Islets were isolated as previously described (22 (link)). After pancreatic digestion, islets were purified using a Ficoll-paque (GE Healthcare, Chalfont St. Giles, U.K.) gradient before overnight culture in Dulbecco's modified Eagle's medium with 11 mmol/l glucose and 10% FCS (Invitrogen, PAISLEY, U.K.). Islets were cultured for a further 48–72 h (chronic culture) with additional 0.4 mmol/l palmitate coupled to 0.92% BSA (lipid) or BSA alone prior to study (18 (link)).
For insulin secretion assays, islets were preincubated for 1 h in Krebs-Ringer buffer containing HEPES (KRBH) containing 0.1% BSA and 2 mmol/l glucose. Batches of five islets were incubated at 37°C for 1 h in 130 μl KRBH containing 0.1% BSA and 2 mmol/l glucose (basal) supplemented with glucose (20 mmol/l) or other additions, as indicated in the text. For inhibition of lipolysis, orlistat (Sigma, St. Louis, MO), used at 200 μmol/l (unless otherwise stated), or vehicle (0.52% DMSO) was included in the KRBH throughout the insulin secretion experiment, but not during chronic culture. Insulin release was determined by radioimmunoassay (Linco/Millipore, Billerica, MA).
For insulin secretion assays, islets were preincubated for 1 h in Krebs-Ringer buffer containing HEPES (KRBH) containing 0.1% BSA and 2 mmol/l glucose. Batches of five islets were incubated at 37°C for 1 h in 130 μl KRBH containing 0.1% BSA and 2 mmol/l glucose (basal) supplemented with glucose (20 mmol/l) or other additions, as indicated in the text. For inhibition of lipolysis, orlistat (Sigma, St. Louis, MO), used at 200 μmol/l (unless otherwise stated), or vehicle (0.52% DMSO) was included in the KRBH throughout the insulin secretion experiment, but not during chronic culture. Insulin release was determined by radioimmunoassay (Linco/Millipore, Billerica, MA).
