Cytosolic Lipid Droplets Research Articles

Active autophagy but not lipophagy in macrophages with defective lipolysis

During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages.

Specific cellular incorporation of a pyrene-labelled cholesterol: lipoprotein-mediated delivery toward ordered intracellular membranes.

In the aim of testing tools for tracing cell trafficking of exogenous cholesterol, two fluorescent derivatives of cholesterol, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), with distinctive chemico-physical characteristics, have been compared for their cell incorporation properties, using two cell models differently handling cholesterol, with two incorporation routes. In the Caco-2 cell model, the cholesterol probes were delivered in bile salt micelles, as a model of intestinal absorption. The two probes displayed contrasting behaviors for cell uptake characteristics, cell staining, and efflux kinetics. In particular, Pyr-met-Chol cell incorporation involved SR-BI, while that of NBD-Chol appeared purely passive. In the PC-3 cell model, which overexpresses lipoprotein receptors, the cholesterol probes were delivered via the serum components, as a model of systemic delivery. We showed that Pyr-met-Chol-labelled purified LDL or HDL were able to specifically deliver Pyr-met-Chol to the PC-3 cells, while NBD-Chol incorporation was independent of lipoproteins. Observations by fluorescence microscopy evidenced that, while NBD-Chol readily stained the cytosolic lipid droplets, Pyr-met-Chol labelling led to the intense staining of intracellular structures of membranous nature, in agreement with the absence of detectable esterification of Pyr-met-Chol. A 48 h incubation of PC-3 cells with either Pyr-met-Chol-labelled LDL or HDL gave same staining patterns, mainly colocalizing with Lamp1, caveolin-1 and CD63. These data indicated convergent trafficking downwards their respective receptors, LDL-R and SR-BI, toward the cholesterol-rich internal membrane compartments, late endosomes and multivesicular bodies. Interestingly, Pyr-met-Chol staining of these structures exhibited a high excimer fluorescence emission, revealing their ordered membrane environment, and indicating that Pyr-met-Chol behaves as a fair cholesterol tracer regarding its preferential incorporation into cholesterol-rich domains. We conclude that, while NBD-Chol is a valuable marker of cholesterol esterification, Pyr-met-Chol is a reliable new lipoprotein fluorescent marker which allows to probe specific intracellular trafficking of cholesterol-rich membranes.

Antitumor 1‐nitroacridine, C‐1748, Decreases Pro‐survival Autophagy and Induces Accumulation of Lipid Droplets Resulting in Apoptosis of Panc‐1 Pancreatic Cancer Cells

Given poor prognosis of pancreatic cancer, there is a strong need for the development of novel therapeutic agents and discovery of new molecular targets. Changes in lipid metabolism can affect numerous cellular processes, including cancer cell growth, proliferation or differentiation. Here, we show for the first time that DNA‐damaging antitumor agent, C‐1748, affects lipid content in pancreatic cancer Panc‐1 cells which may contribute to its antiproliferative effect. Cytotoxic activity of C‐1748 against Panc‐1 cells was similar to clinically used gemcitabine. Compared with unexposed cells, C‐1748 reduced level of pro‐survival autophagy as shown by decrease in Acidic Vesicular Organelles. Staining with Nile Red showed that inhibition of autophagy was accompanied by accumulation of cytosolic lipid droplets (LDs). The pool of LDs following C‐1748 treatment increased in a dose‐ and time‐dependent manner. Importantly, along with decreased autophagy and development of LDs, C‐1748 progressively triggered mitochondrial dysfunction, caspase‐3 activation indicating on induction of apoptotic cell death. Recent studies indicate that autophagy allows cancer cells to maintain energy supply during stress conditions, and inhibition of autophagy increases triglyceride storage in LDs, what prevents them from being used as energy source. Our study suggests that C‐1748 blocks autophagy in pancreatic cancer cells, which disrupts pro‐survival lipid metabolism and leads to cell death.

Roles of Acyl-CoA:Diacylglycerol Acyltransferases 1 and 2 in Triacylglycerol Synthesis and Secretion in Primary Hepatocytes.

Very low-density lipoprotein assembly and secretion are regulated by the availability of triacylglycerol. Although compelling evidence indicates that the majority of triacylglycerol in very low-density lipoprotein is derived from re-esterification of lipolytic products released by endoplasmic reticulum-associated lipases, little is known about roles of acyl-CoA:diacylglycerol acyltransferases (DGATs) in this process. We aimed to investigate the contribution of DGAT1 and DGAT2 in lipid metabolism and lipoprotein secretion in primary mouse and human hepatocytes. We used highly selective small-molecule inhibitors of DGAT1 and DGAT2, and we tracked storage and secretion of lipids synthesized de novo from [(3)H]acetic acid and from exogenously supplied [(3)H]oleic acid. Inactivation of individual DGAT activity did not affect incorporation of either radiolabeled precursor into intracellular triacylglycerol, whereas combined inactivation of both DGATs severely attenuated triacylglycerol synthesis. However, inhibition of DGAT2 augmented fatty acid oxidation, whereas inhibition of DGAT1 increased triacylglycerol secretion, suggesting preferential channeling of separate DGAT-derived triacylglycerol pools to distinct metabolic pathways. Inactivation of DGAT2 impaired cytosolic lipid droplet expansion, whereas DGAT1 inactivation promoted large lipid droplet formation. Moreover, inactivation of DGAT2 attenuated expression of lipogenic genes. Finally, triacylglycerol secretion was significantly reduced on DGAT2 inhibition without altering extracellular apolipoprotein B levels. Our data suggest that DGAT1 and DGAT2 can compensate for each other to synthesize triacylglycerol, but triacylglycerol synthesized by DGAT1 is preferentially channeled to oxidation, whereas DGAT2 synthesizes triacylglycerol destined for very low-density lipoprotein assembly.

Adipose triglyceride lipase is involved in the mobilization of triglyceride and retinoid stores of hepatic stellate cells

Hepatic stellate cells (HSCs) store triglycerides (TGs) and retinyl ester (RE) in cytosolic lipid droplets. RE stores are degraded following retinoid starvation or in response to pathogenic stimuli resulting in HSC activation. At present, the major enzymes catalyzing lipid degradation in HSCs are unknown. In this study, we investigated whether adipose triglyceride lipase (ATGL) is involved in RE catabolism of HSCs. Additionally, we compared the effects of ATGL deficiency and hormone-sensitive lipase (HSL) deficiency, a known RE hydrolase (REH), on RE stores in liver and adipose tissue. We show that ATGL degrades RE even in the presence of TGs, implicating that these substrates compete for ATGL binding. REH activity was stimulated and inhibited by comparative gene identification-58 and G0/G1 switch gene-2, respectively, the physiological regulators of ATGL activity. In cultured primary murine HSCs, pharmacological inhibition of ATGL, but not HSL, increased RE accumulation. In mice globally lacking ATGL or HSL, RE contents in white adipose tissue were decreased or increased, respectively, while plasma retinol and liver RE levels remained unchanged. In conclusion, our study shows that ATGL acts as REH in HSCs promoting the degradation of RE stores in addition to its established function as TG lipase. HSL is the predominant REH in adipocytes but does not affect lipid mobilization in HSCs.

High-resolution imaging of dietary lipids in cells and tissues by NanoSIMS analysis

Nanoscale secondary ion MS (NanoSIMS) imaging makes it possible to visualize stable isotope-labeled lipids in cells and tissues at 50 nm lateral resolution. Here we report the use of NanoSIMS imaging to visualize lipids in mouse cells and tissues. After administering stable isotope-labeled fatty acids to mice by gavage, NanoSIMS imaging allowed us to visualize neutral lipids in cytosolic lipid droplets in intestinal enterocytes, chylomicrons at the basolateral surface of enterocytes, and lipid droplets in cardiomyocytes and adipocytes. After an injection of stable isotope-enriched triglyceride-rich lipoproteins (TRLs), NanoSIMS imaging documented delivery of lipids to cytosolic lipid droplets in parenchymal cells. Using a combination of backscattered electron (BSE) and NanoSIMS imaging, it was possible to correlate the chemical data provided by NanoSIMS with high-resolution BSE images of cell morphology. This combined imaging approach allowed us to visualize stable isotope-enriched TRLs along the luminal face of heart capillaries and the lipids within heart capillary endothelial cells. We also observed examples of TRLs within the subendothelial spaces of heart capillaries. NanoSIMS imaging provided evidence of defective transport of lipids from the plasma LPs to adipocytes and cardiomyocytes in mice deficient in glycosylphosphatidylinositol-anchored HDL binding protein 1.

Diacylglycerol Acyltransferase-2 (DGAT2) and Monoacylglycerol Acyltransferase-2 (MGAT2) Interact to Promote Triacylglycerol Synthesis

Acyl CoA:1,2-diacylglycerol acyltransferase (DGAT)-2 is an integral membrane protein that catalyzes triacylglycerol (TG) synthesis using diacylglycerol and fatty acyl CoA as substrates. DGAT2 resides in the endoplasmic reticulum (ER), but when cells are incubated with fatty acids, DGAT2 interacts with lipid droplets presumably to catalyze localized TG synthesis for lipid droplet expansion. Previous studies have shown that DGAT2 interacts with proteins that synthesize its fatty acyl CoA substrates. In this study, we provide additional evidence that DGAT2 is present in a protein complex. Using a chemical cross-linker, disuccinimidyl suberate (DSS), we demonstrated that DGAT2 formed a dimer and was also part of a protein complex of ∼ 650 kDa, both in membranes and on lipid droplets. Using co-immunoprecipitation experiments and an in situ proximity ligation assay, we found that DGAT2 interacted with monoacylglycerol acyltransferase (MGAT)-2, an enzyme that catalyzes the synthesis of diacylglycerol. Deletion mutagenesis showed that the interaction with MGAT2 was dependent on the two transmembrane domains of DGAT2. No significant interaction of DGAT2 with lipin1, another enzyme that synthesizes diacylglycerol, could be detected. When co-expressed in cells, DGAT2 and MGAT2 co-localized in the ER and on lipid droplets. Co-expression also resulted in increased TG storage compared with expression of DGAT2 or MGAT2 alone. Incubating McArdle rat hepatoma RH7777 cells with 2-monoacylglycerol caused DGAT2 to translocate to lipid droplets. This also led to the formation of large cytosolic lipid droplets, characteristic of DGAT2, but not DGAT1, and indicated that DGAT2 can utilize monoacylglycerol-derived diacylglycerol. These findings suggest that the interaction of DGAT2 and MGAT2 serves to channel lipid substrates efficiently for TG biosynthesis.

The characteristics and potential applications of structural lipid droplet proteins in plants

Plant cytosolic lipid droplets are storage organelles that accumulate hydrophobic molecules. They are found in many tissues and their general structure includes an outer lipid monolayer with integral and associated proteins surrounding a hydrophobic core. Two distinct types can be distinguished, which we define here as oleosin-based lipid droplets (OLDs) and non-oleosin-based lipid droplets (NOLDs). OLDs are the best characterized lipid droplets in plants. They are primarily restricted to seeds and other germinative tissues, their surface is covered with oleosin-family proteins to maintain stability, they store triacylglycerols (TAGs) and they are used as a source of energy (and possibly signaling molecules) during the germination of seeds and pollen. Less is known about NOLDs. They are more abundant than OLDs and are distributed in many tissues, they accumulate not only TAGs but also other hydrophobic molecules such as natural rubber, and the structural proteins that stabilize them are unrelated to oleosins. In many species these proteins are members of the rubber elongation factor superfamily. NOLDs are not typically used for energy storage but instead accumulate hydrophobic compounds required for environmental interactions such as pathogen defense. There are many potential applications of NOLDs including the engineering of lipid production in plants and the generation of artificial oil bodies.

Visualization and analysis of hepatitis C virus structural proteins at lipid droplets by super-resolution microscopy.

Cytosolic lipid droplets are central organelles in the Hepatitis C Virus (HCV) life cycle. The viral capsid protein core localizes to lipid droplets and initiates the production of viral particles at lipid droplet–associated ER membranes. Core is thought to encapsidate newly synthesized viral RNA and, through interaction with the two envelope proteins E1 and E2, bud into the ER lumen. Here, we visualized the spatial distribution of HCV structural proteins core and E2 in vicinity of small lipid droplets by three-color 3D super-resolution microscopy. We observed and analyzed small areas of colocalization between the two structural proteins in HCV-infected cells with a diameter of approximately 100 nm that might represent putative viral assembly sites.

The Epigenetic Drug 5-Azacytidine Interferes with Cholesterol and Lipid Metabolism

DNA methylation and histone acetylation inhibitors are widely used to study the role of epigenetic marks in the regulation of gene expression. In addition, several of these molecules are being tested in clinical trials or already in use in the clinic. Antimetabolites, such as the DNA-hypomethylating agent 5-azacytidine (5-AzaC), have been shown to lower malignant progression to acute myeloid leukemia and to prolong survival in patients with myelodysplastic syndromes. Here we examined the effects of DNA methylation inhibitors on the expression of lipid biosynthetic and uptake genes. Our data demonstrate that, independently of DNA methylation, 5-AzaC selectively and very potently reduces expression of key genes involved in cholesterol and lipid metabolism (e.g. PCSK9, HMGCR, and FASN) in all tested cell lines and in vivo in mouse liver. Treatment with 5-AzaC disturbed subcellular cholesterol homeostasis, thereby impeding activation of sterol regulatory element-binding proteins (key regulators of lipid metabolism). Through inhibition of UMP synthase, 5-AzaC also strongly induced expression of 1-acylglycerol-3-phosphate O-acyltransferase 9 (AGPAT9) and promoted triacylglycerol synthesis and cytosolic lipid droplet formation. Remarkably, complete reversal was obtained by the co-addition of either UMP or cytidine. Therefore, this study provides the first evidence that inhibition of the de novo pyrimidine synthesis by 5-AzaC disturbs cholesterol and lipid homeostasis, probably through the glycerolipid biosynthesis pathway, which may contribute mechanistically to its beneficial cytostatic properties.

575 Intestinal Membrane Transporters Follow the Trail of Fat Into Cytosolic Lipid Droplets During Digestion

Intestinal membrane transporters follow the trail of fat into cytosolic lipid droplets, during digestion. Digestive Disease Week / 102nd Annual AGA meeting

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, ≥10μm(3) 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.

Lipid Droplets as Signaling Platforms Linking Metabolic and Cellular Functions.

The main cells of the adipose tissue of animals, adipocytes, are characterized by the presence of large cytosolic lipid droplets (LDs) that store triglyceride (TG) and cholesterol. However, most cells have LDs and the ability to store lipids. LDs have a well-known central role in storage and provision of fatty acids and cholesterol. However, the complexity of the regulation of lipid metabolism on the surface of the LDs is still a matter of intense study. Beyond this role, a number of recent studies have suggested that LDs have major functions in other cellular processes, such as protein storage and degradation, infection, and immunity. Thus, our perception of LDs has been radically transformed from simple globules of fat to highly dynamic organelles of unexpected complexity. Here, we compiled some recent evidence supporting the emerging view that LDs act as platforms connecting a number of relevant metabolic and cellular functions.

Novel Lipid Droplet–Associated Serine Hydrolase Regulates Macrophage Cholesterol Mobilization

Lipid-laden macrophages or foam cells are characterized by massive cytosolic lipid droplet (LD) deposition containing mostly cholesterol ester (CE) derived from the lipoproteins cleared from the arterial wall. Cholesterol efflux from foam cells is considered to be atheroprotective. Because cholesterol is effluxed as free cholesterol, CE accumulation in LDs may limit free cholesterol efflux. Our objective was to identify proteins that regulate cholesterol trafficking through LDs. In a proteomic analysis of the LD fraction of RAW 264.7 macrophages, we identified an evolutionarily conserved protein with a canonical GXSXG lipase catalytic motif and a predicted α/β-hydrolase fold, the RIKEN cDNA 1110057K04 gene, which we named LD-associated hydrolase (LDAH). LDAH association with LDs was confirmed by immunoblotting and immunocytochemistry. LDAH was labeled with a probe specific for active serine hydrolases. LDAH showed relatively weak in vitro CE hydrolase activity. However, cholesterol measurements in intact cells supported a significant role of LDAH in CE homeostasis because LDAH upregulation and downregulation decreased and increased, respectively, intracellular cholesterol and CE in human embryonic kidney-293 cells and RAW 264.7 macrophages. Mutation of the putative nucleophilic serine impaired active hydrolase probe binding, in vitro CE hydrolase activity, and cholesterol-lowering effect in cells, whereas this mutant still localized to the LD. LDAH upregulation increased CE hydrolysis and cholesterol efflux from macrophages, and, interestingly, LDAH is highly expressed in macrophage-rich areas within mouse and human atherosclerotic lesions. The data identify a candidate target to promote reverse cholesterol transport from atherosclerotic lesions.

Lipid droplet autophagy in the yeastSaccharomyces cerevisiae

Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum-autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast.

Hepatocyte Nuclear Factor 4α and Downstream Secreted Phospholipase A 2 GXIIB Regulate Production of Infectious Hepatitis C Virus

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. The life cycle of HCV is closely associated with the metabolism of lipids, especially very-low-density lipoprotein (VLDL) in hepatocytes. Hepatocyte nuclear factor 4α (HNF4α), the most abundant transcription factor in the liver, regulates the VLDL secretory pathway. However, the effects of HNF4α on the HCV life cycle are unclear. In this study, we investigated the regulatory effects of HNF4α on HCV assembly and secretion. HCV in HNF4α-deficient hepatocytes showed reduced assembly and secretion but unchanged entry and RNA replication. Bezafibrate, a chemical inhibitor of HNF4α, suppressed HCV assembly and secretion. HNF4α downregulation resulted in rearrangement of cytosolic lipid droplets (LDs), as evidenced by the aggregation of large LDs and distorted cytosolic distribution. Phospholipase A2 GXIIB (PLA2GXIIB), an HNF4α-regulated factor involved in VLDL secretion, was found to be crucial in HCV secretion. PLA2GXIIB expression was upregulated in hepatocytes harboring HCV subgenomic replicons or in HCV-infected hepatocytes. This upregulation was transcriptionally controlled in an HNF4α-dependent manner after HCV infection. Furthermore, PLA2GXIIB combined with microsomal triglyceride transfer protein was found to be responsible for the regulation of HNF4α-induced HCV infectivity. These results suggest that HNF4α and its downstream PLA2GXIIB are important factors affecting the late stage of the HCV life cycle and may serve as potential drug targets for the treatment of HCV infection.

Perilipins: Lipid droplet coat proteins adapted for tissue-specific energy storage and utilization, and lipid cytoprotection

Cytosolic lipid storage droplets are primary functional organelles that regulate cellular lipid metabolism and homeostasis. Paradoxically, excess lipid stores are linked to both adaptive (fasting and chronic exercise) and mal-adaptive (obesity and related health complications) conditions. Thus, collective metabolic and physiological processes must balance lipid storage and utilization with prevention of lipocytotoxicity and compounding tissue dysfunctions, urging the need to further define the connection of mammalian lipid droplet function and lipid homeostasis. The perilipins are a multi-protein family that targets lipid droplet surfaces and regulates lipid storage and hydrolysis. Study of perilipin functions has provided insight into the physiological roles of cytosolic lipid droplets and their relationship with obesity-related pathologies. Here, we review the current knowledge of the multiple perilipin proteins in regulating tissue-specific lipid droplets and associations with tissue and systemic energetics.

The effect of simvastatin on lipid droplets accumulation in human embryonic kidney cells and pancreatic cancer cells

BackgroundStatins (HMG-CoA reductase inhibitors) represent a major class of compounds for the treatment of hypercholesterolemia due to their ability to inhibit de novo cholesterol synthesis. In addition to their hypolipidemic effects, chemoprotective properties have been attributed to statins as well. These effects involve multiple mechanisms, which, however, are not known in detail. The aim of our study was to assess in non-malignant as well as cancer cells the impact of simvastatin on the amount of cytosolic lipid droplets (LDs) implicated in many biological processes including proliferation, inflammation, carcinogenesis, apoptosis, necrosis or growth arrest.MethodsHuman embryonic kidney cells HEK-293T and human pancreatic cancer cells MiaPaCa-2 were treated with simvastatin (6 and 12 μM) for 24 and 48 hours respectively. Neutral lipid probe Nile Red was used for detection of LDs by fluorescence microscopy. Cellular cholesterol content was determined by HPLC. Changes in expression of genes related to lipid metabolism in simvastatin-treated MiaPaCa-2 cells were examined by DNA microarray analysis. Validation of gene expression changes was performed using quantitative RT-PCR.ResultsThe treatment of the cells with simvastatin increased their intracellular content of LDs in both non-malignant as well as cancer cells, partially due to the uptake of cholesterol and triacylglyceroles from medium; but in particular, due to enhanced synthesis of triacylglyceroles as proved by significant overexpression of genes related to de novo synthesis of triacylglyceroles and phospholipids. In addition, simvastatin also markedly influenced expression of genes directly affecting cell proliferation and signaling.ConclusionsSimvastatin treatment led to accumulation of cytosolic LDs within the examined cells, a phenomenon which might contribute to the antiproliferative effects of statins.

Crucial Role of Perilipin-3 (TIP47) in Formation of Lipid Droplets and PGE2 Production in HL-60-Derived Neutrophils

Cytosolic lipid droplets (LDs), which are now recognized as multifunctional organelles, accumulate in leukocytes under various inflammatory conditions. However, little is known about the characteristic features of LDs in neutrophils. In this study, we show that perilipin-3 (PLIN3; formerly called TIP47) is involved in LD formation and the inflammatory response in HL-60-derived neutrophils. HL-60, a promyelocytic cell line, was differentiated into neutrophils via treatment with all-trans retinoic acid. After differentiation, cells were stimulated with Porphyromonas gingivalis lipopolysaccharide (P.g-LPS), a major pathogen in adult periodontitis. When HL-60-derived neutrophils were stimulated with P.g-LPS, LDs increased in both number and size. In the differentiated cells, PLIN3 was induced while PLIN1, PLIN2 and PLIN5 were not detected. PGE2 production and the PLIN3 protein level were increased by the P.g-LPS treatment of the cells in a dose-dependent manner. When PLIN3 was down-regulated with siRNA treatment, LDs essentially disappeared and the level of PGE2 secreted in the cell culture medium decreased by 65%. In addition, the suppression of PLIN3 repressed the PGE2 producing enzymes; i.e., microsomal PGE synthase-1, -2 and cyclooxygenase-2. These findings indicate that PLIN3 has a pivotal role in LD-biogenesis in HL-60-derived neutrophils, and that PLIN3 is associated with the synthesis and secretion of PGE2.

Deficiency of liver Comparative Gene Identification-58 causes steatohepatitis and fibrosis in mice

Triglyceride (TG) accumulation in hepatocytes (hepatic steatosis) preludes the development of advanced nonalcoholic fatty liver diseases (NAFLDs) such as steatohepatitis, fibrosis, and cirrhosis. Mutations in human Comparative Gene Identification-58 (CGI-58) cause cytosolic TG-rich lipid droplets to accumulate in almost all cell types including hepatocytes. However, it is unclear if CGI-58 mutation causes hepatic steatosis locally or via altering lipid metabolism in other tissues. To directly address this question, we created liver-specific CGI-58 knockout (LivKO) mice. LivKO mice on standard chow diet displayed microvesicular and macrovesicular panlobular steatosis, and progressed to advanced NAFLD stages over time, including lobular inflammation and centrilobular fibrosis. Compared with CGI-58 floxed control littermates, LivKO mice showed 8-fold and 52-fold increases in hepatic TG content, which was associated with 40% and 58% decreases in hepatic TG hydrolase activity at 16 and 42 weeks, respectively. Hepatic cholesterol also increased significantly in LivKO mice. At 42 weeks, LivKO mice showed increased hepatic oxidative stress, plasma aminotransferases, and hepatic mRNAs for genes involved in fibrosis and inflammation, such as α-smooth muscle actin, collagen type 1 α1, tumor necrosis factor α, and interleukin-1β. In conclusion, CGI-58 deficiency in the liver directly causes not only hepatic steatosis but also steatohepatitis and fibrosis.