Release and metabolism of gastric inhibitory polypeptide

• Main Author: Kieffer, Timothy James
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/6955

This thesis reports methodology that was developed to isolate and enrich intestinal endocrine cell preparations by centrifugal elutriation and short-term culture to enable the study of the regulation of IRGIP secretion at the cellular level. Adherent canine epithelial cell cultures contained ~10% IRGIP cells. The release of IRGIP from these cells was significantly increased by incubation with depolarizing concentrations of K⁺, glucose, somatostatin immunoneutralizing antibody, or GRP, in addition to pharmacological elevations of intracellular cAMP or Ca²⁺. It is concluded that this method provides a means of further investigating the cellular mechanisms controlling GIP release. Enteroendocrine tumor cell lines were also investigated as an alternate source of GIP cells. A cell line derived from intestinal tumors of transgenic mice (STC- 1) was subcloned to produce a cell line with ~30% IRGIP (STC 6-14). HPLC of STC 6-14 extracts indicated that the tumor cell derived IRGIP eluted with synthetic porcine GIP 1-42. Release of IRGIP from STC 6-14 cells; increased in a concentration dependent fashion in response to glucose, was augmented by the addition of somatostatin neutralizing antibody, and attenuated by exogenous somatostatin. Immunoreactive somatostatin (IRSS) release was significantly increased by adding GIP to the incubation medium. It is concluded that this cell line represents a means of rapidly obtaining large numbers of GIP cells, and thus should be useful to investigate stimulus-secretion coupling in the GIP cell. GIP secreted by STC 6-14 cells was metabolized by a serum constituent to biologically inactive GIP 3-42. ¹²⁵I-GIP was purified by HPLC and used as a highly sensitive means to further investigate the degradation of GIP by serum. The removal of the N-terminal dipeptide by serum could be blocked by diprotin A, a competitive inhibitor of dipeptidyl peptidase IV (DPP IV). No GIP 3-42 was produced by incubation of GIP with serum from rats specifically lacking DPP IV. Infusion ¹²⁵I-GIP into rats, followed by HPLC analysis, indicated that 50% was metabolized ¹²⁵I-GIP 3-42 by ~1.5 min. It is concluded that DPP IV is a primary degradative and inactivating enzyme of GIP.