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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 当糖尿病的观点是该回顾性的主题时，我们最近在胰岛中确定了磷脂酶A2（PLA2）活性，我们认为我们认为可能会整合有关葡萄糖诱导的磷脂水解水解的几个观察，这些磷脂水解在胰岛和我们的工作中所产生的胰岛胰岛导致了pair的pairie robertss，start of pair roberberss，start of pair roberbers streat sert streat，start of pair sect sert，start stew，start of pair the。克莱斯·沃尔海姆（Claes Wollheim）以及许多其他人以原始视角引用（1）。 这些观察结果包括以下事实：胰岛浓缩液暴露于刺激胰岛素分泌的D-葡萄糖浓缩物导致磷脂水解，非酯化蛛网膜酸的积累以及蛛网膜氧合产物的产生。葡萄糖诱导的磷脂水解需要代谢葡萄糖，但部分独立于Ca2+影响，而积累的蛛网膜酸似乎是通过支持CA2+的CA2+进入葡萄糖诱导的„ -CELL [Ca2+]的增加来扩大葡萄糖诱导的上升，并通过诱导的CA2+释放的位点（从静脉内释放出来）（1）（1）。 就像类似的活性在心肌细胞质中首次识别，但与其他当时识别的PLA2酶不同，这种胰岛PLA2活性不需要Ca2+用于催化活性，并且通过ATP激活，并且对通过溴烯醇醇（BEL）的抗抑制作用（BEL）的抗抑制作用敏感，这与其他pla2 Enzysemes浓度相当。此外，发现BEL可以抑制葡萄糖诱导的蛛网膜释放，胰岛素分泌以及``-cell celtosolic [Ca2+]的升高，这表明该PLA2可能代表了 - 细胞燃料传感器设备的组成部分（1）。 随后，还发现胰岛素瘤细胞表达了这种PLA2活性，并制备了其色谱分析和纯化（2）。这种表征的表征最终导致了来自84 kDa蛋白的大鼠胰岛cDNA库的克隆，该蛋白包含GXSXG丝氨酸脂肪酶共有序列和8个重复基序的延伸，类似于整合性膜蛋白蛋白质结合结构域（3）。重组蛋白表现出PLA2活性，该酶现在通过PLA2归类为一组，并给定琐碎的名称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 pharmaceutical 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掺入其他细胞中建议的磷脂中（5）。 由于BEL除了IPLA2其他酶外抑制了几种酶，因此IPLA2课程的分子生物学操纵为药物研究提供了重要的补充。培养的胰岛素瘤细胞系通过稳定的转染用含有IPLA2¿CDNA（6）的逆转录病毒载体产生的培养性胰岛素瘤（6）或小的干扰RNA（7）分别表现出比用空载体翻译的细胞高几个或更低的IPLA2活动。使用此类细胞系的研究还支持IPLA2胰岛素分泌的作用，但在� -Cell磷脂重塑中不支持（6，7）。 此类细胞系还表现出与IPLA2表达水平相关的其他特性，包括过表达IPLA2¿（8）的细胞中增殖率提高，以及抑制IPLA2抑制IPLA2¿表达的细胞中增殖率降低（7）。此外，过表达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¿可以参与在给定细胞内或根据特定细胞内的细胞之间或在特定细胞中可能不同的各种细胞生物学过程。