Datensatz

Zusammenfassung

Aims/hypothesis In humans, glucagon-like peptide-1 (GLP-1) is rapidly degraded by dipeptidyl peptidase-4 to a relatively stable metabolite, GLP-1(9-36)NH2, which allows measurement of GLP-1 secretion. However, little is known about the kinetics of the GLP-1 metabolite in mice. We hypothesised that the GLP-1 metabolite is rapidly degraded in this species by neutral endopeptidase(s) (NEP[s]). Methods We administered glucose, mixed meal or water orally to 256 mice, and took blood samples before and 2, 6, 10, 20, 30, 60 or 90 min after stimulation. To study the metabolism of the GLP-1 metabolite, i.v. GLP-1(9-36)NH2 (800 fmol) or saline (154mmol/l NaCl) was administered to 160 mice, some of which had a prior injection of a selective NEP24.11 +/- inhibitor (candoxatril, 5 mg/kg) or saline. Blood was collected before and 1, 2, 4 and 12 min after GLP-1/saline injection. Plasma GLP-1 levels were analysed using a customised single-site C-terminal ELISA, two different two-site ELISAs and MS. Results GLP-1 secretion profiles after oral glucose administration differed markedly when assayed by C-terminal ELISA compared with sandwich ELISAs, with the former showing a far higher peak value and AUC. In mice injected with GLP1(9-36)NH2, immunoreactive GLP-1 plasma levels peaked at approximately 75 pmol/l at 1 min when measured with sandwich ELISAs, returning to baseline (similar to 20 pmol/l) after 12 min, but remained elevated using the C-terminal ELISA (similar to 90 pmol/l at 12 min). NEP 24.11 inhibition by candoxatril significantly attenuated GLP-1(9-36)NH2 degradation in vivo and in vitro. MS identified GLP-1 fragments consistent with NEP 24.11 degradation. Conclusions/interpretation In mice, the GLP-1 metabolite is eliminated within a few minutes owing to endoproteolytic cleavage by NEP 24.11. Therefore, accurate measurement of GLP-1 secretion in mice requires assays for NEP 24.11 meta-bolites. Conventional sandwich ELISAs are inadequate because of endoproteolytic cleavage of the dipeptidyl peptidase-4-generated metabolite.