Endocrinol Metab.  2014 Sep;29(3):320-327. 10.3803/EnM.2014.29.3.320.

A Novel Long-Acting Glucagon-Like Peptide-1 Agonist with Improved Efficacy in Insulin Secretion and beta-Cell Growth

Affiliations
  • 1Division of Endocrinology and Metabolism, Department of Internal Medicine, Korea University College of Medicine, Seoul, Korea.
  • 2Korea University Graduate School of Medicine, Seoul, Korea. mmj34@korea.ac.kr

Abstract

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 beta-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 beta-cells. The effect of xGLP-E4 on beta-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.

Keyword

Glucagon-like peptide 1; Exenatide; xGLP-E4; Analog; Diabetes

MeSH Terms

Cell Count
Diabetes Mellitus
Enzyme-Linked Immunosorbent Assay
Glucagon-Like Peptide 1*
Half-Life
Incretins
Insulin Resistance
Insulin*
Longevity
Luciferases
Pancreas
Proglucagon
Xenopus
Glucagon-Like Peptide-1 Receptor
Glucagon-Like Peptide 1
Incretins
Insulin
Luciferases
Proglucagon

Figure

  • 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).


Reference

1. Moore B. On the treatment of diabetus mellitus by acid extract of duodenal mucous membrane. Biochem J. 1906; 1:28–38.
2. Fehmann HC, Goke R, Goke B. Cell and molecular biology of the incretin hormones glucagon-like peptide-I and glucose-dependent insulin releasing polypeptide. Endocr Rev. 1995; 16:390–410.
3. Orskov C, Holst JJ, Knuhtsen S, Baldissera FG, Poulsen SS, Nielsen OV. Glucagon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are secreted separately from pig small intestine but not pancreas. Endocrinology. 1986; 119:1467–1475.
4. Graziano MP, Hey PJ, Borkowski D, Chicchi GG, Strader CD. Cloning and functional expression of a human glucagon-like peptide-1 receptor. Biochem Biophys Res Commun. 1993; 196:141–146.
5. Shen HQ, Roth MD, Peterson RG. The effect of glucose and glucagon-like peptide-1 stimulation on insulin release in the perfused pancreas in a non-insulin-dependent diabetes mellitus animal model. Metabolism. 1998; 47:1042–1047.
6. Drucker DJ. Minireview: the glucagon-like peptides. Endocrinology. 2001; 142:521–527.
7. Fehmann HC, Habener JF. Insulinotropic hormone glucagon-like peptide-I(7-37) stimulation of proinsulin gene expression and proinsulin biosynthesis in insulinoma beta TC-1 cells. Endocrinology. 1992; 130:159–166.
8. Perfetti R, Merkel P. Glucagon-like peptide-1: a major regulator of pancreatic beta-cell function. Eur J Endocrinol. 2000; 143:717–725.
9. Perfetti R, Zhou J, Doyle ME, Egan JM. Glucagon-like peptide-1 induces cell proliferation and pancreatic-duodenum homeobox-1 expression and increases endocrine cell mass in the pancreas of old, glucose-intolerant rats. Endocrinology. 2000; 141:4600–4605.
10. Drucker DJ. Glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis. Mol Endocrinol. 2003; 17:161–171.
11. Nauck MA, Heimesaat MM, Behle K, Holst JJ, Nauck MS, Ritzel R, Hufner M, Schmiegel WH. Effects of glucagon-like peptide 1 on counterregulatory hormone responses, cognitive functions, and insulin secretion during hyperinsulinemic, stepped hypoglycemic clamp experiments in healthy volunteers. J Clin Endocrinol Metab. 2002; 87:1239–1246.
12. Nauck MA, Niedereichholz U, Ettler R, Holst JJ, Orskov C, Ritzel R, Schmiegel WH. Glucagon-like peptide 1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans. Am J Physiol. 1997; 273(5 Pt 1):E981–E988.
13. Baggio LL, Drucker DJ. Harnessing the therapeutic potential of glucagon-like peptide-1: a critical review. Treat Endocrinol. 2002; 1:117–125.
14. Nauck MA, Sauerwald A, Ritzel R, Holst JJ, Schmiegel W. Influence of glucagon-like peptide 1 on fasting glycemia in type 2 diabetic patients treated with insulin after sulfonylurea secondary failure. Diabetes Care. 1998; 21:1925–1931.
15. Kieffer TJ, McIntosh CH, Pederson RA. Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV. Endocrinology. 1995; 136:3585–3596.
16. Goke R, Fehmann HC, Linn T, Schmidt H, Krause M, Eng J, Goke B. Exendin-4 is a high potency agonist and truncated exendin-(9-39)-amide an antagonist at the glucagon-like peptide 1-(7-36)-amide receptor of insulin-secreting beta-cells. J Biol Chem. 1993; 268:19650–19655.
17. Buse JB, Henry RR, Han J, Kim DD, Fineman MS, Baron AD. Exenatide-113 Clinical Study Group. Effects of exenatide (exendin-4) on glycemic control over 30 weeks in sulfonylurea-treated patients with type 2 diabetes. Diabetes Care. 2004; 27:2628–2635.
18. Al-Sabah S, Donnelly D. A model for receptor-peptide binding at the glucagon-like peptide-1 (GLP-1) receptor through the analysis of truncated ligands and receptors. Br J Pharmacol. 2003; 140:339–346.
19. Edwards CM, Stanley SA, Davis R, Brynes AE, Frost GS, Seal LJ, Ghatei MA, Bloom SR. Exendin-4 reduces fasting and postprandial glucose and decreases energy intake in healthy volunteers. Am J Physiol Endocrinol Metab. 2001; 281:E155–E161.
20. Baggio LL, Holland D, Wither J, Drucker DJ. Lymphocytic infiltration and immune activation in metallothionein promoter-exendin-4 (MT-Exendin) transgenic mice. Diabetes. 2006; 55:1562–1570.
21. Moon MJ, Park S, Kim DK, Cho EB, Hwang JI, Vaudry H, Seong JY. Structural and molecular conservation of glucagon-like peptide-1 and its receptor confers selective ligand-receptor interaction. Front Endocrinol (Lausanne). 2012; 3:141.
22. Hwang JI, Moon MJ, Park S, Kim DK, Cho EB, Ha N, Son GH, Kim K, Vaudry H, Seong JY. Expansion of secretin-like G protein-coupled receptors and their peptide ligands via local duplications before and after two rounds of whole-genome duplication. Mol Biol Evol. 2013; 30:1119–1130.
23. Park CR, Moon MJ, Park S, Kim DK, Cho EB, Millar RP, Hwang JI, Seong JY. A novel glucagon-related peptide (GCRP) and its receptor GCRPR account for coevolution of their family members in vertebrates. PLoS One. 2013; 8:e65420.
24. Irwin DM, Satkunarajah M, Wen Y, Brubaker PL, Pederson RA, Wheeler MB. The Xenopus proglucagon gene encodes novel GLP-1-like peptides with insulinotropic properties. Proc Natl Acad Sci U S A. 1997; 94:7915–7920.
25. Gossrau R. Peptidases II. Localization of dipeptidylpeptidase IV (DPP IV). Histochemical and biochemical study. Histochemistry. 1979; 60:231–248.
26. Burcelin R, Dolci W, Thorens B. Long-lasting antidiabetic effect of a dipeptidyl peptidase IV-resistant analog of glucagon-like peptide-1. Metabolism. 1999; 48:252–258.
27. Kaung HC. Effect of glucose on beta cell proliferation and population size in organ culture of foetal and neonatal rat pancreases. J Embryol Exp Morphol. 1983; 75:303–312.
28. Deacon CF, Nauck MA, Toft-Nielsen M, Pridal L, Willms B, Holst JJ. Both subcutaneously and intravenously administered glucagon-like peptide I are rapidly degraded from the NH2-terminus in type II diabetic patients and in healthy subjects. Diabetes. 1995; 44:1126–1131.
29. Gallwitz B, Ropeter T, Morys-Wortmann C, Mentlein R, Siegel EG, Schmidt WE. GLP-1-analogues resistant to degradation by dipeptidyl-peptidase IV in vitro. Regul Pept. 2000; 86:103–111.
30. Ahren B, Simonsson E, Larsson H, Landin-Olsson M, Torgeirsson H, Jansson PA, Sandqvist M, Bavenholm P, Efendic S, Eriksson JW, Dickinson S, Holmes D. Inhibition of dipeptidyl peptidase IV improves metabolic control over a 4-week study period in type 2 diabetes. Diabetes Care. 2002; 25:869–875.
31. Pauly RP, Demuth HU, Rosche F, Schmidt J, White HA, Lynn F, McIntosh CH, Pederson RA. Improved glucose tolerance in rats treated with the dipeptidyl peptidase IV (CD26) inhibitor Ile-thiazolidide. Metabolism. 1999; 48:385–389.
32. Holst JJ, Deacon CF. Inhibition of the activity of dipeptidyl-peptidase IV as a treatment for type 2 diabetes. Diabetes. 1998; 47:1663–1670.
33. Chen X. Biochemical properties of recombinant prolyl dipeptidases DPP-IV and DPP8. Adv Exp Med Biol. 2006; 575:27–32.
34. Adelhorst K, Hedegaard BB, Knudsen LB, Kirk O. Structure-activity studies of glucagon-like peptide-1. J Biol Chem. 1994; 269:6275–6278.
35. Moon MJ, Kim HY, Kim SG, Park J, Choi DS, Hwang JI, Seong JY. Tyr1 and Ile7 of glucose-dependent insulinotropic polypeptide (GIP) confer differential ligand selectivity toward GIP and glucagon-like peptide-1 receptors. Mol Cells. 2010; 30:149–154.
36. Moon MJ, Kim HY, Park S, Kim DK, Cho EB, Park CR, You DJ, Hwang JI, Kim K, Choe H, Seong JY. Evolutionarily conserved residues at glucagon-like peptide-1 (GLP-1) receptor core confer ligand-induced receptor activation. J Biol Chem. 2012; 287:3873–3884.
37. Underwood CR, Garibay P, Knudsen LB, Hastrup S, Peters GH, Rudolph R, Reedtz-Runge S. Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor. J Biol Chem. 2010; 285:723–730.
38. Mentlein R, Gallwitz B, Schmidt WE. Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7-36)amide, peptide histidine methionine and is responsible for their degradation in human serum. Eur J Biochem. 1993; 214:829–835.
39. Kendall DM, Riddle MC, Rosenstock J, Zhuang D, Kim DD, Fineman MS, Baron AD. Effects of exenatide (exendin-4) on glycemic control over 30 weeks in patients with type 2 diabetes treated with metformin and a sulfonylurea. Diabetes Care. 2005; 28:1083–1091.
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