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A Novel Long-Acting Glucagon-Like Peptide-1 Agonist with Improved Efficacy in Insulin Secretion and β-Cell Growth.
Kim HY
,
Hwang JI
,
Moon MJ
,
Seong JY
.
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BACKGROUND: Glucagon-like peptide-1 (GLP-1) is an incretin hormone produced by cleavage of proglucagon in intestinal L-cells. In the pancreas, GLP-1 stimulates post-prandial insulin secretion, promotes insulin biosynthesis, and improves insulin sensitivity. Because of its insulinotropic activity, GLP-1 has been considered a good candidate drug for treatment of diabetes mellitus. However, clinical use of GLP-1 has been limited by its short half-life, as a result of rapid degradation by dipeptidyl peptidase-IV (DPP-IV).
METHODS: We designed a novel GLP-1 analog, Xenopus GLP-1 (xGLP)-E4. The Ala residue in the second position of xGLP was replaced with a Ser residue to increase the half-life in the body. The C-terminal tail of exendin-4 was added to enhance the binding affinity for the GLP-1 receptor (GLP1R). The potency of GLP-1 and its analogs was determined by luciferase assay. The stability of GLP1R agonists was evaluated by determining the activity of agonists that had been preincubated in the presence of fetal bovine serum, which contains innate DPP-IV activity. The effects of xGLP-E4 on insulin secretion and β-cell growth were investigated using insulin enzyme-linked immunosorbent assay and cell counting.
RESULTS: xGLP-E4 exhibited improved stability against DPP-IV activity and increased potency to GLP1R, compared with GLP-1. An increase in glucose-dependent insulin secretion was observed in xGLP-E4-treated pancreatic β-cells. The effect of xGLP-E4 on β-cell growth was greater than that of GLP-1.
CONCLUSION: We developed a novel GLP-1 analog, xGLP-E4, that shows prolonged longevity and improved efficacy. This analog is a potential candidate for treatment of type 2 diabetes.
Fig. 1. Potency 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. 2. Stability 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. 3. Induction 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. 4. Induction 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).
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