| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
-Mediated Inhibition and Reversal of Adipocyte Differentiation Is Accompanied by Suppressed Expression of PPAR
without Effects on Pref-1 Expression1
Affymax Research Institute (H.X., J.P.N., J.R.G., G.M.R.), Santa Clara, California 95051; and Department of Molecular Sciences (K.E.K., J.-L.S.), GlaxoWellcome Research and Development, Research Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: Hong Xing, Aurora Biosciences Corp., 11149 North Torrey Pines Road, La Jolla, California 92037. E-mail: XingH{at}aurorabio.com
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(TNF) is a polypeptide hormone with
pleiotropic effects on cellular proliferation and differentiation. To
investigate how TNF inhibits and reverses adipocyte differentiation,
we studied the expression of two factors involved in the adipocyte
differentiation process. Peroxisome proliferator-activated receptor 
(PPAR) is a positive regulator of adipogenesis, whereas preadipocyte
factor 1 (Pref-1) inhibits adipocyte differentiation. The expression
patterns of both PPAR and Pref-1 change during early stages of
adipocyte differentiation. Decreased expression of Pref-1 and increased
expression of PPAR occur 1 day and 2 days, respectively, after
3T3-L1 cells reach confluence. During TNF-mediated inhibition of
adipocyte differentiation, PPAR messenger RNA (mRNA) expression
stays at low levels. In contrast, TNF treatment has no effect on the
normal decrease in Pref-1 gene expression that occurs during
adipogenesis. We observed that certain cytokine and growth factors
[such as TNF, basic fibroblast growth factor, transforming growth
factor ß, and protein kinase C-activating agents plus calcium
ionophore], when added to differentiated adipocytes, cause rapid
down-regulation of PPAR mRNA expression with concomitant decrease in
adipocyte-specific gene expression but fail to increase Pref-1 mRNA
expression. Moreover, addition of TNF to fully differentiated
adipocytes results in the rapid disappearance of PPAR protein
expression and the rapid loss of PPAR DNA-binding activity.
Therefore, Pref-1 seems to function as a nonreversible molecular
checkpoint whose expression is insensitive to TNF-generated signals,
whereas PPAR expression remains sensitive to TNF at all stages of
the adipogenesis program. Our results support the notion that
dedifferentiated adipocytes and preadipocytes are not identical, though
they share many similar morphological and gene expression patterns. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(C/EBP) (11, 12, 13), are also
expressed. Because C/EBP is a widely expressed transcription factor
(14), it is unlikely to be the key factor for induction of the
adipocyte phenotype. In contrast, both peroxisome
proliferator-activated receptor (PPAR) (15, 16, 17, 18) [a member of
the nuclear receptor superfamily (reviewed in Refs. 19, 20, 21)] and
preadipocyte factor 1 (Pref-1) (22, 23) [a novel member of the
epidermal growth factor (EGF)-like family with similarity to the
Notch/Delta transmembrane proteins involved in cell fate
determination (reviewed in Refs. 24, 25)] are highly
tissue-specific proteins. PPAR is found only in adipose tissue (16, 17, 26), whereas Pref-1 is expressed only in preadipocytes and in adult
adrenal tissue (22). The highly restricted tissue distribution and
early changes in messenger RNA (mRNA) expression of PPAR and Pref-1
suggest that they may play essential roles in the differentiation
process. Recently, PPAR was identified as a key regulator of at
least two adipocyte genes, aP2 and PEPCK (17, 27). PPAR serves as a
positive regulator of adipocyte differentiation that can cooperate with
C/EBP in stimulating the adipogenic program (16). Although increased
expression of many genes is observed during adipocyte differentiation,
few have been identified that are down-regulated during adipogenesis.
Pref-1 is abundant in preadipocytes but is not expressed in adipocytes,
and constitutive expression of Pref-1 in preadipocytes inhibits
adipogenesis (22). Thus, Pref-1 seems to function as a negative
regulator of adipocyte differentiation.
In mouse adipogenic cell lines, such as 3T3-L1 and TA1 (28, 29, 30), tumor
necrosis factor (TNF) is capable of inhibiting adipocyte
differentiation and of inducing dedifferentiation of fully
differentiated adipocytes (31, 32). Addition of TNF to adipocytes
causes down-regulation of enzymes involved in lipid metabolism and of
adipocyte specific genes, such as aP2 and FSP27, and addition of TNF
to preadipocytes prevents their increased expression (31, 32). Though
it has been demonstrated that rapid down-regulation of C/EBP (33, 34) and induced expression of c-myc (35) are associated with
TNF-mediated reversal of adipocyte differentiation, the exact
mechanism(s) by which TNF affects the adipogenic program remain
obscure.
To investigate the mechanisms by which TNF and certain growth
factors inhibit and reverse adipocyte differentiation, we have studied
the expression of two key regulatory proteins, PPAR and Pref-1. We
show that PPAR mRNAs, their corresponding proteins, and DNA-binding
activity are rapidly diminished after addition of TNF to 3T3-L1
adipocytes. Moreover, PPAR expression stays at low levels during
TNF-mediated inhibition of adipogenesis. Thus, TNF is capable of
regulating PPAR expression in both preadipocytes and adipocytes. In
contrast, Pref-1 message fails to reappear during TNF-mediated
reversal of adipocyte differentiation and is autonomously
down-regulated in differentiating adipocytes in the presence of TNF.
We conclude that the effects of TNF on adipogenesis do not involve a
change in expression of Pref-1 but do elicit specific changes in
PPAR expression.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
complementary DNA was obtained from 3T3-L1
adipocyte total RNA by reverse transcription and PCR using the
following primers: 5'-GGGGAATTCCGTTATGGGTGAAACTCTGGGA-3' and
5'-CTGGTCGATATCACTGGAGAT-3'. The resulting PCR fragment was digested
with EcoR I and EcoR V. A ribovector for PPAR
was constructed by inserting this PCR fragment into pcDNA3 (Invitrogen
Corp., San Diego, CA), digested with EcoR I and
EcoR V, and the insert was confirmed by DNA sequencing. A
riboprobe template for PPAR was generated by StuI digest.
Pref-1 complementary DNA was obtained from 3T3-L1 preadipocyte total
RNA using two sets of primers: 5'-GATTTGGATCCGAGATGATCGCGACCGGAGCCC-3',
5'-GGTCTTGTCGACGAATCCAGCTG-G-3' and 5'-GATTCGTCGACAAGACCTGCAGCC-3',
5'-GTGTATCTA-GAGAATTCTGTCAATCTTCTCGGGGAAGATG-3'. The resulting PCR
fragments were digested with BamHI/SalI and
SalI/XbaI, respectively. A ribovector for Pref-1
was created by subcloning of these two fragments into pcDNA3 and
linearizing with SmaI. The accuracy of the Pref-1 insert was
confirmed by DNA sequencing. Riboprobes for ß-actin and FSP27 have
been described previously (36).
Cell culture
3T3-L1 cells (obtained from ATCC, Rockville, MD) were maintained
in minimum essential medium Eagle (MEM Earle’s salt medium) (Sigma,
St. Louis, MO) supplemented with 10% FCS at 37 C, 5% CO2.
All serum used was heat inactivated at 55 C for 30 min. To induce
adipocyte differentiation, 3T3-L1 cells were treated at confluence with
1 µM dexamethasone (Dex) and 30 µM
indomethacin (Indo) for 6–10 days (8, 37). Alternatively, 2 days after
reaching confluence, cells were treated with 1 µM Dex,
0.5 µM 1-methyl-3-isobutyl-xanthine, and 1.7
µM insulin for 2 days, followed by 2 additional days in
1.7 µM insulin, and finally, 2 days in MEM with 10% FCS
(38). Typically, at this time, approximately 70% of the cells had
differentiated into adipocytes, as judged by cell morphology. No
difference in cell morphology or specific gene expression was observed
between these two differentiation protocols. TNF, basic fibroblast
growth factor (bFGF), and transforming growth factor ß (TGFß) were
purchased from Collaborative Biomedical Products (Bedford, MA). The
phorbol ester tetradecanoyl phorbol-13-acetate (TPA) was from Sigma,
and the calcium ionophore ionomycin was from Calbiochem-Behring Corp.
(La Jolla, CA). Dex, 1-methyl-3-isobutyl-xanthine, and Indo were from
Sigma.
Total RNA preparation and ribonuclease protection assay
Cells were dissolved in guanidium isothiocyanate-containing
solution (Micro RNA Isolation Kit; Stratagene, La Jolla, CA), and total
RNA was isolated according to the manufacturer’s protocol. Riboprobes
for PPAR, Pref-1, FSP27, and ß-actin were generated using a
MAXIscript Kit (Ambion, Austin, TX). Briefly, in vitro
transcription reactions were set up in 10-µl vols containing 1X
transcription buffer; 0.2–0.5 µg template; 500 µM each
ATP, CTP, and GTP; 20 µM dithiothreitol (DTT); 25 µCi
[-32P]UTP (800 Ci/mmol, Amersham, Arlington Heights,
IL); and 5 U of T7 or SP6 RNA polymerase. After incubation at room
temperature for 30–45 min, DNA template was digested with
deoxyribonuclease I at 37 C for 15 min. RNA probes were then separated
from unincorporated nucleotides using S-200 HR columns (Pharmacia,
Alemeda, CA). Ribonuclease protection assays were performed with RPA II
kit (Ambion) and carried out according to manufacturer’s protocol,
with minor modifications. Typically, 2 µg of total RNA and 60,000 cpm
of each riboprobe were included in each assay. Protected RNA fragments
were separated on 5% acrylamide, 8 M urea gels.
Antibody production
Murine monoclonal antibodies to human recombinant PPAR (39)
were generated from immune lymphocytes isolated from a BALB/c mouse
(Charles River, Research Triangle Park, NC). The mouse was immunized a
total of four times with recombinant protein-encoding residues 195–475
of the ligand-binding domain of human PPAR. At 2-week intervals, 50
and 5 µg of recombinant PPAR, emulsified in RIBIs adjuvant (RIBI
Immuno. Chem. Research, Inc., Hamilton, MT), were injected ip. The
mouse was then immunized with 10 µg ip, then 5 µg iv (recombinant
PPAR in 200 µl PBS) 96 and 24 h before death. A polyethylene
glycol-induced somatic fusion of immune splenocytes was performed using
a previously published procedure (40). Monoclonal antibodies
PM4–17.2 and PM2–15.22 produce IgG2a.
Nuclear extract preparation and DNA-binding assay
Nuclear extracts were prepared as described by Schreiber
et al. (41). Briefly, two 150-cm2 confluent
flasks of cells were harvested and cells resuspended in 1 ml of cold
buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1
mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1
mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and
25 µg/ml aprotinin). Then, 62.5 µl of 10% NP-40 solution was
added, and the cell suspension was vortexed vigorously for 10 sec.
Nuclei were recovered by centrifugation for 30 sec at 4 C and
subsequently resuspended in 150 µl of ice-cold buffer C [20
mM HEPES (pH 7.9), 0.4 M NaCl, 10% glycerol, 1
mM EDTA, 1 mM EGTA, 1 mM DTT plus
protease inhibitors, as described above] and rocked vigorously at 4 C
for 30 min. The nuclear lysate was centrifuged at 100,000 rpm for 12
min in Beckman TL-100 ultracentrifuge at 4 C, and the supernatant was
aliquoted and stored at -80 C.
The sequences of double-stranded oligonucleotides used were as follows:
ARE7s, 5'-TCGAGTGTGAACTCTGATCCAGC-3'; ARE7as,
5'-TCGAGCTGGATCAGAGTTCACAC-3'; Histone octamer 1s,
5'-TCGAGTGTAATATGCAAATCATTTGC-3'; Histone octamer 1as,
5'-TCGAGCAAAATGATTTGCATATTACAC-3'. The DNA fragments were allowed to
anneal, labeled with [-32P] ATP (>5000 µci/mmol,
Amersham), filled in the protruding ends with Klenow fragment, and
purified with Bio-Spin 6 chromatography columns (Bio-Rad Laboratories,
Richmond, CA) to dispose of unincorporated [-32P] ATP
and dNTPs.
Electrophoretic mobility shift assay (EMSA) was performed as described by P. Tontonoz et al. (17), with modifications as follows: 5 µg of nuclear extract were incubated with 0.3 µg poly dI-dC for 15 min at 4 C, followed by the addition of 25,000 cpm of 32P-labeled double-stranded oligonucleotide ARE7. The mixture was incubated for 20 min at room temperature and then electrophoresed on a 4% native gel in 0.5 x TBE. When antibody was used, binding reactions were incubated with antibody for 15 min at room temperature before the addition of the probes.
Immunoblotting
Nuclear extracts (20 µg) were added to SDS-PAGE loading
solution, boiled for 8–10 min, and electrophoresed on a denaturing gel
consisting of a 3.8% acrylamide stacking gel and a 12% acrylamide
separating gel, according to Laemmli (42). Proteins were transferred to
Immobilon-P transfer membrane (Millipore, Bedford, MA) and blots
subsequently treated with 5% nonfat milk in TBS [50 mM
Tris-HCl (pH 7.4), 150 mM NaCl], containing 0.2% Tween
20, for 20 min at room temperature. Blocked membranes were incubated
with anti-PPAR monoclonal antibody, PM 2–15.22 at 4 C overnight,
followed by three washes with TBS containing 0.05% tween. Membranes
were then incubated for 1 h with antimouse IgG, conjugated with
horseradish peroxidase, and PPAR proteins visualized with the
enhanced chemiluminescence system (Amersham).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and Pref-1 expression during adipocyte differentiation of
3T3-L1 cells and Pref-1 gene expression, total
cellular RNA was prepared at various stages during the differentiation
process, and PPAR and Pref-1 messages were examined by ribonuclease
protection assay (Fig. 1
). In preadipocytes, PPAR
mRNA was just detectable and remained at low levels as cells reached
confluence (Fig. 1A). In the presence of FCS, Dex, and Indo, PPAR
mRNA was induced at a very early stage in the differentiation process
(within 2 days after cells reached confluence, day zero). Maximal
induction of PPAR message was observed by day 6 (Fig. 1A). Pref-1
mRNA expression level also was altered early during adipocyte
differentiation, decreasing just 1 day after confluence and remaining
at low levels during the entire time course (Fig. 1B). These changes in
expression of PPAR and Pref-1 occurred well before accumulation of
intracellular lipid droplets (data not shown). The induction of PPAR
mRNA and decrease of Pref-1 mRNA expression precede the induction of
adipocyte-specific message FSP27 (3 days post confluence, Fig. 1B) and
expression of transcription factor C/EBP on day 3 (16).
|
and Pref-1 message during TNF-mediated
inhibition of adipocyte differentiation and Pref-1 act as positive and negative regulators
of adipogenesis, respectively, determining the effect of TNF
treatment on their expression is important to understand
TNF-mediated inhibition of adipocyte differentiation. TNF, at
various concentrations, was added to confluent cells in the presence of
Dex and Indo, and cells were cultured in differentiation medium for 10
days. In the absence of TNF, cells differentiated into adipocytes,
and PPAR message increased (Fig. 2A, compare
nontreated preadipocytes and adipocytes). With TNF present in the
differentiation media, PPAR message remained at low levels, even
with the lowest dose of TNF used (Fig. 2A). As expected, levels of
Pref-1 message decreased when cells differentiated into adipocytes
(Fig. 2B). Surprisingly, Pref-1 mRNA decreased significantly, even when
differentiation was blocked by the presence of a wide range of TNF
concentrations (Fig. 2B). In this experiment, TNF-mediated
inhibition of adipocyte differentiation was judged both by inhibition
of adipocyte-specific marker FSP27 expression (Fig. 2B) and by
morphological resemblance to preadipocytes (data not shown).
|
and Pref-1 during TNF-mediated reversal of
adipocyte differentiation to differentiated adipocytes causes
down-regulation of adipocyte specific genes (31, 32, 35), as well as
decreased expression of regulatory factors such as C/EBP (33, 34),
thus substantially reverting these cells to a state resembling the
preadipocyte phenotype. To determine whether TNF has an effect on
the expression pattern of PPAR and Pref-1 in adipocytes, fully
differentiated 3T3-L1 cells were transferred to fresh media, containing
TNF, and analyzed after various time intervals. TNF noticeably
reduced the PPAR steady-state mRNA level between 0.5 and 1 h
after its addition; and after 6 h of TNF treatment, no PPAR
mRNA was detectable (Fig. 3, A and E). FSP27 mRNA
decreased more slowly, reaching a minimal level after 9.5 h of
TNF treatment (Fig. 3, B and E; 35). In contrast, Pref-1 mRNA was
not up-regulated during TNF-mediated reversal of adipocyte
differentiation for up to 3 days of treatment (Fig. 3, C and F).
The reversal of adipocyte differentiation during this time course was
judged both by the loss of adipocyte specific marker FSP27 expression
(Fig. 3, D and E) and by morphological resemblance to preadipocytes
(data not shown).
|
, but not reexpression of Pref-1, is
associated with TGFß, bFGF, and phorbol ester plus calcium
ionophore-mediated reversal of adipocyte differentiation or Pref-1 expression, adipocytes were
treated with TGFß, bFGF, or TPA plus ionomycin, and total cellular
RNA was analyzed. PPAR expression decreased significantly upon
treatment with TGFß, bFGF, or TPA plus ionomycin (Fig. 4, A and C). In contrast, Pref-1 message remained low
during these treatments (Fig. 4, B and C). These results suggest that
down-regulation of PPAR may be one mechanism by which these agents
reverse the adipocyte phenotype. It seems that increased expression of
the negative regulator, Pref-1, may not be involved in their
action.
|
-induced adipocyte dedifferentiation leads to a reversal of
the differentiation-induced profile of PPAR proteins and their
DNA-binding activity protein also was monitored during
TNF-induced adipocyte dedifferentiation by analyzing nuclear protein
extracts prepared at the various times after TNF addition to 3T3-L1
adipocytes, using EMSA and immunoblot assays. As shown in Fig. 5A, no detectable PPAR proteins are present in
preadipocytes (lane Preadipo.). Upon differentiation, both PPAR1
(top) and PPAR2 (bottom) are induced (lane
Adipo.). Addition of TNF into 3T3-L1 adipocytes causes rapid loss of
both PPAR proteins, reaching a level comparable with that of
preadipocytes within 1 h (Fig. 5A).
|
DNA-binding activity is altered during
TNF-induced reversal of adipocyte differentiation, these same
nuclear extracts were examined for PPAR DNA-binding activity (Fig. 5B
, left panel). We used the ARE7-binding site in the aP2
enhancer, previously shown to specifically bind PPAR-RXR
heterodimers, also called ARF6 (18), as probe. Only a small amount of
DNA-binding activity is detected in nuclear extracts from
preadipocytes. Upon differentiation, an increase in ARE7 DNA-binding
activity is seen as expected (Fig. 5B). Treatment of adipocytes with
TNF, rapidly results in decreased PPAR-RXR DNA-binding
activity, reaching a level similar to that observed in preadipocytes
within 1 h (Fig. 5B). As an internal control, the DNA-binding
activity of Oct-1 remains relatively constant during of TNF
treatment (Fig. 5B, right panel). The specificity of the
observed DNA-binding activity was determined using specific and
nonspecific DNA-binding sequence as competitors (Fig. 5C). ARF6
DNA-binding activity is competed by unlabeled ARE7 oligonucleotide, but
not by a histone octamer sequence (Fig. 5C). Evidence of participation
of PPAR in the ARE7-binding complex was obtained by preincubation of
3T3-L1 adipocyte nuclear extracts with monoclonal antibody PM
4–7.22 raised against the ligand-binding domain of PPAR. Addition
of this antibody results in a supershift of the ARE7-PPAR-RXR
complexes, indicating that PPAR is present in the complexes (Fig. 5D). The antibody specificity was confirmed by its failure to
supershift Oct-1 complexes. The rapid disappearance of the PPAR, as
a result of TNF application, strongly suggests that TNF reversal
of the adipose phenotype may, in part, be caused by the rapid loss of
this adipocyte-specific transcription factor. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and Pref-1 changes during early stages of adipocyte
differentiation. This data is in general agreement with that of others
(16, 26). We show that the down-regulation of Pref-1 mRNA expression
and induction of PPAR mRNA occur 1 day and 2 days, respectively,
after cells reach confluence (Fig. 1). These changes not only precede
the expression of adipocyte specific genes, but also precede the
induction of C/EBP (1), which has been shown to play an important
regulatory role in adipogenesis (12, 13, 43, 44). Whereas
glucocorticoid hormones may exert their effects by inducing C/EBP
mRNA expression during adipogenesis (45), the specific actions of Indo
have not previously been defined in this system. Indo, at similar
concentrations to those required for induction of differentiation,
recently has been found to activate PPAR-dependent transcription (H.
Xing, unpublished observations; J. Lehmann, GlaxoWellcome, personal
communication), which may explain why Indo enhances conversion to
adipocytes. The highly restricted tissue distribution and early changes
in mRNA expression of PPAR and Pref-1 suggest that they play an
important role in the adipocyte differentiation process. Pref-1 is a member of a small group of proteins (identified to date) whose expression decreases during adipocyte differentiation. Although Pref-1 expression is rapidly down-regulated during the early stages of adipocyte differentiation, it may be required for initiation of the differentiation process in a permissive and/or instructive manner. The absence of Pref-1 seems to be essential for differentiation (22). Although it remains a formal possibility that Pref-1 participates in some early inductive signals, this seems unlikely, given recent observation that Pref-1 expression is exceedingly low in TA1 preadipocytes that differentiate normally (H. Xing, unpublished observations).
The presence of EGF-like repeats in the extracellular domain of Pref-1 suggests that it may regulate adipogenesis through protein-protein interactions. Studies with Notch and Delta demonstrate that the EGF-like repeats within these proteins govern cell-cell interactions and, consequently, cell fate decisions (24, 25). Recently, the mammalian protein Jagged has been identified as an activating ligand for Notch and, when expressed in myoblasts, prevents muscle cell differentiation (46). Whether Pref-1 acts as a receptor for a Jagged-like protein in preadipocytes or as a ligand for an as yet unidentified adipocyte receptor remains to be determined.
When present in cultures of confluent adipogenic 3T3-L1 or TA1 cells,
TNF inhibits their differentiation to adipocytes. TNF-treated
cells fail to express adipocyte-specific genes and to accumulate
intracellular lipid droplets (Fig. 2B and Refs. 31, 32). We have
found that TNF-mediated inhibition of adipocyte differentiation is
not associated with continued high expression of Pref-1. Rather, Pref-1
expression decreases in a manner indistinguishable from that of
nontreated cells. Thus, the effects of TNF are not mediated by
suppression of the normal autonomous decrease in Pref-1 expression,
which is a prerequisite for differentiation.
In contrast to its lack of effect on Pref-1 expression, TNF prevents
PPAR mRNA expression from increasing in adipogenic cells. The
regulatory roles of PPAR in adipogenesis in general (reviewed in
Ref. 15) and in the expression of some adipocyte-specific genes (17, 27) have been defined. PPAR and C/EBP-binding sites are present
in the promoter regions of the aP2 (17) and PEPCK genes (27). In
addition, TNF causes a rapid decrease in the expression of PPAR
mRNA (Fig. 3A) and corresponding protein (Fig. 5A), as well as a
parallel decrease in DNA-binding activity in adipocytes (Fig. 5B). It
is noteworthy that the time-dependent decrease in both mRNA expression
and DNA-binding activity of PPAR and C/EBP (33, 34) occurs well
before the disappearance of adipocyte-specific genes, such as FSP27 and
aP2 (33, 34, 35), suggesting that the decrease in DNA-binding activities of
PPAR and C/EBP in response to TNF may lead to the decreased
expression of adipocyte-specific genes, thus accounting for the
reversal of the adipocyte phenotype.
TNF is a potent activator of phospholipase A2 in preadipocytes
and adipocytes, leading to formation of a variety of arachidonate
metabolites, notably 5-lipoxygenase products (47). Recently a PG J2
metabolite has been identified as both a PPAR ligand and an inducer
of adipogenesis (48, 49), suggesting a novel mechanism of action for
PGs of the J2 series through a nuclear receptor signaling pathway. It
will be of interest to examine whether any of these TNF-induced
arachidonate metabolites act as antagonists of PPAR activation.
Several reports have indicated that polypeptide hormones, such as
TGFß and bFGF, as well as protein kinase C-activating agents and
calcium ionophores, are capable of reversing adipocyte differentiation
(32, 37). We have shown that TGFß, bFGF, and TPA plus ionomycin
effectively suppress PPAR mRNA expression in adipocytes with
concomitant decrease in adipocyte-specific gene expression. Whether
these agents share similar cellular signaling pathways is unknown at
this time. Consistent with the effects of TNF, none of these agents
are able to cause significant reexpression of Pref-1 mRNA in
adipocytes. Thus, Pref-1 seems to function as a nonreversible molecular
checkpoint at the start of the adipocyte differentiation process whose
expression is independent of all tested agents. These results
demonstrate that dedifferentiated adipocytes and preadipocytes are not
identical, though they share many similar morphological characteristics
and patterns of gene expression.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received December 4, 1996.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Curr Opin Genet Dev 5:571–576[CrossRef][Medline]
2, a lipid-activated
transcription factor. Cell 79:1147–1156[CrossRef][Medline]
2: tissue-specific regulator of an adipocyte
enhancer. Genes Dev 8:1224–1234:
adipose-predominant expression and induction early in adipocyte
differentiation. Endocrinology 135:798–800[Abstract]
2 regulates adipose expression of the phosphoenolpyruvate
carboxykinase gene. Mol Cell Biol 15:351–357[Abstract]
(PPAR ). J Biol Chem 270:12953–12956 promotes the
adipogenic program in a variety of mouse fibroblastic cells. Genes Dev 8:1654–1663 is sufficient to initiate the 3T3–L1 adipocyte differentiation
program. Proc Natl Acad Sci USA 91:8757–8761. Cell 83:803–812[CrossRef][Medline]
and promotes adipocyte
differentiation. Cell 83:813–819[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  J. G. Neels, L. Badeanlou, K. D. Hester, and F. Samad Keratinocyte-derived Chemokine in Obesity: EXPRESSION, REGULATION, AND ROLE IN ADIPOSE MACROPHAGE INFILTRATION AND GLUCOSE HOMEOSTASIS J. Biol. Chem., July 31, 2009; 284(31): 20692 - 20698. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Raff, T. Tholstrup, S. Toubro, J. M. Bruun, P. Lund, E. M. Straarup, R. Christensen, M. B. Sandberg, and S. Mandrup Conjugated Linoleic Acids Reduce Body Fat in Healthy Postmenopausal Women J. Nutr., July 1, 2009; 139(7): 1347 - 1352. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S. Y. Chan, L. J. Schedlich, S. M. Twigg, and R. C. Baxter Inhibition of adipocyte differentiation by insulin-like growth factor-binding protein-3 Am J Physiol Endocrinol Metab, April 1, 2009; 296(4): E654 - E663. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Tejerina, A. De Pauw, S. Vankoningsloo, A. Houbion, P. Renard, F. De Longueville, M. Raes, and T. Arnould Mild mitochondrial uncoupling induces 3T3-L1 adipocyte de-differentiation by a PPAR{gamma}-independent mechanism, whereas TNF{alpha}-induced de-differentiation is PPAR{gamma} dependent J. Cell Sci., January 1, 2009; 122(1): 145 - 155. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L Bouraoui, J Gutierrez, and I Navarro Regulation of proliferation and differentiation of adipocyte precursor cells in rainbow trout (Oncorhynchus mykiss) J. Endocrinol., September 1, 2008; 198(3): 459 - 469. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wu and C. M. Smas Wdnm1-like, a new adipokine with a role in MMP-2 activation Am J Physiol Endocrinol Metab, July 1, 2008; 295(1): E205 - E215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Y. Kim, K. Liu, S. Zhou, K. Tillison, Y. Wu, and C. M. Smas Assessment of fat-specific protein 27 in the adipocyte lineage suggests a dual role for FSP27 in adipocyte metabolism and cell death Am J Physiol Endocrinol Metab, April 1, 2008; 294(4): E654 - E667. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Tchkonia, T. Pirtskhalava, T. Thomou, M. J. Cartwright, B. Wise, I. Karagiannides, A. Shpilman, T. L. Lash, J. D. Becherer, and J. L. Kirkland Increased TNF{alpha} and CCAAT/enhancer-binding protein homologous protein with aging predispose preadipocytes to resist adipogenesis Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1810 - E1819. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Klotz, I. Dani, F. Edenhofer, L. Nolden, B. Evert, B. Paul, W. Kolanus, T. Klockgether, P. Knolle, and L. Diehl Peroxisome Proliferator-Activated Receptor {gamma} Control of Dendritic Cell Function Contributes to Development of CD4+ T Cell Anergy J. Immunol., February 15, 2007; 178(4): 2122 - 2131. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Lacasa, S. Taleb, M. Keophiphath, A. Miranville, and K. Clement Macrophage-Secreted Factors Impair Human Adipogenesis: Involvement of Proinflammatory State in Preadipocytes Endocrinology, February 1, 2007; 148(2): 868 - 877. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Karagiannides, T. Thomou, T. Tchkonia, T. Pirtskhalava, K. E. Kypreos, A. Cartwright, G. Dalagiorgou, T. L. Lash, S. R. Farmer, N. A. Timchenko, et al. Increased CUG Triplet Repeat-binding Protein-1 Predisposes to Impaired Adipogenesis with Aging J. Biol. Chem., August 11, 2006; 281(32): 23025 - 23033. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sastre, I. Dewachter, S. Rossner, N. Bogdanovic, E. Rosen, P. Borghgraef, B. O. Evert, L. Dumitrescu-Ozimek, D. R. Thal, G. Landreth, et al. Nonsteroidal anti-inflammatory drugs repress {beta}-secretase gene promoter activity by the activation of PPAR{gamma} PNAS, January 10, 2006; 103(2): 443 - 448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kudo, A. Sugawara, A. Uruno, K. Takeuchi, and S. Ito Transcription Suppression of Peroxisome Proliferator-Activated Receptor {gamma}2 Gene Expression by Tumor Necrosis Factor {alpha} via an Inhibition of CCAAT/ Enhancer-binding Protein {delta} during the Early Stage of Adipocyte Differentiation Endocrinology, November 1, 2004; 145(11): 4948 - 4956. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Khovidhunkit, M.-S. Kim, R. A. Memon, J. K. Shigenaga, A. H. Moser, K. R. Feingold, and C. Grunfeld Thematic review series: The Pathogenesis of Atherosclerosis. Effects of infection and inflammation on lipid and lipoprotein metabolism mechanisms and consequences to the host J. Lipid Res., July 1, 2004; 45(7): 1169 - 1196. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Brown, M. S. Boysen, S. Chung, O. Fabiyi, R. F. Morrison, S. Mandrup, and M. K. McIntosh Conjugated Linoleic Acid Induces Human Adipocyte Delipidation: AUTOCRINE/PARACRINE REGULATION OF MEK/ERK SIGNALING BY ADIPOCYTOKINES J. Biol. Chem., June 18, 2004; 279(25): 26735 - 26747. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Sitcheran, P. C. Cogswell, and A. S. Baldwin Jr. NF-{kappa}B mediates inhibition of mesenchymal cell differentiation through a posttranscriptional gene silencing mechanism Genes & Dev., October 1, 2003; 17(19): 2368 - 2373. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Tamori, J. Masugi, N. Nishino, and M. Kasuga Role of Peroxisome Proliferator-Activated Receptor-{gamma} in Maintenance of the Characteristics of Mature 3T3-L1 Adipocytes Diabetes, July 1, 2002; 51(7): 2045 - 2055. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Han, S. R. Farmer, J. L. Kirkland, B. E. Corkey, R. Yoon, T. Pirtskhalava, Y. Ido, and W. Guo Octanoate Attenuates Adipogenesis in 3T3-L1 Preadipocytes J. Nutr., May 1, 2002; 132(5): 904 - 910. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Xu, J. Hirosumi, K. T. Uysal, A. D. Guler, and G. S. Hotamisligil Exclusive Action of Transmembrane TNF{alpha} in Adipose Tissue Leads to Reduced Adipose Mass and Local But Not Systemic Insulin Resistance Endocrinology, April 1, 2002; 143(4): 1502 - 1511. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Fruhbeck, J. Gomez-Ambrosi, F. J. Muruzabal, and M. A. Burrell The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation Am J Physiol Endocrinol Metab, June 1, 2001; 280(6): E827 - E847. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Le Roith and Y. Zick Recent Advances in Our Understanding of Insulin Action and Insulin Resistance Diabetes Care, March 1, 2001; 24(3): 588 - 597. [Abstract] [Full Text]
|
|
|
|
 |
|
 R. W. Valyasevi, S. C. Jyonouchi, C. M. Dutton, N. Munsakul, and R. S. Bahn Effect of Tumor Necrosis Factor-{{alpha}}, Interferon-{{gamma}}, and Transforming Growth Factor-{beta} on Adipogenesis and Expression of Thyrotropin Receptor in Human Orbital Preadipocyte Fibroblasts J. Clin. Endocrinol. Metab., February 1, 2001; 86(2): 903 - 908. [Abstract] [Full Text]
|
|
|
|
|
|
A. Valladares, A. M. Alvarez, J. J. Ventura, C. Roncero, M. Benito, and A. Porras p38 Mitogen-Activated Protein Kinase Mediates Tumor Necrosis Factor-{{alpha}}-Induced Apoptosis in Rat Fetal Brown Adipocytes Endocrinology, December 1, 2000; 141(12): 4383 - 4395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Gilbert, X. He, P. Farmer, S. Boden, M. Kozlowski, J. Rubin, and M. S. Nanes Inhibition of Osteoblast Differentiation by Tumor Necrosis Factor-{alpha} Endocrinology, November 1, 2000; 141(11): 3956 - 3964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Waite, E. C. Person, Y. Zhou, K.-H. Lim, T. S. Scanlan, and R. N. Taylor Placental Peroxisome Proliferator-Activated Receptor-{gamma} Is Up-Regulated by Pregnancy Serum J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3808 - 3814. [Abstract] [Full Text]
|
|
|
|
 |
|
 L. Choy, J. Skillington, and R. Derynck Roles of Autocrine TGF-{beta} Receptor and Smad Signaling in Adipocyte Differentiation J. Cell Biol., May 1, 2000; 149(3): 667 - 682. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. E. Law, S. Goetze, X.-P. Xi, S. Jackson, Y. Kawano, L. Demer, M. C. Fishbein, W. P. Meehan, and W. A. Hsueh Expression and Function of PPAR{gamma} in Rat and Human Vascular Smooth Muscle Cells Circulation, March 21, 2000; 101(11): 1311 - 1318. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Sartippour and G. Renier Differential Regulation of Macrophage Peroxisome Proliferator-Activated Receptor Expression by Glucose : Role of Peroxisome Proliferator-Activated Receptors in Lipoprotein Lipase Gene Expression Arterioscler Thromb Vasc Biol, January 1, 2000; 20(1): 104 - 110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Desvergne and W. Wahli Peroxisome Proliferator-Activated Receptors: Nuclear Control of Metabolism Endocr. Rev., October 1, 1999; 20(5): 649 - 688. [Abstract] [Full Text]
|
|
|
|
|
|
H. Xu, J. K. Sethi, and G. S. Hotamisligil Transmembrane Tumor Necrosis Factor (TNF)-alpha Inhibits Adipocyte Differentiation by Selectively Activating TNF Receptor 1 J. Biol. Chem., September 10, 1999; 274(37): 26287 - 26295. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Poretsky, N. A. Cataldo, Z. Rosenwaks, and L. C. Giudice The Insulin-Related Ovarian Regulatory System in Health and Disease Endocr. Rev., August 1, 1999; 20(4): 535 - 582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Smas, L. Chen, L. Zhao, M.-J. Latasa, and H. S. Sul Transcriptional Repression of pref-1 by Glucocorticoids Promotes 3T3-L1 Adipocyte Differentiation J. Biol. Chem., April 30, 1999; 274(18): 12632 - 12641. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. COUSIN, O. MUNOZ, M. ANDRE, A. M. FONTANILLES, C. DANI, J. L. COUSIN, P. LAHARRAGUE, L. CASTEILLA, and L. P ÉNICAUD A role for preadipocytes as macrophage-like cells FASEB J, February 1, 1999; 13(2): 305 - 312. [Abstract] [Full Text]
|
|
|
|
|
|
C. M. Smas, D. Kachinskas, C.-M. Liu, X. Xie, L. K. Dircks, and H. S. Sul Transcriptional Control of the pref-1 Gene in 3T3-L1 Adipocyte Differentiation. SEQUENCE REQUIREMENT FOR DIFFERENTIATION-DEPENDENT SUPPRESSION J. Biol. Chem., November 27, 1998; 273(48): 31751 - 31758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. H. Hansen, B. Madsen, B. Teisner, J. H. Nielsen, and N. Billestrup Characterization of the Inhibitory Effect of Growth Hormone on Primary Preadipocyte Differentiation Mol. Endocrinol., August 1, 1998; 12(8): 1140 - 1149. [Abstract] [Full Text]
|
|
|
|
 |
|
 F. M. GREGOIRE, C. M. SMAS, and H. S. SUL Understanding Adipocyte Differentiation Physiol Rev, July 1, 1998; 78(3): 783 - 809. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Alexander, L. Ganem, P Fernandez-Salguero, F Gonzalez, and C. Jefcoate Aryl-hydrocarbon receptor is an inhibitory regulator of lipid synthesis and of commitment to adipogenesis J. Cell Sci., January 11, 1998; 111(22): 3311 - 3322. [Abstract] [PDF]
|
|
|
|
|
|
P. M. Catalano, S. E. Nizielski, J. Shao, L. Preston, L. Qiao, and J. E. Friedman Downregulated IRS-1 and PPARgamma in obese women with gestational diabetes: relationship to FFA during pregnancy Am J Physiol Endocrinol Metab, March 1, 2002; 282(3): E522 - E533. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
