Lipidomics reveals that adiposomes store ether lipids and mediate phospholipid traffic,

Lipid droplets are accumulations of neutral lipids surrounded by a monolayer of phospholipids and associated proteins. Recent proteomic analysis of isolated droplets suggests that they are part of a dynamic organelle system that is involved in membrane traffic as well as packaging and distributing lipids in the cell. To gain a better insight into the function of droplets, we used a combination of mass spectrometry and NMR spectroscopy to characterize the lipid composition of this compartment. In addition to cholesteryl esters and triacylglycerols with mixed fatty acid composition, we found that ∼10–20% of the neutral lipids were the ether lipid monoalk(en)yl diacylglycerol. Although lipid droplets contain only 1–2% phospholipids by weight, >160 molecular species were identified and quantified. Phosphatidylcholine (PC) was the most abundant class, followed by phosphatidylethanolamine (PE), phosphatidylinositol, and ether-linked phosphatidylcholine (ePC). Relative to total membrane, droplet phospholipids were enriched in lysoPE, lysoPC, and PC but deficient in sphingomyelin, phosphatidylserine, and phosphatidic acid. These results suggest that droplets play a central role in ether lipid metabolism and intracellular lipid traffic. Lipid droplets are accumulations of neutral lipids surrounded by a monolayer of phospholipids and associated proteins. Recent proteomic analysis of isolated droplets suggests that they are part of a dynamic organelle system that is involved in membrane traffic as well as packaging and distributing lipids in the cell. To gain a better insight into the function of droplets, we used a combination of mass spectrometry and NMR spectroscopy to characterize the lipid composition of this compartment. In addition to cholesteryl esters and triacylglycerols with mixed fatty acid composition, we found that ∼10–20% of the neutral lipids were the ether lipid monoalk(en)yl diacylglycerol. Although lipid droplets contain only 1–2% phospholipids by weight, >160 molecular species were identified and quantified. Phosphatidylcholine (PC) was the most abundant class, followed by phosphatidylethanolamine (PE), phosphatidylinositol, and ether-linked phosphatidylcholine (ePC). Relative to total membrane, droplet phospholipids were enriched in lysoPE, lysoPC, and PC but deficient in sphingomyelin, phosphatidylserine, and phosphatidic acid. These results suggest that droplets play a central role in ether lipid metabolism and intracellular lipid traffic. Lipid droplets are recognized by their conserved structural organization, which consists of a hydrophobic matrix of neutral lipid covered by a monolayer of phospholipids and associated proteins (1van Meer G. Caveolin, cholesterol, and lipid droplets? J. Cell Biol. 2001; 152: F29-F34Google Scholar). Although traditionally regarded as a simple repository for stored carbon reserves, emerging evidence suggests that droplets function as dynamic organelles with a central role in cellular lipid metabolism, membrane trafficking, and cell signaling (2Beckman M. Cell biology. Great balls of fat. Science. 2006; 311: 1232-1234Google Scholar). Because lipid droplets can be found in bacteria, yeast, plant, and animal cells, over the years they have acquired a variety of names. Recently, we proposed that this diverse collection of names be replaced with the designation adiposome (3Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 2004; 279: 3787-3792Google Scholar). Thus, an adiposome is an organelle that is specialized for packaging and distributing lipids in cells. In this nomenclature, the droplet is simply the most visible stage in the complex life cycle of an adiposome. During the past few years, a number of reports have focused on the protein composition of lipid droplets isolated from yeast (4Athenstaedt K. Zweytick D. Jandrositz A. Kohlwein S.D. Daum G. Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae. J. Bacteriol. 1999; 181: 6441-6448Google Scholar), plant (5Katavic V. Agrawal G.K. Hajduch M. Harris S.L. Thelen J.J. Protein and lipid composition analysis of oil bodies from two Brassica napus cultivars. Proteomics. 2006; 6: 4586-4598Google Scholar), and animal (3Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 2004; 279: 3787-3792Google Scholar, 6Brasaemle D.L. Dolios G. Shapiro L. Wang R. Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes. J. Biol. Chem. 2004; 279: 46835-46842Google Scholar, 7Fujimoto Y. Itabe H. Sakai J. Makita M. Noda J. Mori M. Higashi Y. Kojima S. Takano T. Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7. Biochim. Biophys. Acta. 2004; 1644: 47-59Google Scholar) cells. A consensus view from these studies is that droplets contain structural proteins, proteins involved in the biosynthesis and breakdown of lipids, and proteins that mediate membrane traffic. Thus, the proteome indicates that droplets are actively engaged in membrane traffic, perhaps for the purpose of maintaining the proper lipid composition of different membrane compartments. In contrast to the proteins, surprisingly little is known about the lipid composition of animal cell droplets. Generally, droplets are rich in neutral lipids such as triacylglycerol (TAG) and cholesteryl esters that have a diverse population of esterified fatty acids (8Murphy D.J. The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog. Lipid Res. 2001; 40: 325-438Google Scholar). Here, we report an analysis of the lipid composition of droplets purified from various types of cultured and tissue cells. We used a combination of NMR spectroscopy and mass spectrometric approaches including high-throughput, direct infusion electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to characterize the neutral lipid and phospholipid composition of isolated droplets. We found that droplets are rich not only in TAG and cholesteryl esters esterified with a variety of different fatty acids but also in the ether neutral lipid monoalk(en)yl diacylglycerol (MADAG). Despite representing only 1–2% of the total lipid in the droplet, the phospholipid composition included diverse molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), ether-linked phosphatidylcholine (ePC), and ether-linked phosphatidylethanolamine (ePE) but very little phosphatidylserine (PS) or sphingomyelin (SM). We identified and quantified >160 phospholipid molecular species, which suggests that the simple phospholipid monolayer surrounding each droplet has an amazingly complex lipid composition. The neutral and phospholipid composition of lipid droplets is consistent with adiposomes having a direct role in lipid metabolism and in the intracellular traffic of membrane lipids. FBS and cosmic calf serum were from Hyclone (Logan, UT). DMEM and oleate were from Sigma (St. Louis, MO). Silica gel TLC plates were from Whatman (Brentford, Middlesex, UK). CHO K2 cells were cultured on 150 mm plates with 25 ml of DMEM (high-glucose; 4.5 g/l) containing 10% cosmic calf serum, 40 μg/ml proline, 100 U/ml penicillin, and 100 μg/ml streptomycin. Immortalized human B lymphocytes were cultured in RPMI 1640 containing 10% FBS as described previously (9Michaely P. Li W.P. Anderson R.G. Cohen J.C. Hobbs H.H. The modular adaptor protein ARH is required for low density lipoprotein (LDL) binding and internalization but not for LDL receptor clustering in coated pits. J. Biol. Chem. 2004; 279: 34023-34031Google Scholar). 3T3-L1 cells, primary human fibroblasts, fibroblasts derived from Zellweger patients (No. GM04340; Coriell Cell Repositories, Camden, NJ), NRK cells, and immortalized human fibroblasts (SV589 cells) were maintained in DMEM (low-glucose; 2.5 g/l) and 10% FBS supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Lipid droplets were induced by incubating confluent cells in the presence of 100 μM oleate for NRK cells or 80 μM oleate for other cells for either 48 h for primary human fibroblasts or 16 h for other cells. 3T3-L1 cells were induced to differentiate by incubating them in the presence of 10 μg/ml insulin, 1 μM dexamethasone, and 200 μM isobutylxanthine (Sigma) for 48 h followed by insulin alone for an additional 4 days with a medium change after 2 days. For mass spectrometry and NMR analysis, lipids were extracted into CHCl3 from droplet fractions or whole cells by a modification of the Bligh and Dyer method (10Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (41848) Google Scholar) in which methanol was replaced with 2-propanol (11Chapman K.D. Moore T.S. N-Acylphosphatidylethanolamine synthesis in plants: occurrence, molecular composition, and phospholipid origin. Arch. Biochem. Biophys. 1993; 301: 21-33Google Scholar). Monophasic extracts were partitioned into two phases, and the organic layer was washed three times with 1 M KCl (12Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 1957; 226: 497-509Google Scholar). Total lipid mass was estimated gravimetrically after removing solvent under a gentle stream of N2 gas. Lipids were extracted from lipid droplets of tissue cultured cells and mouse liver or from 20 mg of adipose tissue for TLC analysis of unknown neutral lipid using CHCl3/acetone (1:1, v/v). The solvent was removed by N2 gas, and the lipids were dissolved in CHCl3 and separated on TLC plates in hexane-diethyl ether-acetic acid (80:20:1, v/v) for 40 min. Lipid classes were visualized by iodine vapor or charring and semiquantified by densitometric scanning (NIH ImageJ software). Neutral lipid classes were quantified (and identified) according to TLC standards (Nu-Chek Prep, Inc., Elysian, MN) and separated under the same conditions at different amounts to generate standard curves for each major lipid class. In some cases, total lipid extracts were dissolved in CHCl3 for LC-MS analysis or phospholipid profiling. In some cases, TLC was used to separate neutral lipid classes that were then recovered from silica gel in acidified CHCl3 without exposure of lipids to iodine vapor. A nonpolar lipid fraction migrating between TAGs and cholesteryl esters was isolated by TLC and recovered in CHCl3 as described above. Fractions were examined for ionization by direct infusion using ESI and atmospheric pressure chemical ionization (APCI) in both positive and negative ion modes. The ionization was with positive ion which was then with LC-MS system with a ion mass was with a 150 particle and of at with major to by direct infusion in min. conditions were as pressure and V. Neutral lipid classes from mg of adiposome lipids were by on silica gel to and with hexane-diethyl ether v/v). major fractions to cholesteryl lipids, and TAGs were under and by NMR and of mass and were acquired on or were mass spectroscopy was on a using acid as the lipid extracts were on a acid in The was with 10 ml of v/v) to a neutral lipid The solvent was and the dissolved in ml of A ml was into ml of in and in the electrospray of an mass as described in were as ion of major TAG and species were using a of V. electrospray ionization-tandem mass spectrometry was and analysis, and were as described previously R. K.D. Identification and of in with metabolic from 40: Scholar, R. Wang ionization mass spectrometry for plant lipids. Biochem. Scholar) with of mg of was for mass spectrometry The lipid was with and such that the of in was and the was 1 and quantified as described previously R. Wang ionization mass spectrometry for plant lipids. Biochem. Scholar), were of of of of of of of of of acid of of of of and of lipid extracts were by infusion into the ESI on a system were using an with the required for the and to the ESI at The pressure was at 2 The with in the were for 40 for PC and for for and for were 100 for and PC and for and were for for PC and and for and were for for for for and for The mass were to a of at For each were in The was the was or was to the electrospray the was at 20 and the two ion were at Lipid species were using the described neutral of in the negative for R. Wang ionization mass spectrometry for plant lipids. Biochem. Scholar, G. R. analysis of membrane lipids at the low by ionization mass Scholar). and neutral of the extracts a of with each a of lipid species containing a was from the same mass as PC of in positive G. R. analysis of membrane lipids at the low by ionization mass Scholar, G. J. G. of phosphatidylcholine and sphingomyelin by electrospray ionization mass spectrometry with Biochim. Biophys. Acta. 2004; Scholar) and by with PC standards using a for with to be The of each was the were and the were using a and were and the lipids in each were quantified in with the two standards of that using curves for the mass Lipid droplets were purified from tissue cells by the method of (3Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 2004; 279: 3787-3792Google Scholar). To droplets from a whole mouse liver was into in 20 ml of containing 20 and 100 μM The was with a on and at for 10 at The fraction was to two ml and with 4 ml of 100 and 2 and at for 1 h at the at the of the was and as described (3Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 2004; 279: 3787-3792Google Scholar). To total cell CHO K2 cells were cultured on 150 mm plates to by in with 100 μM and with a at for on The was at for 10 at The fraction was recovered and at in a for 1 h at to of the The lipid composition of the was as of lipids on TLC plates was by densitometric scanning of plates using ImageJ with TLC standards (Nu-Chek we to characterize the neutral lipid composition of droplets purified from different cell types that in the presence or of The cell for these studies was the CHO K2 which droplets were and the lipids were extracted and separated by TLC with These droplets were rich in cholesteryl esters TAG and an unknown neutral lipid that between cholesteryl esters and amounts of fatty cholesterol, and phospholipids also were The amounts of the major lipid classes little CHO K2 cells were in the presence of 80 μM oleate the same of neutral lipids were found in droplets isolated from NRK and cells that in the presence of oleate We used to the fatty acid species in the TAG fraction The composition was consistent with the fatty acids found in cells, acid acid acid acid and acid on and the of the major TAG species in CHO K2 cell lipid droplets were and spectrometric and NMR and that the cholesteryl fraction at three cholesteryl cholesteryl and cholesteryl oleate not The neutral lipid migrating between cholesteryl esters and TAG by that were different from mass by of major species in this fraction was which the of a containing These with identified by were consistent with the that this neutral lipid was a To the unknown neutral we used NMR spectroscopy and additional mass spectrometry We purified the neutral lipid by silica gel and the The that were of an esterified containing an ether at a carbon was by spectroscopy in which be by of an ether lipid containing and analysis of the neutral lipids from adiposomes species of TAGs and The molecular an of TAGs with of in the same total ion species in mass by which mixed species consistent with TAG and species from LC-MS and A of TAG and species with of and is in ion of TAG and molecular the of the or in the For the derived from the neutral of fatty acid the of the TAG at mass as a of species with of different The were consistent with the TAGs at this mass a of and well as abundant were with ion of the ether neutral lipid species, and a ion derived from the Here, the or with a neutral of fatty acid or fatty to the of species that the were but two major species of that are consistent with the are and The TAGs and identified in were consistent with species identified by The TAG in to the ion with in and the in to the ion with in we the of the purified neutral lipid species with the of major were between the two The of the purified lipid two additional between and and consistent with the presence of an ether in a carbon was in this of The purified lipid also or between and in this to carbon in a has only two in this in this are and oleate only The presence of that the fraction different of mixed We that the unknown neutral lipid is a of neutral molecular species containing and of various and of In addition to fatty such as and the also contain fatty and is by of and ion and was not in cell The in the synthesis of ether lipids is the of to by the H. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). droplets isolated from cells contain amounts of To this is we used primary fibroblasts from patients with Zellweger which to a in lipid metabolism, and 2004; PubMed Scopus Google Scholar). CHO K2 cells as well as and Zellweger human fibroblasts for 48 h in the presence of 80 μM oleate were to lipids from isolated droplets. The was in droplets isolated from cells but in Zellweger cells. We also the of in lipids extracted from adipose tissue and found that was not in either or adipose studies have that the of of the of 3T3-L1 adipocytes. in triacylglycerol J. Biol. Chem. Scholar), which the low amounts of in adipose neutral lipids. very little was found in droplets isolated from 3T3-L1 cells 3T3-L1 cells in the presence of oleate amounts of We also in droplets isolated from liver droplets were also rich in lipid species that with the of and fatty acid we that droplets from NRK cells as well as cells in oleate low amounts of cholesteryl but amounts of which indicates that droplets these esters These results two ether lipid metabolism cell types most is on a between and adiposomes different types of neutral lipids on the specialized function of the cell. Although the of total phospholipids in droplets is to neutral lipids of phospholipids the droplet and as an with other cellular compartments. Recent mass spectrometric have to and phospholipid classes as well as the of molecular species in each R. Wang Lipid species a to lipid and the function of involved in lipid metabolism and Biol. 2004; Scholar). We used direct infusion ESI followed by to different classes of lipids in the droplet and the of the molecular species each class. The species in each are as the by are by and is using standards for each R. K.D. Identification and of in with metabolic from 40: Scholar, R. Wang ionization mass spectrometry for plant lipids. Biochem. Scholar) for phospholipids is in The phospholipids in the major classes were in two different droplet PC and were the most abundant followed by and of and and also were of the different lipid species each major are in of the total PC of a total of molecular was of and the major were and and the was We species of and this was by and we quantified species of and these were and were 10 species and this was by and In with PC and the was with only species We that the phospholipid composition of adiposomes is surprisingly with molecular species in such as and were at low in we not species these We also the of phospholipid classes in droplets total The phospholipid composition of the isolated droplet was to that of cell with the that droplets lysoPC, and and very low of and Because were enriched we the of and species in droplets with total membrane phospholipids were in species and in droplets was in the same fatty acid in and the other we each species in the droplet with the total of that species membrane phospholipid only was enriched in droplets. Thus, droplets contain and to membrane in the cell they are enriched in and in and these results that are and molecular species between the phospholipid monolayer the droplet and the total membrane phospholipid in the cell. These suggest that the phospholipids surrounding each droplet are specialized to metabolic and functions of the adiposome. spectrometry has to about the lipid composition of cellular and compartments. has to be well for lipid analysis direct infusion of a with standards for the of lipid species from the mass analysis of cellular Proteomics. PubMed Scopus Google Scholar). To the lipids by mass spectrometry R. Wang Lipid species a to lipid and the function of involved in lipid metabolism and Biol. 2004; Scholar), we used two standards for each lipid to the species of the from the or neutral on a Although this is be that some lipid species, containing ether have a to a different the with to be better standard Recently, mass spectrometry has used to phospholipids by cells in of cellular and phospholipids from human cells. J. Lipid Res. 2006; Scholar), the of 2 with on the phospholipid and TAG composition of A. L. A. A role for 2 in J. Biol. Chem. 2006; Scholar), and phospholipids in lipid containing receptor are to lipid that contain a of and a J. Biol. Chem. Scholar). analysis has that the phospholipid composition of isolated droplets is PC was the phospholipid class, in with studies K. S. T. R. T. The of lipid droplets is a phospholipid monolayer with a fatty acid composition. J. Biol. Chem. Scholar). We different species of we species of and species of We also a variety of and ether-linked of PC and the other very little or was found in the purified The of species of was in the droplets in total cell For and were enriched in the droplets to total membrane but was cells appear to be enriched in K. S. T. R. T. The of lipid droplets is a phospholipid monolayer with a fatty acid composition. J. Biol. Chem. Scholar), which indicates that droplets be enriched in but the major fatty acid with cell and The rich of phospholipids suggests that the phospholipid monolayer that each droplet is a simple for neutral lipids. The of phospholipids suggests that adiposomes have an function in both phospholipids for membrane lipid and signaling lipids that metabolic H. of lipid intracellular for synthesis in J. 2006; Scholar). In addition to >160 phospholipid species in isolated droplets, we also found a of in droplet was in droplets isolated from both tissue cells and liver but not in lipids extracted from adipose tissue or in droplets isolated from 3T3-L1 cells. The of suggests a role for adiposomes in ether lipid The three in ether lipid biosynthesis are in H. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, of the of 3T3-L1 adipocytes. in triacylglycerol J. Biol. Chem. Scholar, L. H. lipid protein to J. Lipid Res. 1999; 40: Scholar). The of to is to in the lipid metabolism, and 2004; PubMed Scopus Google Scholar). is the that is to by a for in the We that can be to phospholipid major molecular species identified by NMR and and composition to the ether phospholipids in the For we identified with the which be the to or derived from or at the We these phospholipids have an or an at the the adiposome function both as a for ether phospholipid biosynthesis and as a organelle for the of phospholipids and TAGs of triacylglycerol synthesis and their Prog. Lipid Res. 2004; PubMed Scopus Google Scholar, D. Neutral lipid a in triacylglycerol Biochim. Biophys. Acta. PubMed Scopus Google Scholar). the of in droplets also indicates a role for adiposomes in phospholipid The on for ether lipid biosynthesis also suggests a metabolic between the two consistent with studies that in yeast the and droplets D. T. Y. J. Zhao Y. K.D. Anderson R.G. between and lipid J. Cell Biol. 2006; Scholar). In the lipid of the isolated droplet a in phospholipid species as well as a role for the organelle in the of an ether neutral These results suggest that adiposomes have a function in the metabolism and of lipids between compartments. We have found that a insight into the of different classes and species of droplet lipids. method to be an for the function of the adiposome in lipid droplet and intracellular lipid The the of and The for in droplets from and and for with the mass and J. for the of mass and at the were by from the and and the with from the of the and as well as from of from the of of the for was from of of and the and the H. in and of and with