Schöfl C, Börger J, Lange S, von zur Mühlen A, Brabant G
Abteilung Klinische Endokrinologie, Medizinische Hochschule Hannover, Germany.
Endocrinology. 2000 Nov;141(11):4065-71. doi: 10.1210/endo.141.11.7741.
Insulin secretion is under multifactorial control by glucose and neurohumoral factors like acetylcholine (ACH), which activate the Ca2+/phospholipase C signaling pathway. All insulin secretagogues elevate cytosolic free Ca2+ ([Ca2+]i) that is central to the stimulation of insulin secretion. The actions of ACH on [Ca2+]i are glucose dependent but the metabolic steps involved are only partly understood. Here we have characterized the metabolic steps by which glucose exerts its synergistic effects on ACH-linked Ca2+-signals. [Ca2+]i was measured in single fura-2 loaded beta-cells. The ACH analog carbachol (3 microM) caused rise in [Ca2+]i that was strongly dependent on the extracellular glucose concentration ranging from 0-10 mM. Iodoacetate, which blocks glycolysis, thereby preventing the generation of NADH and ATP from glucose metabolism, and rotenone or antimycin, which inhibit complex 1 and 2 of the mitochondrial respiratory chain, respectively, inhibited in glucose (6 mM) the carbachol-induced Ca2+ signal to a similar extent as glucose deprivation. This demonstrates that glucose metabolism and generation of ATP through oxidative phosphorylation of energy rich substrates like NADH and FADH2 are required for carbachol-induced Ca2+ signals. While sodium arsenate, which prevents net glycolytic production of ATP without inhibiting glycolysis, had no significant effect on the carbachol-induced Ca2+-signal, the mitochondrial pyruvate transport inhibitor alpha-cyano-4-hydroxycinnamate and the Krebs cycle inhibitor monofluoroacetate strongly suppressed the rise in [Ca2+]i elicited by carbachol. While pyruvate was ineffective, methyl pyruvate, a membrane-permeant pyruvate analog, and alpha-ketoisocaproate in combination with glutamine, which are both substrates for mitochondrial ATP production, could restore the carbachol-induced Ca2+ signal in glucose-free medium. These data demonstrate for the first time that Krebs cycle metabolism of glucose and ATP formation through oxidative phosphorylation is critical for the glucose dependency of ACH-linked Ca2+-signals in mouse beta-cells, and they suggest that mitochondrial metabolism plays a key role in the interactive regulation of beta-cells by neurohumoral factors activating the Ca2+/phospholipase C signaling pathway.
胰岛素分泌受葡萄糖和神经体液因子（如乙酰胆碱，ACH）的多因素控制，这些因子激活Ca2+/磷脂酶C信号通路。所有胰岛素促分泌剂都会升高胞质游离Ca2+（[Ca2+]i），这对胰岛素分泌的刺激至关重要。ACH对[Ca2+]i的作用依赖于葡萄糖，但其中涉及的代谢步骤仅部分为人所知。在此，我们已明确了葡萄糖对ACH相关Ca2+信号发挥协同作用的代谢步骤。在单个加载了fura-2的β细胞中测量[Ca2+]i。ACH类似物卡巴胆碱（3 microM）使[Ca2+]i升高，这强烈依赖于细胞外葡萄糖浓度，范围为0 - 10 mM。碘乙酸可阻断糖酵解，从而阻止葡萄糖代谢产生NADH和ATP，鱼藤酮或抗霉素分别抑制线粒体呼吸链的复合物1和2，它们在葡萄糖（6 mM）存在时，对卡巴胆碱诱导的Ca2+信号的抑制程度与葡萄糖缺乏时相似。这表明葡萄糖代谢以及通过富含能量的底物（如NADH和FADH2）的氧化磷酸化产生ATP对于卡巴胆碱诱导的Ca2+信号是必需的。虽然砷酸钠可阻止ATP的净糖酵解产生但不抑制糖酵解，对卡巴胆碱诱导的Ca2+信号无显著影响，但线粒体丙酮酸转运抑制剂α-氰基-4-羟基肉桂酸和三羧酸循环抑制剂氟乙酸强烈抑制卡巴胆碱引起的[Ca2+]i升高。丙酮酸无效，但丙酮酸甲酯（一种可透过膜的丙酮酸类似物）以及α-酮异己酸与谷氨酰胺（二者均为线粒体ATP产生的底物）的组合，可在无葡萄糖培养基中恢复卡巴胆碱诱导的Ca2+信号。这些数据首次证明，葡萄糖的三羧酸循环代谢以及通过氧化磷酸化形成ATP对于小鼠β细胞中ACH相关Ca2+信号对葡萄糖的依赖性至关重要，并且表明线粒体代谢在通过激活Ca2+/磷脂酶C信号通路的神经体液因子对β细胞的相互调节中起关键作用。
