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• W2944828463 abstract "In mammals, lipids are selectively transported to specific sites using multiple classes of lipoproteins. However, in Drosophila, a single class of lipoproteins, lipophorin, carries more than 95% of the lipids in the hemolymph. Although a unique ability of the insect lipoprotein system for cargo transport has been demonstrated, it remains unclear how this single class of lipoproteins selectively transports lipids. In this study, we carried out a comparative analysis of the fatty-acid composition among lipophorin, the CNS, and CNS-derived cell lines and investigated the transport mechanism of fatty acids, particularly focusing on the transport of PUFAs in Drosophila. We showed that PUFAs are selectively incorporated into the acyl chains of lipophorin phospholipids and effectively transported to CNS through lipophorin receptor-mediated endocytosis of lipophorin. In addition, we demonstrated that C14 fatty acids are selectively incorporated into the diacylglycerols (DAGs) of lipophorin and that C14 fatty-acid-containing DAGs are spontaneously transferred from lipophorin to the phospholipid bilayer. These results suggest that PUFA-containing phospholipids and C14 fatty-acid-containing DAGs in lipophorin could be transferred to different sites by different mechanisms to selectively transport fatty acids using a single class of lipoproteins. In mammals, lipids are selectively transported to specific sites using multiple classes of lipoproteins. However, in Drosophila, a single class of lipoproteins, lipophorin, carries more than 95% of the lipids in the hemolymph. Although a unique ability of the insect lipoprotein system for cargo transport has been demonstrated, it remains unclear how this single class of lipoproteins selectively transports lipids. In this study, we carried out a comparative analysis of the fatty-acid composition among lipophorin, the CNS, and CNS-derived cell lines and investigated the transport mechanism of fatty acids, particularly focusing on the transport of PUFAs in Drosophila. We showed that PUFAs are selectively incorporated into the acyl chains of lipophorin phospholipids and effectively transported to CNS through lipophorin receptor-mediated endocytosis of lipophorin. In addition, we demonstrated that C14 fatty acids are selectively incorporated into the diacylglycerols (DAGs) of lipophorin and that C14 fatty-acid-containing DAGs are spontaneously transferred from lipophorin to the phospholipid bilayer. These results suggest that PUFA-containing phospholipids and C14 fatty-acid-containing DAGs in lipophorin could be transferred to different sites by different mechanisms to selectively transport fatty acids using a single class of lipoproteins. In mammals, a wide range of lipids is selectively transported between tissues in the form of lipoproteins that are composed mainly of apolipoproteins and cargo lipids [e.g., cholesteryl ester, cholesterol, triacylglycerol (TAG), phospholipids, and lipophilic vitamins]. Multiple classes of lipoproteins are available for selectively transporting the various lipids. For example, VLDLs typically transport TAG to peripheral tissues, while LDLs and HDLs mainly transport cholesteryl ester between the liver and peripheral tissues (1Rosenson R.S. Brewer Jr., H.B. Davidson W.S. Fayad Z.A. Fuster V. Goldstein J. Hellerstein M. Jiang X.C. Phillips M.C. Rader D.J. et al.Cholesterol efflux and atheroprotection: advancing the concept of reverse cholesterol transport.Circulation. 2012; 125: 1905-1919Crossref PubMed Scopus (681) Google Scholar, 2Nagao K. Tomioka M. Ueda K. Function and regulation of ABCA1–membrane meso-domain organization and reorganization.FEBS J. 2011; 278: 3190-3203Crossref PubMed Scopus (45) Google Scholar, 3Hassing H.C. Surendran R.P. Mooij H.L. Stroes E.S. Nieuwdorp M. Dallinga-Thie G.M. Pathophysiology of hypertriglyceridemia.Biochim. Biophys. Acta. 2012; 1821: 826-832Crossref PubMed Scopus (58) Google Scholar). To selectively transport the lipoprotein lipids to specific tissues or cells, several types of receptors and lipid-catabolizing enzymes exist at the cell surface and in the circulation. For example, LDL is recognized by the cell-surface LDL receptor (LDLR) and is endocytosed to take up cargo lipids in the liver and peripheral tissues (4Goldstein J.L. Brown M.S. A century of cholesterol and coronaries: from plaques to genes to statins.Cell. 2015; 161: 161-172Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar), while the cargo lipids of HDL are remodeled in the circulation and transported to the liver via scavenger receptor class B type I (5Lewis G.F. Rader D.J. New insights into the regulation of HDL metabolism and reverse cholesterol transport.Circ. Res. 2005; 96: 1221-1232Crossref PubMed Scopus (824) Google Scholar). Thus, specific lipids can be selectively transported to specific sites using the multiple classes of lipoproteins present in mammals. On the other hand, a single class of lipoproteins, lipophorin, carries more than 95% of the lipids in the hemolymph of Drosophila melanogaster (6Palm W. Sampaio J.L. Brankatschk M. Carvalho M. Mahmoud A. Shevchenko A. Eaton S. Lipoproteins in Drosophila melanogaster–assembly, function, and influence on tissue lipid composition.PLoS Genet. 2012; 8: e1002828Crossref PubMed Scopus (149) Google Scholar). Lipophorin contains apolipophorin I and apolipophorin II, which are apolipoprotein B homologues and produced from a common precursor by proteolytic cleavage (7Kutty R.K. Kutty G. Kambadur R. Duncan T. Koonin E.V. Rodriguez I.R. Odenwald W.F. Wiggert B. Molecular characterization and developmental expression of a retinoid- and fatty acid-binding glycoprotein from Drosophila. A putative lipophorin.J. Biol. Chem. 1996; 271: 20641-20649Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Drosophila lipophorin is enriched in diacylglycerol (DAG), while the core of the mammalian lipoprotein consists of TAG and cholesteryl ester (6Palm W. Sampaio J.L. Brankatschk M. Carvalho M. Mahmoud A. Shevchenko A. Eaton S. Lipoproteins in Drosophila melanogaster–assembly, function, and influence on tissue lipid composition.PLoS Genet. 2012; 8: e1002828Crossref PubMed Scopus (149) Google Scholar). In contrast to mammalian lipoproteins, the surface of which is mainly covered with phosphatidylcholine (PC) (8Cole L.K. Vance J.E. Vance D.E. Phosphatidylcholine biosynthesis and lipoprotein metabolism.Biochim. Biophys. Acta. 2012; 1821: 754-761Crossref PubMed Scopus (221) Google Scholar), Drosophila lipophorin contains phosphatidylethanolamine (PE) as a major phospholipid constituent (6Palm W. Sampaio J.L. Brankatschk M. Carvalho M. Mahmoud A. Shevchenko A. Eaton S. Lipoproteins in Drosophila melanogaster–assembly, function, and influence on tissue lipid composition.PLoS Genet. 2012; 8: e1002828Crossref PubMed Scopus (149) Google Scholar). Lipophorin also carries the Wingless and Hedgehog morphogen proteins to regulate growth and patterning during development (9Panáková D. Sprong H. Marois E. Thiele C. Eaton S. Lipoprotein particles are required for Hedgehog and Wingless signalling.Nature. 2005; 435: 58-65Crossref PubMed Scopus (520) Google Scholar, 10Callejo A. Culi J. Guerrero I. Patched, the receptor of Hedgehog, is a lipoprotein receptor.Proc. Natl. Acad. Sci. USA. 2008; 105: 912-917Crossref PubMed Scopus (65) Google Scholar, 11Khaliullina H. Panakova D. Eugster C. Riedel F. Carvalho M. Eaton S. Patched regulates Smoothened trafficking using lipoprotein-derived lipids.Development. 2009; 136: 4111-4121Crossref PubMed Scopus (71) Google Scholar). It has been reported that lipophorin is produced in fat bodies in a microsomal triglyceride transfer protein-dependent manner and is loaded with lipids that are absorbed from the diet or synthesized de novo in the gut (6Palm W. Sampaio J.L. Brankatschk M. Carvalho M. Mahmoud A. Shevchenko A. Eaton S. Lipoproteins in Drosophila melanogaster–assembly, function, and influence on tissue lipid composition.PLoS Genet. 2012; 8: e1002828Crossref PubMed Scopus (149) Google Scholar). The Drosophila genome contains two LDLR homologue genes, LpR1 and LpR2 (12Parra-Peralbo E. Culi J. Drosophila lipophorin receptors mediate the uptake of neutral lipids in oocytes and imaginal disc cells by an endocytosis-independent mechanism.PLoS Genet. 2011; 7: e1001297Crossref PubMed Scopus (77) Google Scholar). LpR1 and LpR2 each have two putative promoter regions (the distal promoter and the proximal promoter) and many splicing variants (supplemental Fig. S1) (12Parra-Peralbo E. Culi J. Drosophila lipophorin receptors mediate the uptake of neutral lipids in oocytes and imaginal disc cells by an endocytosis-independent mechanism.PLoS Genet. 2011; 7: e1001297Crossref PubMed Scopus (77) Google Scholar). As with the role of LDLR in the uptake of cargo lipids in the mammalian system, Drosophila lipophorin receptors (LpRs) are reported to mediate the endocytosis of lipophorin (10Callejo A. Culi J. Guerrero I. Patched, the receptor of Hedgehog, is a lipoprotein receptor.Proc. Natl. Acad. Sci. USA. 2008; 105: 912-917Crossref PubMed Scopus (65) Google Scholar, 11Khaliullina H. Panakova D. Eugster C. Riedel F. Carvalho M. Eaton S. Patched regulates Smoothened trafficking using lipoprotein-derived lipids.Development. 2009; 136: 4111-4121Crossref PubMed Scopus (71) Google Scholar). However, the insect lipophorin system is also reported to transport lipids to tissues without internalization and degradation of the lipophorin, a mechanism that is coupled with a combination of lipophorin lipase-mediated lipolysis and the subsequent uptake of the free fatty acids (13Canavoso L.E. Jouni Z.E. Karnas K.J. Pennington J.E. Wells M.A. Fat metabolism in insects.Annu. Rev. Nutr. 2001; 21: 23-46Crossref PubMed Scopus (460) Google Scholar). These studies suggest that the insect lipophorin is a reusable shuttle for transporting lipids from sites of absorption or storage to sites of utilization. In Drosophila, LpRs are also shown to mediate the uptake of neutral lipids in oocytes and imaginal disc cells by an endocytosis-independent mechanism (12Parra-Peralbo E. Culi J. Drosophila lipophorin receptors mediate the uptake of neutral lipids in oocytes and imaginal disc cells by an endocytosis-independent mechanism.PLoS Genet. 2011; 7: e1001297Crossref PubMed Scopus (77) Google Scholar). Fatty acids are essential constituents of phospholipids, TAG, cholesteryl esters, and wax esters. Furthermore, fatty acids are also required for signal transduction, the modification of proteins, and the production of energy and metabolic intermediates. Because the physicochemical properties and cellular functions of fatty acids are mainly determined by the number and position of double bonds and the chain length of the fatty acid, the cellular composition of fatty acids needs to be strictly regulated (14Holthuis J.C. Menon A.K. Lipid landscapes and pipelines in membrane homeostasis.Nature. 2014; 510: 48-57Crossref PubMed Scopus (552) Google Scholar, 15van Meer G. Voelker D.R. Feigenson G.W. Membrane lipids: where they are and how they behave.Nat. Rev. Mol. Cell Biol. 2008; 9: 112-124Crossref PubMed Scopus (4481) Google Scholar). In mammals, PUFAs make up about 20% of the dry weight of the brain, in which PUFAs are required for brain processes, including neurotransmission, cell survival, and neuroinflammation, as components of cellular membranes and precursors of bioactive mediators (16Yehuda S. Rabinovitz S. Mostofsky D.I. Essential fatty acids are mediators of brain biochemistry and cognitive functions.J. Neurosci. Res. 1999; 56: 565-570Crossref PubMed Scopus (227) Google Scholar, 17Bazinet R.P. Laye S. Polyunsaturated fatty acids and their metabolites in brain function and disease.Nat. Rev. Neurosci. 2014; 15: 771-785Crossref PubMed Scopus (817) Google Scholar). There has been considerable debate about how PUFAs are transported to the mammalian brain (17Bazinet R.P. Laye S. Polyunsaturated fatty acids and their metabolites in brain function and disease.Nat. Rev. Neurosci. 2014; 15: 771-785Crossref PubMed Scopus (817) Google Scholar, 18Chen C.T. Bazinet R.P. β-oxidation and rapid metabolism, but not uptake regulate brain eicosapentaenoic acid levels.Prostaglandins Leukot. Essent. Fatty Acids. 2015; 92: 33-40Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Studies of mice lacking the receptors for LDL or VLDL have suggested that these receptors are not necessary for maintaining brain PUFA levels (19Rahman T. Taha A.Y. Song B.J. Orr S.K. Liu Z. Chen C.T. Bazinet R.P. The very low density lipoprotein receptor is not necessary for maintaining brain polyunsaturated fatty acid concentrations.Prostaglandins Leukot. Essent. Fatty Acids. 2010; 82: 141-145Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 20Chen C.T. Ma D.W. Kim J.H. Mount H.T. Bazinet R.P. The low density lipoprotein receptor is not necessary for maintaining mouse brain polyunsaturated fatty acid concentrations.J. Lipid Res. 2008; 49: 147-152Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). It is reported that the enzyme lipoprotein lipase is responsible for hydrolyzing circulating plasma lipoproteins and releasing unesterified PUFAs, which are then taken up by the brain (21Goldberg I.J. Eckel R.H. Abumrad N.A. Regulation of fatty acid uptake into tissues: lipoprotein lipase- and CD36-mediated pathways.J. Lipid Res. 2009; 50: S86-S90Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 22Chen S. Subbaiah P.V. Regioisomers of phosphatidylcholine containing DHA and their potential to deliver DHA to the brain: role of phospholipase specificities.Lipids. 2013; 48: 675-686Crossref PubMed Scopus (25) Google Scholar). Experiments using artificial membranes also demonstrated that unesterified fatty acids can diffuse passively into the brain. In Drosophila, PUFAs are reported to activate light-sensitive channels (23Chyb S. Raghu P. Hardie R.C. Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL.Nature. 1999; 397: 255-259Crossref PubMed Scopus (361) Google Scholar), with a lack of dietary PUFAs causing synapse dysfunction in the Drosophila visual system (24Ziegler A.B. Menage C. Gregoire S. Garcia T. Ferveur J.F. Bretillon L. Grosjean Y. Lack of dietary polyunsaturated fatty acids causes synapse dysfunction in the Drosophila visual system.PLoS One. 2015; 10: e0135353Crossref PubMed Scopus (23) Google Scholar). Because lipophorin crosses the blood-brain barrier in Drosophila (25Brankatschk M. Eaton S. Lipoprotein particles cross the blood-brain barrier in Drosophila.J. Neurosci. 2010; 30: 10441-10447Crossref PubMed Scopus (58) Google Scholar), it is likely that lipophorin plays a dominant role in supplying PUFAs to the CNS. Although recent studies have demonstrated that the insect lipoprotein system has a unique ability to selectively transport specific lipids to specific tissues, it remains unclear how the single class of lipoproteins selectively transports PUFAs to the CNS. In this study, we carried out a comparative analysis of fatty acids among lipophorin, CNS, and CNS-derived cell lines and investigated the transport mechanism of fatty acids, focusing particularly on the transport of PUFAs to the CNS in Drosophila. In the course of presenting our results, we discuss how fatty acids are selectively transported using a single class of lipoprotein in Drosophila. The w1118 and Canton-S strains were used as the wild-type control strains. The Df(3R)lpr1/2 strain was obtained from the Bloomington Drosophila Stock Center. Unless otherwise stated, D. melanogaster was reared on standard medium containing glucose, brewer's yeast extract, corn meal, and agar supplemented with propionic acid and butyl p-hydroxybenzoate as preservatives at 25°C under a 12-h light/12-h dark cycle. The medium was supplemented with 2 mM C18:3 (Wako Pure Chemical) to increase the PUFA supply for the flies. The CNS was hand-dissected from third-instar larvae. As reported in previous studies (26Schmidt-Nielsen B.K. Gepner J.I. Teng N.N. Hall L.M. Characterization of an alpha-bungarotoxin binding component from Drosophila melanogaster.J. Neurochem. 1977; 29: 1013-1029Crossref PubMed Scopus (98) Google Scholar), the adult head and thorax/abdomen were separated by putting frozen flies through prechilled stainless-steel sieves. Briefly, 150 frozen adult flies were shaken in a 15 ml tube to break off their heads. The dismembered flies were then tipped onto two stacked stainless-steel sieves. The upper sieve had a 710 μm mesh size and retained the abdomen thoraxes; the lower sieve had a 355 μm mesh size and retained only the heads. Following this process, the fractions were examined using a stereomicroscope to confirm that the heads were completely isolated from the other body parts. Third-instar larvae were bled in PBS containing 5 mM EDTA using a bundle of insect pins. Hemocytes and cell fragments were removed by centrifugation for 15 min at 1,500 g at 4°C and filtered with polyethersulfone syringe filters (pore size: 0.45 μm; Starlab Scientific); 18 ml of hemolymph solution diluted with PBS containing 5 mM EDTA was mixed with 10 g potassium bromide (KBr), and then the density of hemolymph solution was adjusted to 1.3 g/ml. Ten milliliters of the hemolymph solution was overlayed with 9.6 ml 0.9% sodium chloride (NaCl) in a Beckman polycarbonate bottle (25 × 89 mm) and centrifuged in a Beckman L8-60M with Type 60Ti rotor for 22 h at 50,700 rpm at 4°C. Seventeen fractions (1.2 ml each) were collected and analyzed by SDS-PAGE followed by visualization by silver staining. Fractions within the density range of 1.10 to 1.15 g/ml were combined and dialyzed against PBS. The BCA protein assay (Thermo Fisher Scientific) was used to determine the protein concentration of lipophorin. In a typical experiment, 1 mg lipophorin was isolated from approximately 5,000 larvae. Drosophila S2 cells were maintained in Schneider's Drosophila medium supplemented with 10% FBS, 50 units/ml penicillin, and 50 μg/ml streptomycin at 25°C. For the cultivation of BG2-c6 and BG3-c2 cells, 10 μg/ml human insulin was added to the culture medium. Delipidated FBS was prepared as described previously (27Hannah V.C. Ou J. Luong A. Goldstein J.L. Brown M.S. Unsaturated fatty acids down-regulate srebp isoforms 1a and 1c by two mechanisms in HEK-293 cells.J. Biol. Chem. 2001; 276: 4365-4372Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Schneider's Drosophila medium supplemented with 10% delipidated FBS, 10 μg/ml human insulin, 50 units/ml penicillin, and 50 μg/ml streptomycin was used as delipidated medium. The pAc-sgRNA-Cas9 plasmid (Addgene) (28Bassett A.R. Tibbit C. Ponting C.P. Liu J.L. Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9.Biol. Open. 2014; 3: 42-49Crossref PubMed Scopus (84) Google Scholar), harboring common target sequences against the LpR1 and LpR2 genes, was constructed by ligating the synthesized oligonucleotides (5′-TTCGATCGAAAGGGCGGGTATGGA-3′ and 5′-AACTCCATACCCGCCCTTTCGATC-3′) into the BspQI sites of the plasmid. It was subsequently introduced into BG3-c2 cells using TransFectin lipid reagent (Bio-Rad Laboratories) following the manufacturer's instructions. After 2 weeks of puromycin selection (2.5 μg/ml), single cell clones were isolated by limiting dilution. DNA sequencing was then used to confirm the disruption of the LpR1 and LpR2 genes. We identified 11 bp deletions in exon 10 of the LpR1 and LpR2 genes, resulting in frame-shift mutations in both genes. Total lipids were extracted from the samples using the Bligh and Dyer method (29Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42689) Google Scholar) and dissolved in chloroform. Total lipids of the whole body and CNS were extracted from 3 and 10 larvae, respectively. A monolayer of cultured cells in a 60 mm culture dish was used for the extraction of total lipids. Phospholipids, DAGs, TAGs, and free fatty acids were separated from the total lipid extract by TLC using hexane/diethyl ether/acetic acid (60:40:1; v/v/v) as the solvent. PC, PE, phosphatidylserine (PS), phosphatidylinositol (PI), and ceramide phosphoetanolamine (CerPE) were separated from the total lipid extract by two-dimensional TLC using a first solvent system of chloroform/methanol/acetic acid (65:25:10; v/v/v) and a second solvent system of chloroform/methanol/formic acid (65:25:10; v/v/v) on a silica plate. The amount of phospholipids in each spot was determined by inorganic phosphate quantification (30Rouser G. Siakotos A.N. Fleischer S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots.Lipids. 1966; 1: 85-86Crossref PubMed Scopus (1316) Google Scholar). The amount of DAG was determined by using the triglyceride E-test (Wako). The total lipid and phospholipid fractions were incubated with a 5% hydrogen chloride-methanol solution (Nacalai Tesque) at 100°C for 3 h. The fatty-acid methyl esters were extracted into hexane and subjected to GC analysis using a Shimadzu GC-14 A with a flame ionization detector (FID) and a SUPELCO Omegawax Capillary GC column (0.25 μm; 30 m × 0.25 mm) (Sigma-Aldrich) (31Murakami A. Nagao K. Juni N. Hara Y. Umeda M. An N-terminal di-proline motif is essential for fatty acid-dependent degradation of Delta9-desaturase in Drosophila.J. Biol. Chem. 2017; 292: 19976-19986Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The temperature of the injector and the FID were held at 200°C and 280°C, respectively. The column temperature was held initially at 180°C for 5 min and then ramped to 220°C at a rate of 3°C/min, held at 220°C for 7 min, and then finally ramped to 240°C at a rate of 3°C/min and held at that temperature for 10 min. The peak areas of the methyl ester forms of the C12:0, C14:0, C14:1, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:0, C20:4, C20:5, C22:0, and C22:6 fatty acids were determined. The analysis of phospholipids, DAGs, and TAGs was performed on a Shimadzu LC-30AD HPLC system coupled to a triple-quadrupole LCMS-8040 mass spectrometer equipped with an electrospray source (32Suito T. Nagao K. Hatano M. Kohashi K. Tanabe A. Ozaki H. Kawamoto J. Kurihara T. Mioka T. Tanaka K. et al.Synthesis of omega-3 long-chain polyunsaturated fatty acid-rich triacylglycerols in an endemic goby, Gymnogobius isaza, from Lake Biwa, Japan.J. Biochem. 2018; 164: 127-140Crossref PubMed Scopus (6) Google Scholar). Separation was performed on a Kinetex C8 column (2.6 μm; 2.1 × 150 mm; Phenomenex) with a binary mobile phase having the following composition: 10 mM ammonium formate in water (mobile phase A) and 10 mM ammonium formate in 2-propanol/acetonitrile/water (45:45:10; v/v/v) (mobile phase B). The pump controlling the gradient of mobile phase B was programmed as followsμPE and PC analysis: an initial isocratic flow at 20% B for 1 min, a linear increase to 40% B for 1 min, an increase to 92.5% B using a curved gradient for 23 min, a linear increase to 100% B for 1 min, and a hold at 100% B for 4 min; DAG analysis: an initial isocratic flow at 20% B for 1 min, an increase to 100% B using a curved gradient for 24 min, and a hold at 100% B for 3 min; and TAG analysis: a linear increase from 20% B to 93% B for 5 min, a linear increase to 100% B for 25 min, and a hold at 100% B for 10 min. The total flow rate was 0.3 ml/min, the column temperature was 45°C, and the sample temperature was 4°C. The spectrometer parameters were as follows: nebulizer gas flow, 2 l/min; drying gas flow, 15 l/min, interface voltage, 4.5 kV; DL temperature, 250°C; and heat-block temperature, 400°C. The multiple reaction monitoring transition was [M + H]+ → [184.1]+ for PC and [M + H]+ → [M + H − 141.0]+ for PE. For DAG and TAG measurements, [M + NH4]+ was detected. The multiple reaction monitoring transitions used for the detection of cellular C14 fatty acid-containing DAG (28:1) and DAG (28:2) were [M + NH4]+ → [M + H – C14:1]+. The fatty-acid composition of PE, PC, and DAG was determined by product scan analysis of [M − H]−, [M + HCOO]−, and [M + NH4]+ as precursor ions, respectively. Total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific). cDNA was prepared using ReverTra Ace qPCR RT Master Mix (TOYOBO). Expression levels of LpR genes were quantified by the StepOnePlus real-time PCR system (Thermo Fisher Scientific) with PowerUP SYBR Green Master Mix (Thermo Fisher Scientific) and the specific primers (supplemental Table S1) and quantified by the 2-ΔΔCt method. Lipophorin was labeled with Alexa Fluor 488 NHS Ester (Thermo Fisher Scientific). Cells grown on coverslips were washed with Schneider's Drosophila medium containing 0.01% BSA and then incubated with Schneider's Drosophila medium containing 0.01% BSA and Alexa Fluor 488-conjugated lipophorin (20 μg/ml) for 20 min at 25°C. The cells were washed, fixed with 4% paraformaldehyde at room temperature for 30 min, and observed with a confocal microscope (LSM 800; Carl Zeiss). Internalized Alexa Fluor 488-labeled lipophorin was quantified using ImageJ software. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (lissamine rhodamine B sulfonyl; rhodamine-PE) were purchased from Avanti Polar Lipids. A 200:1 DOPC/rhodamine-PE chloroform solution of lipid mixtures was dried at the bottom of a flask for 15 min and evaporated in vacuo overnight. The dried lipid film was hydrated with TBS (10 mM Tris-HCl, pH 7.0; 150 mM NaCl). The resulting multilamellar vesicles were put through 5 freeze-thaw cycles and then extruded 20 times through a polycarbonate filter with a pore diameter of 100 nm using an Avanti Mini-Extruder. Lipophorin (125 μg protein/ml) and LUVs (84 μM phospholipids) were mixed in TBS and incubated at 25°C for 8 h. An aliquot (0.6 ml) of the lipophorin/LUV mixture was combined with 1.4 ml TBS containing 0.2 g/ml KBr and overlayed with 1.92 ml TBS in Beckman Coulter ultra-clear centrifuge tubes (13 × 51 mm). The tubes were centrifuged in a Beckman L8-60M with Sw55Ti rotor for 14 h at 47,800 rpm at 4°C, and 10 fractions (390 μl each) were collected. The fluorescence intensity of rhodamine-PE in each fraction was analyzed with a TECAN Infinite F200 PRO microplate reader (excitation: 535 nm; emission: 590 nm). The BCA protein assay was used to determine the protein concentration of each fraction. The amounts of PE and DAG in lipophorin and LUV fractions were determined by LC-ESI-MS analysis using a Shimadzu LC-30AD coupled to a triple-quadrupole mass spectrometer LCMS-8040 (Shimadzu) equipped with an electrospray source using 12:0/13:0 PE and 1,3-14:0 D5-DAG (Avanti Polar Lipids) as an internal control. The statistical significance of differences between the mean values was analyzed using the nonpaired t-test. Multiple comparisons were performed using Tukey's test following ANOVA. P < 0.05 was considered to be statistically significant. To study the mechanisms underlying the transport of fatty acids between tissues in Drosophila, we first analyzed the lipid composition of lipophorin, the lipoprotein that carries more than 95% of hemolymph lipids (6Palm W. Sampaio J.L. Brankatschk M. Carvalho M. Mahmoud A. Shevchenko A. Eaton S. Lipoproteins in Drosophila melanogaster–assembly, function, and influence on tissue lipid composition.PLoS Genet. 2012; 8: e1002828Crossref PubMed Scopus (149) Google Scholar). We isolated lipophorin from the hemolymph of third-instar larvae by KBr gradient ultracentrifugation. Although minor proteins were also detected, two proteins, with the molecular weights of 260 and 70 kDa, were found to be the major constituents in the fractions within the density range of 1.10 to 1.15 g/ml (supplemental Fig. S2). Consistent with the previous reports (6Palm W. Sampaio J.L. Brankatschk M. Carvalho M. Mahmoud A. Shevchenko A. Eaton S. Lipoproteins in Drosophila melanogaster–assembly, function, and influence on tissue lipid composition.PLoS Genet. 2012; 8: e1002828Crossref PubMed Scopus (149) Google Scholar, 7Kutty R.K. Kutty G. Kambadur R. Duncan T. Koonin E.V. Rodriguez I.R. Odenwald W.F. Wiggert B. Molecular characterization and developmental expression of a retinoid- and fatty acid-binding glycoprotein from Drosophila. A putative lipophorin.J. Biol. Chem. 1996; 271: 20641-20649Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), MALDI-TOF MS analysis revealed that these two proteins were apolipophorin I and apolipophorin II (data not shown). Therefore, we used fractions within the density range of 1.10 to 1.15 g/ml as the lipophorin fraction in our subsequent experiments. We analyzed the fatty-acid composition of lipophorin lipids by GC-FID. Saturated and monounsaturated fatty acids with 14-, 16-, and 18-carbon lengths constituted 94% of the total fatty acids (Table 1), although substantial amounts of PUFAs (C18:2 and C18:3) were also detected. Consistent with previous reports (6Palm W. Sampaio J.L. Brankatschk M. Carvalho M. Mahmoud A. Shevchenko A. Eaton S. Lipoproteins in Drosophila melanogaster–assembly, function, and influence on tissue lipid composition.PLoS Genet. 2012; 8: e1002828Crossref PubMed Scopus (149) Google Scholar), the isolated lipophorin contained phospholipids and DAGs as major lipid constituents (supplemental Fig. S3). The concentration of DAG (393.6 ± 17.0 nmol/mg protein) was higher than that of total phospholipids (257.9 ± 14.3 nmol/mg protein) (Table 2). PE (197.4 ± 11.4 nmol/mg protein) and PC (38.1 ± 2.7 nmol/mg protein) were identified as major phospholipid constituents of lipophorin because they comprised 76.6% and 14.8% of the total phospholipids, respectively (Table 2). Therefore, we analyzed various molecul" @default.
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• W2944828463 modified "2023-09-28" @default.
• W2944828463 title "Different mechanisms for selective transport of fatty acids using a single class of lipoprotein in Drosophila" @default.
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• W2944828463 cites W1537970310 @default.
• W2944828463 cites W1562667733 @default.
• W2944828463 cites W1810253779 @default.
• W2944828463 cites W1877088739 @default.
• W2944828463 cites W1963592957 @default.
• W2944828463 cites W1964649483 @default.
• W2944828463 cites W1966129095 @default.
• W2944828463 cites W1969058208 @default.
• W2944828463 cites W1976336399 @default.
• W2944828463 cites W1977869829 @default.
• W2944828463 cites W1981956598 @default.
• W2944828463 cites W1984785834 @default.
• W2944828463 cites W1989242866 @default.
• W2944828463 cites W2000849147 @default.
• W2944828463 cites W2001010223 @default.
• W2944828463 cites W2011933541 @default.
• W2944828463 cites W2013153343 @default.
• W2944828463 cites W2016434096 @default.
• W2944828463 cites W2018762738 @default.
• W2944828463 cites W2027718994 @default.
• W2944828463 cites W2029042594 @default.
• W2944828463 cites W2033178435 @default.
• W2944828463 cites W2038651535 @default.
• W2944828463 cites W2040571306 @default.
• W2944828463 cites W2041582575 @default.
• W2944828463 cites W2043810467 @default.
• W2944828463 cites W2045297719 @default.
• W2944828463 cites W2046422846 @default.
• W2944828463 cites W2051571980 @default.
• W2944828463 cites W2053337525 @default.
• W2944828463 cites W2054052567 @default.
• W2944828463 cites W2057858725 @default.
• W2944828463 cites W2058584269 @default.
• W2944828463 cites W2068749469 @default.
• W2944828463 cites W2069528220 @default.
• W2944828463 cites W2073463753 @default.
• W2944828463 cites W2076822925 @default.
• W2944828463 cites W2088611132 @default.
• W2944828463 cites W2092474731 @default.
• W2944828463 cites W2094323635 @default.
• W2944828463 cites W2095184060 @default.
• W2944828463 cites W2095777272 @default.
• W2944828463 cites W2097340608 @default.
• W2944828463 cites W2098646234 @default.
• W2944828463 cites W2106863348 @default.
• W2944828463 cites W2111582004 @default.
• W2944828463 cites W2116233205 @default.
• W2944828463 cites W2116890977 @default.
• W2944828463 cites W2125872855 @default.
• W2944828463 cites W2136728173 @default.
• W2944828463 cites W2138590740 @default.
• W2944828463 cites W2144830898 @default.
• W2944828463 cites W2151605966 @default.
• W2944828463 cites W2160347316 @default.
• W2944828463 cites W2300552860 @default.
• W2944828463 cites W2330756258 @default.
• W2944828463 cites W2413630491 @default.
• W2944828463 cites W2759161762 @default.
• W2944828463 cites W2793097736 @default.
• W2944828463 cites W2883177217 @default.
• W2944828463 cites W4239727071 @default.
• W2944828463 cites W4377077114 @default.
• W2944828463 cites W822681412 @default.
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