Prokaryotic Expression Of Irisin And Its Effect On Lipolysis In MRNA Level

Irisin is a hormone involved in blood circulation produced during exercise in skeletal muscle; it comes from the cleavage of membrane protein FDNC5. Recent studies show that irisin can induce white adipocytes transforming into brown adipocytes or beige. Since the main function of brown adipocytes is to consume energy by generating heat, thus irisin was considered a potential drug for the treatment of obesity and diabetes.The purpose of this study is to obtain high quality and quantity of irisin, utilizing the characteristic of E. coli efficiently expressing the exogenous genes, and to study the effects and molecular mechanism of GST-irisin on lipolysis by RT-PCR. Firstly, selecting two plasmids pGEX-2T and pGEX-4T-1 constructed expression vector simultaneously, and transformed them into cloning strain trans5α, and got a large number of recombinant plasmid. Then, we transformed them into the expression strain BL21(DE3) to ferment engineering bacteria. Through the optimization of expression conditions of the induction time, IPTG concentration, considering the efficiency of two expression vectors, we chose pGEX-4T-1-irisin as an expression plasmid. In accordance with the optimized conditions, irisin was expressed. The supernatant was collected and purified by Glutathione Sepharose 4B to obtain the recombinant protein GST-irisin. Expression products were proved to have immunological activity by Western Blot.3T3-L1 cells were insulin-induced cultured into mature adipocytes. Utilizing the different concentrations of irisin(0 nM, 50 nM, 100 nM, 200 nM), we determined the impact of irisin on fat cells at the mRNA level by RT-PCR method for 8 days. Firstly, the browning effect of irisin on white adipocytes was studied; the mRNA of UCP1 in adipocytes was increased significantly after irisin treatment. It can be inferred that irisin promotes white adipocytes to transform into brown adipocytes. Secondly, irisin induced the expression of lipolysis-related proteins, such as ATGL, HSL, and FABP4. Irisin promoted the upregulation of those proteins in different concentrations and time. In addition, irisin improved the secretion of irisin from adipocytes. Irisin increases PPARγ and FNDC5 expression, PPARγ is an activator of irisin expression pathway. From these three aspects, we conclude that irisin can promote white adipocytes to transform into brown adipocytes. It can also increase the rate of lipolysis by increasing the expression of lipolysis-related proteins. Meanwhile irisin has autocrine function, it can increased irisin yield secreted from adipocytes. Due to the close relationships between glucose metabolism and lipid metabolism, we also detected the change of glucose concentration in the medium. We found that glucose concentration decreased in irisin-treated groups at the second day, but increased at the sixth day. Therefore, we did not find the clear relationship between irisin and glucose utilization.This study verified the function of irisin browning white adipocytes. By monitoring the change of mRNA expression in lipolysis, we study of the mechanism of irisin promoting energy metabolism in depth. We provided further proofs of irisin’s potential as an anti-obesity drug.