Accumulation Of Phosphatidylcholine Research Articles

Juvenile hormone-induced microRNA miR-iab-8 regulates lipid homeostasis and metamorphosis in Drosophila melanogaster.

Metamorphosis plays an important role in the evolutionary success of insects. Accumulating evidence indicated that microRNAs (miRNAs) are involved in the regulation of processes associated with insect metamorphosis. However, the miRNAs coordinated with juvenile hormone (JH)-regulated metamorphosis remain poorly reported. In the present study, using high-throughput miRNA sequencing combined with Drosophila genetic approaches, we demonstrated that miR-iab-8, which primarily targets homeotic genes to modulate haltere-wing transformation and sterility was up-regulated by JH and involved in JH-mediated metamorphosis. Overexpression of miR-iab-8 in the fat body resulted in delayed development and failure of larval-pupal transition. Furthermore, metabolomic analysis results revealed that overexpression of miR-iab-8 caused severe energy metabolism defects especially the lipid metabolism, resulting in significantly reduced triacylglycerol (TG) content and glycerophospholipids but enhanced accumulation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In line with this, Nile red staining demonstrated that during the third larval development, the TG content in the miR-iab-8 overexpression larvae was continuously decreased, which is opposite to the control. Additionally, the transcription levels of genes committed to TG synthesis and breakdown were found to be significantly increased and the expression of genes responsible for glycerophospholipids metabolism were also altered. Overall, we proposed that JH induced miR-iab-8 expression to perturb the lipid metabolism homeostasis especially the TG storage in the fat body, which in turn affected larval growth and metamorphosis.

Annexin A2 modulates phospholipid membrane composition upstream of Arp2 to control angiogenic sprout initiation.

The intersection of protein and lipid biology is of growing importance for understanding how cells address structural challenges during adhesion and migration. While protein complexes engaged with the cytoskeleton play a vital role, support from the phospholipid membrane is crucial for directing localization and assembly of key protein complexes. During angiogenesis, dramatic cellular remodeling is necessary for endothelial cells to shift from a stable monolayer to invasive structures. However, the molecular dynamics between lipids and proteins during endothelial invasion are not defined. Here, we utilized cell culture, immunofluorescence, and lipidomic analyses to identify a novel role for the membrane binding protein Annexin A2 (ANXA2) in modulating the composition of specific membrane lipids necessary for cortical F-actin organization and adherens junction stabilization. In the absence of ANXA2, there is disorganized cortical F-actin, reduced junctional Arp2, excess sprout initiation, and ultimately failed sprout maturation. Furthermore, we observed reduced filipin III labeling of membrane cholesterol in cells with reduced ANXA2, suggesting there is an alteration in phospholipid membrane dynamics. Lipidomic analyses revealed that 42 lipid species were altered with loss of ANXA2, including an accumulation of phosphatidylcholine (16:0_16:0). We found that supplementation of phosphatidylcholine (16:0_16:0) in wild-type endothelial cells mimicked the ANXA2 knock-down phenotype, indicating that ANXA2 regulated the phospholipid membrane upstream of Arp2 recruitment and organization of cortical F-actin. Altogether, these data indicate a novel role for ANXA2 in coordinating events at endothelial junctions needed to initiate sprouting and show that proper lipid modulation is a critical component of these events.

PITPNC1 promotes the thermogenesis of brown adipose tissue under acute cold exposure

Brown adipose tissue (BAT) plays an essential role in non-shivering thermogenesis. The phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) is identified as a lipid transporter that reciprocally transfers phospholipids between intracellular membrane structures. However, the physiological significance of PITPNC1 and its regulatory mechanism remain unclear. Here, we demonstrate that PITPNC1 is a key player in thermogenesis of BAT. While Pitpnc1−/− mice do not differ with wildtype mice in body weight and insulin sensitivity on either chow or high-fat diet, they develop hypothermia when subjected to acute cold exposure at 4°C. The Pitpnc1−/− brown adipocytes exhibit defective β-oxidation and abnormal thermogenesis-related metabolism pathways in mitochondria. The deficiency of lipid mobilization in Pitpnc1−/− brown adipocytes might be the result of excessive accumulation of phosphatidylcholine and a reduction of phosphatidic acid. Our findings have uncovered significant roles of PITPNC1 in mitochondrial phospholipid homeostasis and BAT thermogenesis.

Does administration of hydroxychloroquine/amiodarone accelerate accumulation of globotriaosylceramide and globotriaosylsphingosine in Fabry mice?

Drug-induced lysosomal storage disease (DILSD) caused by cationic amphiphilic drugs (CADs), which exhibits toxic manifestations and pathological findings mimicking Fabry disease (α-galactosidase A deficiency), has attracted the interests of clinicians and pathologists. Although the affected region is lysosomes in both the diseases, DILSD is characterized by intralysosomal accumulation of phospholipids and Fabry disease that of globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). However, it is unknown whether administration of CADs affects the catabolism of Gb3 and Lyso-Gb3 in Fabry disease. In this study, we independently administered hydroxychloroquine/amiodarone to wild-type and Fabry mice and examined the effects of the drugs on the enzyme activity and substrates accumulated in organs and tissues. The results revealed that the administration of the drugs induced accumulation of phosphatidylcholine in both the wild-type and Fabry mice. However, reduction of α-galactosidase A activity in the organs and tissues of the wild-type mice was not found, and the storage of Gb3 and Lyso-Gb3 was not accelerated by these drugs in the Fabry mice. This suggests that hydroxychloroquine/amiodarone do not have any significant impact on the catabolism of Gb3 and Lyso-Gb3 in organs and tissues of both wild-type and Fabry mice.

The TOC159 null mutant of Arabidopsis thaliana is impaired in the accumulation of plastid lipids and phosphatidylcholine

We used electrospray ionization tandem mass spectrometry to profile glycerolipids in the TOC159 null mutant of Arabidopsis, which is referred to as plastid protein import 2, or ppi2. The goal was to evaluate the impact of a defective atToc159 receptor in the accumulation of plastid lipids. The ppi2 mutant is severely impaired in the accumulation of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and phosphatidylglycerol (PG), which are major components of the thylakoid membranes. Major molecular species of MGDG and DGDG are drastically decreased, which is consistent with our previous findings of decreased levels of hexadecatrienoic and linolenic acids. Under normal growth conditions, the ppi2 mutant accumulated significantly lower levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). In the cold-acclimated mutant, the amounts of PE and PI were similar to the wildtype level, which indicates that the ER pathway of lipid synthesis was functional in the mutant. The cold-acclimated ppi2 mutant accumulated increased amounts of phosphatidic acid (PA), which was mirrored by an increase in phospholipase Dα (PLDα) transcript levels. These data suggest that PLDα activity contributed to the accumulation of cold-induced PA in the ppi2 mutant. The accumulation of major molecular species in PA indicates that cold-induced PA originated from the degradation of both plastidial and extraplastidial lipids. Compared with the wildtype, the ppi2 mutant had a low double bond index and high acyl chain length, which is indicative of decreased membrane fluidity. Taken together, these data indicate that a defective atToc159 receptor severely impaired the plastid pathway of lipid synthesis, which negatively affected the synthesis and/or accumulation of PC.

Up-regulation of lipid metabolism and glycine betaine synthesis are associated with choline-induced salt tolerance in halophytic seashore paspalum.

Choline may affect salt tolerance by regulating lipid and glycine betaine (GB) metabolism. This study was conducted to determine whether alteration of lipid profiles and GB metabolism may contribute to choline regulation and genotypic variations in salt tolerance in a halophytic grass, seashore paspalum (Paspalum vaginatum). Plants of Adalayd and Sea Isle 2000 were subjected to salt stress (200-mM NaCl) with or without foliar application of choline chloride (1mM). Genotypic variations in salt tolerance and promotive effects of choline application on salt tolerance were associated with both the up-regulation of lipid metabolism and GB synthesis. The genotypic variations in salt tolerance associated with lipid metabolism were reflected by the differential accumulation of phosphatidylcholine and phosphatidylethanolamine between Adalayd and Sea Isle 2000. Choline-induced salt tolerance was associated with of the increase in digalactosyl diacylglycerol (DGDG) content including DGDG (36:4 and 36:6) in both cultivars of seashore paspalum and enhanced synthesis of phosphatidylinositol (34:2, 36:5, and 36:2) and phosphatidic acid (34:2, 34:1, and 36:5), as well as increases in the ratio of digalactosyl diacylglycerol: monogalactosyl diacylglycerol (DGDG:MGDG) in salt-tolerant Sea Isle 2000. Choline regulation of salt tolerance may be due to the alteration in lipid metabolism in this halophytic grass species.

PARL partitions the lipid transfer protein STARD7 between the cytosol and mitochondria.

Intramembrane-cleaving peptidases of the rhomboid family regulate diverse cellular processes that are critical for development and cell survival. The function of the rhomboid protease PARL in the mitochondrial inner membrane has been linked to mitophagy and apoptosis, but other regulatory functions are likely to exist. Here, we identify the START domain-containing protein STARD7 as an intramitochondrial lipid transfer protein for phosphatidylcholine. We demonstrate that PARL-mediated cleavage during mitochondrial import partitions STARD7 to the cytosol and the mitochondrial intermembrane space. Negatively charged amino acids in STARD7 serve as a sorting signal allowing mitochondrial release of mature STARD7 upon cleavage by PARL On the other hand, membrane insertion of STARD7 mediated by the TIM23 complex promotes mitochondrial localization of mature STARD7. Mitochondrial STARD7 is necessary and sufficient for the accumulation of phosphatidylcholine in the inner membrane and for the maintenance of respiration and cristae morphogenesis. Thus, PARL preserves mitochondrial membrane homeostasis via STARD7 processing and is emerging as a critical regulator of protein localization between mitochondria and the cytosol.

Hepatic Steatosis Accompanies Pulmonary Alveolar Proteinosis.

Maintenance of tissue-specific organ lipid compositions characterizes mammalian lipid homeostasis. The lungs and liver synthesize mixed phosphatidylcholine (PC) molecular species that are subsequently tailored for function. The lungs progressively enrich disaturated PC directed to lamellar body surfactant stores before secretion. The liver accumulates polyunsaturated PC directed to very-low-density lipoprotein assembly and secretion, or to triglyceride stores. In each tissue, selective PC species enrichment mechanisms lie at the heart of effective homeostasis. We tested for potential coordination between these spatially separated but possibly complementary phenomena under a major derangement of lung PC metabolism, pulmonary alveolar proteinosis (PAP), which overwhelms homeostasis and leads to excessive surfactant accumulation. Using static and dynamic lipidomics techniques, we compared (1) tissue PC compositions and contents, and (2) in lungs, the absolute rates of synthesis in both control mice and the granulocyte-macrophage colony-stimulating factor knockout model of PAP. Significant disaturated PC accumulation in bronchoalveolar lavage fluid, alveolar macrophage, and lavaged lung tissue occurred alongside increased PC synthesis, consistent with reported defects in alveolar macrophage surfactant turnover. However, microscopy using oil red O staining, coherent anti-Stokes Raman scattering, second harmonic generation, and transmission electron microscopy also revealed neutral-lipid droplet accumulations in alveolar lipofibroblasts of granular macrophage colony-stimulating factor knockout animals, suggesting that lipid homeostasis deficits extend beyond alveolar macrophages. PAP plasma PC composition was significantly polyunsaturated fatty acid enriched, but the content was unchanged and hepatic polyunsaturated fatty acid-enriched PC content increased by 50% with an accompanying micro/macrovesicular steatosis and a fibrotic damage pattern consistent with nonalcoholic fatty liver disease. These data suggest a hepatopulmonary axis of PC metabolism coordination, with wider implications for understanding and managing lipid pathologies in which compromise of one organ has unexpected consequences for another.

Accumulation of phosphatidylcholine on gut mucosal surface is not dominated by electrostatic interactions

The accumulation of phosphatidylcholine (PC) in the intestinal mucus layer is crucial for the protection of colon epithelia from the bacterial attack. It has been reported that the depletion of PC is a distinct feature of ulcerative colitis. Here we addressed the question how PC interacts with its binding proteins, the mucins, which may establish the hydrophobic barrier against colonic microbiota. In the first step, the interactions of dioleoylphosphatidylcholine (DOPC) with two mucin preparations from porcine stomach, have been studied using dynamic light scattering, zeta potential measurement, and Langmuir isotherms, suggesting that mucin binds to the surface of DOPC vesicles. The enthalpy of mucin-PC interaction could be determined by isothermal titration calorimetry. The high affinity to PC found for both mucin types seems reasonable, as they mainly consist of mucin 2, a major constituent of the flowing mucus. Moreover, by the systematic variation of net charges, we concluded that the zwitterionic DOPC has the strongest binding affinity that cannot be explained within the electrostatic interactions between charged molecules.

PHOSPHATIDIC ACID PHOSPHOHYDROLASE Regulates Phosphatidylcholine Biosynthesis in Arabidopsis by Phosphatidic Acid-Mediated Activation of CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE Activity.

Regulation of membrane lipid biosynthesis is critical for cell function. We previously reported that disruption of PHOSPHATIDIC ACID PHOSPHOHYDROLASE1 (PAH1) and PAH2 stimulates net phosphatidylcholine (PC) biosynthesis and proliferation of the endoplasmic reticulum (ER) in Arabidopsis thaliana. Here, we show that this response is caused specifically by a reduction in the catalytic activity of the protein and positively correlates with an accumulation of its substrate, phosphatidic acid (PA). The accumulation of PC in pah1 pah2 is suppressed by disruption of CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE1 (CCT1), which encodes a key enzyme in the nucleotide pathway for PC biosynthesis. The activity of recombinant CCT1 is stimulated by lipid vesicles containing PA. Truncation of CCT1, to remove the predicted C-terminal amphipathic lipid binding domain, produced a constitutively active enzyme. Overexpression of native CCT1 in Arabidopsis has no significant effect on PC biosynthesis or ER morphology, but overexpression of the truncated constitutively active version largely replicates the pah1 pah2 phenotype. Our data establish that membrane homeostasis is regulated by lipid composition in Arabidopsis and reveal a mechanism through which the abundance of PA, mediated by PAH activity, modulates CCT activity to govern PC content.

P4-05-05: Imaging Mass Spectrometry Based Lipid Metabolites Analysis for Breast Cancer.

Abstract Background: Activation of lipid metabolism is an early event of carcinogenesis and a central hallmark of many cancers including breast cancer. Recent findings argue that stearoyl CoA desaturase-1 (SCD1), a key regulator of the fatty acid (FA) composition and the endoplasmic reticulum resident enzyme that converts saturated FA (SFA) into monounsaturated FA (MUFA) is a novel regulator of carcinogenesis. The distinctive lipids composition of membrane in cancer cells and the biological functions of SCD1, however, still remain uncertain. Imaging mass spectrometry (IMS) is a mass spectrometry-based analyzing technique that enables visualization of the individual molecules without requiring antibodies. It allows comprehensive detection of a wide range of biomolecules, such as lipids. We attempted to visualize the localization of lipids in breast cancer by IMS for better understanding of cancer proliferation. Materials and methods: 13 specimens were obtained from the primary breast cancer patients. All were Japanese woman and aged 41–86 years (mean 61.5y.o.). Only one patient received preoperative systematic therapy. 6 were estrogen receptor (ER) and/or progesterone receptor (PgR) positive and human epidermal growth factor receptor 2 (HER2) negative, 2 were ER and/or PgR positive and HER2 positive, 2 were both ER and PgR negative and HER2 positive and 2 were triple negative. IMS: Samples were immediately chilled in liquid Hexan and stored at −80°. All specimens were sliced into 10 mm thin sections, mounted onto one indium-tin oxide-coated glass slides (Bruker Daltonics) and then sprayed by 2,5-Dihydroxybenzoic acid. Matrix assisted laser desorption ionization (MALDI) technique was used as a soft ionization method. We used time of flight (TOF)/TOF type instrument (Ultraflex, Bruker Daltonics) and all the spectrum were acquired automatically using Fleximaging software (Bruker Daltonics). Each spectral intensity at any mass-to-charge ratio (m/z) was measured at 16 regions of interest (ROI); 13 ROI were picked up from cancerous parts and 3 were from non-cancerous parts. Spectral intensities were compared and statistical analysis was performed by Mann Whitney test. The software was also used to create two-dimensional ion-density maps. Results: In the cancerous parts of all the 13 specimens, two distinct peaks of the molecular ions were detected at m/z 798.5 and 810.5, which were not found in the non-cancerous parts. Median intensity of the molecular ions at m/z 798.5 and 810.5 were 38.9 and 3.18 in the cancerous part, while they were 0.84 and 1.02 in the non-cancerous part (p=0.010 and 0.015, respectively). Tandem mass spectrometry analysis for these two molecules revealed that they were two kinds of phosphatidylcholine (PC), PC (16:0/18:1) and PC (18:0/18:1). Localization of the individual PC was visualized by means of IMS, which showed that in cancerous part accumulation of PCs containing MUFA was more pronounced than those containing SFA only. Conclusion: Two kinds of PC containing MUFA were found to highly accumulate in cancerous parts, which may suggest involvement of SCD1 in the membrane composition regulation and cancer proliferation. Further studies may thus be warranted to explore the relation between PC localization and the SCD1 expression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-05-05.

Lipid content disturbance in the arbuscular mycorrhizal, Glomus irregulare grown in monoxenic conditions under PAHs pollution

Most polycyclic aromatic hydrocarbons (PAHs) are ubiquitous natural and/or anthropogenic pollutants that have adverse effects on the human health and the environment. Little is known about their potential effects on arbuscular mycorrhizal fungi (AMF). Thus, using monoxenic cultures, this work aims to study the impact of increasing concentrations (140 and 280 μM) of two PAHs [anthracene and benzo[a]pyrene (B[a]P)] on Glomus irregulare lipid content in relation with its development. Changes in the total lipids [fatty acids (FA), sterols, phospholipids (PL) and their associated FA (PLFA)] compositions and contents as well as [malondialdehyde (MDA)] production, of the AMF G. irregulare were examined. Direct toxic effects of both PAHs on the AMF were shown as compared to the control culture. The extraradical hyphae length and spore production were drastically restricted in the presence of PAHs. Significant decreases of the main membrane constituents, phosphatidylcholine (PC) and sterols (in particular 24-methycholesterol) were shown in G. irregulare grown under PAHs treatment. Moreover, PAHs exposure caused an oxidative stress in the AMF extraradical structures pointed out by an increase of the lipid peroxidation biomarker production (MDA). All the observed changes were less marked in presence of anthracene, which was found to be less toxic than B[a]P. Taken together, our results suggested that the drastic decrease of the AMF growth under PAHs pollution could partially be explained by depletions in sterols, PC and MDA accumulation.

Imaging Mass Spectrometry Reveals Unique Lipid Distribution in Primary Varicose Veins

Background The lipid metabolism of varicose veins (VVs) remains unknown. To elucidate the pathogenesis of VV, we utilized the novel technique of imaging mass spectrometry (IMS). Materials and methods We obtained VV tissues from 10 limbs of 10 VV patients who underwent great saphenous vein stripping. As control vein samples, we harvested segmental vein tissues from 6 limbs of 6 patients with peripheral artery occlusive disease who underwent infra-inguinal bypass with reversed saphenous vein grafting. To identify the localisation of lipid molecules in the VV tissues, we performed matrix-assisted laser desorption/ionization IMS (MALDI-IMS). We also performed MS/MS analyses to identify the structure of each molecule. Results We obtained mass spectra directly from control vein tissues and VV tissues and found a unique localisation of lipid molecules in the VV tissues. We localised lysophosphatidylcholine (LPC) (1-acyl 16:0), phosphatidylcholine (PC) (1-acyl 36:4) and sphingomyelin (SM) (d18:1/16:0) at the site of the VV valve. Conclusion MALDI-IMS revealed the distribution of various lipid molecules in normal veins and VVs both. Accumulation of LPC (1-acyl 16:0), PC (1-acyl 36:4) and SM (d18:1/16:0) in the VV tissues suggested that inflammation associated with abnormal lipid metabolism may contribute to the development of VV.

Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication

BackgroundCellular membranes are crucial host components utilized by positive-strand RNA viruses for replication of their genomes. Published studies have suggested that the synthesis and distribution of membrane lipids are particularly important for the assembly and function of positive-strand RNA virus replication complexes. However, the impact of specific lipid metabolism pathways in this process have not been well defined, nor have potential changes in lipid expression associated with positive-strand RNA virus replication been examined in detail.ResultsIn this study we used parallel and complementary global and targeted approaches to examine the impact of lipid metabolism on the replication of the well-studied model alphanodavirus Flock House virus (FHV). We found that FHV RNA replication in cultured Drosophila S2 cells stimulated the transcriptional upregulation of several lipid metabolism genes, and was also associated with increased phosphatidylcholine accumulation with preferential increases in lipid molecules with longer and unsaturated acyl chains. Furthermore, targeted RNA interference-mediated downregulation of candidate glycerophospholipid metabolism genes revealed a functional role of several genes in virus replication. In particular, we found that downregulation of Cct1 or Cct2, which encode essential enzymes for phosphatidylcholine biosynthesis, suppressed FHV RNA replication.ConclusionThese results indicate that glycerophospholipid metabolism, and in particular phosphatidylcholine biosynthesis, plays an important role in FHV RNA replication. Furthermore, they provide a framework in which to further explore the impact of specific steps in lipid metabolism on FHV replication, and potentially identify novel cellular targets for the development of drugs to inhibit positive-strand RNA viruses.

Depletion of the Membrane-Associated Acyl-Coenzyme A-Binding Protein ACBP1 Enhances the Ability of Cold Acclimation in Arabidopsis

In Arabidopsis (Arabidopsis thaliana), a family of six genes encodes acyl-coenzyme A-binding proteins (ACBPs). A member of this family, ACBP1, contains an amino-terminal transmembrane domain that targets it to the plasma membrane and the endoplasmic reticulum. To investigate ACBP1 function, ACBP1-overexpressing transgenic Arabidopsis plants were characterized using lipid analysis. ACBP1 overexpressors showed reduction in several species of diunsaturated phosphatidylcholine (PC), prompting us to investigate if they were altered in response to freezing stress. ACBP1 overexpressors demonstrated increased freezing sensitivity accompanied by a decrease in PC and an increase in phosphatidic acid (PA), while acbp1 mutant plants showed enhanced freezing tolerance associated with PC accumulation and PA reduction. We also showed binding of a recombinant eukaryotic ACBP (ACBP1) to PA, indicative of the possibility of enhanced PA interaction in ACBP1 overexpressors. Since phospholipase Dalpha1 (PLDalpha1) is a major enzyme promoting the hydrolysis of PC to PA, PLDalpha1 expression was examined and was observed to be higher in ACBP1 overexpressors than in acbp1 mutant plants. In contrast, the expression of PLDdelta, which plays a positive role in freezing tolerance, declined in the ACBP1 overexpressors but increased in acbp1 mutant plants. Given that ACBP1 is localized to the endoplasmic reticulum and plasma membrane, it may regulate the expression of PLDalpha1 and PLDdelta by maintaining a membrane-associated PA pool through its ability to bind PA. Moreover, both genotypes showed no alterations in proline and soluble sugar content or in cold-regulated (COR6.6 and COR47) gene expression, suggesting that the ACBP1-mediated response is PLD associated and is independent of osmolyte accumulation.

Cell Wall Integrity MAPK Pathway Is Essential for Lipid Homeostasis

The highly conserved yeast cell wall integrity mitogen-activated protein kinase pathway regulates cellular responses to cell wall and membrane stress. We report that this pathway is activated and essential for viability under growth conditions that alter both the abundance and pattern of synthesis and turnover of membrane phospholipids, particularly phosphatidylinositol and phosphatidylcholine. Mutants defective in this pathway exhibit a choline-sensitive inositol auxotrophy, yet fully derepress INO1 and other Opi1p-regulated genes when grown in the absence of inositol. Under these growth conditions, Mpk1p is transiently activated by phosphorylation and stimulates the transcription of known targets of Mpk1p signaling, including genes regulated by the Rlm1p transcription factor. mpk1Delta cells also exhibit severe defects in lipid metabolism, including an abnormal accumulation of phosphatidylcholine, diacylglycerol, triacylglycerol, and free sterols, as well as aberrant turnover of phosphatidylcholine. Overexpression of the NTE1 phospholipase B gene suppresses the choline-sensitive inositol auxotrophy of mpk1Delta cells, whereas overexpression of other phospholipase genes has no effect on this phenotype. These results indicate that an intact cell wall integrity pathway is required for maintaining proper lipid homeostasis in yeast, especially when cells are grown in the absence of inositol.

The Secretion and Uptake of Lysosomal Phospholipase A2 by Alveolar Macrophages

Macrophages have long been known to secrete a Phospholipase A(2) with an acidic pH optimum in response to phagocytic stimuli. However, the enzyme or enzymes responsible for this activity have not been identified. We report that mouse alveolar macrophages release lysosomal phospholipase A(2) (LPLA(2)) into the medium of cultured cells following stimulation with zymosan. The release of the enzyme was detected by enzymatic activity assays as well as by Western blotting using an Ab against mouse LPLA(2). LPLA(2) is a high mannose type glycoprotein found in lysosomes, suggesting that the released enzyme might be reincorporated into alveolar macrophages via a mannose or mannose phosphate receptor. Recombinant glycosylated mouse LPLA(2) produced by HEK293 cells was applied to LPLA(2)-deficient (LPLA(2)(-/-)) mouse alveolar macrophages. The uptake of exogenous LPLA(2) into LPLA(2)(-/-) alveolar macrophages occurred in a concentration-dependent manner. The LPLA(2) taken into the alveolar macrophages colocalized with the lysosomal marker, Lamp-1. This uptake was significantly suppressed in the presence of alpha-methyl-mannoside but not in the presence of mannose 6-phosphate. Thus, the predominant pathway for uptake of exogenous LPLA(2) is via the mannose receptor, with subsequent translocation into acidic, Lamp-1-associated compartments. LPLA(2)(-/-) alveolar macrophages are characterized by marked accumulation of phosphatidylcholine and phosphatidylethanolamine. Treatment with the recombinant LPLA(2) rescued the LPLA(2)(-/-) alveolar macrophages by markedly decreasing the phospholipid accumulation. The application of a catalytically inactive LPLA(2) revealed that the enzymatic activity of LPLA(2) was required for the phospholipid reduction. These studies identify LPLA(2) as a high m.w.-secreted Phospholipase A(2).

Positional specificity of lysosomal phospholipase A2

Lysosomal phospholipase A(2) (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A(1) and phospholipase A(2) activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.

Cell Cycle Dependence of Group VIA Calcium-independent Phospholipase A2 Activity

Homeostasis of phosphatidylcholine (PC) is regulated by the opposing actions between CTP:phosphocholine cytidylyltransferase (CT) and the group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)). We investigated this process during the cell cycle. PC mass doubles during late G(1) and early S phase when its rate of catabolism is lowest. We show that iPLA(2) activity is cell cycle-dependent with peak activity during G(2)/M and late S phase. iPLA(2) activity declines during G(1) and is lowest at the G(1)/S transition and early S phase. The accumulation of PC correlates with decreased iPLA(2) activity, suggesting that regulation of this enzyme contributes to phospholipid accumulation. The levels of 80 kDa iPLA(2) protein do not change and thus cannot account for changes in enzyme activity. Reverse transcriptase and real-time PCR experiments show that splice variant iPLA(2) mRNAs are preferentially expressed during G(2)/M. Immunoblot analyses with an antibody directed against the N terminus of iPLA(2) revealed a approximately 50 kDa protein that is of appropriate size to be the truncated protein encoded by the ankyrin-iPLA(2)-1 splice variant mRNA. The levels of truncated iPLA(2) protein were high in cells in late G(1) and S phase cells that had low iPLA(2) activity and low in G(2)/M cells that had high iPLA(2) activity. The truncated protein co-immunoprecipitated with full-length iPLA(2), indicating a physical interaction between the two proteins. Together, these data suggest that truncated iPLA(2) proteins associate with active iPLA(2) and down-regulate its activity during G(1). This down-regulation may contribute to phospholipid accumulation during the cell cycle.

Bezafibrate stimulates canalicular localization of NBD-labeled PC in HepG2 cells by PPARα-mediated redistribution of ABCB4

Fibrates, including bezafibrate (BF), upregulate the expression of ATP binding cassette protein B4 (ABCB4) through gene transcription in mice. To determine the effects of BF on the expression levels of ABCB4 and on the stimulation of biliary phosphatidylcholine (PC) transport in human HepG2 hepatoblastoma cells, mRNA and protein levels as well as subcellular localization were investigated in the cells treated with BF. The canalicular accumulation of a fluorescent PC was assessed by confocal laser scanning microscopy. Treatment with 300 micromol/l BF for 24 h increased levels of ABCB4 mRNA but not protein by up to 151%. BF caused redistribution of ABCB4 into pseudocanaliculi formed between cells. In association with this redistribution, BF accelerated the accumulation of fluorescent PC in bile canaliculi (up to 163% of that in nontreated cells). Suppression of peroxisome proliferator-activated receptor alpha (PPARalpha) expression by either a small interfering RNA duplex or morpholino antisense oligonucleotide attenuated the BF-induced redistribution of ABCB4. These findings suggest that BF may enhance the capacity of human hepatocytes to direct PC into bile canaliculi via PPARalpha-mediated redistribution of ABCB4 to the canalicular membrane. This provides a rationale for the use of BF to improve cholestasis and/or cholangitis that is attributable to hypofunction of ABCB4.