In vitro Insulin Secretion Assay

For in vitro insulin secretion studies, pancreatic islets were isolated from female ICR mice via collagenase digestion of the exocrine pancreas, as described previously (Rackham et al. 2016 (link)). Isolated islets were subsequently maintained at 37°C in RPMI (Sigma) supplemented with 10% (vol/vol) foetal bovine serum, 2 mmol/L glutamine and 100 U/mL penicillin/0.1 mg/mL streptomycin for 24 h before use. Islets were loaded into a multi-channel, temperature-controlled perifusion system, as described previously (Liu et al. 2013 (link)), and pre-perifused for 1 h with physiological salt buffer (Bowe et al. 2019 (link)) containing 2 mmol/L glucose before being exposed to 20 mmol/L glucose in the presence or absence of the CRHR agonists, CRH (50 nmol/L, Sigma), stressin I (100 nmol/L, Tocris) or UCN2 (100 nmol/L, Sigma) at 37°C. Perifusate samples were collected every 2 min and insulin secretion was quantified using an in-house insulin RIA (Jones et al. 1988 (link)).