AIMS/HYPOTHESIS: We designed a chemically modified, enzyme-resistant peptide with triple-acting properties based on human glucagon with amino acid substitutions aligned to strategic positions in the sequence of glucose-dependent insulinotropic polypeptide (GIP).METHODS: Y1-dA2-I12-N17-V18-I27-G28,29-glucagon (termed YAG-glucagon) was incubated with dipeptidylpeptidase IV (DPP-IV) to assess stability, BRIN-BD11 cells to evaluate insulin secretion, and receptor-transfected cells to examine cAMP production. Acute glucose-lowering and insulinotropic properties of YAG-glucagon were assessed in National Institutes of Health (NIH) Swiss mice, while longer-term actions on glucose homeostasis, insulin secretion, food intake and body weight were examined in high-fat-fed mice.RESULTS: YAG-glucagon was resistant to DPP-IV, increased in vitro insulin secretion (1.5-3-fold; p <0.001) and stimulated cAMP production in GIP receptor-, glucagon-like peptide-1 (GLP-1) receptor- and glucagon receptor-transfected cells. Plasma glucose levels were significantly reduced (by 51%; p <0.01) and insulin concentrations increased (1.2-fold; p <0.01) after acute injection of YAG-glucagon in NIH Swiss mice. Acute actions were countered by established GIP, GLP-1 and glucagon antagonists. In high-fat-fed mice, twice-daily administration of YAG-glucagon for 14 days reduced plasma glucose (40% reduction; p <0.01) and increased plasma insulin concentrations (1.8-fold; p <0.05). Glycaemic responses were markedly improved (19-48% reduction; p <0.05) and insulin secretion enhanced (1.5-fold; p <0.05) after a glucose load, which were independent of changes in insulin sensitivity, food intake and body weight.CONCLUSIONS/INTERPRETATION: YAG-glucagon is a DPP-IV-resistant triple agonist of GIP, GLP-1 and glucagon receptors and exhibits beneficial biological properties suggesting that it may hold promise for treatment of type 2 diabetes.