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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。涉及VIA族磷脂酶A2（iPLA 2？）表达的药理学抑制或瞬时降低的研究表明，它是一种管家酶，可调节细胞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，在分泌胰岛素的胰岛细胞和其他一些细胞中，已经提出了iPLA 2信号功能。使用逆转录病毒载体，我们制备了克隆INS-1-细胞系，其中iPLA 2表达被小干扰RNA稳定抑制。两种这样的iPLA 2 <$-敲低（iPLA 2 <$-KD）细胞系表达的iPLA 2 <$不到对照INS-1细胞系的20%。iPLA 2 <$-KD INS-1细胞表现出受损的胰岛素分泌反应和降低的增殖率。在用花生四烯酸培养的INS-1细胞中积累的PC和LPC物质的电喷雾电离质谱（ESI/MS）分析表明，18：0/20：4-甘油磷酸胆碱（GPC）合成涉及sn-2重塑以产生16：0/20：4-GPC，然后通过1-lyso/20：4-GPC中间体进行sn-1重塑。ESI/MS分析还表明，iPLA 2 <$-KD和对照INS-1细胞的PC和LPC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几乎相同。这些发现表明，iPLA 2在细胞分泌和增殖中起信号或效应作用，但即使在LPC通过转化为PC而被积极消耗的条件下，其表达的稳定抑制也不会影响细胞GPC脂质含量或组成。这就质疑了iPLA 2在PC稳态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.