1 Department of Molecular Medicine (C-4), Osaka University Graduate School of
Medicine, Suita, Osaka 565-0871, Japan
2 Department of Internal Medicine, Nishinomiya Municipal Central Hospital,
Nishinomiya, Hyogo 663-8014, Japan
* Author for correspondence (e-mail: kasayama{at}imed3.med.osaka-u.ac.jp)
Accepted 7 July 2002
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Summary |
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Key words: Insulin receptor substrate-2, Glucose transporter 4, Insulin receptor, CCAAT/enhancer-binding protein, Peroxisome proliferator-activated receptor
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Introduction |
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In adipose tissue, the ability of cells to respond to insulin and to
express genes such as fatty-acid-binding protein (422/aP2), lipoprotein lipase
(LPL), adipsin and glucose transporter 4 (GLUT4) is acquired during their
differentiation into mature adipocytes
(Cornelus et al., 1994). Since
C/EBP-binding sites and/or PPAR response elements (PPRE) exist in the promoter
regions of some of these genes, C/EBP proteins and PPAR
are involved to
a large extent in the transcriptional regulation of these genes
(Chaneval et al., 1991
;
Christy et al., 1989
;
MacDougald and Lane, 1995
;
Schoonjans et al., 1996
).
However, it has remained uncertain whether PPAR
or which C/EBP is
involved in the acquisition of these characteristics. It has been recently
shown that C/EBP
-deficient mouse embryonic fibroblasts (MEFs) can
differentiate into adipocytes after the introduction of PPAR
2 and its
subsequent activation (Wu et al.,
1999
). In these adipocytes lacking C/EBP
, the expression
and phosphorylation of insulin receptor (IR) and insulin receptor substrate 1
(IRS-1) are impaired and insulin-stimulated glucose uptake is reduced
(Wu et al., 1999
). These
results indicate the essential roles of C/EBP
in the insulin signals of
adipocytes. In contrast, it has not yet been demonstrated whether C/EBPß
and C/EBP
are involved in the insulin-responsiveness in adipocytes. To
date, it has been difficult to clarify these issues, since
C/EBPß/
-double deficient MEFs cannot differentiate into mature
adipocytes with the aid of a standard adipogenesis induction cocktail
(Tanaka et al., 1997
). To
tackle these issues, we introduced PPAR
2 into
C/EBPß/
-double deficient MEFs and investigated phenotypic
acquisition during adipocyte differentiation.
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Materials and Methods |
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Construction and preparation of adenovirus vector expressing mouse
PPAR2 gene (pAdex-mPPAR
2)
Mouse PPAR2 cDNA was kindly provided by B. M. Spiegelman (Harvard
Medical School, Cambridge, MA). To generate pAdex-mPPAR
2, mouse
PPAR
2 cDNA was inserted into the adenovirus vector (pAdex). The
adenovirus was then expanded by a series of infections to HEK293 cells, and
twice purified by ultracentrifugation on a caesium chloride-gradient followed
by dialysis. The viral titer was determined by plaque assay using HEK293
cells, and calculated as 1.0x109 pfu/ml.
Adipogenic differentiation
Wild-type and C/EBPß/-deficient MEFs were propagated to
confluence. Two days after reaching confluence, the cells were first infected
with pAdex or pAdex-mPPAR
2 for 1 hour and then incubated with 3 µM
15-deoxy-
12,14-prostaglandin J2
(15d-PGJ2) (Cayman Chemical, Ann Arbor, MI) or 10 µM
troglitazone (Sankyo, Tokyo, Japan). The media containing either of the drugs
were renewed every day or every other day, respectively. Eight days after
infection with pAdex or pAdex-mPPAR
2 and stimulation with these drugs,
cytoplasmic lipid accumulation was detected with Oil Red O staining
(Green and Kehinde, 1974
).
Measurement of intracellular triglyceride contents
Cytoplasmic lipid was isolated with 2-propanol, and the triglyceride
content measured with a Triglyceride G-test kit (Wako Junyaku, Osaka, Japan).
Whole cellular protein, prepared from another duplicated well with 1% Triton
X-100, was measured with the BCA Protein Assay Reagent (Pierce, Rockford, IL).
The triglyceride content was then corrected in terms of the protein
content.
Northern blot analyses
Total RNA was prepared from MEFs infected with pAdex-PPAR2 followed
by stimulation with troglitazone on the day indicated. Ten µg of RNA was
electrophoresed by means of denaturing formaldehyde-agarose gel, and
transferred to Hybond-N+ nylon membrane (Amersham Pharmacia
Biotech, Little Chalfont, UK). The cDNA probes for mouse adipsin, 422/aP2,
LPL, GLUT4, C/EBP
, C/EBPß, C/EBP
, PPAR
, IR, IRS-1
and IRS-2, were labeled with [
-32P]dCTP by random primers
and using the Megaprime DNA labeling system (Amersham Pharmacia Biotech).
Northern blot hybridization was performed with QuickHyb Hybridization Solution
(Stratagene, La Jolla, CA). Densitometrical analysis was performed with Immuno
Reader FMBIO II (Hitachi, Kanagawa, Japan).
2-deoxyglucose-uptake analysis
2-deoxyglucose (2-DG)-uptake assay was performed as described before with a
minor modification (Fasshauer et al.,
2000). Cells were serum-starved for 3 hours in DMEM containing 25
mM glucose and 2 mM glutamine, then incubated with 100 nM insulin at 37°C
in serum-starved DMEM without glucose for 30 minutes and finally treated with
[3H] 2-DG (0.33 µCi/ml) for an additional 5 minutes.
[3H] 2-DG uptake was stopped by three washes with ice-cold PBS.
Radioactivity was counted after the cells had been solubilized in 0.1% SDS.
Specific [3H] 2-DG uptake was determined by subtraction of
nonspecific counts in the presence of 10 µM cytochalasin B from each of the
resultant values, which was then corrected in terms of its protein
concentration (BCA Protein Assay Reagent).
Western blot analyses
Cells were harvested on days 0 and 8 during the adipogenic differentiation
course and were lysed in 0.5 ml lysis buffer [10 mM Tris-HCl pH 7.6, 5 mM
EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 0.1 mM Na3
VO4, 1% Triton X-100, 1 mM PMSF and protease inhibitor cocktail
tablet (Roche Molecular Biochemicals, Mannheim, Germany)]. Lysates were
clarified by centrifugation at 15,000 g for 10 minutes. 30
µg of protein was processed for SDS-PAGE, which was performed on 4-20%
gradient gels. The proteins were electrophoretically transferred to Immobilon
P (Millipore, Bedford, MA). The blots were blocked with 5% nonfat milk in
Tris-buffered saline (TBS, pH 7.4) for 1 hour and then incubated with anti-IR
(Santa Cruz Biotechnology, Santa Cruz, CA), anti-IRS-1 (Upstate Biotechnology,
Lake Placid, NY), or anti-IRS-2 (Upstate Biotechnology) antibodies in 5%
nonfat milk in TBS. They were then washed with TBS and incubated with donkey
anti-rabbit IgG conjugated with horseradish peroxidase (1:1000; Amersham
Pharmacia Biotech) in 5% nonfat milk in TBS. After washing with TBS, the bound
antibodies were visualized by enhanced chemiluminescence (Amersham Pharmacia
Biotech) and recorded on X-ray films (Fuji Medical, Tokyo, Japan). The
relative amounts of IR, IRS-1 and IRS-2 were determined by densitometry
scans.
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Results |
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Gene expression in PPAR2-induced adipocytes derived from
C/EBPß/
-deficient MEFs
C/EBPß and C/EBP were expressed in wild-type MEFs. By ectopic
expression and activation of PPAR
2, C/EBPß mRNA levels gradually
increased, whereas C/EBP
mRNA levels were soon reduced in the wild-type
cells (Fig. 3A,B). The
expression of C/EBP
mRNA was detected on day 2 and showed gradually
increasing levels (Fig. 3). By
contrast, in C/EBPß/
-deficient cells the induction of C/EBP
mRNA was lower by
30-40% than that in wild-type cells throughout the
differentiation course (Fig.
3A,B). Mature adipocytes are known to express proteins involved in
fatty acid binding, lipogenesis and lipolysis, and in insulin-sensitive
glucose uptake (MacDougald and Lane,
1995
). Therefore, to define the roles of C/EBPß and
C/EBP
in such genes' expression during adipocyte differentiation, we
compared their expression levels in PPAR
2-induced adipocytes derived
from wild-type MEFs and those from C/EBPß/
-deficient MEFs.
Northern blot analyses demonstrated that fatty-acid-binding protein (422/aP2)
and lipoprotein lipase (LPL) mRNA levels started to increase two days after
ectopic expression and activation of PPAR
2, reaching a plateau on day 5
(Fig. 3A,B). The mRNA levels of
wild-type cells and C/EBPß/
-deficient cells were not significantly
different throughout the experimental course
(Fig. 3A). By contrast, the
levels of GLUT4 and adipsin mRNA, which also increased during the adipogenic
differentiation, were reduced by 40-50% in the C/EBPß/
-deficient
cells compared with those in the wild-type cells throughout the experiment
(Fig. 3A,B).
|
Insulin-stimulated 2-deoxyglucose uptake in PPAR2-induced
adipocytes derived from C/EBPß/
-deficient MEFs
In the next experiments, we analyzed insulin-stimulated 2-DG uptake in the
mature adipocytes. Insulin did not stimulate [3H] 2-DG-uptake in
either wild-type MEFs or C/EBPß/-deficient MEFs before the
adipocyte differentiation (Fig.
4). In adipocytes derived from wild-type MEFs, insulin
significantly enhanced [3H] 2-DG-uptake by a factor of
3.7±0.5 (mean±s.e.m.). In adipocytes derived from
C/EBPß/
-deficient MEFs, the insulin-stimulated [3H]
2-DG-uptake was enhanced 2.1±0.3-fold, which was significantly lower
(P<0.005) than that in adipocytes derived from wild-type MEFs
(Fig. 4).
|
Insulin receptor, IRS-1 and IRS-2 expression in PPAR2-induced
adipocytes derived from C/EBPß/
-deficient MEFs
Western blot analyses demonstrated that the expression of IR and IRS-2, but
not IRS-1, was upregulated during PPAR2-induced adipogenic
differentiation of wild-type MEFs (Fig.
5A). The levels of IR and IRS-1 were similar for the adipocytes
derived from wild-type MEFs and those derived from
C/EBPß/
-deficient MEFs, while the levels of IRS-2 in the
C/EBPß/
-deficient adipocytes were reduced to approximately 50% of
those in the adipocytes derived from wild-type MEFs
(Fig. 5A). Northern blot
analysis (Fig. 5B) also showed
that the mRNA levels for IR and IRS-2, but not IRS-1, increased during
PPAR
2-induced adipogenic differentiation of wild-type MEFs. IRS-2 mRNA
levels were lower in adipocytes derived from C/EBPß/
-deficient
MEFs than those in adipocytes derived from wild-type MEFs, whereas the mRNA
levels for IR and IRS-1 were similar for the both types of adipocytes.
|
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Discussion |
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During adipocyte differentiation, PPAR and each of the C/EBPs are
expressed sequentially. In 3T3-L1 cells treated with the adipogenesis
induction cocktail, C/EBPß and C/EBP
are expressed in an early
phase followed by the induction of C/EBP
and PPAR
(Cao et al., 1991
;
Wu et al., 1996
). We showed
that C/EBP
mRNA induction during PPAR
-induced adipocyte
differentiation was less extensive in C/EBPß/
-deficient cells than
that in wild-type cells, indicating that C/EBPß and C/EBP
are
involved in the induction of C/EBP
during the PPAR
2-induced
adipocyte differentiation. This provides direct evidence that C/EBP
expression is regulated by C/EBPß and C/EBP
as well as
PPAR
.
In PPAR-induced adipocytes derived from
C/EBPß/
-deficient MEFs, the expression of GLUT4 and adipsin mRNA
was reduced compared with that in adipocytes derived from wild-type MEFs. By
contrast, aP2 and LPL mRNA levels were the same in both adipocytes. Thus,
C/EBPß and C/EBP
may be involved in the PPAR
2-induced
expression of GLUT4 and adipsin, whereas they are not required for the
PPAR
2-induced expression of aP2 and LPL. It has also been shown that
PPAR
-expressing Swiss 3T3 and BALB/c-3T3 fibroblasts express aP2 and
GLUT4 whereas PPAR
-expressing NIH-3T3 cells, despite similar adipocyte
morphology and aP2 expression, do not express GLUT4
(El-Jack et al., 1999
).
Together with our results, these findings indicate that such differences may
be derived from differences in the expression levels of the various C/EBP
molecules in these cells.
We showed that insulin-stimulated [3H] 2-DG-uptake was
significantly impaired in C/EBPß/-deficient adipocytes. This
suggests the important roles of C/EBPß and C/EBP
in the
acquisition of insulin-sensitive glucose uptake during adipocyte
differentiation. While it is not clear whether PPAR
or which C/EBP is
involved in the insulin-stimulated glucose uptake in mature adipocytes, it has
been demonstrated in the study using ß/
39 NIH-3T3 fibroblasts
expressing C/EBPß and C/EBP
but not C/EBP
, that the
enhanced expression and activation of PPAR
stimulate synthesis of GLUT4
protein and insulin-responsive glucose uptake
(Wu et al., 1998
). This
finding indicates that PPAR
alone or in combination with C/EBPß
and C/EBP
is capable of activating GLUT4 gene expression. Considering
such data together with our results suggests that C/EBPß and C/EBP
play important roles in PPAR
-induced GLUT4 expression. The reduced
GLUT4 expression may cause the reduction in insulin-stimulated glucose uptake
in C/EBPß/
-deficient adipocytes.
Recently, C/EBP has also been shown to be important for
insulin-stimulated glucose transport activity, as the result of an analysis of
PPAR
-induced adipocytes derived from C/EBP
-deficient MEFs
(Wu et al., 1999
). In the
C/EBP
-deficient adipocytes, gene expression and tyrosine
phosphorylation of IR and IRS-1 are reduced, while those of GLUT4 mRNA are
increased. Our study demonstrated that, while C/EBPß/
-deficient
adipocytes express somewhat lower levels of C/EBP
, these adipocytes
express lower amounts of IRS-2 but not of IR and IRS-1. Therefore,
C/EBP
-deficient and C/EBPß/
-deficient adipocytes are
different in terms of the expression patterns of GLUT4, IR, IRS-1 and IRS-2.
Recently, Fasshauer et al. showed that IRS-2, rather than IRS-1, is critical
for insulin-stimulated GLUT4 translocation and glucose uptake in adipocytes
(Fasshauer et al., 2000
). Thus,
the reduced expression of IRS-2 in C/EBPß/
-deficient adipocytes
also may be responsible for the decrease in the insulin-stimulated glucose
uptake.
PPAR and C/EBP family proteins are expressed at specific times
during adipogenesis. Complicated networks exist among these transcriptional
factors: C/EBPß and C/EBP
turn on the expression of PPAR
(Wu et al., 1996
). PPAR
upregulates C/EBP
and C/EBP
is necessary to elevate the
expression of PPAR
in differentiated adipocytes
(Wu et al., 1999
), and our
study indicates that C/EBPß and C/EBP
are also involved in
C/EBP
expression. Several gene-targeting studies have demonstrated the
biological significance of PPAR
and C/EBP family proteins in adipogenic
differentiation (Barak et al.,
1999
; Kubota et al.,
1999
; Rosen et al.,
1999
; Tanaka et al.,
1997
; Wang et al.,
1995
; Wu et al.,
1999
). In PPAR
-induced adipocytes derived from
C/EBP
-deficient cells, the levels of fatty acid synthase (FAS),
adipsin, LPL and PEPCK decreased whereas those of GLUT4 and aP2 rather
increased (Wu et al., 1999
).
In PPAR
-induced adipocytes deficient of C/EBPß and C/EBP
,
by contrast, we demonstrated a reduced expression of GLUT4 and adipsin mRNA
but normal levels of aP2 and LPL mRNA. Again, C/EBP
deficiency is
associated with reduced expression of IR and IRS-1
(Wu et al., 1999
), whereas
C/EBPß and C/EBP
deficiencies are associated with reduced IRS-2
expression. Thus, each member of the C/EBP family proteins has a distinct
role(s) in the gene regulation during adipogenesis. Activation of PPAR
by thiazolidinediones is known to improve insulin sensitivity
(Saltiel and Olefsky, 1996
).
Our results show that C/EBPß and C/EBP
are involved in the
expression of some insulin-signaling molecules in adipocytes. Since adipose
tissue is an important organ for insulin-stimulated glucose transport in the
body (Abel et al., 2001
), it is
also possible that C/EBPß and C/EBP
are therapeutic targets for
diabetes mellitus.
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Acknowledgments |
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