Am J Physiol Endocrinol Metab 291: E771–E778, 2006.
First published May 16, 2006; doi:10.1152/ajpendo.00560.2005.
Angiotensin II induces monocyte chemoattractant protein-1 expression
via a nuclear factor-␬B-dependent pathway in rat preadipocytes
Kyoichiro Tsuchiya, Takanobu Yoshimoto, Yuki Hirono, Toru Tateno, Toru Sugiyama, and Yukio Hirata
Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Tokyo, Japan
Submitted 15 November 2005; accepted in final form 11 May 2006
adipocyte; obesity
OVER THE LAST DECADE, a growing body of evidence has been
presented that obesity is characterized by a broad inflammatory
response in adipose tissue (41). It has been shown that gene
expression of numerous inflammatory mediators is increased in
adipose tissue during the development of obesity in human and
animal models, and their potential roles in promoting insulin
resistance have been appreciated (14, 28, 40, 41). In addition,
the recent findings indicated that many proinflammatory genes
Address for reprint requests and other correspondence: T. Yoshimoto, Dept.
of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8513, Japan
(e-mail: [email protected]).
http://www.ajpendo.org
are predominantly expressed in the stromal-vascular fraction
that predominantly contains preadipocytes (8, 40, 42).
Monocyte chemoattractant protein-1 (MCP-1), a member
of the chemokine family, induces inflammatory responses
through the production of adhesion molecules (16), inflammatory cytokines (38), and tissue factor (29) and migration of
leucocytes, and their pathophysiological roles in the development of cardiovascular disease have been well characterized
(1, 19, 20). In addition, it has recently been demonstrated that
human adipocytes produce MCP-1, (6, 9), and its expression in
adipose tissue and circulating levels are increased in obese
humans (6). Furthermore, MCP-1 has been shown to decrease
insulin-stimulated glucose uptake and the expression of several
adipogenic genes in adipocytes in vitro (28). These results
suggest that MCP-1 in adipose tissue may be involved in
adipocyte dedifferentiation and partly contributes to hyperinsulinemia and obesity. However, the cellular localization and
the mechanism of MCP-1 expression in adipose tissue still
remain unclear. It is well recognized that angiotensin (Ang) II
plays a pivotal role in the development of cardiovascular
disease by promoting vascular inflammation (3). Furthermore,
ANG II has been shown to be one of the key stimulators of
MCP-1 expression in cardiovascular tissue (5, 43, 44). Adipose
tissue constitutes a local renin-angiotensin-system (RAS) by
producing angiotensinogen (17), a precursor of ANG II, and its
potential role in adipocyte growth (27) and differentiation (15)
has been implicated. In addition, it has been demonstrated that
ANG II stimulates release of plasminogen activator inhibitor-1
(33) and cytokines (IL-6 and IL-8) (34) from adipocytes.
However, the potential role of ANG II in MCP-1 expression in
adipose tissue and its cellular mechanism has not been well
characterized yet.
Thus the present study was designed to determine whether
ANG II induces MCP-1 expression in primary rat preadipocytes and/or differentiated adipocytes in culture, and if so, to
elucidate the cellular mechanism of its expression by ANG II,
and finally, to confirm that ANG II induces MCP-1 gene
expression in rat adipose tissue in vivo.
MATERIALS AND METHODS
Materials. Human ANG II was purchased from Peptide Institutes
(Osaka, Japan); actinomycin D from Biomol Research Laboratories (PA); pyrrolidine dithiocarbamate (PDTC), Dulbecco’s modified Eagle’s medium (DMEM), type II collagenase, 3-isobutyl-1methylxanthine (IBMX), dexamethasone, 3,3⬘,5-triodo-L-thyronine (T3), and bovine serum albumin (BSA) from Sigma-Aldrich
(St. Louis, MO); Bay 11-7085 from Biomol International (Ply-
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.
0193-1849/06 $8.00 Copyright © 2006 the American Physiological Society
E771
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Tsuchiya, Kyoichiro, Takanobu Yoshimoto, Yuki Hirono, Toru
Tateno, Toru Sugiyama, and Yukio Hirata. Angiotensin II induces
monocyte chemoattractant protein-1 expression via a nuclear factor␬B-dependent pathway in rat preadipocytes. Am J Physiol Endocrinol
Metab 291: E771–E778, 2006. First published May 16, 2006;
doi:10.1152/ajpendo.00560.2005.—Both monocyte chemoattractant
protein-1 (MCP-1), a member of chemokine family, and angiotensinogen, a precursor of angiotensin (ANG) II, are produced by adipose
tissue and increased in obese state. MCP-1 has been shown to
decrease insulin-stimulated glucose uptake and several adipogenic
genes expression in adipocytes in vitro, suggesting its pathophysiological significance in obesity. However, the pathophysiological interaction between MCP-1 and ANG II in adipose tissue remains
unknown. The present study was undertaken to investigate the potential mechanisms by which ANG II affects MCP-1 gene expression in
rat primary cultured preadipocytes and adipose tissue in vivo. ANG II
significantly increased steady-state MCP-1 mRNA levels in a timeand dose-dependent manner. The ANG II-induced MCP-1 mRNA and
protein expression was completely abolished by ANG II type 1
(AT1)-receptor antagonist (valsartan). An antioxidant/NF-␬B inhibitor (pyrrolidine dithiocarbamate) and an inhibitor of 1␬B-␣ phosphorylation (Bay 11-7085) also blocked ANG II-induced MCP-1 mRNA
expression. ANG II induced translocation of NF-␬B p65 subunit from
cytoplasm to nucleus by immunocytochemical study. Luciferase assay
using reporter constructs containing MCP-1 promoter region revealed
that two NF-␬B binding sites in its enhancer region were essential for
the ANG II-induced promoter activities. Furthermore, basal mRNA
and protein of MCP-1 during preadipocyte differentiation were significantly greater in preadipocytes than in differentiated adipocytes,
whose effect was more pronounced in the presence of ANG II.
Exogenous administration of ANG II to rats led to increased MCP-1
expression in epididymal, subcutaneous, and mesenteric adipose tissue. In conclusion, our present study demonstrates that ANG II
increases MCP-1 gene expression via ANG II type 1 receptormediated and NF-␬B-dependent pathway in rat preadipocytes as well
as adipose MCP-1 expression in vivo. Thus the augmented MCP-1
expression by ANG II in preadipocytes may provide a new link
between obesity and cardiovascular disease.
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ANG II INDUCES MCP-1 IN PREADIPOCYTES VIA NF-␬B
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(GenBank acc. no. AF079313). Briefly, the MCP-1 promoter and
enhancer region were isolated by PCR on rat genomic DNA prepared
from kidney. To build the pGLM-PRM, the 192-bp rat MCP-1
promoter region between ⫺129 and ⫹63 was PCR amplified with
sense primer 5⬘-GAAACTCGAGTTACTCAGCAGATTCAAACT3⬘ and antisense primer 5⬘-GAAAAAGCTTAGAGAGATCTGGCTTCAGTG-3⬘. This PCR product was gel purified, digested with XhoI
and HindIII, and then ligated into the XhoI-HindIII site of the
pGL3-basic plasmid. To obtain the pGLM-ENH, the 336-bp rat
MCP-1 enhancer region between ⫺2,395 and ⫺2,060 was PCR
amplified with sense primer 5⬘-GAAAGGTACCAGACTATGCCTTTGTTGAGC-3⬘ and antisense primer 5⬘-GAAACTCGAGGAGCCTGGGAGGTCACCATT-3⬘. This PCR product was gel purified, digested with KpnI and XhoI, and then ligated into the KpnI-XhoI site
of the pGL3-PRM. To obtain the mutated constructs pGLM-MA1
and pGLM-MA2, mutated primers were as follows: sense primer
5⬘-GCTAAATATCTCTCCTGAAGGGTCTATCAACTTCCAATACTGCCTC-3⬘ and antisense primer 5⬘-GAGGCAGTAT- TGGAAGTTGATAGACCCTTCAGGAGAGATATTTAGC-3⬘ for mutation in
NF-␬B site A and sense primer 5⬘-CCAATAC- TGCCTCAGAATATCAATTTCCACACTCTTATCCTACTCT- GC-3⬘ and antisense primer
5⬘-GCAGAGTAGGATAAGAG- TGTGGAAATTGATATTCTGAGGCAGTATTGG-3⬘ for mutation in NF-␬B site B (mutated bases are
underlined). PCR mutagenesis was performed using a QuikChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA). All mutations were
confirmed by DNA sequencing.
Transient transfection and luciferase assay. Transient transfection
of 2 ␮g of each MCP-1 reporter plasmid and 0.1 ␮g of pRL-TK
plasmid into preadipocytes were performed using synthetic cationic liposome, (⫹)-N,N-[bis(2-hydroxyethyl)]-N-methyl-N-[2,3di(tetradecanoyloxy)propyl]ammonium iodide with transferrin receptor-operated transfer, as described previously (22). Cells were
then incubated in culture medium containing 0.3% BSA with or
without 10⫺6 M ANG II for 4 h, and luciferase activities were
measured using the Dual Luciferase Reporter Assay System (Promega) using MicroLumatPlus (EG&G Berthold, Wildbad, Germany), as described previously (22). The firefly luciferase activity
of each sample was normalized to an internal reference standard of
Renilla luciferase activity.
Quantification of MCP-1 mRNA and protein. Rat MCP-1 mRNA
levels were quantified with real-time RT-PCR by TaqMan fluorescence methods as described previously (43). mRNA levels for rat
adiponectin, leptin, and ␤-actin were quantified using fluorescent
SYBR Green technology (LightCycler; Roche Molecular Biochemicals, Mannheim, Germany) as described previously (43). In each
experiment, first-strand cDNA was synthesized from the same amount
of total RNA, and the amplification reaction was performed as
described previously (43). PCR primers, TaqMan probes, and size of
each PCR product are listed in Table 1. The mRNA levels of the target
sequence were normalized to those of ␤-actin and used as an endogenous internal control; the relative levels of each mRNA to that of
␤-actin were calculated.
In some experiments, concentrations of MCP-1 in the culture
medium were determined by an enzyme-linked immunosorbent assay
kit (BioSource International, Camarillo, CA).
Statistical analysis. Data are expressed as means ⫾ SE. Differences
between groups were examined for statistical significance using an
unpaired t-test or ANOVA with Dunn’s post hoc test if appropriate.
P ⬍ 0.05 was considered statistically significant.
RESULTS
ANG II induces MCP-1 expression via AT1-receptor in rat
preadipocytes. ANG II (10⫺6 M) increased steady-state
MCP-1 mRNA levels in a time-dependent manner (0.5– 8 h;
Fig. 1A). A significant (P ⬍ 0.05) increase was induced by as
early as 0.5 h, and ⬃3.6-fold increase by 8 h. ANG II
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mouth Meeting, PA); fetal bovine serum (FBS) from Moregate
Biotech (Bulimba, Australia); human insulin from Eli Lilly Japan;
pGL3-basic and pRL-TK plasmid from Promega (Madison, WI);
and valsartan from Novartis Phama (Basel, Switzerland). PCR
primers were synthesized by JbioService (Saitama, Japan) and
Griner Bio-One (Tokyo, Japan). The concentrations of PDTC
(10⫺4 M), Bay 11-7085 (5 ⫻ 10⫺6 M), and actinomycin D (5 ⫻
10⫺6 M) used in the present study were chosen from previous
studies (13, 26, 35).
Animal experiments. All experiments were conducted in accordance with the Tokyo Medical and Dental University Guide for the
Care and Use of Experimental Animals under the National Institutes
of Health Guide for the Care and Use of Laboratory Animals. Male
Sprague-Dawley (SD) rats were obtained from Charles River Japan
(Tsukuba, Japan) at 5 or 8 wk of age. All rats were kept in temperature- and humidity-controlled rooms that were illuminated from 0800
to 2000. They were fed a standard chow ad libitum. For in vivo
experiments, 5-wk-old male SD rats were injected intraperitoneally
with saline or ANG II (1 ␮g/100 g body wt). Three hours later they
were killed under pentobarbital sodium anesthesia, and then epididymal, subcutaneous, and mesenteric adipose tissues were quickly
removed and stored at ⫺80°C.
Isolation and differentiation of preadipocytes. Rat preadipocytes
were isolated and cultured as previously described (32). Briefly,
epididymal adipose tissue from male SD rats (Charles River Japan,
Tsukuba, Japan), aged 8 wk, was quickly removed and cut into
1-mm pieces with DMEM containing 2 mg/ml type II collagenase,
20 mg/ml BSA, 20 mM HEPES, 100 IU/ml penicillin, and 100
␮g/ml streptomycin. After incubating for 60 min at 37°C in a
rocking platform shaker, the dispersed tissue was filtered through
a nylon mesh (pore size 70 ␮m) and centrifuged at 1,500 rpm for
5 min to remove the floating mature adipocytes. The resulting
pellet was resuspended in erythrocyte lysis buffer consisting of 154
mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA for 10 min at
room temperature. After additional washing and centrifugation
three times, the resuspended pellet was filtered (pore size 25 ␮m)
to remove endothelial cells. The cells thus prepared were cultured
until confluent with DMEM supplemented with 10% FBS, 20 mM
HEPES, 100 IU/ml penicillin, and 100 ␮g/ml streptomycin. Preadipocyte differentiation was performed as previously described
(32). Briefly, after preadipocytes reached confluence [Suppl. Fig.
1A (the online version of this article contains the supplemental
figures and table)], differentiation was induced by the addition of
differentiation medium (DMEM supplemented with 10% FBS, 0.5
mM IBMX, 2.0 ␮M dexamethasone, 2.0 ␮M human insulin, and
0.5 nM T3), which was replaced every 2 days. After 5 days of
treatment, preadipocytes were differentiated into mature adipocytes; ⬎70% of cells were filled with multiple lipid droplets
(Suppl. Fig. 1B). The preadipocytes showed negative immnunostaining for CD11b/c and CD31 (data not shown); thus the possible
contamination of endothelial cells and leukocytes, both of which
may constitute cellular components, was negligible in vascularstromal fraction.
Immunohistochemical staining. Rat preadipocytes grown on glass
coverslips were fixed with 70% cold acetone for 30 min and
permeabilized with 0.2% Triton X-100. Then cells were treated
with a rabbit polyclonal antibody specific for NF-␬B p65 subunit
(1:100; Santa Cruz Biotechnology, Santa Cruz, CA) for 12 h at
4°C. Secondary antibody conjugated to Alexa fluor 488 (1:2,000;
Molecular Probes, Eugene, OR) was used to visualize primary
antibody distribution. Laser scanning confocal microscopy was
performed using a Carl Zeiss LSM 510 system (Carl Zeiss Microscopy, Jena, Germany).
Plasmid constructs and mutagenesis. A series of rat MCP-1 reporter constructs (pGL3-PRM, pGL3-ENH, pGL3-MA1, pGL3MA2) was designed and produced by the methods described (18, 36),
with some modifications based on the rat MCP-1 genomic sequence
ANG II INDUCES MCP-1 IN PREADIPOCYTES VIA NF-␬B
E773
Table 1. Sequences of the PCR primers used for real-time
RT-PCR in this study
PCR product
Rat MCP-1
Rat adiponectin
Rat leptin
Rat ␤-actin
Primer Sequence
F
R
T
F
R
F
R
F
R
5⬘-TCTCTTCCTCCACCACTATGCA-3⬘
5⬘-GGCTGAGACAGCACGTGGAT-3⬘
5⬘-TCACGCTTCTGGGCCTGTTGTTCA-3⬘
5⬘-ATGGCAGAGATGGCACTCCT-3⬘
5⬘-CCTGTCATTCCAGCATCTCC-3⬘
5⬘-CCTGTGGCTTTGGTCCTATC-3⬘
5⬘-GATACCGACTGCGTGTGTGA-3⬘
5⬘-CCCTAAGGCCAACCGTGAAA-3⬘
5⬘-AGGGACAACACAGCCTGGAT-3⬘
PCR
Product
Size
91
94
129
101
dose-dependently (10⫺9 to 10⫺6 M) increased MCP-1 mRNA
levels during 3-h incubation (Fig. 1B). A significant (P ⬍ 0.05)
increase was induced by as low as 10⫺8 M and a maximal
increase (2.9-fold) by 10⫺6 M.
To determine whether ANG II-induced MCP-1 expression
is mediated by ANG II type 1 (AT1) receptor, the effect of
the AT1 receptor antagonist valsartan was tested. ANG
II-induced MCP-1 mRNA expression (Fig. 2A) and protein
Fig. 2. Effect of ANG II type 1 (AT1) receptor antagonists on ANG II-induced
MCP-1 expression in rat preadipocytes. Preadipocytes pretreated with valsartan (VAL; 10⫺6 M) for 2 h were stimulated with ANG II (10⫺6 M) for 3 h for
measurement of MCP-1 mRNA levels by real-time RT-PCR (A) and for 24 h
for measurement of protein levels in conditioned medium by ELISA (B). Data
were obtained from 4 independent experiments. Protein levels were expressed
as pg/24 h, and mRNA levels were calculated and plotted as in Fig. 1. *P ⬍
0.05 vs. control (open bars); †P ⬍ 0.05 vs. ANG II.
Fig. 1. Effect of ANG II on monocyte chemoattractant protein-1 (MCP-1)
mRNA expression in rat preadipocytes. Rat preadipocytes were incubated with
ANG II (10⫺6 M) for the indicated time (A) and in the indicated concentrations
for 3 h (B). MCP-1 mRNA levels were measured by real-time RT-PCR. Data
obtained from 4 independent experiments were expressed as fold increase over
control (open bar). *P ⬍ 0.05 vs. control.
AJP-Endocrinol Metab • VOL
secretion (Fig. 2B) were completely blocked by valsartan
(10⫺6 M).
ANG II regulates MCP-1 gene transcription via redoxsensitive and NF-␬B-dependent pathway. To determine
whether ANG II-induced MCP-1 mRNA expression is mediated by a redox-sensitive and NF-␬B-dependent pathway,
the effects of PDTC, a potent antioxidant (12, 24, 31) and
NF-␬B inhibitor (31), and Bay 11-7085, an inhibitor of
I␬B-␣ phosphorylation (23), were examined (Fig. 3). Both
PDTC (10⫺4 M) and Bay 11-7085 (5 ⫻ 10⫺6 M) completely
blocked the ANG II-stimulated MCP-1 mRNA expression;
treatment with PDTC alone did not show any effect on the
MCP-1 mRNA level, whereas Bay 11-7085 alone significantly decreased the steady-state MCP-1 mRNA level. ANG
II-stimulated MCP-1 mRNA expression was blocked by a
transcriptional inhibitor, actinomycin D (5 ⫻ 10⫺6 M),
suggesting transcriptional regulation of MCP-1 gene by
ANG II (data not shown). Collectively, these data suggest
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MCP-1, monocyte chemoattractant protein-1; F, forward primer; R, reverse
primer; T, TaqMan probe.
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ANG II INDUCES MCP-1 IN PREADIPOCYTES VIA NF-␬B
Fig. 3. Effects of pyrrolidine dithiocarbamate
(PDTC) and Bay 11-7085 (Bay) on ANG IIinduced MCP-1 expression in rat preadipocytes. Preadipocytes pretreated without or with
PDTC (10⫺4 M; A) and Bay 11-7085 (5 ⫻
10⫺6 M; B) for 1 h were stimulated with ANG
II (10⫺6 M) for 3 h. MCP-1 mRNA levels were
measured by real-time RT-PCR. Data from 4
independent experiments were calculated and
plotted as in Fig. 2. *P ⬍ 0.05 vs. control
(open bar); †P ⬍ 0.05 vs. ANG II.
increase in the MCP-1 promoter activity to the same extent as
did TNF-␣ (10 ng/ml) in pGL3-ENH-transfected preadipocytes
(Fig. 5B); neither pGL3-MA1 nor -MA2 constructs were responsive to ANG II. These results suggest that the two distinct
NF-␬B binding sites (A1, A2) in the MCP-1 enhancer region
are essential for ANG II-induced MCP-1 promoter activity in
rat preadipocytes.
Differential expression of MCP-1 mRNA during preadipocytes differentiation. Because many proinflammatory genes are
predominantly expressed in the stromal-vascular fraction cells
containing preadipocytes predominantly in adipose tissue from
experimental obese mice (42), we examined the changes of
gene expression of several adipokines during preadipocytes
differentiation. Along with preadipocyte differentiation, basal
expression of both adiponectin and leptin mRNAs was significantly (P ⬍ 0.05) greater in differentiated adipocytes than in
preadipocytes (Fig. 6A), whereas ANG II (10⫺6 M) did not
affect adiponectin and leptin gene expression in either preadipocytes or differentiated adipocytes (Suppl. Fig. 2). In contrast,
basal expression of both MCP-1 mRNA (Fig. 6B) and protein
(Fig. 6C) was significantly (P ⬍ 0.05) greater in preadipocytes
than in differentiated adipocytes, whose effect was more pronounced in the presence of ANG II (10⫺6 M). These data
suggest that MCP-1 expression declines during preadipocyte
Fig. 4. Immunohistochemical staining for NF-␬B p65 subunit after ANG II treatment in rat preadipocyte. A: nontreated cells as control. B and C: preadipocytes
pretreated without (B) or with (C) valsartan (10⫺6 M) for 2 h were stimulated with ANG II (10⫺6 M) for 30 min. Representative microscopic images of
immunohistochemical staining for NF-␬B p65 subunit are shown. Scale bar, 10 ␮m.
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that the redox-sensitive pathway is involved in ANG IIinduced MCP-1 gene expression in rat preadipocytes and
that the NF-␬B signaling pathway is involved in the basal as
well as ANG II-stimulated MCP-1 gene expression.
To confirm that ANG II induces the activation of NF-␬B, we
examined by immunocytochemical analysis whether nuclear
translocation of the p65 subunit of NF-␬B takes place after
ANG II treatment. p65 Immunoreactivity was located exclusively in the cytoplasm in nonstimulated cells (Fig. 4A),
whereas it was concentrated in the nucleus of the cells after
stimulation with ANG II (10⫺6 M) for 30 min (Fig. 4B), whose
effect was prevented by pretreatment with valsartan (Fig. 4C).
These results clearly indicate that ANG II activates NF-␬B by
translocation of its p65 subunit to the nucleus in rat preadipocytes.
ANG II enhances MCP-1 promoter activity via NF-␬Bdependent pathway. We then determined whether NF-␬B is
actually involved in the ANG II-induced activation of the
MCP-1 promoter by luciferase assay. Because the rat MCP-1
promoter possesses two distinct NF-␬B binding sites (A1 and
A2) crucial for enhancer activity in the distal enhancer region
(39), we used the MCP-1 promoter/enhancer-driven reporter
construct pGL3-ENH and two mutated (A1, A2) reporter
constructs, pGL3-MA1 and -MA2, respectively (Fig. 5A).
ANG II (10⫺6 M) caused a significant (P ⬍ 0.05) 1.5-fold
ANG II INDUCES MCP-1 IN PREADIPOCYTES VIA NF-␬B
E775
DISCUSSION
differentiation and that preadipocytes are the major site of
MCP-1 expression after ANG II stimulation.
ANG II induces MCP-1 expression in rat adipose tissue in
vivo. Finally, we examined whether ANG II induces MCP-1
expression in various adipose tissues from SD rats. After a
3-h intraperitoneal injection of ANG II, MCP-1 mRNA
levels significantly increased (P ⬍ 0.05) in various adipose
tissues (epididymal, subcutaneous, and mesenteric), whose
effect was blocked by pretreatment with valsartan (Fig. 7);
plasma ANG II concentrations (134.8 ⫾ 45.9 pg/ml) increased markedly from basal levels (11.5 ⫾ 2.5 pg/ml) after
ANG II administration. However, systolic blood pressure
did not significantly change after ANG II treatment (Suppl.
Table 1). Furthermore, ANG II (10⫺6 M) significantly (P ⬍
0.05) induced almost comparable MCP-1 mRNA expression
in preadipocytes from various fat pads, including epididymal, subcutaneous, and mesenteric adipose tissues (Suppl.
Fig. 3), suggesting that ANG II equally induces MCP-1 gene
expression in any preadipocytes from different adipose
tissues.
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Fig. 5. MCP-1 promoter activity by ANG II in rat preadipocytes. A: schematic
representation of rat MCP-1 reporter constructs showing proximal promoter
region (PRM) and distal enhancer region (ENH), indicated by open and filled
boxes, respectively. Mutation of A1 (MA1) and A2 (MA2) site of the enhancer
region is indicated by crossout (X) in pGL3-MA1 and pGL3-MA2, respectively. B: preadipocytes transfected with MCP-1 reporter constructs and
pRL-TK plasmid were stimulated without (open bars) or with ANG II (10⫺6
M; filled bars) and TNF-␣ (10 ng/ml, gray bar) for 4 h. Each luciferase (luc⫹)
activity was normalized by the ratio of the indicated luciferase activity to that
of cotransfected pRL-TK. Relative luciferase activities expressed as fold
increase over control are shown. Each bar represents data from 4 independent
experiments. *P ⬍ 0.05 vs. control.
We have clearly demonstrated for the first time that ANG II
induces MCP-1 mRNA and protein expression in rat primary
cultured preadipocytes via the AT1 receptor-mediated and
NF-␬B-dependent pathways. In addition, MCP-1 expression in
rat adipose tissue was augmented by exogenous ANG II
administration in vivo. The present results are consistent with
the previous findings that ANG II stimulates gene expression
of MCP-1 as well as other proinflammatory factors in cardiovascular cells (3, 5, 43, 44). Taken together with the notion that
adipose tissue produces angiotensinogen (17), it is assumable
that ANG II locally generated in adipose tissue may participate
in the inflammatory response in adipose tissue.
In cardiovascular cells, ANG II increases reactive oxygen
species (ROS) formation mainly through NADPH oxidase
activation via AT1 receptor and subsequently activates various
redox-sensitive transcription factors, such as NF-␬B, AP-1,
and Sp-1, thereby leading to increased expression of a series of
redox-sensitive proinflammatory genes, including MCP-1 (3,
5, 43, 44). These observations are consistent with our results
that AT1 receptor antagonist (valsartan), a potent antioxidant
(PDTC), and a transcription inhibitor (actinomycin D) all
abolished ANG II-induced MCP-1 expression in primary cultured rat preadipocytes. Although it remains unknown whether
PDTC alters redox state in adipocytes, this reagent has been
widely used as a potent antioxidant as well as an NF-␬B
inhibitor without affecting other redox-sensitive transcriptional
factors (AP-1, AP-2, Sp-1) in various cell culture experiments
(12, 24, 31). It is therefore suggested that the inhibitory effect
of PDTC on MCP-1 expression in rat preadipocytes is mediated through its antioxidant as well as NF-␬B inhibition effects.
NF-kB is a key transcriptional factor primarily involved in
the process of inflammation. NF-kB, a heterodimeric complex
usually composed of the p50/p65 subunit, associates with a
cytoplasmic inhibitor, I␬B-␣, to form an inactive ternary complex (2). Activation of NF-␬B requires proteasome-mediated
degradation of I␬B-␣ after phosphorylation by serine/threonine
kinases, termed I␬B kinases (IKK), thereby causing NF-␬B
p50/p65 heterodimer translocate to the nucleus and bind to
NF-␬B sites of its target genes, and finally leading to activation
of its target gene transcription (10, 30).
In the present study, we clearly demonstrated the involvement of NF-kB in MCP-1 gene expression by ANG II in
preadipocytes. First, PDTC, an antioxidant and NF-kB inhibitor, and Bay 11-7085, an irreversible inhibitor of I␬B-␣
phosphorylation, abolished the ANG II-induced MCP-1 gene
expression. Second, immunohistochemical analysis revealed
that ANG II stimulates nuclear translocation of NF-kB p65
subunit, a hallmark of NF-␬B activation. Third, luciferase
assay using a series of rat MCP-1 promoter constructs revealed
that two NF-kB binding sites (A1, A2) in the MCP-1 enhancer
region were essential for ANG II-induced MCP-1 transcriptional activation. It has been shown that redox-signaling molecules, including ROS, stimulate IKK␤ to activate the downstream NF-␬B pathway (30). Taken together, it is conceivable
that ANG II-stimulated redox-signaling, and possibly ROS
generation (11, 43, 44), induces IKK␤ activation in preadipocytes, finally leading to transcriptional activation of the MCP-1
promoter via the NF-␬B pathway. However, the possibility that
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ANG II INDUCES MCP-1 IN PREADIPOCYTES VIA NF-␬B
other putative binding motifs located in the MCP-1 promoter
may also contribute to the ANG II-induced MCP-1 transcriptional activation remains to be elucidated. It should also be
noted that the NF-␬B pathway may be involved in the constitutive MCP-1 gene expression in rat preadipocytes, since Bay
11-7085 also decreased the steady-state MCP-1 mRNA expression.
Fig. 7. Effect of ANG II administration on MCP-1 mRNA expression in
various rat adipose tissues in vivo. After pretreatment without or with valsartan
(100 mg/kg body wt) for 7 days, saline (control), or ANG II (1 ␮g/100 g body
wt) was administrated ip. After 3 h, MCP-1 mRNA levels in epididymal,
subcutaneous, and mesenteric adipose tissue were measured by real-time
RT-PCR. Data from 6 animals were calculated and plotted as in Fig. 2. *P ⬍
0.05 vs. control (open bar); †P ⬍ 0.05 vs. ANG II.
AJP-Endocrinol Metab • VOL
The present study also revealed that MCP-1 gene expression changes during cell differentiation; steady-state MCP-1
mRNA levels drastically decreased after preadipocyte differentiation, whereas those of leptin and adiponectin markedly increased. Our data are consistent with previous studies
using 3T3L-1 and human primary preadipocytes in which
MCP-1 mRNA expression level is greater in preadipocytes
than in mature adipocytes (7, 9). Furthermore, ANG IIstimulated MCP-1 mRNA expression was markedly blunted
after preadipocyte differentiation. These findings lend support to the assumption that preadipocytes, rather than matured adipocytes, are the major site of MCP-1 expression,
especially in response to ANG II.
The present study also showed that exogenous administration of ANG II in rats increased MCP-1 mRNA expression in
epididymal, subcutaneous, and mesenteric fat pads, whose
effects were blocked by the AT1 receptor antagonist. Thus our
in vivo data complement the in vitro study showing that
adipose MCP-1 gene expression is induced by ANG II. Recent
findings indicated that cells from the vascular-stromal fraction
rather than the adipocyte fraction from adipose tissue with
obesity is the primary source for a series of proinflammatory
mediators, such as MCP-1 and TNF-␣ (42). Because ANG
II-induced MCP-1 mRNA expression was greater in preadipocytes than in differentiated adipocytes in vitro, our in vivo
findings suggest that preadipocytes in the whole adipose tissue
could largely contribute to MCP-1 expression by ANG II.
However, our in vivo results could not exclude the possibility
that other cells in the vascular-stromal fraction, especially
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Fig. 6. Changes in adiponectin, leptin, and
MCP-1 gene expressions during preadipocyte
differentiation. Preadipocytes (open bars) and
differentiated adipocytes (filled bars) were cultured for measurement of adiponectin and leptin mRNA levels (A) and were stimulated with
or without ANG II (10⫺6 M) for measurements
of MCP-1 mRNA (B) and MCP-1 protein (C),
respectively, in medium. Data from 4 independent experiments are calculated and plotted as
in Fig. 2. *P ⬍ 0.05 vs. preadipocytes; †P ⬍
0.05 vs. ANG II.
ANG II INDUCES MCP-1 IN PREADIPOCYTES VIA NF-␬B
GRANTS
This study was supported in part by grants-in-aid from the Ministry of
Education, Science, Sports, Culture and Technology, and the Ministry of
Health, Labor and Welfare, of Japan.
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increases MCP-1 expression in rat adipose tissue in vivo as
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disease and obesity.
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