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Am J Physiol Endocrinol Metab 291: E771-E778, 2006. First published May 16, 2006; doi:10.1152/ajpendo.00560.2005
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Angiotensin II induces monocyte chemoattractant protein-1 expression via a nuclear factor-{kappa}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


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 time- and 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-{kappa}B inhibitor (pyrrolidine dithiocarbamate) and an inhibitor of 1{kappa}B-{alpha} phosphorylation (Bay 11-7085) also blocked ANG II-induced MCP-1 mRNA expression. ANG II induced translocation of NF-{kappa}B p65 subunit from cytoplasm to nucleus by immunocytochemical study. Luciferase assay using reporter constructs containing MCP-1 promoter region revealed that two NF-{kappa}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 receptor-mediated and NF-{kappa}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.

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 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
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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-1-methylxanthine (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 (Plymouth 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 x 10–6 M), and actinomycin D (5 x 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 µg/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 µg/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 µm) 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 µm) 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 µg/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 µM dexamethasone, 2.0 µM 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 vascular-stromal fraction.


Figure 1
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  		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.

 
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-{kappa}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, pGL3-MA2) was designed and produced by the methods described (18, 36), with some modifications based on the rat MCP-1 genomic sequence (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'-GAAACTCGAGTTACTCAGCAGATTCAAACT-3' 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'-GCTAAATATCTCTCCTGAAGGGTCTATCAACTTCCAAT-ACTGCCTC-3' and antisense primer 5'-GAGGCAGTAT- TGGAAGTTGATAGACCCTTCAGGAGAGATATTTAGC-3' for mutation in NF-{kappa}B site A and sense primer 5'-CCAATAC- TGCCTCAGAATATC-AATTTCCACACTCTTATCCTACTCT- GC-3' and antisense primer 5'-GCAGAGTAGGATAAGAG- TGTGGAAATTGATATTCTGAGGCAGTATTGG-3' for mutation in NF-{kappa}B site B (mutated bases are underlined). PCR mutagenesis was performed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All mutations were confirmed by DNA sequencing.

Transient transfection and luciferase assay. Transient transfection of 2 µg of each MCP-1 reporter plasmid and 0.1 µg of pRL-TK plasmid into preadipocytes were performed using synthetic cationic liposome, (+)-N,N-[bis(2-hydroxyethyl)]-N-methyl-N-[2,3-di(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 beta-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 beta-actin and used as an endogenous internal control; the relative levels of each mRNA to that of beta-actin were calculated.


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  		Table 1. Sequences of the PCR primers used for real-time RT-PCR in this study

 
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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
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 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 secretion (Fig. 2B) were completely blocked by valsartan (10–6 M).


Figure 2
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  		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); {dagger}P < 0.05 vs. ANG II.

 
ANG II regulates MCP-1 gene transcription via redox-sensitive and NF-{kappa}B-dependent pathway. To determine whether ANG II-induced MCP-1 mRNA expression is mediated by a redox-sensitive and NF-{kappa}B-dependent pathway, the effects of PDTC, a potent antioxidant (12, 24, 31) and NF-{kappa}B inhibitor (31), and Bay 11-7085, an inhibitor of I{kappa}B-{alpha} phosphorylation (23), were examined (Fig. 3). Both PDTC (10–4 M) and Bay 11-7085 (5 x 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 x 10–6 M), suggesting transcriptional regulation of MCP-1 gene by ANG II (data not shown). Collectively, these data suggest that the redox-sensitive pathway is involved in ANG II-induced MCP-1 gene expression in rat preadipocytes and that the NF-{kappa}B signaling pathway is involved in the basal as well as ANG II-stimulated MCP-1 gene expression.


Figure 3
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  		Fig. 3. Effects of pyrrolidine dithiocarbamate (PDTC) and Bay 11-7085 (Bay) on ANG II-induced MCP-1 expression in rat preadipocytes. Preadipocytes pretreated without or with PDTC (10–4 M; A) and Bay 11-7085 (5 x 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); {dagger}P < 0.05 vs. ANG II.

 
To confirm that ANG II induces the activation of NF-{kappa}B, we examined by immunocytochemical analysis whether nuclear translocation of the p65 subunit of NF-{kappa}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-{kappa}B by translocation of its p65 subunit to the nucleus in rat preadipocytes.


Figure 4
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  		Fig. 4. Immunohistochemical staining for NF-{kappa}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-{kappa}B p65 subunit are shown. Scale bar, 10 µm.

 
ANG II enhances MCP-1 promoter activity via NF-{kappa}B-dependent pathway. We then determined whether NF-{kappa}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-{kappa}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 increase in the MCP-1 promoter activity to the same extent as did TNF-{alpha} (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-{kappa}B binding sites (A1, A2) in the MCP-1 enhancer region are essential for ANG II-induced MCP-1 promoter activity in rat preadipocytes.


Figure 5
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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-{alpha} (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.

 
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 differentiation and that preadipocytes are the major site of MCP-1 expression after ANG II stimulation.


Figure 6
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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; {dagger}P < 0.05 vs. ANG II.

 
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.


Figure 7
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  		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 µg/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); {dagger}P < 0.05 vs. ANG II.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
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-{kappa}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-{kappa}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-{kappa}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-{kappa}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{kappa}B-{alpha}, to form an inactive ternary complex (2). Activation of NF-{kappa}B requires proteasome-mediated degradation of I{kappa}B-{alpha} after phosphorylation by serine/threonine kinases, termed I{kappa}B kinases (IKK), thereby causing NF-{kappa}B p50/p65 heterodimer translocate to the nucleus and bind to NF-{kappa}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{kappa}B-{alpha} 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-{kappa}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 IKKbeta to activate the downstream NF-{kappa}B pathway (30). Taken together, it is conceivable that ANG II-stimulated redox-signaling, and possibly ROS generation (11, 43, 44), induces IKKbeta activation in preadipocytes, finally leading to transcriptional activation of the MCP-1 promoter via the NF-{kappa}B pathway. However, the possibility that 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-{kappa}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.

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 II-stimulated 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-{alpha} (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 macrophages, also contribute to MCP-1 expression by ANG II in the whole adipose tissue.

Because local RAS is augmented in adipose tissue in obesity (25), augmented MCP-1 expression by ANG II, in vitro as well as in vivo, as demonstrated in this study, lends strong support to the contention that ANG II-induced MCP-1 expression in adipose tissue may partly contribute to the development of insulin resistance and the metabolic syndrome. Indeed, it has been shown that plasma MCP-1 levels are increased and correlate with body weight in diet-induced obese human models (6). MCP-1 has been shown to decrease insulin-stimulated glucose uptake and expression of adipogenic genes, such as peroxisome proliferator-activated receptor-{gamma}, adipocyte lipid-binding protein, adipsin, and lipoprotein lipase (28), suggesting its anti-insulin action on adipose tissue as well as its inhibitory effect on adipocyte differentiation. In primary culture of human adipocytes, it has been shown that ANG II inhibits adipocyte differentiation via the AT1 receptor, whereas its blockade markedly enhances adipogenesis (15). Thus ANG II-induced MCP-1 expression in preadipocytes may be partly responsible for its inhibitory effect on adipocyte differentiation.

In the obese state, both angiotensinogen and MCP-1 genes in adipose are overexpressed (25, 28). There have been no studies demonstrating that ANG II-induced MCP-1 expression is responsible for the recruitment of macrophages to adipose tissue. However, it has been well established that ANG II enhances focal inflammation and macrophage recruitment in cardiovascular tissue (3, 21, 37). Therefore, it is reasonable to assume that ANG II-induced MCP-1 expression in adipose tissue may be involved in focal inflammation and macrophage recruitment, as is the case in cardiovascular tissue. However, the exact pathophysiological role of adipose tissue-derived MCP-1 in the development of insulin resistance and cardiovascular disease associated with obesity awaits further study.

In conclusion, our present study demonstrates that ANG II induces MCP-1 expression via AT1 receptor- and NF-{kappa}B-dependent pathways in rat preadipocytes in vitro and that it increases MCP-1 expression in rat adipose tissue in vivo as well. Thus augmented MCP-1 expression by ANG II in preadipocytes may provide a new link between the cardiovascular disease and obesity.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
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.


   FOOTNOTES
 

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: tyoshimoto.cme{at}tmd.ac.jp)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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