The Surface of Lipid Droplets Is a Phospholipid Monolayer with a Unique Fatty Acid Composition

Abstract

We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079–1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2β and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain. We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079–1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2β and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain. Lipid droplets have been regarded as a depot of neutral lipids. They exist most abundantly in adipose cells and steroid-producing cells but can be found in virtually any kind of cell. The core of lipid droplets is occupied by triacylglycerol and cholesterol ester in various ratios depending on the cell type (1Zweytick D. Athenstaedt K. Daum G. Biochim. Biophys. Acta. 2000; 1469: 101-120Crossref PubMed Scopus (278) Google Scholar), but information on the lipid droplet surface has been scarce. Recently we as well as others showed that caveolins can exist in the lipid droplet surface (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar, 3Ostermeyer A.G. Paci J.M. Zeng Y. Lublin D.M. Munro S. Brown D.A. J. Cell Biol. 2001; 152: 1071-1078Crossref PubMed Scopus (213) Google Scholar, 4Pol A. Luetterforst R. Lindsay M. Heino S. Ikonen E. Parton R.G. J. Cell Biol. 2001; 152: 1057-1070Crossref PubMed Scopus (278) Google Scholar). Caveolins, i.e. caveolin-1, 2, 3, are membrane proteins that are incorporated to the sphingolipid/cholesterol-enriched membrane microdomain and form the framework of caveolae (5Parton R.G. Curr. Opin. Cell Biol. 1996; 8: 542-548Crossref PubMed Scopus (495) Google Scholar). Furthermore, lipid droplets were reported to contain other microdomain proteins,i.e. Lyn and mitogen-activated protein kinase, as well as abundant free cholesterol (6Yu W. Bozza P.T. Tzizik D.M. Gray J.P. Cassara J. Dvorak A.M. Weller P.F. Am. J. Pathol. 1998; 152: 759-769PubMed Google Scholar, 7Yu W. Cassara J. Weller P.F. Blood. 2000; 95: 1078-1085Crossref PubMed Google Scholar, 8Prattes S. Horl G. Hammer A. Blaschitz A. Graier W.F. Sattler W. Zechner R. Steyrer E. J. Cell Sci. 2000; 113: 2977-2989Crossref PubMed Google Scholar). These results suggest that the lipid droplet surface is a kind of membrane and that it might have some similarity to the microdomain. However, electron microscopy of conventional resin-embedded ultrathin sections cannot visualize any membranous structure around the lipid droplet. In the ultrathin section of specimens fixed by aldehydes and then by osmium tetroxide, the lipid droplet content appears vacant, and its periphery is usually seen as a thin intermittent line. In many diagrams, the lipid droplet surface has been depicted as a phospholipid monolayer with the hydrophilic headgroup facing the cytoplasm and the hydrophobic acyl chains extending into the lipid droplet content (9Murphy D.J. Vance J. Trends Biochem. Sci. 1999; 24: 109-115Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar,10Brown D.A. Curr. Biol. 2001; 11: R446-R449Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). It has also been assumed that lipid droplets form by accumulation of neutral lipids between the two leaflets of the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; ADRP, adipose differentiation-related protein; Cap-LC/ESI, capillary liquid chromatography/electrospray ionization; PC, phosphatidylcholine; TIFF, Triton X-100-insoluble floating fraction membrane. However, evidence to support the above assumptions is scarce. Only freeze-fracture electron microscopy showed that the fracture plane along the lipid droplet surface is occasionally continuous with the cytoplasmic leaflet of the ER membrane (11Blanchette Mackie E.J. Dwyer N.K. Barber T. Coxey R.A. Takeda T. Rondinone C.M. Theodorakis J.L. Greenberg A.S. Londos C. J. Lipid Res. 1995; 36: 1211-1226PubMed Google Scholar, 12Peixoto de Menezes A. Pinto da Silva P. Lab. Invest. 1979; 40: 545-553PubMed Google Scholar). We wanted to examine whether the lipid droplet surface is really a phospholipid monolayer. Because it is difficult to retain lipid-rich structures by conventional morphological methods, we took advantage of cryoelectron microscopy that can observe biological specimens at the atomic resolution without fixation or staining (13Fujiyoshi Y. Mizusaki T. Morikawa K. Yamagishi H. Aoki Y. Kihara H. Harada Y. Ultramicroscopy. 1991; 38: 241-251Crossref Scopus (175) Google Scholar). Furthermore, to gain information about the lipid droplet phospholipids, we analyzed the fatty acid composition of phospholipids by mass spectrometry. The microscopy showed that the lipid droplet surface is indeed a hemi-membrane, or a phospholipid monolayer, and mass spectrometry revealed that the fatty acid composition of the lipid droplet phospholipids is distinct from that of rough ER and cholesterol/sphingolipid-rich microdomain. The result not only questions the current hypothesis on the mechanism of lipid droplet formation but also provides a firm basis for further studies on the physiological function of lipid droplets. Mouse anti-caveolin-2 antibody (BD Transduction Laboratories), mouse anti-adipose differentiation-related protein (ADRP) antibody (Progen), and colloidal gold-conjugated secondary antibody (Amersham Biosciences) were obtained from respective suppliers. HepG2 cells were grown in Dulbecco's modified Eagle's medium added with 10% fetal calf serum. A HepG2 cell line stably expressing human caveolin-2β (clone A-8) (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar), kept in the presence of 200 μg/ml G418, was also used for some experiments. Cells were disrupted by nitrogen cavitation and subjected to sucrose density gradient ultracentrifugation as described (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar). The lipid droplet layer floating on the surface was used in subsequent studies. Absence of contamination by other organelles was confirmed by Western blotting of marker molecules. Triton X-100-insoluble floating fraction (TIFF) was obtained by treating cells with 1% Triton X-100; a light scattering band at the interface of 5%/35% sucrose solutions after ultracentrifugation was collected by aspiration (14Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2658) Google Scholar). In some experiments, 1% Triton X-100 was substituted with either 0.025% Triton X-100 (15Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar) or 500 mm sodium carbonate (pH 11) (16Song K.S., Li Shengwen Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar), and fractions were obtained from the top. In this report, only the fraction obtained by the 1% Triton X-100 procedure is called TIFF, and the others are referred to by separate names. Rough microsome, representing rough ER, was obtained from cycloheximide-treated cells as a fraction at the interface of 1.5 m/1.8 msucrose (17Morimoto T. Sabatini D.D. Spector. D.L. Goldman. R.D. Leinwand. L.A. Cells: A Laboratory Manual. 1. Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1998Google Scholar). Crude microsome was prepared by centrifuging a postmitochondrial supernatant for 60 min at 140,000 ×g. Lipid droplet suspension placed on microgrid was rapidly plunged into liquid ethane cooled by liquid nitrogen (18Adrian M. Dubochet J. Lepault J. McDowall A.W. Nature. 1984; 308: 32-36Crossref PubMed Scopus (1008) Google Scholar). The specimen was transferred into a JEOL 3000SSF cryoelectron microscope using a cryotransfer device and observed at a stage temperature of 4.2 K. Micrographs were taken using the minimum dose system to alleviate the radiation damage (19Fujiyoshi Y. Kobayashi T. Ishizuka T. Uyeda N. Ishida Y. Harada Y. Ultramicroscopy. 1980; 5: 459-468Crossref Scopus (94) Google Scholar). A defocus value was set at 1–2 μm to increase the image contrast. The electron radiation to the specimen was less than 5,000 electrons/nm2, which does not seriously damage the vitrified biological specimen at the temperature. Lipid droplet suspension prepared from clone A-8 was placed between thin copper plates and rapidly frozen by the metal sandwich method (20Fujimoto T. Fujimoto K. J. Histochem. Cytochem. 1997; 45: 595-598Crossref PubMed Scopus (29) Google Scholar) and then freeze-fractured in a Balzers BAF400D. Platinum/carbon replicas were treated with 1% SDS and labeled for caveolin-2 by the procedure described previously (21Fujimoto K. J. Cell Sci. 1995; 108: 3443-3449Crossref PubMed Google Scholar). The total lipids were extracted from lipid droplets and crude microsome Y. J. Biol. Chem. Full Text PDF Google Scholar). the from lipid droplets was subjected to to separate and lipids S. J. Lipid Res. 24: Full Text PDF PubMed Google Scholar). They were on plates by and then by acid and by cholesterol and total phospholipids in lipid were and the was obtained G. S. PubMed Scopus Google Scholar, W. M. J. J. Lipid Res. Full Text PDF PubMed Google Scholar). The lipids extracted from isolated lipid droplets were subjected to to the content of neutral which the of phospholipids by mass spectrometry. The mass spectrometry was as described previously R. J. Y. Ishida M. J. 2000; PubMed Scopus Google Scholar). after of phospholipids by Nomura the was analyzed by a mass with of of phospholipid was by the mass electron microscopy of resin-embedded sections not membrane structure in the of lipid droplets in HepG2 cells not To visualize the lipid droplet surface we cryoelectron microscopy that can visualize at or a phospholipid was observed as two by this method without any fixation or staining Y. Y. PubMed Scopus Google Scholar). By isolated lipid droplets were in thin in microgrid On the stage cooled to 4.2 K, the specimen was at a low with a minimum electron in a from the were taken by using the minimum dose system (19Fujiyoshi Y. Kobayashi T. Ishizuka T. Uyeda N. Ishida Y. Harada Y. Ultramicroscopy. 1980; 5: 459-468Crossref Scopus (94) Google Scholar). The of isolated lipid droplets observed by cryoelectron microscopy was It was than which was observed as the of lipid droplets in resin-embedded ultrathin sections of HepG2 cells by conventional electron The indicates that lipid droplets were disrupted into Lipid droplets were observed as structures the content was of low electron but the was observed as a single line of about in The result that a single of or a phospholipid monolayer, the lipid droplet A of content showed a electron density than lipid droplets, were with two of the are to be a membrane a membrane. lipid droplets were seen to have The line was the the and A structure that to to the was seen by freeze-fracture of isolated lipid lipid droplets with some showed fracture or Furthermore, anti-caveolin-2 antibody labeled not only the fracture but also the fracture we that the lipid droplets with the a we assumed that may be phospholipid and the of lipid D.J. R.G. J. Cell Biol. PubMed Scopus Google Scholar, G. J. Cell Biol. 2001; 152: PubMed Scopus Google Scholar). However, in to membrane observed by conventional electron the are with without any and in cells were rapidly frozen and the was found only not we that the be by temperature the However, further studies be to the of the in isolated lipid droplets. In HepG2 expressing caveolin-2β without caveolin-1, caveolin-2β was found around lipid droplets by The as or not as reported previously for other cell S. D. Sargiacomo M. K. Lisanti M.P. 1998; PubMed Scopus Google Scholar). the cell was in the presence of 1% Triton X-100 and subjected to sucrose density gradient caveolin-2β in fractions and was not from the fractions not However, by 1% Triton X-100 with 500 mm sodium carbonate or 0.025% Triton caveolin-2β was from the low density fractions By lipid droplets were to contain phosphatidylcholine and free cholesterol as well as abundant cholesterol ester and On the other hand, other phospholipids, and not be by the method we used not showed that the of total phospholipids and free cholesterol is for lipid droplet and for postmitochondrial crude The crude microsome contain the membrane and the ER as well as other that most free cholesterol in the membrane Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the result indicates that the content of free cholesterol in the lipid droplet surface is than that in the membrane but than that in the In to the result in S. Horl G. Hammer A. Blaschitz A. Graier W.F. Sattler W. Zechner R. Steyrer E. J. Cell Sci. 2000; 113: 2977-2989Crossref PubMed Google Scholar), not lipid droplets in HepG2 cells not The result suggests that the free cholesterol content of lipid droplets may be different between and Lipid droplets have been hypothesized to by accumulation of neutral lipids between the two leaflets of the ER membrane (9Murphy D.J. Vance J. Trends Biochem. Sci. 1999; 24: 109-115Abstract Full Text Full Text PDF PubMed Scopus (491) Google may either from the ER or to it. The surface of lipid droplet is assumed to be derived from the cytoplasmic leaflet of the ER membrane. the lipid droplet surface is continuous with the ER composition of phospholipids in the lipid droplet surface may be to that of the accumulation of caveolin-2β and its in low density might the lipid droplet surface is to the microdomain. To examine we the fatty acid composition of and in lipid droplets, rough microsome rough and the by mass spectrometry. The fatty acid of obtained by a low was between lipid droplet and rough of and were observed as In in was enriched with fatty with a of and were The obtained by this the mass of and not fatty On the other hand, by using a acyl chains are from by and can be as a By this lipid droplets and rough microsome showed distinct for the for lipid droplets, but the of and were seen for rough The result indicates that the of was of in lipid droplets, the in rough microsome a of By the the of that the of in lipid droplets may contain than in rough microsome, the may be enriched with Furthermore, analyzed by the also distinct for lipid droplets and rough microsome of and were in lipid droplets, were low or in rough microsome and TIFF, and were in the that the lipid droplet surface is distinct from the rough ER is it to the sphingolipid/cholesterol-rich microdomain. The surface of lipid droplets has been to be a phospholipid monolayer hydrophobic lipid may exist stably in the cytoplasm only with with hydrophilic facing most the only result that the has been a freeze-fracture the of a ER membrane leaflet and the lipid droplet surface (11Blanchette Mackie E.J. Dwyer N.K. Barber T. Coxey R.A. Takeda T. Rondinone C.M. Theodorakis J.L. Greenberg A.S. Londos C. J. Lipid Res. 1995; 36: 1211-1226PubMed Google Scholar, 12Peixoto de Menezes A. Pinto da Silva P. Lab. Invest. 1979; 40: 545-553PubMed Google Scholar). In the we a cryoelectron which is to visualize biological specimens with damage and has been used to structures at atomic resolution C. Y. K. K. M. A. Y. Nature. 2001; PubMed Scopus Google Scholar). The cryoelectron microscopy has previously that the of the A is a phospholipid monolayer with a protein layer Y. K. J. PubMed Scopus Google Scholar). By this a single line representing a of was observed in the surface of isolated lipid droplets. It with two seen in the membrane Y. Y. PubMed Scopus Google Scholar), which is a phospholipid and for the that the lipid droplet surface is a phospholipid monolayer. The presence of a phospholipid monolayer in the surface is consistent with the current that lipid droplets are by lipid ester between the two leaflets of the ER membrane and may to it (9Murphy D.J. Vance J. Trends Biochem. Sci. 1999; 24: 109-115Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, D.A. Curr. Biol. 2001; 11: R446-R449Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). of a that cholesterol in the ER J. D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google and not to that lipid droplets may along the membrane. However, mass spectrometry showed that fatty acid of and in lipid droplets are distinct from in rough microsome, which is to be to the rough The result does not the that the lipid droplet surface is from the ER membrane but indicates that the is a differentiated lipid droplets may exist as a structure of the the lipid droplet may be to the ER, but some mechanism may the lipid droplet surface from the bulk ER membrane as for other ER Vance J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). is the we that in of the ER not but are to to lipid droplets. or other lipid proteins may be in the A of lipid droplets revealed by mass spectrometry is the of fatty in and the was is not but reported to exist in lipid droplets of (6Yu W. Bozza P.T. Tzizik D.M. Gray J.P. Cassara J. Dvorak A.M. Weller P.F. Am. J. Pathol. 1998; 152: 759-769PubMed Google Scholar), may be in lipid droplets is derived from a of with acyl in 1. The of with two acyl chains and in lipid droplets is in line with the The of the is also They may be in the ER and to lipid with neutral lipids might be in the accumulation of acyl However, it is also that the were in lipid in this the continuous of lipid and the and of fatty are to be to the surface contamination in the lipid droplet fraction is by the of marker of other organelles (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar), but a of contamination by phospholipids may to be However, using the cell as a with two acyl chains are found abundantly only in the lipid droplet and less in the microsome or Furthermore, using the mass obtained from cell and membrane not the characteristics found with the lipid droplet. These results it that the found in the lipid droplet in a and to any membrane In of caveolins to lipid droplets was found to be or cells were treated with A (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar, 3Ostermeyer A.G. Paci J.M. Zeng Y. Lublin D.M. Munro S. Brown D.A. J. Cell Biol. 2001; 152: 1071-1078Crossref PubMed Scopus (213) Google Scholar). The result was to that caveolins in the ER membrane and then to the lipid droplet by the membrane However, two questions were this was caveolins be in lipid droplets and the other was caveolins to lipid droplets were not after A was The may be by that the lipid droplet surface is not continuous to the ER membrane. the can be only by a mechanism of caveolins appears to in lipid droplets. a showed that cholesterol are incorporated to droplets containing G. B. M.P. S. J. Lipid Res. 2001; Full Text Full Text PDF PubMed Google Scholar). It is that lipid along with phospholipids, may be from the ER to lipid droplets by and caveolins might the some mass spectrometry also showed that the lipid droplet surface is different from the sphingolipid/cholesterol-rich microdomain in the fatty acid a of fatty than rough microsome, lipid droplet The result indicates that not only between and cholesterol but also between phospholipid and cholesterol are in the as from membrane studies R. E. Brown D. Sci. S. A. PubMed Scopus Google Scholar). on the of we previously that the lipid droplet surface might have a to the microdomain (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar). However, the result does not support the in of the fatty acid Furthermore, showed that the of and free cholesterol in with is less in lipid droplets than in not lipid composition does not to be the of to lipid droplets. as HepG2 cells are caveolin-2β was found around lipid droplets, but was found in the and only in lipid was not to lipid droplets treated with A (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar). The of caveolins may a to elucidate the mechanism of to lipid droplets. obtained by the 1% Triton X-100 method has been as of the sphingolipid/cholesterol-rich microdomain D.A. E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). We found that caveolin-2β in the lipid droplet was with 1% Triton X-100 in the but to low density fractions 500 mm sodium carbonate or 0.025% Triton X-100 was used in cell The low density fractions obtained by the sodium carbonate were reported to be enriched with the as (16Song K.S., Li Shengwen Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar). However, whether the two really the membrane has not been On the other hand, the fractions collected by the 0.025% Triton X-100 method were to contain with less to the sphingolipid/cholesterol-rich microdomain (15Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). The result showed that although the lipid composition is in the lipid droplet surface are enriched in the fraction as the sphingolipid/cholesterol-rich microdomain. It further studies to whether the result indicates some similarity in the of the two or indicates that the low density fractions obtained by the sodium carbonate and 0.025% Triton X-100 contain In the showed that the lipid droplet surface is a phospholipid monolayer with a fatty acid Lipid droplets been regarded as a of neutral lipids and as However, in of indicating accumulation of proteins to various D.D. T. S. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google H. PubMed Scopus Google Scholar), it is to lipid droplets are and The revealed a firm basis for the studies. We are most to Y. for the use of cryoelectron and the We also M. and T. for