Interferon-
-induced Regulation of Peroxisome
Proliferator-activated Receptor
and STATs in Adipocytes*
Kyle J.
Waite,
Z. Elizabeth
Floyd,
Patricia
Arbour-Reily, and
Jacqueline M.
Stephens
From the Department of Biological Sciences, Louisiana State
University, Baton Rouge, Louisiana 70803
Received for publication, August 29, 2000, and in revised form, October 24, 2000
 |
ABSTRACT |
Interferon-
(IFN-
) is known primarily for
its roles in immunological responses but also has been shown to
affect fat metabolism and adipocyte gene expression. To further
investigate the effects of IFN-
on fat cells, we examined the
effects of this cytokine on the expression of adipocyte transcription
factors in 3T3-L1 adipocytes. Although IFN-
regulated the expression
of several adipocyte transcription factors, IFN-
treatment resulted
in a rapid reduction of both peroxisome proliferator-activated receptor (PPAR) protein and mRNA. A 48-h exposure to IFN-
also
resulted in a decrease of both CCAAT/enhancer-binding
and sterol
regulatory element binding protein (SREBP-1) expression. The short
half-life of both the PPAR
mRNA and protein likely contributed
to the rapid decline of both cytosolic and nuclear PPAR
in the
presence of IFN-
. Our studies clearly demonstrated that the
IFN-
-induced loss of PPAR
protein is partially inhibited in the
presence of two distinct proteasome inhibitors. Moreover, IFN-
also
inhibited the transcription of PPAR
, which was accompanied by a
decrease in PPAR
mRNA accumulation. In addition, exposure to
IFN-
resulted in a substantial increase in STAT 1 expression and a
small increase in STAT 3 expression. IFN-
treatment of 3T3-L1
adipocytes (48-96 h) resulted in a substantial inhibition of
insulin-sensitive glucose uptake. These data clearly demonstrate that
IFN-
treatment results in the development of insulin resistance,
which is accompanied by the regulation of various adipocyte
transcription factors, in particular the synthesis and degradation of
PPAR
.
 |
INTRODUCTION |
The adipocyte plays an active role in a variety of physiological
and pathological processes regulating energy metabolism. The recent
consideration of adipose tissue as an endocrine organ that secretes a
variety of unrelated bioactive molecules has broadened our
understanding of adipocyte function to exceed its previously considered
passive role in lipid metabolism. A number of cell lines are available
for studying adipocytes. The 3T3-L1 cell line differentiates under the
controlled conditions of cell culture from fibroblasts, or
preadipocytes, to cells with the morphological and biochemical
properties of adipocytes (1, 2). The 3T3-L1 adipocytes are comparable
with native adipocytes as they have the ability to accumulate lipid,
respond to insulin, and secrete leptin. The major transcription factors
involved in adipocyte gene regulation include peroxisome
proliferator-activated receptor
, proteins belonging to the
CCAAT/enhancer-binding protein family, and adipocyte determination and
differentiation-dependent factor 1, also known as sterol
regulatory element-binding protein (reviewed in Refs. 3 and 4).
Recent studies have also suggested that the signal
transducer and activator of
transcription
(STAT)1 family of
transcription factors may also be important in fat cells. The STAT
family of transcription factors is comprised of seven family members
(STATs 1, 2, 3, 4, 5A, 5B, and 6) that, in response to the stimulation
of various receptors, mainly those for cytokines, are phosphorylated on
tyrosine residues, which causes their translocation to the nucleus.
Each STAT family member shows a distinct pattern of activation by
cytokines and upon nuclear translocation can regulate the transcription
of particular genes in a cell- or tissue-specific manner (5). In fat
cells, the expression of STATs 1, 5A, and 5B is highly induced during
differentiation and correlates with lipid accumulation (6, 7). The
regulation of STAT expression has also been investigated in NIH 3T3
cells ectopically overexpressing C/EBPs
and
, a condition
that results in adipogenesis (8). In these studies, the expression of
STATs 1, 5A, and 5B was induced in a PPAR
ligand-dependent fashion during adipogenesis (9). STATs 3 and 6 are also expressed in adipocytes, but the expression of these
proteins does not change during differentiation. However, the tyrosine
phosphorylation of STAT 3 occurs following the induction of
differentiation, and studies with antisense STAT 3 suggest that this
protein may be important in adipogenesis (10). Although the functions
of STATs in fat cells have not been identified, numerous studies
suggest that these transcription factors may be important regulators of adipocyte gene expression.
Interferon-
(IFN-
) is primarily known for its roles in
immunological responses but also has been shown to affect fat
metabolism and adipocyte gene expression. In adipocytes, IFN-
treatment results in a decrease of lipoprotein lipase activity and
increased lipolysis (11). In 3T3-F442 adipocytes, exposure to IFN-
results in a decreased expression of lipoprotein lipase and fatty acid synthase. Also in various rodent preadipocyte cell lines, IFN-
inhibits the differentiation of preadipocytes (12-14). We have recently shown that acute IFN-
treatment of cultured and native adipocytes results in a dose- and time-dependent activation
of STATs 1 and 3 (15). Exposure of adipocytes to IFN-
results in the
tyrosine phosphorylation and nuclear translocation of STATs 1 and 3 in
fat cells. Because IFN-
has effects on adipocyte gene expression, we
examined the effects of this cytokine on the expression of a variety of
adipocyte transcription factors.
Although we observed that IFN-
affected the expression of a
number of adipocyte transcription factors, the most profound effect of
IFN-
was on the expression of PPAR
. PPAR
is a member of the
nuclear hormone superfamily and exists as two isoforms, PPAR
1 and
PPAR
2, which are transcribed from the same gene by the use of
alternative promoters (16). PPAR
2 is 30 amino acids longer than
PPAR
1 and is largely adipocyte-specific. Although expressed in a
variety of other tissues, PPAR
1 is also predominately expressed in
fat (17). Thiazolidinediones (TZDs) are high affinity synthetic ligands
of PPAR
and have recently been shown to affect the
degradation of this transcription factor (18). Our studies with IFN-
also indicate that PPAR
is targeted to the proteasome for
degradation, but this is not the only mechanism for the substantial effect that IFN-
has on PPAR
expression. Our findings indicate that a newly identified inhibitor of PPAR
expression, IFN-
, results in a substantial loss of PPAR
expression by regulating two
cellular events as follows: 1) targeting PPAR
to the proteasome for
degradation and 2) inhibiting the synthesis of PPAR
. Prolonged IFN-
treatment of 3T3-L1 adipocytes also results in the development of insulin resistance and regulation of other adipocyte transcription factors and supports the hypothesis that PPAR
is involved in conferring insulin sensitivity.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Dulbecco's modified Eagle's medium (DMEM) was
purchased from Life Technologies, Inc. Bovine and fetal bovine serum
was obtained from Sigma and Life Technologies, Inc., respectively.
Murine interferon-
(IFN-
) was purchased from Roche Molecular
Biochemicals. Actinomycin D was purchased from Calbiochem.
Cycloheximide was purchased from Sigma. The nonphospho STAT antibodies
were either monoclonal IgGs purchased from Transduction Laboratories or
polyclonal IgGs from Santa Cruz Biotechnology Inc. A highly
phospho-specific polyclonal antibody for STAT 1 (Y701) was
provided by Quality Controlled Biochemicals. PPAR
was a mouse
monoclonal antibody from Santa Cruz Biotechnology Inc. SREBP-1, C/EBP
, and ERK1/ERK2 were rabbit polyclonal antibodies from Santa Cruz Biotechnology Inc.
Cell Culture--
Murine 3T3-L1 preadipocytes were plated and
grown to 2 days postconfluence in DMEM with 10% bovine serum. Medium
was changed every 48 h. Cells were induced to differentiate by
changing the medium to DMEM containing 10% fetal bovine serum, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM
dexamethasone, and 1.7 µM insulin. After 48 h this
medium was replaced with DMEM supplemented with 10% fetal bovine
serum, and cells were maintained in this medium until utilized for experimentation.
Preparation of Whole Cell Extracts--
Monolayers of 3T3-L1
adipocytes were rinsed with phosphate-buffered saline and then
harvested in a nondenaturing buffer containing 150 mM NaCl,
10 mM Tris, pH 7.4, 1 mM EGTA, 1 mM
EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 1 µM
phenylmethylsulfonyl fluoride, 1 µM pepstatin, 50 trypsin inhibitory milliunits of aprotinin, 10 µM
leupeptin, and 2 mM sodium vanadate. Samples were extracted for 30 min on ice and centrifuged at 15,000 rpm at 4 °C for 15 min.
Supernatants containing whole cell extracts were analyzed for protein
content using a BCA kit (Pierce) according to the manufacturer's instructions.
Preparation of Nuclear/Cytosolic Extracts--
Cell monolayers
were rinsed with phosphate-buffered saline and then harvested in a
nuclear homogenization buffer (NHB) containing 20 mM Tris
(pH 7.4), 10 mM NaCl, and 3 mM
MgCl2. Nonidet P-40 was added to a final concentration of
0.15%, and cells were homogenized with 16 strokes in a Dounce
homogenizer. The homogenates were centrifuged at 1500 rpm for 5 min.
Supernatants were saved as cytosolic extract, and the nuclear pellets
were resuspended in 0.5 volume of NHB and centrifuged as before. The
pellet of intact nuclei was resuspended again in 0.5 of the original
volume of NHB and centrifuged again. A small portion of the nuclei was
used for trypan blue staining to examine the integrity of the nuclei. The majority of the pellet (intact nuclei) was resuspended in an
extraction buffer containing 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol. Nuclei were extracted for 30 min on ice and then placed at room temperature for 10 min. Two hundred
units of DNase I were added to each sample, and tubes were inverted and
incubated an additional 10 min at room temperature. Finally, the sample
was subjected to centrifugation at 15,000 rpm at 4 °C for 30 min.
Supernatants containing nuclear extracts were analyzed for protein content.
Gel Electrophoresis and Immunoblotting--
Proteins were
separated in 5, 7.5, or 12% polyacrylamide (acrylamide from National
Diagnostics) gels containing sodium dodecyl sulfate (SDS) according to
Laemmli (19) and transferred to nitrocellulose (Bio-Rad) in 25 mM Tris, 192 mM glycine, and 20% methanol.
Following the transfer, the membrane was blocked in 4% milk for 1 h at room temperature. Results were visualized with horseradish
peroxidase-conjugated secondary antibodies (Sigma) and enhanced
chemiluminescence (Pierce).
RNA Analysis--
Total RNA was isolated from cell monolayers
with TriZOL (Life Technologies, Inc.) according to the
manufacturer's instruction with minor modifications. For Northern blot
analysis, 20 µg of total RNA was denatured in formamide and
electrophoresed through a formaldehyde-agarose gel. The RNA was
transferred to Zeta Probe-GT (Bio-Rad), cross-linked, hybridized, and
washed as previously described (20). Probes were labeled by random
priming using the Klenow fragment (Promega) and
[
-32P]dATP (PerkinElmer Life Sciences).
Determination of 2-Deoxyglucose--
The assay of
2-[3H]deoxyglucose was performed as previously
described (21). Prior to the assay, fully differentiated 3T3-L1 adipocytes were serum-deprived for 2-4 h. Uptake measurements were
performed in triplicate under conditions where hexose uptake was
linear, and the results were corrected for nonspecific uptake and
absorption determined by 2-[3H]deoxyglucose uptake in the
presence of 5 µM cytochalasin B (Sigma). Nonspecific
uptake and absorption were always less than 10% of the total uptake.
Nuclei Isolation and Run-on Transcription Assays--
Following
exposure of fully differentiated adipocytes to IFN-
for 1 h,
the cell monolayers (six 10-cm plates per time point) were washed once
with ice-cold phosphate-buffered saline and nuclei were isolated, and
run-on transcription assays were performed as we have previously
described (20).
 |
RESULTS |
The expression of adipocyte transcription factors was examined
following a time course of IFN-
treatment on fully differentiated 3T3-L1 adipocytes. As shown in Fig. 1,
immunoblotting of whole cell extracts demonstrated that IFN-
treatment resulted in a significant decrease in PPAR
2 (upper
band) and -
1(lower band) within 24 h and
resulted in a notable decline in C/EBP
. The expression of STATs 1 and 3 increased following a 24-h IFN-
treatment. The expression of
STATs 5A, 5B, and 6 was not regulated by exposure to IFN-
treatment.
Also, the expression of SREBP-1 decreased after a 48-h treatment. The
spliced 67-kDa form of SREBP-1 was similarly decreased with IFN-
treatment (data not shown).

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 1.
The effects of IFN-
on the expression of adipocyte transcription factors. Whole
cell extracts were prepared from fully differentiated 3T3-L1 adipocytes
following a treatment with 100 units/ml IFN- for 0, 24, 48, 72, or
96 h. Cells were treated every 24 h with a fresh bolus of
IFN- . Extracts were prepared as described under "Experimental
Procedures." One hundred µg of each extract was separated by
SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot
analysis. The molecular mass of each protein is indicated to the
left of the blot in kilodaltons. The detection system was
horseradish peroxidase-conjugated secondary antibodies (Sigma) and
enhanced chemiluminescence (Pierce). This is a representative
experiment independently performed three times.
|
|
As shown in Fig. 1, a 24-h treatment of IFN-
resulted in a
substantial loss of PPAR
2 and -
1 protein expression. Therefore, we examined the effects of IFN-
over a 24-h time course. Whole cell
extracts were isolated from fully differentiated 3T3-L1 adipocytes that
were treated with IFN-
for the various times indicated in Fig.
2. Interestingly, IFN-
resulted in a
substantial loss of PPAR
2 and -
1 expression within 6 h. In
addition, we observed a striking increase in STAT 1 expression between
8 and 12 h and a small increase in STAT 3 during this time period.
There was no change in STAT 5A during this time course. JAK 1, the kinase that activates STAT 1 in adipocytes, increases slightly with
IFN-
treatment. In addition, fatty acid synthase expression
decreased with IFN-
treatment.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 2.
IFN- treatment
results in a rapid loss of PPAR expression in
adipocytes. Whole cell extracts were prepared from fully
differentiated 3T3-L1 adipocytes following a treatment with 100 units/ml IFN- as indicated at the top of the figure. One
hundred µg of each extract was separated by SDS-PAGE, transferred to
nitrocellulose, and subjected to Western blot analysis. The molecular
mass of each protein is indicated to the left of the blot in
kilodaltons. Samples were processed and results were visualized as
described in the legend to Fig. 1. This is a representative experiment
independently performed three times. (JAK1, the
kinase that activates STAT1 in adipocytes.
|
|
Clearly, an analysis of whole cell extracts reveals a substantial loss
of PPAR
2 and -
1 expression in adipocytes following IFN-
treatment. However, it was unclear whether IFN-
had any effect on
the amount of PPAR
proteins present in the adipocyte nucleus. To
further examine the inhibition of PPAR
by IFN-
, we performed
another time course in which adipocytes were fractionated to isolate
cytosolic and nuclear extracts. As shown in the top panel of
Fig. 3, the majority of PPAR
2 and
-
1 protein was present in the nucleus, and the amount of nuclear
PPAR
protein was substantially reduced after 6 h. A darker
exposure of this blot indicates the presence of PPAR
proteins in the
cytosol in untreated adipocytes and cells that were exposed to IFN-
for 30 min. However, following a 6-h or greater IFN-
treatment,
there was no detectable PPAR
2 or -
1 in the cytosol and a
significant loss of both PPAR
isoforms in the nucleus. We also
observed an increase in STAT 1 in the cytosol between 6 and 12 h
and the presence of activated STAT 1 in the nucleus following a 30-min
treatment with IFN-
. Detection of the phosphorylated form of STAT 1 was performed with an antibody specific for phosphorylation on tyrosine
701 (STAT 1 Y701). Analysis with either one of these STAT 1 antibodies demonstrates the presence of STAT 1 in the nucleus following
a 30-min IFN-
stimulation. However, the STAT 1 Y701
antibody is more sensitive, and we observed this protein in the nucleus
even after a 12-h IFN-
treatment. We have previously reported that
STAT 5A is present in the nucleus of adipocytes under basal conditions
(15), and IFN-
treatment does not cause a redistribution of this
protein. Therefore, STAT 5A (Fig. 3, bottom panel) is
shown to indicate the even loading of both cytosolic and nuclear
samples.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 3.
IFN- treatment
results in a decrease of nuclear PPAR and an
increase in cytosolic STAT 1 in adipocytes. Cytosolic and nuclear
extracts were isolated from fully differentiated 3T3-L1 adipocytes
following treatment with IFN- as indicated at the top of
the figure. One hundred µg of each extract was separated by SDS-PAGE,
transferred to nitrocellulose, and subjected to Western blot analysis.
Samples were processed and results were visualized as described in the
legend to Fig. 1. This is a representative experiment independently
performed two times.
|
|
The rapid loss of PPAR
1 and -
2 proteins in the presence of
IFN-
suggested that the PPAR
proteins may be labile. Therefore, we examined the decay of PPAR
and STATs in 3T3-L1 adipocytes. Whole
cell extracts were isolated from 3T3-L1 adipocytes at various times
following the addition of 5 µM cycloheximide
(+CH) or ethanol (
CH), a vehicle control. As
shown in Fig. 4, the inhibition of protein synthesis resulted in the loss of PPAR
by 12 h with
over half of the protein decayed by 6 h. A log plot of the
remaining protein versus time was used to estimate the
half-life of PPAR
and of adipocyte-expressed STAT proteins.
The estimated half-life of these proteins is indicated in Fig. 4 and is
an average calculation of three independent experiments. PPAR
1 and
-
2 are labile compared with the STAT proteins, which have half-lives
at least twice as long as the PPAR
proteins. We also investigated
the effect of IFN-
on PPAR
in the presence of cycloheximide.
Because of experimental variability, it was difficult to quantitate the
decrease in the half-life of the PPAR
proteins in the presence of
IFN-
. However, in each experiment, the decay of the PPAR
was
quicker in the presence of IFN-
, as shown in Fig.
5. Adipocytes were treated with 5 µM cycloheximide in the presence or absence of IFN-
,
and whole cell extracts were isolated at 0, 1, and 4 h. As shown
in Fig. 5, the decay of both PPAR
2 and -
1 is increased in the
presence of IFN-
with a complete loss of PPAR
1 at 4 h.

View larger version (62K):
[in this window]
[in a new window]
|
Fig. 4.
The turnover of PPAR
and adipocyte-expressed STAT proteins. Whole cell extracts
were prepared from 3T3-L1 adipocytes following various periods of
treatment with 5 µM cycloheximide (+CH) or
ethanol ( CH), a vehicle control. One hundred µg of each
extract was separated by SDS-PAGE, transferred to nitrocellulose, and
subjected to Western blot analysis. Samples were processed and results
were visualized as described in the legend to Fig. 1. This is a
representative experiment independently performed three times.
|
|

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 5.
PPAR turnover is
increased in the presence of IFN- . Whole
cell extracts were prepared from 3T3-L1 adipocytes following various
periods of treatment with 5 µM cycloheximide or ethanol
in the presence or absence of IFN- . One hundred µg of each extract
was separated by SDS-PAGE, transferred to nitrocellulose, and subjected
to Western blot analysis. Samples were processed and results were
visualized as described in the legend to Fig. 1. This is a
representative experiment independently performed three times.
|
|
TZD treatment has also been shown to decrease PPAR
expression.
Therefore, we compared the effects of IFN-
and englitazone (ENG), a
TZD, on the expression of PPAR
in adipocytes. As shown in Fig. 6,
fully differentiated adipocytes were
exposed to IFN-
or ENG alone or in combination. In the first
combination, adipocytes were treated with IFN-
1 h prior to the
addition of englitazone. In the second combination, adipocytes were
treated with englitazone 1 h prior to the addition of IFN-
. For
each combination, whole cell extracts were isolated 5 h after
initiation of the experiment. These results demonstrate that the
combination of both inhibitors of PPAR
expression resulted in an
even greater decrease in PPAR
expression than one agonist alone.

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 6.
The IFN- -induced
increase of PPAR expression is even greater in
the presence of englitazone. Whole cell extracts were prepared
from fully differentiated 3T3-L1 adipocytes following a 5-h treatment
of IFN- , ENG, IFN- + ENG (added 1 h after the addition of
IFN- ), and ENG + IFN- (added 1 h after the addition of ENG).
One hundred µg of each extract was separated by SDS-PAGE, transferred
to nitrocellulose, and subjected to Western blot analysis. Samples were
processed and results were visualized as described in the legend to
Fig. 1. CTL, control.
|
|
The results of the cycloheximide experiments in Fig. 5 suggest that the
decay of PPAR
2 and -
1 is increased in the presence of IFN-
.
Therefore, we examined PPAR
expression in the presence of proteasome
inhibitors. As shown in Fig. 7, a 6-h
treatment of either epoxomicin or lactacystin had little effect on the
levels of PPAR
2 or -
1 protein. A 6-h IFN-
treatment resulted
in a substantial loss of PPAR
protein, but the IFN-
-induced loss of PPAR
2 and -
1 was inhibited in the presence of either
epoxomicin or lactacystin. Notably, the presence of these two different
proteasome inhibitors did not restore PPAR
2 and -
1 to the levels
found in untreated adipocytes, suggesting that protein degradation is only one manner in which IFN-
regulates PPAR
expression. In the
presence of the two proteasome inhibitors, there were no differences in
the levels of any STATs or ERK1/ERK2. Interestingly, the
IFN-
-induced increase in STAT 1 was blunted in the presence of
epoxomicin or lactacystin, suggesting that the IFN-
-induced increase
in STAT 1 may be dependent on the degradation of some protein(s).

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 7.
The IFN- -induced
decrease of PPAR is partially inhibited in the
presence of proteasome inhibitors. Whole cell extracts were
prepared from fully differentiated 3T3-L1 adipocytes following a 6-h
treatment of either 100 nM epoxomicin or 5 µM
lactacystin in the presence or absence of IFN- (100 units/ml). One hundred µg of each extract was separated by SDS-PAGE,
transferred to nitrocellulose, and subjected to Western blot analysis.
Samples were processed and results were visualized as described in the
legend to Fig. 1. This is a representative experiment independently
performed three times. CTL, control.
|
|
These experiments indicate that, in addition to having an effect on the
turnover of the PPAR
proteins, there is presumably another means by
which IFN-
causes a decrease in PPAR
expression. Therefore, we
examined the effect of IFN-
on PPAR
mRNA accumulation. As
shown in Fig. 8, a 2-h IFN-
treatment
resulted in a substantial loss of PPAR
mRNA. Northern blot
analysis cannot distinguish between the two forms of PPAR
. A
decrease in C/EBP
and GLUT4 was also observed
following a 20-h IFN-
treatment. In addition, we observed an
increase in the levels of both C/EBP
and C/EBP
following an
IFN-
treatment. A notable decrease in aP2/422 was observed after a 12-h treatment with IFN-
. The expression of glycerol phosphate dehydrogenase (GPD), a gene whose
expression is elevated in adipocytes, was substantially decreased
following a 20-h treatment with IFN-
. Following a 24-h IFN-
treatment, there was also a slight decline in adipsin
mRNA. The hybridization of
-actin is shown to represent the even
loading of the samples.

View larger version (78K):
[in this window]
[in a new window]
|
Fig. 8.
IFN- treatment
results in a rapid loss of PPAR mRNA and a
decrease in expression of other adipocyte markers. Total RNA was
isolated from fully differentiated 3T3-L1 adipocytes following
treatment with IFN- as indicated at the top of the
figure. Twenty µg of total RNA was electrophoresed, transferred to
nylon, and subjected to Northern blot analysis. This is a
representative experiment independently performed two times.
GPD, glycerol phosphate dehydrogenase.
|
|
Because the IFN-
-induced loss of PPAR
mRNA was relatively
rapid, we predicted that the decay of the PPAR
mRNA would be brief compared with C/EBP
. Therefore, we investigated the turnover of these two transcription factor mRNAs. Total RNA was isolated from cells at various times after treatment with actinomycin D. As
shown in Fig. 9A, the PPAR
mRNA decayed rapidly compared with the C/EBP
mRNA. We
estimated the half-life of the PPAR
mRNA to be less than 3 h. We also examined the decay of PPAR
in the presence of IFN-
to
determine whether this growth factor had any effect on the stability of
the PPAR
mRNA. We found that the decay of PPAR
mRNA was
not altered in the presence of IFN-
as indicated in Fig.
9B. These results strongly suggested that IFN-
would have
an effect on the transcription of PPAR
.

View larger version (85K):
[in this window]
[in a new window]
|
Fig. 9.
PPAR mRNA is
more labile than C/EBP mRNA in adipocytes,
and the decay of these mRNAs is not affected by
IFN- . A, total RNA was
isolated from fully differentiated 3T3-L1 adipocytes following
treatment with 5 µg/ml actinomycin D for the various periods of time
indicated at the top of the figure. Control samples
( actinomycin D) were isolated at the start and finish of the
experiment. B, total RNA was isolated from fully
differentiated 3T3-L1 adipocytes following treatment with 5 µg/ml
actinomycin D for the various periods of time indicated at the
top of the figure in the absence or presence of IFN- . In
each experiment, 20 µg of total RNA was electrophoresed, transferred
to nylon, and subjected to Northern blot analysis for C/EBP and
PPAR . This is a representative experiment independently performed
two times.
|
|
To determine whether the IFN-
-induced changes in PPAR
and
C/EBP
mRNA accumulation shown in Fig. 8 were attributable to the
effects on synthesis, we measured the transcription rates of these
genes in nuclei isolated from control and IFN-
-treated adipocytes.
Fully differentiated 3T3-L1 adipocytes were exposed to cycloheximide
(±IFN-
) for 1 h. As shown in Fig.
10, a substantial suppression of both
PPAR
and C/EBP
was observed following IFN-
treatment,
indicating that the effect of IFN-
on the transcription of these
genes was independent of new protein synthesis. IFN-
had no effect
on
-actin transcription (data not shown).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 10.
IFN- treatment
results in a suppression of PPAR and
C/EBP transcription in adipocytes in a manner
that is independent of new protein synthesis. Nuclei were isolated
from fully differentiated adipocytes that were exposed to 5 mM cycloheximide in the presence or absence of IFN- for
1 h. Nuclei were subjected to run-on analysis, and the
autoradiogram displayed isa representative of an experiment
performed twice with independent preparations of nuclei.
CTL, control.
|
|
IFN-
is known to have effects on both lipolysis and lipogenesis, so
we investigated the effect of this growth factor on basal and
insulin-sensitive glucose uptake. As shown in Table
I, serum-deprived 3T3-L1 adipocytes had a
6.7-fold increase in glucose uptake following a 10-min treatment of 100 nM insulin. After a 24-h treatment of IFN-
, cultured
adipocytes were still responsive to insulin (6.13-fold increase).
However, following a 48-h treatment of IFN-
, when both PPAR
and
C/EBP
were substantially decreased (Fig. 1), there was a
discernible decrease in insulin-stimulated glucose uptake (4.42-fold
increase). Exposure to IFN-
for 72 and 96 h had no effect on
basal glucose uptake but resulted in a substantial decrease in
insulin-sensitive glucose uptake. Following a 96-h IFN-
exposure, there was only a 2.2-fold increase following insulin treatment. IFN-
treatment for more than 96 h did not result in a further decline
of insulin-sensitive glucose uptake (data not shown). The
IFN-
-induced effects on insulin sensitivity do not appear to be a
result of any significant lipid loss as there were no distinguishable
differences in Oil Red O staining from control and
IFN-
-treated (96 h) adipocytes (data not shown).
 |
DISCUSSION |
IFN-
affected the expression of many adipocyte
transcription factors, including PPAR
2 and -
1, C/EBP
,
C/EBP
, C/EBP
, SREBP-1, STAT 1, and STAT 3. However, the
most profound effect of IFN-
was on PPAR
expression. These
studies have also revealed that both the PPAR
mRNA and protein
are labile compared with other adipocyte transcription factors. IFN-
treatment of adipocytes leads to a decrease in PPAR
that is the
result of the inhibition of transcription coupled with an increase in
the degradation of PPAR
2 and -
1. Interestingly, recent studies
have revealed that thiazolidinedione treatment of the 3T3-F442A
adipocytes results in a reduction of PPAR
protein that is distinct
from mRNA regulation (18). In that study, the data indicated that
the TZD treatment of adipocytes resulted in the ubiquitination
of PPAR
and subsequent degradation that was dependent on the
proteasome complex (18). These results are comparable with the effects
we observed with IFN-
. In our studies, two distinct proteasome
inhibitors affected PPAR
2 and -
1 protein levels in the
presence of IFN-
but had little effect in adipocytes lacking
cytokine stimulation. In summary, both TZD and IFN-
treatments of
adipocytes appear to target PPAR
to the proteasome for degradation.
Moreover, our observations on the lability of PPAR
proteins suggest
that PPAR
turnover is an important event.
The inhibition of proteasome activity in the presence of IFN-
did
not restore the PPAR
proteins to normal cellular levels, and we
observed a potent effect of IFN-
on PPAR
transcription and
mRNA accumulation. These results have led us to hypothesize that
IFN-
-induced STAT 1 activation in fat cells may be responsible for
the transcriptional suppression of PPAR
. Cross-talk between STATs
and PPARs has been demonstrated in liver cells (22). We are currently
initiating studies to identify the IFN-
-sensitive element in the
PPAR
promoters and determine whether STAT 1 is directly involved in
the transcriptional suppression of PPAR
. Although STATs are
generally thought to be transcriptional activators, there is evidence
that this family of transcription factors can also act as repressors of
transcription (23). These studies have led us to hypothesize that
IFN-
-induced STAT 1 dimers directly bind to the PPAR
promoters
and result in an inhibition of transcription.
The effects of IFN-
on PPAR
degradation are less expected.
Numerous studies have shown that serine phosphorylation of PPAR
on
Ser112 by mitogen-activated protein kinases
(ERK1/ERK2 and stress-activated protein kinase/c-Jun
NH2-terminal kinase) results in a strong suppression of
PPAR
activity (24-27), which in part appears to involve ligand
binding (28). Our previous studies in the 3T3-L1 adipocytes have
demonstrated that IFN-
resulted in both STAT 1 and STAT 3 tyrosine phosphorylation and nuclear translocation (15). However,
unlike other cytokines, IFN-
did not result in the activation of
ERK1/ERK2 in adipocytes. Therefore, it does not appear that
ERK1/ERK2-induced serine phosphorylation of PPAR
could be involved
in the effects of IFN-
that we observed on PPAR
degradation. Our
results are supported by the findings of Spiegelman and
co-workers (18), which indicate that the phosphorylation of
PPAR
on Ser112 is not required for its down-regulation.
However, we have not examined the role of serine phosphorylation in the
IFN-
-induced PPAR
degradation or the effect of IFN-
on the
activation of c-Jun NH2-terminal kinase in adipocytes.
Although the mechanism by which IFN-
directs PPAR
to the
proteasome for degradation is not known, it is clear that the turnover of PPAR
is further increased when both IFN-
and a PPAR
ligand are present. Perhaps IFN-
could either modulate the phosphorylation state of PPAR
or have an effect on the synthesis of an endogenous PPAR
ligand. Alternatively, IFN-
-induced PPAR
degradation
could occur via a pathway that is independent of ligand-induced
degradation. It is interesting to note that the analysis of PPAR
mutants by the Spiegelman laboratory demonstrated that the
TZD-induced PPAR
decay was not strictly dependent on its
transcriptional activity but was dependent upon the ligand-gated
activation function (AF-2) domain. In these studies, ligand
binding and the activation of the AF-2 domain not only increased the
transcriptional function of PPAR
but also induced ubiquitination and
subsequent proteasomal degradation.
Unlike TZDs, which are insulin sensitizers, IFN-
treatment of
adipocytes resulted in a condition of insulin resistance, as measured
by insulin-sensitive glucose uptake and a decrease in the expression of
adipocyte genes, such as GLUT4, aP2/422,
GPD, and adipsin. PPAR
has been implicated in
the regulation of systemic insulin sensitivity, and some PPAR
mutations are associated with severe insulin resistance and diabetes
mellitus (29). In our studies, the most profound effect of IFN-
was
on PPAR
expression, which was significantly decreased after only
6 h. Interestingly, we did not observe any substantial effects on
insulin-sensitive glucose uptake even after a 24-h treatment of IFN-
despite the dramatic loss of PPAR
expression. Following a 48-h
treatment, we did observe a substantial inhibition of insulin-sensitive
glucose uptake. At this time, there was also a marked effect on
C/EBP
expression. These studies suggest that the loss of PPAR
may
be insufficient to confer insulin resistance in 3T3-L1 adipocytes. However, the low levels of PPAR
observed after a 24- and 48-h IFN-
treatment may be sufficient levels of PPAR
expression to account for the insulin responsiveness of these cells. Alternatively, the primary role of PPAR
may be to regulate the expression of other
transcription factors, such as C/EBP
. Nonetheless, the increase in
PPAR
turnover and the inhibition of PPAR
synthesis induced by
IFN-
are prominent because of the relatively rapid decay of both the
PPAR
mRNA and the protein. Because the regulation of PPAR
is
the first observed effect of IFN-
on adipocyte transcription factor
expression, this event is likely very important in the development of
IFN-
-induced insulin resistance. IFN-
treatment also results in a
decrease of GLUT4, aP2, GPD, and
adipsin expression in adipocytes. However, there is
no notable difference in the morphology of the cells, and there is no
observable difference in Oil Red O staining from untreated
3T3-L1 fully differentiated adipocytes and those that have been treated
for 96 h with IFN-
(data not shown). In conclusion, the tightly
controlled regulation of PPAR
synthesis and degradation that we
observed in the presence of IFN-
suggests that the cellular levels
of PPAR
are a meaningful effector of gene expression.
 |
FOOTNOTES |
*
This work was supported by Grant R01DK52968-02 from the
National Institutes of Health and by a Career Development Award from the American Diabetes Association (to J. M. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biological
Sciences, Louisiana State University, 508 Life Sciences Bldg., Baton
Rouge, LA 70803; Tel.: 225-388-1749; Fax: 225-388-2597; E-mail:
jsteph1@unix1.sncc.lsu.edu.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M007894200
 |
ABBREVIATIONS |
The abbreviations used are:
STAT, signal
transducer and activator of transcription;
C/EBP, CCAAT/enhancer-binding protein;
ERK, extracellular signal-regulated
kinase;
TZD, thiazolidinedione;
DMEM, Dulbecco's modified Eagle's
medium;
ENG, englitazone;
SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis;
PPAR, peroxisome
proliferator-activated receptor;
SREBP-1, sterol regulatory
element-binding protein.
 |
REFERENCES |
1.
|
Green, H.,
and Kehinde, O.
(1976)
Cell
7,
105-113[Medline]
[Order article via Infotrieve]
|
2.
|
Green, H.,
and Kehinde, O.
(1975)
Cell
5,
19-27[Medline]
[Order article via Infotrieve]
|
3.
|
Morrison, R. F.,
and Farmer, S. R.
(1999)
J. Cell. Biochem.
Suppl. 32-33,
59-67[CrossRef]
|
4.
|
Rosen, E. D.,
Walkey, C. J.,
Puigserver, P.,
and Spiegelman, B. M.
(2000)
Genes Dev.
14,
1293-1307[Free Full Text]
|
5.
|
Darnell, J. E. J.
(1997)
Science
277,
1630-1635[Abstract/Free Full Text]
|
6.
|
Stephens, J. M.,
Morrison, R. F.,
and Pilch, P. F.
(1996)
J. Biol. Chem.
271,
10441-10444[Abstract/Free Full Text]
|
7.
|
Stewart, W. C.,
Morrison, R. F.,
Young, S. L.,
and Stephens, J. M.
(1999)
Biochim. Biophys. Acta
1452,
188-196[Medline]
[Order article via Infotrieve]
|
8.
|
Wu, Z.,
Bucher, N. L.,
and Farmer, S. R.
(1996)
Mol. Cell. Biol.
16,
4128-4136[Abstract]
|
9.
|
Stephens, J. M.,
Morrison, R. F.,
Wu, Z.,
and Farmer, S. R.
(1999)
Biochem. Biophys. Res. Commun.
262,
216-222[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Deng, J.,
Hua, K.,
Lesser, S. S.,
and Harp, J. B.
(2000)
Endocrinology
141,
2370-2376[Abstract/Free Full Text]
|
11.
|
Doerrler, W.,
Feingold, K. R.,
and Grunfeld, C.
(1994)
Cytokine
6,
478-484[Medline]
[Order article via Infotrieve]
|
12.
|
Gregoire, F.,
De Broux, N.,
Hauser, N.,
Heremans, H.,
Van Damme, J.,
and Remacle, C.
(1992)
J. Cell. Physiol.
151,
300-309[Medline]
[Order article via Infotrieve]
|
13.
|
Keay, S.,
and Grossberg, S. E.
(1980)
Proc. Natl. Acad. Sci. U. S. A.
77,
4099-4103[Abstract]
|
14.
|
Grossberg, S. E.,
and Keay, S.
(1980)
Ann. N. Y. Acad. Sci.
350,
294-300[Abstract]
|
15.
|
Stephens, J. M.,
Lumpkin, S. J.,
and Fishman, J. B.
(1998)
J. Biol. Chem.
273,
31408-31416[Abstract/Free Full Text]
|
16.
|
Zhu, Y.,
Qi, C.,
Korenberg, J. R.,
Chen, X. N.,
Noya, D.,
Rao, M. S.,
and Reddy, J. K.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
7921-7925[Abstract]
|
17.
|
Chawla, A.,
Schwarz, E. J.,
Dimaculangan, D. D.,
and Lazar, M. A.
(1994)
Endocrinology
135,
798-800[Abstract]
|
18.
|
Hauser, S.,
Adelmant, G.,
Sarraf, P.,
Wright, H. M.,
Mueller, E.,
and Spiegelman, B. M.
(2000)
J. Biol. Chem.
275,
18527-18533[Abstract/Free Full Text]
|
19.
|
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
|
20.
|
Stephens, J. M.,
and Pekala, P. H.
(1992)
J. Biol. Chem.
267,
13580-13584[Abstract/Free Full Text]
|
21.
|
Stephens, J. M.,
and Pekala, P. H.
(1991)
J. Biol. Chem.
266,
21839-21845[Abstract/Free Full Text]
|
22.
|
Zhou, Y. C.,
and Waxman, D. J.
(1999)
J. Biol. Chem.
274,
2672-2681[Abstract/Free Full Text]
|
23.
|
Luo, G.,
and Yu-Lee, L.
(1997)
J. Biol. Chem.
272,
26841-26849[Abstract/Free Full Text]
|
24.
|
Hu, E.,
Kim, J. B.,
Sarraf, P.,
and Spiegelman, B. M.
(1996)
Science
274,
2100-2103[Abstract/Free Full Text]
|
25.
|
Zhang, B.,
Berger, J.,
Zhou, G.,
Elbrecht, A.,
Biswas, S.,
White-Carrington, S.,
Szalkowski, D.,
and Moller, D. E.
(1996)
J. Biol. Chem.
271,
31771-31774[Abstract/Free Full Text]
|
26.
|
Camp, H. S.,
Tafuri, S. R.,
and Leff, T.
(1999)
Endocrinology
140,
392-397[Abstract/Free Full Text]
|
27.
|
Camp, H. S.,
and Tafuri, S. R.
(1997)
J. Biol. Chem.
272,
10811-10816[Abstract/Free Full Text]
|
28.
|
Shao, D.,
Rangwala, S. M.,
Bailey, S. T.,
Krakow, S. L.,
Reginato, M. J.,
and Lazar, M. A.
(1998)
Nature
396,
377-380[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Barroso, I.,
Gurnell, M.,
Crowley, V. E.,
Agostini, M.,
Schwabe, J. W.,
Soos, M. A.,
Maslen, G. L.,
Williams, T. D.,
Lewis, H.,
Schafer, A. J.,
Chatterjee, V. K.,
and O'Rahilly, S.
(1999)
Nature
402,
880-883[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.