AbstractThe importance of Glucagon like peptide 1 (GLP-1) for metabolic control and insulin release sparked the evolution of genes mimicking GLP-1 action in venomous species (e.g. Exendin-4 in Heloderma suspectum (gila monster)). We discovered that platypus and echidna express a single GLP-1 peptide in both intestine and venom. Specific changes in GLP-1 of monotreme mammals result in resistance to DPP-4 cleavage which is also observed in the GLP-1 like Exendin-4 expressed in Heloderma venom. Remarkably we discovered that monotremes evolved an alternative mechanism to degrade GLP-1. We also show that monotreme GLP-1 stimulates insulin release in cultured rodent islets, but surprisingly shows low receptor affinity and bias toward Erk signaling. We propose that these changes in monotreme GLP-1 are the result of conflicting function of this peptide in metabolic control and venom. This evolutionary path is fundamentally different from the generally accepted idea that conflicting functions in a single gene favour duplication and diversification, as is the case for Exendin-4 in gila monster. This provides novel insight into the remarkably different metabolic control mechanism and venom function in monotremes and an unique example of how different selective pressures act upon a single gene in the absence of gene duplication.