Metabolic stability, receptor binding, cAMP generation, insulin secretion and antihyperglycaemic activity of novel N-terminal Glu⁹-substituted analogues of glucagon-like peptide-1

Glucagon-like peptide-1(7-36)amide (GLP-1) is an incretin hormone with therapeutic potential for type 2 diabetes. Rapid removal of the N-terminal dipeptide, His⁷-Ala⁸, by the ubiquitous enzyme dipeptidyl peptidase IV (DPP IV) curtails the biological activity of GLP-1. Chemical modifications or substitutions of GLP-1 at His⁷ or Ala⁸ improve resistance to DPP-IV action, but this often reduces potency. Little attention has focused on the metabolic stability and functional activity of GLP-1 analogues with amino acid substitution at Glu⁹, adjacent to the DPP IV cleavage site. We generated three novel Glu⁹-substituted GLP-1 analogues, (Pro⁹)GLP-1, (Phe⁹)GLP-1 and (Tyr⁹)GLP-1 and show for the first time that Glu⁹ of GLP-1 is important in DPP IV degradation, since replacing this amino acid, particularly with proline, substantially reduced susceptibility to degradation. All three novel GLP-1 analogues showed similar or slightly enhanced insulinotropic activity compared with native GLP-1 despite a moderate 4-10-fold reduction in receptor binding and cAMP generation. In addition, (Pro⁹)GLP-1 showed significant ability to moderate the plasma glucose excursion and increase circulating insulin concentrations in severely insulin resistant obese diabetic (ob/ob) mice. These observations indicate the importance of Glu⁹ for the biological activity of GLP-1 and susceptibility to DPP IV-mediated degradation.