OBJECTIVE: Phosphatidylinositol 3-OH kinase (PI3K) has a long-recognized role in beta-cell mass regulation and gene transcription and is implicated in the modulation of insulin secretion. The role of nontyrosine kinase receptor-activated PI3K isoforms is largely unexplored. We therefore investigated the role of the G-protein-coupled PI3Kgamma and its catalytic subunit p110gamma in the regulation of insulin granule recruitment and exocytosis.
RESEARCH DESIGN AND METHODS: The expression of p110gamma was knocked down by small-interfering RNA, and p110gamma activity was selectively inhibited with AS605240 (40 nmol/l). Exocytosis and granule recruitment was monitored by islet perifusion, whole-cell capacitance, tot... More
OBJECTIVE: Phosphatidylinositol 3-OH kinase (PI3K) has a long-recognized role in beta-cell mass regulation and gene transcription and is implicated in the modulation of insulin secretion. The role of nontyrosine kinase receptor-activated PI3K isoforms is largely unexplored. We therefore investigated the role of the G-protein-coupled PI3Kgamma and its catalytic subunit p110gamma in the regulation of insulin granule recruitment and exocytosis.
RESEARCH DESIGN AND METHODS: The expression of p110gamma was knocked down by small-interfering RNA, and p110gamma activity was selectively inhibited with AS605240 (40 nmol/l). Exocytosis and granule recruitment was monitored by islet perifusion, whole-cell capacitance, total internal reflection fluorescence microscopy, and electron microscopy in INS-1 and human beta-cells. Cortical F-actin was examined in INS-1 cells and human islets and in mouse beta-cells lacking the phosphatase and tensin homolog (PTEN).
RESULTS: Knockdown or inhibition of p110gamma markedly blunted depolarization-induced insulin secretion and exocytosis and ablated the exocytotic response to direct Ca(2+) infusion. This resulted from reduced granule localization to the plasma membrane and was associated with increased cortical F-actin. Inhibition of p110gamma had no effect on F-actin in beta-cells lacking PTEN. Finally, the effect of p110gamma inhibition on granule localization and exocytosis could be rapidly reversed by agents that promote actin depolymerization.
CONCLUSIONS: The G-protein-coupled PI3Kgamma 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, p110gamma activity plays an important role in maintaining a membrane-docked, readily releasable pool of secretory granules in insulinoma and human beta-cells.
