The in vitro selection of the DNA-templated library8 (link) used 20 µg His6-tagged mouse IDE immobilized on cobalt magnetic beads (Invitrogen). IDE inhibition was assayed using the fluorogenic peptide Mca-RPPGFSAFK(Dnp)-OH (R&D), confirmed using an anti-insulin antibody time-resolved FRET assay (Cysbio), and a LCMS assay for CGRP cleavage fragments in plasma9 (link). Macrocyclic inhibitors were synthesized by Fmoc-based solid-phase synthesis and purified by HPLC. LCMS quantitation of 6bK in biological samples was performed using 6bK synthesized with 13C6,15N2 lysine (Sigma-Aldrich).
Wild-type lean and DIO C57BL/6J age-matched male mice (Jackson Laboratories) were used at 14–16, and 24–26 weeks respectively (> 20 weeks of high-fat diet). Gcgr−/− and Ide−/− mice were fully backcrossed to the C57BL/6J line, bred from heterozygous mice, and used between 11 and 21 weeks. Animals were fasted overnight 14 h for all experiments, except for the insulin tolerance test, which required 5 h of fasting during the morning. Blood glucose was measured from tail nicks using AccuCheck (Aviva) meters. Trunk blood was obtained for plasma hormone measurements using the Multiplexed Mouse Metabolic Hormone panel (Milliplex, EMD Millipore) on a Luminex FlexMap 3D instrument.
Wild-type lean and DIO C57BL/6J age-matched male mice (Jackson Laboratories) were used at 14–16, and 24–26 weeks respectively (> 20 weeks of high-fat diet). Gcgr−/− and Ide−/− mice were fully backcrossed to the C57BL/6J line, bred from heterozygous mice, and used between 11 and 21 weeks. Animals were fasted overnight 14 h for all experiments, except for the insulin tolerance test, which required 5 h of fasting during the morning. Blood glucose was measured from tail nicks using AccuCheck (Aviva) meters. Trunk blood was obtained for plasma hormone measurements using the Multiplexed Mouse Metabolic Hormone panel (Milliplex, EMD Millipore) on a Luminex FlexMap 3D instrument.