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Lipids orchestrate biological processes by acting remotely as signaling molecules or locally as membrane components that modulate protein function. Detailed insight into lipid function requires knowledge of the subcellular localization of individual lipids. We report an analysis of the subcellular lipidome of the mammalian macrophage, a cell type that plays key roles in inflammation, immune responses, and phagocytosis. Nuclei, mitochondria, endoplasmic reticulum (ER), plasmalemma, and cytoplasm were isolated from RAW 264.7 macrophages in basal and activated states. Subsequent lipidomic analyses of major membrane lipid categories identified 229 individual/isobaric species, including 163 glycerophospholipids, 48 sphingolipids, 13 sterols, and 5 prenols. Major subcellular compartments exhibited substantially divergent glycerophospholipid profiles. Activation of macrophages by the Toll-like receptor 4-specific lipopolysaccharide Kdo2-lipid A caused significant remodeling of the subcellular lipidome. Some changes in lipid composition occurred in all compartments (e.g., increases in the levels of ceramides and the cholesterol precursors desmosterol and lanosterol). Other changes were manifest in specific organelles. For example, oxidized sterols increased and unsaturated cardiolipins decreased in mitochondria, whereas unsaturated ether-linked phosphatidylethanolamines decreased in the ER. We speculate that these changes may reflect mitochondrial oxidative stress and the release of arachidonic acid from the ER in response to cell activation. Lipids orchestrate biological processes by acting remotely as signaling molecules or locally as membrane components that modulate protein function. Detailed insight into lipid function requires knowledge of the subcellular localization of individual lipids. We report an analysis of the subcellular lipidome of the mammalian macrophage, a cell type that plays key roles in inflammation, immune responses, and phagocytosis. Nuclei, mitochondria, endoplasmic reticulum (ER), plasmalemma, and cytoplasm were isolated from RAW 264.7 macrophages in basal and activated states. Subsequent lipidomic analyses of major membrane lipid categories identified 229 individual/isobaric species, including 163 glycerophospholipids, 48 sphingolipids, 13 sterols, and 5 prenols. Major subcellular compartments exhibited substantially divergent glycerophospholipid profiles. Activation of macrophages by the Toll-like receptor 4-specific lipopolysaccharide Kdo2-lipid A caused significant remodeling of the subcellular lipidome. Some changes in lipid composition occurred in all compartments (e.g., increases in the levels of ceramides and the cholesterol precursors desmosterol and lanosterol). Other changes were manifest in specific organelles. For example, oxidized sterols increased and unsaturated cardiolipins decreased in mitochondria, whereas unsaturated ether-linked phosphatidylethanolamines decreased in the ER. We speculate that these changes may reflect mitochondrial oxidative stress and the release of arachidonic acid from the ER in response to cell activation. Lipids regulate and modify protein function and thus various biological processes by two distinct mechanisms. Signaling lipids, including free fatty acids, eicosanoids, sphingosine-1-phosphate, and lysophosphatidic acid, may be released from the sites of their generation in membranes and can subsequently affect receptors located remotely throughout tissues and cells e.g., see (1Wymann M.P. Schneiter R. Lipid signalling in disease.Nat. Rev. Mol. Cell Biol. 2008; 9: 162-176Crossref PubMed Scopus (972) Google Scholar, 2Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2484) Google Scholar, 3Nicholls D.G. The physiological regulation of uncoupling proteins.Biochim. Biophys. Acta. 2006; 1757: 459-466Crossref PubMed Scopus (151) Google Scholar, 4Jenkins C.M. Cedars A. Gross R.W. Eicosanoid signalling pathways in the heart.Cardiovasc. Res. 2009; 82: 240-249Crossref PubMed Scopus (131) Google Scholar) for reviews. Structural lipids, which represent the bulk of the lipid in the organism, affect membrane-bound enzymes, transporters, and receptors in a local fashion by altering membrane properties or by specific binding to target proteins reviewed in (5van 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 (4618) Google Scholar, 6Andersen O.S. Koeppe II, R.E. Bilayer thickness and membrane protein function: an energetic perspective.Annu. Rev. Biophys. Biomol. Struct. 2007; 36: 107-130Crossref PubMed Scopus (635) Google Scholar, 7Dowhan W. Mileykovskaya E. Bogdanov M. Diversity and versatility of lipid-protein interactions revealed by molecular genetic approaches.Biochim. Biophys. Acta. 2004; 1666: 19-39Crossref PubMed Scopus (110) Google Scholar, 8Lee A.G. How lipids affect the activities of integral membrane proteins.Biochim. Biophys. Acta. 2004; 1666: 62-87Crossref PubMed Scopus (948) Google Scholar). A fundamentally accepted general concept of cell biology is the compartmentalization of biological processes within subcellular structures, termed organelles. Detailed information about the location of biochemical reactions is crucial for understanding their roles in cellular function and dysfunction. By the same token, knowing how different signaling and structural lipids affect cellular responses requires knowledge of their subcellular distribution. Traditional lipid analysis involves organic extraction of whole cells or tissues followed by separation and measurement of lipid classes and subclasses. When these approaches are taken, information regarding the subcellular distribution of lipid classes and the molecular identity of individual species of lipids is lost. Because the physical properties of individual membranes are determined not only by lipid head groups (lipid classes) but also by their acyl chain composition (6Andersen O.S. Koeppe II, R.E. Bilayer thickness and membrane protein function: an energetic perspective.Annu. Rev. Biophys. Biomol. Struct. 2007; 36: 107-130Crossref PubMed Scopus (635) Google Scholar, 8Lee A.G. How lipids affect the activities of integral membrane proteins.Biochim. Biophys. Acta. 2004; 1666: 62-87Crossref PubMed Scopus (948) Google Scholar), we sought to overcome these limitations by combining subcellular fractionation approaches we developed for the macrophage (9Andreyev A.Y. Shen Z. Guan Z. Ryan A. Fahy E. Subramaniam S. Raetz C.R. Briggs S. Dennis E.A. Application of proteomic marker ensembles to subcellular organelle identification.Mol. Cell. Proteomics. 2010; 9: 388-402Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) with sophisticated mass spectrometric analysis of lipid species. In choosing a cell to apply these approaches to, we took advantage of the extraordinary biology of the macrophage. This cell type is ubiquitous throughout the mammalian kingdom and undergoes drastic transformation in response to activation (10Karnovsky M.L. Lazdins J.K. Biochemical criteria for activated macrophages.J. Immunol. 1978; 121: 809-813PubMed Google Scholar, 11North R.J. The concept of the activated macrophage.J. Immunol. 1978; 121: 806-809PubMed Google Scholar, 12Cohn Z.A. Activation of mononuclear phagocytes: fact, fancy, and future.J. Immunol. 1978; 121: 813-816PubMed Google Scholar, 13Mosser D.M. Edwards J.P. Exploring the full spectrum of macrophage activation.Nat. Rev. Immunol. 2008; 8: 958-969Crossref PubMed Scopus (6343) Google Scholar). The LIPID MAPS Consortium has developed quantitative methods for comprehensively evaluating the composition, biosynthesis, and function of macrophage lipids (14Schmelzer K. Fahy E. Subramaniam S. Dennis E.A. The lipid maps initiative in lipidomics.Methods Enzymol. 2007; 432: 171-183Crossref PubMed Scopus (115) Google Scholar) and has now applied these methods to subcellular fractions. The classic pathway of macrophage activation is triggered by molecular components of pathogens, e.g., lipopolysaccharide (LPS), a complex structural lipid found in the outer membranes of most gram-negative bacteria (15Raetz C.R. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar, 16Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zahringer U. Seydel U. Di Padova F. et al.Bacterial endotoxin: molecular relationships of structure to activity and function.FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar). Macrophages and other immune cell types detect submicromolar amounts of LPS (17Raetz C.R. Garrett T.A. Reynolds C.M. Shaw W.A. Moore J.D. Smith Jr., D.C. Ribeiro A.A. Murphy R.C. Ulevitch R.J. Fearns C. et al.Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4.J. Lipid Res. 2006; 47: 1097-1111Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 18Aderem A. Ulevitch R.J. Toll-like receptors in the induction of the innate immune response.Nature. 2000; 406: 782-787Crossref PubMed Scopus (2632) Google Scholar, 19Visintin A. Halmen K.A. Latz E. Monks B.G. Golenbock D.T. Pharmacological inhibition of endotoxin responses is achieved by targeting the TLR4 coreceptor, MD-2.J. Immunol. 2005; 175: 6465-6472Crossref PubMed Scopus (130) Google Scholar, 20Akashi S. Saitoh S. Wakabayashi Y. Kikuchi T. Takamura N. Nagai Y. Kusumoto Y. Fukase K. Kusumoto S. Adachi Y. et al.Lipopolysaccharide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity than that with MD-2 or CD14.J. Exp. Med. 2003; 198: 1035-1042Crossref PubMed Scopus (332) Google Scholar) via the binding of this molecule to Toll-like receptor 4 (TLR-4) (15Raetz C.R. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3423) Google Scholar, 18Aderem A. Ulevitch R.J. Toll-like receptors in the induction of the innate immune response.Nature. 2000; 406: 782-787Crossref PubMed Scopus (2632) Google Scholar, 21Gangloff M. Gay N.J. MD-2: the Toll 'gatekeeper’ in endotoxin signalling.Trends Biochem. Sci. 2004; 29: 294-300Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). It stimulates macrophages via its lipid anchor, which is termed lipid A. In our studies, we have employed Kdo2-lipid A (KLA), a homogenous subspecies of LPS that is specific for the TLR-4 receptor (17Raetz C.R. Garrett T.A. Reynolds C.M. Shaw W.A. Moore J.D. Smith Jr., D.C. Ribeiro A.A. Murphy R.C. Ulevitch R.J. Fearns C. et al.Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4.J. Lipid Res. 2006; 47: 1097-1111Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). of LPS to TLR-4 and its activates an signaling that cells of the immune to the This is by significant changes in the cellular levels of signaling and structural lipids and in their pathways et In the we isolated the major subcellular from and macrophages and determined the lipid composition of organelle by We found that lipid changes occurred and subcellular levels and lipid cells were from the and were from with endotoxin from and lipid were from from from were and from other were from were from were and by of were and as (9Andreyev A.Y. Shen Z. Guan Z. Ryan A. Fahy E. Subramaniam S. Raetz C.R. Briggs S. Dennis E.A. Application of proteomic marker ensembles to subcellular organelle identification.Mol. Cell. Proteomics. 2010; 9: 388-402Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). cells were 4 and and The of and with and For an of cells were a of in of cells were with or for followed by subcellular were by in of for in of and to For cells were to by in of and The the cell to a of the and the by with a The with the to this in cell and organelle separation and decreased The to an by the of of the and with to were as for to cells for to mitochondria, and for to and and were by and for to and The and mitochondrial were by and in and The from the as the The and were to of in an were to the the the for with 5 the to were from the in 4 in and for the The mitochondrial and were in and to and by for in The with 4 4 and of the in the and endoplasmic reticulum the and A from the the most and termed (9Andreyev A.Y. Shen Z. Guan Z. Ryan A. Fahy E. Subramaniam S. Raetz C.R. Briggs S. Dennis E.A. Application of proteomic marker ensembles to subcellular organelle identification.Mol. Cell. Proteomics. 2010; 9: 388-402Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). were and of and composition of determined as (9Andreyev A.Y. Shen Z. Guan Z. Ryan A. Fahy E. Subramaniam S. Raetz C.R. Briggs S. Dennis E.A. Application of proteomic marker ensembles to subcellular organelle identification.Mol. Cell. Proteomics. 2010; 9: 388-402Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). of analyses were to lipid levels as (9Andreyev A.Y. Shen Z. Guan Z. Ryan A. Fahy E. Subramaniam S. Raetz C.R. Briggs S. Dennis E.A. Application of proteomic marker ensembles to subcellular organelle identification.Mol. Cell. Proteomics. 2010; 9: 388-402Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) proteomic marker ensembles from analysis of fractions. analysis from the of with the of cardiolipins see were and as by and an of and to of the fractions. of were by for 5 and the the extraction and of The lipid in of The mass spectrometric analysis and were as Y. and by mass Enzymol. 2007; 432: PubMed Scopus Google Scholar). The with the of A mass for the and to a of a two and a were a 5 a A of and for the The of the mass and were as in Y. and by mass Enzymol. 2007; 432: PubMed Scopus Google Scholar). achieved by of of the to of defined and as in S. J. H. an analysis of cellular lipids by 2006; PubMed Scopus Google Scholar). were to a mass with an an an A of of of The of a A to and for followed by a 5 to and for A 5 to for and to A 5 and for an The A of the to the of the mass with as and and were as The The The The The of these were and for and were as T.A. Guan Z. Raetz C.R. of and by Enzymol. 2007; 432: PubMed Scopus Google Scholar). were and to the LIPID MAPS S. E. H. Y. quantitative methods for analysis of by mass Enzymol. 2007; 432: PubMed Scopus Google Scholar). of with of and of with of and The were in a and For of in were and for to were and with of of and of were were and The the The a and for which a 5 and the 5 in subcellular were and methods in et and analysis of sterols in biological by mass Enzymol. 2007; 432: PubMed Scopus Google Scholar). a of sterols as to and a were and in were by and by an in The with a The A and the A and 5 The mass in for lipids were and LIPID MAPS T.A. Guan Z. Raetz C.R. of and by Enzymol. 2007; 432: PubMed Scopus Google Scholar) with as and were by a a two and a to a mass with a a of with a as of A for and increased to and for 4 A of of A from in the with as and cell The for and in the subcellular of RAW 264.7 a of as an The for and are and In these the are the and the is the major to a of the of The of in the with as and The for The for and were and The for and were and In these the were the and the were the of lipidomic as as the are the of the LIPID analyses were the major categories of mammalian lipids that were most to lipid protein function glycerophospholipids, sphingolipids, sterols, and For our studies, we to not the two lipid categories that and lipids fatty and lipids as eicosanoids, and their and fatty were from these lipids are to be released from membranes of and are thus to subcellular these lipids function remotely as signaling molecules of that are distinct from a local of membrane composition, have in lipids are within as lipid and have membrane properties of the major A of the of lipid species in the analysis of the major subcellular and the whole cell is in quantitative lipid species are in of the subcellular the of lipid molecular species that were and in the subcellular in the or activated states. in a the of lipid molecular species that were and in the subcellular in the or activated states. Lipid for subcellular were from the analyses of subcellular a (9Andreyev A.Y. Shen Z. Guan Z. Ryan A. Fahy E. Subramaniam S. Raetz C.R. Briggs S. Dennis E.A. Application of proteomic marker ensembles to subcellular organelle identification.Mol. Cell. Proteomics. 2010; 9: 388-402Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). an for the subcellular distribution of different lipid species in the and in the ER Lipid the of various lipid are also and in general the lipid of the whole analyses in the subcellular distribution of individual lipids and classes and lipid of organelles. For example, as the subcellular localization of the mitochondrial were from the membrane and in the ER and ether-linked were in all membranes but were than other glycerophospholipid classes in and distribution of lipids with specific ether-linked are of for molecular species, the of individual lipids were but be by glycerophospholipid of individual For example, the two most species in were and whereas in ER they were phosphatidylethanolamines and ether-linked and in membrane they were and The subcellular lipidome are for analysis as lipid maps The most in the analysis of the subcellular lipidome is the of lipid distribution. localization of lipid or individual lipids and information localization of their of lipid (5van 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 (4618) Google Scholar, E. M. and Cell Biol. 2008; PubMed Scopus Google Scholar). in the lipid (e.g., this information is by two other and species an of lipid in an organelle is a function not only of various of lipid in of the of membrane in the which we the of protein is as the of the organelle lipid of the organelle is as the this be in the and in two most and It is to that these in the are determined by the protein than the lipid of the In with this all major lipid classes of the subcellular that lipid distribution we to this from the The subcellular distribution of the to the be a distribution (e.g., and (e.g., that lipid subcellular from the general of the these specific the of a lipid in an organelle is a function of as and that are of their an example, the of fatty is the of fatty are in the of the lipid in the whole cell that may be as a of this lipid in the lipid of the This is a target for lipidomic analysis of but may be to from the subcellular lipidomic is determined by that are localization of the and species from the lipidomic we the The of a lipid in an organelle may be as a of the the species most a of the for lipid and organelle where is of lipid in organelle is lipid in organelle is lipid species and is a of lipid The and are determined from the lipidome and For organelle is by all lipid in the organelle For lipid is by of the lipid in the whole cell the only in the of the of and this can be For is to 4 and For lipid The of of an in an organelle and its from the whole cell of the and the of lipid in the organelle in other a from the general of lipid distribution in the to from the general of of specific in a this is with in other organelles. of this analysis applied to the of lipids is in is by is to the ER the specific localization is for specific mitochondrial of in of different species of and the of their as by the of is the the interaction of with TLR-4 the macrophages to an activated that the This is by the release of eicosanoids, (e.g., and (17Raetz C.R. Garrett T.A. Reynolds C.M. Shaw W.A. Moore J.D. Smith Jr., D.C. Ribeiro A.A. Murphy R.C. Ulevitch R.J. Fearns C. et al.Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4.J. Lipid Res. 2006; 47: 1097-1111Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), and of (e.g., and not In with the transformation of the macrophage, the subcellular lipidome remodeling to that all lipid categories and a of lipid which maps for the activation of the various lipid species Some changes in lipid were to a in all subcellular membranes (e.g., other changes were to a specific organelle (e.g., changes were in all lipids lipids of the same with acyl in a of higher not all changes the of is by the of in the maps in and we the by as information in of in acid an are significant from to groups are by the of and of lipidome changes the of ceramides This M.L. of in lipopolysaccharide LPS increases than acting as a structural Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) and the whole cell of the subcellular lipidome that ceramides activation all major cellular membranes may biological throughout the a also for two cholesterol and desmosterol of a local but lipidome is in species of acid increases in the whereas the changes were in the other and in the whole The mitochondrial lipid a complex response to cell activation species with or whereas unsaturated species to sterols not an of the of these lipids to in the of which decreased throughout the ether-linked a complex of these lipids increased in all major cellular most of of this to in the with the most by unsaturated that for ether-linked other the ER is the subcellular location these may have biological are significant in in in are in unsaturated ether-linked lipids. an are significant from of of groups are by the of and are The a analysis of the subcellular lipidome of a mammalian a macrophage The of the major species of membrane lipids were determined in all major cellular and compartments in and activated the lipidome remodeling that macrophage we that is to in the of by in a lipid as is in the maps and This is a lipid is to whereas an lipid is to a in but is to the properties of a to this is found in signaling lipids as which may via affinity interactions with specific proteins and thus biological a Because is a to signaling properties of a we have found to two types of the and and the lipid The changes the activated of may be that changes with TLR-4 signaling were the It also be acyl chain composition may be by fatty acid the cell or in the in our that the lipid composition is a function of as the fatty acyl lipid and subcellular and and is for the lipidome that we is but also than a a analysis to that may in the understanding of various of lipid and macrophage stress to as the is a of the response by macrophages in response to and A major of this response is the release of species from the activation of an of the F. J. The of molecular and J. Biochem. PubMed Scopus Google Scholar). The released in the of the in by membrane lipid In macrophage and cell are also of of A.Y. A.A. of 2005; PubMed Scopus Google Scholar). A of mitochondrial is inhibition Y. Murphy A. species by and J. 2002; PubMed Scopus Google Scholar), a that may also be in activated In the by to not which that may have to the oxidative stress by the activated In most mitochondrial a to the oxidative stress is to analysis of the subcellular lipidome for the of The of increases in and and a in oxidized sterols are to be of reactions and as are a of oxidative stress in their In the amounts of these oxidized sterols not in from the in levels are also with local oxidative The of unsaturated species may be to fatty are to of the of It is that in that have acyl we as of the acyl are these be to an of the of of fatty to oxidative stress and of as in the of the the of mitochondrial oxidative stress in activated macrophages and may be to from these our subcellular lipidome has only major lipid classes and e.g., information about oxidized and other the species the of groups not about fatty acyl composition of the lipids. levels of analysis that and species oxidized H. A.A. et analysis of and of and in cells and tissues by and Sci. 2009; PubMed Scopus Google and individual molecular species the analysis are in the ER and these lipids, a and unsaturated is of the most components We speculate that a of this lipid arachidonic acid the this lipid is with this the lipid by a of two activation of macrophages a in which of arachidonic acid are released into the or are into R.C. A. Dennis E.A. TLR-4 and of protein in Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). our regarding the of as a of arachidonic acid in macrophages Gross R.W. is the major for arachidonic acid in and is Sci. PubMed Scopus Google Scholar, Major of in the Biol. Full Text PDF PubMed Google Scholar), and not the the of in arachidonic acid release in the macrophage cell RAW Biophys. Acta. 2008; PubMed Scopus Google Scholar). in the levels of acid species may activation of a in with a we are this of the subcellular macrophage we the major lipid species and the major organelles. local of the lipids that modulate protein may be the of and manifest the of membranes (e.g., outer membranes of of membranes of or of lipids (lipid This report the a to the subcellular lipid of the
Andreyev et al. (Thu,) studied this question.