Properties of native and in vitro glycosylated forms of the glucagon-like peptide-1 receptor antagonist exendin (9–39)

Abstract The intestinal hormone glucagon-like peptide-1-(7–36)-amide (GLP-1) has recently been implicated as a possible therapeutic agent for the management of type 2 non—insulin-dependent diabetes mellitus (NIDDM). However, a major difficulty with the delivery of peptide-based agents is their short plasma half-life, mainly due to rapid serum clearance and proteolytic degradation. Using a peptide analog of GLP-1, the GLP-1 receptor antagonist exendin(9–39), we investigated whether the conjugation of a carbohydrate structure to exendin(9–39) would generate a peptide with intact biological activity and improved survival in circulation. The C-terminal portion of exendin(9–39) was reengineered to generate an efficient site for enzymatic O -glycosylation. The modified exendin(9–39) peptide (exe-M) was glycosylated by recombinant UDP-GalNAc:polypeptide N -acetylgalactosaminyltransferase 1 (GaINAc-T1) alone or in conjunction with a recombinant GaINAc α2,6-sialyltransferase (Sialyl-T), resulting in exe-M peptides containing either the monosaccharide GaINAc or the disaccharide NeuAcα2,6GaINAc. The nonglycosylated and glycosylated forms of exe-M competed with nearly equal potency (> 90% of control) with the binding of [ 125 I]GLP-1 to human GLP-1 receptors expressed on stably transfected COS-7 cells. In addition, each peptide was equally effective for inhibiting GLP-1—induced cyclic adenosine monophosphate (cAMP) production in vitro. Studies with rats demonstrated that the modified and glycosylated forms of exendin(9–39) could antagonize exogenously administered GLP-1 in vivo. Interestingly, glycosylated exendin(9–39) homologs were more than twice as effective as the nonglycosylated peptide for inhibiting GLP-1—stimulated insulin production in vivo, suggesting a longer functional half-life in the circulation for glycosylated peptides. Results from in vivo studies with 3 H-labeled peptides suggest that the glycosylated peptides may be less susceptible to modification in the circulation.