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J. Biol. Chem., Vol. 275, Issue 49, 38486-38493, December 8, 2000
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From the
Received for publication, August 11, 2000
The perilipins are the most abundant proteins at
the surfaces of lipid droplets in adipocytes and are also found in
steroidogenic cells. To investigate perilipin function, perilipin A,
the predominant isoform, was ectopically expressed in fibroblastic
3T3-L1 pre-adipocytes that normally lack the perilipins. In control
cells, fluorescent staining of neutral lipids with Bodipy 493/503
showed a few minute and widely dispersed lipid droplets, while in cells
stably expressing perilipin A, the lipid droplets were more numerous
and tightly clustered in one or two regions of the cytoplasm.
Immunofluorescence microscopy revealed that the ectopic perilipin A
localized to the surfaces of the tiny clustered lipid droplets;
subcellular fractionation of the cells using sucrose gradients
confirmed that the perilipin A localized exclusively to lipid droplets.
Cells expressing perilipin A stored 6-30-fold more triacylglycerol
than control cells due to reduced lipolysis of triacylglycerol stores. The lipolysis of stored triacylglycerol was 5 times slower in lipid-loaded cells expressing perilipin A than in lipid-loaded control
cells, when triacylglycerol synthesis was blocked with 6 µM triacsin C. This stabilization of
triacylglycerol was not due to the suppression of triacylglycerol
lipase activity by the expression of perilipin A. We conclude that
perilipin A increases the triacylglycerol content of cells by forming a
barrier that reduces the access of soluble lipases to stored
lipids, thus inhibiting triacylglycerol hydrolysis. These studies
suggest that perilipin A plays a major role in the regulation of
triacylglycerol storage and lipolysis in adipocytes.
Lipid droplets in adipocytes store the body's major energy
reserves as triacylglycerols. These structures contain a large core of
neutral lipid, primarily triacylglycerol, covered by a phospholipid
monolayer. The intracellular mechanisms that control the storage and
release of triacylglycerols are largely uncharacterized, yet are likely
to be fundamental to understanding the regulation of energy metabolism
in the body. Recent studies have shown that lipid droplets are covered
with a proteinaceous coat; the functions and identities of the
component proteins have not been fully elucidated. The first identified
lipid droplet-specific proteins are the perilipins (1-7), a family of
proteins coating the surfaces of lipid droplets of adipocytes and
steroidogenic cells of adrenal cortex, testes, and ovaries, but lacking
in other types of cells and in other cellular compartments. The
perilipins are encoded by a single copy gene that gives rise to
multiple mRNAs by alternative splicing mechanisms1; these mRNAs
are translated to yield the three described protein isoforms (2, 4).
Perilipin A is the predominant isoform in both adipocytes and
steroidogenic cells, perilipin B is found primarily in adipocytes, and
perilipin C is unique to steroidogenic cells. Perilipin A is the most
abundant protein on highly purified lipid droplets isolated from fully
differentiated cultured 3T3-L1 adipocytes and from murine primary
adipocytes,2 as assessed by
Coomassie staining and silver staining of lipid-droplet proteins
separated on denaturing gels. Based on the localization of perilipins
at the surfaces of lipid droplets, we hypothesized that the perilipins
may function in regulating the packaging and storage of neutral lipids.
The mechanisms of lipid droplet formation are poorly understood. Early
studies showed that all cells in culture take up free fatty acids and
lipoproteins provided by serum in the culture medium, and use the
lipids as a source of energy and building components for membrane
synthesis (8). Many cells have been observed to accumulate tiny
cytoplasmic inclusions of triacylglycerols or cholesterol esters during
exposure to lipid-rich medium (8, 9), while cultured adipocytes form
relatively huge droplets (3) that occupy the majority of the cell
volume. Additionally, analysis of histological sections reveal enormous
lipid droplets in adipocytes and smaller lipid droplets in various
non-adipose tissues including liver, heart, adrenal gland, testes,
ovary, muscle, intestine, kidney, and mammary gland (10-12).
Nonetheless, few details of how or where lipid droplets are formed are
known. Enzyme activities for neutral lipid synthesis have been isolated in microsomes from fractionated cells, thus implying localization to
the endoplasmic reticulum
(ER).3 Both diacylglycerol
acyltransferase, which catalyzes the final step in triacylglycerol
synthesis, and acyl coenzyme A-cholesterol acyltransferase, which adds
fatty acids to cholesterol, have been localized to ER by subcellular
fractionation (13, 14) and immunofluorescence microscopy (15, 16). The
mechanisms leading to the nucleation of lipid droplet formation are
currently uncharacterized; a hypothetical model includes the formation
of a lens of neutral lipids between the lumenal and cytoplasmic
leaflets of the membrane bilayer of the ER that either is retained as a
bleb associated with the ER (17), or pinches off and takes a
cytoplasmic location.
Assuming that lipid droplets are formed within the ER membrane,
proteins unique to lipid droplets may originate as integral ER proteins
that selectively target to the patches of the ER that contain the
growing accumulation of neutral lipids, or alternatively, may be
translated on free ribosomes and then inserted into the lipid droplet
following its dissociation from the ER. Several sterol and phospholipid
biosynthetic enzymes and fatty acyl-coenzyme A ligases in yeast are
found on both the ER and isolated lipid droplets (18-21), and hence
may be examples of lipid droplet-associated proteins that are
synthesized on the ER; similarly, phosphatidylethanolamine N-methyltransferase has been localized to lipid droplets
isolated from rat hepatocytes (22), as well as to an ER-like
compartment termed the mitochondria-associated membrane fraction (23),
that is enriched in numerous lipid biosynthetic enzymes (24). By contrast, the perilipins are translated on free, and not ER-bound, ribosomes,4 and thus, traffic
to and associate with lipid droplets post-translationally. Although the
perilipins appear to be associated only with lipid droplets, their
functions are uncharacterized.
To study the function of perilipins in lipid metabolism, perilipin A
was ectopically and stably expressed in fibroblastic 3T3-L1
pre-adipocytes that lack endogenous perilipins prior to differentiation. The ectopic perilipin A was found exclusively on tiny
lipid droplets in these cells, which stored significantly more
triacylglycerol than control cells lacking perilipins. The metabolic
basis for the increased storage of triacylglycerol was investigated.
Materials
Fetal bovine serum, fatty acid-free bovine serum albumin,
triolein, oleic acid, diethyl-p-nitrophenyl phosphate, and
p-chloromercuribenzoate were purchased from Sigma. Geneticin
was purchased from Life Technologies, Inc. or Mediatech, Inc. (Herndon,
VA). [9,10-3H]Oleic acid and
[9,10-3H]triolein were purchased from PerkinElmer Life
Sciences. Triacsin C (Biomol, Plymouth Meeting, PA) was
generously donated by Dr. Peter Gillies and Dr. Sandie Germain at
DuPont (Wilmington, DE). Diethylumbelliferyl phosphate was synthesized
by Chem-Master International, Inc. (East Setauket, NY). Ammonium
sulfate-impregnated silica gel H thin layer chromatography plates were
purchased from Analtech (Newark, DE). An anti-calnexin polyclonal
antibody was purchased from StressGen Biotechnologies Corp. (Victoria,
British Columbia, Canada). Bodipy 493/503 and Alexa Fluor 546 goat
anti-rabbit polyclonal IgGs were obtained from Molecular Probes, Inc.
(Eugene, OR).
Methods
Cell Culture--
3T3-L1 pre-adipocytes were cultured in Corning
100-mm dishes in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum and 2 mM glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin, and the cells were grown in a
5% CO2 atmosphere at 37 °C.
Expression of Perilipin A in Cells--
The cDNA for mouse
perilipin A was amplified by the polymerase chain reaction using
oligonucleotide primers corresponding to the 5' and 3' ends of the
perilipin A coding sequence with added HindIII sites, and
was subcloned into the HindIII site of the pSR Microscopy--
Control 3T3-L1 pre-adipocytes and cells
expressing perilipin A were grown on 12-mm glass coverslips in 100-mm
culture dishes. Cells were prepared for immunofluorescence microscopy
(3) and stained with antiserum raised against a recombinant
6-histidine-tagged amino-terminal peptide of perilipin A (29), and an
Alexa Fluor 546 secondary antibody. Neutral lipids were stained with
Bodipy 493/503 (30). Cells were viewed with a Nikon Eclipse E800
fluorescence microscope equipped with a Hamamatsu Orca digital camera
interfaced with a Power Macintosh G4 computer; images were processed
using Improvision Openlab software.
Subcellular Fractionation of Cells and Characterization of
Fractions--
Confluent monolayers of 3T3-L1 pre-adipocytes stably
expressing perilipin A were incubated with 400 µM oleic
acid complexed to fatty acid-free bovine serum albumin (6:1, moles of
oleate:moles of albumin) in culture medium for 16 h to increase
the storage of triacylglycerols. Cells were harvested and lysed in a
hypotonic medium containing 10 mM Tris, pH 7.4, 1 mM EDTA, 10 mM sodium fluoride, 20 µg/ml
leupeptin, 1 mM benzamidine, and 100 µM
[4-(2-aminoethyl)-benzenesulfonylfluoride] hydrochloride for 10 min
at 4 °C, followed by 10 strokes in a Teflon/glass homogenizer. The
homogenate was centrifuged for 10 min at 1000 × g at
4 °C, and the supernatant was adjusted to 35% sucrose, and layered
over a 0.5-ml cushion of 50% sucrose. A linear 0-30% sucrose
gradient was then layered over the density-adjusted supernatant, and
the tubes were centrifuged for 4 h at 154,000 × g
at 4 °C. The floating lipid droplet layer was harvested in approximately 1 ml by slicing off the top of the tube using a Beckman
tube slicer; 11 additional fractions were collected. Equal portions of
each fraction were solvent-extracted for lipid analysis, as described
previously (7); the proteins in an additional portion of each fraction
were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, transferred electrophoretically to nitrocellulose membranes and immunoblotted with antibodies against perilipin A and
calnexin, an ER integral membrane protein; enhanced chemiluminescence was used to detect bound antibodies. Two separate fractionations of
cells were compared and found to be nearly identical.
Lipid Analysis--
Cellular lipid content was determined
following solvent extraction of cells, as described previously (7).
Lipid content was expressed relative to total cellular protein content
measured by the bicinchoninic acid method (Pierce; Ref. 31).
Measurement of the Rate of Triacylglycerol Hydrolysis in Intact
Cells--
Confluent monolayers of 3T3-L1 pre-adipocytes stably
expressing perilipin A and control cells were incubated with 400 µM oleic acid complexed to bovine serum albumin for
16 h to increase the storage of triacylglycerols. The medium was
then removed and 6 µM triacsin C (from a 1 mg/ml stock in
Me2SO) was added in fresh culture medium without
supplemental fatty acids. Cells were rinsed with phosphate-buffered
saline and harvested by scraping at various times; lipids were
extracted and quantified (7), and the mass of triacylglycerol was
expressed relative to cell protein (31). Confluent monolayers of 3T3-L1
pre-adipocytes were used for these experiments since cell division is
inhibited by contact inhibition in these cells, and the cell number
remained constant throughout the chase incubation. Triacsin C, an
acyl-coenzyme A synthetase inhibitor, was added to the chase medium to
inhibit the re-utilization of fatty acids released from hydrolyzed
triacylglycerols (32, 33). To test for the effectiveness of triacsin C
in the inhibition of triacylglycerol synthesis, 3T3-L1 cells stably
expressing perilipin A and control cells were incubated with 400 µM oleate complexed to albumin for 16 h in the
presence or absence of 6 µM triacsin C; lipids were
solvent-extracted and quantified (7).
Measurement of Cellular Lipolytic Activity against
Triacylglycerols in Vitro--
Twelve 100-mm dishes of confluent
3T3-L1 pre-adipocytes stably expressing perilipin A or control cells
were rinsed with phosphate-buffered saline, harvested by scraping, and
pooled. Cells were centrifuged at 500 × g to yield
pellets of approximately 250 µl, and were lysed in 750 µl of cold
20 mM Tris, pH 7.4, 1 mM EDTA by incubation on
ice for 10 min, followed by homogenization in a cold Teflon/glass homogenizer. Homogenates were centrifuged at 15,000 × g for 15 min at 4 °C. Triplicate supernatant fractions of
200 µl, each corresponding to approximately 2.8 mg of total cell
protein, were assayed for the ability to hydrolyze emulsified
[3H]triolein by the methods of Khoo and Steinberg (34).
Released [3H]oleic acid was partitioned into solvent (35)
and quantified by liquid scintillation counting.
Measurement of Triacylglycerol Synthesis--
To measure
triacylglycerol synthesis without the complications of the simultaneous
hydrolysis of triacylglycerols, several inhibitors of triacylglycerol
or cholesterol ester hydrolysis, including
diethyl-p-nitrophenyl phosphate (E600)3
(36, 37), p-chloromercuribenzoate (PCMB) (37), and
diethylumbelliferyl phosphate (DEUP) (38, 39) were tested for the
ability to inhibit the hydrolysis of triacylglycerols in 3T3-L1
pre-adipocytes. Inhibitor stocks were made up as follows. E600 was
dissolved in water (2.5 mg/ml), PCMB was dissolved in 0.1 M
sodium hydroxide to make a 1000-fold stock, and DEUP was solubilized in
Me2SO (50 mg/ml). Confluent monolayers of 3T3-L1
pre-adipocytes were incubated with 400 µM oleate
complexed to albumin for 16 h to increase the storage of
triacylglycerols. The lipid-loading medium was removed, and fresh
culture medium without supplemental fatty acids but containing 6 µM triacsin C and the lipolysis inhibitors was added.
Cells were rinsed with phosphate-buffered saline and harvested by
scraping; lipids were extracted and the mass of triacylglycerol was
quantified (7).
The ability of cells to synthesize triacylglycerols from
[3H]oleate was measured in confluent monolayers of 3T3-L1
cells stably expressing perilipin A and control cells; both types of
cells were incubated with 0.6 mM DEUP to inhibit
triacylglycerol hydrolysis. The cells were incubated with 400 µM [3H]oleate (1 µCi/60-mm culture dish)
complexed to fatty acid-free bovine serum albumin, harvested at various
times, and the cellular lipids extracted and analyzed by thin-layer
chromatography (7). Incorporation of radioactivity into
triacylglycerols was determined by liquid scintillation counting
(Beckman LS 6800) of silica gel bands containing triacylglycerols.
[3H]Oleate incorporated into triacylglycerols was
expressed relative to cellular protein content (31). A paired
Student's t test was used to assess differences in
triacylglycerol synthesis between samples from cells expressing
perilipin A relative to samples from control cells.
The Ectopic Expression of Perilipin A Causes Lipid Droplets to
Cluster in One or Two Regions of the Cytoplasm in Fibroblastic
Cells--
To facilitate study of the functions of perilipins in lipid
metabolism, perilipin A was ectopically and stably expressed in fibroblastic 3T3-L1 pre-adipocytes using an efficient retroviral expression system (25). Untransfected and transfected control cells
normally lack perilipins, but store small quantities of cholesterol
esters and triacylglycerols; the staining of neutral lipids with Bodipy
493/503 (30) revealed that control cells have a few minute lipid
droplets dispersed throughout the cytoplasm (Fig.
1A). When oleic acid was added
to the culture medium to provide substrate for triacylglycerol
synthesis, more numerous lipid droplets were observed dispersed
throughout the cytoplasm, but excluded from a perinuclear region of the
control cells (Fig. 1B). Expressed perilipin A targets to
and associates with lipid droplets; immunostaining for perilipins
revealed tightly aggregated clusters of tiny spherical structures in
one or two areas of the cytoplasm (Fig. 1C); the cores of
these spherical structures were stained with Bodipy 493/503, thus
identifying the structures as lipid droplets. Perilipin staining was
either coincident with the neutral lipid staining or appeared as
distinct rings around the perimeters of minute lipid droplets (Fig.
1C), due, at least in part, to the imaging of the clustered
lipid droplets at various focal planes. When oleic acid was added to
the culture medium, the number and sizes of lipid droplets in the
clusters increased, and a distinct staining pattern of rings of
perilipin surrounding cores of neutral lipid became apparent (Fig.
1D). No staining for perilipins was observed in control
3T3-L1 pre-adipocytes when the cells lacked an expression construct
(see Fig. 8I in Ref. 29), or were infected with a retroviral
vector lacking the cDNA for the coding sequence of perilipin A
(Fig. 1, A and B). Comparable results were
obtained in Chinese hamster ovary (CHO) fibroblasts stably expressing
perilipin A and control cells (data not shown); the expression of
perilipin A altered the distribution of lipid droplets to one or two
tight clusters of numerous lipid droplets covered with perilipin A,
compared with a dispersed arrangement of a few minute lipid droplets in
control CHO cells lacking perilipin A.
Ectopic Perilipin A Localizes Exclusively to Lipid
Droplets--
To confirm the localization of ectopic perilipin A on
lipid droplets, homogenates of 3T3-L1 pre-adipocytes stably expressing perilipin A were fractionated on sucrose gradients. The isolation of
lipid droplets in the most buoyant fraction was confirmed by the
detection of greater than 99% of the total cellular triacylglycerol in
the uppermost fraction of the sucrose gradients (Fig.
2). Immunoblotting of the proteins in the
gradient fractions revealed that perilipin A was quantitatively
collected in the buoyant lipid droplet fraction (Fig. 2). Massive
overexposure of the immunoblots failed to reveal perilipin A in any
other subcellular fractions (data not shown). The nitrocellulose
membranes were also probed for calnexin, an integral ER protein. Most
of the calnexin was found in fractions of intermediate density that
also contained the highest levels of cholesterol, another marker for
cellular membranes (Fig. 2); however, approximately 3% of the total
calnexin was found in the floating lipid droplet fraction, thus
indicating potential contamination of this fraction with a small amount
of ER.
The Ectopic Expression of Perilipin A Drives Triacylglycerol
Storage in 3T3-L1 Pre-adipocytes--
Since cells expressing perilipin
A showed more staining with Bodipy 493/503 than control cells, the
neutral lipid content was quantified in control and perilipin
A-expressing 3T3-L1 pre-adipocytes. Cells expressing perilipin A showed
a higher triacylglycerol content than control cells, while showing no
detectable differences in the extremely low levels of stored
cholesterol esters (Table I). The
triacylglycerol content of both control and perilipin A-expressing cells varied depending upon when the samples were harvested for lipid
analysis after the addition of fresh culture medium containing serum
lipids; when the lipid content was measured 24 h after feeding the
cells, 3T3-L1 pre-adipocytes expressing perilipin A stored 6.4-fold, or
more than 30-fold, more triacylglycerol than control cells in two
separate transfection experiments. Similar results were obtained when
comparing CHO fibroblasts stably expressing perilipin A and control
cells (data not shown); cells expressing perilipin A stored 6-7-fold
more triacylglycerol than control cells, while cholesterol ester levels
were significantly higher than the levels found in 3T3-L1
pre-adipocytes and were unaltered by perilipin expression.
We reasoned that the increased storage of triacylglycerol in cells
expressing perilipin A was due to either increased synthesis of
triacylglycerols, or decreased hydrolysis of stored triacylglycerols by
cytosolic lipases. Given the observations that triacylglycerols are
synthesized on the ER (13) and that perilipins coat the surfaces of
lipid droplets, and have not been found on the ER, we hypothesized that
the most likely mechanism for the increased storage of triacylglycerols
in cells expressing perilipin A was via the decreased turnover of
stored triacylglycerols, since the hydrolysis of triacylglycerol most
likely occurs at the surfaces of the lipid droplets.
The Expression of Perilipin A Inhibits the Hydrolysis of
Triacylglycerols--
To assess the consequences of the expression of
perilipin A on the hydrolysis of triacylglycerols without complications
due to the recycling of newly released fatty acids back into
triacylglycerols, we used triacsin C, an inhibitor of acyl-coenzyme A
synthetase (32, 33). To test the efficacy of triacsin C, 3T3-L1
pre-adipocytes expressing perilipin A and control cells were incubated
with 400 µM oleate complexed to albumin in the presence
or absence of 6 µM triacsin C for 16 h, and the
triacylglycerol content of the cells quantified (Fig.
3). The addition of oleate to both types of cells increased the mass of stored triacylglycerol by 20-fold or
more. Cells expressing perilipin A stored 30% more triacylglycerol than cells lacking perilipins (p < 0.05). The addition
of 6 µM triacsin C reduced triacylglycerol synthesis by
85-90% (p < 0.0001).
To measure the rates of triacylglycerol hydrolysis in intact 3T3-L1
pre-adipocytes expressing perilipin A and control cells, the cells were
first incubated with oleic acid complexed to albumin for 16 h to
increase the content of stored triacylglycerol. The supplemental fatty
acids were then removed, and chase medium containing 6 µM
triacsin C was added to the cells to inhibit further triacylglycerol synthesis. The cells were harvested at various times, the lipids were
extracted, and the triacylglycerol remaining in the cells was
quantified. The total mass of triacylglycerol decreased with a
t1/2 = 6.8 h in control cells and a
t1/2 = 30.5 h in perilipin A-expressing cells
(Fig. 4); a second experiment showed a
t1/2 = 4.2 h for control cells and
t1/2 = 22.2 h for perilipin A-expressing cells
(data not shown). Additional experiments on untransfected 3T3-L1
pre-adipocytes (lacking perilipins) showed rates of triacylglycerol
hydrolysis of t1/2 = 5.1, 6.2, 3.2, and 8.5 h
(data not shown). In the absence of triacsin C, the total mass of
triacylglycerols decreased more slowly for both types of cells (control
cells, t1/2 = 12.5 h, perilipin A-expressing
cells, t1/2 = 70.3 h; data not shown), likely
reflecting the recycling of newly released fatty acids back into
triacylglycerols. In the experiment shown, the perilipin A-expressing
cells hydrolyzed 9 µg of triacylglycerol/mg of cell protein in the
first 4 h of the incubation; by contrast, the control cells
hydrolyzed 61 µg of triacylglycerol/mg of cell protein in the first
4 h. Thus, over the chase period, triacylglycerols were hydrolyzed
in both types of cells, but the triacylglycerol in cells expressing
perilipin A was more resistant to hydrolysis.
The Expression of Perilipin A Does Not Alter the Activity of
Soluble Lipases--
The reduced hydrolysis of triacylglycerols in
cells expressing perilipin A may be a consequence of 1) the reduction
of the expression or activity of soluble lipases when perilipin A is expressed, or 2) the sequestration of triacylglycerols into a perilipin-covered lipid droplet that reduces the access of soluble lipases to the underlying stored neutral lipids. To address the first
of these possibilities, post-mitochondrial supernatants were prepared
from 3T3-L1 pre-adipocytes expressing perilipin A and control cells.
The cell extracts were incubated with [3H]triolein
emulsified with gum arabic, and the released fatty acids were
quantified. In two experiments, extracts from both cell types were
demonstrated to hydrolyze exogenous triacylglycerol equally (Table
II); thus, the expression of perilipin A
has no effect on the total activity of lipases in the cells.
Triacylglycerol Hydrolysis in 3T3-L1 Pre-adipocytes Is Inhibited by
DEUP and E600, but Not by PCMB--
Cells stably expressing ectopic
perilipin A stored approximately 30% more triacylglycerol than control
cells when incubated with 400 µM oleic acid for 16 h
(Fig. 3). We investigated whether this increased storage of
triacylglycerol was due solely to the increased rate of turnover of
triacylglycerols in the control cells, or could be due, in part, to an
increased rate of triacylglycerol synthesis in the cells expressing
perilipin A. To measure triacylglycerol synthesis without the
complications of simultaneous turnover, we sought conditions to inhibit
triacylglycerol hydrolysis.
Since the soluble triacylglycerol hydrolases of 3T3-L1 pre-adipocytes
have not yet been identified or characterized, we tested several known
inhibitors of triacylglycerol or cholesterol ester hydrolysis (36-39)
for the ability to inhibit the hydrolysis of triacylglycerols in 3T3-L1
pre-adipocytes. To increase stored triacylglycerols, the cells were
incubated for 16 h with 400 µM oleate complexed to
albumin (Fig. 5A,
"lipid-loaded" conditions). Supplemental fatty acids were withdrawn
and chase medium containing 6 µM triacsin C and PCMB,
E600, DEUP, or medium without hydrolysis inhibitors was added to the
cells for an additional 15 h. The cells were harvested, lipids
extracted, and the remaining triacylglycerol quantified. Over 15 h, the triacylglycerol content of cells incubated with chase medium
lacking hydrolysis inhibitors was reduced by 75% (Fig. 5A),
consistent with previous observations (Fig. 4). PCMB failed to
effectively inhibit the hydrolysis of triacylglycerols when tested at
concentrations up to 50 µM (Fig. 5A); higher
concentrations of PCMB led to cell death. Both E600 and DEUP inhibited
triacylglycerol hydrolysis; almost complete inhibition of cellular
lipases was observed with 0.6 mM E600 or DEUP in the chase
media. DEUP (0.6 mM) inhibited greater than 90% of
triacylglycerol hydrolysis for up to 18 h (Fig. 5B). No
obvious signs of toxicity were observed when cells were incubated with
E600 or DEUP up to 0.8 mM for 24 h (data not
shown).
Cells Expressing Perilipin A Do Not Synthesize Significantly More
Triacylglycerol than Control Cells--
The incorporation of
[3H]oleate into triacylglycerols was measured in the
presence and absence of 0.6 mM DEUP in 3T3-L1
pre-adipocytes stably expressing perilipin A and control cells. For all
conditions, the rate of triacylglycerol synthesis increased after a lag
of approximately 2 h. Consistently more triacylglycerol synthesis was measured in cells incubated with DEUP than in cells incubated without lipolysis inhibitors (Fig. 6;
p < 0.05). While cells stably expressing perilipin A
displayed a trend toward increased synthesis of triacylglycerols over
8 h when compared with control cells, the differences in
triacylglycerol synthesis were not considered to be statistically
significant when data obtained from three separate experiments was
pooled and compared. We observed no consistent differences in the
incorporation of radiolabeled oleic acid into phospholipids when
perilipin A-expressing and control cells were compared (data not
shown). Similar results were obtained by using E600 to inhibit
triacylglycerol hydrolysis (data not shown). These data also
demonstrate that under lipid-loading conditions and without the
addition of hydrolysis inhibitors, the rate of triacylglycerol synthesis exceeds the rate of hydrolysis resulting in the accumulation of triacyglycerols in both types of cells.
Ectopically expressed perilipin A associates exclusively with
lipid droplets in cultured 3T3-L1 pre-adipocytes. Although most cultured cells store a small mass of neutral lipids in a few minute lipid droplets when provided with serum containing fatty acids and
cholesterol, few cells express the perilipins. To date, the perilipins
have been found only in differentiated, but not undifferentiated, adipocyte cell lines, such as 3T3-L1 adipocytes (1, 3, 4), and
steroidogenic Y-1 adrenal cortical cells (4, 7) and MA-10 Leydig cells
(4, 29); additionally, perilipins coat the lipid droplets of adipocytes
in white and brown adipose tissue, and in mammary tissue (3). Previous
studies have demonstrated that epitope-tagged perilipin A targets to
lipid droplets when expressed in Y-1 adrenal cortical cells (7) that
have a background of perilipins A and C (4, 7). Furthermore, it is
probable that exogenous perilipins A and B expressed via an adenoviral expression system targeted to lipid droplets in fully differentiated 3T3-L1 adipocytes (40), although these constructs lacked an epitope tag
to distinguish the expressed perilipins from endogenous perilipins in
immunoblotting and microscopy experiments. The present study is the
first to demonstrate the expression of perilipin A in cells that lack
all perilipins. Clearly, these cells contain all of the cellular
machinery necessary for newly synthesized perilipin A to target to and
assemble onto lipid droplets. Additionally, ectopic perilipin A targets
to and assembles onto lipid droplets in CHO fibroblasts, human Hep G2
hepatoma cells and rat McArH7777 hepatoma cells (data not shown). Thus,
many cultured cells are able to synthesize lipid droplets containing
perilipins when provided with the appropriate expression constructs;
hence, the assembly of perilipins onto lipid droplets appears to
require no cell-specific machinery. Furthermore, we have found no
evidence of perilipins associated with subcellular fractions containing
ER markers, thus suggesting that, if lipid droplets are initially
formed within the membrane bilayer of the ER, then the perilipins
likely add to the lipid droplets following their dissociation from the
ER.
The ectopic expression of perilipin A in fibroblasts induces two
notable changes; 1) cells expressing perilipin A store more triacylglycerol than control cells grown in the same culture
conditions, and 2) the organization of perilipin A-containing lipid
droplets into tight aggregates within the cytoplasm is very different
than the dispersed pattern of lipid droplets in control cells lacking perilipins. The increased triacylglycerol content was demonstrated to
be the consequence of decreased triacylglycerol hydrolysis in perilipin
A-expressing cells. Cells stably expressing perilipin A were shown to
retain nascent triacylglycerols in lipid droplets for 5-fold longer
than control cells lacking perilipins, but both control and perilipin
A-expressing cells were shown to have similar levels of activity of
soluble lipases. Therefore, the most likely explanation for the
decreased rate of triacylglycerol hydrolysis in intact cells expressing
perilipin A is that the perilipin A forms a barrier at the surfaces of
lipid droplets that shields the underlying lipids from the action of
soluble lipases. Thus, a control cell lacking perilipins synthesizes
triacylglycerols, but degrades them relatively quickly; when perilipin
A is present, the triacylglycerols are sequestered in a protected pool
to which the lipases have restricted access.
We hypothesize that perilipin A shields stored triacylglycerols from
soluble, cytosolic lipases, yet the cytosolic lipases of 3T3-L1
pre-adipocytes and most other cells are uncharacterized. The existence
of cytosolic neutral lipid hydrolases in cells such as fibroblasts has
long been postulated as an essential element in the control of
cholesterol homeostasis to regulate the flux of cholesterol between an
esterified storage pool in lipid droplets and free cholesterol in
membranes. Likewise, fatty acids released from triacylglycerol-rich
lipid droplets, such as those of skeletal muscle, provide a source of
energy, yet the mechanisms controlling this lipolysis have not been
elucidated. To date, only two candidate lipases have been identified:
1) hormone-sensitive lipase, which hydrolyzes stored triacylglycerols
in response to the stimulation of cell surface Although the present study demonstrates reduced hydrolysis of
triacylglycerols in 3T3-L1 pre-adipocytes stably expressing ectopic
perilipin A, the perilipins are naturally abundant in differentiated
3T3-L1 adipocytes where triacylglycerol lipase activity has been shown
to be 19-fold higher than that of undifferentiated cells (49). Support
for a role for the perilipins in the protection of adipose
triacylglycerol stores from lipolysis derives from a recent study
investigating the lipolysis induced by the chronic treatment of
cultured 3T3-L1 adipocytes with tumor necrosis factor- The aggregation of lipid droplets into tight clusters in cells
expressing perilipin A, but not in control cells, implies that perilipins may play a role in bringing small lipid droplets together, potentially through protein-protein interactions between perilipins, or
between perilipins and other proteins, on adjacent lipid droplets. This
aggregation of lipid droplets may serve an important function in
differentiating adipocytes where many small lipid droplets appear to
fuse into a few larger droplets that eventually coalesce into a single
droplet with reduced surface area relative to volume. Although
perilipins may play a role in droplet fusion, perilipins alone appear
to be insufficient to mediate fusion, since in all cells that we have
investigated to date, the addition of fatty acids to cells ectopically
expressing perilipin A results in the formation of larger clusters of
relatively small lipid droplets, rather than the formation of very
large droplets such as those found in adipocytes.
We do not yet know the significance of the selective increase in
triacylglycerol content in cells ectopically expressing perilipin A. Although the lipid droplets of control 3T3-L1 pre-adipocytes contain
primarily triacylglycerol, the droplets of control CHO fibroblasts are
relatively enriched in cholesterol esters. The serum-containing culture
medium provides both fatty acids and cholesterol for the synthesis of
neutral lipids; however, the droplets of both 3T3-L1 pre-adipocytes and
CHO fibroblasts expressing ectopic perilipin A become selectively
enriched in triacylglycerols, with no detectable change in the mass of
stored cholesterol esters. Future studies will determine whether the
perilipin A binds preferentially to, or selectively protects, droplets
containing a triacylglycerol core.
These studies suggest that the perilipins play a major role in
regulating lipolysis and the storage of triacylglycerols in adipocytes
and steroidogenic cells. Perilipin A has been shown to be a major
substrate for cAMP-dependent protein kinase in
lipolytically stimulated adipocytes (1, 50). Further study is required to determine whether or not the phosphorylation of the six consensus sites for cAMP-dependent protein kinase of perilipin A (2) following the lipolytic stimulation of adipocytes plays a role in
attenuating the barrier to lipolysis to permit hormone-sensitive lipase
access to the lipid droplet.
We thank Dr. Peter Gillies and Dr. Sandie
Germain for generously providing the triacsin C used in these studies,
and Dr. Nathan Wolins, Dr. Judith Storch, Dr. Susan Fried, Dr. Anne
Garcia, Dr. Ginny Kellner-Weibel, Dr. George Rothblat, Dr. Michael
Phillips, and Dr. Brian Oliver for helpful discussions or critical
review of the manuscript.
*
This work was supported by American Heart
Association-Southeastern Pennsylvania Affiliate Grant B98429E (to
D. L. B.) and an American Diabetes Association research grant
(to D. L. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Nutritional Sciences, Rutgers, The State University of New Jersey, 96 Lipman Dr., New Brunswick, NJ 08901. Tel.: 732-932-6524; Fax: 732-932-6837; E-mail: brasaemle@aesop.rutgers.edu.
**
Present address: Amersham Pharmacia Biotech, Inc., Piscataway, NJ 08855.
Published, JBC Papers in Press, August 17, 2000, DOI 10.1074/jbc.M007322200
1
X. Lu, J. Gruia-Gray, C. Londos, and A. Kimmel,
manuscript in preparation.
2
N. E. Wolins and C. Londos, unpublished data.
4
N. E. Wolins, C. J. Schultz, and C. Londos, unpublished data.
The abbreviations used are:
ER, endoplasmic
reticulum;
CHO, Chinese hamster ovary;
DEUP, diethylumbelliferyl
phosphate;
E600, diethyl-p-nitrophenyl phosphate;
PCMB, p-chloromercuribenzoate;
TNF-
Perilipin A Increases Triacylglycerol Storage by Decreasing the
Rate of Triacylglycerol Hydrolysis*
§¶,§,
**,, and
Department of Biochemistry, MCP Hahnemann
University, Philadelphia, Pennsylvania 19129, the
§ Department of Nutritional Sciences, Rutgers, The State
University of New Jersey, New Brunswick, New Jersey 08901, and the
Molecular Mechanisms of Development Section and
Membrane Regulation Section, Laboratory of
Cellular and Developmental Biology, NIDDK, National Institutes of
Health, Bethesda, Maryland 20892
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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MSVtkneo
retroviral expression vector (25). The retroviral construct was
purified using an endotoxin-free plasmid preparation kit (Qiagen, Santa
Clarita, CA) and was co-transfected by the calcium phosphate method
(26, 27) with the pSV-
-E-MLV vector (25) containing cDNAs for
retroviral packaging proteins into 293T cells (28) for assembly of the
retrovirus. Medium containing the retrovirus was collected from the
cultures of 293T cells, filtered, and transferred to subconfluent
3T3-L1 pre-adipocytes. Cells containing the retrovirus were selected
for by the addition of 0.6 mg/ml active geneticin to the culture media;
typically, more than 95% of cells survive the selection conditions.
Cells for control conditions stably incorporated the retroviral vector lacking the perilipin cDNA.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (51K):
[in a new window]
Fig. 1.
The stable, ectopic expression of perilipin A
alters the number, size, and arrangement of lipid droplets in
fibroblastic cells. Control 3T3-L1 pre-adipocytes (A
and B) and cells stably expressing perilipin A (C
and D) were fixed and co-stained with Bodipy 493/503
(indicated in red) to detect neutral lipids, and
simultaneously, with polyclonal antibodies raised against perilipins
followed by an Alexa Fluor 546 secondary antibody (in
green); coincident staining is indicated in
yellow. Cells in panels A and
C were cultured in standard low lipid-containing culture
medium, while cells in panels B and D
were incubated for 16 h with 400 µM oleic acid
complexed to albumin prior to staining. Each panel contains portions of
two, three, or four representative cells. Bars, 10 µm. Peri A, perilipin A.
View larger version (62K):
[in a new window]
Fig. 2.
Ectopic perilipin A localizes exclusively to
buoyant lipid storage droplets containing the majority of cellular
triacylglycerol on sucrose gradients of cell homogenates. 3T3-L1
pre-adipocytes stably expressing ectopic perilipin A were incubated for
16 h with 400 µM oleic acid complexed to albumin
before being harvested, homogenized, and fractionated by the
centrifugation of 0-30% sucrose gradients; 12 equal fractions were
collected following centrifugation. The top panel
shows a thin layer chromatography plate of lipid extracts from each of
the 12 fractions developed to separate phospholipids, cholesterol,
triacylglycerols, and cholesterol esters, as indicated. The
lower panel shows a single immunoblot of proteins
from each of the 12 fractions resolved by SDS-polyacrylamide gel
electrophoresis, and probed with polyclonal antibodies raised against
perilipin A and calnexin, a marker for endoplasmic reticulum.
The expression of perilipin A in 3T3-L1 pre-adipocytes selectively
increases the cellular storage of triacylglycerols, and not
cholesterol esters
View larger version (10K):
[in a new window]
Fig. 3.
Triacsin C inhibits the synthesis of
triacylglycerols in 3T3-L1 pre-adipocytes stably expressing perilipin A
(filled bars) and control cells lacking
perilipins (open bars) incubated with
oleate. Cells were incubated in culture medium in the presence or
absence of 400 µM oleate complexed to albumin, and in the
presence or absence of 6 µM triacsin C, an inhibitor of
acyl-coenzyme A synthetase, for 16 h. Triacylglycerol content was
determined by quantitative thin layer chromatography. The
triacylglycerol content of control cells lacking perilipins and
incubated without supplemental oleic acid was at the limit of detection
( 
0.5 µg/mg of protein). Data represent the means ± standard
deviations from three (no oleate samples and + oleate + triacsin
samples) or six (+ oleate samples) independent determinations.
View larger version (14K):
[in a new window]
Fig. 4.
The expression of perilipin A increases
triacylglycerol storage by decreasing the rate of triacylglycerol
hydrolysis. Confluent monolayers of 3T3-L1 pre-adipocytes
expressing perilipin A ( 
) and control cells (
) were incubated
with 400 µM oleic acid complexed to albumin for 16 h
to increase the synthesis and storage of triacylglycerols. The
supplemental fatty acids were withdrawn, and the cells were incubated
with 6 µM triacsin C to inhibit further triacylglycerol
synthesis. The cellular lipid content was determined by quantitative
thin layer chromatography; the triacylglycerol content is expressed as
a percentage of the mass of triacylglycerol in the cells immediately
following the incubation with exogenous oleic acid. Data are means ± standard deviations for triplicate samples for a representative
experiment; where error bars are not visible, they are contained within
the symbol. Triacylglycerol hydrolysis occurred with a
t1/2 = 6.8 h for control cells lacking
perilipins and a t1/2 = 30.5 h for cells stably
expressing perilipin A.
The rate of hydrolysis of emulsified triacylglycerols is equivalent for
post-mitochondrial supernatants from 3T3-L1 pre-adipocytes expressing
perilipin A and control cells
View larger version (14K):
[in a new window]
Fig. 5.
Triacylglycerol hydrolysis is inhibited by
E600 and DEUP, but not PCMB, in 3T3-L1 pre-adipocytes. Confluent
monolayers of 3T3-L1 pre-adipocytes were incubated with 400 µM oleic acid complexed to albumin for 16 h to
increase stored triacylglycerols. A, supplemental fatty
acids were removed and chase medium containing 6 µM
triacsin C and PCMB, E600, or DEUP at the indicated concentrations was
added for an additional 15 h before cells were harvested and the
triacylglycerol quantified. Data are the means and standard deviations
for the triacylglycerol content of triplicate samples expressed as the
percentage of lipid-loaded samples before the 15-h chase period. Cells
chased for 15 h in culture medium without hydrolysis inhibitors
(no inhibitor) lost approximately 75% of the
starting triacylglycerol. B, chase medium containing 600 µM DEUP ( ) or no inhibitors () was added to the
cells; triplicate samples were removed at various times, lipids
extracted, and triacylglycerol quantified. Data are means and standard
deviations of triplicate determinations; where error bars are not
visible, they are contained within the symbol.
View larger version (17K):
[in a new window]
Fig. 6.
3T3-L1 pre-adipocytes stably expressing
perilipin A ( 
, 
) do not synthesize significantly more
triacylglycerol than control cells lacking perilipins (, 
).
Confluent monolayers of 3T3-L1 pre-adipocytes stably expressing
perilipin A and control cells were incubated with
[3H]oleic acid complexed to albumin in the presence (,
) or absence (, ) of 0.6 mM DEUP to inhibit
triacylglycerol hydrolysis. Cells were harvested at various times, the
lipids extracted, and the incorporation of [3H]oleate
into triacylglycerols determined, as a measure of triacylglycerol
synthesis. Data represent the means and standard deviations of the
average values from quadruplicate samples from three separate
experiments; where error bars are not visible, they are contained
within the symbol.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic receptors
in adipocytes, and has also been found at low abundance in cells of the
adrenal cortex, testes, and ovaries, as well as in heart, skeletal
muscle, peritoneal macrophages (41-44), and the cultured CHO cell line
(45); and 2) an unrelated triacylglycerol hydrolase that has recently
been found on isolated lipid droplets and in microsomal subcellular fractions from liver, and in homogenates of kidney and intestine (22,
46, 47). Many unidentified lipases appear to be members of the serine
esterase class of enzymes and are irreversibly inhibited by
organophosphorous reagents such as E600 and DEUP. The activity of the
newly identified liver triacylglycerol hydrolase (22, 46, 47) is
inhibited by E600, as is a hydrolase of human skin fibroblasts that
cleaves triacylglycerols with short and medium chain fatty acids (37).
DEUP has been reported to irreversibly inhibit the cholesterol esterase
of homogenates of rat Fu5AH hepatoma cells (38, 39) and murine MA10
Leydig cells (48). The sulfhydral reagent PCMB inhibits the long chain
triacylglycerol hydrolase of human skin fibroblasts (37). We now report
that the hydrolysis of triacylglycerols in 3T3-L1 pre-adipocytes is
inhibited by E600 and DEUP, but not by PCMB.
(TNF-)
(40). In this study, TNF- increased glycerol release, and hence
lipolysis, during 24-h treatments concomitant with a reduction in the
expression of perilipins A and B. The expression of perilipins A or B
in the cultured 3T3-L1 adipocytes using an adenoviral expression vector
prevented the TNF--induced increase in glycerol release while
maintaining perilipin levels on the lipid droplets. The authors propose
that the overexpression of perilipins limited TNF--mediated lipid
hydrolysis (40). Here, we have used a completely different approach of
determining the rates of triacylglycerol hydrolysis and synthesis in
the presence and absence of perilipin A, yet we have also concluded
that perilipin A protects triacylglycerols from lipolysis. The high
levels of soluble lipase activity in adipocytes may necessitate the
presence of the perilipins at the surfaces of lipid droplets to protect the vast stores of triacylglycerol from hydrolysis.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
, tumor necrosis
factor-.
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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A. W. Cohen, B. Razani, W. Schubert, T. M. Williams, X. B. Wang, P. Iyengar, D. L. Brasaemle, P. E. Scherer, and M. P. Lisanti Role of Caveolin-1 in the Modulation of Lipolysis and Lipid Droplet Formation Diabetes, May 1, 2004; 53(5): 1261 - 1270. [Abstract] [Full Text] [PDF]
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S. Nagai, C. Shimizu, M. Umetsu, S. Taniguchi, M. Endo, H. Miyoshi, N. Yoshioka, M. Kubo, and T. Koike Identification of a Functional Peroxisome Proliferator-Activated Receptor Responsive Element within the Murine Perilipin Gene Endocrinology, May 1, 2004; 145(5): 2346 - 2356. [Abstract] [Full Text] [PDF]
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M. Paciga, C. R. McCudden, C. Londos, G. E. DiMattia, and G. F. Wagner Targeting of Big Stanniocalcin and Its Receptor to Lipid Storage Droplets of Ovarian Steroidogenic Cells J. Biol. Chem., December 5, 2003; 278(49): 49549 - 49554. [Abstract] [Full Text] [PDF]
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N. B. Cole, D. D. Murphy, T. Grider, S. Rueter, D. Brasaemle, and R. L. Nussbaum Lipid Droplet Binding and Oligomerization Properties of the Parkinson's Disease Protein alpha -Synuclein J. Biol. Chem., February 15, 2002; 277(8): 6344 - 6352. [Abstract] [Full Text] [PDF]
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T. Raclot, C. Holm, and D. Langin Fatty acid specificity of hormone-sensitive lipase: implication in the selective hydrolysis of triacylglycerols J. Lipid Res., December 1, 2001; 42(12): 2049 - 2057. [Abstract] [Full Text] [PDF]
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A. Rudich, S. Vanounou, K. Riesenberg, M. Porat, A. Tirosh, I. Harman-Boehm, A. S. Greenberg, F. Schlaeffer, and N. Bashan The HIV Protease Inhibitor Nelfinavir Induces Insulin Resistance and Increases Basal Lipolysis in 3T3-L1 Adipocytes Diabetes, June 1, 2001; 50(6): 1425 - 1431. [Abstract] [Full Text]
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J. T. Tansey, C. Sztalryd, J. Gruia-Gray, D. L. Roush, J. V. Zee, O. Gavrilova, M. L. Reitman, C.-X. Deng, C. Li, A. R. Kimmel, et al. Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity PNAS, May 22, 2001; 98(11): 6494 - 6499. [Abstract] [Full Text] [PDF]
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A. G. Ostermeyer, J. M. Paci, Y. Zeng, D. M. Lublin, S. Munro, and D. A. Brown Accumulation of Caveolin in the Endoplasmic Reticulum Redirects the Protein to Lipid Storage Droplets J. Cell Biol., March 5, 2001; 152(5): 1071 - 1078. [Abstract] [Full Text] [PDF]
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N. E. Wolins, B. Rubin, and D. L. Brasaemle TIP47 Associates with Lipid Droplets J. Biol. Chem., February 9, 2001; 276(7): 5101 - 5108. [Abstract] [Full Text] [PDF]
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L. Izem and R. E. Morton Cholesteryl Ester Transfer Protein Biosynthesis and Cellular Cholesterol Homeostasis Are Tightly Interconnected J. Biol. Chem., July 6, 2001; 276(28): 26534 - 26541. [Abstract] [Full Text] [PDF]
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