Glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal hormone, which regulates insulin release and glucose homeostasis, but is rapidly inactivated by enzymatic N-terminal truncation. Here we report the enzyme resistance and biological activity of several Glu(3) -substituted analogues of GIP namely; (Ala(3))GIP, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))GIP. Only (Lys(3))- GIP demonstrated moderately enhanced resistance to DPP-IV (p < 0.05 to p < 0.01) compared to native GIP. All analogues demonstrated a decreased potency in cAMP production (EC50 1.47 to 11.02 nM; p < 0.01 to p < 0.001) with (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated cAMP production (p < 0.05). In BRIN-BD11 cells, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))- GIP did not stimulate insulin secretion with both (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated insulin secretion (p < 0.05). Injection of each GIP analogue together with glucose in oblob mice significantly increased the glycaemic excursion compared to control (p < 0.05 to p < 0.001). This was associated with lack of significant insulin responses. (Ala(3))GIP, (Phe(3))GIP and (Tyr(3))GIP, when administered together with GIP, significantly reduced plasma insulin (p < 0.05 to p < 0.01) and impaired the glucose-lowering ability (p < 0.05 to p < 0.01) of the native peptide. The DPP-IV resistance and GIP antagonism observed were similar but less pronounced than (Pro(3))GIP. These data demonstrate that position 3 amino acid substitution of GIP with (Ala(3)), (Phe(3)), (Tyr(3)) or (Pro(3)) provides a new class of functional GIP receptor antagonists. (C) 2007 Elsevier Inc. All rights reserved.