Bioactivity Of A Novel GLP-1 Analog GlGLP-1

Glucagon-like peptide 1(GLP-1)is an incretin hormone released from the L cells.GLP-1 could promote the proliferation of beta cells and prevent beta cells from apoptosis.GLP-1 shows potent ability in lowering blood glucose with glucose-dependent manner.And GLP-1 could inhibit gastric emptying and decrease appetite.However,it's reported that the half-life of GLP-1 is less than 2 min in vivo because of the degradation of DPPIV at the 8th amino acid of GLP-1.Many efforts were paid on developing new GLP-1 long-acting analogs.Exendin-4 is one of drugs approved by FDA for therapy of T2DM.By comparing the structure of GLP-1 with Exendin-4,we found that the amino acid of Exendin-4 corresponding with the 8th amino acid of GLP-1 was G.It's indicated that replacement from A to G could contribute the stability of GLP-1 in vivo.It's also reported that adding a cys to the C-terminal could form disulfide bond between two GLP-1 monomers.And this new GLP-1 analog showed long stability in vivo.However,the disulfide bond could be broken in vivo which weaked its stability.Based the present study,we designed a new long-acting GLP-1 analog named glGLP-1.Through chemical method,two GLP-1 monomers were dimerized with a Lys.Compared with the disulfide bond,covalent binding is more stable.This study aimed to test the bioactivity of glGLP-1 from experiments in vitro and in vivo including 3 model mice(healthy KM mice,STZ induced T2DM mice,db/db mice).The results of are as follows:1,the cell assay of glGLP-1After transfection of GLP-1 R and cAMP response plasmids into HEK 293T cell line,we built a recombination cell which expressed GLP-1R at its membrane.When treated by glGLP-1 and GLP-1,HEK 293T cell expressed related protein which refleted the activation of GLP-1 R.From the GFP assay,it was shown that glGLP-1 could activate the GLP-1 R with dose-dependent manner.glGLP-1 could activate cotransfected GLP-1R and cAMP response SEAP plasmid cell to express SEAP.And This method was used to calculate the EC50 of glGLP-1 and GLP-1.Based on our result,the glGLP-1 showed a little better EC50 of 2.947nmol compared to GLP-1(3.181nmol).It indicated that the modification didn't change the activation of GLP-1R.In the insulin secretion assay of Min6 cell,we found that glGLP-1 could rapidly promote beta cell to release insulin in 15 min.In details,10nmol/kg and 100nmol/kg glGLP-1 could stimulate insulin secreation for 5.95?g/L and 9.69?g/L in 15min.The control group was 0.023?g/L.2,the anti-hyperglycemia ability of glGLP-1 in the assay in vivo1)bioactivity in healthy KM miceglGLP-1 showed potent ability in improving IPGTT of KM mice in the IPGTT assay(P<0.001).glGLP-1 could effect on lowing blood glucose for 10h in vivo,which showed no difference in IP injection and IH injection.In the glucose-dependent anti-hyperglycemia assay,glGLP-1 could rapidly stimulate the secretion of insulin to low blood glucose till the normal blood glucose.Compared with A-GLP-1,it implied thatt the dimerization of glGLP-1 contributed most to ITS long-acting time in vivo.2)acute assay in T2DM miceBased on the short-term assay in KM mice,we studied the hypoglycemic ability of glGLP-1 in T2DM mice including db/db mice and STZ-induced T2DM mice.In two model mice,glGLP-1 could significantly improved the IPGTT(P<0.001)and effectively low blood glucose for 10h.3)long-term assay in T2DM miceSince glGLP-1 could keep 10h at its active form,we designed a twice daily injection of glGLP-1 to treat T2DM mice for a long term.a)Long-term assay for STZ induced T2DM miceC57 BL/6J mice were accepted injection of STZ with 50mg/kg for 5days into T2DM model.Then this model mice received administration of glGLP-1 for 8 weeks and victoza was used as positive control.The result showd that glGLP-1 could significantly improve the FBG(P<0.001),food intake(P<0.05)and water comsuption(P<0.001),HbAlc level(P<0.001)and blood lipid(P<0.05)vs control group.b)Long-term assay for db/db micedb/db mice were accepted injection of glGLP-1 for 6 weeks.The result showed that glGLP-1 could significantly improved random blood glucose(P<0.001),food intake(P<0.01)and water comsuption(P<0.001).After 6 weeks,the body weight of control group increased for 27%and the glGLP-1 group increased for 11%.It indicated that glGLP-1 showed ability in decreasing body weight.3,glGLP-1 could inhibit gastric emptyingglGLP-1 showed significantly potent ability in inhibiting gastric emptying comparing with saline and Victoza(P<0.001,VS control group)in KM mice.In details,the ink progradation rate of Victoza group and glGLP-1 group were 24%and 19%respectively.Above all,glGLP-1 shows potent ability in improving IPGTT and resisting degradation in vivo for 10h.glGLP-1 based therapy could improve the condition of T2DM mice and decrease the HbAlc level.glGLP-1 is a promising candidate in developing into a new therapy for type 2 diabetic mellitus.