The structure-function relationship of glucose-dependent insulinotropic polypeptide

Glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide (GIP) is a 42 amino acid endocrine gut hormone which exhibits several direct and indirect effects on fat and glucose metabolism. The first known, and most scrutinized, metabolic function of the hormone was the potentiation of insulin release from pancreatic 13-cells in the presence of threshold glucose levels. In order to determine the region(s) of the molecule involved in mediating the insulin response at the beta cell, synthetic and proteolytic fragments of the molecule were generated and tested for their ability to potentiate the insulin response in the isolated, perfused rat pancreas. Previous work by others suggested that GIP amino acids 15 to 30 might be necessaly for biological activity yet a synthetic 15-30 fragment was biologically-inactive. However, enterokinase treatment of a synthetic 15 to 30 fragment restored approximately 30% of the integrated insulinotropic activity over a 25 mm perfusion of the isolated rat pancreas. The hypothesis that the restoration of biological activity was due to the enzymatic removal of the amino terminus aspartic acid/lysine residues of 1G513P0 was supported by the observation that a synthetic fragment lacking these two residues was also insulinotropic. There was no apparent difference in the insulin responses to the synthetic fragment or the enterokinase-derived fragment. Further fractionation of the molecule generated a 19-30 fragment which was also biologically active suggesting that the residues necessaiy for the insulin response were contained within this region. Two recombinant prokaiyotic expression systems for GIP were developed to further define the bioactive residues across the 19-30 region and to establish a system for the generation of large amounts of biologically-active material. Site-directed mutagenesis of a recombinant GIP clone was used to generate a full-length GIP molecule with alanine residues substituted across the 23 to 26 region of the polypeptide. This substitution resulted in the loss of biological activity suggesting that one or more of these residues is critical to the insulin response. An innate intramolecular interaction associated with the expression system precluded the isolation of sufficient quantities of alanine substituted material in the 19-22 and 27-30 regions of the molecule.Medicine, Faculty ofCellular and Physiological Sciences, Department ofGraduat