Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland.
Biochem J. 2009 Dec 10;424(3):459-66. doi: 10.1042/BJ20090729.
Transfer of reducing equivalents between cytosolic compartments and the mitochondrial matrix is mediated by NADH shuttles. Among these, the malate-aspartate shuttle has been proposed to play a major role in beta-cells for the control of glucose-stimulated insulin secretion. AGC1 or Aralar1 (aspartate-glutamate carrier 1) is a key component of the malate-aspartate shuttle. Overexpression of AGC1 increases the capacity of the malate-aspartate shuttle, resulting in enhanced metabolism-secretion coupling, both in INS-1E cells and rat islets. In the present study, knockdown of AGC1 was achieved in the same beta-cell models, using adenovirus-mediated delivery of shRNA (small-hairpin RNA). Compared with control INS-1E cells, down-regulation of AGC1 blunted NADH formation (-57%; P<0.05), increased lactate production (+16%; P<0.001) and inhibited glucose oxidation (-22%; P<0.01). This correlated with a reduced secretory response at 15 mM glucose (-25%; P<0.05), while insulin release was unchanged at intermediate 7.5 mM and basal 2.5 mM glucose. In isolated rat islets, efficient AGC1 knockdown did not alter insulin exocytosis evoked by 16.7 mM glucose. However, 4 mM amino-oxyacetate, commonly used to block transaminases of the malate-aspartate shuttle, inhibited glucose-stimulated insulin secretion to similar extents in INS-1E cells (-66%; P<0.01) and rat islets (-56%; P<0.01). These results show that down-regulation of the key component of the malate-aspartate shuttle AGC1 reduced glucose-induced oxidative metabolism and insulin secretion in INS-1E cells, whereas similar AGC1 knockdown in rat islets did not affect their secretory response.
胞质腔室和线粒体基质之间的还原当量的转移是由 NADH 穿梭介导的。在这些穿梭中，苹果酸-天冬氨酸穿梭被认为在β细胞中对于控制葡萄糖刺激的胰岛素分泌起主要作用。AGC1 或 Aralar1（天冬氨酸-谷氨酸载体 1）是苹果酸-天冬氨酸穿梭的关键组成部分。AGC1 的过表达增加了苹果酸-天冬氨酸穿梭的能力，导致 INS-1E 细胞和大鼠胰岛中的代谢-分泌偶联增强。在本研究中，使用腺病毒介导的 shRNA（短发夹 RNA）在相同的β细胞模型中实现了 AGC1 的敲低。与对照 INS-1E 细胞相比，AGC1 的下调削弱了 NADH 的形成（-57%；P<0.05），增加了乳酸的产生（+16%；P<0.001）并抑制了葡萄糖氧化（-22%；P<0.01）。这与 15 mM 葡萄糖时的分泌反应降低（-25%；P<0.05）相关，而在中间的 7.5 mM 和基础的 2.5 mM 葡萄糖时胰岛素释放没有变化。在分离的大鼠胰岛中，有效的 AGC1 敲低不会改变 16.7 mM 葡萄糖引起的胰岛素胞吐作用。然而，4 mM 氨基氧乙酸，通常用于阻断苹果酸-天冬氨酸穿梭的转氨酶，在 INS-1E 细胞（-66%；P<0.01）和大鼠胰岛（-56%；P<0.01）中对葡萄糖刺激的胰岛素分泌的抑制作用相似。这些结果表明，苹果酸-天冬氨酸穿梭的关键组成部分 AGC1 的下调降低了 INS-1E 细胞中葡萄糖诱导的氧化代谢和胰岛素分泌，而在大鼠胰岛中类似的 AGC1 敲低并未影响其分泌反应。
