Dynamics of Insulin Secretion from EndoC-βH1 β-Cell Pseudoislets in Response to Glucose and Other Nutrient and Nonnutrient Secretagogues

The dynamics of insulin secretion were characterized in response to a variety of physiological and pharmacological stimulators and other compounds in perifused pseudoislets generated from cells of the EndoC-βH1 β-cell line. Perifusion of EndoC-βH1 pseudoislets with the physiological stimulus glucose (16.7 mM) induced sustained insulin secretion, which was inhibited by mannoheptulose. The adenylate cyclase activators IBMX and forskolin strongly potentiated this secretion. Glibenclamide, a Kir 6.2 potassium channel blocker, and Bay K 8644, an opener of the voltage-sensitive Ca2+ channel, also potentiated glucose-induced insulin secretion. The dynamics of insulin secretion from EndoC-βH1 pseudoislets were characterized by an insulin secretory response to glucose starting within 1-2 min and passing over without interruption into a sustained phase of insulin release for the whole stimulation period. This lack of a transient decline between the first and the second phases of insulin release is an indication for a quick supply of insulin secretory granules from the reserve pool to the docking sites below the plasma membrane. Thereby, new secretory granules are directly made available for sustained exocytosis of insulin in EndoC-βH1 β-cells. The study shows that EndoC-βH1 β-cell pseudoislets are well suited for kinetic analyses of insulin secretion.

Comparison of the effects of perchlorate and Bay K 8644 on the dynamics of cytoplasmic Ca2+ concentration and insulin secretion in mouse β-cells

Non-inbred ob/ob mice were used to study the dynamics of cytoplasmic Ca2+ concentration ([Ca2+]i) in isolated pancreatic β-cells using microfluorimetry with fura 2/AM as probe, and the dynamics of insulin secretion in isolated pancreatic islets. D-Glucose (20 mM) caused a transient peak increase in [Ca2+]i which changed to either an oscillating or a flat, elevated phase. The lag-time before the first peak increase in [Ca2+]i was markedly shortened by 12 mM ClO4- and the glucose-stimulated level of [Ca2+]i after the first peak was clearly elevated by the anion. ClO4- also amplified K+-stimulated (20 mM) [Ca2+]i. ClO4- did not change the basal [Ca2+]i at 3 mM glucose. Extracellular Ca2+ deficiency abolished the effect of high glucose and ClO4- on [Ca2+]i. This suggests that ClO4- acts as an amplifier of transmembrane Ca2+ inflow. The L-type Ca2+ channel agonist, Bay K 8644 (0.01–1.0 μM), strictly reproduced all the effects of perchlorate on the glucose-stimulated β-cell [Ca2+]i. Both phases of insulin release (20 mM glucose) were markedly enhanced by ClO4- (12 mM) or Bay K 8644 (1.0 μM). The lag-time for glucose-stimulated insulin release was shortened by both agents. Taken together, these data strengthen the idea that perchlorate amplifies the glucose-stimulation of [Ca2+]i and insulin release by directly modifying the function of the L-type Ca2+ channel. This effect can induce both a more prompt onset of and an amplified level of β-cell secretory activity.