Fig. 1Potency of glucagon-like peptide-1 (GLP-1) analogs toward GLP-1 receptor (GLP1R). Ligand potencies of GLP-1 analogs were examined using HEK293T cells expressing GLP1R. Cells were treated with increasing concentrations of GLP-1 analogs for 6 hours, and luciferase activity was measured. The data on the sigmoidal curves and EC50 values are presented as means±standard error of the mean of at least three independent experiments. CRE-luc, cAMP response element-luciferase; xGLP, Xenopus GLP-1.
Fig. 2Stability of glucagon-like peptide-1 (GLP-1) analogs. The stability of GLP-1 and GLP-1 analogs against dipeptidyl peptidase-IV (DPP-IV) activity was evaluated by incubating the individual peptides in medium containing 10% fetal bovine serum (FBS) (A) or in 100% FBS (B). Peptides were incubated at an initial concentration of 100 nM in Dulbecco's Modified Eagle's medium containing 10% FBS or 100% FBS at 37℃ in the presence or absence of 0.2 mM of the peptidase inhibitor diprotin A. Peptide activity was then assessed by measuring luciferase activity in cells expressing GLP1R and CRE-luc. Results are presented as mean±standard error of the mean of at least three independent experiments.
Fig. 3Induction of glucose-dependent insulin secretion by (xGLP-E4). (A) INS-1 cells and (B) β-TC-6 cells were cultured in 24-well plates until 90% confluence. Cells were washed and incubated in Krebs-Ringer Bicarbonate (KRB) buffer and treated with glucose for 2 hours at 37℃. Cells were incubated with 10 nM glucagon-like peptide-1 (GLP-1) analog in KRB buffer containing low and high glucose for another 2 hours, and insulin secretion into the buffer was then determined. CTL, control; GLP-1, glucagon-like peptide-1; Exe-4, exendin-4; xGLP, Xenopus GLP-1.
Fig. 4Induction of β-cell growth by (xGLP-E4). INS-1 cells were seeded in 12-well plates at a density of 4×104 cells/well. Every 2 days, the respective peptides were added to the cultures in fresh medium containing the appropriate concentration of glucose. (A) Six and (B) 10 days after cell seeding, cells were washed, harvested, and counted. Results are presented as mean±standard error of the mean of at least three independent experiments. GLP-1, glucagon-like peptide-1; Exe-4, exendin-4; xGLP, Xenopus GLP-1. a vs. control (CTL) (P<0.05); b vs. CTL (P<0.001).
Table 1Comparison of Amino Acid Sequences of GLP1R Agonists