LIPID MESSENGERS FROM A PHOSPHOLIPASE A2 ENZYME AND BETA CELL BIOLOGY

来自磷脂酶 A2 酶和 β 细胞生物学的脂质信使

• 批准号: 7721463
• 负责人: JOHN W TURK
• 金额: $ 1.07万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721463
• 关键词: 6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; Alternative Splicing; Ankyrins; Arachidonic Acids; Beta Cell; Cell Line; Cells; Cellular biology; Cloning; Complement; Complementary DNA; Complex; Computer Retrieval of Information on Scientific Projects Database; Consensus Sequence; Cytosol; Diabetes Mellitus; Endoplasmic Reticulum; Enzymes; Event; Exhibits; Exons; Extracellular Space; Funding; Generations; Glucose; Grant; Housekeeping; Human; Hydrolysis; Induction of Apoptosis; Institution; Integral Membrane Protein; Islets of Langerhans; Lipase; Lipids; Messenger RNA; Molecular; Myocardial; Phospholipase A2; Phospholipids; Play; Process; Property; Protein Binding Domain; Protein Isoforms; Protein Overexpression; Proteins; Rate; Rattus; Recombinant Proteins; Research; Research Personnel; Resources; Retroviral Vector; Role; Serine; Site; Small Interfering RNA; Source; Stress; Stretching; Supporting Cell; Transfection; United States National Institutes of Health; Work; Writing; arachidonate; cDNA Library; enzyme activity; insulin secretion; insulinoma; islet; sensor; suicide substrates; vector

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在撰写本回顾性研究的主题《糖尿病展望》时，我们最近发现了胰岛中的磷脂酶A2（PLA 2）活性，我们认为这可能整合了我们和R. Paul Robertson、Stewart梅斯、Marjorie Dunlop、Claes Wollheim和许多其他人在最初的《观点》（1）中引用。 这些观察结果包括这样的事实，即暴露于刺激胰岛素分泌的D-葡萄糖浓度导致磷脂水解，非酯化花生四烯酸的积累和花生四烯酸氧化产物的产生。葡萄糖诱导的磷脂水解需要葡萄糖被代谢，但在一定程度上不依赖于Ca 2+内流，积累的花生四烯酸似乎通过促进Ca 2+从细胞外空间进入和诱导Ca 2+从细胞内螯合位点释放来放大葡萄糖诱导的细胞[Ca 2 +]升高（1）。 类似的活动首先在心肌胞质溶胶中识别，但不像其他然后识别的PLA 2酶，这种胰岛PLA 2活性不需要Ca 2+的催化活性，被ATP激活，并敏感的抑制溴烯醇内酯（BEL）自杀底物，不抑制其他PLA 2酶在可比浓度。此外，发现BEL抑制葡萄糖诱导的花生四烯酸释放、胰岛素分泌和<$-细胞胞质[Ca 2 +]的升高，这表明PLA 2可能代表<$-细胞燃料传感器装置的一个组件（1）。 随后，还发现胰岛素瘤细胞表达这种PLA 2活性，这有助于其色谱分析和纯化（2）。这种特征的？- 细胞活性最终导致从大鼠胰岛cDNA文库中克隆出84 kDa蛋白，该蛋白含有GXSXG丝氨酸脂肪酶共有序列和8段重复基序，该重复基序类似于锚蛋白的整合膜蛋白结合结构域中的重复基序（3）。重组蛋白表现出PLA 2活性，这种酶现在被归类为VIA PLA 2组，并被简单命名为iPLA 2 <$（4）。 后来发现人类胰岛表达编码两种不同亚型iPLA 2 <$的mRNA，这两种亚型是由选择性剪接的外显子跳跃机制产生的（4），在胰岛素瘤细胞中对BEL进行的药理学抑制研究支持iPLA 2 <$在胰岛素分泌中的磷脂水解事件中的作用，但未能提供iPLA 2 <$在胰岛素分泌中的作用的证据。在花生四烯酸掺入磷脂中起管家作用（5），这在其他细胞中已经被提出。 由于BEL除了抑制iPLA 2外还抑制几种酶，因此iPLA 2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iPLA 2 <$cDNA（6）或小干扰RNA（7）的逆转录病毒载体稳定转染培养的胰岛素瘤细胞系，其iPLA 2 <$活性水平分别比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iPLA 2在胰岛素分泌中的作用，但不在细胞磷脂重塑中（6，7）。 这些细胞系还表现出与iPLA 2 <$表达水平相关的其他特性，包括过表达iPLA 2 <$的细胞中增殖率增加（8）和iPLA 2 <$表达受到抑制的细胞中增殖率降低（7）。此外，过表达iPLA 2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），表明iPLA 2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因特定情况而异。