From the Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201-1180 and the Program of Oncology, Marlene and Stewart Greenebaum Cancer Center of the University of Maryland, Baltimore, Maryland 21201-1180
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ABSTRACT |
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Adipose differentiation related protein (ADRP) is
a 50-kDa novel protein cloned from a mouse 1246 adipocyte cDNA
library, rapidly induced during adipocyte differentiation. We have
examined ADRP function, and we show here that ADRP facilitates fatty
acid uptake in COS cells transfected with ADRP cDNA. We demonstrate that uptake of long chain fatty acids was significantly stimulated in a
time-dependent fashion in ADRP-expressing COS-7 cells
compared with empty vector-transfected control cells. Oleic acid uptake velocity increased significantly in a dose-dependent manner
in ADRP-expressing COS-7 cells compared with control cells. The
transport Km was 0.051 µM, and
Vmax was 57.97 pmol/105 cells/min
in ADRP-expressing cells, and Km was 0.093 µM and Vmax was 20.13 pmol/105 cells/min in control cells. The oleate uptake
measured at 4 °C was only 10% that at 37 °C. ADRP also
stimulated uptake of palmitate and arachidonate but had no effect on
uptake of medium chain fatty acid such as octanoic acid and glucose.
These data suggest that ADRP specifically enhances uptake of long chain
fatty acids by increasing the initial rate of uptake and provide novel
information about ADRP function as a saturable transport component for
long chain fatty acids.
Long chain non-esterified free fatty acids
(FA)1 and their derivatives
have multiple functions as either essential components of membrane,
efficient sources of energy, or important effective molecules that
regulate metabolism and mediate gene expression (1, 2). Adipose tissue
is the main source of lipids and fatty acids in the body where they
play key roles in the regulation of energy balance in mammals. Proteins
involved in FA and triglyceride synthesis and accumulation as well as
utilization of exogenous lipid are induced during adipocyte
differentiation (3, 4). Increase of enzymatic activities regulating
lipogenesis and lipolysis is also ongoing at this stage. These multiple
roles of FA suggest that careful regulation of cellular aspects of FA
metabolism including cellular uptake in liver, fat, cardiac and
skeletal muscles, and other organs is essential.
The mechanism by which long chain free FA enter the cells is not
completely understood. It has long been postulated that the movement of
long chain FA across the cell membrane is invariably passive (5, 6).
Studies (5, 6) have suggested that FA penetrate cardiac myocytes by a
passive unregulated mechanism rather than by a specific facilitated
process and that saturation of an intracellular metabolism step is the
cause for apparent saturation of uptake in other studies. Others (7)
have also shown that the entry of FA into hepatocytes reflected their
passive partitioning into the lipid component of the cell membrane.
However, recent observations indicated that at least a portion of FA
uptake might occur by a carrier-mediated transport system (8, 9).
Studies with liver, fat cells, cardiac tissue, and skeletal myocytes
have shown that free FA uptake exhibited many of the kinetic properties
of a facilitated process, namely saturation, trans-stimulation,
cis-inhibition, stereospecificity, and counter transport (8-12).
Several studies have indicated that FA uptake by adipocytes was
phloretin-inhibitable and blocked by antibody raised against specific
binding proteins (10, 13). These features could not be explained by
diffusion. It is then possible to assume that facilitated and passive
uptake processes both occur simultaneously in cells (12), with the
facilitated transport being the predominant process. In support of
these findings, five putative mammalian free FA transporters have been
identified so far in the plasma membrane of several tissues (14-18).
cDNA clones have been isolated for three of them, specifically
plasma membrane fatty acid-binding protein (FABPpm) (14), fatty acid
translocase (FAT) (15), and fatty acid transport protein (FATP) (16).
In addition, small molecular weight cytosolic fatty acid-binding
proteins have been characterized, cloned, and extensively studied (for
review see Ref. 19).
Adipose differentiation related protein (ADRP) is a novel 50-kDa
protein originally cloned by differential hybridization from a cDNA
library of differentiated mouse 1246 adipocytes (20). The 1.7-kilobase
pair ADRP mRNA was induced 50-100-fold a few hours after the onset
of adipose differentiation in 1246 cells, thus making ADRP an early
marker of the adipose differentiation program (20). It has been shown
that ADRP mRNA was expressed at high levels in adipose tissue (20)
and also in many different types of cells and tissues where lipids were
accumulated or synthesized, although at lower levels than in adipocytes
(21). Sequencing of ADRP did not provide any information about its
possible function in adipocytes. Immunolocalization studies done at
different times during the adipose differentiation program in 1246 and
3T3-L1 adipocytes indicated that ADRP was localized in the vicinity of the plasma membrane in cells that started to differentiate and was
found on the surface of lipid droplets in mature adipocytes (20-21).
Moreover, ADRP expression was found to be induced in the liver of mice
treated with the carnitine palmitoyltransferase I inhibitor, etomoxir,
which caused neutral lipid accumulation in the organ (22). These
various studies suggested that ADRP might be involved in the formation
or stabilization of lipid droplets in adipocytes. As a first step to
investigate this hypothesis, we have examined here whether ADRP was
involved in FA uptake using ADRP-transfected COS-7 cells as an
experimental model system. The results presented in this paper show
that ADRP plays a role in facilitated FA transport in COS-7 cells
expressing the protein.
Construction of ADRP-pcDNA3 Expression Vector and Transient
Transfection in COS-7 Cells--
COS-7 cells (CRL 1651, American Type
Culture Collection, Manassas, VA) were cultivated in DME-F12 medium
(1:1 mixture of Dulbecco's modified Eagle's medium and Ham's
nutrient F12 medium) supplemented with 10% fetal bovine serum (FBS)
(Life Technologies, Inc.) in T-75-cm2 plates. Cell stocks
were cultivated in these conditions until nearly confluent and
subcultured at 1:20 dilution or plated for an experiment. For
expression into COS-7 cells, ADRP cDNA was cloned into the
mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA).
Mouse ADRP cDNA fragment containing the 1.3-kilobase pair ADRP open
reading frame was cloned in the sense orientation in XbaI
and BamHI sites of pcDNA3. Expression of green
fluorescent protein (GFP, CLONTECH, Palo Alto, CA)
was used to monitor transfection efficiency. GFP plasmid DNA was
co-transfected into COS-7 cells with pcDNA3 empty vector (control
cells) or pcDNA3-ADRP vector plasmid DNA.
Transient Transfection of Plasmid cDNA into COS-7
Cells--
Transient transfection of plasmid DNA into COS-7 cells was
carried out by the DEAE-dextran method (23) established for COS cells.
Briefly, 2 ml of a solution containing plasmid DNA and DEAE-dextran was
prepared by mixing 0.2 ml of DEAE-dextran at 10 mg/ml, 5 pmol (about 10 µg) of plasmid DNA for each vector into 1.8 ml of phosphate-buffered
saline (PBS) and incubated for 1 h at 37 °C with COS-7 cells
cultivated in T-75-cm2 flasks. After 1 h, the cells
were treated with 100 µM chloroquine for 4 h at
37 °C in serum-free culture medium followed by treatment with 10%
dimethyl sulfoxide for 2 min. Cells were then washed twice with culture
medium and then cultivated in DME/F12 medium supplemented with 10% FBS
for 48 h for maximal ADRP expression in the transfected cells.
Control cells were co-transfected with empty vector pcDNA3 and with
GFP plasmid DNA using a similar method.
Transfection efficiency was determined by counting the number of cells
expressing GFP (co-transfected with either ADRP-pcDNA3 or with
pcDNA3 empty vector) when compared with the total number of cells
counted with a hemocytometer.
Determination of ADRP Expression in Transfected Cells--
ADRP
protein expression in transfected COS cells was examined in cell
lysates prepared from transfected COS-7 cells by Western blot analysis
using rabbit anti-ADRP antibody. Briefly, cells were washed once with
PBS and lysed in 1 ml of SDS sample buffer (62.5 mM
Tris-HCl, pH 6.8, 2% SDS, 10% glycerol) without Immunolocalization of ADRP in Transfected COS-7 Cells--
COS-7
cells were plated in chamber slides (Becton Dickinson) in DME/F12
medium supplemented with 10% FBS. Transfection of ADRP or empty vector
plasmid DNA was carried out as described above. After 48 h, cells
were washed with PBS, fixed with 2% paraformaldehyde, and
permeabilized with 0.2% Triton X-100 using a previously described method (16) followed by several washes with PBS. Cells were incubated
with affinity purified anti-ADRP antibody for 1 h at room
temperature followed by incubation with FITC-conjugated goat anti-rabbit secondary antibody (Bio-Rad). After extensive washing with
PBS, slides were mounted in buffered glycerol solution and examined
with an Olympus BX40 fluorescence microscope equipped with an Olympus camera.
Preparation of Albumin-bound Fatty
Acids--
[9,10-3H]Oleic acid (1.2 µCi/nmol) (NEN
Life Science Products) and unlabeled oleic acid sodium salts were
dissolved in 10 ml of water at 40 °C to give a concentration of
about 320 µM (48 µCi/ml). When the solution was
completely clear after ~10 min, fatty acid-free BSA (Sigma) from a
concentrated stock solution (20%) was added with gentle mixing to
obtain a final concentration of 80 µM BSA and an oleic
acid/BSA molar ratio of 4.0. For the assays, an aliquot of the stock
solution of FA/BSA (320 µM) was diluted 1:8 with PBS
containing or not containing additional fatty acid-free BSA to obtain
the final desired concentrations of oleic acid (40 µM)
and of fatty acid-free BSA (10, 13, 20, 40, and 80 µM)
and oleic acid/BSA molar ratios of 4.0, 3.0, 2.0, 1.0, and 0.5, respectively. The final concentration of fatty acids in the assay was
20 µM [3H]oleic acid (3 µCi/ml) with
various concentrations of fatty acid-free BSA. The unbound oleic acid
concentration in the presence of BSA was determined from the oleate/BSA
molar ratio according to the calculation of Abumrad et al.
(13), using the association constants of Spector and Fletcher (25) and
the model of stepwise equilibrium developed by Klotz et al.
(26). [9,10-3H]Palmitic acid (43 µCi/nmol),
[5,6,8,11,12,14,15-3H]arachidonic acid (222 µCi/nmol),
and [1-14C]octanoic acid (55 µCi/µmol) (NEN Life
Science Products) were diluted to 40 µM with a fatty
acid-free BSA concentration of 10 µM. The final working
concentration in the tubes was 20 µM
[3H]palmitic acid (3.1 µCi/ml),
[3H]arachidonic acid (1.3 µCi/ml), and
[14C]octanoic acid (12 nCi/ml), respectively, with 5 µM fatty acid-free BSA.
Radiolabeled Fatty Acid Uptake by Transfected COS-7
Cells--
Transfected COS-7 cells were resuspended in PBS (5 × 105 cells/ml) in 15 ml of polypropylene centrifuge tubes.
200-µl aliquots of cell suspension (105 cells) were
placed in 5-ml polypropylene centrifuge tubes. Cell suspensions in PBS
were preincubated for 5 min at 37 °C in a shaking water bath. An
equal volume of 2-fold concentrated stock fatty acid/BSA solutions was
added to each tube to perform fatty acid uptake. At specific intervals,
uptake was stopped by adding 5 ml of ice-cold PBS containing 0.1% BSA
and 200 µM phloretin (wash solution) into each assay
tube. Then the solutions and cell suspensions were filtered through
GF/C filters (Whatman, Maidstone, UK) which had been presaturated with
15 ml of 0.1% BSA in PBS. Cells retained by filters were rapidly
washed 3 times with 5 ml of cold wash solution. Filters were soaked
overnight in 10 ml of scintillation mixture prior to counting with a
liquid scintillation counter. Nonspecific fatty acid adsorbed to
filters lacking cells was routinely measured and subtracted from
experimental values. Background radioactivity representing isotope
trapped extracellularly and bound nonspecifically by the cells was
measured from zero time incubation determined by adding stop solution
to the cells before adding radiolabeled fatty acid-BSA complex.
Fatty acid uptake data were normalized with the transfection efficiency
that had been determined by counting the number of fluorescent cells
expressing GFP compared with the total cell number. Since
non-transfected cells could also uptake fatty acid, it was necessary to
remove their contribution to the total FA uptake in order to determine
the contribution due to the expression of ADRP in the cells. To achieve
this, the uptake of FA by non-transfected cells was determined by
multiplying the value of total FA uptake in non-transfected control
cells by the percentage of non-transfected cells (100% Bligh-Dyer Extraction and Thin Layer Chromatography of
Lipids--
The distribution of labeled oleate in intracellular lipids
in ADRP-transfected or empty vector-transfected COS-7 cells was examined after uptake was performed. Uptake was carried out as described above. At various time points after uptake was stopped, cells
from triplicate samples (6 × 105 cells) were washed
by centrifugation. Lipids were immediately extracted by the method of
Bligh and Dyer (27) from cells resuspended in 0.8 ml of PBS. Extracted
lipids were resuspended in 100 µl of CHCl3/MeOH (2:1) and
separated by thin layer chromatography (TLC) on a silica gel G plate
(Analtech, Newark, DE) in the solvent mixture of hexane/ether/formic
acid (60:40:1). Triolein, diolein, mono-olein, oleic acid, and
1-palmityl,2-oleyl lecithin (Sigma) were used as standards for the TLC.
Lipid spots were visualized by iodine vapors and immediately scraped
with a razor blade into scintillation vials to count radioactivity
incorporated into intracellular lipids in ADRP and empty
vector-transfected COS-7 cells.
2-[14C]Deoxyglucose Uptake by Transfected COS-7
Cells--
ADRP and empty vector-transfected COS-7 cells were
transferred from T-75 flasks to 24-well plates, 24 h after
transfection. After incubation at 37 °C for another 24 h, media
were changed to DME/F12 without FBS. Cells were incubated at 37 °C
for 3 h. The cells were then washed in Krebs-Ringer phosphate
(KRP) buffer (137 mM NaCl, 4.7 mM KCl, 0.4 mM MgCl2, 1 mM CaCl2,
10 mM
Na2HPO4/NaH2PO4 buffer,
pH 7.3, 0.2% BSA) and incubated in KRP buffer for 2 h prior to
performing the uptake. 200 µM
2-[14C]deoxyglucose (1.6 µCi/ml) (NEN Life Science
Products) was freshly added into KRP buffer. After 10, 20, and 30 min
incubation at 37 °C, cells were washed 2 times with ice-cold KRP
buffer and then dissolved in 200 µl of 0.1 M NaOH. 5 ml
of scintillation mixture was added into each tube for scintillation counting.
Calculation and Statistical Analysis--
All experiments were
repeated at least three times. The data presented here are expressed as
mean ± S.D. Differences between experimental groups were
evaluated with two-tailed Student's t tests. Differences
were considered significant if p < 0.05.
ADRP Expression in COS-7 Cells Transiently Transfected with
ADRP-pcDNA3--
To test whether ADRP promoted FA transport, the
pcDNA3 expression vector containing ADRP cDNA insert was
transiently transfected into COS-7 cells. Plasmid DNA from empty
pcDNA3 vector was transfected into COS-7 cells as control. Cells
were harvested 48 h after transfection, and cell lysates were
prepared as described under "Experimental Procedures," in order to
measure ADRP protein expression by Western blot analysis. As shown in
Fig. 1, ADRP protein expression was very
low in empty vector-transfected COS-7 cells. In contrast, cells
transfected with the ADRP-pCDNA3 expression vector expressed high
levels of ADRP protein equivalent to the one in 1246 adipocytes known
to express high level of ADRP (19, 24) and used as positive controls.
These results demonstrated that ADRP protein was effectively expressed
in COS-7 cells and that the transfected COS-7 cells could be used as a
model of ADRP protein overexpression to examine the function of ADRP in
mammalian cells.
Localization of ADRP in transfected COS-7 cells was examined by
immunofluorescence staining of fixed cells with anti-ADRP antibody
followed by FITC-conjugated goat anti-rabbit secondary antibody.
Fluorescence microscopy revealed a specific pattern of staining at the
cell periphery in ADRP-transfected COS-7 cells (Fig.
2 top panel) which was not
observed in empty vector-transfected COS-7 cells (Fig. 2 bottom
panel) or in cells incubated with preimmune IgG used as negative
control (data not shown). Additional immunostaining was also found
associated with the nucleus in ADRP-transfected cells as well as in
control cells although with a lesser degree. These data show that ADRP
is preferentially found associated with the plasma membrane in the
transfected COS-7 cells.
Time Course of [3H]Oleic Acid Uptake in
ADRP-transfected and in Control COS-7 Cells--
Assays of oleic acid
transport were performed in control and in ADRP-expressing COS-7 cells.
[3H]Oleate at a final concentration of 20 µM corresponding to an oleate/BSA molar ratio of 4:1 was
incubated with 105 cells at 37 °C from 15 s to 30 min. Uptake was stopped by adding 5 ml of an ice-cold stop solution
containing 200 µM phloretin and 0.1% BSA in PBS. Since
the stop solution removed surface-bound [3H]oleate while
blocking efflux from the cells of oleate already internalized (8),
accurate quantitation of cumulative cellular oleate uptake could be
obtained after stop solution treatment. The [3H]oleate
uptake measured as the total [3H]oleate accumulated in
ADRP or empty vector-transfected cells was normalized to the
transfection efficiency as described under "Experimental
Procedures." Fig. 3 showed the time
course of oleate uptake in transfected COS-7 cells determined at 2.30 µM unbound oleic acid with an oleate/BSA ratio of 4:1
(mol/mol). The time course was biphasic with a fast early phase linear
for up to 0.5 min (Fig. 3A) followed by a slower phase (Fig.
3B) both for control and ADRP-transfected cells. In the
later slower phase, the oleate uptake rate decreased although the cells
continued to accumulate oleate. ADRP increased [3H]oleate
uptake by about 3-fold over the uptake in control cells at all time
points measured. Each of the two phases were significantly faster in
ADRP-transfected cells than in control cells (p < 0.01 in each case). By 30 min, ADRP-transfected COS-7 cells had incorporated 401.0 ± 57.6 pmol/105 cells and control cells
194.6 ± 42.6 pmol/105 cells, respectively. These
values were significantly different from each other (p < 0.01). The initial uptake rate determined by the slope of the linear
portion of the uptake curve over the initial 0.5-min period (Fig.
2A) increased from 21.15 ± 0.46 pmol/105
cells/min in control cells to 58.01 ± 9.48 pmol/105
cells/min in ADRP-transfected cells (p < 0.01).
Accurate quantitative measure of influx velocity could be obtained by
measuring the initial uptake rate (8). These data indicated that ADRP
promoted [3H]oleate uptake in COS-7 cells by increasing
the influx velocity of oleic acid.
The distribution of the label in intracellular lipids in ADRP and
control COS-7 cells at different time points of the uptake reaction was
examined. For this purpose, cell-associated lipids were extracted by
the Bligh-Dyer method and analyzed by thin layer chromatography as
described under "Experimental Procedures." Initially, the majority
of the label was found in the free fatty acid pool. At 5 min, 35% of
the label was found in the free fatty acids with 31% in the
phospholipids and the rest in the neutral lipids. At 15 min, less than
10% of the label was found associated with free fatty acids, whereas
30% of the label was still found in the phospholipids and 60% in the
neutral lipid pools. The same distribution of label over time was
observed in empty vector-transfected cells indicating that ADRP did not
change the distribution of label in the intracellular lipids but simply
increased the level of uptake of fatty acids.
Uptake of [3H]Oleic Acid as a Function of Oleic Acid
Concentration--
The kinetics of [3H]oleic acid uptake
by ADRP was assessed by examining oleate uptake over a 15-s period
within a range of unbound oleic acid concentrations from 0.043 to 2.30 µM corresponding to a molar ratio of oleate/BSA of 0.5 to
4, respectively. As shown in Fig. 4, the
uptake in control and ADRP-transfected cells was saturable.
[3H]Oleate uptake velocity in control cells and
ADRP-transfected cells reached a plateau at concentrations above 0.11 µM unbound oleic acid. After a 15-s incubation, the
[3H]oleic acid uptake velocity in ADRP-transfected COS-7
cells was significantly higher than that of control cells at this range of oleic acid concentrations (p < 0.01). From a
double-reciprocal plot of these data, the transport
Km of ADRP was determined to be 0.051 µM and Vmax was 57.97 pmol/105 cells/min, whereas Km was 0.093 µM and Vmax was 20.13 pmol/105 cells/min in control cells. These results
suggested that ADRP had the function of facilitating the uptake of
fatty acid by increasing fatty acid uptake velocity.
Uptake of [3H]oleate was also examined at 4 °C and
compared with the one measured at 37 °C. After 2 min incubation,
oleic acid uptake at the unbound oleic acid concentration of 2.30 µM obtained from 5 replicate determinations was 90%
lower in ADRP-transfected cells at 4 °C (5.17 ± 0.33 pmol per
105 cells) than in ADRP-transfected cells at 37 °C
(61.52 ± 11.08 pmol per 105 cells). This low uptake
value was similar to the one measured with control transfected cells at
4 °C.
Specificity of Fatty Acids Uptake--
Experiments were carried
out to examine the uptake of other fatty acids by COS cells transfected
with ADRP or with empty vector. Uptake was performed at 37 °C with a
4:1 molar ratio of fatty acid/BSA and a total of 20 µM
fatty acid. We measured the uptake of medium chain fatty acid such as
octanoic acid and long chain fatty acids either saturated such as
palmitic acid or polyunsaturated such as arachidonic acid. As shown in
Table I, uptake of palmitic acid and
arachidonic acid was significantly increased in ADRP-transfected cells
when compared with control groups similarly to what was observed for
oleic acid. In contrast, there was no significant difference between
ADRP and control groups in the uptake of radiolabeled medium chain
octanoic acid. These data indicate that ADRP only regulated long chain
fatty acid uptake.
Uptake of 2-[14C]Deoxy-D-glucose--
In
contrast to FA transport, transport of 2-deoxyglucose (Fig.
5) was not significantly altered by ADRP
expression. 2- [14C]Deoxy-D-glucose uptake
per 105 cells at a concentration of 200 µM
glucose was comparable between the control group and ADRP transfection
group, indicating that glucose uptake was not regulated by ADRP.
In this study, we examined whether ADRP expression stimulated
fatty acid uptake. These studies were carried out using COS-7 cells
expressing high level of ADRP by transient transfection of ADRP
cDNA as an experimental system. Immunolocalization of ADRP in
transfected COS-7 cells indicated that ADRP was found preferentially
associated with the plasma membrane. This was also confirmed by
analyzing the distribution of ADRP immunoreactivity by SDS-PAGE and
Western blot analysis of fractions obtained after subcellular
fractionation of transfected COS cell lysates. ADRP was found in the
105,000 × g particulate fraction, whereas cytosol fraction was negative (data not shown).
Uptake studies presented here show that ADRP expression resulted in
increased FA uptake in transfected COS-7 cells. We observed saturation
of oleate uptake velocity as a function of the unbound FA concentration
in the medium. The data indicate that ADRP expression resulted in a
3-fold increase in the initial velocity of oleic acid uptake when
compared with empty vector-transfected COS-7 cells. There was a
2.5-fold increase in the Vmax for the saturable component of oleate uptake in ADRP-expressing COS-7 cells. The Km of the uptake component contributed by ADRP
expression decreased by 2-fold when compared with control cells. These
data suggested that the saturable component of fatty acid uptake could be facilitated by ADRP expression. It has been shown that this influx
of non-esterified FA into adipocytes is phloretin-inhibitable, saturable, and independent of cellular metabolism (9, 13).
The oleate uptake mediated by ADRP expressed into COS cells was
temperature-dependent, since the uptake at 4 °C was only
10% that at 37 °C. This suggested that a specific carrier-mediated process was involved in FA transport systems in COS-7 cells. The results presented here suggest that this process is facilitated by ADRP
expression. Analysis of the distribution of label in intracellular lipids showed no difference after oleate uptake in ADRP and empty vector control cells suggesting that ADRP did not influence the fate of
fatty acid in intracellular pools but merely facilitated its uptake.
Comparison of uptake of various fatty acids indicated that ADRP
facilitated uptake of long chain fatty acids whether they were
saturated, mono-unsaturated, or polyunsaturated. In contrast, ADRP had
no effect on the uptake of medium chain FA octanoic acid and on the
uptake of glucose. These data suggested that ADRP mediates specifically
uptake of long chain fatty acid and that uptake of medium chain fatty
acid occurs by a different mechanism.
All of the results demonstrated that ADRP indeed specifically enhanced
uptake of long chain fatty acids by increasing the initial uptake rate.
Together with localization studies showing that ADRP localized near the
membrane in transfected COS-7 cells and in cells starting to
differentiate into adipocytes (20), these data strongly support the
idea that ADRP acts as a fatty acid carrier protein.
Although it has long been considered that the movement of long chain FA
across the cell membrane is invariably passive (7, 23, 26), increasing
evidence suggests that a specific and selective facilitated transport
mechanism is responsible for the transmembrane flux of free FA (10, 13,
28). FA-binding protein of plasma membrane (FABPpm) (14), fatty acid
translocase (FAT) (15), fatty acid transport protein (FATP) (16), and
two other putative proteins (17, 18) have been identified as FA
transporters in several tissues. In Escherichia coli, a
peripheral membrane FA acceptor and a distinct transmembrane
transporter, which composes the facilitated FA transport system, have
been well characterized (29, 30). The features of FABPpm, FAT, and FATP
as FA transporters have been investigated extensively in adipocytes,
hepatocytes, and myocytes (8, 10, 11, 16).
Preliminary studies carried out in our laboratory have shown that
inhibition of ADRP expression in adipocyte precursors by ADRP antisense
oligodeoxynucleotides resulted in inhibition of lipid droplet
accumulation with a concomitant decrease in triglyceride accumulation.2
The studies presented in this report provide for the first time a
function for ADRP as a protein involved in carrier-mediated FA influx
into cells. These results are important since ADRP sequence did not
provide any clue as to its functional properties and since ADRP does
not have sequence similarity with the well known small molecular weight
fatty acid-binding proteins (21) and with other fatty acid transporters
recently cloned from adipocytes (15-16). It is presently unclear
whether in the adipocytes ADRP acts solely as a mediator of FA uptake,
whether it is involved in the transport systems in cooperation with
other cellular proteins also involved in lipid metabolism, or whether
it has some other regulatory role not directly involved in transport systems.
Hormonal stimulation of ADRP expression in adipocyte precursors shows
that it is stimulated by cyclooxygenase inhibitors (24) and also by
long chain FA.3 Regulation of
gene activities by substrate is common in mammalian cells. The
expression of the glucose transporter (GLUT1) is modulated by glucose
deprivation (31). Enzymes involved in glucose metabolism are also
regulated by glucose (32, 33). It has been shown that the transcription
of the gene coding for hydroxymethylglutaryl-CoA reductase is modulated
by cholesterol (34). Expression of the fatty acid-binding protein gene
aP2 is stimulated by fatty acids (35). Thus, the fact that a
protein involved in FA transport and/or metabolism is modulated by FA
could provide a regulatory mechanism to control FA transport and/or metabolism.
In conclusion, the present investigation provides a function for ADRP,
identifies a new protein involved in fatty acid uptake, and suggests
that ADRP could play a role in regulating lipid accumulation in
adipocytes by increasing FA uptake. Adipocytes have the ability to
uptake or synthesize fatty acids for esterification into
triacylglycerol and to hydrolyze these triglycerides in response to
lipolytic stimuli. Based on the results presented here and on the
localization of ADRP during adipose differentiation, it is then
tempting to speculate that ADRP may serve as a "shuttling" protein
of lipid substrate to the lipid droplets. Additional studies are
underway to investigate this hypothesis and to understand structurally the mechanism by which ADRP may act as a FA transport-associated protein.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol and bromphenol blue (all chemicals were from Bio-Rad). The cell lysate
was then sonicated at 40%, 20-watt output for 10 s using a Vibra
Cell sonicator (Sonics & Materials Inc., Danbury, CT), and centrifuged
at 10,000 × g for 10 min, and the supernatant was
collected. The protein concentration of cell lysate was measured by
using micro-BCA protein assay reagent kit (Pierce). After adding 1/10
volume of 10× loading dye (50% 2-Me, 1% bromphenol blue), equal
amounts of protein (20 µg) from ADRP- and empty vector-transfected COS-7 cells were analyzed by polyacrylamide gel electrophoresis in the
presence of sodium dodecyl sulfate on a 10% gel followed by Western
blot analysis to measure ADRP expression in both cell types. Conditions
for SDS-PAGE and Western blot analysis were similar to the ones
described previously to measure ADRP expression in adipocytes (24).
transfection
efficiency). Non-transfected control cells were treated similarly to
transfected cells but without adding plasmid DNA of vectors used for
transfection. The value for the remaining FA uptake contributed by the
transfected cells was obtained by subtracting the nontransfected cells
uptake from the total uptake measured experimentally. Then the
remaining FA uptake of transfected cells was normalized to the
transfection efficiency. The final amount of uptake was expressed as
pmol per 105 transfected cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ADRP protein expression in COS-7 cells
transiently transfected with ADRP cDNA. COS-7 cells cultivated
in DME-F12 medium supplemented with 10% FBS were transiently
transfected either with plasmid DNA of pcDNA3-ADRP expression
vector or with pcDNA3 empty vector as control by the DEAE-dextran
method as described under "Experimental Procedures." Cell lysates
were collected 48 h after transfection. 20 µg of proteins from
either lysate of transfected cells were examined for ADRP expression by
Western blot analysis using anti-mouse ADRP polyclonal antibody.
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Fig. 2.
Immunofluorescent localization of ADRP in
transfected COS-7 cells. ADRP-transfected COS-7 cells (top
panel) and empty vector-transfected COS-7 cells (bottom
panel) were cultivated in microscope chamber slides. 48 h
after transfection, the cells were fixed with 2% paraformaldehyde in
PBS and 0.2% Triton X-100 followed by incubation with anti-ADRP
antibody and FITC-conjugated secondary antibody as described under
"Experimental Procedures." Slides were observed under a
fluorescence microscope equipped with a camera.
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Fig. 3.
Time course of oleic acid uptake by
ADRP-transfected COS-7 cells. ADRP (closed squares) and
control empty vector (open squares)-transfected COS-7 cells
were harvested 48 h after transfection. A suspension of 5 × 105 cells/ml in PBS was prepared and incubated at 37 °C
with a 4:1 molar ratio of [3H]oleate/BSA containing 2.30 µM unbound oleic acid. At the indicated times, aliquots
were treated with phloretin and filtered on glass fiber filters as
described under "Experimental Procedures." Values were mean ± S.D. of six replicates. Results were normalized for transfection
efficiency as described under "Experimental Procedures."
A, 0-2-min period; B, 0-30-min period.
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Fig. 4.
[3H]Oleic acid uptake by ADRP-
and empty vector-transfected COS-7 cells as a function of unbound oleic
acid concentration. ADRP- (closed squares) and control
empty vector (open squares)-transfected COS-7 cells were
harvested 48 h after transfection. A suspension of 5 × 105 cells/ml in PBS was incubated at 37 °C for 15 s
with various ratios of [3H]oleate/BSA at a total of 20 µM oleic acid corresponding to a fatty acid/BSA ratio
from 0.5 to 4 and unbound fatty acid from 0.043 µM to
2.30 µM. Results were normalized for transfection
efficiency as described under "Experimental Procedures" and were
expressed as the mean ± S.D. of six replicates.
Uptake of saturated and unsaturated fatty acids with various carbon
chain lengths
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Fig. 5.
Time course of glucose uptake in ADRP and
empty vector-transfected COS-7 cells. ADRP- (closed
squares) and control empty vector (open
squares)-transfected cells were kept at 37 °C for 3 h in
serum-free DME and then switched to KRP buffer for a 2-h stabilization
period. Uptake of 2-[14C]deoxy-glucose was measured at
37 °C by adding 200 µM
2-[14C]deoxyglucose into KRP buffer for various times.
Data for each treatment were mean ± S.D. of six replicates.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Bernadette Condon and Hong Ye for establishing the transfection conditions and Dr. Jun Hayashi for help with the immunofluorescence experiments.
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FOOTNOTES |
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* This work was supported in part by Grants RO1 DK 51463 from the National Institutes of Health and Grant 194174 from the Juvenile Diabetes Foundation International.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.
Current address: Dept. of Central Nervous System and
Cardiovascular Research, Schering-Plough Research Institute, 2015 Galloping Hill Rd., Kenilworth, NJ 07033.
§ To whom correspondence should be addressed: Dept. of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N. Pine St., Baltimore, MD 21201-1180. Tel.: 410-706-6639; Fax: 410-706-0346; E-mail: gserrero{at}pharmacy.ab.umd.edu.
2 B. Condon and G. Serrero, manuscript in preparation.
3 J. Gao, H. Ye, and G. Serrero, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: FA, fatty acids; ADRP, adipose differentiation related protein; BSA, fatty acid-free bovine serum albumin; DME-F12, 1 to 1 mixture of Dulbecco's modified Eagle's and Ham's F12 media; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; KRP, Krebs-Ringer phosphate buffer; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; FBS, fetal bovine serum; FAT, fatty acid translocase; FATP, fatty acid transport protein; FABPpm, plasma membrane fatty acid-binding protein.
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