STABLE SUPPRESSION OF IPLA2B EXPRESSION ON PHOSPHOLIPID CONTENT, INSULIN, INS-1

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 有关药物抑制或通过磷脂酶A2(IPLA2)表达一过性降低基团的研究表明，iPLA2是一种管家酶，调节细胞2-溶血磷脂酰胆碱(LPC)水平、花生四烯酸掺入磷脂的速率以及过量磷脂酰胆碱(PC)的降解。相反，在胰岛素分泌的胰岛细胞和其他一些细胞中，iPLA2的信号功能已经被提出。利用逆转录病毒载体，我们制备了克隆的INS-1细胞系，其中iPLA2的表达可被小干扰RNA稳定抑制。两个这样的iPLA2基因敲除(iPLA2？KD)细胞系表达的iPLA2不到对照INS-1细胞系的20%。IPLA2？-KD INS-1细胞表现出胰岛素分泌反应受损和增殖率降低。对花生四烯酸培养的INS-1细胞中PC和LPC的电喷雾电离质谱分析表明，18：0/20：4-甘油磷胆碱(GPC)的合成涉及sn-2重塑产生16：0/20：4-GPC，然后通过1-lyso/20：4-GPC中间体重塑sn-1。ESI/MS分析还表明，iPLA2？-KD和对照INS-1细胞的PC和LPC的含量和组成几乎相同，花生四烯酸掺入PC的速率以及其他磷脂类的组成和重塑也是如此。这些发现表明，iPLA2在细胞分泌和增殖中起信号或效应作用，但稳定抑制其表达并不影响细胞GPC的脂质含量或组成，即使在LPC被转化为PC而被积极消耗的情况下也是如此。这让人质疑拟议的iPLA2在PC动态平衡和重塑中的管家功能的普遍性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Studies involving pharmacologic inhibition or transient reduction of Group VIA Phospholipase A2 (iPLA2¿) expression have suggested that it is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels, rates of arachidonate incorporation into phospholipids, and degradation of excess phosphatidylcholine (PC). In insulin-secreting islet ¿-cells and some other cells, in contrast, iPLA2¿ signaling functions have been proposed. Using retroviral vectors, we prepared clonal INS-1 ¿-cell lines in which iPLA2¿ expression is stably suppressed by small interfering RNA. Two such iPLA2¿-knockdown (iPLA2¿-KD) cell lines express less than 20% of the iPLA2¿ of control INS-1 cell lines. The iPLA2¿-KD INS-1 cells exhibit impaired insulin secretory responses and reduced proliferation rates. Electrospray ionization mass spectrometric (ESI/MS) analyses of PC and LPC species that accumulate in INS-1 cells cultured with arachidonic acid suggest that 18:0/20:4-glycerophosphocholine (GPC) synthesis involves sn-2 remodeling to yield 16:0/20:4-GPC and then sn-1 remodeling via a 1-lyso/20:4-GPC intermediate. ESI/MS analyses also indicate that the PC and LPC content and composition of iPLA2¿-KD and control INS-1 cells are nearly identical, as are the rates of arachidonate incorporation into PC and the composition and remodeling of other phospholipid classes. These findings indicate that iPLA2¿ plays signaling or effector roles in ¿-cell secretion and proliferation but that stable suppression of its expression does not affect ¿-cell GPC lipid content or composition even under conditions in which LPC is being actively consumed by conversion to PC. This calls into question the generality of proposed housekeeping functions for iPLA2¿ in PC homeostasis and remodeling.