Abstract
Impairment in beta-cell exocytosis is associated with reduced insulin secretion and diabetes. Here we aimed to investigate the dynamics of Ca2+-dependent insulin exocytosis with respect to pool depletion and Ca2+-current inactivation. We studied exocytosis, measured as increase in membrane capacitance (ΔCm), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis. The observed linear relationship between ΔCm and Q suggests that Ca2+-channel inactivation rather than granule pool restrictions is responsible for the decline in exocytosis observed at longer depolarizations. INS-1 832/13 cells possess an immediately releasable pool (IRP) of ∼10 granules and most exocytosis of granules occurs from a large pool. The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected. These findings suggest that most insulin release occurs away from Ca2+-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.
Highlights
Insulin is secreted from the pancreatic beta-cells following an increase in glucose concentration to mediate uptake of glucose into target tissue
Influx does not deviate in presence of ethylene glycol tetraacetic acid (EGTA) First, we were interested in investigating kinetics of exocytosis using an experimental setting used previously [8,33]
It might be that the biphasic pattern of the increase in membrane capacitance is caused by current inactivation rather than immediately releasable pool (IRP) depletion, and to investigate this question one should relate DCm to Q rather than to pulse length [18,22]
Summary
Insulin is secreted from the pancreatic beta-cells following an increase in glucose concentration to mediate uptake of glucose into target tissue. Failure of the beta-cells to release enough insulin is an important factor in the development of type-2 diabetes. Recent genetic data have demonstrated that the expression of genes involved in the final steps of insulin secretion is reduced in patients with type-2 diabetes [1,2]. These final steps include influx of Ca2+ through voltage-dependent Ca2+ channels triggering exocytosis of insulin containing granules and release of insulin [3]. Four of the top genetic risk variants associated with human type-2 diabetes is associated with reduced exocytosis [4]. A better understanding of the exocytotic process will have important clinical implications
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