Radiolabeling

¹²⁵I-labeling. The radioiodinated reference ligand [Nle¹⁴, [¹²⁵I]Tyr(3-I)⁴⁰]exendin-4 ([¹²⁵I]Tyr(3-I)⁴⁰–3) was prepared via the Iodogen method (Salacinski et al., 1981), with some minor modifications. The precursor [Nle¹⁴, Tyr⁴⁰]exendin-4 (150 μg, 30.6 nmol, 122 eq.) was dissolved in 20 μL DMSO and 280 μL TRIS buffer (25 mM TRIS HCl, 0.04 M NaCl, pH = 7.5). The solution was transferred to a vial, which has been precoated with 15.0 μg Iodogen (34.7 nmol, 139 eq.). Afterwards, 15 ± 5 MBq [¹²⁵I] NaI (74 MBq/nmol, 3.7 GBq/mL in 40 mM NaOH, Hartmann Analytic, Braunschweig, Germany) (5.00 μL, 250 pmol, 1.00 eq.) were added, the reaction solution was incubated for 15 min at r.t. and 150 μL thereof were purified by radio-RP-HPLC (0% B (2 min) ➔ 0–37.5% B (3 min) ➔ 37.5% B (35 min) ➔ 38% B (10 min), Method A*, 1 mL/min) to afford 3.15 MBq (48.8% RCY, > 99% RCP) of product [¹²⁵I]Tyr(3-I)⁴⁰–3. The radioligand was stored in the HPLC solvent (stock solution) at − 80 °C until further use. The radioligand stock solution was used to a maximum of 14 days to ensure a RCP of > 92% (Table 1 in the Additional file 1) and replaced by freshly prepared radioligand afterwards. Prior to the in vitro experiments, the radioligand was dissolved in HBSS (with or w/o 1% BSA) to achieve a final concentration of 0.41 nM in the respective assays. *Method A: solvent A = water + 0.1% TFA, solvent B = acetonitrile + 2% water + 0.1% TFA.