LIPID MESSENGERS FROM A PHOSPHOLIPASE A2 ENZYME AND BETA CELL BIOLOGY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。在撰写这篇回顾文章的《糖尿病视角》时，我们最近发现了胰岛中磷脂酶A2 （PLA2）的活性，我们认为这可能整合了我们和R. Paul Robertson、Stewart Metz、Marjorie Dunlop、Claes Wollheim以及其他许多人在原始视角(1)中引用的关于胰岛中葡萄糖诱导磷脂水解的观察结果。这些观察结果包括，胰岛暴露于刺激胰岛素分泌的d -葡萄糖浓度下，导致磷脂水解、未酯化花生四烯酸积累和花生四烯酸氧合产物的产生。葡萄糖诱导的磷脂水解需要葡萄糖代谢，但部分独立于Ca2+内流，而积累的花生四烯酸似乎通过促进Ca2+从细胞外空间进入和诱导Ca2+从细胞内封存位点释放来放大葡萄糖诱导的细胞[Ca2+]升高(1)。与最初在心肌细胞质中发现的类似活性一样，与其他已知的PLA2酶不同，这种胰岛PLA2活性不需要Ca2+催化活性，由ATP激活，并且对溴烯醇内酯（BEL）自杀底物的抑制敏感，该底物在相同浓度下不抑制其他PLA2酶。此外，BEL被发现抑制葡萄糖诱导的花生四烯酸释放、胰岛素分泌和细胞胞浆[Ca2+]的升高，这表明PLA2可能是细胞燃料传感器装置的一个组成部分(1)。随后，胰岛素瘤细胞也被发现表达PLA2活性，这有助于其色谱分析和纯化(2)。这种细胞活性的表征最终导致从大鼠胰岛cDNA文库中克隆出一个84 kDa的蛋白，该蛋白包含GXSXG丝氨酸脂肪酶一致序列和8段重复基序，与锚蛋白的整体膜蛋白结合域相似(3)。重组蛋白表现出PLA2活性，这种酶现在被归类为VIA PLA2，并被命名为iPLA2¿(4)。后来发现人类胰岛表达了编码iPLA2¿两种不同亚型的mRNA物种，这两种亚型是由选择性剪接的外显子跳跃机制产生的(4)，胰岛素瘤细胞中BEL的药理学抑制研究支持iPLA2¿在胰岛素分泌的磷脂水解事件中发挥作用，但未能提供证据证明iPLA2¿在花生四烯酸掺入磷脂中发挥“清洁”作用(5)，这在其他细胞中已被提出。由于BEL除抑制iPLA2¿外还抑制多种酶，因此对iPLA2¿活性的分子生物学操作为药理学研究提供了重要的补充。用含有iPLA2¿cDNA(6)或小干扰RNA(7)的逆转录病毒载体稳定转染培养的胰岛素瘤细胞系，其iPLA2¿活性水平分别比用空载体转染的细胞高或低几倍。对这些细胞系的研究也支持iPLA2在胰岛素分泌中发挥作用，但在细胞磷脂重塑中不起作用（6,7）。这些细胞系还表现出与iPLA2¿表达水平相关的其他特性，包括在过度表达iPLA2¿的细胞中增殖率增加(8)，在iPLA2¿表达受到抑制的细胞中增殖率降低(7)。此外，过度表达iPLA2¿的胰岛素瘤细胞对引起内质网应激的药物诱导凋亡的敏感性增加(9)，这表明iPLA2¿可能参与多种复杂的细胞生物学过程，这些过程可能在细胞之间或特定细胞内根据特定情况而有所不同。与这种可能性相一致的是，一些由mRNA的选择性剪接产生的iPLA2¿同种异构体现在被识别出来(4)，同样也被识别出来的是由蛋白水解加工产生的同种异构体（10）。在¿-细胞中，主要的异构体是通过蛋白水解去除c端氨基酸序列的14 kDa，以产生催化活性的70 kDa的参与信号传导的蛋白（10）。蛋白-蛋白相互作用和iPLA2¿的亚细胞位置也会影响其作用的后果，细胞中一个主要的相互作用蛋白是Ca2+/钙调素依赖性蛋白激酶II¿，它在细胞Ca2+信号传导中起重要作用（11）。升高cAMP和增加胰岛素分泌的药物也能诱导iPLA2¿的亚细胞再分配(4)。操纵iPLA2¿在整个动物中的表达水平也可以深入了解其潜在的生物学功能。早期对转基因小鼠的研究表明，与野生型小鼠相比，这些小鼠的空腹和受刺激血糖水平较低，胰岛素水平较高（图1）。对同源重组破坏iPLA2¿基因的全敲除小鼠的代谢表型的表征也在进行中。雄性iPLA2¿敲除小鼠的生育能力大大降低，精子活力受损，这可能反映了细胞内[Ca2+]调节受损（12），并且这些小鼠的巨噬细胞在暴露于双链RNA病毒时不表现出诱导型一氧化氮合酶基因转录增加的正常反应（13），这表明iPLA2¿在这一过程中也起着信号传导作用。早期研究还表明，iPLA2基因敲除小鼠的死亡率低于野生型小鼠，这表明iPLA2基因可能会影响寿命（图2），最近也对其他人提出的iPLA2基因的其他作用进行了综述（14,15）。

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. When the Perspective in Diabetes that is the subject of this retrospective was written, we had recently identified a phospholipase A2 (PLA2) activity in pancreatic islets that we believed might integrate several observations about glucose-induced phospholipid hydrolysis in pancreatic islets that had resulted from our work and that of R. Paul Robertson, Stewart Metz, Marjorie Dunlop, Claes Wollheim, and many others cited in the original Perspective (1). Such observations included the facts that exposure of islets to concentrations of D-glucose that stimulated insulin secretion resulted in hydrolysis of phospholipids, accumulation of nonesterified arachidonic acid, and generation of arachidonate oxygenation products. Glucose-induced phospholipid hydrolysis required that glucose be metabolized but was in part independent of Ca2+ influx, and the arachidonic acid that accumulated appeared to amplify the glucose-induced rise in ¿-cell [Ca2+] by facilitating Ca2+ entry from the extracellular space and by inducing Ca2+ release from intracellular sequestration sites (1). Like a similar activity first recognized in myocardial cytosol but unlike other then recognized PLA2 enzymes, this islet PLA2 activity did not require Ca2+ for catalytic activity, was activated by ATP, and was sensitive to inhibition by a bromoenol lactone (BEL) suicide substrate that did not inhibit other PLA2 enzymes at comparable concentrations. Moreover, BEL was found to suppress glucose-induced arachidonate release, insulin secretion, and the rise in ¿-cell cytosolic [Ca2+], suggesting that this PLA2 might represent a component of the ¿-cell fuel sensor apparatus (1). Subsequently, insulinoma cells were also found to express this PLA2 activity, and this facilitated its chromatographic analysis and purification (2). Such characterization of the ¿-cell activity eventually resulted in the cloning from a rat islet cDNA library of an 84 kDa protein that contained a GXSXG serine lipase consensus sequence and 8 stretches of a repetitive motif similar to that in the integral membrane protein-binding domain of ankyrin (3). The recombinant protein exhibited PLA2 activity, and this enzyme is now classified as a group VIA PLA2 and given the trivial designation iPLA2¿ (4). Human islets were later found to express mRNA species encoding two distinct isoforms of iPLA2¿ that arise by an exon-skipping mechanism of alternative splicing (4), and pharmacologic inhibition studies with BEL in insulinoma cells supported a role for iPLA2¿ in phospholipid hydrolytic events in insulin secretion but failed to provide evidence that iPLA2¿ plays a housekeeping role in arachidonic acid incorporation into phospholipids (5) that had been suggested in other cells. Because BEL inhibits several enzymes in addition to iPLA2¿, molecular biologic manipulations of iPLA2¿ activity provide an important complement to pharmacologic studies. Cultured insulinoma cell lines generated by stable transfection with retroviral vectors containing either iPLA2¿ cDNA (6) or small interfering RNA (7) express several-fold higher or lower levels of iPLA2¿ activity, respectively, than do cells transfected with empty vectors. Studies with such cell lines also support a role for iPLA2¿ in insulin secretion but not in ¿-cell phospholipid remodeling (6, 7). Such cell lines also exhibit other properties that correlate with iPLA2¿ expression level, including increased proliferation rates in cells that overexpress iPLA2¿ (8) and reduced proliferation rates in cells in which iPLA2¿ expression is suppressed (7). Moreover, insulinoma cells that overexpress iPLA2¿ exhibit increased sensitivity to induction of apoptosis by agents that cause endoplasmic reticulum stress (9), suggesting that iPLA2¿ could participate in a complex variety of cell biologic processes that might differ among cells or within a given cell depending on specific circumstances. Consistent with that possibility, several iPLA2¿ isoforms that arise from alternative splicing of mRNA are now recognized (4), as are isoforms that arise from proteolytic processing (10). A predominant isoform in ¿-cells arises from proteolytic removal of 14 kDa of the C-terminal amino acid sequence to yield a catalytically active 70 kDa protein involved in signaling (10). Protein-protein interactions and the subcellular location of iPLA2¿ could also affect the consequences of its action, and a major interacting protein in ¿-cells is the Ca2+/calmodulin-dependent protein kinase II¿ that plays important role in ¿-cell Ca2+ signaling (11). Agents that elevate cAMP and amplify insulin secretion also induce subcellular redistribution of iPLA2¿ (4). Manipulation of iPLA2¿ expression level in whole animals can also provide insight into its potential biological functions. Early studies with transgenic mice that overexpress iPLA2¿ by several-fold specifically in ¿-cells indicate that these mice have lower fasting and stimulated blood glucose levels and higher insulin levels than do wild-type mice (Figure 1). Characterization of the metabolic phenotype(s) of global knockout mice homozygous for an iPLA2¿ gene disrupted by homologous recombination is also ongoing. Male iPLA2¿ knockout mice have greatly reduced fertility and impaired motility of spermatozoa that might reflect impaired intracellular [Ca2+] regulation (12), and macrophages from such mice do not exhibit the normal response of increasing transcription of the inducible nitric oxide synthase gene when exposed to double-stranded RNA virus (13), suggesting that iPLA2¿ also plays a signaling role in that process. Early studies also suggest that iPLA2¿ knockout mice have lower mortality rates than wild-type mice, suggesting that iPLA2¿ might affect longevity (Figure 2), and additional roles for iPLA2¿ proposed by others have recently been reviewed (14, 15).