A buffer temperature controlled perifusion system to study temperature dependence and kinetics of insulin secretion in MIN6 pseudoislets

Introduction; The perifusion of pancreatic islets is a well-known method to investigate the kinetics of insulin secretion. Nevertheless, little interest has been attributed to a precise temperature control in perifusion systems. Insulin secretion from MIN6 cells, cultured as monolayers, differs substantially from pancreatic islets, at least partly due to missing β-to-β cell contacts. These cellular contacts are abundant in MIN6 pseudoislets, which show a more pronounced glucose-induced insulin release. Here, a perifusion system that directly and dynamically controls the perifusion buffer temperature inside the reaction chamber is described. Additionally, the influence of small temperature changes, glucagon-like peptide 1 (GLP-1) and tolbutamide on insulin release from MIN6 pseudoislets is examined. Methods: MIN6 cells were cultured in suspension culture dishes to generate MIN6 pseudoislets. The pseudoislets were perifused using a newly developed 12-channel perifusion system. The buffer temperature inside the reaction chambers was dynamically controlled by a programmable proportional plus integral plus differential (PID) controller. Insulin was determined by radioimmunoassay. Results; After adjusting the PID controller, the temperature inside the reaction chambers was constant in a very narrow range. The first phase of the glucose-induced insulin secretion was enhanced from 1.0±0.1 to 2.8±0.2 ng insulin/ml and the second phase from 5.4±0.9 to 17.8±1.3 ng insulin/ml, when the temperature was elevated by 1 °C, from 37 to 38 °C. GLP-1 concentration dependently increased insulin release at 15.0 mM and was ineffective at 0.0 mM glucose. Tolbutamide induced a concentration-dependent increase in both phases of the insulin secretion. Discussion: MIN6 pseudoislets are a useful tool to study insulin secretion from β-cells, which are arranged in clusters like pancreatic β-cells in the islet. The strong influence of temperature on insulin release from these pseudoislets requires a perifusion system, which precisely controls the buffer temperature.