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Abstract
Background
Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), which act as a coactivator of nuclear receptors and several other transcription factors. This study was performed to evaluate the expressional regulation of insulin and inflammatory response genes by PGC-1α.
Methods
Transient transfection assays were performed to measure the promoter activity of the insulin and CXCL10 gene. The insulin gene expression levels in INS-1 cells were determined by Northern blot analysis. Differentially expressed genes by PGC-1α overexpression in HASMCs were confirmed using DNA microarray, real-time PCR and Northen blot analysis.
Results
Insulin promoter activity and mRNA levels were suppressed by GR and Ad-PGC-1α. Northern blot analysis of the INS-1 cells revealed that infection with Ad-PGC-1α markedly reduced the amount of insulin mRNA and treatment of Dex enhanced this effect in an additive manner. The PGC-1α-specific siRNA decreased insulin expression that was induced by Dex in the GR-expressing INS-1 cells was nearly restored by this siRNA treatment. We found that when vascular smooth muscle cells (VSMCs) overexpressed PGC-1α, immune or inflammatory response genes were highly expressed. For example, promoter activity and mRNA level of CXCL10 gene were increased by PGC-1α.
Figures and Tables
Fig. 1
Insulin gene promoter is inhibited by PGC-1α. INS-1 and HepG2 cells were infected 50 MOI of adenoviral PGC-1α. 2 h later, the cells were co-transfected with pCMV-β Gal together with 600 ng of the human insulin promoter reporter phINS-362Luc (A), plus the GR (300 ng) expression plasmids (B). 4 h later, the cells in Bwere incubated with 10% FBS for 12 h and then serum-starved for 6 h. Subsequently, the cells were treated with 500 nM Dex for 24 h. Luciferase activity was then assayed and the luciferase results were normalized with respect to the transfection efficiency, which was assessed by β-galactosidase assays of co-transfected pCMV-β Gal vector. The results are presented as relative luciferase activities. Values are presented as means SEM of 3 independent experiments. *P < 0.05, **P < 0.01 vs basal values.
Fig. 2
Effect of the GR and PGC-1α on the insulin gene expression in INS-1 cell. INS-1 cells were cultured with 11.1 mM glucose and co-transfected GR expression vector and then treated for 24 h with 0, 10 or 50 MOI of adenoviral PGC-1α with (B) or without (A) Dex. Alternatively, the INS-1 cells were transfected with siRNA specific for PGC-1α and then treated with Dex (C). As a negative control, the cells were transfected with non-specific (scrambled) siRNA (C). In (D), we evaluated the effect of the siRNAs on endogenous PGC-1α expression using realtime RT-PCR. In A-C cases, the total RNAs of the cell lysates were subjected to Northern blot analysis (10 µg per lane) to evaluate the expression of the insulin and RNA quantity was normalized by 18S rRNA. Fig. D, The expression levels of PGC-1α were quantified relative to GAPDH expression. The values shown are means SEM of three independent experiments. *P < 0.05 and **P < 0.01 vs basal values.
Fig. 3
Expression levels of TNFLSF10, CXCL10 and CXCL11 gene by PGC-1α overexpression in VSMCs. VSMCs were treated 50 or 100 MOI of ad-PGC-1α or Null virus for 24 h, after which their total RNAs were harvested and subjected to realtime RT-PCR to determine TNFLSF10 (A), CXCL10 (B) and CXCL11 (C) expression. The expression levels were quantified relative to GAPDH expression. The data (n = 9) are expressed as means The TNFLSF10/GAPDH, CXCL10/GAPDH and CXCL11/GAPDH values were normalized relative to the control (C). *P < 0.05, **P < 0.01 compared to the control.
Fig. 4
Messenger RNA level and promoter activity are increased by PGC-1α overexpression in VSMCs. A, Vascular smooth muscle cells (VSMCs) were treated with 100 MOI of adenoviral PGC-1α for 6, 12 or 24 h. The total RNAs of the cell lysates were subjected to Northern blot analysis (10 µg per lane) to evaluate the expression of the insulin and RNA quantity was normalized by 18S rRNA; B, VSMCs were co-transfected with pCMV-β Gal together with 600 ng of the CXCL10-Luc reporter, plus the PGC-1α expression plasmids (50, 100 or 300 ng of PGC-1α). 4 hours later, the cells were incubated with DMEM-Low medium contained 0.5% FBS for 24 h. 10 ng/mL of TNFα treatment as a positive control. Luciferase activity was then assayed and the luciferase results were normalized with respect to the transfection efficiency, which was assessed by β-galactosidase assays of co-transfected pCMV-β Gal vector. The results are presented as relative luciferase activities. Values are presented as means ± SEM of 3 independent experiments. *P < 0.05 vs basal values.
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