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INTRODUCTION |
Because of its ability to store extra energy as triacylglycerol
(lipogenesis) and to release fatty acids and glycerol (lipolysis), adipose tissue plays a crucial role in energy balance. In obesity, excessive accumulation of triacylglycerol in adipocytes (hypertrophy) results from an alteration in the balance between lipogenic and/or lipolytic activities of the adipocytes.
It is now recognized that, beside their involvement in lipid
homeostasis, adipocytes also produce and secrete numerous factors. Among them are endocrine peptides (leptin, adiponectin,
angiotensinogen, etc.) which may play an important role in the
development of morbid complications of obesity such as cardiovascular
diseases, hypertension, diabetes, and cancer. Other adipocyte-secreted
factors (tumor necrosis factor, fatty acids, eicosanoids,
lysophosphatidic acid, etc.) are produced locally and may influence
adipose tissue development and/or metabolism by exerting
autocrine/paracrine effects on the different cells composing adipose
tissue (adipocytes, preadipocytes, and endothelial cells) (1-3). Of
particular interest is the ability of some adipocyte-secreted factors
to exert a paracrine control on preadipocyte proliferation and
differentiation, cellular processes leading to the recruitment of new
fat cells in adipose tissue (adipogenesis) and a further increase in
adipose tissue mass (3, 4).
Our group has demonstrated that lysophosphatidic acid
(LPA)1 is released from
adipocytes in vitro and is present in vivo in the
extracellular fluid of adipose tissue collected by microdialysis (5).
In parallel, LPA is able to activate preadipocyte motility and
proliferation by interacting preferentially with the LPA1 receptor
(LPA1-R) (6). Therefore LPA may participate to the paracrine control of adipogenesis.
LPA is a bioactive phospholipid regulating a wide range of cellular
responses (proliferation, survival, motility, ion flux, and secretion)
through the activation of the G-protein-coupled receptors LPA1-, LPA2-,
and LPA3-R (7, 8). Bioactive LPA was initially found in culture serum
(9) and was further detected in other biological fluids such as plasma
(10, 11), ascitic fluid (12), follicular fluid (13), aqueous humor
(14), and the extracellular fluid of adipose tissue (5). Whereas serum LPA mainly originates from aggregating platelets (15, 16), the precise
cellular origin of LPA in other biological fluids still remains unclear.
We recently showed that, in parallel to LPA, adipocytes also release a
LPA-synthesizing activity that was characterized as a lysophospholipase
D activity (LPLDact), catalyzing transformation of
lysophosphatidylcholine into LPA (17). Adipocyte LPL Dact is
soluble and sensitive to cobalt ions (17). In addition, adipocyte lysophospholipase D activity is insensitive to primary alcohol, suggesting that it could be catalyzed by a non-conventional
phospholipase D that remains to be identified.
In the present study, adipocyte LPLD activity was purified and
identified as the type II ecto-nucleotide pyrophosphate
phosphodiesterase autotaxin (ATX). ATX was found to be expressed by and
released from adipocytes, activates preadipocyte proliferation, and its expression was strongly up-regulated during adipocyte differentiation and in a model of genetic obesity. Because of its LPLDact,
adipocyte-ATX leads to LPA synthesis and could be involved in the
control of adipose tissue development as well as in obesity-associated pathologies.
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EXPERIMENTAL PROCEDURES |
Culture and Transfection Studies--
The mouse preadipose cell
line 3T3F442A (18) was grown and differentiated as described previously
(19). Briefly, cells were grown to confluence (day 0 of
differentiation) in DMEM supplemented with 10% donor calf serum and
then shifted in a differentiating medium consisting in DMEM
supplemented with 10% fetal calf serum plus 50 nM insulin.
In these culture conditions, quiescent preadipocytes differentiate into
functional adipocytes. COS-7 monkey cells (American Type Culture
Collection) were grown in DMEM supplemented with 10% fetal calf serum
and transfected using DEAE-dextran as reported previously (20).
Preparation of Conditioned Media--
3T3F442A cells or COS-7
cells were washed twice with phosphate-buffered saline to remove serum
and incubated (5 ml for a 10-cm diameter plate; 1 ml for a 3-cm
diameter plate) in serum-free DMEM supplemented (for LPA release) or
not (for LPLDact release) with 1% free fatty acid BSA at 37 °C in a
humidified atmosphere containing 7% CO2. Conditioned media
were also prepared from adipose tissue by incubating 300-500 mg of
finely cut out subcutaneous adipose tissue from db/+ or
db/db mice in 3 ml of serum-free DMEM to measure
released LPLDact. After various time of incubation (0-5 h for
measurement of LPLD activity; 7 h for quantification of LPA and
lysophosphatidylcholine (LPC)), conditioned medium (CM) was separated
from the cells or tissue, centrifuged to eliminate cell debris, and
stored at
20 °C before measurement of LPLDact or quantification of
LPA. In some experiments, conditioned media were concentrated (about
50-fold) using an Amicon Ultra 10,000 (Millipore). After adjustment of
the protein concentration, concentrated conditioned media (CCM) were
aliquoted and stored at
20 °C before use.
Measurement of Lysophospholipase D Activity--
LPLDact was
measured by conversion of radiolabeled LPC into radiolabeled LPA as
described previously (21) with minor modifications. A solution of
[14C]palmitoyl-lysophosphatidylcholine (PerkinElmer Life
Sciences; 55.8 mCi/mmol) at 0.0025 µCi/µl in DMEM
supplemented with 1% free fatty acid BSA was first prepared, and 20 µl of this solution was incubated with 500 µl of thawed CM plus 1 µl of sodium orthovanadate (0.5 mM) for 90 min at
37 °C. At the end of the incubation period, phospholipids were
extracted with 500 µl of 1-butanol, evaporated, spotted on a silica
gel 60 TLC glass plate (Merck), and separated using
CHCl3/MeOH/NH4OH (60:35:8) as the migration
solvent. The plate was autoradiographed overnight at
80 °C using a
Biomax-MS film (Kodak) to localize radiolabeled LPA spots, which were
scraped and counted with 3 ml of scintillation mixture.
Purification of Lysophospholipase D Activity--
All the
procedures were performed at 0-4 °C. One hundred twenty milliliters
of 3T3F442A adipocyte-conditioned medium containing LPLDact was
centrifuged for 1 h at 100,000 × g to remove cell debris and concentrated 8-fold on polyethylene glycol 20,000 (PEG 20,000). After overnight dialysis against 10 liters of purification buffer (PB) using Spectra-Por 1.7 ml/cm tubing (Pierce Chemicals, Interchim) to remove salts and small proteins, 16 ml of the
concentrated medium was applied (60 ml/h) onto a heparin-Sepharose
column (CL+6B, Amersham Biosciences; 15-ml packed volume) equilibrated
with 100 ml of PB. The column was washed with 150 ml of PB and eluted
with 125 ml of PB containing 0.5 M NaCl. The elution
fraction containing LPLDact was dialyzed overnight against 10 liters of
PB using Spectra-Por 1.7 ml/cm tubing (Pierce Chemicals, Interchim) to
remove NaCl and concentrated on PEG 20,000 to obtain a volume of 40 ml.
This concentrate was applied (60 ml/h) on a phosphocellulose column (P11, Whatman, 15 ml packed volume) equilibrated with 100 ml PB. The
column was washed with 150 ml of PB and eluted with 125 ml of PB
containing 0.5 M NaCl. The elution fraction containing
LPLDact was dialyzed overnight against 10 liters of purification buffer using Spectra-Por 1.7 ml/cm tubing (Pierce Chemicals, Interchim) to
remove NaCl, and concentrated on PEG 20,000 to obtain a final volume of
5 ml. This concentrate was separated (30 ml/h) over a gel filtration
column (HiLoad 16/60, Superdex 200 PrepGrade, Amersham Biosciences) on
an Amersham Biosciences FPLC system previously equilibrated with PB.
The column was eluted with the same buffer. Aliquots of the collected
fractions were assayed for LPLDact. All the fractions containing
LPLDact were pooled and concentrated 10 times on dialysis tubing (0.5 ml/cm) in PEG 20,000. The protein concentration was determined by the
Bradford assay (Protassay, Bio-Rad) with bovine serum albumin as standard.
SDS-PAGE Separation--
SDS-PAGE (4-20%) was performed
according to Laemmli (22) followed by SYPRO Ruby or colloidal blue
staining. After the addition of sample buffer (Novex, Invitrogen),
concentrated fractions from gel filtration were boiled at 100 °C for
5 min. Electrophoretic separation of proteins was carried out on a 1-mm
thick 8 × 6-cm gel 10% acrylamide. A 40-µg portion of total
protein in sample buffer was loaded into a 4-mm well of the gel and
separated at 40 mA. A total of 30 µg of standards (Mark12,
Invitrogen) migrated in a neighboring lane. After coloration with
colloidal Coomassie Blue (Biosafe, Bio-Rad), the 110-kDa protein was
cut, reduced, and alkylated using dithiothreitol and iodoacetamide,
respectively, and subjected to digestion with trypsin overnight
following the protocol of Shevchenko et al. (23). Gel pieces
were successively washed with ammonium bicarbonate and dehydrated with
acetonitrile. After drying in a Speedvac (Heto), the pieces were
incubated at 56 °C with 10 mM dithiothreitol and then at
room temperature with 55 mM iodoacetamide. After successive
washing steps with ammonium bicarbonate and acetonitrile, the gel
pieces were completely dried in the Speedvac. Trypsin solution
(Promega) was added, and digestion was performed overnight at 37 °C.
Peptide digests were extracted successively with acetonitrile and 5%
formic acid. Before analysis, the peptides were dried in the Speedvac,
diluted in 5% formic acid, and desalted on Poros R-2 resin. Peptides
were eluted with 2 µl of 60% methanol/1% formic acid directly into
nanospray capillary needles (Micromass, Manchester, UK).
Tandem Mass Spectrometry--
Peptides were analyzed
on a Q-TOF 2 mass spectrometer (Micromass). MS acquisitions were
performed within the mass range of 550-1300 m/z
and MS/MS within 50-2000 m/z.
Data Base Searching and Sequence Analysis--
Amino acid
sequences were used to search Swiss-Prot and TREMBL with BLAST
interface (United States National Center for Biotechnology Information). Partial amino acid sequences (sequence TAG) were used to
search the data bases with PeptideSearch interface (European Molecular
Biology Laboratory, Heidelberg, Germany). Hits were confirmed by
performing a theoretical trypsin digestion with GPMAW (Lighthouse data,
Odense, Denmark) and comparing the resulting peptide mass and sequence
to those obtained experimentally.
Real Time RT-PCR Analysis--
Total RNAs were isolated using
the RNeasy mini kit (Qiagen). Total RNAs (1 µg) were reverse
transcribed for 60 min at 37 °C using Superscript II reverse
transcriptase (Invitrogen) in the presence of a random hexamer. A minus
RT reaction was performed in parallel to ensure the absence of genomic
DNA contamination. Real time RT-PCR was performed starting with 25 ng
cDNA and 900 nM (human ATX and mouse ATX) or 900 nM (mouse ap2) concentration of both sense and antisense
primers in a final volume of 25 µl using the SYBR green TaqMan
Universal PCR Master Mix (Applied Biosystems). Fluorescence was
monitored and analyzed in a GeneAmp 5700 detection system instrument
(Applied Biosystems). Analysis of the 18 S ribosomal RNA was performed
in parallel using the ribosomal RNA control Taqman Assay Kit (Applied
Biosystem) to normalize gene expression. Results are expressed as
2(Ct18S
Ctgene), where Ct corresponds to the
number of cycles needed to generate a fluorescent signal above a
predefined threshold.
Oligonucleotide primers were designed using the Primer Express software
(PerkinElmer Life Sciences). Oligonucleotides used were as follows:
mouse aP2, sense 5'-TTCGATGAAATCACCGCAGA-3' and antisense
5'-GGTCGACTTTCCATCCCACTT-3'; mouse ATX, sense
5'-GACCCTAAAGCCATTATTGCTAA-3' and antisense 5'-GGGAAGGTGCTGTTTCATGT-3';
mouse HSL, sense 5'-GGCTTACTGGGCACAGATACCT-3' and antisense
5'-CTGAAGGCTCTGAGTTGCTCAA-3'.
Cloning of Autotaxin from 3T3F442A Adipocytes--
Total RNA
from 15 days post-confluent 3T3F442A adipocytes was reverse transcribed
using random hexamers and Superscript II reverse transcriptase
(Invitrogen). First strand cDNA (corresponding to 1 µg of total
RNA) was amplified using a program consisting of 30 cycles of 95 °C
for 30 s, 55 °C for 30 s, and 72 °C for 3 min with a
pre- and post-incubation of 95 °C for 2 min and 72 for 10 min,
respectively. PCR amplification utilized oligonucleotide primers
based on the GenBankTM entry for mouse autotaxin
(forward 59-64, reverse 2630-2651, accession number BC003264). The
PCR fragment was isolated and ligated into pcDNA3 (Invitrogen)
downstream of the FLAG peptide sequence (Sigma). The recombinant
plasmid, designated pcDNA-mATX-FLAG, was sequenced on both strands
by automated sequencing.
Quantification of LPA and LPC--
LPA was butanol-extracted
from conditioned medium and quantified using a radioenzymatic assay as
described previously (11). The amount of lysophosphatidylcholine
present in conditioned media was determined as described previously
(17) after 30 min of treatment with bacterial phospholipase D followed
by LPA quantification.
Housing and Treatment of Animals--
Male C57BL/KsJ
db/db and db/+ mice (Jackson
Laboratory) were housed in an animal room maintained at 22 °C on a
12:12 h light/dark cycle with ad libitum access to food and
water. Animals were handled in accordance with the principles and
guidelines established by the National Institute of Medical Research
(INSERM). On the day of sacrifice, mouse blood was collected on heparin
and glucose was immediately measured with a glucose meter (Glucometer
4; Bayer Diagnosis, Puteaux, France). Perigonadic and inguinal white
adipose tissues were removed, and adipocytes were isolated as described above and immediately processed for RNA extraction using an RNeasy mini
kit (Qiagen). The other tissues (interscapular brown adipose tissue,
brain, kidney, and liver) were removed and snap frozen in liquid
nitrogen before RNA isolation using RNA STAT kit (AMS Biotechnology
Ltd., Oxon, UK).
Isolation and Culture of Preadipocytes and Adipocytes from Mouse
Adipose Tissue--
Adipose tissue was dissected from mice and
digested in 5 ml of DMEM supplemented with 1 mg/ml collagenase, 1%
BSA, and 2 µg/ml gentamycin for 45-60 min at 37 °C under shaking.
Digestion was followed by filtration through a 100-µm screen and
centrifugation at 800 × g for 10 min at room
temperature. This phase allowed the separation of floating adipocytes
from the pellet containing the stromal-vascular fraction.
Adipocytes were washed twice in DMEM and further processed for RNA
extraction using the RNeasy mini kit (Qiagen). The stromal-vascular
pellet was washed twice in DMEM and resuspended in 1 ml of lysis buffer
(7 mM Tris, 0.83% NH4Cl, pH 7.6) for 3 min to
get rid of erythrocytes and then seeded in 12-well plates (150,000 cells per plate) in DMEM/Ham's F-12 medium supplemented with 10%
fetal calf serum. After overnight culture, the medium was replaced by a
serum-free differentiating medium consisting of DMEM/Ham's F12
supplemented with transferrin (10 µg/ml), biotin (33 µM), insulin (66 nM), T3 (1 nM),
and pantothenate (17 µM). That time point is considered
as day 0. In these conditions, adipocyte differentiation occurs a few
days later (24). Total RNAs were prepared at different time points
after induction of the differentiation using the RNeasy mini kit (Qiagen).
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RESULTS |
Co-purification of Adipocyte Lysophospholipase D Activity with
Autotaxin--
To identify the enzyme responsible for LPLDact present
in adipocyte CM (17), its purification from 3T3F442A adipocyte CM was
undertaken. LPLDact hydrolyzes the phosphocholine bound in LPC to
generate LPA and free choline. In the present work, LPLDact was
measured using [14C]lysopalmitoyl-phosphatidylcholine as
substrate, followed by TLC separation of synthesized
[14C]LPA (see "Experimental Procedures"). The initial
release of LPLDact in adipocyte CM was observed in an Hepes buffer
incubation medium (17). As shown in Fig.
1, a stronger (3.3-fold after 5 h of
incubation) LPLDact was measured when using DMEM as the incubation
medium. In both incubation media, LPLDact was detectable after a 30-min
incubation and reached a maximum after 5 h. Based upon these
results, purification of LPLDact was undertaken from 3T3F442A adipocyte
CM prepared after 5 h of incubation in DMEM.

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Fig. 1.
Release of LPLDact from 3T3F442A
adipocytes. Differentiated 3T3F442A adipocytes were incubated in
serum-free DMEM, and LPLDact present in the culture medium after
various times of incubation (0-5 h) was measured as described under
"Experimental Procedures" using
[14C]palmitoyl-lysophosphatidylcholine
([14C]LPC) as the substrate and
[14C]lysophosphatidic acid
([14C]LPA) as the product. Representative of
three separated experiments. HB, Hepes buffer.
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After 10-fold concentration, CM was first applied onto a heparin
affinity chromatography column (Fig. 2,
section 1), washed and eluted with 0.5 M NaCl at once (lane e), and
concentrated again. This allowed the recovery of LPLDact, but no
specific band was identified by SDS-PAGE (lane
f). As a second purification step, concentrated fraction
eluted from the heparin column was applied onto a phosphocellulose
column (Fig. 2), eluted at 0.2 M NaCl (lane
i), concentrated 25 times (lane j),
applied on a gel filtration column (Fig. 2, section
3), and eluted (Fig. 2, section 3,
fractions 52 to 82). LPLDact was
detected in fractions 68 to 76. SDS-PAGE analysis revealed the presence
of a band (molecular mass between 116 and 97 kDa) in the
fractions exhibiting maximal LPLD activity (fractions 70, 72, and 74).
Fraction 70 was concentrated 100 times, separated by 4-20%
SDS-polyacrylamide gel electrophoresis, stained with Coomassie Blue,
and cut out from the gel. Tryptic cleavage followed by tandem mass
spectrometry of the specific band of fraction 70 led to the generation
of 27 peptides exhibiting 100% homology (Table
I) with mouse ecto-nucleotide
pyrophosphatase phosphodiesterase (ENPP2), also called autotaxin (25,
26). These results showed that adipocyte-LPLDact was co-purified with ATX.

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Fig. 2.
Purification of LPLDact from 3T3FF42A
adipocyte conditioned medium. Conditioned media from 3T3F442A
adipocytes (lane a) was concentrated 10 times on PEG 20,000 (lane b) and applied on a heparin agarose column
(section 1), washed with Tris buffer
(lanes c and d), eluted with 0.5 M NaCl (lane e), concentrated 10 times on polyethylene glycol (lane f) and washed with Tris
buffer (lanes g, h). Lane f
was applied on a phosphocellulose agarose column (section
2), eluted in NaCl gradient at 0.2 M NaCl
(lane i), concentrated 25 times (lane j) on
polyethylene glycol, applied on a gel filtration column (3), and
fractionated in Tris buffer into fractions
52-82 (fractions 52-56
are flow-through fractions). The protein content of each fraction was
analyzed on SDS-PAGE (A) in parallel to the measurement of
LPLDact (B). Wm, weight marker. Arrow
corresponds to the band that was analyzed by mass spectrometry (Table
I) in fractions 70 and 72 (*).
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Table I
Peptides and mouse autotoxin sequence
Sequence of the peptides derived from the 97-116 kDa protein
co-purified with adipocyte LPLDact. The specific band of 97-116 kDa in
fraction 70 of Fig. 2 was submitted to tryptic cleavage and analyzed by
tandem mass spectrometry, allowing the identification of 27 peptides
exhibiting 100 % homology with mouse ATX. Underlined and bold
characters indicate the peptides identified by mass spectrometry. indicates potential proteolytic cleave site.
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Cloning of 3T3F442A Adipocyte Autotaxin--
A unique cDNA
encoding ATX was cloned from 3T3F442A adipocytes and subcloned in a
pcDNA-FLAG vector. This cDNA exhibited 100% identity with
previously identified mouse-ATX cDNA present in the
GenBankTM data base (BC003264, NM_015744, AF123542) (Table
I). The corresponding protein is composed of 863 amino acids (molecular mass of ~100 kDa) which, by analogy with human, corresponds to the
previously described
-form (27-29). In human ATX the site of
cleavage has been located between Ser-48 and Asp-49
in the amino acid sequence EGPPTVLS
DSPWTN, which is entirely
conserved in mouse (BC003264, NM_015744, AF123542, and herein the 3T3F442A-cDNA sequence). Based upon mouse ATX sequence, the
expected cleavage site of mouse ATX should be between Ser-47 and
Asp-48. This should lead to a secreted protein of 816 amino
acids (molecular mass of ~94 kDa). Purification of
LPLD-activity from 3T3F442A adipocytes led to
identification of a protein with a molecular mass between 97 and 116 kDa (Fig. 1). This mass is slightly higher than that calculated for the
secreted form of mouse-ATX. Such a discrepancy could be attributed to
the presence, in the mouse sequence, of at least four glycosylation
sites (Net Nglyc 1.0 prediction analysis) that would increase the
apparent molecular weight of the secreted protein.
ATX Behaves as a Secreted Lysophospholipase D--
To determine
whether ATX indeed behaved as a secreted lysophospholipase D,
3T3F442A adipocyte ATX cDNA was transiently transfected in COS-7
cells. After transfection with empty pcDNA vector, COS-7 cells did
not release LPLDact in culture medium (Fig.
3). Conversely, transfection with
pcDNA-mATX-FLAG vector led to the release of LPLDact in the culture
medium (Fig. 3). Similar results were obtained when transfecting COS-7
cells with a pcDNA vector expressing human ATX (30, 31).

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Fig. 3.
Recombinant expression of ATX in COS-7
cells. COS-7 cells were transiently transfected with empty
pcDNA (lane 2), pcDNA-mATX-FLAG (lane 3),
or pcDNA-human ATX (lane 4). Twenty-four hours after
transfection, COS-7 cells were incubated for 5 h in serum-free
DMEM before measuring the LPLDact in the culture medium. Lane
1 corresponds to LPLDact measured in 3T3F442A adipocyte
conditioned medium as described in Fig. 1. The figure is representative
of at least three separate experiments.
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Up-regulation of ATX with Adipocyte Differentiation--
We
previously observed that adipocytes release more LPLDact than
preadipocytes (17). We therefore tested the existence of an adipocyte
differentiation-dependent regulation of ATX. Adipocyte differentiation of 3T3F442A cells was generated by treating confluent preadipocytes (day 0) with a differentiation medium (see
"Experimental Procedures"). This treatment led to a rapid (4 days
after confluence) emergence of specific genetic markers of
differentiation such as adipocyte fatty acid-binding protein (aP2) and
hormone sensitive lipase (HSL) mRNAs (Fig.
4B), followed (7 days after
confluence) by the accumulation of triglycerides (Fig. 4A),
the final adipocyte phenotype. Whereas aP2 and HSL mRNA levels
reached a plateau after 10 days (Fig. 4B), triglyceride
content continued to increase regularly thereafter (Fig.
4A).

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Fig. 4.
Up-regulation of ATX expression with adipose
differentiation of 3T3F442A preadipocytes. Confluent 3T3F442A
preadipocytes (day 0) were induced to differentiation as described
under "Experimental Procedures." At different times after
induction, cells were either collected to measure triglyceride content
(µg/mg cell protein) (A) and mRNA expression (/18 S
RNA × 104) (B) or placed in a serum-free
medium to measure LPLDact (pmol of LPA/mg of cell protein)
(C) and the amount of LPA and LPC (pmol/mg cell protein)
(D) in the culture medium as described under "Experimental
Procedures." Values are means ± S.E. of three
experiments.
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Under these conditions, ATX mRNAs were detected 7 days after
confluence, and their level increased regularly thereafter (Fig. 4B). Interestingly, the emergence of ATX mRNAs was
delayed by 3-4 days as compared with that of aP2 and HSL mRNAs.
Up-regulation of ATX mRNA was tightly associated with the increase
in LPLDact (Fig. 4C) and LPA concentration (Fig.
4D) in the culture medium. In parallel, the culture medium
also contained LPC (the substrate for LPLDact), which was present at
higher concentration than LPA but which was not significantly modified
during adipocyte differentiation (Fig. 4D).
Up-regulation of ATX was also observed after adipocyte differentiation
of the primary culture of preadipocytes. As described previously (24),
the preadipocytes present in the stroma vascular fraction of adipose
tissue can be differentiated into adipocytes when cultured for 7-10
days in the presence of a differentiating mixture (see "Experimental
Procedures"). As shown in Fig. 5, after 10 days the expression of ATX mRNA was increased 10-fold as
compared with the first day of culture. These results clearly show the existence of a regulation of ATX with adipocyte differentiation. ATX
mRNA was also present in mature adipocytes isolated from mouse adipose tissue, and its expression was lower than in 10-day-old differentiated adipocytes (Fig. 5).

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Fig. 5.
Differentiation-dependent
regulation of ATX in primary mouse preadipocytes. Preadipocytes
were isolated from the stroma vascular fraction of the adipose tissue
of a 4-week-old C57BL6J mouse as described under "Experimental
Procedures." ATX mRNA expression was measured after overnight
attachment (day 0) and after 10 days of culture (day 10) in a
differentiating medium (see "Experimental Procedures"). ATX
mRNA expression was also measured in mature adipocytes freshly
isolated from mouse adipose tissue. Values are means ± S.E. of
three separate experiments.
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Recombinant ATX Allowed the Release of LPA by
Preadipocytes--
Our group has demonstrated previously that LPA is
able to increase preadipocyte proliferation (5, 6). Conversely to adipocytes, preadipocytes did not release LPLDact (see Fig.
4C) or LPA (Fig. 4D) but released LPC (see Fig.
4D), the substrate of the LPLDact of ATX. Therefore, we tested
whether ATX could exert an LPA-dependent increase in
preadipocyte proliferation.
To test this hypothesis, conditioned media from COS-7 cells transfected
with empty pcDNA or pcDNA-mATX-FLAG were concentrated and used
to treat 3T3F442A preadipocytes. As shown in Fig.
6A, 5 h of treatment with
concentrated conditioned medium from pcDNA-mATX-FLAG transfected
COS-7 cells (ATX-CCM) led to a dilution-dependent increase
in LPA concentration in 3T3F442A preadipocyte culture medium. LPA was
significantly detected with a 10 µl/ml dilution and reached a maximum
at 100 µl/ml with a range of LPA concentration varying from 13 to 130 nM. Conversely no significant concentration LPA was
generated after treatment with concentrated conditioned medium from
empty pcDNA transfected COS-7 cells (sham CCM) (Fig. 6A).

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Fig. 6.
Influence of recombinant ATX on
preadipocytes. A, serum starved 3T3F442A preadipocytes
were treated with decreasing dilutions of CCM from COS-7 cells
transiently transfected with either empty pcDNA
(sham-CCM) or pcDNA-mATX-FLAG (ATX-CCM) in
DMEM supplemented with 1% BSA. After 5 h of treatment, LPA was
quantified in preadipocyte culture medium using a radioenzymatic assay.
B, serum-starved 3T3F442A preadipocytes were treated with 50 µl/ml sham-CCM or ATX-CCM in DMEM supplemented with 1% BSA.
Treatments were performed in the presence or absence of 0.1 units/ml
phospholipase B (PLB). After 72 h of treatment,
preadipocyte number was measured using a cell counter.
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To test the influence of recombinant ATX on preadipocyte proliferation,
3T3F442A preadipocytes were exposed to 50 µl/ml sham CCM or ATX CCM,
and the cell number was determined after 72 h of treatment. As
shown in Fig. 6B, sham CCM led to an increase in cell
number, suggesting the presence of a growth factor different from ATX
in sham CCM. Nevertheless, treatment with ATX CCM led to an additional
increase in cell number (Fig. 6B), which was completely
abolished by co-treatment phospholipase B, a lysophospholipase that, as
we and others have demonstrated previously, hydrolyzes and inactivates
LPA (5, 6, 9, 32). These results strongly supported the idea that
recombinant ATX was able to increase preadipocyte proliferation
according to an LPA-dependent mechanism.
Up-regulation of Adipocyte ATX in db/db Mice--
To
test the existence of a regulation of ATX expression with obesity,
adipocyte ATX expression was measured in genetically obese diabetic
mice (db/db). As expected, 12-week-old
db/db mice were obese as attested by higher body
weight (1.7-fold) and higher adipose tissue weight (9.5 and 10-fold in
perigonadic and inguinal depot, respectively) when compared with their
lean siblings, db/+ mice (Table
II). As was also expected,
db/db mice were diabetic as attested by their
higher (3.6-fold) non-fasting glycemia when compared with
db/+ mice (Table II). As shown in Fig.
7A, ATX mRNA expression
was significantly higher in isolated adipocytes from db/db (3.8 and 4.8-fold in perigonadic and
inguinal adipocytes, respectively) as compared with db/+. A
significant increase in ATX mRNA expression was also observed in
interscapular brown adipose tissue (2.4-fold) and kidney (2.2-fold)
(Fig. 7B). Conversely, no changes were observed in ATX
mRNA expression in liver and brain (Fig. 7B). In a
separate experiment, small explants of subcutaneous adipose tissue from
db/+ and db/db mice were incubated in
serum-free DMEM, and released LPLDact was measured. After 5 h of
incubation, LPLDact released in the incubation medium was 18.2 ± 2 and 29.3 ± 4 pmol of LPA/mg of protein in db/+
(n = 5) and db/db
(n = 5) adipose tissue explants (p < 0.01). This result showed that up-regulation of ATX mRNA in adipose
tissue of db/db mice was accompanied by up-regulation of its activity. Taken together, the above results showed
that db/db genetic obesity was associated with
up-regulation of ATX expression in some tissues, including adipose
tissue.
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Table II
Weights and glycemia of 3-month-old male db/db (+/ )
and db/db ( / ) mice
Values are means ± S.E. of four animals for each group.
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Fig. 7.
ATX expression in obese diabetic
db/db mice. Total RNA was
extracted from different tissues of lean db/+ and obese
db/db mice. Adipocytes were isolated from
perigonadic (PG) and inguinal (ING) white adipose
tissues (panel A) and whole inguinal white adipose tissue
(WAT), brown adipose tissue (BAT), liver, kidney,
and brain (panel B). ATX mRNA level (/18S
RNA × 104) was measured by real time RT-PCR. Values
are mean ± S.E. of four animals.
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DISCUSSION |
The initial objective of the present study was to identify the
enzyme responsible for LPLDact released in the culture medium of
adipocytes. Our results show the following: (i) that adipocyte LPLDact
is co-purified with ATX; (ii) that ATX is expressed in adipocytes;
(iii) that recombinant expression of ATX in COS-7 cells permits the
release of LPLDact in their culture medium; (iv) that ATX expression
and activity are strongly increased during adipocyte differentiation;
(v) that recombinant ATX is able to increase preadipocyte proliferation
via a LPA-dependent mechanism; and (vi) that adipocyte-ATX
is substantially over-expressed in a genetic model of obesity.
ATX is an ecto-nucleotide pyrophosphatase phosphodiesterase initially
discovered as a motility-stimulating protein in the culture medium of
A2058 melanoma cells (25). ATX was then found to be expressed in
several cell lines and tissues (28). The motility-stimulating action of
ATX is blocked by pretreatment with pertussis toxin, suggesting the
involvement of a G-protein-coupled receptor in its action (31). ATX is
an ecto-protein anchored to the plasma membrane by a short N-terminal
sequence and likely released in the extracellular medium after
proteolytic cleavage. In its extracellular domain, ATX contains a large
phosphodiesterase catalytic site involved in the hydrolysis of
phosphodiester and pyrophosphate bonds present in a classical substrate
such as ATP or ADP (26, 30, 33).
Our results strongly support the idea that ATX is responsible for the
LPLDact released from 3T3F442A adipocytes. LPLDact was indeed
co-purified with a protein exhibiting 100% homology with previously
cloned mouse ATX. In addition, a release of LPLDact in culture medium
was obtained after recombinant expression of mouse ATX cDNA in
COS-7, a cell line that does not normally release LPLDact. Finally, ATX
expression was clearly demonstrated in adipocytes and was tightly
co-regulated with the release of LPLDact and LPA in culture medium
during differentiation of 3T3F442A preadipocytes. Our conclusions are
in agreement with recent publications that appeared during the
completion of the present study, reporting that ATX was responsible for
a LPLDact present in bovine and human sera (34, 35).
ATX expression is strongly up-regulated during adipocyte
differentiation. Terminal differentiation of adipocytes is a complex process that requires the emergence of specific genes such as aP2 and
HSL (analyzed in the present work), which are necessary for the
adipocytes to ensure its specific metabolic functions such as
triglyceride accumulation. When comparing with aP2 and HSL, ATX
expression is rather late during adipose conversion and is more closely
associated with the accumulation of triglycerides. These observations
suggest that ATX expression is a consequence of, rather than a
requirement for, adipocyte differentiation. Further experiments will be
necessary to precisely identify the factor(s) involved in the
regulation of ATX expression with adipocyte differentiation.
ATX expression in adipocytes is substantially increased in genetically
obese diabetic db/db mice. These mice bear a
mutation in the leptin receptor resulting in hyperphagia and the
massive accumulation of adipose tissue associated with type II diabetes and, ultimately, type I diabetes. Up-regulation of adipocyte ATX in
db/db suggests that this protein could either be
associated with obesity or diabetes or both. ATX belongs to the
ecto-nucleotide pyrophosphate phosphodiesterase (ENPP) family. This
family encompasses the membrane glycoprotein plasma cell 1 (PC-1), a
protein with a high structure homology with ATX (36). It is interesting
to note that PC-1 expression was found to be increased in adipose tissue of obese diabetic Zucker fatty rats (37) which, like db/db mice, bear a mutation on the leptin
receptor. Alterations in PC-1 expression have been associated with
alterations in whole body insulin sensitivity as well as insulin
receptor-tyrosine kinase activity (38, 39). Further experiments will be
necessary to identify the factor(s) involved in regulation of ATX in
obesity and/or diabetes.
What could be the role of adipocyte-ATX? In adipose tissue, adipocytes
and preadipocytes are present in the same environment and communicate
with each other via paracrine mediators. As demonstrated in the present
study, ATX catalyzes a LPLDact, which is responsible for the presence
of LPA in adipocyte culture medium. We demonstrated previously that LPA
is present in adipocyte culture medium and can activate
preadipocyte growth via activation of the LPA1 receptor (6). In
contrast to adipocytes, preadipocytes do not release ATX but release
high concentration of LPC (see Fig. 4), the substrate of ATX for LPA
synthesis. It can be speculated that, in intact adipose tissue, one of
the possible roles of adipocyte ATX would be to exert a paracrine
control on preadipocyte growth because of the generation of LPA at the
extracellular face of the preadipocytes in close contact with
adipocytes. This hypothesis is supported by our results showing that
recombinant ATX is able to activate preadipocyte proliferation. In
addition, the blocking effect of phospholipase B strongly suggests that
the action of ATX on preadipocytes is very likely to be mediated by
synthesis of LPA and the activation of the LPA receptor present at the
surface of the preadipocytes. This hypothesis is in agreement with
previous reports showing that ATX activates cell motility and cell
proliferation in several cell lines via a G-protein-coupled receptor
that could be a LPA-receptor (31, 34).
What could be the consequence of the proliferative activity of ATX on
preadipocytes? Preadipocyte proliferation is a key step in adipose
tissue development, because it conditions the number of potential new
adipocytes in the adipose tissue. In most obesity models such as
db/db mice (where we observed an over-expression in adipocyte-ATX), the increase in adipose tissue mass results from an
excess accumulation of triglycerides in existing adipocytes (hypertrophy), followed, above a certain level of hypertrophy, by the
recruitment of new adipocytes (hyperplasia). It could be proposed that
adipocyte ATX plays a role in the recruitment of new adipocytes by
exerting a paracrine control on preadipocyte growth. ATX could
therefore play a role in obesity-associated hyperplasia. This
hypothesis is reinforced by very recent report (40) showing that LPA
behaves as an agonist of the peroxisome proliferator activated receptor
, a crucial transcriptional factor involved in the adipocyte
differentiation program.
Obesity-associated increase in fat mass is also dependent on its
vasculature and therefore requires angiogenesis (41). ATX has
previously been demonstrated to behave as a pro-angiogenic mediator
(42). Adipocyte ATX could then play a role in the paracrine control of
angiogenesis in adipose tissue. Finally, an endocrine action of
adipocyte ATX can also be proposed. As pointed out earlier, ATX has
recently been identified in blood (serum and plasma) (34, 35), but,
despite the presence of ATX mRNA in different tissues (28), the
origin of circulating ATX remains unknown. When comparing it with
other tissues, ATX mRNA appears to be fairly abundant in adipose
tissue. In addition, considering the high mass of adipose tissue in the
body, particularly in obese individuals, its significant contribution
in circulating ATX can be proposed. Future development of transgenic
animals exhibiting altered expression of adipocyte ATX will help to
verify these different hypotheses.
In conclusion, the present work identifies ATX as a new adipocyte
mediator exerting its biological activities via neo-synthesis of the
bioactive phospholipid LPA. Its close association with adipocyte
differentiation and obesity and/or diabetes suggests a possible role of
ATX in the normal or pathological development of adipose tissue.