Okadaic acid-induced decrease in the magnitude and efficacy of the Ca²⁺ signal in pancreatic β cells and inhibition of insulin secretion

Abstract

1. Phosphorylation by kinases and dephosphorylation by phosphatases markedly affect the biological activity of proteins involved in stimulus-response coupling. In this study, we have characterized the effects of okadaic acid, an inhibitor of protein phosphatases 1 and 2A, on insulin secretion. Mouse pancreatic islets were preincubated for 60 min in the presence of okadaic acid before their function was studied.

2. Okadaic acid dose-dependently (IC₅₀∼200 nM) inhibited insulin secretion induced by 15 mM glucose. At 0.5 μM, okadaic acid also inhibited insulin secretion induced by tolbutamide, ketoisocaproate and high K⁺, and its effects were not reversed by activation of protein kinases A or C.

3. The inhibition of insulin secretion did not result from an alteration of glucose metabolism (estimated by the fluorescence of endogenous pyridine nucleotides) or a lowering of the ATP/ADP ratio in the islets.

4. Okadaic acid treatment slightly inhibited voltage-dependent Ca²⁺ currents in β cells (perforated patch technique), which diminished the rise in cytoplasmic Ca²⁺ (fura-2 method) that glucose and high K⁺ produce in islets. However, this decrease (25%), was insufficient to explain the corresponding inhibition of insulin secretion (90%). Moreover, mobilization of intracellular Ca²⁺ by acetylcholine was barely affected by okadaic acid, whereas the concomitant insulin response was decreased by 85%.

5. Calyculin A, another inhibitor of protein phosphatases 1 and 2A largely mimicked the effects of okadaic acid, whereas 1-norokadaone, an inactive analogue of okadaic acid on phosphatases, did not alter β cell function.

6. In conclusion, okadaic acid inhibits insulin secretion by decreasing the magnitude of the Ca²⁺ signal in β cells and its efficacy on exocytosis. The results suggest that, contrary to current concepts, both phosphorylation and dephosphorylation of certain β cell proteins may be involved in the regulation of insulin secretion.

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