Human islet culture
Human islets were obtained from the Nordic Network for Clinical Islet Transplantation (Uppsala University Hospital, Uppsala, Sweden). In total, human islets from 26 brain-dead non-diabetic donors were used in this study (age: 60.1 ± 2.0 years, male/female: 14/12; BMI: 27.4 ± 0.9 kg/m2, HbA1c: 5.5 ± 0.1 %). Human islets were cultured in CMRL 1066 medium (Invitrogen, Paisley, UK) containing 5.5 mmol/l glucose (Sigma, St. Louis, MO) and supplemented with 10 % fetal bovine serum (Invitrogen), 1 % glutamine (Invitrogen), 100 units/ml penicillin (Invitrogen) and 100 μg/ml streptomycin (Invitrogen) at 37 °C in humidified air containing 5 % CO2. Islets were used within 10 days after isolation.
Fatty acid preparation
Fatty acids were prepared as previously described . Briefly, 100 mmol/l stock solutions containing palmitate, stearate, or oleate (all from Sigma Aldrich, St. Louis, MO, USA) were prepared by dissolving fatty acids in 50 % ethanol. Stock solution of palmitoleate (Sigma Aldrich) was prepared in 100 % ethanol to a concentration of 200 mmol/l. Stock solutions were then diluted in incubation medium containing 5 mg/ml of fatty acid-free BSA (Boehringer Mannheim GmbH, Mannheim, Germany) to a final concentration of 0.5 mmol/l. Fatty acids were allowed to complex with BSA at 37 °C for at least 30 min.
Human islet perifusion and static incubation
Human islets were perifused as described previously . Briefly, 25–30 human islets were hand-picked and placed into a perifusion chamber. Islets were perifused for 60 min at 37 °C in KRBH buffer consisting of 130 mmol/l NaCl, 4.8 mmol/l KCl, 1.2 mmol/l MgSO4, 1.2 mmol/l KH2PO4, 1.2 mmol/l CaCl2, 5.0 mmol/l NaHCO3 and 5.0 mmol/l HEPES, titrated to pH 7.4 with NaOH and supplemented with 5 mg/ml fatty acid-free BSA and 5.5 mmol/l glucose or without glucose. After this initial perifusion period, samples were collected every 5 min for 20 min at the same concentration of glucose. This was followed by another 20-min perifusion with the same buffer containing 5 mg/ml fatty acid-free BSA and 0.5 mmol/l palmitate (16:0), palmitoleate (16:1), stearate (18:0), or oleate (18:1). In addition, 10 μM triacsin C (Sigma Aldrich) or 10 μM DC260126 (Tocris Bioscience, Bristol, UK) were added during the perifusion to inhibit long-chain fatty acyl CoA synthetase or FFAR1/GPR40 signaling, respectively. Perifusates were collected at 2, 4, 6, 8, 10, 15, and 20 min. The perifusion rate was 170 μl/min and collected perifusates were used to measure the amounts of secreted insulin. Insulin released during the first 6 min after the introduction of fatty acids was referred to as the first phase and that during the subsequent 14 min was regarded as the second phase.
Human islets were hand-picked in batches of 50 and statically incubated for 60 min in 0.5 ml KRBH buffer, identical to the one used for the perifusion, supplemented with 0.5 mmol/l of the different fatty acids and 5.5 mmol/l glucose or without glucose for 20 min.
The amount of insulin secreted was measured by enzyme-linked immunosorbent assay as described previously . For each perifusion, total fatty acid-induced insulin secretion was normalized to insulin secretion in the absence of the fatty acids. For static incubation total insulin secretion during 20 min was normalized to total protein content and insulin secretion was expressed as fold change between insulin secretion from fatty acid-treated and untreated islets for each donor.
Oxygen consumption and extracellular acidification rates
The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of isolated human pancreatic islets were determined by Extracellular Flux Analyzer XFe96 (Seahorse Bioscience, MA, USA) as previously reported . Ten hand-picked human islets were placed into the wells of the XFe96 cell culture microplate pre-coated with poly-D-lysine. Islets were pre-incubated with assay medium (Seahorse Bioscience) composed of 143 mmol/l NaCl, 5.4 mmol/l KCl, 0.91 mmol/l NaH2PO4, 0.8 mmol/l MgSO4, 1.8 mmol/l CaCl2, 2 mmol/l Glutamax, 3 mg/l Phenol Red, and supplemented with 5 mg/ml fatty acid-free BSA and 0.5 mmol/l palmitate (16:0), palmitoleate (16:1), stearate (18:0), or oleate (18:1) in the presence or absence of 5.5 mmol/l glucose (pH adjusted to 7.4) for 1 h at 37 °C before the microplate was inserted into the Analyzer. For each donor, 6–8 replicates of each treatment condition were used. OCR and ECAR were then measured in parallel for 40 min followed by the injection of 5 μmol/l rotenone and 5 μmol/l antimycin A to inhibit mitochondrial respiration. The remaining OCR was considered as non-mitochondrial respiration. To calculate the mitochondrial respiration, non-mitochondrial OCR was subtracted from the total OCR. Data were normalized to total islet area calculated by the Image J software (National Institutes of Health, USA) from pictures (40×) taken with camera (Olympus) mounted onto an inverted Olympus CKX41 microscope. Calculated basal OCR and ECAR were expressed as fold changes from fatty acid-treated islets compared with that from untreated islets for each experiment.
Human islets were washed with phosphate buffer saline (PBS) (Sigma) and lysed in PBS containing 1 % Triton-X 100 (Sigma). Total protein content in the lysates was determined by DC protein assay according to the manufacturer’s instructions (Bio-Rad Laboratories, USA).
Results were presented as means ± SEM. Statistical analysis was performed using GraphPad Prism Version 6.0b (GraphPad software, CA, USA). Statistical significance among several groups was analyzed by using one-way ANOVA followed by Holm-Sidak multiple comparison test. P < 0.05 was considered statistically significant.