DhHP-6Structure Identification And Its Protective Effect On STZ-induced T2DM Wistar Rats

Type2diabetes mellitus (Type2diabetes mellitus T2DM) is a type of the insulinindependent diabetes mellitus and can be treated with many other anti-diabetic drugsbeside insulin. The characteristic of T2DM is many tissues and organs e.g. liver,muscle, adipose tissuewith decreased insulin sensitivity and insulin secretion abilitydeficiency. In the pathogenesis of type2diabetes and related complications, reactiveoxygen species not only affect the insulin signal transduction pathway, butalso areinvolved in the insulin resistance. The insulin resistance of hepatic cells, muscle cellsand adipose cells caused by accumulated ROS production is a key feature of type2diabetes mellitus. Nicholas et al established two kinds of cell model for insulinresistance which are adipocytes by TNF-alpha and dexamethasone3T3-L1, and theydetermined the cell oxidation reduction state of the insulin resistant cell models.Theyfound ROS levels are increased significantly in these two insulin resistant cell models.The obese mice were given MnTBAP which is a mimic of MnSOD can improve theglucose tolerance in obese mice. This indicated that MnTBAP can effectively improveinsulin sensitivity statusof insulin target cells by scavenging free radicals. ALL theserevealed that the removal of ROS can be a potential treatment strategy for T2DM.We designed new APX mimics-DhHP-6base on the structure of catalyticactivity center of MP-8, MP-9and MP-11.We modified the heme structure of DhHP-6to make it more easily to synthesis without losing enzymatic activity according to thebasic principle of the solid phase peptide synthesis.First we change the hemin todeuteron-hemin by removing the vinyl functional group in the hemin molecules whichcan easily be oxidized by H2O2. Secondly, we use the deuteron-hemin as the targetmolecule of phage display peptide library to get a high-affinity short peptidesequence.Due to the asymmetry of the DhHP-6molecule, DhHP-6is actually two isomericmixture. And the ferric iron of detero-hemin in DhHP-6is at high spin state, so thestructure of DhHP-6can not be identified by1H NMR. We determined the1H NMRof DhHP-6by adding potassium cyanide in the DhHP-6solution to change the highspin state of Fe3+to the low spin state of Fe3+of DhHP-6.We found a newfragmentation mechanism of peptide Mass spectrometry due to the functional group of deuteron-hemin in DhHP-6when determining the mass spectrometry of DhHP-6.Through the determination of activity of both isomers of DhHP-6, the resultsshowed that both isomers can effectivelyscavengereactive oxygen free radicals incells, stimulatethe proliferation of pancreatic islet beta cell, inhibit the apoptosis ofislet beta cell induced by high concentration of glucose and palmitate andrestore theimpared insulin secretion ability of pancreatic beta cells caused by ROS accumulation.And Both isomers have no significant difference on the free radical scavengingactivity.DhHP-6can also increase the glucose absorption rate of C2C12muscle cells bythe removal of excess ROS in C2C12cells. By subcutaneous injection of DhHP-6, theblood glucose level can be effectively decreased in STZ-induced type2diabetic ratsthrough the clearance of excess ROS in type2diabetic rats. ALL these indicated thatDhHP-6can be a potential lead compound for drug development of type2diabetictreatment.