The Primary Mechanism And Effects Of Insulin Releasing Function Of The Antibacterial Peptide AWRK6in Vivo And Vitro

Antimicrobial peptides (AMPs) is a low molecular weight peptide produced byorganism itself to resist pathogen, with the features of broad-spectrum antibacterialactivity, low immunogenicity and strong thermal stability. Amphibian inhabits theenvironment which is complex, and their moist and bare skin can secrete theantibiotic active substance. With the developing of the research about the function ofAMPs, it is found that AMPs also have insulin-releasing activity. Recently, it isreported that there are various kinds of AMPs with the function of insulin secretion(J. Michael Conlon etc.). And comparing to traditional hypoglycemic agents, AMPscan’t be decomposed and also its hypoglycemic action is only in high glucoseconcentration as well as it can promote islet cell proliferation, which could make itbecome a hopefully efficient and safe new type therapeutic for Type ⅡDiabetesmellitus.Dybowskin-2CDYa (SAVGRHGRRFGLRKHRKH) is a new naturalantibacterial peptide which is found and named by our laboratory in frog skin,however for its lower thermal stability, we reformed the peptides as6basesreplacement and named it as AWRK6(SWVGKHGKKFGLKKHKKH). In theprevious research, AWRK6showed strong activity of insulin-secretion. In order toexplore its mechanism for insulin releasing, in this study we used mouse islethybridoma cell line MIN6and synthetic AWRK6, combining with techniques ofELISA, calcium probes Fluo3-AM and Western Blot and so forth to analyze theprimary mechanism of insulin releasing function. We studied how AWRK6causedthe flow of calcium when MIN6secreted insulin through protein level andMicroscopic imaging techniques, respectively. The result of ELISA showed thatverapamil could not block the insulin-releasing effect of AWRK6, and afteradministration of EGTA, it was clear to see AWRK6could also induce insulinsecretion. So the concentration increase of calcium which was caused by AWRK6was from the intracellular. And the result of Lactic Dehydrogenase (LDH) and Cellcounting Kit showed that AWRK6maintained the membrane integrity, promotedcell multiplication and also remained the activity.We also dected the cell cycle ofMIN6treated by AWRK6through Flow cytometry, and the result showed that the percentage of S phase had been increased comparing with the control group. Theresult of western blot showed the expression of Epac2increased.In this research, we utilized streptozotocin (STZ) to build T2DM animal modelfor exploring the hypoglycemic action of AWRK6in vivo. It was showed that byintraperitoneal injection AWRK6(100nm/L) could degrease the blood glucose after30min, and the value of blood glucose dropped to ideal levels（≤11.1mM/L）.However, the same concentration of AWRK6did not cause thecontrol group blood glucose dropping, which meant the antidiabetic effects ofAWRK6only happened under high glucose condition,and also could not causehypoglycemic reaction.This study laid a further basic theoretical and experimentalfoundation for exploring the AMPs AWRK6as a new type therapeutic for T2DM.