The molecular mechanism of sterol regulatory element binding protein-1c suppressing insulin receptor substrate-1 in skeletal muscle

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. Key words: Sterol regulatory element binding protein-1c; Palmitic acid; Skeletal muscle; Insulin receptor substrate-1; Molecular mechanism