Inhibition of dipeptidyl peptidase 4 allows accurate measurement of GLP-1 secretion in mice

Abstract

Dipeptidyl peptidase (DPP)-4 and neprilysin (NEP) within few minutes degrade glucagon-like peptide 1 (GLP-1) in mice, generating small, inactive fragments of the peptide. Commercially available sandwich ELISA kits may not accurately detect intact GLP-1 (that is GLP-1[7-36]NH2) and these moieties, leading to underestimation of secretion and potentially misleading results. Single-site antibody approaches may pick up some fragments, yet require large plasma volumes and the accuracy is uncertain. We aimed to find a way to stabilize GLP-1 in mice allowing reliable measurement with sensitive commercially available ELISA kits. Non-anesthetized male C57Bl/6JRj mice were subjected to an oral glucose tolerance test (OGTT; 2 g/kg glucose via oral gavage). Blood was drawn from the retrobulbar plexus before and repeatedly during the OGTT and total and intact GLP-1 were measured by commercially available sandwich ELISA kits (Mercodia and Alpco, respectively). In blood samples taken 15 minutes after the glucose load, there were no increase in plasma GLP-1 concentration. There was a small insignificant increase in total GLP-1 (1-2 pmol/L) in samples taken at t=5 and t=10 minutes following the OGTT, but no rise in intact GLP-1. We then administered control (saline), or a DPP-4 inhibitor (valine pyrrolidide, 0.1 {micro}mol/g or sitagliptin, 10 mg/kg) with or without a NEP-inhibitor (sacubitril, 0.3 mg/kg) 30 minutes before the OGTT. In the four inhibitor groups, intact GLP-1 increased during the OGTT (levels ranging between 4.04 [SD 2.87] and 15.53 [SD 6.76] pmol/L). The combination of sitagliptin with sacubitril gave the largest increase in intact GLP-1 levels. Finally, after injecting male C57Bl/6JRj mice with a known dose of GLP-1(7-36)NH2, the peak GLP-1 levels were significantly higher during sitagliptin, but not with the combination of sitagliptin/sacubitril. Both inhibitor groups, however, showed prolonged half-life of the GLP-1 plasma disappearance. We conclude that for measurements of GLP-1 secretion in mice with commercially available sandwich ELISA kits, it is necessary to consider both timing of blood sampling and in vivo inhibition of DPP-4. The described approach allows improved estimates of GLP-1 secretion for future studies. It is a limitation that DPP-4 and NEP inhibition may have metabolic effects by stabilizing the intact GLP-1 peptide (e.g. influencing levels of insulin and glucagon).