Hepatic stellate cell lipid droplets: A specialized lipid droplet for retinoid storage

The majority of retinoid (vitamin A and its metabolites) present in the body of a healthy vertebrate is contained within lipid droplets present in the cytoplasm of hepatic stellate cells (HSCs). Two types of lipid droplets have been identified through histological analysis of HSCs within the liver: smaller droplets bounded by a unit membrane and larger membrane-free droplets. Dietary retinoid intake but not triglyceride intake markedly influences the number and size of HSC lipid droplets. The lipids present in rat HSC lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Retinyl ester and triglyceride are present at similar concentrations, and together these two classes of lipid account for approximately three-quarters of the total lipid in HSC lipid droplets. Both adipocyte-differentiation related protein and TIP47 have been identified by immunohistochemical analysis to be present in HSC lipid droplets. Lecithin:retinol acyltransferase (LRAT), an enzyme responsible for all retinyl ester synthesis within the liver, is required for HSC lipid droplet formation, since Lrat-deficient mice completely lack HSC lipid droplets. When HSCs become activated in response to hepatic injury, the lipid droplets and their retinoid contents are rapidly lost. Although loss of HSC lipid droplets is a hallmark of developing liver disease, it is not known whether this contributes to disease development or occurs simply as a consequence of disease progression. Collectively, the available information suggests that HSC lipid droplets are specialized organelles for hepatic retinoid storage and that loss of HSC lipid droplets may contribute to the development of hepatic disease.     

Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets

The biosynthesis and storage of triglyceride (TG) is an important cellular process conserved from yeast to man. Most mammalian cells accumulate TG in lipid droplets, most prominent in adipocytes, which are specialized to store large amounts of the TG over long periods. In this study, we followed TG biosynthesis and targeting by fluorescence imaging in living 3T3-L1 adipocytes and COS7 fibroblasts. Key findings were (i) not only TG but also its direct metabolic precursor diacylglycerol, DG, accumulates on lipid droplets; (ii) the essential enzyme diacylglycerol acyltransferase 2 associates specifically with lipid droplets where it catalyzes the conversion of DG to TG and (iii) individual lipid droplets within one cell acquire TG at very different rates, suggesting unequal access to the biosynthetic machinery. We conclude that at least part of TG biosynthesis takes place in the immediate vicinity of lipid droplets. In vitro assays on purified lipid droplets show that this fraction of the biosynthetic TG is directly inserted into the growing droplet.     

Controlling the size of lipid droplets: lipid and protein factors

Recent advances have transformed our understanding of lipid droplets (LDs). Once regarded as inert lipid storage granules, LDs are now recognized as multi-functional organelles that affect many aspects of cell biology and metabolism. However, fundamental questions concerning the biogenesis and growth of LDs remain unanswered. Recent studies have uncovered novel modes of LD growth (including rapid/homotypic as well as slow/atypical LD fusion), and identified key proteins (e.g. Fsp27, seipin, FITM2 and perilipin 1) and lipids (e.g. phosphatidylcholine and phosphatidic acid) that regulate the size of LDs. Phospholipids appear to have an evolutionarily conserved role in LD growth. Protein factors may regulate LD expansion directly and/or indirectly through modulating the level and composition of phospholipids on LD surface.     

The life of lipid droplets

Lipid droplets are the least characterized of cellular organelles. Long considered simple lipid storage depots, these dynamic and remarkable organelles have recently been implicated in many biological processes, and we are only now beginning to gain insights into their fascinating lives in cells. Here we examine what we know of the life of lipid droplets. We review emerging data concerning their cellular biology and present our thoughts on some of the most salient questions for investigation.     

Cell biology of lipid droplets

Lipid storage has attracted much attention in the past years, both by the broader public and the biomedical scientific community. Driven by concerns about the obesity epidemic that affects most industrialized countries and even substantial parts of the population in less and least developed countries, work from researchers of many disciplines has shed light on the genetics, the physiology, and the cellular mechanisms of fat accumulation. This review focuses on the actual organelle of fat deposition, the lipid droplet (LD), and on the recent progress in mechanistic understanding of processes like LD biogenesis, LD growth and degradation, protein targeting to LDs and LD fusion.     

HOT models in flux: mitochondrial glucose oxidation fuels glioblastoma growth

Cancer cells in culture obtain ATP and biosynthetic precursors primarily by aerobic glycolysis, not by mitochondrial glucose oxidation. In this issue of Cell Metabolism, Marin-Valencia et?al. (2012) demonstrate that glioblastoma, an aggressive and, in culture, highly glycolytic cancer, primarily uses glucose oxidation to meet energetic and biosynthetic demands in?vivo.     

Proteins under new management: lipid droplets deliver

Lipid droplets are ubiquitous organelles that store neutral lipids and have crucial roles in lipid metabolism. Recent studies have uncovered many examples of lipid droplets recruiting proteins from other cellular compartments, in a cell type-specific and regulated manner. Some droplet-recruited proteins are destined for destruction, whereas others are released and reused when conditions change. Droplets might therefore have a general role in managing the availability of proteins, and they have been proposed to serve as generic sites of protein sequestration. The implications of this emerging role of lipid droplets include regulated inactivation of proteins, prevention of toxic protein aggregates and localized delivery of signaling molecules.     

Hydrophobic sequences target and anchor perilipin A to lipid droplets

Perilipins regulate triacylglycerol storage and hydrolysis in adipocytes. The central 25% of the perilipin A sequence, including three hydrophobic sequences (H1, H2, and H3) and an acidic region, targets and anchors perilipins to lipid droplets. Thus, we hypothesized that H1, H2, and H3 are targeting and anchoring motifs. We now show that deletion of any single hydrophobic sequence or combinations of H1 and H3 or H2 and H3 does not prevent targeting of the mutated perilipin to lipid droplets. In contrast, mutated perilipin lacking H1 and H2 showed reduced targeting, whereas perilipin lacking H1, H2, and H3 targeted poorly to lipid droplets; thus, H3 is a weak targeting signal and either H1 or H2 is required for optimal targeting. Complete elimination of perilipin targeting was observed only when all three hydrophobic sequences were deleted in combination with either the acidic region or N-terminal sequences predicted to form amphipathic beta-strands. Unlike intact perilipin A, mutated perilipin lacking either H1 and H2 or H1, H2, and H3 was released from lipid droplets after alkaline carbonate treatment, suggesting that these forms are loosely associated with lipid droplets. The three hydrophobic sequences play a major role in targeting and anchoring perilipins to lipid droplets.     

Transfer cells and lipid droplets of someValerianaceae

Development, structure and the axial distribution of transfer cells and their lignification were investigated inValerianella locusta, Valeriana officinalis, andV. tuberosa (Valerianaceae). Fundamental new results are: (1) Transfer cells often contain numerous lipid droplets. Within the stem the distribution of cells containing lipid droplets correlates to that of transfer cells. (2) InValeriana officinalis persisting protuberances are frequently found on pit membranes of xylem transfer cells. Lignified transfer cells can undergo a second modification: a layer covering the secondary wall forms wall ingrowths similar to those of transfer cells. (3) Peripheral pith cells, abuting transfer cells, are able to modify into transfer cells. Cambial derivatives are only temporarily developed as transfer cells. (4) Phloem transfer cells are found in vascular bundles of the whole axis. (5) In roots, xylem transfer cells are poorly developed or absent. (6) Oil cells with oil bodies are present in the rape ofValeriana tuberosa. They are absent however in the stem of the species investigated. (7) Tannins occur in elements of the primary cortex, phloem and secondary xylem ofValeriana officinalis.     

Deformation of lipid droplets in fixed samples

Nile red, Sudan III, and oil red O have been used to stain lipid droplets (LDs) for fluorescence microscopy. We noticed that LDs labeled by Nile red are different in appearance from those stained by the latter two dyes. To understand the cause of the difference, we used sequential labeling procedures (first LD stain-photography-quenching-second LD stain-photography), and examined the effect of several factors. Immunofluorescence labeling for adipose differentiation-related protein (ADRP), an LD marker, was also observed comparatively with the lipid stains. As a result, we found that ethanol and isopropanol used for Sudan III and oil red O staining, respectively, and glycerol used for mounting, cause fusion of adjacent LDs even in glutaraldehyde-fixed samples. By the same treatment, immunofluorescence labeling for ADRP was dislocated to the rim of large LDs that were formed as a result of the artifactual fusion. The result indicates that the LD structure can be better observed with Nile red than with Sudan III or oil red O.     

Isolation of hepatocellular lipid droplets: the separation of distinct subpopulations

A new method for the isolation of lipid droplets from rat liver has been devised. The procedure involves tissue homogenization and discontinuous density gradient centrifugation. Six discrete bands of lipid particles, rich in triglyceride and cholesterol, are visible following 30 min of centrifugation at 25,770 g (max) in a swinging bucket rotor. An entire rat liver can be processed in 1-2 hr. Differences in the density of these liver lipid particles correlate with their triglyceride, sterol, phospho-lipid, and protein contents. The separation of these distinct populations of intracellular particulate neutral lipids provides an approach to the study of their origin and metabolism. This procedure may also be useful in the isolation of various populations of lipid-rich particles from other tissues.     

Cytosolic lipid droplets: From mechanisms of fat storage to disease

Abstract

The lipid droplet (LD) is a phylogenetically conserved organelle. In eukaryotes, it is born from the endoplasmic reticulum, but unlike its parent organelle, LDs are the only known cytosolic organelles that are micellar in structure. LDs are implicated in numerous physiological and pathophysiological functions. Many aspects of the LD has captured the attention of diverse scientists alike and has recently led to an explosion in information on the LD biogenesis, expansion and fusion, identification of LD proteomes and diseases associated with LD biology. This review will provide a brief history of this fascinating organelle and provide some contemporary views of unanswered questions in LD biogenesis.     

Rab-regulated interaction of early endosomes with lipid droplets

Recent studies indicate that lipid droplets isolated from a variety of different cells are rich in proteins known to regulate membrane traffic. Among these proteins are multiple Rab GTPases. Rabs are GTP switches that regulate intracellular membrane traffic through an ability to control membrane-membrane docking as well as vesicle motility. Here we present evidence that the multiple Rabs associated with droplets have a function in regulating membrane traffic. Droplet Rabs are removed by Rab GDP-dissociation inhibitor (RabGDI) in a GDP-dependent reaction, and are recruited to Rab-depleted droplets from cytosol in a GTP-dependent reaction. Rabs also control the recruitment of the early endosome (EE) marker EEA1 from cytosol. We use an in vitro reconstitution assay to show that transferrin receptor positive EEs bind to the droplet in a GTP/Rab-dependent reaction that appears not to lead to membrane fusion. This docking reaction is insensitive to ATP(gamma s) but is blocked by ATP. Finally, we show that when GTP bound active or GDP bound inactive Rab5 is targeted to the droplet, the active form recruits EEA1. We conclude that the Rabs associated with droplets may be capable of regulating the transient interaction of specific membrane systems, probably to transport lipids between membrane compartments.     

Accumulation of myocardial lipid droplets in dexamethasone-treated mice

Summary Conventionally fixed and plastic-embedded myocardial tissue from mice treated with 1 mg/kg body weight dexamethasone for 48 h was examined in the electron microscope. The dexamethasone-treated mice showed a marked increase in the accumulation of lipid droplets as compared with control animals. The per cent volume of lipid droplets, calculated by morphometric analysis, showed a significant increase in the dexamethasone-treated mice. No other ultrastructural difference between dexamethasone-treated mice and controls was observed.     

Degradation of lipid droplets by chimeric autophagy-tethering compounds

Degrading pathogenic proteins by degrader technologies such as PROTACs (proteolysis-targeting chimeras) provides promising therapeutic strategies, but selective degradation of non-protein pathogenic biomolecules has been challenging. Here, we demonstrate a novel strategy to degrade non-protein biomolecules by autophagy-tethering compounds (ATTECs), using lipid droplets (LDs) as an exemplar target. LDs are ubiquitous cellular structures storing lipids and could be degraded by autophagy. We hypothesized that compounds interacting with both the LDs and the key autophagosome protein LC3 may enhance autophagic degradation of LDs. We designed and synthesized such compounds by connecting LC3-binding molecules to LD-binding probes via a linker. These compounds were capable of clearing LDs almost completely and rescued LD-related phenotypes in cells and in two independent mouse models with hepatic lipidosis. We further confirmed that the mechanism of action of these compounds was mediated through LC3 and autophagic degradation. Our proof-of-concept study demonstrates the capability of degrading LDs by ATTECs. Conceptually, this strategy could be applied to other protein and non-protein targets.Subject terms: Macroautophagy, Molecular biology