Studies of phospholipid metabolism, proliferation, and secretion of stably transfected insulinoma cells that overexpress group VIA phospholipase A2
Studies of phospholipid metabolism, proliferation, and secretion of stably transfected insulinoma cells that overexpress group VIA phospholipase A2
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DOI：
10.1007/s11745-001-0774-9
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发表时间：
2001-07-01
期刊：
影响因子：
1.9
通讯作者：
Turk, J
中科院分区：
文献类型：
--
作者：
Ma, ZM;Bohrer, A;Turk, J
从中国仓鼠卵巢（CHO）细胞、小鼠P388D1细胞、胰岛β细胞和其他来源克隆了一种不需要催化的胞浆性磷脂酶A(2) (iPLA(2) β)，提出的iPLA(2) β功能包括通过降解CHO细胞中过量表达CTP的PC来参与磷脂酰胆碱（PC）稳态：磷脂酰胆碱转移酶（CT），该酶催化PC生物合成的速率限制步骤；通过生成溶血磷脂酰胆碱（LPC）受体，参与P388D1细胞中含有花生四烯酸酯的PC物种的生物合成；并参与胰岛细胞分泌胰岛素的信号事件。为了进一步研究iPLA(2) β在β细胞中的功能，我们制备了稳定转染的INS-1胰岛素瘤细胞系，与亲代INS-1细胞或转染了不含iPLA(2) β cDNA的空逆转录病毒载体的INS-1细胞相比，这些细胞系过表达了8倍的iPLA(2) β活性。与亲代细胞相比，iPLA(2) β -过表达细胞的CT活性增加了两倍，但[H-3]胆碱并入PC或从PC中消失的速率几乎没有变化。电喷雾电离（ESI）串联质谱测量表明，iPLA(2) β -过表达细胞的LPC水平比亲本INS-1细胞高1.5倍，但未表现出[H-3]花生四烯酸酯掺入磷脂的增加率，并且掺入不受iPLA(2) β的溴烯醇内酯（BEL）自杀底物抑制剂的影响。在添加花生四烯酸孵育的iPLA(2) β -过表达细胞和亲本INS-1细胞中，ESI质谱显示含有花生四烯酸的磷脂酰乙醇胺物种的出现率也相似，并且这一过程不受BEL的影响。与亲本INS-1细胞相比，iPLA(2) β -过表达细胞增殖更快，并且对蛋白激酶c激活的酚酯、葡萄糖和cAMP类似物表现出放大的胰岛素分泌反应。这些发现表明iPLA(2) β在β -细胞中发挥信号作用，不同于P388D1和CHO细胞中PC生物合成和降解的内务功能。
A cytosolic 84 kDa Croup VIA phospholipase A(2) (iPLA(2)beta) that does not require Call for catalysis was cloned from Chinese hamster ovary (CHO) cells, murine P388D1 cells, pancreatic islet beta -cells, and other sources, Proposed iPLA(2)beta functions include participation in phosphatidylcholine (PC) homeostasis by degrading excess PC generated in CHO cells that overexpress CTP:phosphocholine cytidylyltransferase (CT), which catalyzes the rate-limiting step in PC biosynthesis; participation in biosynthesis of arachidonate-containing PC species in P388D1 cells by generating lysophosphatidylcholine (LPC) acceptors for arachidonate incorporation; and participation in signaling events in insulin secretion from islet beta -cells. To further examine iPLA(2)beta functions in beta -cells, we prepared stably transfected INS-I insulinoma cell lines that overexpress iPLA(2)beta activity eightfold compared to parental INS-1 cells or to INS-1 cells transfected with an empty retroviral vector that did not contain iPLA(2)beta cDNA. The iPLA(2)beta -overexpressing cells exhibit a twofold increase in CT activity compared to parental cells but little change in rates of [H-3]choline incorporation into or disappearance from PC. Electrospray ionization (ESI) tandem mass spectrometric measurements indicate that iPLA(2)beta -overexpressing cells have 1.5-fold higher LPC levels than parental INS-1 cells but do not exhibit increased rates of [H-3]arachidonate incorporation into phospholipids, and incorporation is unaffected by a bromoenol lactone (BEL) suicide substrate inhibitor of iPLA(2)beta. The rate of appearance of arachidonate-containing phosphatidylethanolamine species visualized by ESI mass spectrometry is also similar in iPLA(2)beta -overexpressing and parental INS-1 cells incubated with supplemental arachidonic acid, and this process is unaffected by BEL. Compared to parental INS-1 cells, iPLA(2)beta -overexpressing cells proliferate more rapidly and exhibit amplified insulin secretory responses to a protein kinase C-activating phorbol ester, glucose, and a cAMP analog. These findings suggest that iPLA(2)beta plays a signaling role in beta -cells that differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells.