Abstract

Evidence that group VIA cytosolic calcium-independent phospholipase A₂ (iPLA₂β) participates in β-cell signal transduction includes the observations that inhibition of iPLA₂β with the bromoenol lactone suicide substrate suppresses glucose-stimulated insulin secretion and that overexpression of iPLA₂β amplifies insulin secretory responses in INS-1 insulinoma cells. Immunofluorescence analyses also reveal that iPLA₂β accumulates in the perinuclear region of INS-1 cells stimulated with glucose and forskolin. To characterize this phenomenon further, iPLA₂β was expressed as a fusion protein with enhanced green fluorescent protein (EGFP) in INS-1 cells so that movements of iPLA₂β are reflected by changes in the subcellular distribution of green fluorescence. Stimulation of INS-1 cells overexpressing iPLA₂β-EGFP induced greater insulin secretion and punctate accumulation of iPLA₂β-EGFP fluorescence in the perinuclear region. To determine the identity of organelles with which iPLA₂β might associate, colocalization of green fluorescence with fluorophores associated with specific trackers targeted to different subcellular organelles was examined. Such analyses reveal association of iPLA₂β-EGFP fluorescence with the ER and Golgi compartments. Arachidonate-containing plasmenylethanolamine phospholipid species are abundant in β-cell endoplasmic reticulum (ER) and are excellent substrates for iPLA₂β. Arachidonic acid produced by iPLA₂β-catalyzed hydrolysis of their substrates induces release of Ca²⁺ from ER stores—an event thought to participate in glucose-stimulated insulin secretion.