Rapid Turnover of Phosphatidylinositol-4,5-Bisphosphate in Insulin-Secreting Cells Mediated by Ca²⁺ and the ATP-to-ADP Ratio

Abstract

Phosphatidylinositol-4,5-bisphosphate (PIP₂) is important for a variety of cellular processes as a precursor for second messengers and by regulating ion channels, the cytoskeleton, and vesicle traffic in many types of cells, including insulin-secreting β-cells. Here, we applied evanescent wave microscopy and the PIP₂-binding pleckstrin homology domain from phospholipase C (PLC)-δ fused to the green fluorescent protein to characterize the regulation of plasma membrane PIP₂ in individual insulin-secreting MIN6 β-cells. Elevation of the glucose concentration from 3 to 11 mmol/l evoked antisynchronous oscillations of [PIP₂] and cytoplasmic Ca²⁺concentration, consistent with PLC being periodically activated by the voltage-dependent Ca²⁺ influx. The effect of adenine nucleotides on [PIP₂] was studied in cells permeabilized with α-toxin. ATP dose- dependently stimulated PIP₂ synthesis with half-maximal effect at 300 μmol/l. Omission of the nucleotide resulted in rapid loss of PIP₂ with t_1/2 < 40 s. ADP also stimulated PIP₂ formation, but this effect reflected local ATP formation and was prevented by the adenylate kinase inhibitor diadenosine-pentaphosphate. The ATP-induced PIP₂ synthesis was counteracted by the ADP analog adenosine-5′-O-2-thiodiphosphate. We conclude that plasma membrane PIP₂ is dynamically regulated by intracellular Ca²⁺ and the ATP-to-ADP ratio in insulin-secreting cells. The rapid turnover allows maintenance of PIP₂ levels while generating second messengers of critical importance for insulin secretion.