Islet isolation and glucose-stimulated insulin secretion assay (GSIS)

Primary islets were isolated from the pancreas of 8–13-week-old C57BL/6N male mice via collagenase P (Roche) digestion as described above, followed by a centrifugation step using Optiprep density gradient (Sigma). Isolated islets were handpicked twice and incubated in RPMI supplemented with 10 % v/v FBS and 1 % v/v P/S O/N for recovery. The next day, islets were treated with various concentrations of the commercially available bacterial FNDC4 (Adipogen), the in-house produced mammalian FcsFNDC4, or the corresponding negative controls PBS and Fc-peptide for 24 h. To gain sufficient islet numbers, islets of two mice were pooled for each biological replicate. For the GSIS assay, 9 islets of comparable size were transferred per well into a low-attachment V-shaped 96-well plate. Islets were incubated in modified Krebs Ringer phosphate HEPES buffer (KRPH; 115 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄·7H₂O, 20 mM NaHCO₃ 20 mM, 16 mM HEPES, 2.56 mM CaCl₂·2H₂O) supplemented with 0.1 % BSA (RIA grade) with various glucose concentrations in the presence of the proteins described above. Exendin-4 served as positive control. After incubation in the presence of 1 mM glucose for 1 h, islets were sequentially incubated with 2.8 mM glucose (low glucose) and 16.7 mM glucose (high glucose) for 30 min each. In between the incubation steps, islets were washed twice using KRPH with 2.8 mM glucose. Insulin concentration in the supernatant was assessed using the mouse insulin ELISA kit from ALPCO.