Increased expression of CCAAT/enhancer binding protein-β and -δ and monocyte chemoattractant protein-1 genes in aortas from hyperinsulinaemic rats
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- Sato, Y., Nishio, Y., Sekine, O. et al. Diabetologia (2007) 50: 481. doi:10.1007/s00125-006-0480-4
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We evaluated whether hyperinsulinaemia stimulates the expression of transcription factor CCAAT/enhancer binding protein (C/EBP)-β and C/EBP-δ and leads to the induction of the chemokine (C-C motif) ligand 2 gene (Ccl2, also known as MCP-1) expression in aortas.
Hyperinsulinaemia was induced by feeding rats a high-fructose diet. CCL2 production was analysed by ELISA. The expression of Ccl2, Cebpb and Cebpd mRNAs was investigated by quantitative RT-PCR. The binding of C/EBP-β to Ccl2 was assessed by chromatin immunoprecipitation (ChIP) assay.
Insulin at a concentration of 10 nmol/l significantly stimulated the expression of Cebpb,Cebpd and Ccl2 mRNAs, depending on activation of phosphatidylinositol 3-kinase (PI3K) in cultured vascular smooth muscle cells. The knock-down of C/EBP-β with siRNA abolished the insulin-induced Ccl2 mRNA expression. In the aortas from fructose-fed rats, the levels of phosphorylation of Akt/protein kinase B, a downstream effector of PI3K, were also increased. The expression of Cebpb, Cebpd and Ccl2 mRNAs in the aortas from fructose-fed rats were significantly elevated, by 330, 300 and 300%, respectively, compared with those of control-fed rats. The induction Ccl2 mRNA expression in the aortas was significantly correlated with the expression of Cebpb and Cebpd mRNAs in the aortas. Furthermore, the ChIP assay showed elevated binding of C/EBP-β to the 5′ upstream region of Ccl2 in the aortas from fructose-fed rats.
These findings clearly indicate the role of C/EBPs in the mechanism of upregulation of CCL2, an inflammation-related protein, observed in the hyperinsulinaemic state, which may initiate the process of atherosclerosis.
KeywordsAtherosclerosis C/EBP ChIP Inflammation Hyperinsulinaemia CCL2 PI3K MCP-1
CCAAT/enhancer binding protein
chemokine (C-C motif) ligand 2 (also known as monocyte chemoattractant protein-1)
nuclear factor-kappa B
vascular smooth muscle cell
Atherosclerosis is the result of excessive proliferative and inflammatory responses of vascular tissues, including migration and proliferation of smooth muscle cells and infiltration of inflammatory cells . In the atherosclerotic lesion, vascular cells release a number of inflammatory cytokines, such as IL-6, IL-1, TNF-α and chemokine (C-C motif) ligand 2 (CCL2, also known as monocyte chemoattractant protein-1) . The role of insulin in atherogenesis has long been a matter of debate, with many epidemiological studies  suggesting that hyperinsulinaemia is a strong independent risk factor for a cardiovascular event [4, 5]. Several clinical studies, including the UKPDS , have shown that insulin treatment does not increase the risk of cardiovascular disease, therefore, it may be that a decrease in insulin action in the vascular tissue is responsible for increased cardiovascular risk in the hyperinsulinaemic state. We recently reported that in the presence of physiological concentrations of insulin and continuous activation of membrane-targeted phosphatidylinositol 3-kinase (PI3K) through the overexpression of p110CAAX, Ccl2 expression is increased through the activation of transcription factors known as CCAAT/enhancer binding proteins (C/EBPs) . In addition to CCL2, C/EBPs are responsible for the induction of a wide array of other genes, including those encoding proteins with a role in innate immunity, cell proliferation, adipogenesis, inflammatory response and the acute-phase response, such as IL-6, IL-8, IL-12, TNF-α and C-reactive protein (CRP) [8, 9, 10]. Several reports have shown that C/EBPs play an important role in atherosclerosis [11, 12, 13]. Thus, it might be possible that insulin induces the expression of genes involved in inflammation through activation of the C/EBPs in the cardiovascular tissues. While several lines of epidemiological evidence indicate that hyperinsulinaemia is associated with increased levels of inflammatory proteins such as serum CRP [14, 15], to our knowledge, there are no data on the expression of C/EBP genes or the role of C/EBPs in the expression of genes encoding inflammatory cytokines in cardiovascular tissues in the hyperinsulinaemic state. Use of a hyperinsulinaemic rat model to elucidate the role of C/EBPs in the gene expression of inflammatory cytokines in the cardiovascular tissues may provide insights into cardiovascular abnormalities in hyperinsulinaemic insulin-resistant patients and possible molecular targets for therapeutic amelioration of the development of atherosclerosis in these patients.
The aim of this study was to investigate the expression of Cebpb, Cebpd and Ccl2 in cardiovascular tissues from fructose-feeding-induced hyperinsulinaemic rats and in cultured vascular smooth muscle cells (VSMCs) exposed to physiological concentrations of insulin.
Materials and methods
Human recombinant insulin was kindly provided by Eli Lilly & Co. (Indianapolis, IN, USA). DMEM and FCS were from Invitrogen (Grand Island, NY, USA). Akt/protein kinase B and phospho-Akt antibodies were from Cell Signaling Technology (Beverly, MA, USA). Antibodies to C/EBP-β and C/EBP-δ were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to CCL2 was from Chemicon (Temecula, CA, USA). LY294002 was purchased from Sigma (St Louis, MO, USA). XAR-5 film was purchased from Eastman Kodak (Rochester, NY, USA).
VSMCs were isolated from the aortas of male Sprague–Dawley rats (200–300 g) by enzymatic digestion, and maintained in DMEM supplemented with 10% FCS, 80 U/ml penicillin G and 80 μg/ml streptomycin in 100-mm plates (5 × 106 cells per dish), as described previously . VSMCs were used between the 6th and 12th passages. Cell growth was arrested for 48 h in DMEM supplemented with 0.1% FCS before the experiments.
siRNA analysis To silence C/EBP-β, siRNA against rat Cebpb mRNA was utilised (SMART pool Reagent; Dharmacon, Lafayette, CO, USA). A control siRNA was also utilised (non-silencing control siRNA; Qiagen, Valencia, CA, USA). Rat VSMCs were transfected with 10 nmol/l siRNA using DharmaFECT 1 (Dharmacon), and the silencing effect was confirmed by real-time PCR and western blotting 48 h after transfection. VSMCs transfected with siRNA were starved for 48 h and then stimulated by the addition of 10 nmol/l insulin for 4 h at 37°C.
Six-week-old male Sprague–Dawley rats (Japan SLC, Shizuoka, Japan) were housed in an environmentally controlled room with a 12 h light–dark cycle. The animals were divided into a control diet group and a high-fructose diet group. The rats in each group were fed for 4 weeks. The control diet (Oriental Yeast, Tokyo, Japan) consisted of 58% carbohydrate (no fructose), 12% fat and 30% protein (energy % of diet). The diet high in fructose (Oriental Yeast) contained 67% carbohydrate (98% of which was fructose), 13% fat and 20% protein. The rats were pair-fed. The day before the experiment, food was withdrawn from the cages of all animals at 20.00 hours. Rats were re-fed in the dark from 06.00–08:00 hours, at which time the chow was removed. After 2 h of food deprivation, rats were anaesthetised, and the aorta and the heart were removed and immediately frozen in liquid nitrogen and stored at −80°C until use. All experiments were approved by the Shiga University of Medical Science Animal Care Committee.
Western blot analysis
The aortas from experimental animals were powdered in liquid nitrogen and lysed in a solubilising buffer containing 20 mmol/l Tris–HCl, pH 7.5, 1 mmol/l EDTA, 140 mmol/l NaCl, 1% Nonidet P-40 (Nakarai Tesque, Kyoto, Japan), 50 U/ml aprotinin and 1 mmol/l phenylmethylsulfonyl fluoride (PMSF) for 10 min at 4°C. For western blot analysis, whole-tissue lysates were denatured by boiling in Laemmli sample buffer containing 100 mmol/l dithiothreitol, resolved by SDS-PAGE, and then transferred to nitrocellulose by electroblotting. Blots were then incubated with a rabbit anti-phospho-Akt primary antibody, a rabbit anti-Akt primary antibody, a rabbit anti-CCL2 primary antibody, a rabbit anti-C/EBP-β primary antibody or a rabbit anti-C/EBP-δ primary antibody plus a horseradish peroxidase-linked secondary antibody (GE Healthcare Bio-Sciences, Piscataway, NJ, USA), followed by chemiluminescence detection, according to the instructions of the manufacturer (PerkinElmer Life Sciences, Boston, MA, USA).
Chromatin immunoprecipitation assay
The chromatin immunoprecipitation (ChIP) assay protocol was adapted from methods described elsewhere [17, 18]. In brief, rat VSMCs (1 × 108 cells/condition) were cross-linked with 1% formaldehyde in PBS at 37°C for 15 min. Cells were harvested with cell scraping buffer (1 ml/plate of ice-cold PBS with the protease and phosphatase inhibitors 1 mmol/l PMSF, protease inhibitor cocktail [Roche Diagnostics, Tokyo, Japan]). Cells were then pelleted, resuspended in SDS lysis buffer (1% SDS, 10 mmol/l EDTA, 50 mmol/l Tris–HCl, pH 8, with protease and phosphatase inhibitors), and incubated on ice for 10 min. To shear chromatin, the lysate was sonicated on ice, yielding chromatin fragments of around 600 bp in size. Samples were centrifuged at 17,000 g for 10 min at 4°C and the supernatant fraction was divided into aliquots for subsequent tenfold dilution in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mmol/l EDTA, 16.7 mmol/l Tris–HCl, pH 8, with protease and phosphatase inhibitors). To provide the same amount of DNA for each sample, DNA concentration was measured using Hoechst 33258 dye (Invitrogen, Carlsbad, CA, USA). To reduce non-specific binding, each 1-ml chromatin sample was precleared with 30 μl of protein A-agarose slurry (Upstate, Charlottesville, VA, USA), supplemented with 400 μg/ml sonicated E. coli DNA (Invitrogen, Carlsbad, CA, USA), for 1 h at 4°C. Chromatin complexes were immunoprecipitated for 12–18 h at 4°C with 1–2 μg of primary antibody or non-specific antibody (control) to provide negative controls. Immune complexes were collected with 30 μl of protein A-agarose for 3 h at 4°C, followed by centrifugation at 17,000 g for 1 min at 4°C. The precipitates were washed with 1 ml of each of the following buffers twice in succession for 5 min at 4°C: low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mmol/l EDTA, 20 mmol/l Tris–HCl, pH 8.0, 150 mmol/l NaCl), high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mmol/l EDTA, 20 mmol/l Tris–HCl, pH 8.0, 500 mmol/l NaCl), LiCl wash buffer (250 mmol/l LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mmol/l EDTA, 10 mmol/l Tris–HCl, pH 8.0), and TE buffer (10 mmol/l Tris–HCl, pH 8.0, 1 mmol/l EDTA). Chromatin complexes were eluted with 200 μl of elution buffer (1% SDS, 0.1 mol/l NaHCO3, 10 mmol/l dithiothreitol) at 65°C. To reverse cross-linking, NaCl (200 mmol/l final concentration) was added, and the samples were incubated at 65°C for over 6 h. To digest proteins, samples were incubated with 10 mmol/l EDTA, 40 mmol/l Tris–HCl, pH 6.5, and 50 μg/ml proteinase K at 45°C for 90 min. Samples were extracted with phenol–chloroform, resuspended in 20 μl of TE buffer, and stored at −80°C until quantitative real-time PCR analysis was performed.
For the ChIP assay using tissue samples, the aortas were incubated with 1% formaldehyde in PBS at room temperature for 15 min, followed by incubation with PBS supplemented with 125 mmol/l glycine for 5 min. Samples were then frozen in liquid nitrogen and stored at −80°C until use. Frozen tissues were powdered in liquid nitrogen, and 2 ml ice-cold PBS was added. After homogenisation, samples were resuspended in 500 μl cell lysis buffer (5 mmol/l 1,4-piperazinediethanesulfonic acid [PIPES], 85 mmol/l KCl, 0.5% Nonidet P-40) with phosphatase inhibitors, the protease inhibitor cocktail and PMSF (1 mmol/l), and incubated on ice for 15 min. They were then centrifuged at 17,000 g for 1 min at 4°C. The pellet was resuspended in 300 μl SDS lysis buffer with the same protease inhibitors and incubated on ice for 10 min. Approximately 0.1 g of glass beads (G-1277; Sigma) was added to each sample. The mixture of nuclei and glass beads was sonicated on ice until the average length of chromatin was about 600 bp, and then microfuged at 17,000 g for 10 min at 4°C to remove debris.
RNA extraction and real-time PCR analysis
Frozen tissues were powdered in liquid nitrogen, and total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using 1 μg of total RNA and reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Real-time quantitative PCR was performed using the fluorescent dye SYBR Green I (Roche Molecular Biochemicals, Mannheim, Germany) and real-time PCR system (Roche). To exclude the contamination of non-specific PCR products, melting curve analysis was applied to all final PCR products after the cycling protocol. Finally, the quality of the PCR products was analysed by conventional agarose gel electrophoresis. All the quantitative data were normalised by the intensity of 18s rRNA signals.
Immunohistochemical analysis was performed as described previously [19, 20]. Briefly, rats were perfused with a fixative containing 4% paraformaldehyde, 0.5% glutaraldehyde and 0.2% picric acid in 0.1 mol/l phosphate buffer. The aortic tissue was dissected out and immersed for 24 h in a postfixative containing 4% paraformaldehyde, 0.2% picric acid in 0.1 mol/l phosphate buffer at 4°C. The sections were incubated for 2 days with antibody against CCL2, or C/EBP-β and α-smooth muscle actin (α-SMA), diluted in accordance with the manufacturer’s instructions. The sections were then incubated with the species-specific secondary antibodies conjugated to either fluorescein isothiocyanate (FITC) or Texas Red and viewed using fluorescence microscopy (Olympus IX70, Japan). Images were taken with a CCD camera (Cool SNAP/HQ; Nippon Roper, Osaka, Japan).
Northern blot analysis
Total RNA (30 μg) was denatured and electrophoresed on a 1% agarose gel and blotted onto a Nytran N membrane. The membrane was incubated with specific probes, washed, and visualised by autoradiography. All the quantitative data were corrected by the intensity of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA signals.
The sets of PCR primers were designed as shown in Electronic supplementary material (ESM) Table 1.
Concentrations of CCL2 in plasma and conditioning media were determined using an ELISA kit (BioSource International, Camarillo, CA, USA).
Values are expressed as means ± SEM. The Tukey–Kramer comparison test was used to determine the significance of differences between four or more groups. The non-paired t test was used to determine the significance of differences between two groups. Pearson correlations and scatter plots were used to evaluate bivariate relationships. A p value of less than 0.05 was considered significant.
Insulin induces Ccl2 gene expression and secretion of the protein through the PI3K pathway in VSMCs
Insulin induces the expression of Cebpb and Cebpd and increases the binding of C/EBP-β to the Ccl2 promoter in VSMCs
Characteristics of experimental animals
Characteristics of the experimental groups
Control diet-fed rats (n = 8)
Fructose diet-fed rats (n = 10)
Body weight (g)
352.8 ± 9.7
343.2 ± 7.3
Blood glucose (mmol/l)
6.14 ± 0.26
6.17 ± 0.20
330 ± 52
670 ± 95a
Total cholesterol (mmol/l)
1.34 ± 0.08
1.69 ± 0.14b
0.69 ± 0.07
1.90 ± 0.34a
50.0 ± 5.9
63.4 ± 7.2
Phosphorylation of Akt in the aorta
Aortic mRNA expression and protein production
Binding of C/EBP-β to the 5′ upstream region of the Ccl2 gene in the aortas from hyperinsulinaemic rats
Expression of Ccl2, Cebpb and Cebpd in the heart
In the present study we found that the expression of Cebpb and Cebpd was increased in the aortas from fructose diet-induced hyperinsulinaemic rats. C/EBP family members, especially C/EBP-β and C/EBP-δ, are known to play an important role in the regulation of the expression of proinflammatory genes, including CCL2 [8, 23]. We found that, in aortas, the levels of Cebpb and Cebpd mRNAs correlated very closely with the levels of Ccl2 mRNA. The ChIP assay demonstrated increased binding of C/EBP-β to the C/EBP response element located at −3123 to −3099 bp from the starting point of rat Ccl2 transcription in the aortas from hyperinsulinaemic rats. In addition, inhibition of Cebpb gene expression with siRNA completely blocked the insulin-induced expression of Ccl2 in VSMCs. These findings clearly indicate that the increased expression of Cebpb and Cebpd mRNAs in the aorta is responsible for the enhanced expression of Ccl2 in the aortas from hyperinsulinaemic rats. Importantly, CCL2 is reported to be play a role in most of the processes involved in the formation of atherosclerotic lesions  and is strongly expressed in human atheromatous lesions but not in normal arteries . Furthermore, the selective knockout of CCR2, the receptor for CCL2, prevents the initiation of the atherosclerotic process in vivo . Therefore, our present findings suggest a significant role for C/EBPs as regulators of CCL2 production in the development of atherosclerosis. However, our results do not necessarily imply that the induction of Ccl2 gene expression observed in the aorta in the hyperinsulinaemic state is exclusively due to an enhanced activity of C/EBPs. It has been reported that several transcription factors, including nuclear factor-κB, have a role in the regulation of Ccl2 expression [27, 28]. Using an electrophoretic gel retardation assay, we have previously shown increased binding activity of NF-κB in the aorta and in the heart of fructose-feeding-induced hyperinsulinaemic rats using an electrophoretic gel retardation assay . In addition to the effect on Ccl2 gene, C/EBP-β, and C/EBP-δ regulate the transcription of a number of genes involved in the inflammatory response [8, 29]. Furthermore, other reports have revealed a role for C/EBPs in the development of atherosclerosis [11, 12, 13]. Thus, increased expression of Cebpb and Cebpd in aortas from hyperinsulinaemic rats may be a significant factor to induce vascular inflammation leading to atherosclerosis.
Consistent with the results of a previous study , insulin increased levels of C/EBP-β, and C/EBP-δ in cultured VSMCs in a PI3K/Akt-dependent manner. In the hyperinsulinaemic rats, Cebpb mRNA expression in the aortas was strongly correlated with plasma insulin levels. Furthermore, the level of phosphorylation of Akt, a downstream molecule in the PI3K pathway, in the aortas were increased in the fructose-fed hyperinsulinaemic rats, suggesting chronic activation of PI3K in the aortas of these rats. These observations suggest that insulin stimulates the expression of Cebpb and Cebpd genes in a PI3K/Akt-dependent manner in the aortas, as well as in cultured VSMCs. On the other hand, we were not able to rule out the possibility that some unknown factors related to the hyperinsulinaemic state caused the activation of the PI3K pathway observed in the present study. Instead of the activation of PI3K, Jiang et al. showed the activation of the Ras/mitogen-activated protein kinase (MAPK) pathway in response to insulin in the vascular tissues of genetically hyperinsulinaemic Zucker fatty rats . In addition, it has been reported that insulin promotes numerous deleterious vascular effects by stimulating the actions of various growth factors acting through the MAPK signaling pathway . Therefore, further investigation is needed to clarify the expression of Ccl2 through the MAPK pathway in the insulin-resistant state.
In conclusion, the expression of Ccl2, one of the proinflammatory cytokines, is stimulated by enhanced expression of C/EBP genes in the aorta of the insulin-resistant rat. The overexpression of Cebpb and Cebpd in aortas of these rats is caused by chronic hyperinsulinaemia.
This work supported by research grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan.