Gastric Inhibitory Polypeptide (3-42) (human)

Gastric inhibitory polypeptide (GIP), an incretin, is an important subject in endocrinology. Some LC-MS assays have been proposed; however, their sensitivities are insufficient for the study of endogenous human incretin. Here, we describe a nanoflow LC hybrid triple quadrupole/linear ion trap MS assay for the simultaneous quantification of GIP1-42 and GIP3-42 from human plasma. We selected the surrogate peptide to avoid oxidative modification, and the endoproteinase Asp-N was selected for the proteolysis of GIP1-42 and GIP3-42. The phenylalanine residue at position 6 in both GIP1-42 and GIP3-42 was substituted with (13)C9,(15)N-labeled phenylalanine, and these substituted GIPs were used as the internal standards. This facilitated accurate and precise quantification because large corrections are possible at all steps of sample pretreatment and ionization efficiency. The lower limit of quantification was 1 pM for GIP1-42 and 10 pM for GIP3-42 by using 200 μL of plasma. Quantification of GIP1-42 and GIP3-42 in plasma from patients with type 2 diabetes was possible using this method, which included protein precipitation, Asp-N proteolysis, solid-phase extraction, nanoflow LC, and positive-ion multiple reaction monitoring cubed (MRM(3)) for GIP1-8, and MRM for GIP3-8 to achieve accurate, precise, and quantitative analysis that can be validated to support large clinical trials.

Gastric inhibitory polypeptide (GIP) is susceptible to degradation, but only recently has dipeptidyl peptidase IV been identified as the enzyme responsible. Most RIAs recognize both intact GIP-(1-42) and the noninsulinotropic N-terminally truncated metabolite, GIP-(3-42), hampering measurement of plasma concentrations. The molecular nature of GIP was examined using high pressure liquid chromatography and a newly developed RIA specific for the intact N-terminus of human GIP. In healthy subjects after a mixed meal, intact GIP (N-terminal RIA) accounted for 37.0+/-2.5% of the total immunoreactivity determined by C-terminal assay. High pressure liquid chromatographic analysis of fasting samples by C-terminal assay revealed one major peak (73.8+/-2.9%) coeluting with GIP-(3-42). One hour postprandially, two major peaks were detected, corresponding to GIP-(3-42) and GIP-(1-42) (58.1+/-2.7% and 35.7+/-4.2%, respectively). GIP-(3-42) was not detected by N-terminal assay; the major peak coeluted with intact GIP (86.4+/-5.8% and 81.3+/-0.9%, 0 and 1 h, respectively). After iv infusion, intact GIP constituted 37.1+/-4.1% and 41.3+/-3.4% of the total immunoreactivity in healthy and type 2 diabetic subjects, respectively. The plasma t1/2 was shorter (P < 0.0001) when determined by N-terminal compared with C-terminal assay (7.3+/-1.0 vs. 16.8+/-1.6 and 5.2+/-0.6 vs. 12.9+/-0.9 min, healthy and diabetic subjects, respectively), and both t1/2 were shorter in the diabetic group (P < 0.05). We conclude that dipeptidyl peptidase IV is important in GIP metabolism in humans in vivo, and that an N-terminally directed assay is required for determination of plasma concentrations of biologically active GIP.

The present review focuses initially on experimental studies that were designed to identify acid inhibitory factors, referred to as 'enterogastrones,' that ultimately led to the isolation of gastric inhibitory polypeptide (GIP), a 42-amino acid polypeptide. GIP was shown to inhibit acid secretion in animal models, as well as stimulating gastric somatostatin secretion. However, its role in human gastric physiology is unclear. Further studies showed that GIP strongly stimulated the secretion of insulin, in the presence of elevated glucose, and this 'incretin' action is now considered to be its most important; an alternative for the GIP acronym, glucose-dependent insulinotropic polypeptide, was therefore introduced. In the 1970s, GIP purified by conventional chromatography was shown by high-performance liquid chromatography to consist largely of GIP 1-42 and GIP 3-42. It was later shown that dipeptidyl peptidase 4 was a physiologically relevant enzyme responsible for this conversion, as well as the similar metabolism of the second incretin, glucagon-like peptide-1. Dipeptidyl peptidase-4 inhibitors are currently in use as type 2 diabetes therapeutics, and studies on islet transplantation in rodent models of type 1 diabetes have shown that dipeptidyl peptidase-4 inhibitor treatment reduces graft rejection. Additional studies on C-terminally shortened forms of GIP have shown that GIP 1-30 and a dipeptidyl peptidase-4-resistant form (D-Ala(2) GIP 1-30) are equipotent to the intact polypeptide in vitro, and administration of D-Ala(2) GIP 1-30 to diabetic rodents greatly improved glucose tolerance and reduced apoptotic cell death in islet β-cells. There are probably therefore further clinically useful effects of GIP that require investigation.