Stimulus-secretion coupling in pancreatic β-cells of healthy and diabetic rats in tissue slice preparation

A primary event in the development of type 2 diabetes is a decrease in the capacity of pancreatic β-cells to secrete insulin in response to glucose stimulation. The Goto Kakizaki (GK) rat is a widely used animal model to study this defective glucose-stimulated insulin release. Similar as recently shown for pancreatic islets of type 2 diabetic patients, the expression of several proteins involved in Ca2+-dependent exocytosis of insulin-containing large dense-core vesicles (LDCVs) is dysregulated in this animal model. Unexpectedly, previous studies failed to demonstrate a defect in late, Ca2+-dependent steps of insulin secretion. These studies, however, either lacked the temporal and spatial resolution to reveal subtle kinetic alterations of LDCV release, or were performed in vitro on isolated β-cells in primary cell culture, an invasive preparation that is known to alter the secretory function of β-cells.To resolve the apparent contradiction between biochemical data and the physiological studies performed so far we established a novel tissue slice preparation of rat pancreas. With its higher degree of preservation of cellular interaction this preparation resembles more closely the in vivo situation of pancreatic physiology. We studied the coupling of stimulus-induced Ca2+ influx and insulin secretion of healthy and diabetic rat β-cells by assessing exocytosis with high time-resolution measurements of membrane capacitance and whole-cell currents.β-cells in fresh pancreatic tissue slices of GK rats responded to glucose stimulation with a normal oscillatory increase in the cytosolic Ca2+ concentration. However, secretion from GK rat β-cells was defective in spite of putatively compensatory mechanisms like prominently upregulated cell size and increased voltage-activated Ca2+ currents. This impairment presented itself as a depressed initial release rate in response to trains of depolarizing pulses and a reduction in the efficacy of Ca2+ to trigger secretion. This was neither due to a decrease of functional vesicle pool sizes nor due to different kinetics of pool refilling. Unexpectedly, strong stimulation with two successive trains of depolarizing pulses led to a prominent facilitation of release in GK rat β-cells whereas secretion in controls was unaffected. The latter finding is in line with the paradoxical hypersecretion due to strongly depolarizing non-nutrient stimulation found in GK rats as well as individuals suffering from type 2 diabetes. Using a pharmacological approach to inhibit protein kinase activity in healthy and diabetic rat β-cells, our data furthermore indicates that the underlying cause for the observed phenomena in β-cells of diabetic GK rats might be chronically enhanced protein kinase C (PKC) activity. Broad-range inhibition of PKC increased the apparent Ca2+ sensitivity of exocytosis whereas it prevented the activity-dependent facilitation in GK rat β-cells.We conclude that a decrease in the apparent Ca2+ sensitivity of the GK rat β-cell release machinery is involved in defective glucose-stimulated insulin secretion of this animal model. Furthermore, we propose a role for constitutively increased activity of one ore more PKC isoenzymes in the diabetic phenotype of GK rat β-cells.