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    • The first hint that GLP might

    2020-04-06

    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 (μmolar) 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 SAR-302503 australia 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 SAR-302503 australia 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].