A buffer temperature controlled perifusion system to study temperature dependence and kinetics of insulin secretion in MIN6 pseudoislets.
BACKGROUND
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 beta-to-beta 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 degrees C, from 37 to 38 degrees 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.


CONCLUSIONS
MIN6 pseudoislets are a useful tool to study insulin secretion from beta-cells, which are arranged in clusters like pancreatic beta-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.
Brenner
Martin B
MB
Lilly Research Laboratories, Essener Strasse 93, D-22419 Hamburg, Germany.
Mest
Hans-Juergen
HJ
D000818:Animals; D019898:Autocrine Communication / Q000502:physiology; D002021:Buffers; D002460:Cell Line; D015194:Diffusion Chambers, Culture; D005934:Glucagon / Q000494:pharmacology; D052216:Glucagon-Like Peptide 1; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D010446:Peptide Fragments / Q000494:pharmacology; D010477:Perfusion / Q000379:methods*; D011498:Protein Precursors / Q000494:pharmacology; D011863:Radioimmunoassay; D013696:Temperature; D014044:Tolbutamide / Q000494:pharmacology
D002021:Buffers; D007004:Hypoglycemic Agents; D007328:Insulin; D010446:Peptide Fragments; D011498:Protein Precursors; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D014044:Tolbutamide