipid interaction. Liposomes, which consist of small enclosed lipid droplets resembling chylomicrons, contained triglycerides of specific activity similar to microsomal triglyceride. While the evidence that liposome triglyceride may be microsomal in origin is indirect, the results do indicate that the liposome fraction represents a phase in the transport and(or) storage of new glyceride. At least two forms of compartmentation of newly synthesized lipids occurred. The first, termed "structural," refers to localization of lipids to organelle fractions. The second type of compartmentation, termed "chemical," concerns the intracellular segregation of a specific lipid class. The accumulation and segregation of newly synthesized diglyceride in the bulk storage pool are examples of the latter form of compartmentation.     

Structural determinants that target the hepatitis C virus core protein to lipid droplets

Hepatitis C virus core protein is targeted to lipid droplets, which serve as intracellular storage organelles, by its C-terminal domain, termed D2. From circular dichroism and nuclear magnetic resonance analyses, we demonstrate that the major structural elements within D2 consist of two amphipathic alpha-helices (Helix I and Helix II) separated by a hydrophobic loop. Both helices require a hydrophobic environment for folding, indicating that lipid interactions contribute to their structural integrity. Mutational studies revealed that a combination of Helix I, the hydrophobic loop, and Helix II is essential for efficient lipid droplet association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from lipid droplet association, membrane interaction of D2 is necessary for folding and stability of core following maturation at the endoplasmic reticulum membrane by signal peptide peptidase. These studies identify critical determinants within a targeting domain that enable trafficking and attachment of a viral protein to lipid droplets. They also serve as a unique model for elucidating the specificity of protein-lipid interactions between two membrane-bound organelles.     

The vacuole as central element of the lytic system and sink for lipid droplets in maturing appressoria ofMagnaporthe grisea

Summary Histochemical and ultrastructural studies were carried out on a wild-type strain (Guyll) and a melanin-deficient mutant(büβ) of the rice-blast pathogen,Magnaporthe grisea (=Pyricularia oryzae), in order to investigate the destination of lipid storage reserves during appressorium development. Lipid droplets were abundant in conidia and were mobilised upon germination, accumulating in the appressorial hook which developed at the tip of each germ tube. Following the formation of a septum at the base of the nascent appressorium, one or a few closely appressed central vacuoles became established and were observed to enlarge in the course of appressorium maturation. On unyielding artificial surfaces such as glass or plastic, appressoria matured to completion within 36–48 h, by which time the enlarged vacuole filled most of the inside volume of the appressorium. Light and transmission electron microscopical observations revealed that the lipid droplets entered the vacuole by autophagocytosis and were degraded therein. Histochemical approaches confirmed the vacuole as the key lytic element in maturing appressoria. Endocytosis of a vital dye, Neutral Red, progressed via endosomes which migrated into the vacuole and lysed there, releasing their dye content into the vacuolar lumen. Furthermore, activity of the lysosomal marker enzyme, acid phospho-monoesterase, was strongly localised in the vacuole at all stages of appressorium maturation. It is therefore envisaged that vacuoles are involved in the degradation of lipid storage reserves which may act as sources of energy and/or osmotically active metabolites such as glycerol, which generate the very high turgor pressure known to be crucial for penetration of hard surfaces. On softer surfaces such as onion epidermis, appressoria ofM. grisea were able to penetrate before degradation of lipid droplets had been completed.