Cell-Specific Ca2+ Response in Pancreatic ß-cells

Abstract

Pancreatic ß-cells are heterogeneous in their secretory responsiveness, glucose sensitivity and metabolic rate. A diminished and delayed first-phase insulin release is an early sign of failing ß-cells in diabetes. Mechanisms controlling functional characteristics, such as lag time for insulin release or magnitude of the response in each individual cell are unknown. To find out whether the heterogeneity represents a random phenomenon in ß-cell or is a manifestation of reproducible characteristics, we compared parameters of Ca2+ response in Fura-2 labelled ob/ob mouse ß-cells during two consecutive stimulations with glucose. Lag times, as well as peak heights and nadirs of initial lowering showed a strong correlation between the first and second stimulation. Thus, timing and magnitude of the early Ca2+ response were specific for each cell. ß-Cells from lean mice, diabetic db/db mice and rats also showed cell-specific responses characteristics. This indicates that a cell-specific Ca2+ response to glucose is common in rodent ß-cells, both normal and diabetic. Another question was whether aggregated ß-cells show cell-specific responses. Using the same protocol as for dispersed ß-cells, we analysed Ca2+ responses in clusters of different size and in intact islets from ob/ob and lean mice. Correlations were found between the first and second stimulation for timing and magnitude of [Ca2+]i rise, and for the initial lowering. Next, we tested if the ß-cell response is cell-specific, when induced at different steps of the stimulus-secretion coupling. The glycolytic intermediate glyceraldehyde, the mitochondrial substrate KIC, the KATP-channel blocker tolbutamide and arginine were used as tools. [Ca2+]i changes were studied in dispersed ß-cells from lean, ob/ob and db/db mice. NADH responses to glucose and KIC were analyzed as a measure of metabolic flux. The correlation between Ca2+ and insulin response from individual ß-cells was tested using Fluo-3 and Fluozin-3. Both timing and magnitude of calcium responses were cell-specific in lean mouse ß-cells with all tested secretagogues. ß-Cells from ob/ob and db/db mice showed cell-specific timing of Ca2+ responses to glyceraldehyde but not to KIC, tolbutamide or arginine. However, ob/ob mouse ß-cells within intact islets showed cell-specific timing of tolbutamide-induced response. NADH responses to glucose were cell-specific in all three mouse models, but the timing of NADH responses to KIC was cell-specific only in lean mice. Thus, a cell-specific response can be induced in normal ß-cells at several steps of stimulus-secretion coupling for nutrient-stimulated insulin release. Cell-specific properties of ß-cell ion channels and the mitochondrial metabolism are affected in db/db and ob/ob mice. The relation between mitochondrial mass and parameters of Ca2+ responses were investigated in Mitotracker Red and Fluo-3 labelled ß-cells using confocal microscopy. Data show that ß-cell mitochondrial state may play an important role in determining the timing of [Ca2+]i changes. In summary, the early Ca2+ response pattern in ß-cells, including the lag time, the nadir of initial lowering and the height of the first peak response is cell-specific. Isolated and functionally coupled ß-cells show cell-specific timing of Ca2+ responses when stimulated with metabolic and non-metabolic agents. This may be a robust mechanism of importance for the adequate function of ß-cells and a basis for the pacemaker function of some cells. A disturbed cell specificity of the mitochondrial metabolism and ion channel function appears to be a marker of ß-cell dysfunction in hyperglycemia and diabetes and may explain the delayed insulin release in ß-cells from diabetic subjects