RESUMEN
Glucagon-like peptide-1 (GLP-1) is an incretin hormone that reduces postprandial glycemic excursions by enhancing insulin secretion. In this study, a new dimeric GLP-1 analogue (GLP-1cpGLP-1) was designed by inserting human insulin C-peptide (CP) in the middle of a dimer of [Gly8] GLP-1 (7-36). Then, the dimeric incretin (GLP-1cpGLP-1) was ligated to human αB-crystallin (αB-Cry) to create a hybrid protein, abbreviated as αB-GLP-1cpGLP-1. The constructed gene was well expressed in the bacterial host system. After specific chemical release from the hybrid protein, the dimeric incretin was purified by size exclusion chromatography (SEC). Finally, the RP-HPLC analysis indicated a purity of >99 % for the dimeric incretin. The secondary structure assessments by various spectroscopic methods, and in silico analysis suggested that the dimeric incretin has α-helical rich structure. The dynamic light scattering (DLS) analysis indicates that our dimeric incretin forms large oligomeric structures. This incretin analogue significantly reduced blood glucose levels in both healthy and diabetic mice while effectively triggering insulin release. The size exclusion HPLC also indicates the interaction of the new incretin analogue with human serum albumin, the main carrier protein in the bloodstream. Consistent with the results obtained from the biological activity assessments, this significant interaction indicates its potential as a viable therapeutic agent with a long-lasting effect. The results of our research represent a significant breakthrough in the successful design of an active incretin dimer capable of effectively controlling blood sugar levels and inducing insulin secretion in the realm of diabetes treatment.
RESUMEN
Nowadays, a small number of incretin mimics are used to treat type 2 diabetes mellitus (T2DM) due to their longer half-life. The present study aimed to introduce a novel method for producing the liraglutide precursor peptide (LPP) and developing a potentially new incretin mimic. Here, human αB-crystallin (αB-Cry) was ligated to the LPP at the gene level, and the gene construct was expressed in Escherichia coli with a relatively good efficiency. The hybrid protein (αB-lir) was then purified by a precipitation method followed by anion exchange chromatography. After that, the peptide was released from the carrier protein by a chemical cleavage method yielding about 70%. The LPP was then purified by gel filtration chromatography, and HPLC estimated its purity to be about 98%. Also, the molecular mass of the purified peptide was finally confirmed by mass spectroscopy analysis. Assessment of the secondary structures suggested a dominant α-helical structure for the LPP and a ß-sheet rich structure for the hybrid protein. The subcutaneous injection of the LPP and the αB-lir hybrid protein significantly reduced the blood sugar levels in healthy and diabetic mice and stimulated insulin secretion. Also, the hybrid protein exerts its bioactivities more effectively than the LPP over a relatively longer period of time. The results of this study suggested a novel method for the easy and cost-effective production of the LPP and introduced a new long-acting incretin mimic that can be potentially used for the treatment of T2DM patients.