Promote The Development Of Type 2 Diabetes And Its Mechanism Of Glp-1 Receptor Cloning And Expression Of P58

The onset of type 2 diabetes is a complicated process which is affected by environmental and genetic factors.CDC2L2 is a recently reported susceptibility gene of type 2 diabetes of Han people in northern China,P58 is one of proteins coded by CDC2L2.Our previous study on cell lines showed p58 can induce apoptosis ofβcell and then promote the further development of type 2 diabetes.In this study,using the established type 2 diabetic rat model,we detected the expression of p58 and the proteins related to apoptosis of the rat pancreatic tissues in different phases of type 2 diabetes,then discussed the relationship between p58 and apoptosis of 13 cell and explored the role of p58 in the development of type 2 diabetes.We detected the mRNA and protein level of p58 of rat pancreatic tissues in different phases of type 2 diabetes by RT-PCR and Western Blot,and then we detected the level of anti-apoptotic factor Bcl-2,pro-apoptotic factor Bax and especially the cleaved PARP,an important maker protein of apoptosis,by Western Blot.The results showed the mRNA and protein level of p58 were increased in the whole process of type 2 diabetes.The expression of Bcl-2 was decreased and the expression of Bax was increased at late phase of type 2 diabetes.We also saw the cleaved PARP in this phase.The decreased expression of Bcl-2 and the increased expression of Bax and the appearance of cleaved PARP indicate that apoptosis is occurred in the pancreatic tissues of type 2 diabetic rats.The increase of p58 occurred earlier than apoptosis indicate p58 may induce apoptosis inβ-cell.The present study showed the high glucose condition of type 2 diabetes can increase the expression of p58 inβ-cell,and then alter the expression of apoptosis-related factors to promoteβ-cell apoptosis and the development of type 2 diabetes.The study also validated the results we got from the study of cell lines. Glucagon-like peptide -1(GLP-1) is a bioactive peptide which is secreted by intestinal L cells(Langerhans cells).Recently,it has become a new target of drug research because of its significant effect on improving the related symptoms of type 2 diabetes.GLP-1 plays biological role mainly by combining with its receptor(GLP-1R), so the study on GLP-1R also has important significance.In this study,through the cloning and expression of glucagon-like peptide -1 receptor(GLP-1R),we provided an experimental basis for the establishment of eukarytic cell line and the screen of the analogue of GLP-1.Firstly,the cDNA of GLP-1R was amplified through RT-PCR from the total RNA which was extracted from the INS-1 cell.Secondly,the cDNA of GLP-1R was inserted into the prokaryotic expression vector pGEX-4T-1 and eukaryotic expression vector pCMV—Tag2B respectively and identified by DNA sequencing.Finally,the prokaryotic expression vector pGEX-4T-1-GLP-1R was transferred into BL-21(D3).The fusion protein GLP-1R/GST was obtained via IPTG inducing and then confirmed by SDS-PAGE which showed the expected band.Collectively,we cloned the cDNA of GLP-1R successfully and constructed the prokaryotic expression vector pGEX-4T-1-GLP-1R and eukaryotic expression vector pCMV—Tag2B-GLP-1R.We also identified the fusion protein GLP-1R/GST which exists in the supernatant with a molecular weight of 83 kD after expressing the pGEX-4T-1-GLP-1R in BL21(D3).This study would make it possible to prepare large amounts of protein samples and produce GLP-1R antibody,and the constructed pCMV-Tag2B-GLP-1R also makes it possible to create a platform for screening the new drugs of type 2 diabetes.