Hughes William E, Elgundi Zehra, Huang Ping, Frohman Michael A, Biden Trevor J
Cell Signalling Group, The Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010, Australia.
J Biol Chem. 2004 Jun 25;279(26):27534-41. doi: 10.1074/jbc.M403012200. Epub 2004 Apr 14.
Phospholipase D (PLD) has been strongly implicated in the regulation of Golgi trafficking as well as endocytosis and exocytosis. Our aim was to investigate the role of PLD in regulating the biphasic exocytosis of insulin from pancreatic beta-cells that is essential for mammalian glucose homeostasis. We observed that PLD activity in MIN6 pancreatic beta-cells is closely coupled to secretion. Cellular PLD activity was increased in response to a variety of secretagogues including the nutrient glucose and the cholinergic receptor agonist carbamoylcholine. Conversely, pharmacological or hormonal inhibition of stimulated secretion reduced PLD activity. Most importantly, blockade of PLD-catalyzed phosphatidic acid formation using butan-1-ol inhibited insulin secretion in both MIN6 cells and isolated pancreatic islets. It was further established that PLD activity was required for both the first and the second phase of glucose-stimulated insulin release, suggesting a role in the very distal steps of exocytosis, beyond granule recruitment into a readily releasable pool. Visualization of granules using green fluorescent protein-phogrin confirmed a requirement for PLD prior to granule fusion with the plasma membrane. PLD1 was shown to be the predominant isoform in MIN6 cells, and it was located at least partially on insulin granules. Overexpression of wild-type or a dominant negative catalytically inactive mutant of PLD1 augmented or inhibited secretagogue-stimulated secretion, respectively. The results suggest that phosphatidic acid formation on the granule membrane by PLD1 is essential for the regulated secretion of insulin from pancreatic beta-cells.
磷脂酶D（PLD）与高尔基体运输以及胞吞作用和胞吐作用的调节密切相关。我们的目的是研究PLD在调节胰腺β细胞胰岛素双相胞吐作用中的作用，这对哺乳动物的葡萄糖稳态至关重要。我们观察到MIN6胰腺β细胞中的PLD活性与分泌密切相关。细胞PLD活性会因多种促分泌剂而增加，包括营养物质葡萄糖和胆碱能受体激动剂氨甲酰胆碱。相反，对刺激分泌的药理学或激素抑制会降低PLD活性。最重要的是，使用丁醇阻断PLD催化的磷脂酸形成会抑制MIN6细胞和分离的胰岛中的胰岛素分泌。进一步证实，葡萄糖刺激的胰岛素释放的第一阶段和第二阶段都需要PLD活性，这表明PLD在胞吐作用的非常远端步骤中发挥作用，超出了颗粒募集到易于释放的池的过程。使用绿色荧光蛋白 - 嗜铬蛋白对颗粒进行可视化证实，在颗粒与质膜融合之前需要PLD。PLD1被证明是MIN6细胞中的主要亚型，并且它至少部分位于胰岛素颗粒上。野生型或PLD1的显性负性催化失活突变体的过表达分别增强或抑制了促分泌剂刺激的分泌。结果表明，PLD1在颗粒膜上形成磷脂酸对于胰腺β细胞中胰岛素的调节分泌至关重要。
