A Phospholipase D-dependent Process Forms Lipid Droplets Containing Caveolin, Adipocyte Differentiation-related Protein, and Vimentin in a Cell-free System*

Denis Marchesan {ddagger}, Mikael Rutberg {ddagger}, Linda Andersson {ddagger} §, Lennart Asp {ddagger}, Thomas Larsson §, Jan Borén {ddagger}, Bengt R. Johansson ¶ and Sven-Olof Olofsson {ddagger} ||

From the {ddagger}Department of Medical Biochemistry and the Wallenberg Laboratory for Cardiovascular Research, the §Swegene Proteomic Center, and the Department of Anatomy and Cell Biology, Göteborg University, SE-413 45 Göteborg, Sweden

Received for publication, February 10, 2003 , and in revised form, April 29, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We developed a microsome-based, cell-free system that assembles newly formed triglyceride (TG) into spherical lipid droplets. These droplets were recovered in the d <= 1.055 g/ml fraction by gradient ultracentrifugation and were similar in size and appearance to those isolated from rat adipocytes and 3T3-L1 cells. Caveolin 1 and 2, vimentin, adipocyte differentiation-related protein, and the 78-kDa glucose regulatory protein were identified on the droplets from the cell-free system. The caveolin was soluble in 1% Triton X-100, as was the caveolin on lipid droplets from 3T3-L1 cells. The lipid droplets from the cell-free system, like those from 3T3-L1 cells, contained TG, diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The assembly of these TG-containing structures was dependent on the rate of TG biosynthesis and required an activator present in the 160,000 x g supernatant from homogenized rat adipocytes. The activator induced phospholipase D (PLD) activity, and its effect on the release of the TG-containing structures from the microsomes was inhibited by 1-butanol (but not 2-butanol) or 2,3-diphosphoglycerate. The activator could be replaced by a constitutively active PLD or phosphatidic acid. These results indicate that PLD and the formation of phosphatidic acid are important in the assembly of the TG-containing structures.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Within the cell, triglycerides (TG)1 are stored in cytosolic lipid droplets. Little is known about how these structures are assembled. Several proteins have been identified on their surface, including adipocyte differentiation-related protein (ADRP or adipophilin), perilipins (reviewed in Refs. 1 and 2), and caveolin (see Refs. 35 and reviewed in Refs. 2 and 6), but their roles in lipid droplet assembly are unknown.

ADRP and perilipins are abundant on lipid droplets. ADRP is found mostly on smaller droplets; when overexpressed, it stimulates lipid droplet formation (7), suggesting a role in the assembly process. Perilipins are present on large lipid droplets (7) and have a central role in the turnover of TG within these structures (1, 8, 10), perhaps affording protection against hormone-sensitive lipase (11). Perilipin-null mice have smaller lipid droplets and a higher rate of basal lipolysis than wild-type mice and are resistant to diet-induced obesity (12).

Caveolin is a 21-kDa membrane protein with a hairpin structure whose N and C termini face the cytosol (13, 14). In mammals, there are three caveolin genes. Caveolin 1 and 2 are expressed in adipocytes, whereas caveolin 3 is present in muscle. Caveolin plays a key role in intracellular lipid transport (15, 16), binding fatty acid (17), and transporting cholesterol (16) in a manner reminiscent of the way plasma apolipoproteins transport lipids in the blood (16, 18).

Phospholipase D (PLD) catalyzes the conversion of phosphatidylcholine to phosphatidic acid (PA) and appears to be important in intracellular transport and sorting processes (1922), either as an intracellular messenger or as a cone-shaped lipid that alters the curvature of the membrane (2325). The formation of PA is important for the fission of transport vesicles (26, 27). Two mammalian isoforms of phosphatidylcholine-specific PLD (PLD1 and PLD2) have been identified and extensively investigated (2830). Although they exhibit about 50% identity, the two enzymes have different biochemical properties and depend on different cofactors (2831). Our studies (32) suggest that PLD is important for the second step in the assembly of very low density lipoproteins (reviewed in Refs. 33 and 34), which appears to involve the formation of a lipid droplet in the microsomal lumen (35).

Cell-free systems are extremely useful for studies of sorting processes, such as the budding of transport vesicles (reviewed in Refs. 36 and 37) and are the only method for determining the mechanism by which such complex structures are formed. We developed a microsome-based, cell-free system to study the transport of TG from microsomes to the cytosol. This system assembles TG-containing lipid droplets similar to the small lipid droplets isolated from 3T3-L1 cells. Here we report that caveolin, ADRP, vimentin, and the 78-kDa glucose-regulated protein (GRP-78) are present on these lipid droplets and that the release of the droplets from microsomes is dependent on PLD and the formation of PA.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Dulbecco's modified Eagle's medium (containing glucose and pyridoxine but no phenol red), 1,2-dioleoyl-sn-glycerol, 3-isobutyl-1-methylxanthine, dexamethasone, pepstatin A, fatty acid-free bovine serum albumin, phenylmethylsulfonyl fluoride, oleic acid, ATP, GTP, creatine phosphate, creatine kinase, palmitoyl-CoA, and cabbage PLD were obtained from Sigma. Dulbecco's minimum essential medium (with high glucose and L-glutamine but without sodium pyruvate), fetal calf serum, nonessential amino acids, glutamine, penicillin, and streptomycin were purchased from PAA Laboratories (Linz, Austria). Trasylol (aprotinin) was from Bayer Leverkusen (Leverkusen, Germany). N-Acetyl-Leu-Leu-norleucinal, [14C]palmitoyl-CoA (55 mCi/mmol), 1-stearoyl, 2-[1-14C]arachidonyl-sn-glycerol (56 mCi/mmol), Rainbow molecular weight markers, PD-10 columns, and the enhanced chemiluminescence Western blotting system were from Amersham Biosciences. Ready Safe was from Beckman Instruments (Fullerton, CA). The µMacs separation system was from Miltenyi Biotec (Bergisch Gladbach, Germany). Insulin (100 IU/mg) was from Novo Nordisk (Copenhagen, Denmark). Leupeptin was from Chemicon (Temecula, CA).

Antibodies—Rabbit immunoglobulin was purchased from Dako (Glostrup, Denmark). Caveolin antibodies were from Transduction Laboratories (Lexington, KY) and Affinity Bioreagents (Golden, CO). Adipophilin (ADRP) antibodies and antibodies to perilipins were obtained from Research Diagnostics (Flanders, NJ). Antibodies to Na/K-ATPase and vimentin were from Abcam (Cambridge, UK). Antibodies to GRP-78 were from Affinity Bioreagents (Golden, CO). Golgin antibodies were from Molecular Probes (Leiden, Netherlands). Gold-conjugated anti-rabbit antibodies were from British Biocell International (Cardiff, UK).

Isolation of Microsomes from 3T3 Cells—3T3-L1 cells were cultured and differentiated for 2 days (unless stated otherwise) as described (38). The cells (2 x 106 cells/incubation) were homogenized (30 strokes in a Dounce glass homogenizer in 10 mM Tris-HCl, pH 7.5, containing 250 mM sucrose, 1 mM EDTA, 100 KIU/ml Trasylol, 0.1 mM leupeptin, 0.1 µM pepstatin, and 1 mM phenylmethylsulfonyl fluoride), and the microsomes were isolated essentially as described (39). To remove loosely bound (nonintegral) proteins from the microsomes, the pellet was resuspended in 500 mM Tris-HCl, pH 7.5, containing 1.2 M potassium chloride (high salt wash; modified from Ref. 40) and incubated for 30 min at 4 °C with head-over-tail mixing. The microsomes were then recovered by ultracentrifugation at 160,000 x g for 70 min at 12 °C. The washed microsomes were resuspended in 10 mM Tris-HCl, pH 7.5, containing 250 mM sucrose.

The Cell-free System—The in vitro system used to investigate the formation of lipid droplets was based on microsomes that had been subjected to a high salt wash, an activator from the 160,000 x g supernatant from homogenized rat adipocytes, and a substrate for DGAT.

To prepare the DGAT substrate, 0.25 mg of diacylglycerol was dissolved in 40 µl of 10 mM Tris-HCl, pH 7.5, with 1.0 mM palmitoyl-CoA by gentle vortexing; 1.25 µl of [14C]palmitoyl-CoA (55 mCi/mmol; total 0.06 µCi) and 360 µl of 10 mM Tris-HCl, pH 7.5, were added, and the mixture was vortexed again. In some experiments the [14C]palmitoyl-CoA was replaced with diacylglycerol (1-stearoyl, 2-[1-14C]arachidonyl-sn-glycerol).

To start the reaction, 600 µl of the partially purified cytosolic activator in 10 mM Tris-HCl, pH 7.5, with 250 mM sucrose, 150 µl of 1.4 M MgCl2 (in the same buffer), and 50 µl of the microsome solution (0.5–1 mg of microsomes) was added to the DGAT substrate. Incubation was carried out at 37 °C for 60 min (the production of lipid droplets plateaued between 30 and 60 min).

After the incubation, the total production of radioactive triglycerides was measured, and the incubation mixture was subjected to gradient ultracentrifugation as described below. Before centrifugation, the mixture was supplemented with (final concentrations) Trasylol (100 KIU/ml), leupeptin (0.1 mM), phenylmethylsulfonyl fluoride (1 mM), pepstatin A (1 µM), N-acetyl-Leu-Leu-norleucinal (5 µM), and EDTA (0.5 mM).

Partial Purification of the Cytosolic Activator—To form lipid droplets, the cell-free system needed an activator present in the cytosol of rat adipocytes. Adipocytes were prepared essentially as described (41), suspended in 10 mM Tris-HCl, pH 7.4, with 250 mM sucrose, homogenized by 15 strokes in a Dounce homogenizer, and centrifuged at 160,000 x g for 2 h at 4 °C. The centrifuge tube was punctured 1 cm from the bottom, and the supernatant was aspirated, carefully avoiding contamination from the fat cake and the pellet. The supernatant was concentrated (final volume, 0.5 ml) and subjected to gel chromatography on a Superdex 200 column (HR 10/30; Amersham Biosciences) equilibrated with 10 mM Tris-HCl, pH 7.4, containing 250 mM sucrose, using an Äkta prime chromatography system (Amersham Biosciences). The flow rate was 0.3 ml/min, and the fraction size was 0.5 ml. The optical density (280 nm) was recorded, and the eluted peaks were assayed for their ability to induce lipid droplet formation. This activity eluted from the column with an apparent molecular mass of 180 kDa.

This 180-kDa fraction was concentrated (final volume, 2 ml) and subjected to anion-exchange chromatography with a Q-resource column (and the Äkta prime system) equilibrated with 10 mM Tris-HCl, pH 8.0. The column was eluted first with 18 ml of the same buffer and then with a 0–0.5 M NaCl gradient (in 10 mM Tris-HCl, pH 8.0; total volume 37 ml). Optical density (280 nm) and conductivity were recorded, and all fractions were analyzed for their ability to induce lipid droplet formation. Fractions containing this activity (eluted between 0.17 and 0.23 M NaCl) were combined, desalted, and concentrated. They are referred to as the Q-fraction.

The Q-fraction was adjusted to pH 7.0 and chromatographed on an S-resource column using the Äkta prime system. The column was equilibrated with 10 mM Tris-HCl, pH 7.0. The elution was started with 30 ml of the same buffer, followed by a 0–0.5 M NaCl gradient (in 10 mM Tris-HCl, pH 7.0; total volume 20 ml). Optical density and conductivity were recorded, and each fraction (2 ml) was analyzed for its ability to induce lipid droplet formation. The activity was eluted between 0.1 and 0.5 M NaCl. This fraction is referred to as the partially purified cytosolic activator or S-fraction.

Gradient Ultracentrifugation—For gradient I the samples were adjusted to 25% sucrose, and 1 ml was layered under 2 ml of 50 mM Tris-HCl, pH 7.5, with 10% sucrose and 10 mM EDTA, which in turn was overlaid with 2 ml of 50 mM Tris-HCl, pH 7.5, and 10 mM EDTA. Centrifugation was carried out at 160,000 x g for 17 h at 4 °C in a Beckman SW55 Ti rotor. Homogenates of 3T3-L1 cells (induced for 10 days) were used to establish the gradient. The gradient was divided into four fractions with mean densities of <=1.018 (2 ml), 1.034 (1 ml), 1.055 (1 ml), and 1.098 (1 ml) g/ml, respectively. The major amount of TG (62.6 ± 9.7%) was recovered in the d <= 1.018 g/ml fraction; 17.7 ± 3.0% was recovered in the 1.034 g/ml fraction; 8.3 ± 2.0% was recovered in the 1.055 g/ml fraction, and 9.7 ± 9.2% was recovered in the 1.098 g/ml fraction. The pellet (microsomes and cell debris) contained only 2.7 ± 0.5% of the total amount of TG recovered from the gradient (all data are mean ± S.D.; n = 3). Caveolin was present in all fractions and in the pellet.

For gradient II the samples were adjusted to 40% sucrose, and 1.2 ml was layered under 3 ml of 50 mM Tris-HCl, pH 7.5, with 25% sucrose and 10 mM EDTA, which was in turn overlaid with 1 ml of 50 mM Tris-HCl, pH 7.5, and 10 mM EDTA. Centrifugation was carried out at 160,000 x g for 17 h at 4 °C in a Beckman SW55 Ti rotor. The gradient was arbitrarily divided into five fractions with mean densities of 1.055 (1.2 ml), 1.099 (1 ml), 1.112 (1 ml), 1.141 (1 ml), and 1.161 (1 ml) g/ml, respectively. Most (75%) of the TG was present in the top fraction (d <= 1.055 g/ml). Caveolin was present in the top fraction and in the two bottom fractions (mean densities, 1.141 and 1.161 g/ml, respectively).

Lipid Analysis—Lipids were extracted as described (42), with modifications (43). The lipid extract was separated by TLC using a two-solvent system: chloroform/methanol/H2O (60:30:5 by volume) followed by petroleum ether/diethyl ether/acetic acid (80:20:1.5 by volume). Lipids were visualized by iodine vapor and identified by standards run in parallel. Radioactive TG was separated, counted as described (43), and expressed as a percentage of the total amount of radioactive TG recovered in the d <= 1.055 g/ml fraction.

The PLD assay was performed as described (44), except that labeled 1-butanol was used. The labeled phosphatidylbutanol formed was isolated by TLC with ethyl acetate/trimethyl pentane/acetic acid (8:5:2 by volume) as the solvent. Using the partially purified cytosolic activator, we observed a substrate-dependent reaction velocity with a Vmax at 420 mM 1-butanol (not shown). We therefore carried out the assay at that concentration.

Other Methods—Samples for electron microscopy were pelleted and fixed in 2.5% glutaraldehyde in 0.05 M sodium cacodylate, pH 7.2. The fixed sample was postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate and stained with 0.5 M uranyl acetate. Immunoelectron microscopy was performed with a Zeiss transmission electron microscope (model 902A) as described (45).

Treatment with 1% Triton X-100 was carried out essentially as described (46). The Triton-treated d <= 1.055 g/ml fraction from the cell-free system was centrifuged (46) in a Beckman TLS-55 rotor at 55,000 rpm (258,000 x g) at 4 °C for 13 h. Seven fractions (300 µl each) were recovered from the top and analyzed for caveolin. The Triton-treated d <= 1.018 fraction from the homogenized 3T3-L1 cells was centrifuged (46) at 35,000 rpm in a Beckman SW40 rotor at 4 °C for 12 h. Fractions of 2.4 ml were collected and analyzed for caveolin.

Immunoprecipitation was performed with the µMacs system essentially as recommended by the manufacturer, except that the beads were recovered by centrifugation for 15 min at 16,000 x g after the incubation with the antibody-reacted sample. The bound material was eluted with hot sample buffer. To analyze the phospholipids present on the immunoaffinity-purified lipid droplets, the beads were removed from the column and extracted with chloroform.

Protein was quantified with the BCA kit (Pierce). SDS-PAGE and immunoblotting were carried out as described (43, 47). For analysis by mass spectrometry, the gels were silver-stained as described (48). Protein bands were cut out, treated with trypsin, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) as described (47), except that Mascot software (available at www.matrixscience.com) was used for protein assignment. If the spectra were not good enough, the samples were cleaned up and concentrated with a ZipTip containing C18 material (Millipore) before the MS run. When the MALDI-TOF analysis was inconclusive, tandem MS/MS was performed with the quadrupole time-of-flight technique as described (49), and proteins were identified with Mascot software.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Release of TG and Caveolin from Microsome—Immunoblot analysis indicated that the microsomes used in the cell-free system contained GRP-78 (a marker for the ER) and Golgin (a marker for the Golgi apparatus) but not Na/K-ATPase (a marker for the plasma membrane) (Fig. 1). These results indicate that the isolated microsomal fraction is mainly derived from the ER and the Golgi apparatus. The homogenate from the 3T3-L1 cells contained caveolin, vimentin, and ADRP but not perilipins (Fig. 2); perilipins were detected in cells induced for 8 days (not shown). Caveolin, vimentin, and ADRP were present on the high salt-washed microsomes used in the cell-free system (Fig. 2).



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FIG. 1.
Characterization of the microsomal fraction isolated from 3T3-L1 cells. Microsomes were isolated from 3T3-L1 cells (induced for 2 days) and immunoblotted against antibodies to Na/K-ATPase (marker for the plasma membrane), GRP-78 (marker for the ER), and Golgin (marker for the Golgi apparatus).

 


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FIG. 2.
Immunoblotting of total cell homogenate or microsomes from uninduced and induced 3T3-L1 cells. Uninduced and induced (2 days) 3T3-L1 cells were homogenized, and the microsomes were isolated from the induced cells and subjected to a high salt wash. The total cell homogenate and the microsomes were then blotted against polyclonal antibodies against caveolin, ADRP, vimentin, and perilipins.

 

Upon incubation of the complete cell-free system (high salt-washed microsomes, DGAT substrate, and partially purified activator), newly formed (labeled) TG and caveolin were released from the microsomes and appeared in the less dense fractions obtained by gradient ultracentrifugation (Fig. 3A). Most of the TG and the caveolin were in the three bottom fractions of the gradient; relatively small amounts were present in the top fraction. We therefore combined the radioactive TG in the d <= 1.055 g/ml fractions in all calculations. The bottom fraction was excluded because its proximity to the membrane pellet increased the risk of contamination with microsomes. In the absence of the activator, TG and caveolin were not released (Fig. 3B).



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FIG. 3.
Accumulation of TG and caveolin. The cell-free system was incubated with the partially purified cytosolic activator (A) or buffer alone (B). After incubation, the samples were ultracentrifuged (gradient I). TG were recovered from each fraction by organic solvent extraction and TLC, and the radioactivity was determined. The results are given as the percentage of the total pool of radioactive TG formed in the cell-free system that was recovered in the different density fractions (mean ± S.D.; n = 3). Caveolin was detected by immunoblot.

 

Electron Microscopy of the d <= 1.055 g/ml Fraction—The d <= 1.055 g/ml fraction consisted of rounded filled structures (Fig. 4A). Most were 100–400 nm in diameter and were similar in size and appearance to the lipid droplets recovered from the d <= 1.018 g/ml fraction of homogenized 3T3 L1 cells and the smaller lipid droplets isolated from rat adipocytes lysed with hypotonic buffer (Fig. 4A). Immunoelectron microscopy showed that caveolin was present on filled structures of the same size as those isolated by gradient ultracentrifugation (Fig. 4B). Control experiments demonstrated that lipid droplets from the cell-free system did not derive from the partially purified cytosolic activator.



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FIG. 4.
Ultrastructural findings. A, 3T3-L1 cells were differentiated for 10 days and homogenized, and the d <= 1.018 fraction was recovered by ultracentrifugation (gradient I). Two regions are shown to illustrate the different particle sizes. The d <= 1.055 g/ml fraction was isolated (gradient I) from the cell-free system after incubation in the presence of the partially purified cytosolic activator. Rat adipocytes were lysed in 10 mM Tris-HCl, pH 7.5, and the d <= 1.018 fraction was recovered (gradient I). Bar represents 600 nm. B, the cell-free system was incubated with the partially purified cytosolic activator. Incubation with a gold-labeled polyclonal antibody to caveolin, fixation, and staining were carried out as described under "Experimental Procedures." Bar represent 600 nm. The samples were viewed with a Zeiss 902A transmission electron microscope.

 

Protein Composition of the d <= 1.055 g/ml Fraction—As shown by immunoblotting, caveolin 1 and 2, but not 3, were present on the lipid droplets (d <= 1.055 g/ml fraction) isolated from the cell-free system (Fig. 5A). Virtually all of the caveolin was in the denser fractions obtained by gradient ultracentrifugation after treatment with 1% Triton (Fig. 5B), indicating that the caveolin was detergent-soluble (14). This was also the case for the caveolin in lipid droplets from 3T3-L1 cells that had been induced for 10 days (Fig. 5C). The lipid droplets from the cell-free system also contained ADRP (Fig. 5A) but not perilipins (Fig. 5A) or Tip 47 (not shown).



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FIG. 5.
Immunoblot analysis of lipid droplets from the cell-free system and from 3T3-L1 cells. A, the cell-free system was incubated with partially purified cytosolic activator, and the d <= 1.055 g/ml fraction was isolated by ultracentrifugation (gradient I), subjected to SDS-PAGE with 12% gels, and blotted against antiserum to caveolin 1–3, perilipins, and ADRP. B, the d <= 1.055 g/ml fraction was recovered (gradient I) from the cell-free system and treated with 1% Triton and subjected to gradient ultracentrifugation as described under "Experimental Procedures." C, 3T3-L1 cells were induced for 10 days and homogenized, and the d <= 1.018 g/ml fraction was recovered by ultracentrifugation (gradient I) and treated with 1% Triton and subjected to gradient ultracentrifugation as described under "Experimental Procedures." Each density fraction was blotted against a polyclonal antibody to caveolin.

 

To analyze further the protein composition of the formed lipid droplets, we incubated the cell-free system in the presence or absence of the partially purified cytosolic activator and analyzed the d <= 1.055 g/ml fraction by SDS-PAGE (Fig. 6). In the presence of the activator (Fig. 6I), seven major silver-staining bands were present. In its absence, no proteins were detected (Fig. 6II). Incubation of the purified activator with the DGAT substrate alone showed that none of the silver-stained bands in the d <= 1.055 g/ml fraction derived from the interaction between proteins in the activator and this substrate (Fig. 6III).



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FIG. 6.
SDS-PAGE of the d <= 1.055 g/ml fraction. The cell-free system was incubated in the presence (I) or absence (II) of the partially purified cytosolic activator, and the partially purified cytosolic activator was incubated with the DGAT substrate (III). The d <= 1.055 g/ml fractions recovered by ultracentrifugation (gradient II) were subjected to SDS-PAGE with 12% gels, and the gels were stained with silver. The migration of standards with known molecular weight is indicated in the figure. 1–7 indicate silver-stained bands.

 

Bands 6 and 7 (Fig. 6I) reacted with antibodies to caveolin 1 and 2 on immunoblot. Band 5 migrated on the gel with an estimated molecular mass of ~50 kDa and reacted with ADRP antibody. Band 4 (~54 kDa) was cut out, trypsinized, and analyzed by MALDI-TOF. Identified trypsin fragments (with no or only one missed cleavage) were used to search the complete non-redundant data base at NCBI. Vimentin (mouse) got a probability score (Mascot) of 104 (scores above 74 are significant at the 95% level). Band 3 (~70 kDa) was identified by tandem MS/MS (quadrupole time-of-flight). In a search of the NCBI data base, GRP-78 (mouse) got a score of 172. No spectra were obtained from bands 1 and 2.

In summary, the lipid droplets formed in the cell-free system contained caveolin 1 and 2, ADRP, vimentin, and GRP-78. Immunoblot demonstrated that all these proteins were present on the high salt-washed microsomes but not in the partially purified cytosolic activator. MALDI-TOF analysis after SDS-PAGE confirmed that the activator did not contain caveolin, ADRP, vimentin, or GRP-78. Thus, the proteins on the lipid droplets generated in the cell-free system derived from the microsomes and not from the fraction containing the cytosolic activator.

Lipid Composition of the d <= 1.055 g/ml Fraction—As shown by TLC, the lipid droplets from 3T3-L1 cells (Fig. 7A) and those from the cell-free system (Fig. 7B) contained TG, diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, and a small amount of phosphatidylserine. Diacylglycerol was relatively more abundant in the d <= 1.055 g/ml fraction from the cell-free system than in lipid droplets from 3T3-L1 cells. By using antibodies to ADRP, we were able to immunoprecipitate 60–70% of the d <= 1.055 g/ml fraction (measured as radioactive TG). TLC of the bound fraction demonstrated TG, diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (Fig. 7C).



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FIG. 7.
TLC of lipid extracts. A, 3T3-L1 cells were homogenized, and the d <= 1.018 g/ml fraction was recovered (gradient I). B, the cell-free system was incubated in the presence of the partially purified cytosolic activator, and the d <= 1.055 fraction was recovered (gradient II). C, the d <= 1.055 fraction from the cell-free system was immunoaffinity-purified with antibodies to ADRP and protein G-coated µMacs beads. The bound material was eluted with hot sample buffer. The sample buffer gave rise to an artifact that completely obscured the phospholipids. We therefore removed the µMacs beads from the column and extracted them with chloroform. All fractions were extracted in a two-phase system and analyzed by TLC using a two-solvent system; "1" indicates the starting solvent, and "2" indicates the second solvent. The chromatogram was stained with iodine vapor. The extraction of the d <= 1.018 and the d <= 1.055 g/ml fractions gave rise to an artifact (not shown) migrating near the front of solvent 1 (Rf value of 0.84), whereas the chloroform extraction of the µMacs beads resulted in another artifact (not shown) near the front of solvent 1 Rf value of 0.81). Lipids were identified by standards run in parallel. CE, cholesterol esters; DG, diacylglycerol; PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine.

 

Lipid Droplet Formation Requires DGAT Substrates and TG Biosynthesis—In the absence of diacylglycerol and palmitoyl-CoA, TG biosynthesis in the cell-free system was virtually abolished, and no caveolin accumulated in the d <= 1.055 g/ml fraction (Fig. 8A). Removing these DGAT substrates also abolished the accumulation of TG in the d <= 1.055 g/ml fraction, even when expressed as the proportion of the very small amount of TG synthesized. Thus, the formation of lipid droplets from the microsomes is dependent on the DGAT substrate and on the rate of TG biosynthesis. To address this further, we removed either the cold palmitoyl-CoA or the cold diacylglycerol from the DGAT substrate (Fig. 8B). In the absence of palmitoyl-CoA, TG formation in the cell-free system and the accumulation of caveolin and TG in the lipid droplets (d <= 1.055 g/ml fraction) were significantly reduced. In the absence of diacylglycerol, only a small amount of TG was formed, and no caveolin and virtually no TG accumulated in the droplets.



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FIG. 8.
The formation of lipid droplets in the cell-free system is dependent on DGAT substrate and on TG biosynthesis. The cell-free system was incubated with the partially purified cytosolic activator in the presence or absence of DGAT substrate (A) or with the complete DGAT substrate (diamond) or a DGAT substrate lacking palmitoyl-CoA (square) or diacylglycerol (triangle) (B). Labeled palmitoyl-CoA was used as a tracer in the experiments shown in A; radiolabeled diacylglycerol was used in the experiments shown in B. When diacylglycerol was omitted from the DGAT substrate, only the tracer (but no cold diacylglycerol) was present in the system. After the incubation, the d <= 1.055 g/ml fraction was isolated by sucrose gradient ultracentrifugation (gradient I); TG was recovered by TLC, and the radioactivity was determined. TG values are expressed as a percentage of the total pool of radioactive TG formed in the cell-free system. Data are mean ± S.D. (n = 3) in A and mean ± S.E. (n = 3) in B. Caveolin in the d <= 1.055 g/ml fractions was determined by immunoblot. Some error bars are smaller than the symbol.

 

Lipid Droplet Formation in the Cell-free System Requires PLD—1-Butanol, but not 2-butanol, inhibited lipid droplet formation in the cell-free system, as measured by the appearance of caveolin and TG in the d <= 1.055 g/ml fraction (Fig. 9A). Because1-butanol acts as a nucleophile during PLD-catalyzed hydrolysis of phosphatidylcholine, phosphatidylbutanol is formed instead of PA. 2-Butanol, which is less nucleophilic, had no effect. Thus, the assembly of lipid droplets in the cell-free system involves the formation of PA and PLD. Another PLD inhibitor, 2,3-diphosphoglycerate, also inhibited droplet formation (Fig. 9B).



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FIG. 9.
The effect of PLD inhibitors on the formation of lipid droplets. The cell-free system was incubated with partially purified cytosolic activator and 1-butanol (filled diamonds) or 2-butanol (filled squares) (A), or 2,3-diphosphoglycerate (DPG) (B). A, accumulation of radioactive TG in the d <= 1.055 g/ml density fraction as a percentage of the value observed in the untreated cell-free system. B, accumulation of newly formed TG in the d <= 1.055 g/ml fraction as a percentage of total radioactive pool of TG formed during the incubation. Caveolin was detected by immunoblotting.

 

Next we investigated the effects of the cytosolic activator on PLD activity. The partially purified activator increased both PLD activity and lipid droplet formation in the cell-free system (Table I). Moreover, droplet formation and PLD activity increased after the partial purification (Table I). If the cytosolic activator activates a PLD involved in the formation of the lipid droplets, it should be possible to replace the activator with constitutively active PLD or PA. This was the case. After addition of active PLD or exogenous PA in the absence of the activator, newly formed TG and caveolin accumulated in the d <= 1.055 g/ml fraction (Fig. 10). Thus, PLD activity in the microsomes is essential for lipid droplet formation in the cell-free system.


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TABLE I
Effect of the cytosolic activator on the lipid droplet formation and on PLD activity

 


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FIG. 10.
Effect of a constitutively active PLD or exogenous PA on lipid droplet formation. A, the cell-free system was incubated with cabbage PLD in the absence of the cytosolic activator. B, the cell-free system was incubated in the presence of buffer, the partially purified cytosolic activator, or the indicated amount of PA. PA was added with the DGAT substrate. After incubation, the accumulation of newly formed TG in the d <= 1.055 g/ml fraction was determined as percentage of the total radioactive pool of TG formed during the incubation. Caveolin was detected by immunoblotting.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We developed a microsome-based, cell-free system that assembles lipid droplets containing newly formed TG. These droplets are similar in size, appearance, and lipid composition to those isolated from 3T3-L1 cells and rat adipocytes. The assembly of the droplets was dependent on the TG biosynthesis and on a PLD activity and the production of PA. The droplets contain caveolin, vimentin, ADRP, and GRP-78. Vimentin and ADRP on the lipid droplets were derived from a membrane-associated pool of these proteins.

The formation of lipid droplets required the DGAT substrate and ongoing biosynthesis of TG. However, the release of the lipid droplets was dependent on the formation of PA and on PLD activity. This conclusion is based on several observations. First, release of the droplets was inhibited by 1-butanol but not by 2-butanol, an indication that a PLD and the formation of PA are essential to the process (21, 32). Moreover, 2,3-diphosphoglycerate inhibited the release of the TG-containing structures. Second, lipid droplet formation required an activator present in the 160,000 x g supernatant of rat adipocytes. This activator also induced PLD activity in the microsomes of the cell-free system. Third, the activator could be replaced by a constitutively active PLD or by PA. PLDs are highly regulated by ADP-ribosylation factor 1 (44, 50, 51), protein kinase C (5153), and phosphatidylinositol 4,5-bisphosphate (5456), for example, and may participate in intracellular membrane "budding" processes (1922). However, the role of PLD in the formation of the lipid droplets within microsomes remains to be elucidated.

All proteins present on the lipid droplets formed in the cell-free system have been identified on lipid droplets in intact cell.

Caveolin participates in the transport of lipids, particularly cholesterol (15), and binds fatty acids (17). Its presence on cytosolic lipid droplets (35) and its induction by hepatic overexpression of PPAR{gamma}1, which causes adipogenic steatosis (57), suggest that caveolin is involved in TG storage. Consistent with this notion, caveolin-1-deficient mice are lean and resistant to diet-induced obesity (58). It has been proposed, based on its intracellular sorting (18, 59) and its lipid binding capacity, that caveolin serves as a shuttle for intracellular lipids (18). In support of this model, our findings in this study that caveolin is released from microsomes with lipid droplets suggest that caveolin may help sort newly formed TG from microsomes into a cytosolic form. Our results also support the suggestion (2, 6) that caveolin "buds off" with lipid droplets from the microsomal membranes (35).

Although vimentin is a cytosolic protein, the vimentin on the lipid droplets was derived from the microsomal membrane. Vimentin-type intermediate filaments interact with lipid bilayers (60), and this interaction is mediated by the highly positively charged N-terminal domain (61). Vimentin has also been shown to be associated with both ER (62) and the Golgi apparatus (6264). Vimentin forms a cage of intermediate filaments around cytosolic lipid droplets (65). In 3T3-L1 cells, expression of a dominant-negative vimentin reduces the formation of lipid droplets (66), suggesting that this protein is essential for their assembly. Our observations are compatible with such a conclusion. However, vimentin-deficient mice do not have any obvious phenotype (67). Thus, vimentin may promote lipid droplet formation but not be essential for this process. Alternatively, lipid droplets may be formed through more than one mechanism.

The ADRP on the lipid droplets was also derived from a membrane-associated pool, indicating that it can be targeted to the microsomal membranes. ADRP can be acylated (1, 68), and these fatty acids may take part in the interaction between ADRP and the membrane, because covalently linked fatty acids participate in the membrane targeting of other proteins (e.g. ADP-ribosylation factor 1) (6971). The cell-free system was based on microsomes from cells induced for 2 days. Such cells assemble mostly small lipid droplets, like those formed in the cell-free system. ADRP is mostly present on smaller lipid droplets and, when overexpressed, seems to stimulate lipid droplet formation (7). Together our observations and the results from the literature may suggest that ADRP binds to the microsomal membrane and participate in the budding of the small lipid droplets.

Perilipins were not expressed in the 3T3-L1 cells used to prepare the microsomes and were not present in the partially purified cytosolic activator. Therefore perilipins were not present on the lipid droplets assembled in the cell-free system. Thus, small lipid droplets can be assembled in the absence of perilipins. However, perilipins are dominant proteins on cytosolic lipid droplets (1, 2). Because these proteins interact with hormone-sensitive lipase (810), they may protect the droplets against lipolysis (11) and perhaps thereby promote the formation of the large droplets seen in the adipocytes.

GRP-78 is a chaperon protein that contains a KDEL sequence and is therefore restricted to the ER/Golgi region of the secretory pathway. Because GRP-78 is a major protein in the ER, its presence on the lipid droplets from the cell-free system may reflect leakage of the ER-derived microsomes. The released protein may adhere to the assembled particles. However, in one study, GRP-78 appeared to be present on the cytosolic side of the surface layer of lipid droplets (72), suggesting that the surface layer is derived from the membrane of the ER (72). This is in agreement with the model proposed for the assembly of lipid droplets (2, 9, 73). According to this model the triglycerides will during the biosynthesis "oil-out" between the leaflets of the bilayers, forming lens-shaped structures in the membrane. The lipid droplet, covered by a monolayer, will be formed by the budding of such a lens from the membrane. The structure will be stabilized by proteins bound to both the luminal and cytosolic surface of the membrane.

The lipid droplets that were assembled in the cell-free system differed somewhat in density from those isolated from 3T3-L1 cells (1.055–1.018 versus <=1.018 g/ml, respectively). This may be due to a difference in the load of TG, perhaps reflecting a difference in the efficiency of TG biosynthesis in the two systems. If so, we would expect to find at least a small amount of denser droplets in 3T3-L1 cells. Such droplets were found in the 1.055 and 1.034 g/ml fractions isolated from the 3T3-L1 cells (not shown). A difference in the triglyceride load of the droplets is supported by the qualitative analysis which suggested that the lipid droplets in the d <= 1.018 g/ml fraction of 3T3-L1 cells have a higher TG:phospholipid ratio than the d <= 1.055 g/ml fraction from the cell-free system. Another possibility is that the lipid droplets formed in the cell-free system had a higher diacylglycerol:TG ratio than the droplets isolated from the cells; however, a portion this diacylglycerol could represent contamination from the DGAT substrate.

In conclusion, we propose that lipid droplets are formed from regions of the microsomal membrane that contains caveolin, vimentin, and ADRP. The process is dependent on DGAT activity and is driven by the activity of a PLD and the formation of PA.


    FOOTNOTES
 
* This work was supported by Grant 7142 from the Swedish Medical Research Council, the Swedish Heart and Lung Foundation, Novo Nordic Foundation, the Söderberg Foundation, Nestlé, and the Swedish Strategic Funds (National Network and Graduate School for Cardiovascular Research). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Wallenberg Laboratory, Sahlgrenska University Hospital/S, SE-413 45 Göteborg, Sweden. Tel.: 46-31-342 1956; Fax: 46-31-82 37 62; E-mail: Sven-Olof.Olofsson{at}medkem.gu.se.

1 The abbreviations used are: TG, triglyceride(s); ADRP, adipocyte differentiation-related protein; DGAT, diacylglycerol acyltransferase; ER, endoplasmic reticulum; GRP-78, 78-kDa glucose regulatory protein; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; PA, phosphatidic acid; PLD, phospholipase D. Back


    ACKNOWLEDGMENTS
 
We thank Stephen Ordway for excellent editing of the manuscript.



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