From the
Whitehead Institute for Biomedical
Research, Cambridge, Massachusetts 02142, the
Section of Atherosclerosis and Lipoprotein
Research, and Program of Cardiovascular Sciences, the Department of Medicine,
Baylor College of Medicine and The Methodist Hospital, Houston, Texas 77030,
and the ||Department of Biology, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02142
Received for publication, March 26, 2003 , and in revised form, May 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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Treatments of type 2 diabetes, such as correcting relative insulin deficiency, inhibiting hepatic glucose production, and delaying glucose absorption from the gastrointestinal tract, lower plasma glucose levels but do little to improve insulin sensitivity. In time, these interventions often fail to restore metabolic homeostasis and to prevent the development of most of the complications of type 2 diabetes. Thus, there remains a great need to restore insulin responsiveness in the clinical management of type 2 diabetes.
Thiazolidinediones
(TZD),1 a class of
anti-diabetic medications and synthetic ligands for PPAR, decrease
plasma free fatty acid concentrations as well as fasting and postprandial
plasma glucose levels in patients with type 2 diabetes by improving insulin
sensitivity in major insulin-target tissues. In addition, TZD reduce plasma
triglyceride levels, improve the plasma lipoprotein profile, lower blood
pressure in diabetic hypertensive patients, and correct the proinflammatory
and procoagulant state (5).
Taken together, TZD target insulin resistance and restore metabolic
homeostasis while improving the cluster of abnormalities that occur in type 2
diabetes. However, the direct target tissue(s) of TZD and the molecular
mechanism(s) by which TZD sensitize the major insulin-responsive tissues
in vivo remain elusive.
TZD are high affinity ligands for peroxisome proliferator receptor
activator- (PPAR-
). PPAR-
has two protein isoforms,
PPAR-
1 and PPAR-
2
(6,
7). Whereas PPAR-
1 is
ubiquitously expressed at low levels in many tissues including muscle,
PPAR-
2 is most highly expressed in adipose tissue
(8,
9). Notably, adipose tissue is
not only essential for maintaining the overall in vivo insulin
sensitivity but is also a source of endocrine activity that modulates several
physiological processes that include systemic energy metabolism, inflammation,
blood coagulation, blood vessel tone, and reproduction. A number of
adipocyte-derived factors have been implicated in insulin resistance in
obesity and obesity-linked type 2 diabetes. Thus, it is likely that TZD could
directly target molecular mediator(s) of insulin resistance in adipocytes and
restore the metabolic function and endocrine signals of adipose tissue, and
thereby contribute to the improved systemic insulin sensitivity.
Tumor necrosis factor- (TNF-
), an autocrine/paracrine factor
that is highly expressed in adipose tissues of obese animals and human
subjects, is a potential molecular mediator of insulin resistance that is of
physiological importance. Although many factors may trigger the development of
insulin resistance in humans
(10), TNF-
-regulated
autocrine or paracrine pathways in adipose tissue have been implicated in
mediating the metabolic consequences of obesity to cause insulin resistance
(11). One proposed mechanism
for TNF-
induction of insulin resistance involves inhibition of
insulin-stimulated tyrosine phosphorylation of the insulin receptor and
insulin receptor substrate-1
(1214).
More recently, the inhibitor
B kinase-
has been implicated in
this process (15). However,
other mechanisms by which TNF-
induces insulin resistance have also
been described, including down-regulation of protein levels of insulin
receptor substrate-1 (16),
GLUT4 (16), CEBP-
(17), PPAR-
(18), perilipin
(19,
20), and ACRP30
(2123)
in adipocytes.
We recently substantiated the critical role of TNF--regulated gene
expression in adipocytes in the development of systemic insulin resistance by
association of gene expression profiles in major insulin-responsive tissues
with overall in vivo insulin sensitivity in rats infused with
TNF-
(24). We also
demonstrated that NF-
B activation by TNF-
is obligatory in the
repression of key adipocyte genes and induction of many proinflammatory and
acute phase proteins (22).
These data assert the importance of TNF-
and TNF-
-induced
NF-
B activation in the etiology of insulin resistance in
adipocytes.
It is well established that TZD counteract a number of effects of
TNF- on adipocytes, including adipocyte differentiation
(25) and gene expression
(17,
18,
22,
26,
27), insulin signaling
(28), insulin-stimulated
glucose uptake (26),
lipogenesis (29), and
lipolysis (19). Yet the
molecular mechanisms for these effects of TZD are not known. Interestingly,
TZD-induced PPAR-
activation in monocytes or macrophages suppresses the
induction of many inflammatory and immune response mediators such as
TNF-
, IL-1
, IL-6, metalloproteases, and inducible nitric-oxide
synthase in part by inhibiting the activities of NF-
B, signal
transducers and activators of transcription, and AP-1
(30,
31). In addition, PPAR-
agonists inhibit cytokine-induced VCAM-1 and IL-6 expression in endothelial
cells and vascular smooth muscle cells through interference with NF-
B
and AP-1 action by protein-protein interactions and cofactor squelching
(32,
33). However, experimental
data demonstrating a functional interaction between NF-
B and
PPAR-
in the context of adipocytes are lacking.
Herein we have used a number of complementary experimental approaches to
determine whether ligand-induced PPAR- activation can override the
transcription-repressive effects of NF-
B activation by TNF-
on
key adipocyte genes, and whether NF-
B family members can directly
inhibit the transcriptional regulatory activity of PPAR-
.
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EXPERIMENTAL PROCEDURES |
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Identification of adipocyte-abundant genes was determined by selecting those with 3-fold or higher expression levels in adipocytes than fibroblasts. We then excluded genes expressed at a very low level by setting an arbitrary threshold value for the array measurements (Average Differences, according to Affymetrix), and genes whose expression levels were below 150 at all time points and under all conditions were excluded. We also excluded genes whose differences in expression levels (maximal value-minimal value) between any two time points were less than 150. Thus, these filters allow us to include adipocyte-abundant genes that exhibit robust changes in steady state mRNA levels in response to at least one treatment.
Next we used a modified score system, originally described by Hacohen and
co-workers (36), to identify
genes regulated by TNF-, troglitazone, or both. Briefly, let
Ri and Ci be the
steady state mRNA levels of treated samples and control samples, respectively,
at the ith time point. Define µC to be the
mean expression level of samples of the control time course and
C as the standard deviation of expression levels in
the control samples. Then we can define a score,
Si = (Ri
µC)/
C, to measure the
statistical significance of the changes in gene expression in the treated
samples at each time point Ri. Genes with low
scores are a consequence of large variation in mRNA levels in the control time
course (high noise) or small differences between the control and treated
samples. By setting a threshold value for Si (see
below), we can exclude genes whose expression levels were not significantly
affected by a treatment or fluctuated over time under control conditions.
Identification of up-regulated genes was determined by requiring one of the
following: 1) Si 4 for at least 1 time
point; 2) Si
1.4 for at least two
consecutive time points. Down-regulated genes were selected by requiring
Si
1.4 for at least three consecutive
time points or Si
3 for at least one
time point. We used this score system to identify TNF-
-regulated
transcripts from the 175 adipocyte-abundant known genes, and we then compared
the list of identified genes with our master list of 64 TNF-
-affected
adipocyte-enriched genes that have been verified previously by Northern
Blotting, semi-quantitative RT-PCR, or literature search
(22,
24). The score system
identified 102 TNF-
-affected genes
(Table I) including 51 genes
from the master list. The scores of the rest of the 13 master list genes are
very close to but did not pass the score threshold and were excluded from the
TNF-
-regulated gene list in this study. The 13 genes are as follows: CD
36; lactate dehydrogenase 2; apoE; sterol carrier protein 2;
11-
-hydroxysteroid dehydrogenase; cytochrome P450; carbonic anhydrase 3;
catalase; adenylate kinase isozyme 3; complement component C3; amyloid
(A4) precursor-like protein 2; GADD 45; and Rho B. This indicates that the
score system might give false-negative results. However, the inclusion of
these 13 genes would not have changed our interpretation of the results, and
we therefore used this score system to assess genes regulated by other
treatments.
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Fold changes in gene expression in response to a treatment relative to control were calculated as the following: Fup = max{L1, L2,... Ln-1} for genes up-regulated by a treatment, and Fdown = 1/min{L1, L2,... Ln-1} for down-regulated genes, where Li = geomean{Ri Ri+1}/geomean{Ci Ci+1}.
Oligonucleotide microarray data were also collected from wild-type 3T3-L1
adipocytes and adipocytes expressing a dominant negative inhibitor of
NF-B, I
B-
-DN, treated with TNF-
(1 nmol/liter) for
0, 0.5, 1, and 2 h (22).
Identification of TNF-
-repressed genes was determined by requiring one
of the following: 1) mRNA level decreases 30% or more for at least two
consecutive time points; and 2) mRNA level decreases 50% or more at the end of
the 2-h incubation.
Tissue Culture3T3-L1 cells were purchased from ATCC (Manassas, VA), maintained as fibroblasts, and differentiated into adipocytes as described previously (37). HeLa cells were provided by Dr. C. C. Zhang (Whitehead Institute, Cambridge, MA), and were propagated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
Western Blot Analysis3T3-L1 adipocytes were incubated in
growth media or media containing TNF- (1 nmol/liter), troglitazone (1
µmol/liter), or both together for various times. Cell lysates were
separated by SDS-PAGE and electroblotted onto a nitrocellulose membrane
(Amersham Biosciences). The filter was incubated with various primary
antibodies, washed, and incubated with the appropriate horseradish
peroxidase-conjugated secondary antibody. Bound antibodies were detected using
the enhanced chemiluminescence Western blotting analysis system (Amersham
Biosciences). Blots were stained with Ponceau S solution to visualize the
amount of total protein in each lane.
PlasmidsThe reporter gene PEPCK-Luc was generated by
subcloning the 2100-bp fragment of the PEPCK gene promoter
(38) (provided by Dr. D.
Granner, Vanderbilt, TN) into pTAL-Luc (Clontech, La Jolla, CA). The
NF-B-responsive luciferase reporter gene was from Clontech. The
PPAR-
-responsive luciferase reporter gene (PPRE-luc) was constructed by
subcloning 6 tandem repeats of PPAR-
-response elements into the
pTAL-luc vector. The expression plasmids p65 (RelA), p65 (S276A) mutant, and
p50 were provided by Dr. D. Granner
(39); c-Rel was a gift from
Dr. W. Tam (Whitehead Institute, Cambridge, MA). Murine full-length
PPAR-
2 was subcloned into pIRES2-GFP expression vector (Clontech), and
the sequence was verified by DNA sequencing.
Transfection AssaysAll transfections were performed using
Fu-GENE 6 (Roche Applied Science) according to the manufacturer's
instructions. Forty eight hours after transfection, cell lysates were prepared
and analyzed for luciferase activity using a kit from Promega (Madison, WI).
The luciferase activity was normalized using an internal CMV--Gal
control plasmid. All experiments were done in triplicate and were repeated at
least three times.
ELISA3T3-L1 adipocytes were incubated in growth media or
media containing TNF- (1 nmol/liter), troglitazone (1 µmol/liter),
or both together, for the indicated times. Nuclear extracts were prepared and
assayed for NF-
B activities using an ELISA kit (Active Motif, Carlsbad,
CA) following the manufacturer's instructions. Briefly, the NF-
B
consensus sequence (5'-GGGACTTTCC-3') has been immobilized on a
96-well plate. Various nuclear extracts were then added to the plate and
incubated for1hat room temperature. The plate was washed and incubated with
anti-p65 antibody for 1 h. Following a thorough wash and incubation with a
horseradish peroxidase-conjugated secondary antibody, the plate was washed
extensively and developed, and the A450 was determined by
spectrophotometry. The specificity of the ELISA was verified by measuring the
binding of p65 to the ELISA plate in the absence or the presence of a
saturating amount (20 pmol) of oligonucleotides containing either wild-type or
mutant NF-
B consensus sequences.
Statistical AnalysisComparisons were performed using a
two-tailed Student's t test assuming unequal variances. p
0.05 was considered significant.
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RESULTS |
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TNF- treatment for up to 24 h significantly altered the expression
levels of 102 adipocyte-abundant genes (58%, see
Fig. 1 and
Table I). Among them, the mRNA
levels of 86 genes (84%) were repressed by TNF-
, and the expressions of
16 genes (16%) were highly induced by TNF-
treatment
(Fig. 1 and
Table I). In contrast, TGZ had
only a moderate effect on the expression of adipocyte-abundant genes
(Fig. 1 and
Table I). Strikingly, the
presence of TGZ decreased the maximal fold repression of 78 genes (91%) by
TNF-
over a period of 24 h. Of these 78, 64 genes showed at least a 50%
increase in mRNA levels compared with those in cells treated with TNF-
alone for 24 h (Table I, in
boldface type). We then assessed the expression levels of the 64 genes that
are protected by TGZ from TNF-
-induced down-modulation
(Table I, boldface type) in
adipocytes expressing a dominant inhibitor of NF-
B activation
(I
B
-DN) (22) in
the absence or the presence of TNF-
. We found that the mRNA levels of
28 genes were significantly repressed by TNF-
in control 3T3-L1
adipocytes after 2 h of incubation, whereas the expression levels of these
genes were unaffected by TNF-
in cells expressing I
B
-DN
(Table I, underlined). The mRNA
levels of the rest of the 36 genes were not significantly repressed by
TNF-
in control cells during the 2-h incubation. Because TNF-
induced extensive apoptosis in adipocytes expressing I
B
-DN after
2 h of incubation, we could not determine whether the repression of the other
genes by TNF-
depended on NF-
B. However, the steady state mRNA
levels of many of the 36 genes increased significantly in cells expressing
I
B
-DN, indicating a potential NF-
B-mediated inhibitory
effect on the expression of these genes. Thus, the repressive effect of
TNF-
on adipocyte gene expression was NF-
B-dependent, and TGZ
may protect adipocyte genes that are essential for adipocyte function through
inhibition of NF-
B.
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To assess the potential impact of the antagonistic actions between TGZ and
TNF- on adipocyte biology, we grouped the genes that are differentially
regulated by TGZ and TNF-
according to the biological functions of
their encoded proteins (Table
I). These are discussed below.
Troglitazone Prevents TNF--induced Down-regulation of
Adipocyte Genes Implicated in the Suppression of Free Fatty Acid Release from
3T3-L1 AdipocytesIncreased free fatty acid release from adipocytes
is a key feature in type 2 diabetes. The balance between cellular triglyceride
(TG) synthesis, free fatty acid re-esterification, and lipolysis determines
the amount of FFA release from adipocytes
(40,
41). As we reported previously
(22,
24), TNF-
significantly
repressed adipocyte-abundant genes implicated in the suppression of FFA
release from adipocytes. Among them, glycerol-3-phosphate acyltransferase
(Fig. 2A, filled
circles), 1-acylglycerol-3-phosphate acyltransferase
(Fig. 2B, filled
circles), diacylglycerol acyltransferase
(Fig. 2C, filled
circles), and long chain fatty acyl-CoA synthase
(Fig. 2D, filled
circles), are enzymes essential for cellular TG synthesis.
Phosphoenolpyruvate carboxykinase (PEPCK,
Fig. 2E, filled
circles), also down-regulated by TNF-
, generates glycerol
3-phosphate, a 3-carbon glycolytic intermediate essential for long chain fatty
acyl CoA incorporation into TG. Although sufficient glycerol 3-phosphate can
be generated from glucose, during the fasting state or lipolysis in adipocytes
the supply of glucose is significantly reduced, and glycolysis is inhibited.
Thus, the PEPCK-dependent glycerol 3-phosphate production becomes especially
important because fat cells lack the enzyme glycerol kinase that
phosphorylates glycerol to generate glycerol 3-phosphate. The supply of
glycerol 3-phosphate is critical for maintaining adipocyte TG synthesis, which
limits the amount of free fatty acids released into plasma. Pyruvate
carboxylase (Fig. 2F)
is also involved in cellular lipogenesis. Transaldolase
(Fig. 2G) and malic
enzyme (Fig. 2H)
generate NADPH required for lipid biosynthesis. S3-12
(Fig. 2I) was
originally identified as a plasma membrane-associated protein that is highly
induced upon 3T3-L1 adipocyte differentiation
(42). Although the exact
function of S3-12 remains unclear, it contains the signature domain found in
the perilipin family of lipid droplet-associated proteins and thus may
potentially interact with cellular fat droplets as well. Adipose
differentiation-related protein (Fig.
2J) is also a member of the perilipin family and is
highly induced upon adipocyte differentiation. The perilipin family of
proteins has been implicated in interfering with the interaction between lipid
droplets and cellular lipid hydrolases and thus reducing hormone-sensitive
lipase (HSL)- or other TG hydrolase-mediated TG hydrolysis
(19,
20,
43). These proteins act
together favoring cellular TG synthesis and suppressing FFA mobilization and
release from adipocytes, and are all repressed by TNF-
treatment.
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Whereas TGZ induces the mRNA levels of PEPCK
(Fig. 2E, open
circles) and S3-12 (Fig.
2I, open circles), it does not significantly
alter the expression of the rest of the genes discussed above
(Fig. 2, open
circles). However, TGZ prevented, at least partially, TNF--induced
and NF-
B-mediated repression of expression of all of these genes
(Fig. 2, filled
triangles). This suggests a possible molecular mechanism by which TGZ
antagonizes the actions of TNF-
and suppresses FFA production from
adipocytes. Notably, TGZ also blocked the down-regulation of mRNA levels of
HSL by TNF-
(Table I),
consistent with previous reports
(19,
44). HSL is an
adipocyte-specific protein that is induced 166-fold upon 3T3-L1 adipocyte
differentiation (Table I). The
HSL-mediated TG hydrolysis is mostly dependent on the regulation of its
activity by phosphorylation state rather than HSL protein mass. Thus the
down-regulation of HSL mRNA may not contribute, significantly, to the amount
of FFA released from fat cells. On the other hand, the down-regulation of HSL
mRNA by TNF-
and its prevention by TGZ supports our hypothesis that TGZ
and TNF-
antagonize each other in the regulation of adipocyte-abundant
genes.
Troglitazone Antagonizes the Effects of TNF- on
Expression of Secreted Protein and Genes Encoding Transcription Factors and
Signaling Molecules That Are Essential for Adipocyte Phenotype and
FunctionThe profile of secreted proteins changes dramatically upon
3T3-L1 adipocyte differentiation. Adipocytes secrete a variety of bioactive
molecules that affect multiple biological processes such as energy
homeostasis, appetite control, blood vessel tone, extracellular matrix
composition, blood coagulation, and immune and inflammatory responses. To
evaluate whether TGZ antagonizes the actions of TNF-
on the
endocrine/paracrine functions of adipocytes, we first examined the effects of
TGZ on genes encoding secreted proteins that were repressed by TNF-
. As
we reported previously (22),
TNF-
repressed the mRNA levels of ACRP30
(Fig. 3A), an
adipocyte hormone that sensitizes the actions of insulin in liver and muscle.
In addition, expression of angiotensinogen
(Fig. 3B), resistin
(Fig. 3C), and adipsin
(Fig. 3D), which are
highly induced upon 3T3-L1 adipocyte differentiation and regulate blood vessel
tone, energy homeostasis, and complement activation, respectively, were
repressed by TNF-
as well. The presence of TGZ reversed or partially
blocked the repressive effect of TNF-
on expression of these
adipocyte-derived hormones.
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Adipocyte differentiation is also associated with induction of many
transcription factors and proteins that are essential for adipocyte phenotype
and response to insulin. TNF- down-regulated the mRNA levels of several
lipogenic and adipogenic transcription factors such as CEBP-
(Fig. 3E, filled
circles), PPAR-
(Fig.
3F, filled circles), and spot 14
(Table I), whereas TGZ potently
blocked the action of TNF-
on CEBP-
and spot 14 but not for
PPAR-
, whose mRNA level was further repressed in the presence of TGZ
(Fig. 3F, open
circles). This is consistent with a previous report
(45) in which rosiglitazone,
another member of the thiazolidinediones family, down-regulates PPAR-
gene expression in fully differentiated 3T3-L1 adipocytes. The significance of
this observation is not known, and yet troglitazone did cause PPAR-
activation as evidenced by the robust induction of several PPAR-
target
genes including PEPCK (Fig.
2E). In addition, TNF-
repressed many genes
encoding proteins implicated in cell signaling, such as c-Cbl-associated
protein (Cap, Fig.
3G), which is implicated in insulin-stimulated glucose
transport, ADP-ribosylation factor-like 4
(Fig. 3H), and
homeodomain-interacting protein kinase 2 and 3
(Table I). TGZ prevented the
down-regulation of c-Cbl-associated protein and partially blocked the
repression of many signaling molecules
(Table I).
Troglitazone Blocks TNF--mediated and
NF-
B-dependent Repression of Genes Implicated in Cell Cycle
ArrestUpon 3T3-L1 adipocyte differentiation, the expression of
many preadipocyte-derived growth factors such as proliferin, mitogen-regulated
protein/proliferin 3, and fibroblast growth factor 7 are shut
down,2 whereas many
anti-proliferative proteins including Cdk4 and Cdk6 inhibitor p18 and
G0S2-like protein are highly induced (Table
I). As a result, terminally differentiated adipocytes virtually
cease proliferation and completely withdraw from cell cycle progression.
TNF-
treatment of 3T3-L1 adipocytes strongly induced pre-adipocyte
growth factors and repressed anti-proliferative proteins that constrain cell
cycle progression including G0S2-like protein, transducer of ERBB2, p18, and
apoptosis-associated tyrosine kinase (Table
I). TGZ potently blocked the repression of these genes by
TNF-
(Table I). Because
NF-
B activation is obligatory for TNF-
repression of these
adipocyte genes, the impaired transcription repressive activity of NF-
B
in the presence of TGZ specifically indicates that TGZ-induced PPAR-
activation may directly target NF-
B in adipocytes treated with
TNF-
.
Troglitazone Modulates the Induction of Gene Expression in Adipocytes
by TNF-To determine whether TNF-
-mediated
induction of gene expression in adipocytes would be affected by TGZ treatment,
we examined the effects of troglitazone on the expression kinetics of
TNF-
-induced secreted factors. TNF-
treatment caused a robust
induction of many genes encoding cytokines, chemokines, acute phase reactants,
protease inhibitors, and antioxidants. Many of the TNF-
-induced
molecules such as macrophage colony-stimulating factor, monocyte chemotactic
protein 1 (MCP1, Fig.
4A), monocyte chemotactic protein 3 (MCP3,
Fig. 4B), vascular
cell adhesion molecule-1 (Vcam-1,
Fig. 4C), hemopexin,
ceruloplasmin (Fig.
4D), haptoglobin, Spi 2 proteinase inhibitor, and PAI-1
are risk factors for atherosclerosis
(4649),
indicating that adipocytes may be a significant contributor to these
characteristic features of type 2 diabetes. Strikingly, TGZ partially
prevented the induction of some of these pro-inflammatory genes
(Fig. 4, AD,
and Table I) but had no
significant effects on a number of genes induced by TNF-
, such as PAI-1
and haptoglobin (Table I).
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To determine the effect of TGZ on TNF- induction of
preadipocyte-abundant transcription factors and growth phase proteins, we
assessed the mRNA levels of high mobility group protein I-C (HMGI-C,
Fig. 5A), CEBP-
(Fig. 5B), and
Fos-like antigen-1 (Fra-1, Fig.
5C) following TGZ and/or TNF-
treatment.
TNF-
treatment rapidly induced these transcription factors
(Fig. 5,
AC, filled circles). Notably, TNF-
induced the expression of CEBP-
and Fra-1 in cells expressing
I
B
-DN to the same extent as in control
cells,2 indicating
that the induction of these genes by TNF-
is NF-
B-independent.
There was no immediate induction of HMGI-C mRNA in either control or
I
B
-DN cells after2hof TNF-
incubation, and thus we could
not determine whether the induction of HMGI-C depends on NF-
B. TGZ
alone also caused a transient and small increase in the mRNA levels of
CEBP-
(Fig.
5B, open circles) and Fra-1
(Fig. 5C, open
circles), but it did not affect the expression of HMGI-C, nor did it
impair the induction of their expression by TNF-
(Fig. 5,
AC, filled triangles).
|
Next, we assessed the effects of TGZ on the expression levels of three
immediate early genes, IB-
, p65, and NF-
B1 (p105/p50),
which are induced by TNF-
in an NF-
B-dependent manner
(Fig. 5, DF).
I
B-
is rapidly induced by TNF-
treatment, and its mRNA
levels increased 3.5- and 11-fold within 1 and 2 h of TNF-
treatment,
respectively (Fig.
5D). The steady state mRNA levels of I
B-
in
TNF-
-treated cells were 3.5-fold of the control cells at the end of the
24-h incubation. Interestingly, TGZ did not interfere with the induction of
I
B-
mRNA by TNF-
, as the mRNA levels as well as the
kinetics of I
B-
expression in response to TNF-
were
essentially identical in the presence or the absence of TGZ in a period of 24
h. In addition, TGZ alone did not affect the expression of I
B-
.
We then determined the effect of TGZ on the mRNA levels of two NF-
B
subunits, p65 and NF-
B1 (p105/p50). TNF-
induced medium-early
and sustained increase in the mRNA levels of both NF-
B subunits
(Fig. 5, E and
F). Notably, there was no significant effect on
NF-
B expression when TGZ was added together with TNF-
, and TGZ
alone did not alter basal NF-
B expression. Thus, our data indicate that
although TGZ generally blocks TNF-
-induced and NF-
B-mediated
repression of adipocyte-abundant genes, it interferes with only a subset of
genes induced by TNF-
in 3T3-L1 adipocytes.
Troglitazone Antagonizes TNF--induced Repression of
Adipocyte-abundant Genes by Inhibiting NF-
B Trans-repression
and Not by Interfering with NF-
B Activation, Nuclear
Translocation, or DNA Binding ActivityTo establish whether TGZ
protects the adipocyte-abundant genes from down-regulation by TNF-
through inhibition of NF-
B, we first examined the effect of TGZ on
protein levels of I
B-
and its phosphorylation state in the
absence or presence of TNF-
(Fig.
6A). Various NF-
B-inducing signals cause the
phosphorylation, poly-ubiquitination, and degradation of I
B proteins
and result in NF-
B activation. I
B-
is a major isoform of
the inhibitor of
B proteins (I
Bs), and is a well characterized,
NF-
B-induced immediate early gene. Thus, I
B-
is a
naturally occurring molecular marker of NF-
B activation. As shown in
Fig. 6A, TNF-
induced rapid reduction of I
B-
protein within 15 min. Also, no
phosphorylated I
B-
was detected 15 min after TNF-
addition, indicating complete degradation of any presumably phosphorylated
I
B-
proteins. After 30 min, I
B-
protein started to
re-accumulate and the newly synthesized I
B-
proteins continue to
increase 60 min after TNF-
addition. Some of the newly synthesized
I
B-
was also phosphorylated 30 and 60 min after TNF-
addition. In contrast, TGZ did not alter the phosphorylation states or the
amount of I
B-
proteins. Importantly, TGZ did not affect the
degradation, reaccumulation, or phosphorylation of I
B-
proteins
in response to TNF-
, consistent with the lack of effect of TGZ on the
kinetics of induction of I
B-
mRNA by TNF-
(Fig. 5D).
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To determine whether the DNA binding activity of NF-B would be
inhibited by TGZ, we used ELISA analysis of p65 activity in 3T3-L1 adipocytes
following TNF-
stimulation. p65 was chosen because it is a major
NF-
B family member that shows increased nuclear accumulation in
response to TNF-
in 3T3-L1 adipocytes
(22).
Fig. 6B shows that
upon TNF-
stimulation, p65 activity in nuclear extract increased
14.8-fold (p = 0.01) within 15 min and was maintained at the similar
level until the end of the 60 min of incubation. In contrast, TGZ alone did
not activate p65 nor did it impair p65 activation by TNF-
at all time
points examined. The specificity of the ELISA was confirmed by measuring p65
activity in the absence or the presence of a saturating amount (20 pmol) of
oligonucleotides containing either the wild-type or mutant NF-
B
consensus sequences. As shown in Fig.
6C, the wild-type competitive oligonucleotides completely
abolished p65 binding to the ELISA plate, whereas the p65 binding was
essentially unaffected by the same amount of competing oligonucleotides with
mutant NF-
B consensus sequences.
We then examined the effect of TGZ-induced PPAR- activation on the
transcription regulatory activity of p65 using reporter gene assays in HeLa
cells. Fig. 7A shows
that ectopic expression of p65 induced the expression of a luciferase reporter
gene driven by multiple copies of NF-
B response elements
(NF-
B-luc) in a dose-dependent manner, with a maximal induction of
10.5-fold (p = 0.005). p50 was ineffective (data not shown). To test
whether TGZ-induced PPAR-
activation would interfere with p65-mediated
gene transcription, we co-transfected HeLa cells with NF-
B-luc, p65
(0.1 or 0.3 µg), together with PPAR-
(0.3 µg) or the control
vector (Fig. 7B).
Transfected HeLa cells were then incubated with TGZ or Me2SO for 48
h. p65 (0.1 µg) induced transcription of the NF-
B-luc promoter
9.1-fold (p = 0.003), whereas PPAR-
abolished induction by
p65, and the inhibition was enhanced by the addition of TGZ (1 µmol/liter).
Increasing the amount of p65 to 0.3 µg was able to partially override the
inhibitory effect of PPAR-
both in the absence and the presence of TGZ
(Fig. 7B). Notably,
PPAR-
inhibits the basal activity of NF-
B-luc in HeLa cells,
indicating that PPAR-
targets the endogenous NF-
B activity as
well (Fig. 7B). The
inhibition of the NF-
B-luc promoter transcription by PPAR-
was
specific to p65, because little effect was seen with a reporter gene driven by
a basic TATA box promoter or the cytomegalovirus promoter (data not shown).
Taken together, these data indicate that TGZ does not inhibit NF-
B
activation, nuclear translocation, or gene expression but is consistent with a
potential function for TGZ in antagonizing the transcriptional regulatory
activity of NF-
B and/or other cofactors of NF-
B on adipocyte
gene transcription.
|
p65 Inhibits PPAR--dependent Gene
Transcription PPAR-
2 is essential for the expression of
adipocyte-specific genes. On the other hand, NF-
B activation is
required for TNF-
-induced repression of key adipocyte genes. To confirm
that NF-
B could directly inhibit PPAR-
-dependent
adipocyte-specific gene transcription, we assessed the effects of p65 on
PPAR-
-mediated gene expression using reporter gene assays in HeLa
cells. As shown in Fig.
8A, transcription of a luciferase reporter gene driven by
the 2100-bp PEPCK promoter containing a functional PPAR-
-response
element (PEPCK-luc) (50) was
induced by TGZ-activated PPAR-
in a dose-dependent manner, with a
maximal induction of 11.5-fold (p < 0.02). PPAR-
was
ineffective in the absence of TGZ. Similar induction was observed when a
reporter gene driven by multiple copies of PPAR-
-response elements
(PPRE-luc) was used (Fig.
8B). To determine the involvement of individual
NF-
B family members in PPAR-
-mediated transcription, we
co-transfected HeLa cells with PEPCK-luc, p50, p65, p65 (S276A) mutant, c-Rel,
or the control vector. Fig.
8C shows that p65 significantly inhibited the basal
activity of PEPCK-luc, indicating that p65 represses the endogenous
PPAR-
activity and/or other endogenous PEPCK promoter-inducing signals
(left panel). In contrast, p50, another NF-
B family member,
had little or no effect on basal transcription of PEPCK-luc, whereas c-Rel
caused a small but statistically significant inhibition of basal PEPCK-luc
transcription. Notably, the p65 (S276A) mutant, which contains a serine to
alanine mutation at amino acid residue 276 of p65 protein, inhibited the basal
PEPCK-luc activity less effectively than its wild-type counterpart. Previous
studies have established that phosphorylation of p65 on serine 276 by protein
kinase A is essential for p65 interaction with the transcription co-activator
CBP/p300 (51,
52); this interaction enhances
the transcriptional activity and efficiency of p65. Importantly, CBP/p300 has
also been implicated in positively regulating PPAR-
- and other nuclear
receptor-mediated gene transcription
(53). Our data thus indicate
that the association of p65 with CBP/p300 is likely to be involved in the
inhibitory effects of p65 on basal PEPCK-luc transcription.
|
Next, we examined the effects of the above NF-B family members on
PPAR-
-mediated gene transcription. Transcription of the PEPCK promoter
was induced 3.3-fold (p = 0.004) by PPAR-
in the presence of
TGZ, whereas PPAR-
was ineffective in the absence of TGZ
(Fig. 8C, right
panel). p65 abolished induction of the PEPCK promoter by activated
PPAR-
, whereas c-Rel and p50 only partially blocked induction by
activated PPAR-
(Fig.
8C, right panel). The S276A mutant of p65
inhibited induction of the PEPCK promoter by activated PPAR-
less
effectively than did wild-type p65 but did retain some inhibitory activity.
The repression by p65 was specific to the PEPCK promoter, as reporter genes
driven by a TATA-like promoter or the cytomegalovirus promoter were not
significantly affected by p65 (data not shown), and p65 inhibited
PPAR-
-mediated transcription in a dose-dependent manner
(Fig. 8D).
To determine whether p65 inhibits PEPCK-luc transcription by directly
binding to the NF-B-response element(s) in the 2100-bp PEPCK promoter
region, we constructed a PPAR-
-responsive luciferase reporter gene
driven only by multiple copies of PPAR-
-response elements (PPRE-luc).
As shown in Fig. 8E
(left panel), p65 abolished basal PPRE-luc transcription, whereas
c-Rel and the S276A mutant of p65 partially blocked the basal PPRE-luc
transcription. p50 was ineffective. PPAR-
up-regulated the PPRE-luc
promoter 2.1-fold (p = 0.001) in the absence of TGZ, and PPRE-luc was
induced 3.4-fold (p = 0.0001) by PPAR-
when TGZ is present
(Fig. 8E, right
panel). When co-transfected with PPAR-
, p65 prevented induction of
the PPRE-luc promoter by PPAR-
both in the absence and presence of TGZ
(Fig. 8E, right
panel). In contrast, the S276A mutant of p65 blocked the induction of
PPRE-luc by activated PPAR-
but had little effect on
PPAR-
-mediated PPRE-luc transcription in the absence of TGZ. p50 and
c-Rel inhibited PPAR-
-mediated transcription less effectively than p65.
Thus, a functional NF-
B-binding site was not required for the
repressive effects of NF-
B on PPAR-
-dependent gene
transcription.
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DISCUSSION |
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TGZ improves insulin sensitivity in patients with type 2 diabetes and
antagonizes the actions of TNF- both in cell culture
(19) and in whole animals
(54). By using 3T3-L1
adipocytes expressing the non-degradable NF-
B inhibitor
I
B
-DN, we previously showed that NF-
B activation is
obligatory for TNF-
-induced repression of most of adipocyte-specific
genes, whereas only 6070% of the genes induced by TNF-
are
NF-
B-dependent (5).
Those investigations demonstrated that NF-
B-mediated transcriptional
inhibition is a mechanism by which TNF-
induces insulin resistance in
adipocytes. The seemingly opposing effects of TGZ and TNF-
on adipocyte
biology prompted us to look at their potential interactions on the expression
of adipocyte-abundant genes.
Here we identified 64 known adipocyte-abundant genes that are normally
repressed by TNF- in an NF-
B-dependent manner but are protected,
at least partially, by TGZ. These genes form clusters with distinct biological
functions (Table I), suggesting
that TGZ improves insulin sensitivity in part through antagonizing the actions
of TNF-
on adipocyte gene expression. For example,
Fig. 2 shows a selected group
of genes encoding proteins implicated in triglyceride synthesis and/or
suppression of FFA release from adipocytes. Although in many cases TGZ only
partially prevented the down-regulation of the mRNA levels of these genes by
TNF-
, their encoded proteins reside in a common pathway of FFA
metabolism and lipogenesis. Thus, within adipocytes, the aggregate effect of
even a moderate increase in each protein has a significant impact that
increases FFA incorporation into triglyceride and thereby reduces FFA
release.
Another group of genes regulated simultaneously by TGZ and TNF-
encodes proteins involved in cell growth and proliferation. Terminally
differentiated adipocytes permanently withdraw from cell cycle progression.
TNF-
-mediated reprogramming of adipocyte gene expression includes
induction of genes involved in cell cycle reentry and progression and
NF-
B-dependent repression of genes implicated in cell cycle arrest. TGZ
is sufficient to prevent this loss of cell cycle constraints through
antagonizing the transcriptional repressive activity of NF-
B on cell
cycle inhibitors and thus contributes to the maintenance of the adipocyte
phenotype.
Although TNF--induced repression of adipocyte-specific genes are
mediated mainly through NF-
B
(22), the induction of gene
expression by TNF-
in adipocytes is mediated by multiple mediators
through various pathways that may or may not be equally affected by TGZ. For
example, TGZ partially blocked the induction of acute phase proteins and
atherosclerotic risk factors that are normally induced by TNF-
(Fig. 4), whereas it had no
effect on the induction of I
B-
(Fig. 5D), p65
(Fig. 5E),
NF-
B1 (p105/p50, Fig.
5F), and several adipocyte secreted proteins including
PAI-1 by TNF-
. These data reinforce the notion that TGZ is not a
general inhibitor of TNF-
; rather, TGZ may specifically target and
inhibit NF-
B that appears to play a major role in TNF-
-induced
repression of key adipocyte genes and modulates the induction of a subset of
genes by TNF-
.
By using reporter gene assays in HeLa cells, we found that p65
significantly inhibited the transcriptional activity of PPAR- and that
the repressive effect of p65 is independent of the presence of any
NF-
B-binding sites in the promoter region of PPAR-
-responsive
genes. Thus, p65 may potentially inhibit PPAR-
activity by directly
binding to PPAR-
and inhibiting its DNA binding and/or transcriptional
activity, or by sequestering key transcriptional co-activators such as
CREB-binding protein (CBP/p300) or steroid receptor coactivator-1 (SRC-1).
PPAR-
requires interaction with transcription co-activators for full
transcriptional activity (55),
and CBP/p300 and SRC-1 have been implicated in positively regulating
PPAR-
-dependent gene transcription
(53,
56). Importantly, both
CBP/p300 and SRC-1 also interact with NF-
B and stimulate its
transcriptional activity (51,
52,
57). Thus, a competition for
limiting amounts of co-activators could result in selective inhibition of
CBP/p300- and/or SRC-1-dependent gene transcription and account for the
transcriptional repressive effect of NF-
B on key adipocyte genes.
As an initial step to explore the role of co-factor squelching on the
inhibitory effect of p65 on adipocyte gene expression, we compared the effect
of wild-type and the S276A mutant of p65, which has lost the ability to
interact with co-activator CBP/p300. Our reporter gene assays in HeLa cells
showed that the S276A mutant p65 blocked the PPAR--mediated
transcription less effectively than its wild-type counterpart, but it did
retain some inhibitory activity. This indicates that co-factor squelching
contributes to some of the repressive effect of p65 but other unidentified
mechanisms probably also play a role. To ascertain whether exogenous
expression of CBP/p300 or SRC-1 can rescue the inhibitory effect of p65 on
PPAR-
-stimulated gene expression, we co-transfected CBP/p300 together
with p65, PPAR-
, and a PPAR-
-responsive reporter gene. However,
we were unable to see a significant increase in PPAR-
-mediated gene
transcription in the presence of CBP/p300.2 This may simply relate
to the technical limitations in our assay system or the suboptimal ratios
between PPAR-
, p65, and CBP/p300. Nevertheless, our reporter gene
assays using the S276A mutant p65 provided evidence, at least indirectly, that
the interaction with co-factors might be involved in p65-mediated repression
of PPAR-
activity. Supporting this notion, p65 has already been shown
to interact with CBP/p300 to inhibit glucocorticoid and cAMP-mediated
induction of PEPCK gene expression
(39).
On the other hand, PPAR- activation blocked p65-mediated gene
transcription, and the inhibition is also independent of the presence of any
PPAR-
-response elements in the promoter region of NF-
B-activated
genes. Thus, p65 and PPAR-
antagonize the transcriptional activity of
each other. The in vivo significance of the functional antagonism
between p65 and PPAR-
is that these two major transcription factors may
lie in a common pathway that integrates multiple signals regulating adipocyte
gene expression and function. The balance between the activities of p65,
PPAR-
, and possibly other DNA-binding proteins therefore is likely
important for adipocyte function and response to insulin.
In this study, we identified the common target genes of PPAR- and
NF-
B and thus provided a basis for further investigation of the exact
mechanisms by which PPAR-
and NF-
B antagonize each other. It
will be intriguing to test whether endogenous NF-
B activity is truly
induced in obesity and type 2 diabetes, and whether endogenous NF-
B is
indeed a fundamental target for the treatment of type 2 diabetes and its
complications. It will also be critical to identify the
NF-
B-independent mediator(s) that are induced by TNF-
and thus
contribute to the reprogramming of adipocyte gene expression and loss of
insulin responsiveness.
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FOOTNOTES |
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¶ Supported by a postdoctoral fellowship from American Diabetes Association
and currently holds a Postdoctoral Fellowship for Physician Scientists from
the Howard Hughes Medical Institute. Present address: Whitehead Institute for
Biomedical Research, Cambridge, MA 02142.
** To whom correspondence should be addressed: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Rm. 601, Cambridge, MA 02142. Tel.: 617-258-5216; Fax: 617-258-6768; E-mail: lodish{at}wi.mit.edu.
1 The abbreviations used are: TZD, thiazolidinediones; TGZ, troglitazone;
TNF-, tumor necrosis factor-
; PPAR-
, peroxisome
proliferator activator receptor-
; ELISA, enzyme-linked immunosorbent
assay; PPRE-luc, The PPAR-
-responsive luciferase reporter gene; PEPCK,
phosphoenolpyruvate carboxykinase; HSL, hormone-sensitive lipase; I
Bs,
inhibitor of
B proteins; IL, interleukin; TG, triglyceride; FFA, free
fatty acid.
2 H. Ruan and H. F. Lodish, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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