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  • The first hint that GLP might not

    2020-11-16

    The first hint that GLP-1 might not be completely stable came from a meeting abstract in 1992, reporting that GLP-1 could be degraded in plasma incubations [16], although the enzyme(s) involved was not identified. A year later, Mentlein and colleagues [17], in a search for peptide substrates of the serine protease DPP-4, used high performance liquid chromatography to demonstrate that pharmacological (╬╝molar) concentrations GLP-1, and the other incretin hormone glucose-dependent insulinotropic polypeptide (GIP), were degraded in vitro by the purified enzyme and in plasma to form an N-terminally truncated metabolite, and that this degradation was prevented in the presence of a DPP-4 inhibitor. Although the degradation of physiological peptide concentrations or endogenous peptides were not examined, given that the N-terminus of members of the glucagon/VIP peptide family, to which GLP-1 and GIP belong, is important for receptor activation [18], the authors speculated that DPP-4 action could lead to the loss of biological activity as well as potentially interfere with their measurement by immunoassays [17]. Subsequently, more physiologically relevant (picomolar) levels of GLP-1 were also shown to be degraded by DPP-4 in plasma, and the N-terminally truncated metabolite generated by DPP-4 action was identified as the major circulating form of endogenous GLP-1 in humans, suggesting that DPP-4 was likely to play a physiological role in the metabolism of the peptide in vivo [19]. This suggestion was supported by the finding that exogenously administered radio-labelled GLP-1 and GIP were degraded in wild-type, but not in DPP-4 deficient rats by Kieffer et al., [20] who, like Mentlein and colleagues [17], also concluded that DPP-4-mediated degradation of the incretin dna pk could interfere with their accurate measurement whilst also being responsible for their inactivation. Conventional immunoassays of the time mostly employed antisera directed towards epitopes in the mid-region or C-terminal end of GLP-1; such assays are insensitive to modifications at the N-terminus of the peptide, meaning that they will also react with any peptides which are either N-terminally elongated or truncated. Therefore, in order to assess the relevance of DPP-4-mediated degradation of GLP-1 in humans, we developed a novel immunoassay for the intact N-terminus of GLP-1. By comparing GLP-1 concentrations measured with this assay against those determined with the conventional type of assay, we showed that exogenously administered GLP-1 underwent substantial degradation in both healthy individuals and subjects with T2DM; after subcutaneous injection, less than 10% of the peptide survived in the intact state, thereby verifying that the commonly used conventional GLP-1 assays considerably over-estimated concentrations of the biologically active peptide (because the inactive metabolite was also detected) [1]. Moreover, this thereby provided an explanation for the short-lived effects of GLP-1 in the earlier clinical studies (because the intact biologically active peptide was rapidly degraded by DPP-4), and led to the initial proposal that inhibiting the activity of DPP-4 to reduce this degradation and enhance levels of active GLP-1 may be a novel therapeutic approach to manage T2DM [1]. For this approach to be successful, the DPP-4 enzyme would need to play a key role in the metabolism of GLP-1. From in vitro kinetic studies, Mentlein et al. [17] had already predicted that GLP-1 was a particularly good substrate for the enzyme, and in vivo studies in rats [20] and humans [1], [19] had indicated that the N-terminally truncated metabolite arising from DPP-4-mediated cleavage seemed to be the predominant endogenous [19] and exogenous [1], [20] metabolite, and was formed rapidly after exogenous administration of the peptide [1], [20]. Endogenous GLP-1 is also exquisitely susceptible to DPP-4-mediated degradation. Although the peptide is stored predominantly in its intact form, over half of the GLP-1 which is secreted from the perfused intestine was found to be already degraded to the N-terminally truncated metabolite by the time it had reached the local blood vessels draining the preparation, and it was concluded that DPP-4, which was shown to be present on the vascular endothelium, including capilliaries adjacent to the GLP-1-producing L-cells, was responsible [21]. Together, these observations suggested that DPP-4 was likely to be the initial and primary route of degradation of GLP-1. Similar conclusions were reached regarding the role of DPP-4 in GIP metabolism. Accordingly, observations from in vitro studies [17] and from in vivo administration of radio-labelled GIP in rats [20] showing that GIP was degraded by DPP-4 were followed by the development of an N-terminally directed immunoassay to reveal that the metabolite generated by DPP-4 cleavage was the major circulating form of endogenous GIP in humans [22].