Insulin granule recruitment and exocytosis is dependent on p110γ in insulinoma and human β-cells

Abstract

Objective: Phosphatidylinositol 3-OH kinase (PI3K) has a long recognized role in β-cell mass regulation and gene transcription and is implicated in the modulation of insulin secretion. The role of non-tyrosine kinase receptor activated PI3K isoforms is largely unexplored. We therefore investigated the role of the G-protein coupled PI3K (p110γ) in the regulation of insulin granule recruitment and exocytosis.

Research Design and Methods: The expression of p110γ was knocked-down by siRNA and p110γ activity was selectively inhibited with AS605240 (40 nM). Exocytosis and granule recruitment was monitored by islet perifusion, whole-cell capacitance, total internal reflection fluorescence (TIRF) microscopy and electron microscopy in INS-1 and human β-cells. Cortical F-actin was examined in INS-1 cells and human islets, and in mouse β-cells lacking the phosphatase and tensin homolog (PTEN).

Results: Knock-down or inhibition of p110γ markedly blunted depolarization-induced insulin secretion and exocytosis, and ablated the exocytotic response to direct Ca²⁺ infusion. This resulted from reduced granule localization to the plasma membrane and was associated with increased cortical F-actin. Inhibition of p110γ had no effect on F-actin in β-cells lacking PTEN. Finally, the effect of p110γ inhibition on granule localization and exocytosis could be rapidly reversed by agents that promote actin de-polymerization.

Conclusions: The G-protein coupled PI3K-γ is an important determinant of secretory granule trafficking to the plasma membrane, at least in part through the negative regulation of cortical F-actin. Thus, p110γ activity plays an important role in maintaining a membrane-docked readily releasable pool of secretory granules in insulinoma and human β-cells. 248 words