 |
INTRODUCTION |
Because of the detergent and regulatory properties of unesterified
fatty acid, a variety of organisms including animals, plants, and yeast
have evolved mechanisms to regulate closely the intracellular level of
unesterified fatty acids (1, 2). One such mechanism is the storage in
lipid droplets of fatty acids in esterified form as triacylglycerides
and sterol esters. Lipid droplets are especially prevalent in adipose
tissue (including mammary gland) and steroidogenic tissues, whereas
lower levels are found in cells of liver, kidney, intestine, and muscle
(heart and skeletal muscle) (reviewed in Ref. 3). Normally lipid
droplets represent a storage site for energy (fatty acids), membrane
phospholipid (triacylglycerol), and steroidogenesis (cholesterol,
cholesteryl-ester) (reviewed in Refs. 4-7). In contrast, abnormalities
in intracellular lipid storage are associated with obesity (reviewed in
Ref. 8), cardiovascular disease (reviewed in Refs. 9 and 10), diabetes
(reviewed in Ref. 11), neutral lipid storage disease (4), and Niemann Pick C disease (reviewed in Refs. 12-14). Despite the importance of
lipid droplets in normal lipid metabolism and disease, relatively little is known about the structure, lipid, and protein composition or
factors that regulate these parameters in lipid droplets.
It is generally assumed that the lipid components of lipid droplets are
assembled as a core of neutral lipid (triacylglycerol and/or
cholesterol ester) surrounded by a surface monolayer composed of polar
lipids (cholesterol, phospholipids, and fatty acids) (reviewed in Refs.
3 and 5). Two major types of lipid droplets are recognized in animal
cells. Lipid droplets from steroidogenic cells, rich in cholesteryl
esters, supply a ready source of cholesterol for steroid hormone
synthesis (7, 15, 16). Steroidogenic cells are rich in sterol carrier
protein-2 (17). SCP-21
stimulates cholesteryl ester formation in vitro, in intact
cells, and animals (reviewed in Ref. 17). SCP-2 may thereby also
contribute to the composition of lipid droplets in steroidogenic cells
(15) and to cholesterol transfer between lipid droplets and
mitochondria in steroidogenic cells (7, 16, 18). In contrast, lipid droplets from adipocytes, rich in triacylglycerides, are either secreted into milk or retained within the cell to provide a source of
fatty acids for signaling/gene regulation (1), fatty acids and
glycerides for membrane phospholipid synthesis (4), as well as fatty
acids for mitochondrial and peroxisomal oxidation to produce energy
(reviewed in Refs. 3 and 19). Adipose tissue is poor in sterol carrier
protein-2, a factor that correlates with the paucity of cholesterol and
cholesteryl esters in adipocyte lipid droplets (17). In contrast,
neither the properties nor regulation of lipid droplets in other cell
types have been reported.
Increasingly it is recognized that a unique group of lipid
droplet-specific proteins are localized as a protein "capsule" localized at the surface monolayer of the lipid droplet (reviewed in
Ref. 5). These proteins include the perilipins (5), adipose differentiation-related protein (ADRP) (5, 20-22), P200
capsule protein (23, 24), and vimentin (23). Other proteins, such as
sterol carrier protein-2 (15) and hormone-sensitive lipase (25), have
been shown to translocate to lipid droplets of steroidogenic cells or
steroidogenic cells from animals upon stimulation with lipolytic
hormones. However, it is only recently that the function of proteins
associated with the lipid droplet surface capsule have begun to be
resolved. Upon lipolytic stimulation, the intracellular hormone-sensitive lipase is phosphorylated and translocated to the
lipid droplet surface where it catalyzes the rate-limiting step in the
release of fatty acids from interior core triacylglycerides and
cholesteryl esters (25). However, accessibility of the triacylglycerols and cholesteryl esters for lipolysis by hormone-sensitive
lipase/cholesteryl esterase is closely correlated with decreased
association of perilipin (26, 27) and of P200 capsule
protein (24) with the lipid droplet surface. In contrast, ADRP
expression appears to be linked to the conversion of small to larger
lipid droplets (3, 5). ADRP also binds fatty acids (28) and enhances
their transport (20). SCP-2 has high affinity for not only fatty acids
(29-31) and fatty acyl-CoAs (32) but also for other lipid
droplet-associated lipids such as cholesterol (32-34) and
phospholipids (35, 36). SCP-2 accelerates the intracellular transfer of
fatty acids (37, 38) and differentially targets lipid droplet
cholesterol away from HDL-mediated efflux through the plasma membrane
(21) while enhancing lipid droplet cholesterol transfer toward the
mitochondria for steroidogenesis (7, 16). Although these data suggest an intricate balance of protein association with the lipid droplet, relatively little is known regarding either the lipid composition of
lipid droplets (7) or if their lipid composition may be determined by
the expression of the lipid droplet-associated proteins.
The nature of lipid droplets from cell types other than adipocytes and
steroidogenic cells is essentially unknown. The purpose of the present
investigation was 2-fold as follows: first, to examine the protein and
lipid structure of such lipid droplets; second, to determine the effect
of SCP-2 expression on modulating the lipid and protein components of
the lipid droplets. L cells provided a useful model for
these purposes based on the following data. (i) L cells
have substantial lipid droplets (21, 22, 38). (ii) SCP-2 expression,
transport of cholesterol from lipid droplets (for efflux to cell
surface bound HDL), and cellular levels of a lipid droplet-specific
protein (adipose differentiation related protein, ADRP) are
interrelated in L cells (21, 22). (iii) L cells
express low levels of SCP-2 (0.008 ± 0.001% of total protein)
(39) comparable to most peripheral tissues (i.e. <0.01% of
soluble protein) (reviewed in Refs. 17 and 34). (iv) Immunofluorescence confocal microscopy revealed that the intracellular localization of
endogenous SCP-2 in L cells (40, 41) reflected that
observed in other cells and tissues (reviewed in Refs. 17 and 34). (v)
L Cells posttranslationally process SCP-x/pro-SCP-2 gene
products (38, 39, 42, 43) in a similar manner as in animal tissues (reviewed in Ref. 17). Transfection of L cells with a
plasmid construct containing the cDNA encoding 15-kDa pro-SCP-2
(the normal SCP-2 gene translation product is the 15-kDa pro-SCP-2
precursor protein) resulted in overexpression and complete
posttranslational processing to the 13-kDa SCP-2. This was in contrast
to transfected Chinese hamster ovary cells (44) and hepatoma cells (45)
where incomplete posttranslational processing of 15-kDa pro-SCP-2 to the mature 13-kDa SCP-2 was observed. (vi) The intracellular
distribution of SCP-2 in transfected L cells exhibited a
pattern similar to that of SCP-2 in untransfected or mock transfected
L cells (40, 41, 46, 47) as well as in animal tissues
(reviewed in Refs. 17 and 34). In summary, these data suggested that
L cells provide a useful model to study the protein and
lipid components of lipid droplets as well as the effect of increased
SCP-2 expression thereon in intact cells.
In the present work, cellular subfractionation together with confocal
and multiphoton laser scanning microscopy as well as environmental
scanning electron microscopy showed the following. 1) The lipid
droplets from L cells exhibited properties intermediate to
those of lipid droplets from adipose and steroidogenic cells. 2) SCP-2
was not tightly associated with lipid droplets. 3) The expression of
SCP-2 significantly altered the lipid and fatty acyl composition of
purified lipid droplets, the association of several proteins (important
to lipid droplet metabolism) with purified lipid droplets in
vitro as well as in intact cells, the targeting of fatty acids and
cholesterol to lipid droplets, and/or the association of these lipids
with ADRP in intact cells. The results presented herein provide new
insights into the structure and regulation of lipid storage droplet
lipids on a molecular and functional level.
 |
MATERIALS AND METHODS |
Materials Sources--
Lab-Tek chamber coverglass slides and
organic solvents including petroleum ether, diethyl ether, glacial
acetic acid, and methanol were from Fisher; silica gel G plates were
purchased from Analtech (Newark, DE); lipid standards were purchased
from Nu-Chek-Prep (Elysian, MN) and Avanti Polar Lipids, Inc.
(Alabaster, AL). Complete Mini Protease Inhibitor Mixture was from
Roche Molecular Biochemicals. Rat and rabbit polyclonal anti-human
SCP-2 were obtained and purified as described earlier (40). Rabbit
polyclonal antiserum to ADRP was prepared as described (21). Rabbit
polyclonal antibodies against perilipin A and B and hormone-sensitive
lipase were a generous gift from Drs. A. S. Greenberg, Tufts
University (Boston, MA), and Dr. F. Kraemer, Stanford University
Medical Center (Stanford, CA), respectively. Monoclonal anti-vimentin was from Accurate Chemical and Scientific Corp. (Westbury, NY). The
F(ab')2 fragment of Alexa Fluor 594 goat anti-rabbit IgG (H + L chain-specific), Alexa Fluor 488 goat anti-rat IgG (H + L chain-specific), and NBD-stearate were purchased from Molecular Probes
(Eugene, OR). Data obtained on the environmental scanning electron
microscope was obtained with the technical support of Dr. Helga
Sittertz-Bhatkar (Electron Microscopy Center, Texas A & M University,
TX). Visualization of protein bands on the SDS-PAGE gel was
accomplished using the Silver Stain Plus kit from Bio-Rad. All reagents
and solvents used were of the highest grade available and were cell
culture tested as necessary.
L Cell Culture--
Cells were grown to confluency in Higuchi
medium (48) supplemented with 10% fetal bovine serum (HyClone, Logan,
UT) as described previously (39). Murine L cell fibroblasts
(L arpt
tk
) were obtained and stably
transfected with the cDNA encoding the 15-kDa pro-SCP-2 as
described (42). As expected, Western blotting showed that the 15-kDa
pro-SCP-2 was completely and posttranslationally processed to the
mature 13.2-kDa SCP-2 protein. SCP-2 composed 0.036 ± 0.002% of
the total proteins in stably overexpressing cells (34). In contrast,
SCP-2 expression was very low (0.008 ± 0.001% of total protein)
in control cells (i.e. untransfected and mock-transfected)
(39). Thus, the levels of SCP-2 expression in both SCP-2 overexpressing
and in control cells were in the range of those reported for murine
tissues (0.01-0.08% of cytosolic proteins) (49). For
immunocytochemistry experiments, cells were seeded at a density of
50,000 cells/chamber onto Lab-Tek chamber coverglass slides. For lipid
droplet isolation studies, the cells were grown to confluency on 10-cm
dishes and 245 × 245 × 25 mm trays (Nunc, Naperville, IL).
Isolation of Lipid Droplets from Mouse L Cell
Fibroblasts--
Lipid droplets were isolated from control and
SCP-2-expressing cells as described by Chanderbhan et al.
(7). To avoid potential proteolytic degradation during homogenization
and isolation of lipid droplets, Complete Mini Protease Inhibitor
Mixture (Roche Molecular Biochemicals) was included in the isolation
buffer as recommended by the manufacturer. Briefly, cells scraped from
8-10 confluent trays were homogenized in 50 mM
NaH2PO4 buffer, pH. 7.4, containing 154 mM NaCl, 5 mM MgCl2, and protease
inhibitor mixture, followed by centrifugation at 800 × g for 10 min. In order to sediment mitochondria the
resulting supernatant was centrifuged at 5,000 × g for
20 min, followed by a second centrifugation step at 35,000 rpm in a SW
40.1 rotor for 2 h at 4 °C. The lipid droplet fraction, forming
a distinct white band on the surface of the preparation, was removed
for lipid analysis. Details of the purity of the lipid droplets are
provided under "Results."
Lipid Mass Determination--
Lipid droplets, isolated from
control and SCP-2-expressing cells, were extracted with
n-hexane/2-propanol 3:2 (v/v), and lipid classes were
resolved by lipid class on Silica Gel G thin layer chromatography
plates developed in the following solvent system: petroleum
ether/diethyl ether/methanol/acetic acid 90:7:2:0.5 (50). Each lipid
sample was divided into two portions for mass and fatty acid
composition determination. Total cholesterol, free fatty acid,
triglyceride, and cholesteryl ester content were determined by the
method of Marzo et al. (51). Total phospholipid content was
determined by digesting the phospholipid fraction in deionized water
and perchloric acid for 1 h at 180 °C followed by addition of
ammonium molybdate and ascorbic acid (52). The sample was further
heated for 5 min in a boiling water bath and cooled, and the absorbance
read at 797 and 660 nm to quantify total phosphorous. Proteins were
determined by the method of Bradford (53) from the dried protein
extract residue digested overnight in 0.2 M KOH. Lipids
were stored under an atmosphere of N2 to limit oxidation, and all glassware was washed with sulfuric acid-chromate before use.
Transesterification--
Base-catalyzed transesterification was
performed on one-half of each lipid fraction (i.e.
cholesteryl esters, triglycerides, and phospholipids), isolated from
the above lipid extraction procedure, to convert the lipid acyl chains
to fatty acid methyl esters (FAME) (52). FAME were extracted into
n-hexane and were separated by gas-liquid chromatography
(GLC) on a GLC-14A (Shimadzu Kyoto, Japan) equipped with a SP-2330
capillary column (0.32 mm inner diameter × 30 m length,
Supelco, Bellefonte, PA). The injector and detector temperatures were
set at 220 °C with the column temperature maintained at 185 °C. A
Dionex UI-120 analytical-to-digital interface was used to collect the
peak area data that were converted to peak area using Dionex PeakNet software.
SDS-PAGE and Western Blot Analysis--
Cell homogenates and
lipid droplets from control and SCP-2-expressing cells were subjected
to SDS-PAGE using 12% Tricine gels (20 µg per lane) analyzed by
Western blot analysis to determine the content of specific proteins
such as SCP-2, ADRP, perilipin, hormone-sensitive lipase, and vimentin.
The cellular level of ADRP was shown previously to be decreased 70% in
the SCP-2-expressing cells (21). Samples run on the Tricine gels were
transferred to nitrocellulose membranes, blocked in 3% gelatin in TBST
(10 mM Tris-HCl, pH 8, 100 mM NaCl, 0.055 Tween
20), and incubated with antisera against SCP-2, perilipin,
hormone-sensitive lipase, and vimentin. Alkaline-phosphatase conjugates
of goat anti-rabbit or mouse IgG and Sigma Fast
5-bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolim tablets
(Sigma) were used to visualize the bands of interest.
Double Label Indirect Immunofluorescence
Microscopy--
Indirect immunolabeling of SCP-2 expression clones was
performed on cells grown to confluence on Lab-Tek chamber coverglass slides. The cells were fixed using cold acetone/ethanol (70:30 v/v)
followed by permeabilization with 0.05% saponin in Hanks' solution
(Life Technologies, Inc.). After washing with Hanks' solution, the
cells were blocked with 2% BSA (Sigma) for 1 h at room
temperature. The cells were then incubated in a mixture of anti-ADRP
(rabbit polyclonal anti-mouse ADRP purified by a subtractive Tricine
gel method as described earlier (39)) and anti-SCP-2 (rat polyclonal
anti-human SCP-2 also purified by the subtractive method) in 1% BSA in
Hanks' for 1 h at room temperature using a titer below
saturation. After extensive washing with 1% BSA in Hanks', a mixture
of secondary reagents consisting of a F(ab')2 fragment of
Alexa Fluor 594 goat anti-rabbit IgG (H + L chain-specific) and Alexa
Fluor 488 goat anti-rat IgG (H + L chain-specific) in 1% BSA in
Hanks' medium was added at a titer below saturation and incubated for
1 h at room temperature. Finally, the cells were washed with
Hanks' medium and examined. For the colocalization of ADRP and
NBD-stearic acid experiment, cells were incubated with 0.005%
NBD-stearic acid in serum-free medium for 1 h at 37 °C before
proceeding as described above with Alexa Fluor 594 goat anti-rabbit IgG
as the secondary antibody.
Cells were examined using a Bio-Rad MRC-1024MP laser scanning system
(Bio-Rad) equipped with three photomultiplier tubes for fluorescence
detection in separate channels, a 15-milliwatt krypton-argon laser
(American Laser, Salt Lake City, UT) with a 5-milliwatt output measured
at the microscope stages, and a Zeiss Axiovert 135 inverted
epifluorescence microscope (Zeiss, Thornwood, NY) with a 63× oil
immersion objective with a numerical aperture of 1.4. Fluorescence
imaging was performed simultaneously using 488 nm excitation, 540/30
emission, (green channel) and 568 nm excitation, HQ598/40 emission (red
channel). The confocal images from the green and red channels were
merged in order to see the extent of colocalization (red and green are
additive and yield yellow to orange in RGB color space). Pixel
fluorograms were constructed, and correlation coefficients generated
from the fluorograms were derived as described earlier (21). Image
files were analyzed using LaserSharp image software (Bio-Rad) and
Metamorph software (West Chester, PA).
Morphology of Isolated Lipid Droplets--
The morphology of
lipid droplets isolated from control and SCP-2-expressing cells was
determined by environmental scanning electron microscopy and
multiphoton laser scanning microscopy techniques. High resolution
secondary images were taken on an E3 Electroscan Environmental Scanning
Electron Microscope equipped with a Peltier cooling stage and
micro-manipulator/injector at 15 kV and pressures of 5 torr.
Multiphoton laser scanning microscopy was performed on lipid droplets
incubated with Nile Red (0.05%) on an MRC-1024MP multiphoton laser
scanning system (Bio-Rad) in combination with a Axiovert 135 inverted
microscope fitted with a 63× oil-immersion objective with a numerical
aperture of 1.4. A SABRE argon ion laser operating at 12 watts provided
the pump source for a femtosecond pulsed MIRA-900-F Ti:Sapphire laser
(Coherent, Inc., Sunnyvale, CA) that was used to provide multiphoton
excitation at 875 nm. Fluorescence of Nile Red was detected through an
external detector system (Bio-Rad) using a HQ575/150 bandpass filter
(Chroma Technology Corp., Brattleboro, VT).
Ligand Binding to ADRP, Displacement Assay--
ADRP binds
NBD-stearic acid with high affinity and 1:1 molar stoichiometry (28).
To determine the relative affinity of ADRP for nonfluorescent ligands,
the above assay was modified as a displacement assay as follows. ADRP
(50 nM) was incubated with NBD-stearic acid (180 nM) in 25 mM potassium phosphate buffer for 5 min at 37 °C to allow maximal binding. Increasing concentrations of
non-fluorescent ligand (stearic acid, oleic acid, oleoyl CoA, and
cholesterol) were added, and fluorescence intensity was monitored and
corrected for the blank and background. Integrative fluorescence intensity was measured for emission wavelengths higher than 500 nm, in
a ratio mode for NBD-stearate excited at 473 nm using a PC1 Photon
Counting Fluorometer (ISS Instruments, Champaign, IL). The data were
fitted using a simple, single binding site model (29).
Statistics--
All values were expressed as the mean ± S.E. with n and p indicated under "Results."
Statistical analyses were performed using Student's t test
(GraphPad Prism, San Diego, CA). Values with p < 0.05 were considered statistically significant.
 |
RESULTS |
Lipid Droplet Purification--
Although the lipid composition of
very large lipid droplets (up to 15 µm diameter) from
steroidogenic or adipose tissue has been examined (5, 7), almost
nothing is known regarding the lipid content/composition of the smaller
lipid droplets (near 1 µm diameter) more typical of most other
mammalian cells. L cell fibroblasts contain small lipid
droplets in this size range (21, 22). Furthermore, confocal microscopy
studies indicate that in intact cells the lipid (cholesterol) and
protein (ADRP) dynamics of these lipid droplets are influenced by SCP-2
expression (21). In the present investigation, these results were
extended to examine the structure of isolated lipid droplets from
L cell fibroblasts.
The relative purification of lipid droplets was assessed by comparison
of lipid composition and protein content markers of isolated lipid
droplets versus cell homogenate. Whereas lipid droplets do
not contain specific lipid species unique to lipid droplets, as
compared with cell homogenate, lipid droplets are relatively deficient
in surface lipids (cholesterol and phospholipid) and rich in core
lipids (triacylglycerol and cholesterol) (7). As shown in Table
I, L cell lipid droplets had
29.4-, 647-, 30.1-, and 10.8-fold lower cholesterol, phospholipid,
surface lipid, and total lipid mass (nmol/mg protein), respectively,
than the cell homogenate. The lipid droplets also exhibited 20.9-, 6.8-, and 17.9-fold lower molar ratios of phospholipid/cholesterol, cholesterol/cholesteryl ester, and surface lipid to core lipid, respectively (Table I). Indeed, the low levels of phospholipid and
cholesterol found within lipid droplets served as a marker of purity
since the membranes of other organelles have much higher levels of
these lipids. Contamination from other membranes would have led to
increased levels of cholesterol and phospholipid in the isolated lipid
droplet fractions. In summary, these data showed that lipid droplets
were highly purified as compared with cell homogenate.
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Table I
Purification of lipid droplets from L cell fibroblasts
Values represent means (nmol/mg protein or nmol/nmol) ± S.E.,
n = 3-5 separate isolations. Surface lipids (SL)
include cholesterol, phospholipids, and free fatty acids (FFA). Core
lipids (CL) include triacylglycerols (TG) and cholesteryl esters. Total
lipids include cholesterol, phospholipids, free fatty acids,
triacylglycerols, and cholesterol esters.
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Lipid Droplet Lipid Mass and Composition in L Cell
Fibroblasts--
Two classes of lipids were resolved from purified
L cell lipid droplets as follows: hydrophobic core lipids
and more polar surface lipids. The hydrophobic core lipids,
triacylglycerols and cholesteryl esters, represented 68% of the total
lipids in L cell lipid droplets (Fig.
1). L cells exhibited higher
triacylglyceride (10.6 ± 2.1 nmol/mg protein) than cholesteryl
ester (5.30 ± 0.47 nmol/mg protein) content, representing
45.2 ± 8.8 and 22.2 ± 2.0 mol % of total lipids,
respectively. The surface lipids (fatty acids, cholesterol, and
phospholipids) represented 32% of total lipids in the lipid droplets
(Fig. 1). Fatty acids were present at 5.2 ± 1.7 nmol/mg lipid
droplet protein and represented 21.6 ± 6.9 mol % of total
lipids in the lipid droplet. In contrast, the fatty acid content of
cell homogenate, 13.9 nmol/mg protein, represented only 5.4 ± 1.9 mol % of the lipids in the cell homogenate. Cholesterol and
phospholipids were present at 2.2 ± 0.3 and 0.23± 0.03 nmol/mg
lipid droplet protein, respectively, and represented 9.2 ± 1.1 and 0.1 ± 0.02 mol % of total lipids in the lipid droplet, respectively.

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Fig. 1.
Effect of SCP-2 expression on lipid droplet
lipid mass. Lipid droplets isolated from transfected L cells were
extracted and resolved into separate lipid classes to determine lipid
mass and composition. Values represent the means ± S.E.,
n = 3-5 separate isolations. Surface lipids
(SL) include cholesterol (C), phospholipids
(PL), and free fatty acids (FFA). Core lipids
(CL) include triacylglycerol (TG) and cholesteryl
esters (CE). * indicates significance as compared with the
control, p < 0.05.
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In summary, the smaller lipid droplets isolated from L
cells exhibited a unique lipid composition. The proportion of
triacylglycerol and cholesteryl ester was intermediate between that of
the larger triacylglyceride-rich lipid droplets from adipose tissue and
the cholesteryl ester-rich lipid droplets from steroidogenic tissue. Finally, the data showed for the first time that the lipid droplet was
4-fold enriched in unesterified fatty acids as compared with the cell homogenate.
Effect of SCP-2 Expression on the Lipid Composition of Lipid
Droplets Isolated from L Cell Fibroblasts--
As indicated in the
Introduction, relatively little is known regarding regulation of the
lipid composition of intracellular lipid droplets. SCP-2 binds many of
the lipids detected in lipids droplets (fatty acids, cholesterol, and
phospholipids) (reviewed in Ref. 34), influences the uptake,
intracellular transport, and/or metabolism of these lipids (reviewed in
Refs. 34 and 37), and is significantly localized in the cytoplasm (40, 41, 54). Therefore, it was important to establish whether increased
intracellular levels of SCP-2 could influence the composition of core
and surface lipids in lipid droplets isolated from L cell fibroblasts.
As with the control cells, the predominant hydrophobic lipid core
lipids in lipid droplets isolated from SCP-2-expressing L
cells were triacylglyceride and cholesteryl esters, 13.65 ± 1.53 and 2.20 ± 0.75 nmol/mg protein, respectively (Fig. 1). Lipid droplets isolated from SCP-2-expressing cells more closely resembled those of adipocyte-type lipid droplets with an increased
triacylglycerol content and a 2.4-fold decrease in cholesterol ester,
p < 0.05 n = 3-5 (Fig. 1). The trend
toward a more adipocyte lipid droplet was also reflected in nearly
3-fold increased relative ratio of triacylglycerides/cholesteryl esters
in the core lipids.
SCP-2 expression equally dramatically altered the surface lipids of the
lipid droplets. SCP-2 expression resulted in 11.8-fold reduction of
surface lipid mass from 7.6 ± 1.7 to 0.65 ± 0.04 nmol/mg
lipid droplet protein (Fig. 1). This reduction in surface lipid mass
was selective. Unesterified fatty acids were decreased 5.2-fold, below
the level of detection (p < 0.01, n = 3-5). Since SCP-2 expression did not significantly decrease the
unesterified fatty acid content of L cell homogenate
(15.3 ± 8.6 versus 14.0 ± 4.9 nmol/mg protein,
n = 4-5), this suggested that the majority of
unesterified fatty acids were redistributed away from the lipid droplet
in SCP-2-expressing cells. SCP-2 expression also decreased the lipid
droplet, but not cell homogenate, cholesterol content (nmol/mg lipid
droplet protein) by 6.6-fold (p < 0.01, n = 3-5), which resulted in a redistribution of the
majority of cholesterol away from the lipid droplet (Fig. 1). Although
SCP-2 expression did not significantly alter the lipid droplet
phospholipid mass, the cell homogenate phospholipid mass was decreased
nearly 2-fold from 148.8 ± 16.5 to 73.6 ± 12.4 (nmol/mg protein).
In summary, SCP-2 expression differentially altered the lipid
composition of isolated lipid droplets as compared with the cell
homogenate. The relative loss of fatty acids and cholesterol from lipid
droplets of SCP-2-expressing cells suggested that, due to its high
affinity for fatty acids (29-31) and cholesterol (31, 34, 55), SCP-2
may partition these lipids away from the lipid droplets. In contrast,
the lower affinity of SCP-2 for phospholipids (36, 56) may account for
the lack of change in lipid droplet phospholipid mass.
Fatty Acid Composition of Surface and Core Lipids--
Since SCP-2
binds fatty acyl-CoAs with high affinity (32, 57) as well as
differentially stimulates fatty acyl-CoA utilization for microsomal
phosphatidic acid and phospholipid synthesis in vitro and in
intact cells (41, 52), the possibility that SCP-2 expression may alter
the fatty acids esterified to lipids from lipid droplets was examined.
Four major fatty acids (16:0, 18:0, 18:1(n-9), and
22:4(n-6)) were associated with the phospholipid component
of lipid droplets (Fig. 2). SCP-2
expression significantly decreased only the 16:0 content in
phospholipids from 35.3 nmol/nmol phospholipid to below the level of
detection (p < 0.01, n = 3-4) (Fig.
2). This was consistent with the greater ability of SCP-2 to stimulate
in vitro microsomal incorporation of unsaturated fatty
acyl-CoAs into phosphatidic acid as compared with 16:0-CoA (41).

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Fig. 2.
Effect of SCP-2 expression on L cell lipid
droplet phospholipid fatty acid mass. Four fatty acids from the
phospholipid class of lipid droplets isolated from transfected L cells
were resolved as FAME by GLC as described under "Materials and
Methods." Values represent the means ± S.E., n = 3-5 separate isolations. * indicates significance as compared with
the control, p < 0.01.
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Six fatty acids (16:0, 18:0, 18:1(n-9),
20:1(n-7), 22:4(n-6), and 22:6(n-3))
represented the major fatty acid species esterified to cholesterol
(Fig. 3). Four of the six were
unsaturated fatty acids with the 18:1(n-9) being in largest
concentration in control cells. SCP-2 expression significantly
(p > 0.05, n = 3-5) decreased the
levels of 16:0, 18:1, and 20:1 in the cholesteryl ester fraction of the
lipid droplets. Concomitantly, the mass of 22:6(n-3) was increased 10.5-fold (p > 0.05, n = 3-5) in the SCP-2-expressing cells (Fig. 3). Overall, the saturated
fatty acids were significantly decreased (2-fold, p < 0.05, n = 3-5) in lipid droplet cholesteryl esters
from SCP-2-expressing cells, and polyunsaturated fatty acids were
increased 5-fold (p < 0.5, n = 3-5),
and the ratio of unsaturated to saturated fatty acids was increased
2-fold (p < 0.025, n = 3-5). These
results were consistent with the ability of SCP-2 to stimulate
cholesterol esterification in vitro (43, 58) as well as
cholesterol esterification and cholesterol cycling in transfected cells
(43, 45).

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Fig. 3.
Effect of SCP-2 expression on L cell lipid
droplet cholesteryl ester fatty acid mass. Six fatty acids from
the cholesteryl ester class of lipid droplets isolated from transfected
L cells were resolved as FAME by GLC as described under "Materials
and Methods." Values represent the means ± S.E.,
n = 3-5 separate isolations. * indicates significance
as compared with the control, p < 0.01.
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Seven fatty acids (16:0, 18:0, 18:1(n-9),
18:3(n-6), 20:3(n-6), 22:4(n-6), and
22:6(n-3)) were detected at significant levels in the
triglyceride fraction of lipid droplets (Fig.
4). The 18:0, 16:0, and
22:6(n-3) fatty acids represented the largest components. Except for the absence of 18:3(n-6) in the triacylglycerols
from lipid droplets of SCP-2-expressing cells, no significant
difference in the rest of the fatty acids isolated from the
triglyceride fraction of lipid droplets was observed between the
control and SCP-2-expressing cells.

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Fig. 4.
Effect of SCP-2 expression on L cell lipid
droplet triacylglycerol fatty acid mass. Seven fatty acids from
the cholesteryl ester class of lipid droplets isolated from transfected
L cells were resolved as FAME by GLC. Values represent the means ± S.E., n = 3-5 separate isolations. * indicates
significance as compared with the control, p < 0.01.
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In summary, a comparison of the fatty acids detected in the
triglycerides, cholesteryl esters, and phospholipids showed that polyunsaturated fatty acids were 12- and 4.5-fold higher in
concentration in the lipid droplet phospholipid fraction
(p > 0.05, n = 3-5) than in the
cholesteryl esters and triacylglycerols fractions, respectively. SCP-2
expression differentially targeted unsaturated/polyunsaturated fatty
acids toward phospholipids and cholesterol esters but had little effect
on the fatty acids esterified to triacylglycerols.
Cholesteryl Ester, Triglyceride, and Phospholipid Fatty Acid Mass
of Fetal Bovine Serum--
In order to determine if the fatty acids
targeted to the different lipid fractions of the lipid droplet simply
reflected those of the corresponding lipids in the fetal bovine serum
added to the cell culture medium, the fatty acid masses from the
cholesteryl ester, triglyceride, and phospholipid fractions isolated
from fetal bovine serum were determined. Whereas the same fatty acids esterified in the lipid fractions of lipid droplets were also qualitatively present in the fetal bovine serum, important quantitative differences were evident in all three fractions.
The major fatty acids present in serum phospholipids were 18:0 > 18:1 > 16:0 > 22:0 >24:1 > 22:6. In contrast, the
major fatty acids in phospholipids from lipid droplets were 18:1 > 18:0 >16:0 > 22:4 (Fig. 2). The ratio of serum and lipid
droplet unsaturated/saturated fatty acids in the phospholipid fraction,
0.91 ± 0.04 and 0.96 ± 0.67 (Fig. 2), respectively, did not
differ. However, the ratio of serum and lipid droplet phospholipid
polyunsaturated/monounsaturated fatty acids, 0.37 ± 0.02 and
0.15 ± 0.08 (Fig. 2), respectively, differed by 2.5-fold.
The major fatty acids present in serum cholesteryl esters were
18:1 > 16:0 > 20: 4 > 16:0 = 18:2 > 16:1 > 18:0. In contrast, the major fatty acids in cholesteryl
esters from lipid droplets were 18:1 > 18:0 >16:0 = 22:4 > 20:1 = 22:6 (Fig. 3). The ratio of serum and lipid
droplet unsaturated/saturated fatty acids in the cholesteryl ester
fraction, 4.36 ± 0.32 and 3.50 ± 1.19 (Fig. 3),
respectively, did not differ significantly. The ratio of serum and
lipid droplet in the cholesteryl ester fraction
polyunsaturated/monounsaturated fatty acids, 0.59 ± 0.05 and
0.22 ± 0.11 (Fig. 3), respectively, differed 2.7-fold.
The major fatty acids present in serum triacylglycerols were 18:1 > 20:4 > 16:0 > 18:2 > 20:2 > 18:0. In
contrast, the major fatty acids in triacylglycerols esters from lipid
droplets were 18:0 > 16:0 > 22:6 > 18:1 > 22:4 > 20:3 (Fig. 4). The ratio of serum and lipid droplet
unsaturated/saturated fatty acids in the triacylglycerol fraction,
3.48 ± 0.40 and 0.61 ± 0.21 (Fig. 4), respectively,
differed 5.7-fold. The ratio of serum and lipid droplet triacylglycerol
polyunsaturated/monounsaturated fatty acids, 1.54 ± 0.20 and
4.04 ± 1.61 (Fig. 4), respectively, differed severalfold.
In summary, the pattern of fatty acid esterification to phospholipids,
cholesteryl esters, and triacylglycerols of intracellular lipid
droplets differed substantially from those of the same lipid components
in the serum lipids.
Effect of SCP-2 Expression on Cholesterol to Phospholipid
Ratio--
Although cells closely regulate the
phospholipid/cholesterol mass ratio (nmol/nmol) of their surface
membranes (reviewed in Ref. 59), it is not known if this is also true
for the surface monolayer of lipid droplets. Because of the paucity of
phospholipids in the polar surface of the lipid droplet, the ratio of
cholesterol/phospholipid was very low, 0.11 ± 0.018 (Fig.
5), and much lower than that typical of
cell surface and intracellular membranes (59). Due to the 6.6-fold
decrease in cholesterol mass, the phospholipid/cholesterol ratio was
increased 8.7-fold (p < 0.01, n = 3-5) to 0.96 ± 0.13 in SCP-2-expressing cells.

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Fig. 5.
Effect of SCP-2 expression on lipid droplet
lipid ratios. Lipid ratios were determined from lipid droplets
isolated from transfected L cells. Surface lipids (SL)
include cholesterol (C), phospholipids (PL), and
free fatty acids. Core lipids (CL) include triacylglycerol
and cholesteryl esters (CE). Neutral lipids (NL) include
cholesterol, free fatty acids, triacylglycerol, and cholesterol esters.
Prot indicates protein (mg). * indicates significance
as compared with the control, p < 0.05.
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In summary, SCP-2 expression dramatically increased the
phospholipid/cholesterol ratio in the surface of lipid droplets. In bilayer membranes such alterations in phospholipid/cholesterol ratio
are typically accompanied by significant fluidization and altered
function of membrane proteins (reviewed in Refs. 60-63).
Effect of SCP-2 Expression on the Relative Proportion of Esterified
Cholesterol in the Lipid Droplet--
It is important to note that
SCP-2 expression induced a loss of cholesterol not only from the
surface lipid but also from the interior core wherein the mass of
cholesteryl esters was decreased 2.4-fold (Fig. 1). The relatively
greater loss of cholesterol than cholesteryl ester in lipid droplets of
SCP-2-expressing cells (Fig. 1) decreased the ratio of
cholesterol/cholesterol esters 2.7-fold from 0.41 ± 0.06 to
0.15 ± 0.05 nmol/nmol (Fig. 5). Thus, SCP-2 expression
dramatically mobilized/shifted cholesterol, and less so cholesteryl
esters, away from the lipid droplets.
Effect of SCP-2 Expression on Surface to Core Lipid
Ratio--
Since the ratio of surface area to volume determines
particle size, the ratio of surface to core lipid (surface lipids/core lipids) ratio in lipid droplets may be a predictor of the average size
of lipid droplets. Surface lipids include cholesterol, phospholipids, and free fatty acids, whereas the core lipids are the triglycerides and
cholesteryl esters. Although no significant difference was observed in
the amount of core lipids, the 11.6-fold decrease (p < 0.01, n = 3-5) in surface lipids in lipid droplets
isolated from SCP-2-expressing cells resulted in an 11.7-fold decrease (p < 0.01, n = 3-5) in the surface
lipids/core lipids ratio (Fig. 5). If the lipids components were the
exclusive determinants of the surface/volume relationship, these data
suggested that the lipid droplets from SCP-2-expressing cells were
smaller or that the amount and/or type of proteins associated with
these lipid droplets was different.
Effect of SCP-2 Expression on Lipid Droplet Morphology--
A
random sampling of lipid droplets derived from control and
SCP-2-expressing cells was measured from confocal images obtained as
described under "Materials and Methods" to determine lipid droplet
size. SCP-2 expression did not significantly alter the mean diameter of
droplets (1.47 ± 0.037 µm, n = 70, versus 1.57 ± 0.037 µm, n = 100)
that ranged in size from 0.5 to 3.0 µm. Since the decreased ratio of
surface/interior core lipid in the lipid droplets noted in
SCP-2-expressing cells did not result in increased lipid droplet size,
these data suggested that SCP-2 expression also altered the protein
components of the lipid droplets.
In order to characterize further the lipid droplets, environmental
scanning electron microscopy (Fig.
6A) and multiphoton laser
scanning microscopy (Fig. 6B) were performed on lipid
droplets isolated from control and SCP-2-expressing cells.
Environmental scanning electron microscopy allowed a high resolution,
three-dimensional view of isolated lipid droplets in solution. Whereas
some dehydration of sample occurred resulting in flattened lipid
droplets with concave centers, the size distribution of the purified
lipid droplets ascertained by environmental scanning electron
microscopy was within that observed by confocal microscopy within
intact cells (0.5-3.0 µ). Multiphoton laser scanning microscopy was
performed on isolated lipid droplets labeled with Nile Red as described under "Materials and Methods." The solution was allowed to
partially dry on a coverslip before viewing. This resulted in a
concentrated collection of lipid droplets with concave centers similar
to those seen by environmental scanning electron microscopy. Although
both techniques allowed good resolution of the droplets and showed good
agreement in size and overall shape, the results from the two
microscopy techniques revealed that the lipid droplets isolated from
the control (Fig. 6) and SCP-2-overexpressing cells (data not shown)
were not morphologically different.

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Fig. 6.
Environmental scanning electron microscopy
and multiphoton laser scanning microscopy of lipid droplets isolated
from L cells. A, high resolution secondary images of
lipid droplets isolated from control L cells were taken on an
Electroscan E3 Environmental Scanning Electron Microscope equipped with
a Peltier cooling stage and micro-manipulator/injector at 15 kV and
pressures of 5 torr. B, multiphoton laser scanning
microscopy was performed on lipid droplets isolated from control cells
and incubated with Nile Red (0.05%). Nile Red in lipid droplets was
excited at 875 nm with a Bio-Rad MRC-1024MP Multiphoton Laser Scanning
Microscopy system as described under "Materials and Methods."
Fluorescence emission of Nile Red was detected at 63× (objective)
through an external detector system using a HQ575/150 bandpass
filter.
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Effect of SCP-2 Expression on Lipid Droplet Protein Content and
Profile--
As indicated in the Introduction, relatively little is
known regarding the function of the protein capsule of the lipid
droplet surface. To avoid potential proteolytic degradation during
isolation of lipid droplets, a protease inhibitor mixture (Complete
Mini protease inhibitor mixture) was included in the homogenization and
lipid droplet isolation buffer as described under "Materials and
Methods." SCP-2 expression increased the mass ratio of protein/lipid 1.4-fold in the lipid droplets. Furthermore, the profile/composition of
the proteins associated with the lipid droplet surface was significantly altered in the SCP-2-expressing cells (Fig.
7). When 20 µg of proteins from lipid
droplets isolated from control cells were loaded on the SDS-PAGE
gel and stained using Silver Stain Plus (Bio-Rad), less than 10 prominent bands ranging from 27 to 100 kDa molecular mass were
observed (Fig. 7, lane 2), including an unknown
protein of >94 kDa as well as bands in the molecular weight range of
hormone-sensitive lipase (84 kDa), perilipin A (57 kDa), vimentin (58 kDa), ADRP (53 kDa), and perilipin B (46 kDa). Several minor additional
bands are as yet unidentified. When equal amounts (20 µg) of proteins
from lipid droplets isolated from SCP-2-overexpressing cells were
concomitantly loaded on the SDS-PAGE gel and stained using Silver Stain
Plus (Bio-Rad) (Fig. 7, lane 3), the relative
proportion of the above proteins was clearly different. There was some
loss of the higher molecular weight protein (i.e. >94 kDa)
as well as significant loss of protein in the molecular weight range of
hormone-sensitive lipase. Concomitantly, there was a significant
increase in proteins located just below the 67-kDa protein marker
(i.e. proteins with molecular weights in the range of
perilipin A, vimentin, and ADRP).

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Fig. 7.
Effect of SCP-2 expression on lipid droplet
protein profile. A silver-stained Tricine gel (12%) was loaded as
follows: lane 1, lipid droplets (20 µg) isolated from
control cells; lane 2, lipid droplets (20 µg) isolated
from transfected L cells overexpressing SCP-2; and lane 3,
molecular weight marker. The approximate molecular masses where
hormone-sensitive lipase, perilipin A, vimentin, ADRP, and perilipin B
are expected to appear were indicated from top to
bottom.
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In summary, the reduced surface/core lipid ratio in lipid droplets of
SCP-2-expressing cells was at least in part compensated by more protein
associating with the lipid droplet surface. However, this increase in
proteins was selective. The lipid droplets from SCP-2-expressing cells
exhibited a relative paucity of high molecular weight protein (>94
kDa), with some increases in middle molecular weight proteins (53-58
kDa). It should be noted that lipid droplet proteins are tightly
associated with the lipid droplets and are not dislodged by alkaline
carbonate or the mild techniques used herein to isolate lipid droplets
(5).
Western Blotting to Determine the Effect of SCP-2 Expression on the
Levels of Specific Lipid Droplet-associated Proteins--
Although the
silver-stained SDS-PAGE gels in the preceding section were informative,
they did not identify specific proteins associated with the lipid
droplets. Therefore, the effect of SCP-2 expression on the level of
several functionally important proteins associated with lipid droplets
was quantitated by Western blotting. The level of ADRP, a 53-kDa lipid
droplet-specific protein (5, 64), was reduced 1.8-fold from 952 ± 34 to 538 ± 23 ng/mg protein (n = 4) in
SCP-2-expressing cells as compared with controls. Since ADRP expression
in intact cells was similarly reduced 1.7-fold in SCP-2-expressing
cells, this difference in isolated lipid droplets was not due to
differential loss of ADRP upon lipid droplet isolation. Perilipins A
and B, 57- and 46-kDa lipid droplet-associated proteins (27), were
differentially affected by SCP-2 expression. Western analysis showed
that perilipin A (Fig. 8B, band
1) was increased by 3.1-fold in lipid droplets isolated from SCP-2
expression cells (p < 0.0008, n = 3).
Perilipin B (Fig. 8B, doublet band 2) was unchanged.
Hormone-sensitive lipase, a 84-kDa protein whose active form is
associated with lipid droplets, was detected in lipid droplets isolated
from the control cells but not from SCP-2-expressing cells. Finally,
vimentin, a 58-kDa protein associated with lipid droplets of cells
undergoing adipose conversion (23), was found to be decreased by
4.5-fold in SCP-2-expressing cells (Fig. 8D). These results
were consistent with SCP-2 expression altering protein composition of
lipid droplet-associated proteins.

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Fig. 8.
Western blot analysis on lipid droplets
isolated from control and transfected L cells overexpressing
SCP-2. Tricine gels were loaded as follows: lane 1, L
cell homogenate (20 µg); lane 2, lipid droplets isolated
from L cells (20 µg); lane 3, transfected L cell
homogenate overexpressing SCP-2 (20 µg); lane 4, lipid
droplets isolated from transfected L cells overexpressing SCP-2 L cells
(20 µg); and lane 5, SCP-2 standard (A),
perilipin B from 3T3-L1 adipocyte lysate (B), and prestained
molecular weight markers (C and D). The blots
were probed against anti-SCP-2 (A), anti-perilipin
(B), anti-hormone-sensitive lipase (C), and
anti-vimentin (D) as described under "Materials and
Methods."
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Is SCP-2 Directly Bound to the Surface of Isolated Lipid
Droplets?--
The possibility that the above effects of SCP-2
expression were due to direct association of SCP-2 with lipid droplets
was examined. First, Western blots showed that SCP-2 was detectable in
cell homogenates (Fig. 8A, lanes 1 and 3) but not
in isolated lipid droplets (Fig. 8A, lanes 2 and
4). Second, SCP-2-expressing L cells were
simultaneously immunolabeled with antisera to SCP-2 and ADRP, a protein
closely associated with lipid droplets (Fig. 9A). A punctate pattern
(green) indicative of peroxisomes (where SCP-2
primarily resides) was clearly evident, and the large, circular droplets (red) were characteristic of lipid droplets (where
ADRP is located). At a first glance, little to no colocalization was evident. In order to delineate graphically the extent of
colocalization, a pixel fluorogram (Fig. 9B) was generated.
The low level of colocalization was confirmed by the small correlation
numbers (red:green = 0.11 and green:red = 0.12) and by the
fluorogram shape showing divergent populations of red and green pixels.
Colocalization of stain would have resulted in a significant population
of data points falling along a diagonal line between the red and green
axis and much higher correlation coefficients. Thus, both Western
blotting of isolated lipid droplets and immunofluorescence of intact
cells confirmed that little, if any, SCP-2 was associated with lipid droplets.

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Fig. 9.
Double label immunofluorescence with
SCP-2 and ADRP and intracellular distribution of ADRP and NBD-stearic
acid in transfected L cells. Colocalization patterns of SCP-2 and
ADRP were shown using pseudo-coloring derived from confocal image
acquisition from red- and green-specific
photomultiplier tubes channels. A 24-bit RGB image was created from the
red plus green plus blue (null)
channels. L cells expressing the SCP-2 protein were
simultaneously labeled for SCP-2 and ADRP (A) with Alexa
Fluor 488-conjugated goat anti-rat IgG and Alexa Fluor 594-conjugated
goat anti-rabbit IgG as described under "Materials and Methods."
Superimposition of the probes was graphically demonstrated in a pixel
fluorogram (B) to determine the extent of overlap.
Correlation coefficients were 0.11 (red) and 0.12 (green) indicating little to no overlap of probes. The
intracellular distribution of ADRP and NBD-stearic acid was shown with
control (C) and transfected L cells overexpressing SCP-2
(D) simultaneously labeled for ADRP and NBD-stearic acid as
described under "Materials and Methods." The cells were examined
using a Bio-Rad MRC-1024 confocal system. Objective 63×.
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Ligand Binding Specificity of ADRP--
Three factors suggested
that ADRP may, at least in part, account for the association of
specific lipids with the lipid droplet surface. (i) Little, if any,
SCP-2 was directly associated with lipid droplets. (ii) SCP-2
expression reduced the lipid droplet ADRP content. (iii) ADRP binds
NBD-cholesterol and NBD-stearic acid with high affinity (2.0 and 145 nM, respectively) (22, 28). However, it is not known if
these lipids bind to ADRP simply because of the unique properties of
the NBD fluorophore attached to the cholesterol and fatty acid.
Likewise, it is not known if ADRP can bind the activated forms of fatty
acids, i.e. fatty acyl-CoAs. Therefore, a displacement assay
was developed, and the ability of stearic acid, cholesterol, oleic
acid, and oleoyl-CoA to displace NBD-labeled stearic acid bound to ADRP
was determined (Fig. 10). Inhibition
constants (Ki) were calculated from nonlinear fits
to an exponential decay curve of NBD-stearate fluorescence versus the competitor concentration. The
Ki values for displacement by stearic acid,
oleic acid, oleoyl-CoA, and cholesterol were 41.7 ± 6.9, 23.4 ± 1.2, 8.6 ± 0.5, and 10.2 ± 0.9, respectively (Table II).

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Fig. 10.
Displacement of ADRP-bound NBD-stearic acid
by nonfluorescent ligands. ADRP (50 nM) was
preincubated with stearic acid (180 nM), followed by
addition of several displacing ligands as follows: stearic acid
(A), oleic acid (B), oleoyl-CoA (C),
and cholesterol (D). Fluorescence intensity decay in the
presence of competitive nonfluorescent ligand was monitored as
described under "Materials and Methods."
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Table II
Competitive inhibition of NBD-stearate binding to ADRP by
nonfluorescent ligands
Values represent means ± S.E. (n = 3).
Displacement assays were performed as described under "Materials and
Methods." Inhibition constants (Ki) were
calculated from a nonlinear fit to an exponential decay curve of
NBD-stearate fluorescence versus the competitor
concentration. Ki was equal to the ligand
concentration at Fmax/2.
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In summary, the displacement assay clearly showed that all of these
naturally occurring lipids displaced the ADRP-bound NBD-stearic acid.
The displacement assay indicated that ADRP bound the unsaturated oleic
acid (18:1) with nearly 2-fold higher affinity than the saturated
stearic acid (18:0). Comparison of cholesterol and oleoyl-CoA binding
showed that ADRP bound both with nearly equal affinity. Finally, ADRP
bound cholesterol with 4- and 2.2-fold higher affinity than stearic
acid and oleic acid, respectively. These data suggested that the ADRP
may be important for determining the specific distribution of fatty
acids, fatty acyl-CoAs, and cholesterol associated with the lipid droplet.
Lipid Droplet Populations--
Because both SCP-2 and ADRP bind
the same ligands (i.e. fatty acids, fatty acyl-CoAs,
and cholesterol), the effect of SCP-2 expression on the distribution of
ADRP and fluorescent fatty acid in lipid droplets was examined in
intact cells. Control cells (Fig. 9C) and SCP-2-expressing
cells (Fig. 9D) were double-labeled with NBD-stearic acid
followed by fixation and labeling with anti-ADRP. It became evident
that the lipid droplets were not uniform populations with regard to
NBD-stearic acid content and the presence of ADRP. At least two
populations of lipid droplets were observed in both control (Fig.
9C) and SCP-2-expressing (Fig. 9D) cells. One
population of lipid droplets was rich in ADRP (red
droplets), and the other had both ADRP and NBD-stearic acid
colocalized (yellow droplets). A possible third set of lipid
droplets had only NBD-stearic acid present (green droplets).
It should be noted, that NBD-stearate might also be present in other
organelles such as mitochondria and peroxisomes. However, at this level
of NBD-stearate concentration the heaviest staining was with lipid droplets.
To quantitate the effect of SCP-2 expression on association of ADRP and
NBD-stearate with lipid droplets, the staining patterns of a large
number of lipid droplets from many randomly selected cells were
examined (Table III). In the control
cells, ADRP and NBD-stearic acid were found in 69.8 ± 4.9 and
23.8 ± 4.4% of lipid droplets, respectively. Colocalization of
both ADRP and NBD-stearic acid in control cells was very low, 12.6 ± 1.8% (Table III). SCP-2 expression dramatically altered this
pattern by decreasing the percentage of lipid droplets containing ADRP
by 34% (p < 0.01). Concomitantly, the percentage of
lipid droplets stained with only with NBD-stearic acid was increased
2-fold (p < 0.05). Interestingly, SCP-2 expression
increased the colocalization of ADRP and NBD-stearic acid by 3-fold
(p < 0.05).
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Table III
Lipid droplet populations immuno-labeled with ADRP and/or
NBD-stearic acid
Values reflect the mean ± S.E. A total of 155 and 94 lipid
droplets for control and SCP-2-expressing cells, respectively, were
used to determine the percentage stained with each probe.
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In summary, these data suggest that SCP-2 expression may reduce, at
least in part, the association of ADRP with the lipid droplet surface
by stripping away ADRP-bound ligands (fatty acids and cholesterol) for
which SCP-2 competes. The data further suggest that ADRP-containing
bound ligand (NBD-stearic acid) is more closely/tightly associated with
the lipid droplet. Consistent with these possibilities, SCP-2
expression dramatically reduced the content of unesterified fatty acid
(Fig. 1), cholesterol (Fig. 1), and ADRP (see above) associated with
the lipid droplet.
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DISCUSSION |
Although much progress has been made in our understanding of the
structure, function, and regulation of serum lipoproteins, relatively
little is known regarding the processes governing intracellular lipid
droplets. Cells store lipids as lipid droplets composed of a surface
monolayer (cholesterol, phospholipids, fatty acids, and proteins)
encasing a neutral lipid core (cholesterol esters and triglycerides)
(5). At least three types of lipid droplets are recognized as follows.
(i) Large (up to 15 µm diameter) triacylglyceride-rich lipid
droplets, found in adipose tissue (adipose and mammary), contain almost
no esterified cholesterol (reviewed in Ref. 5). These
triacylglycerol-rich lipid droplets provide an energy storage site for
secretion in milk (mammary tissue) or for release of fatty acids (from
triacylglycerides by hormone-sensitive lipase) to be transported to and
oxidized in peroxisomes and mitochondria. (ii) Small (~1 µm
diameter) lipid droplets, found in steroidogenic tissues (adrenal,
Leydig, and luteal) (15, 65), are cholesteryl ester-rich (81% of core
lipids), and almost all cholesterol (97%) is esterified (7).
Cholesterol and cholesteryl esters (hydrolyzed by hormone-sensitive
lipase) stored in the lipid droplet provide a ready source of
cholesterol for transfer, via the plasma membrane (66), to mitochondria
for steroidogenesis (6, 16, 65, 68). (iii) Almost all other mammalian
tissues examined contain small (near 1 µm diameter), less well
characterized lipid droplets (5, 69, 70). One potential function of
these smaller lipid droplets was suggested by a recent report showing
that plasma membrane caveolar SRB1 mediates reversible uptake and
efflux of unesterified cholesterol between HDL and intracellular lipid
droplets in non-adipose, non-steroidogenic cells (21, 22). Thus, the smaller lipid droplets common to nearly all cells may be essential for
"reverse cholesterol transport" (10). However, almost nothing is
known of the structure, composition, or function of the ubiquitous smaller lipid droplets found in nearly all mammalian cells. The results
presented herein demonstrate for the first time several new
observations characterizing these smaller lipid droplets from L cells and examine potential roles of SCP-2 in regulating
their lipid and protein composition.
First, the lipid composition of these lipid droplets was unique. Key
differences from lipid droplets of adipose and steroidogenic tissues
are as follows. (i) The core lipid profile, 66% triacylglycerol and
33% cholesteryl ester, was intermediate between that of the large
lipid droplets found in adipose tissues (5) and that of the small lipid
droplets typical of steroidogenic cells (7). (ii) The ratio of surface
lipids/core lipids of the lipid droplets (0.50), while much lower than
that of adipose lipid droplets, was in the range of the reported for
lipid droplets of steroidogenic cells, i.e. 0.70 (7),
possibly reflecting the similarity in size of these lipid droplets.
(iii) The surface monolayer of the L cell lipid droplets
exhibited a very high cholesterol/phospholipid ratio, almost 19-fold
higher than that of lipid droplets from steroidogenic cells (7). This
ratio was also 3-6-fold higher than ratios typical of plasma membrane
bilayers, normally containing the highest level of cholesterol in the
cell (59). Consistent with the high degree of localization of
cholesterol to lipid droplets, fluorescent sterols are rapidly taken up
(NBD-cholesterol) and preferentially targeted (NBD-cholesterol,
dehydroergosterol) to lipid droplets in living cells (21, 22, 70).
Thus, the data presented herein indicate that the high localization of
NBD-cholesterol and dehydroergosterol to lipid droplets of living cells
reflects the high proportion of lipid droplet cholesterol. (iv) The
surface lipids of the isolated lipid droplets contained significant
amounts of unesterified fatty acids, i.e. 5.2 nmol/mg
protein (21.6% of total lipid droplet lipids). This represented a
>4-fold enrichment of unesterified fatty acids in the lipid droplet as
compared with the cell homogenate. The appearance of unesterified fatty
acid in the lipid droplet surface lipids was consistent with confocal microscopic imaging of living cells which showed that fluorescent fatty
acids were rapidly targeted to the lipid droplet (69). Equally
important, it demonstrated that the targeting of the fluorescent labeled fatty acids (e.g. NBD-stearic acid) to the lipid
droplets was not an artifact induced by the insertion of the NBD group into the fatty acid. (v) The fatty acids esterified to the lipid droplet lipid species (phospholipids, triacylglycerols, and cholesteryl esters), while qualitatively similar to those of the serum lipoproteins in the culture medium, exhibited distinct quantitative differences. Lipid droplet phospholipids, cholesteryl esters, and triacylglycerols were enriched 2-3-fold in polyunsaturated fatty acids as compared with
the corresponding serum lipoprotein lipids. This suggests that the
lipid droplets may be important not only for storage of fatty acids in
esterified form to be used as future energy sources but also for
storage of polyunsaturated fatty acid signaling molecules or precursors
thereof (2).
Second, overexpression of SCP-2 significantly altered the lipid
composition of lipid droplets by decreasing their cholesteryl ester,
cholesterol, and unesterified fatty acid content. The loss of lipid
droplet sterol was consistent with observations showing that SCP-2
enhances cholesterol transfer from lipid droplets to mitochondria for
oxidation (7) as well as reports that SCP-2 expression may play an
important role in cholesterol secretion into bile (71-73).
Overexpression of SCP-2 in mice increased hepatic cholesterol content
and increased enterohepatic circulation of bile (74), whereas treatment
of rats with SCP-2 antisense reduced and delayed the appearance of
biliary cholesterol (75). Contrary to expectation, SCP-2/SCPx gene
ablation caused biliary cholesterol hypersecretion and gallstone
formation, an effect due to concomitant up-regulation of liver fatty
acid-binding protein, another cholesterol transport molecule (76). The
relatively smaller loss of cholesteryl ester and lower unesterified
fatty acid levels were consistent with the reduced association of
hormone-sensitive lipase with lipid droplets of SCP-2-expressing cells
(Fig. 7). Taken together with the high affinity of SCP-2 for
cholesterol (33, 34, 40) and fatty acids (29, 33), these data suggested
that SCP-2 expression enhanced the efflux of cholesterol as well as
fatty acids from the lipid droplets.
Third, the protein profile of the lipid droplets was intermediate with
that of triglyceride-rich and cholesteryl-ester rich lipid droplets.
Tricine/SDS-PAGE gels of the isolated lipid droplet proteins resolved
over 27 protein bands, some of which were in the high molecular weight
range of P200, a lipid droplet surface capsule protein that
prevents access to hormone-sensitive lipase (77). Western blotting
showed that the isolated lipid droplets contained ADRP, perilipin A,
perilipin B, vimentin, hormone-sensitive lipase, but not SCP-2.
Immunofluorescence confocal microscopy also detected ADRP, but not
SCP-2, in the lipid droplets. The distribution of several of these
proteins was intermediate with that of triglyceride-rich and
cholesteryl ester-rich lipid droplets. ADRP is absent from large
triglyceride-rich lipid droplets of mature adipocytes (3). Perilipins
are present in high amounts in adipocytes and steroidogenic cells
(reviewed in Ref. 3). Both ADRP and perilipins were present in
L cell lipid droplets. These data suggested that many of
the proteins thought to be important for lipid droplet function are
detected in isolated L cell lipid droplets.
Fourth, SCP-2 expression dramatically altered the proteins associated
with the lipid droplet. Lipid analysis showed that SCP-2 expression
increased the protein/lipid ratio in the lipid droplet. However, the
profile of lipid droplet proteins was significantly altered. The
isolated lipid droplets were reduced in the proportion of high
molecular weight proteins with concomitant slight increases in
intermediate molecular weight proteins. Loss of the high molecular weight capsular proteins such as P200 makes the lipid
droplet accessible to hormone-sensitive lipase (24). Consistent with this possibility, the lipid droplet of cholesteryl ester content was
reduced in SCP-2-expressing cells. Furthermore, levels of perilipin A,
but not perilipin B, was increased 3.1-fold, whereas levels of ADRP and
vimentin were decreased. An inverse relationship between perilipins and
ADRP has also been reported for the lipid droplets of differentiating
adipocytes (3).
Fifth, although a direct interaction of SCP-2 tightly associated with
lipid droplets was not observed in the present study, a link between
the two has nevertheless been suggested from data reported herein and
earlier by this and other laboratories as follows. (i) The observation
that SCP-2 can affect transfer of cholesterol from lipid droplets in
intact cells (16, 18, 21), without SCP-2 being detected in Western
blots of isolated lipid droplets has precedent. SCP-2 enhanced the
transfer of cholesterol from plasma membranes for esterification in the
endoplasmic reticulum in intact cells, even though SCP-2 was not
detected in purified plasma membranes (43, 76). (ii) SCP-2 expression
in transfected cells redirected the transfer of cholesterol from lipid
droplets away from the plasma membrane (i.e. inhibited
efflux to HDL) (21) and toward intracellular sites, especially
mitochondria and endoplasmic reticulum (reviewed in Refs. 17, 47,
78-80). (iii) Since SCP-2-mediated intermembrane cholesterol transfer
required interaction of SCP-2 with a donor membrane surface (81), it
seems likely that a similar, albeit transitory requirement may exist
for SCP-2-mediated cholesterol transfer from lipid droplets. Indeed,
the small lipid droplets of L cells present a surface ideal
for electrostatic interactions with SCP-2 (67, 82). The lipid droplet
surface is rich in anionic lipid (unesterified fatty acids), exhibits a
high cholesterol/phospholipid ratio, and has a high radius of
curvature. (iv) Microinjection of steroidogenic cells with antisera to
SCP-2 inhibited cholesterol transfer from lipid droplets for oxidation
to steroids in mitochondria (16, 18). (v) Immunogold electron
microscopy showed a close association of SCP-2 with lipid droplets in
luteal cells derived from hormone-stimulated, but not control, rats
(15). As indicated in the Introduction, endocrine stimulation alters
the pattern of multiple proteins (e.g. hormone-sensitive
lipase) associated with lipid droplets. In summary, the above data
suggest that SCP-2 may interact with lipid droplets through
electrostatic interactions that are weak, transient, and/or require
endocrine stimulation.
Sixth, since levels of ADRP, a protein closely associated with lipid
droplets, were decreased in transfected L cells expressing SCP-2, the
ability of ADRP to affect lipid content was further investigated.
Several lines of evidence suggest ADRP has a regulatory role in lipid
metabolism as follows. (i) Indirect immunofluorescence imaging studies
have shown that ADRP is localized to the surface of lipid droplets (3,
21). (ii) In the present study, the surface lipid content of
transfected L cells was observed to be significantly lower (7.7-fold,
p < 0.01) than in control cells. Surface lipid to core
lipid ratios were also decreased (7.2-fold, p < 0.01).
(iii) A concomitant decrease (1.7-fold) in ADRP levels was observed in
transfected cells (21). The above observations suggest that ADRP
functions as a regulatory protein, governing the deposition and release
of lipid stores from droplets. Results from several binding and
displacement studies help support this theory. Although ADRP doesn't
bind cholesteryl esters, it exhibited saturable binding of NBD-labeled
cholesterol and stearic acid to high affinity (2.0 and 145 nM, respectively) (22, 28). Inhibition constants
(Ki) calculated from displacement data in the
present study were in agreement with the cholesterol and stearic acid
binding values, indicating that cholesterol binds tightly to ADRP and
is able to displace readily fatty acids. The results from the binding
and displacement data were further confirmed by immunofluorescence
imaging studies where colocalization of ADRP and NBD-labeled stearic
acid in transfected L cells showed up to three populations of lipid
droplets. One population was rich in ADRP; another set had both ADRP
and the fatty acid colocalized, and the last retained only the fatty
acid probe. Although it is possible the last set might not be lipid
droplets, morphologically it appeared the fatty acid probe localized in
storage droplets. Evidence for different populations of lipid droplets
have been reported elsewhere where changing levels of ADRP and
perilipins, another lipid droplet-associated protein, were found when
differentiation was induced in 3T3-L1 adipocytes (3). Consistent with
the binding and displacement data and the fact that SCP-2 also binds
both fatty acids and cholesterol with affinities very similar (22, 29,
34) to those of ADRP, the lipid droplets from SCP-2-expressing cells
lost much less cholesterol than unesterified fatty acid. Furthermore,
the data suggested that the liganded ADRP was preferentially retained
in the lipid droplets. In summary, given the results from the binding,
displacement, and imaging experiments, along with the concomitant low
levels of surface unesterified fatty acid, cholesterol, and ADRP in
SCP-2-expressing cells, the results presented herein point to a
potential functional role for SCP-2 and ADRP in regulating lipid
content in lipid storage droplets.
In conclusion, the data presented here demonstrate for the first time
that expression of SCP-2 in transfected L cells affected the lipid
content, esterified fatty acid composition, and protein content/profile
of lipid storage droplets. This is not surprising given the huge body
of evidence supporting a role for SCP-2 in cholesterol, fatty acid, and
phospholipid metabolism and intracellular trafficking (reviewed in Ref.
34). While the interaction was shown not to be due to permanent
association of SCP-2 with the lipid droplet, clearly the cellular
expression of SCP-2 alters the structure of the lipid droplet. A
concomitant displacement and immunological imaging study of ADRP, whose
levels were decreased in the presence of SCP-2, provided evidence for
regulatory roles for both SCP-2 and ADRP in maintaining lipid stores.
In summary, the results presented herein provide new insights into the
functional significance of these proteins associated with lipid droplets.