Bao Shunzhong, Jin Chun, Zhang Sheng, Turk John, Ma Zhongmin, Ramanadham Sasanka
Department of Medicine, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Diabetes. 2004 Feb;53 Suppl 1(0 1):S186-9. doi: 10.2337/diabetes.53.2007.s186.
Evidence that group VIA cytosolic calcium-independent phospholipase A(2) (iPLA(2)beta) participates in beta-cell signal transduction includes the observations that inhibition of iPLA(2)beta with the bromoenol lactone suicide substrate suppresses glucose-stimulated insulin secretion and that overexpression of iPLA(2)beta amplifies insulin secretory responses in INS-1 insulinoma cells. Immunofluorescence analyses also reveal that iPLA(2)beta accumulates in the perinuclear region of INS-1 cells stimulated with glucose and forskolin. To characterize this phenomenon further, iPLA(2)beta was expressed as a fusion protein with enhanced green fluorescent protein (EGFP) in INS-1 cells so that movements of iPLA(2)beta are reflected by changes in the subcellular distribution of green fluorescence. Stimulation of INS-1 cells overexpressing iPLA(2)beta-EGFP induced greater insulin secretion and punctate accumulation of iPLA(2)beta-EGFP fluorescence in the perinuclear region. To determine the identity of organelles with which iPLA(2)beta might associate, colocalization of green fluorescence with fluorophores associated with specific trackers targeted to different subcellular organelles was examined. Such analyses reveal association of iPLA(2)beta-EGFP fluorescence with the ER and Golgi compartments. Arachidonate-containing plasmenylethanolamine phospholipid species are abundant in beta-cell endoplasmic reticulum (ER) and are excellent substrates for iPLA(2)beta. Arachidonic acid produced by iPLA(2)beta-catalyzed hydrolysis of their substrates induces release of Ca(2+) from ER stores-an event thought to participate in glucose-stimulated insulin secretion.
ⅥA 族胞质钙非依赖性磷脂酶 A2（iPLA2β）参与β细胞信号转导的证据包括以下观察结果：用溴代烯醇内酯自杀底物抑制 iPLA2β可抑制葡萄糖刺激的胰岛素分泌，而 iPLA2β的过表达可增强 INS-1 胰岛素瘤细胞中的胰岛素分泌反应。免疫荧光分析还显示，iPLA2β在葡萄糖和福斯可林刺激的 INS-1 细胞的核周区域积聚。为了进一步表征这一现象，iPLA2β在 INS-1 细胞中作为与增强型绿色荧光蛋白（EGFP）的融合蛋白表达，从而 iPLA2β的运动通过绿色荧光的亚细胞分布变化得以反映。对过表达 iPLA2β-EGFP 的 INS-1 细胞的刺激诱导了更多的胰岛素分泌以及 iPLA2β-EGFP 荧光在核周区域的点状积聚。为了确定 iPLA2β可能与之相关的细胞器的身份，研究了绿色荧光与靶向不同亚细胞器的特定追踪剂相关的荧光团的共定位。此类分析揭示了 iPLA2β-EGFP 荧光与内质网（ER）和高尔基体区室的关联。含花生四烯酸的缩醛磷脂酰乙醇胺磷脂种类在β细胞内质网（ER）中丰富，并且是 iPLA2β的优良底物。iPLA2β催化其底物水解产生的花生四烯酸诱导内质网储存的 Ca2+释放——这一事件被认为参与葡萄糖刺激的胰岛素分泌。
