Stiernet Patrick, Guiot Yves, Gilon Patrick, Henquin Jean-Claude
Units of Endocrinology and Metabolism, University of Louvain Faculty of Medicine, B-1200 Brussels, Belgium.
Unit of Pathology, University of Louvain Faculty of Medicine, B-1200 Brussels, Belgium.
J Biol Chem. 2006 Aug 4;281(31):22142-22151. doi: 10.1074/jbc.M513224200. Epub 2006 Jun 7.
Glucose-induced insulin secretion requires a rise in beta-cell cytosolic Ca2+ ([Ca2+]c) that triggers exocytosis and a mechanistically unexplained amplification of the action of [Ca2+]c. Insulin granules are kept acidic by luminal pumping of protons with simultaneous Cl- uptake to maintain electroneutrality. Experiments using patched, dialyzed beta-cells prompted the suggestion that acute granule acidification by glucose underlies amplification of insulin secretion. However, others found glucose to increase granular pH in intact islets. In this study, we measured islet granular pH with Lysosensor DND-160, a fluorescent dye that permits ratiometric determination of pH < 6 in acidic compartments. Stimulation of mouse islets with glucose reversibly decreased granular pH by mechanisms that are dependent on metabolism and Cl- ions but independent of changes in [Ca2+]c and protein kinase A or C activity. Granular pH was increased by concanamycin (blocker of the vesicular type H+-ATPase) > methylamine (weak base) > Cl- omission. Concanamycin and methylamine did not alter glucose-induced [Ca2+]c increase in islets but strongly inhibited the two phases of insulin secretion. Omission of Cl- did not affect the first phase but decreased the second phase of both [Ca2+]c and insulin responses. Neither experimental condition affected the [Ca2+]c rise induced by 30 mM KCl, but the insulin responses were inhibited by concanamycin > methylamine and not affected by Cl- omission. The amplification of insulin secretion by glucose was not suppressed. We conclude that an acidic granular pH is important for insulin secretion but that the acute further acidification produced by glucose is not essential for the augmentation of secretion via the amplifying pathway.
葡萄糖诱导的胰岛素分泌需要β细胞胞质Ca2+（[Ca2+]c）升高，这会触发胞吐作用以及对[Ca2+]c作用的一种机制不明的放大。胰岛素颗粒通过腔内质子泵入同时摄取Cl-以维持电中性而保持酸性。使用膜片钳技术、透析β细胞的实验提示，葡萄糖引起的颗粒急性酸化是胰岛素分泌放大的基础。然而，其他人发现葡萄糖会使完整胰岛中的颗粒pH升高。在本研究中，我们使用溶酶体传感器DND-160测量胰岛颗粒pH，这是一种荧光染料，可用于在酸性区室中进行pH<6的比率测定。用葡萄糖刺激小鼠胰岛会通过依赖于代谢和Cl-离子但独立于[Ca2+]c以及蛋白激酶A或C活性变化的机制使颗粒pH可逆性降低。颗粒pH因巴弗洛霉素（囊泡型H+-ATP酶阻滞剂）>甲胺（弱碱）>去除Cl-而升高。巴弗洛霉素和甲胺不会改变葡萄糖诱导的胰岛中[Ca2+]c升高，但强烈抑制胰岛素分泌的两个阶段。去除Cl-不影响第一阶段，但会降低[Ca2+]c和胰岛素反应的第二阶段。两种实验条件均不影响30 mM KCl诱导的[Ca2+]c升高，但胰岛素反应受到巴弗洛霉素>甲胺的抑制，且不受去除Cl-的影响。葡萄糖对胰岛素分泌的放大作用未被抑制。我们得出结论，酸性颗粒pH对胰岛素分泌很重要，但葡萄糖引起的急性进一步酸化对于通过放大途径增强分泌并非必不可少。
