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The molecular mechanism of sterol regulatory element binding protein-1c suppressing insulin receptor substrate-1 in skeletal muscle.
- Source :
- Chinese Journal of Diabetes Mellitus; Dec2015, Vol. 7 Issue 12, p754-758, 5p
- Publication Year :
- 2015
-
Abstract
- Objective To investigate the molecular mechanism of sterol regulatory element binding protein-1c (SREBP- 1c) suppressing insulin receptor substrate-1(IRS-1) in skeletal muscle cells. Methods Luciferase plasmid for rat IRS- 1 promoter, expression plasmid of SREBP-1c and pRL-TK renilla plasmid were cotransfected into L6 cells using Lipofectamine 2000 (Invitrogen). After transfection for 36 h, luciferase activity was measured using the dual-luciferase reporter assay system following the manufacturer' s instructions. L6 myotubes were infected with adenoviral vectors expressing SREBP-1c. Adenovirus expressing green fluorescent protein (GFP) was used as control. The cells were harvested for nucleus protein after infection for 48 h. Electrophoretic mobility shift assay (EMSA) was performed using a Light Shift Chemiluminescent EMSA Kit. Chromatin immunoprecipitation assay was performed by CHIP Kit. The interaction between the transcription factor SREBP-1c and the promoter region of IRS-1 was assessed by above experiments. Differences among the groups were determined using two-way ANOVA. Results The luciferase deletion studies suggested the region from -450 to -210 bp on the IRS-1 promoter was a potential target region for SREBP-1c. Sequence analysis showed that the target region of the rat IRS-1 promoter gene contained a potential binding site (GCCTCCCGAG), which was located between -302 and -292 bp. PCR site- directed mutagenesis (TGTTAAATTA) was generated and analyzed using a luciferase assay. The transcriptional activity of wild-type IRS-1 promoter was dramatically down-regulated by SREBP-1c, whereas SREBP-1c had no effect on the IRS-1 promoter bearing mutation of SREBP-1c binding site(7.03±1.28 vs 19.09±2.45,3.55±1.68 vs 3.96±1.09,F=114.437,0.251,all P>0.05). The EMSA results showed that the labeled wild- type probe successfully formed a complex with nuclear proteins. But the unlabelled mutant probe did not interfere the complex. The results from CHIP assay showed that SREBP-1c could bind directly to the IRS- 1 promoter region. The quantity of SREBP- 1c protein binding to the IRS- 1 promoter was two times of control under PA conditions in L6 cells or five times of control when SREBP-1c was over-expressed (2.15±0.03 vs 1.07±0.24,5.48±1.28 vs 0.86±0.19,t=6.877,5.495,all P<0.05). Conclusion SREBP- 1c could directly bind to the atypical SRE sequence in the promoter region of IRS- 1, suppressing IRS- 1 expression and the subsequent insulin signaling pathway. [ABSTRACT FROM AUTHOR]
Details
- Language :
- Chinese
- ISSN :
- 16745809
- Volume :
- 7
- Issue :
- 12
- Database :
- Complementary Index
- Journal :
- Chinese Journal of Diabetes Mellitus
- Publication Type :
- Academic Journal
- Accession number :
- 114310409
- Full Text :
- https://doi.org/10.3760/cma.j.issn.1674-5809.2015.12.008