Effect of Metformin-Induced Stimulation on the Expression of Insulin Receptor Substrate 1 through Negative Regulation of P70S6k

Abstract

Objective: The aim is to study the effects of metformin on the expression of 70 kDa ribosomal protein S6 kinase (P70S6k), insulin receptor substrate 1 (IRS-1), and IRS-1Ser307 phosphorylation in human luteinized granulosa cells. Methods: Granulosa cells in the experimental group were cultured in M199 medium containing 0.1 mmol/L metformin for 24 h and those in control group were cultured in M199 medium. The expression levels of P70S6k and IRS-1 mRNA were detected by reverse-transcriptiom polymerase chain reaction (RT-PCR) and real-time PCR. P70S6k, IRS-1, p-ser307-IRS-1, and p-thr389-P70S6k protein expression levels were detected by immunofluorescence and western blotting. Results: P70S6k mRNA level was higher and IRS-1 was significantly lower in the experimental group than those in the control group. IRS-1 and p-ser307-IRS-1 were expressed in cell plasma, and P70S6k and p-thr389-P70S6k were expressed in cell nucleus. The results of Western blot analysis indicated that the expression levels of P70S6k, p-thr389-P70S6k, IRS-1, and p-ser307-IRS-1 proteins had significant difference between the experimental group and the control group. Compared to the control group, the relative intensity illustrated that the expression levels of P70S6K and p-thr389-P70S6k significantly increased in the experimental group; however, those of IRS-1 and p-ser307-IRS-1 proteins significantly decreased. Conclusion: Metformin can inhibit the P70S6k mRNA and protein expression levels in the granulosa cells and improve insulin sensitivity by regulating IRS-1 expression through Akt/P70S6k/IRS-1-dependent pathway.