The USDA Human Nutrition Research Center on Aging at Tufts University and Division of Endocrinology Tupper Medical Research Institute New England Medical Center Boston, Massachusetts 02111
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ABSTRACT |
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INTRODUCTION |
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TNF has been shown to alter adipose tissue metabolism and
decrease the expression of several adipocyte gene products such as
aP2 (the fattyacid-binding protein), GLUT 4 (the insulin-responsive
glucose transport protein), C/EBP
(the CAAT enhancer-binding
protein), and PPAR
2 (peroxisome proliferator-activated receptor, a
nuclear transcription factor) (PPAR
2) (8, 9). PPAR
2 is critical
for the terminal differentiation of precursor cells into adipocytes.
Although PPAR
2 is expressed in many different cells at low levels,
its predominant expression is in adipocytes, where it is expressed
early in the differentiation pathway (10). The expression of PPAR
2
increases during the differentiation of preadipocytes to adipocytes
(10, 11). This transcription factor is activated by fatty acids,
peroxisome proliferators, and disparate lipids such as clofibric acid,
linoleic acid, and 5,8,11-eicosatetraynoic acid (the synthetic
analog of arachidonic acid) as well as the arachidonic acid metabolite,
15 deoxy-
12,
14-prostaglandin J2
(15d-PGJ2) (12, 13, 14).
The thiazolidinediones, a family of antidiabetic compounds that
includes BRL 49653, have been shown to bind strongly to and activate
PPAR2 (14). The ability of these compounds to improve insulin
sensitivity in obese animals was directly related to their binding
affinity for PPAR
2 (14). When a PPAR
2 ligand is incubated with
preadipocytes or fibroblasts that have been transfected with PPAR
2,
PPAR
2 expression increases, and an increase in adipocyte
differentiation is seen (14).
The precise mechanism by which PPAR2 regulates adipocyte
differentiation has not yet been defined. However, the evidence
strongly suggests a role for PPAR
2 in the regulation of many
adipocyte genes (15).
Previously, TNF was shown to reduce the expression of PPAR
2 (9).
Zhang et al. (9) suggested that down-regulation of PPAR
2
by TNF
was an important part of the mechanism by which TNF
reduces expression of several other adipocyte genes. With low level
constitutive expression of PPAR
2, Zhang and co-workers were able to
partially prevent TNF
from decreasing the expression of aP2 and
adipsin in adipocytes, suggesting that the level of PPAR
2 expression
is important for TNF
to exert its effects on adipocyte gene
expression.
In this paper, we use the 3T3-L1 adipoblast cell line to examine
the mechanisms by which TNF and BRL 49653 interact to regulate
adipocyte gene expression in differentiated adipocytes (16). Most other
studies have focused on gene expression during differentiation of
preadipocytes to adipocytes (17, 18). For the most part, our results
concur with previous findings, but some of our results were unexpected
and quite surprising. Although BRL 49653 very effectively opposed
TNF
s down-regulation of several adipocyte genes such as perilipin
(the lipid droplet-associated protein), aP2, and hormone-sensitive
lipase (HSL) (the rate-limiting enzyme in lipolysis) during incubation
periods of up to 24 h, it did not prevent the down-regulation of
PPAR
2 at this time. In fact, BRL 49653 alone from 624 h decreased
PPAR
2 mRNA and protein expression in differentiated cells, and, in
combination with TNF
, decreased PPAR
2 expression further. Longer
incubation times with BRL 49653 resulted in a smaller down-regulation
of the PPAR
2 protein but also less inhibition of the effects of
TNF
on perilipin protein levels. Therefore, the contrasting effects
of BRL 49653 and TNF
on gene expression in differentiated adipocytes
are not simply the result of alterations in the level of PPAR
2
expression. Understanding the mechanism by which the thiazolidinediones
antagonize TNF
s actions may increase understanding of how the
thiazolidinediones such as BRL49653 ameliorate insulin resistance.
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RESULTS |
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Incubation with TNF alone resulted in a significant decrease
in steady state mRNA of perilipin, PPAR
2, and aP2 after 6 h of
treatment; the decrease at 1 h was minimal (Fig. 1
). Both PPAR
2
and perilipin decreased more than 70% by 6 h, whereas the
decrease in aP2 mRNA was slightly delayed, but by 24 h it was also
decreased by 70%. Thus, TNF
greatly reduced the expression of the
mRNAs for several adipocyte- specific genes throughout the 24-h
time course.
When the effects of BRL 49653 on mRNA levels were examined, the results
were unexpected. The effect of BRL 49653 on PPAR2 differed from its
effects on perilipin and aP2 mRNA expression in 3T3-L1 adipocytes,
especially during the first 24 h of treatment. BRL 49653 induced
both perilipin and aP2 mRNA levels about 200% at 24 h with little
effect at 1 or 6 h of treatment compared with untreated controls
(Fig. 1
). Surprisingly, the only message examined that was not induced
by BRL 49653 was PPAR
2. In fact, the effect of BRL 49653 on PPAR
2
mRNA was similar to the effect of TNF
; the thiazolidinedione
decreased the mRNA of its own receptor; by 6 h BRL 49653 reduced
the PPAR
2 mRNA by 50%, and this decrease was seen through 24
h.
When TNF and BRL 49653 were incubated together for 6 h, the
thiazolidinedione greatly inhibited the ability of TNF
to
down-regulate perilipin and, to a greater extent, aP2 mRNA, as
determined by densitometry (Fig. 1c
). After 24 h of
coincubation, BRL 49653 completely prevented the ability of TNF
ability to decrease aP2, and it prevented the ability of TNF
to
decrease perilipin mRNA by 50%, as compared with TNF
alone. By
contrast, BRL 49653 did not prevent TNF
from decreasing the level of
PPAR
2 mRNA expression; in fact, the PPAR
2 message declined
further in the presence of both agents than with either one alone (Fig. 1
). Whereas TNF
decreased PPAR
2 by 65% at 24 h and BRL
49653 decreased PPAR
2 by 50%, together they decreased PPAR
2 mRNA
by 90% at 24 h. In conclusion, both TNF
and BRL 49653
decreased the PPAR
2 mRNA throughout the time course, with the
greatest effect occurring at 24 h.
TNF Decreases Adipocyte Gene Transcription
The decrease in steady state message of several adipocyte genes by
TNF (including the nuclear transcription factor PPAR
2) suggested
that the effects of TNF
were mediated in part at the level of
transcription. The effects of TNF
on adipocyte gene expression at
the level of transcription were examined (Fig. 2
). First, 3T3-L1 adipocytes were treated
with TNF
for 1, 3, or 6 h and nuclei were isolated. The nuclei
were subsequently used in a nuclear run-on assay (Fig. 2
). TNF
dramatically decreased the transcription of PPAR
2, perilipin, and
HSL by 1 h. Bluescript plasmid without insert was undetected and
served as a negative control. Actin transcription decreased slightly at
1 h but increased slightly at 3 and 6 h (
2-fold over
control levels) consistent with what others have seen (8). We also saw
a similar increase in steady-state levels of actin mRNA in the presence
of TNF
(data not shown). Therefore, the effects of TNF
on
transcription were specific, affecting all of the adipocyte-specific
genes examined.
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Ectopic expression of the transcription factor C/EBPß, alone or in
combination with C/EBP in fibroblasts, followed by treatment with
steroids, has been reported to stimulate expression of PPAR
2 and
adipocyte differentiaton (20). We therefore investigated whether
down-regulation of PPAR
2 by TNF
and BRL 49653 reflected a
decrease in the transcription of C/EBPß and C/EBP
. The results
indicate that the 8590% reduction in PPAR
2 transcription in the
presence of TNF
and BRL 49653 is not accompanied by significant
reductions in C/EBPß or
. In fact, C/EBPß and C/EBP
transcription levels are actually increased by 38% and 59%,
respectively, in the presence of BRL (Fig. 3
and Table 1
).
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Both TNF and BRL 49653 Reduce PPAR
2 Protein Levels
Since both TNF and BRL 49653 separately and together reduce
PPAR
2 mRNA levels, their effects on protein expression were
examined. Consistent with the data from both the Northern and nuclear
run-on studies, both TNF
and BRL 49653 decreased PPAR
2 protein
levels (Fig. 4a
). With TNF
treatment,
PPAR
2 protein decreased by 45% at 1 h, 70% at 4 h, and
greater than 90% at 24 h. BRL 49653 alone reduced PPAR
2
protein level by 45% at 1 h and 4 h and by greater than 50%
at 24 h. When BRL 49653 was coincubated with TNF
, the effects
were similar to BRL 49653 alone; by 24 h PPAR
2 protein was
reduced by greater than 80%. The combination of the two agents did not
reduce the expression of PPAR
2 protein to a greater extent than
either one alone.
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The Arachidonic Acid Metabolite 15d-PGJ2
Has a Similar Mechanistic Effect to the Synthetic Agent BRL 49653
The arachidonic acid metabolite 15d-PGJ2 is a
naturally occurring ligand for PPAR2. The effects of
15d-PGJ2 on PPAR
2 protein expression were similar to the
efects of both TNF
and BRL49653. The 15d-PGJ2 compound
caused PPAR
2 to decline significantly (Fig. 5
). Also, 15d-PGJ2 or BRL 49653,
in combination with TNF
, caused a further decline in PPAR
2
protein expression compared with any of the three agents alone.
PPAR
1, which is also recognized by the PPAR
antibody, was
similarly decreased by 15d-PGJ2, BRL 49653, and TNF
(Fig. 5
, lower band). In summary, both 15d-PGJ2 and BRL
49653 decreased PPAR
2 protein expression at 24 h in
differentiated adipocytes, which is contrary to their previously
reported actions in preadipocytes (21).
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DISCUSSION |
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A direct relationship between high levels of adipocyte secretion of
TNF and the insulin resistance of obesity has been strongly
suggested (3, 22). BRL 49653 has been effective in ameliorating insulin
resistance in obese individuals. The exact mechanisms used by BRL 49653
are unknown, but it appears to have an effect on adipocyte metabolism,
and its antidiabetic actions appear to be mediated through PPAR
2
(23). The evidence connecting PPAR
2 activity to the antidiabetic
activities of the thiazolidinediones (TZDs) includes the data
showing that the TZDs with the greatest antidiabetic activity also
possessed the strongest binding affinity for PPAR
2. In addition,
non-TZD ligands with strong affinitity to PPAR
2 have also been shown
to possess antidiabetic activity (23). We therefore
examined the effects of TNF
and BRL 49653 on the expression of
PPAR
2 and other adipocyte genes.
In our run-on experiments, we also examined whether TNF or BRL 49653
alters the transcription of the transcription factors,
C/EBPß and C/EBP
. Recent experiments by Wu
et. al. (20) have shown that conditional expression of
C/EBPß and C/EBP
in the presence of
steroid directly stimulates the appearance of PPAR
2, and that if the
stimulus for C/EBPß and C/EBP
expression is removed, the levels of
expression of PPAR
2 and aP2 mRNA subsequently fall. The run-on
experiments indicated that neither TNF
nor BRL 49653 decreased
C/EBP
/
transcription levels significantly. Thus, our data suggest
that the actions of TNF
and BRL on PPAR
2 expression are probably
not mediated through C/EBPß/
in differentiated adipocytes.
In our experiments with 3T3-L1 adipocytes, BRL 49653 prevented the
TNF- associated decrease in perilipin A mRNA and protein expression.
Interestingly, BRL 49653 was more effective at counteracting the
effects of TNF
on perilipin protein expression at 24 h of
treatment than it was at 6 h, suggesting that BRL 49653 may need
time to overcome the actions of TNF
. BRL 49653 did not, however,
prevent the TNF
-associated decrease in PPAR
2 expression. In fact,
TNF
and BRL 49653 together decreased PPAR
2 mRNA expression to a
greater extent than either agent alone. It is also possible that the
PPAR
2 mRNA is degraded faster than the PPAR
2 protein, which may
be more stable during the longer treatments. Recently, Wu et.
al. (24) reported that, in adipocyte-like cells, the induction of
GLUT 4 mRNA expression (the insulin-dependent glucose transporter) by
PPAR
2 required 2448 h of exposure to the TZD, ciglitazone,
suggesting that the effects of PPAR
2 are not immediate and occur
over a period of time.
In addition to altering the transcription of several adipocyte genes,
TNF has also been shown to exert posttranscriptional control by
increasing mRNA turnover (25, 26). The amount of time it takes BRL
49653 to work maximally may be the result of its actions at may
different levels, some transcriptional and some posttranscriptional.
Surprisingly, BRL 49653 was less effective in preventing TNF
from
decreasing perilipin protein (but not mRNA) at 72 h than 24
h, even though PPAR
2 protein expression was elevated compared with
its 24 h level, suggesting that increased amounts of PPAR
2 do
not necessarily lead to an increase in BRL 49653 activity.
Since both TNF and BRL 49653 decrease PPAR
2 expression, yet have
disparate effects on the expression of other adipocyte-specific genes,
we speculate that a low level of PPAR
2 protein expression may be
sufficient for activity. The ability of PPAR
2 to exert its actions
on gene expression in the presence of one of its ligands,
i.e. BRL 49653, may depend on factors other than the amount
of PPAR
2 present.
Recent studies have also suggested that phosphorylation of PPAR2 by
kinases such as mitogen-activated protein kinase reduces its ability to
bind to its target DNA binding sequences, and therefore reduces its
activity (27). TNF
is a weak activator of mitogen-activated protein
kinase, and the ability of PPAR
2 to bind to its target DNA sequence
may be decreased by phosphorylation as a result of the actions of
TNF
in adipocytes (27). BRL 49653 may then overcome this block. The
reduction in mRNA and protein levels of PPAR
2 and other
adipocyte-specific genes after TNF
treatment may be important for
the antiadipogenic effects of TNF
that could be due to changes in
complex interactions secondary to phosphorylation (9).
Recently, a coactivator of PPAR2, called mouse steroid receptor
coactivator-1 (mSRC-1), has been isolated. This coactivator binds to
PPAR
2 in a ligand-independent fashion. When mSRC-1 is coexpressed
with PPAR
2, and a PPAR
2 ligand is present, the transcriptional
activity of mPPAR
2 increases (28). One may speculate that BRL 49653
is a potent stimulator of this coactivator.
In summary, the work presented in this paper indicates that both TNF
and BRL 49653 decrease the transcriptional and translational regulation
and expression of PPAR
2. However, BRL 49653 antagonizes the actions
of TNF
on the expression of other adipocyte-specific genes and,
alone, induces expression of these genes. Understanding the
interactions between TNF
and BRL 49653 on adipocyte gene expression
may help us determine how the thiazolidinediones enhance insulin
sensitivity in vivo.
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MATERIALS AND METHODS |
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Reagents
TNF was purchased from Genzyme Corp. (Cambridge, MA). BRL
49653 was originally purchased from Biomol Corp. (Plymouth Meeting, PA)
but recently was generously provided by Hamish Ross from SmithKline
Beecham Pharmaceuticals (King of Prussia, PA). 15d-PGJ2 was
purchased from Cayman Chemical (Ann Arbor, MI).
RNA Analysis
RNA was isolated using TRIZOL (GIBCO/BRL Life Technologies,
Gaithersburg, MD) following the instructions provided by the
manufacturer. Briefly, the 3T3-L1 cells were rinsed twice with PBS and
3 ml of TRIZOL were added to each 100-mm tissue culture plate. The
cells were scraped, transferred to a 15-ml tube, and allowed to
incubate for 5 min at room temperature. Then, 0.6 ml of chloroform was
added, and the tubes were shaken vigorously and centrifuged for 10 min
at 4 C. The aqueous phase was removed, and the RNA was precipitated
with isopropyl alcohol and centrifuged for 20 min at 4 C. The pellet
was washed with 70% ethanol, dried, and dissolved in sterile,
distilled water. Northern blot analysis was performed using HYBOND-N
nylon transfer membrane (Amersham, Arlington Heights, IL), which was
cross-linked, incubated in Express Hyb hybridization solution
(CLONTECH, Palo Alto, CA), and hybridized to cDNA probes labeled with
[32P]dCTP using the random primer method (29).
Probes
The cDNA probes used were: murine perilipin A, a generous gift
from the laboratories of Drs. Jasmine Gruia-Gray, Alan Kimmel, and C.
Londos; murine aP2 was kindly provided by Dr. David Bernlohr; rat HSL
was kindly provided by Drs. Cecilia Holm and Michael Schotz; C/EBPß
and C/EBP were kindly provided by Dr. Steve Farmer; ß-actin was
kindly provided by Dr. Phil Pekala; and murine PPAR
was supplied by
Drs. Peter Tontonoz and Bruce Spiegelman. The PPAR
probe recognizes
both PPAR
1 and
2; however, the predominant isoform expressed in
3T3-L1 adipocytes is
2.
Two mPPAR2 antibodies were used: one was the generous gift of
Dr. Mitchell Lazar and the other was purchased from Affinity
Bioreagents (Golden, CO). Both gave similar results.
Western Analysis
For perilipin Western blots, 50 µg of protein from 3T3-L1
adipocytes were separated on a 10% SDS polyacrylamide gel, and
transferred to nitrocellulose using a semidry transfer apparatus from
Owl Scientific (Woburn, MA). The membrane was blocked overnight in Tris
buffer containing 5% BSA, incubated with the same, fresh buffer
containing the primary antibody for 1 h, washed, incubated with
the horseradish peroxidase-linked secondary antibody, and subjected to
enhanced chemiluminescence (ECL) from Amersham. Autoradiography was
then performed. With the mPPAR2 antibody, nonfat dried milk replaced
the BSA as a blocking agent to avert the formation of background bands
resulting from the primary antibody cross-reacting with BSA.
Also, 100150 µg of total protein were loaded in each lane since the
PPAR
2 protein is much less abundant than perilipin (30).
Perilipin antibodies were generated using a peptide that is identical to amino acids 923 of the rat perilipin (31) (CLLDGDLPEQENVLQ) to immunize rabbits (Quality Controlled Biochemicals, Inc., Hopkinton, MA). The serum was subsequently affinity purified with a peptide column and used at a dilution of 1:1500. For HSL, a peptide KDLSFKGNSEPSDSPEMC, based upon the rat (GeneBank) HSL sequence, was used to immunize rabbits (QCB). The antisera were subsequently affinity purified over a peptide column and used for Western blotting at a dilution of 1:1500.
Nuclear Run-On Analysis
This procedure was performed following the protocol of Greenberg
and Ziff (32) with minor modifications. Briefly, cells were washed with
ice-cold PBS, scraped, and lysed in buffer containing 0.5% NP-40, 10
mM Tris, 10 mM NaCl, 3 mM
MgCl2, 0.001 M dithiothreitol, and 0.25
M sucrose. The nuclei were pelleted, washed once with lysis
buffer without NP-40, and quickly frozen in glycerol storage buffer in
liquid N2 until needed. The nuclei were then thawed on ice
and incubated with an equal volume of 2x reaction buffer containing
cold ribonucleotides and 100 µCi [-32P]UTP for 30
min at 30 C. The radioactive RNA was isolated through a series of
purification steps and then hybridized to cDNA plasmids (1015
µg/slot) that had been previously slot blotted onto a positively
charged nylon membrane. The hybridization was allowed to continue for 2
days, and the membranes were then washed and exposed to x-ray film, or
the radioactivity was detected on the PhosphorImager. The film
was then scanned with a Personal Densitometer from Molecular Dynamics
(Sunnyvale, CA) and was analyzed using Imagequant software provided by
the manufacturer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grants T32DK-07704 and P30 DK-40561.
In preliminary studies, S. C. Souza and A. S. Greenberg in our laboratory have found that 5-day treatment of 3T3-L1 cells with TNF plus BRL increases the protein expression of perilipin A approximately 2-fold compared to TNF alone.
Received for publication June 12, 1997. Revision received April 17, 1998. Accepted for publication April 23, 1998.
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REFERENCES |
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