[Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo

• Published in: The Journal of biological chemistry Vol. 279; no. 6; p. 3998
• Main Authors: Hinke, Simon A, Manhart, Susanne, Kühn-Wache, Kerstin, Nian, Cuilan, Demuth, Hans-Ulrich, Pederson, Raymond A, McIntosh, Christopher H S
• Format: Journal Article
• Language: English
• Published: United States 06.02.2004
• Subjects: Animals; Blood Glucose - metabolism; CHO Cells; Cricetinae; Cyclic AMP - metabolism; Dipeptidyl Peptidase 4; Drug Stability; Gastric Inhibitory Polypeptide - chemical synthesis; Gastric Inhibitory Polypeptide - metabolism; Gastric Inhibitory Polypeptide - pharmacology; Gastrointestinal Hormones - chemical synthesis; Gastrointestinal Hormones - metabolism; Gastrointestinal Hormones - pharmacology; Glucagon - chemical synthesis; Glucagon - metabolism; Glucagon - pharmacology; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; In Vitro Techniques; Male; Peptide Fragments - chemical synthesis; Peptide Fragments - metabolism; Peptide Fragments - pharmacology; Phosphoserine - chemistry; Protein Precursors - chemical synthesis; Protein Precursors - metabolism; Protein Precursors - pharmacology; Rats; Rats, Wistar; Receptors, Gastrointestinal Hormone - metabolism; Receptors, Glucagon - metabolism; Serine - chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M311304200

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16-0.52 nm) and IC50s (4.3-8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)2]incretins > or = [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.