Expression And Biological Activity Of GLP-1 Fusion Protein And Betatrophin

Diabetes is a kind of metabolic disease characterized by hyperglycemia,resulted from relative insulin deficiency caused by carbohydrate metabolism disorder;how to solve the contradiction between the treatment and hypoglycemia,and how to protect islet beta cell function from progressive decline are the two major challenges of the current treatment of diabetes.For gut-derived hormone,glucagon-like peptide-1?GLP-1?,regulates blood sugar in a glucose level-dependent manner by promoting insulin secretion from islet beta cells,it will not cause severe hypoglycemia,which has drawn widespread attention.However,once GLP-1 is produced and released into the circulation,it can be easily degraded by dipeptidyl peptidase-4?DPP-IV?,resulting to a plasma half-life of less than 2 minutes,which greatly limits the clinical application of natural GLP-1 molecule.Moreover,oral GLP-1 receptor agonists have not yet been obtained.Also,Betatrophin,secreted by human liver and adipose tissue,is a recently discovered animal hormone that has been shown to be involved in the lipid metabolism,but has aroused disagreements on promotion beta cell proliferation.The loss of beta cell function in the elderly is the main cause of diabetes mellitus,and whether Betatrophin can play a glucose-regulatory role in older diabetic mice remains to be seen.Therefore,structural modification of GLP-1,chemical synthesis and bio-activity analysis of mGLP-1,fusion expression and biological activity of 6×mGLP-1,recombinant expression and functional research in Betatrophin have been studied in order to construct foundation for the development of new therapies to cure diabetes,and we has made such progress as follows:1.mGLP-1 is made from site-specific mutation of amino acid residues in native GLP-1 sensitive to DPP-IV and Trypsin degradation(His⁷-Ala⁸ to His⁷-Gly⁸,Lys^26,34 to Gln²⁶,Asp³⁴,respectively)and chemical synthesis.Compared with natural GLP-1,mGLP-1 can effectively resist to the degradation of Trypsin and Pancreatin in vitro,still can stimulate islet beta cell proliferation,promote insulin secretion,improve glucose tolerance in mice and lower the blood glucose level in type 2 diabetic mice?resulted from STZ induction?,without causing severe hypoglycemia;moreover,its bio-activity is more potent,however,it remains to be studied further on the way of oral administration;2.On the basis of previous research,6×mGLP-1 is modified by replacing amino acid residues in native GLP-1 sensitive to DPP-IV and Trypsin degradation(His⁷-Ala⁸ to His⁷-Ser⁸/Gly⁸,Lys^26,34 to Gln²⁶,Asn³⁴/Asp³⁴,respectively),adding Cys residues in the C-terminal,making 6 tandem repeats,adding His-tag at the C-terminal,optimizing the codons according to E.Coli,and obtained from prokaryotic expression and step-by-step purification subsequently.The result of HPLC analysis demonstrated that its purity fully met the requirements of the following experiments.Our study found that in vitro,6×mGLP-1 can form dimers and trimers,can be degraded by Trypsin into monomers resistant to Trypsin,and whether 6×mGLP-1 as a whole or monomers resulted from Trypsin hydrolysis,can stimulate the proliferation of pancreatic beta cells;in vivo,6×mGLP-1,administrated either by intraperitoneal injection or via oral gavage,can greatly improve glucose tolerance in normal mice,6×mGLP-1 administrated by intraperitoneal injection can effectively lower the blood glucose level in type 2 diabetic mice and the effect of time can last for up to 16.7 hours,without causing hypoglycemia;moreover,the blood glucose concentrations of db/db mice were decreased in nearly 4 hours after oral administration of 6×mGLP-1.The results of serum kinetics showed that 6×mGLP-1 could be stable for as long as 24 hours in mice in its integrity form;3.mGLP-1 and 6×mGLP-1 as natural analogues of GLP-1 or GLP-1 receptor agonists are resulted from structural modification and have prolonged half-life,so the blood glucose-lowering activity are more potent than natural GLP-1.Improvement on the pharmacokinetics of 6×mGLP-1 and its oral administration mode will make it beyond the current commercially available GLP-1 receptor agonists which are administrated by subcutaneous injection;4.Mature forms of recombinant Betatrophin were obtained by codon optimization,prokaryotic expression and purification,and the purity of the product detected by SDS-PAGE analysis was completely in line with the experimental requirements.Our study found that in vitro,r-Betatrophin cannot stimulate beta cell proliferation,while can stimulate the secretion of triglyceride in HepG2 cells by up-regulating the expression level of Egr1 gene and down-regulating the expression level of ATGL gene;in vivo,r-Betatrophin,can increase the concentration of plasma triglyceride both in adult and aged KM mice,can improve the glucose tolerance of aged KM mice by intraperitoneal injection,but had no glucose-regulatory effect in db/db mice.Hence,it was shown that r-Betatrophin plays dual roles in glucose and lipid metabolism.Effects of Betatrophin on lipid and glucose metabolism are more and more widely accepted,but the effect on carbohydrate metabolism is only just starting soon.It is believed that with the improvement on the molecular mechanism of the effect of Betatrophin on lipid metabolism and researches to fill the gaps in the relationship between Betatrophin and carbohydrate metabolism,the treatment of metabolic syndrome will take one more step.In this study,a great deal of bio-active 6×mGLP-1 fusion protein and human Betatrophin recombinant protein were obtained by using genetic engineering technology.The prolonged half-life of6×mGLP-1 fusion protein and the feasibility of oral administration have laid a theoretical and material basis for the development of new drugs for type 2 diabetes mellitus.Betatrophin recombinant protein can enhance the glucose tolerance in old KM mice possibly by enhancing the activity of insulin,which provides new experimental evidence for its involvement in carbohydrate metabolism.