Lys9 for Glu9 substitution in glucagon-like peptide-1(7–36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9–36)amide and exendin (9–39)

doi:10.1016/j.metabol.2003.09.015

Metabolism

Volume 53, Issue 2, February 2004, Pages 252-259

https://doi.org/10.1016/j.metabol.2003.09.015 Get rights and content

Abstract

The incretin hormone glucagon-like peptide-1(7–36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLP-1’s action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His⁷ and Ala⁸, can greatly improve resistance to this enzyme. Little research has assessed what effect Glu⁹-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu⁹ of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue, (Lys⁹)GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9–39) and GLP-1(9–36)amide. We investigated the in vivo antagonistic actions of (Lys⁹)GLP-1 in comparison with GLP-1(9–36)amide and exendin (9–39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor.

Section snippets

Reagents

High-performance liquid chromatography (HPLC) grade acetonitrile, diethyl ether, and dichloromethane (DCM) were obtained from Rathburn (Walkersburn, Scotland). Sequencing grade trifluoroacetic acid (TFA), DPP IV, forskolin (FSK), isobutylmethylxanthine (IBMX), cAMP, and adenosine 5′-triphosphate (ATP) were purchased from Sigma (Poole, Dorset, UK). Fmoc protected amino acids were purchased from Calbiochem Novabiochem (Beeston, Nottingham, UK). RPMI 1640 and Dulbecco’s modified Eagles’s medium

Confirmation of the identities of peptides by ESI-MS

Table 1 shows the monoisotopic masses obtained for GLP-1 and related peptides using ESI-MS. Prominent multiply-charged species (M+2H)²⁺ and (M+3H)³⁺ were obtained for GLP-1, corresponding to M_r of 3,297.3 Da; for GLP-1(9–36)amide of 3,088.4 Da; for (Lys⁹)GLP-1 of 3,297.1 Da, and for exendin 4 (1–39) of 4,186.6 dalton (d). Finally, for exendin (9–39), (M+3H)³⁺ and (M+4H)⁴⁺ charged species were observed corresponding to of 3,369.4 d. Values measured compared closely to the theoretical molecular

Discussion

Much interest has been generated by attempts to develop DPP IV-resistant analogues of GLP-1 using N-terminal modifications, such as Ala⁸-substitution14, 15, 16, 17, 18, 29 and His⁷-modification.19, 20 These studies have met with varying degrees of success with many analogues suffering from reduced receptor affinity and diminished insulinotropic potency.14, 20, 29 Other analogues have displayed similar biologic activity to native GLP-1.14, 16, 17, 18, 19 As yet, no study has investigated how

Acknowledgements

We thank Professor Bernard Thorens (Institute of Pharmacology, University of Lausanne, Switzerland) for kindly providing the Chinese hamster lung (CHL) fibroblast cell transfected with the human GLP-1 receptor. We also thank Dr Andrew Young (Amylin Corporation, San Diego, CA) for generously donating exendin 4 (1–39) and exendin (9–39).

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^☆: Supported by the University of Ulster Strategy Funding.

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Lys9 for Glu9 substitution in glucagon-like peptide-1(7–36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9–36)amide and exendin (9–39)☆

Abstract

Section snippets

Reagents

Confirmation of the identities of peptides by ESI-MS

Discussion

Acknowledgements

Eur J Pharmacol

Regul Pept

Regul Pept

Biochim Biophys Acta

Regul Pept

Anal Biochem

Biochem Pharmacol

Metabolism

J Biol Chem

Metabolism

The glucagon-like peptides

Endocr Rev

Biological actions and therapeutic potential of the glucagon-like peptides

Gastroenterology

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

Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats

Diabetes

Glucagon-like peptide 1 induces differentiation of islet duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting cells

Diabetes

Truncated GLP-1 (proglucagon 78–107-amide) inhibits gastric and pancreatic functions in man

Dig Dis Sci

Lys⁹ for Glu⁹ substitution in glucagon-like peptide-1(7–36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9–36)amide and exendin (9–39)☆