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Immunoassays for the incretin hormones GIP and GLP-1

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The measurement of the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), using immunologically based assays is made difficult by the fact that the processing of the precursor molecules gives rise to a number of different peptides which cross-react with antisera raised against the two hormones. For GLP-1, the picture is further complicated because of the necessity to differentiate between the intestinal and pancreatic proglucagon products. Finally, once secreted, both incretins are rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP-4) to generate metabolites which have lost their insulinotropic activities. These metabolites are the major circulating forms of the incretins, accounting for 60–80% of total immunoreactive GLP-1 and GIP in the peripheral plasma, while concentrations of the intact forms can be very low and, in some cases, barely detectable. The use of highly specific assays using well-characterised antisera and careful sample handling is therefore required for a reliable determination of incretin hormone concentrations.

Section snippets

Measurement of proglucagon-derived peptides

The proglucagon (PG) gene is expressed in the pancreatic α-cells, the intestinal L-cells and in the brain.1, 2 Following the cloning of the gene in 19835, it was discovered that the way in which it is processed differs in a tissue-specific way, giving rise to a number of peptides which are secreted in parallel (Fig. 1). In the pancreatic α-cells, cleavage by prohormone convertase (PC) 26 gives rise to glicentin-related pancreatic peptide (GRPP) and glucagon, corresponding to PG (1–30) and PG

Measurement of preproGIP-derived peptides

The immunological determination of GIP concentrations generally does not present the same difficulties as GLP-1 since the processing pattern of GIP is much less complicated (Fig. 2). GIP is derived by proteolytic processing of the 153-residue precursor, preproGIP, which is expressed in the intestinal K-cells, and consists of three domains.29 The 42-amino-acid-long GIP is flanked at either side by an N-terminal domain, which contains the signal peptide, and a C-terminal domain. Cleavage by PC

Practical considerations

It is noteworthy that a comparison of incretin hormone levels determined in earlier studies with those reported in more recent publications shows that there is a clear trend for the measured concentrations to have become lower with time. Part of the explanation is that the interference from cross-reacting peptides has been eliminated (or at least reduced) by the selection of antisera directed against well-defined epitopes, based upon the better understanding of the processing patterns of the

Conclusions

The determination of incretin hormone concentrations by immunological methods is complicated because the processing of the precursor molecules and subsequent degradation of the products in the plasma give rise to a plethora of related peptides which can cross-react to varying degrees with the assays being used.

It is also worthy of comment that commercially available antisera and assay kits are, in general, poorly characterised, and, in many cases, the epitope which is being detected is

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