[Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo.
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.
Hinke
Simon A
SA
Department of Physiology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Manhart
Susanne
S
Kühn-Wache
Kerstin
K
Nian
Cuilan
C
Demuth
Hans-Ulrich
HU
Pederson
Raymond A
RA
McIntosh
Christopher H S
CH
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D018819:Dipeptidyl Peptidase 4; D004355:Drug Stability; D005749:Gastric Inhibitory Polypeptide / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D005768:Gastrointestinal Hormones / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D005934:Glucagon / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D052216:Glucagon-Like Peptide 1; D000067757:Glucagon-Like Peptide-1 Receptor; D066298:In Vitro Techniques; D008297:Male; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D010768:Phosphoserine / Q000737:chemistry; D011498:Protein Precursors / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism; D018027:Receptors, Glucagon / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001786:Blood Glucose; D005768:Gastrointestinal Hormones; C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D010446:Peptide Fragments; D011498:Protein Precursors; D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D010768:Phosphoserine; D012694:Serine; D005749:Gastric Inhibitory Polypeptide; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; C041086:gastric inhibitory polypeptide receptor; D000242:Cyclic AMP; D018819:Dipeptidyl Peptidase 4