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. 2010 May 4;285(29):22174–22185. doi: 10.1074/jbc.M109.085464

FIGURE 4.

FIGURE 4.

ADD1/SREBP1c and C/EBPs up-regulate Orm1 promoter activity in adipocytes. A, Orm1 promoter activity is enhanced by ADD1/SREBP1c. h293 cells were co-transfected with luciferase reporter containing Orm1 promoter and β-galactosidase DNA with or without ADD1/SREBP1c (ADD1; 100 ng) expression plasmid. 24 h after transfection, cells were harvested. Luciferase activities were measured and normalized by β-galactosidase activity. Ctl, control. B, in adipocytes, expression of Orm1 mRNA is stimulated by ADD1/SREBP1c. 3T3-L1 adipocytes were infected with mock (−) or ADD1/SREBP1c (ADD1) expressing adenovirus. 36 h after adenoviral infection, cells were harvested and subjected to Q-PCR analysis. FAS, fatty-acid synthase. C, insulin augments binding of ADD1/SREBP1c to the Orm1 promoter. 3T3-L1 adipocytes were incubated with or without insulin (100 nm) for 24 h and subjected to chromatin immunoprecipitation with preimmune serum or anti-ADD1/SREBP1c antibody. D, Orm1 promoter activity is stimulated by C/EBPα and C/EBPβ. h293 cells were co-transfected with luciferase reporter containing the Orm1 promoter and β-galactosidase DNA with or without C/EBPα (10 and 100 ng) or C/EBPβ (10 and 100 ng) expression plasmid. 24 h after transfection, cells were harvested. Luciferase activities were measured and normalized by β-galactosidase activity. *, p < 0.05; **, p < 0.01. E, high glucose-induced Orm1 promoter activity is suppressed by dominant negative C/EBP (dnC/EBP). h293 cells were co-transfected with the Orm1 promoter luciferase reporter and β-galactosidase with or without dominant negative C/EBPβ (500 ng) expression plasmid. 12 h after transfection, cells were incubated in low (5.5 mm) or high glucose (25 mm) media for additional 24 h and subjected to luciferase activity assays. F, high glucose condition promotes binding of C/EBPα and C/EBPβ to the Orm1 promoter in adipocytes. 3T3-L1 adipocytes were incubated in low (5.5 mm) and high glucose (25 mm) conditions for 24 h and subjected to chromatin immunoprecipitation assays using anti-C/EBPα or anti-C/EBPβ antibodies.