Increased expression of CCAAT/enhancer binding protein-beta and -delta and monocyte chemoattractant protein-1 genes in aortas from hyperinsulinaemic rats.
OBJECTIVE
We evaluated whether hyperinsulinaemia stimulates the expression of transcription factor CCAAT/enhancer binding protein (C/EBP)-beta and C/EBP-delta and leads to the induction of the chemokine (C-C motif) ligand 2 gene (Ccl2, also known as MCP-1) expression in aortas.


METHODS
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-beta to Ccl2 was assessed by chromatin immunoprecipitation (ChIP) assay.


RESULTS
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-beta 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-beta to the 5' upstream region of Ccl2 in the aortas from fructose-fed rats.


CONCLUSIONS
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.
Sato
Y
Y
Division of Endocrinology and Metabolism, Department of Medicine, Shiga University of Medical Science, Seta-Tsukinowa-Cho, Otsu, Shiga, 520-2192, Japan.
Nishio
Y
Y
Sekine
O
O
Kodama
K
K
Nagai
Y
Y
Nakamura
T
T
Maegawa
H
H
Kashiwagi
A
A
D000818:Animals; D001011:Aorta / Q000503:physiopathology*; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics*; D051743:CCAAT-Enhancer-Binding Protein-delta / Q000235:genetics*; D018932:Chemokine CCL2 / Q000235:genetics*; D002843:Chromatin / Q000235:genetics / Q000648:ultrastructure; D017931:DNA Primers; D005786:Gene Expression Regulation; D006801:Humans; D006946:Hyperinsulinism / Q000235:genetics*; D007328:Insulin / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000503:physiopathology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction
D022782:CCAAT-Enhancer-Binding Protein-beta; C491766:Ccl2 protein, rat; D018932:Chemokine CCL2; D002843:Chromatin; D017931:DNA Primers; D007328:Insulin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D051743:CCAAT-Enhancer-Binding Protein-delta; D012313:RNA; D019869:Phosphatidylinositol 3-Kinases