Investigation of metabolism of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) by dipeptidyl peptidase IV (DP IV)

The incretins glucose-dependent insulinotropic polypeptide (GTP1-42) and truncated forms of glucagon-like peptide-1 (GLP-I7-36 and GLP-I7.37) are hormones released from the gut in response to ingested nutrients and act on the endocrine pancreas to potentiate glucose-induced insulin secretion. GTP1.42 and GLP-I7.36 are known substrates of the circulating exopeptidase dipeptidyl peptidase IV (DP IV, CD26, EC 3.4.14.5) which selectively hydrolyzes peptides after penultimate N-terminal proline or alanine. Hydrolysis of GJJP1.42 and GLP-I7.36 by DP IV yields the biologically inactive polypeptides GTP3-42 and GLP-I9.36, and the dipeptides Tyr-Ala and His-Ala respectively. It has been speculated that DP IV-catalyzed incretin hydrolysis is the primary step in the metabolism of these hormones. This thesis reports the use of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to study incretin hydrolysis in vitro, including enzyme kinetics, and the establishment of a protocol for the inhibition of DP IV in vivo, allowing study of the influence of endogenous DP IV on the enteroinsular axis. Analysis of MS spectra indicated that human serum-incubated GUP 1-42 and GLP-17.36 were cleaved by DP IV with only minor secondary degradation due to other serum protease activity. Kinetic constants of incretin hydrolysis by purified porcine kidney-derived enzyme and by human serum DP IV activity suggest that DP IV-mediated hydrolysis of these peptides is significant at physiological incretin concentrations. Ile-thiazolidide, one of a new class of competitive reversible transition state analogue inhibitors of DP IV was used to block DP IV activity in vitro and in vivo. Endogenous DP IV inhibition resulted in an earlier rise and peak of plasma insulin and more rapid clearance of blood glucose in response to an intraduodenal glucose challenge. High performance liquid chromatography (HPLC) analysis revealed that inhibition of DP IV in vivo was able to prevent the hydrolysis of radiolabelled GLP-17.36, indicating that the altered insulin profile is likely an incretin-mediated response. On the basis of the studies described in this thesis it was concluded that DP IV is the principal protease responsible for the degradation of GIP1-42 and GLP-I7.36 and manipulation of endogenous DP IV activity was able to improve glucose tolerance in the rat.Medicine, Faculty ofCellular and Physiological Sciences, Department ofGraduat