OSCILLATIONS AND THE CONTROL OF GLYCOLYSIS

振荡和糖酵解的控制

• 批准号: 3230166
• 负责人: KEITH TORNHEIM
• 金额: $ 21.47万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3230166
• 关键词: 6 phosphofructokinase; acidity /alkalinity; adenosine diphosphate; adenosine triphosphate; calcium flux; carbachol; citrates; disease /disorder model; enzyme mechanism; fluorescence microscopy; fluorescent dye /probe; fructose phosphate; glucose phosphate; glyceraldehyde; glycolysis; hormone regulation /control mechanism; laboratory rat; membrane potentials; nicotinamide adenine dinucleotide; noninsulin dependent diabetes mellitus; pancreatic islets; potassium channel; sulfonylurea; tissue /cell culture

This proposal addresses the hypothesis that oscillatory behavior of glycolysis may underlie glucose-stimulated insulin secretion in pancreatic islets. According to the proposed model, the increased glycolytic flux in the islets due to increased glucose concentration initiates oscillations in glycolysis and the ATP/ADP ratio. The oscillations in the ATP/ADP ratio in turn cause oscillations in the activity of ATP-sensitive K-channels, the membrane potential, and intracellular free Ca2+ that lead to insulin release. The model could account for the observed pulsatile release of insulin in vivo and in isolated islets, and the loss of this pulsatility in Type II diabetes. It could also explain why glucose stimulation of insulin secretion requires metabolism of the glucose, yet no specific-metabolite has been pinpointed as the effector--because the oscillatory process itself provides the coupling. The model is supported by recent observations of (a) oscillations in intracellular free Ca2+ in single rat islets, (b) NADH oscillations (indicative of glycolytic oscillations) in islet extracts, and (c) induction by an oscillating ATP/ADP ratio of oscillations in free Ca2+ of a suspension of permeabilized clonal pancreatic B-cells and in the activity of ATP-sensitive K-channels. The proposed studies will test and further refine this model. Cultured rat islets will be examined using fluorescence microscopy. Intracellular free Ca2+ and pH will be monitored with fluorescent probes, and NADH by endogenous fluorescence. At different phases of the Ca2+ oscillation cycle, islets will be assayed for glycolytic intermediates and adenine nucleotides. The dependence of the amplitudes and/or frequencies of these oscillating parameters on the glucose concentration will be examined. The mechanism of the regulation of the metabolic oscillations will be determined from detailed analysis of changes in metabolite profiles in islet extracts, together with studies of the kinetic properties of islet phosphofructokinase and pyruvate kinase. The effects of factors shown to modulate glycolytic oscillations in muscle extracts (e.g., fructose-2,6-P2, glucose-1,6-P2, citrate) will be examined. Finally, studies. will be extended to the effects of non-glucose secretagogues, such an glyceraldehyde, carbamylcholine and sulfonylurea, on glycolytic oscillations in islets.

该提议解决了以下假设 糖酵解可能是葡萄糖刺激的胰岛素分泌的基础 胰岛。 根据提议的模型，增加了 由于葡萄糖浓度升高，胰岛中的糖酵解通量 启动糖酵解和ATP/ADP比的振荡。 这 ATP/ADP比率的振荡反过来引起振荡 ATP敏感的K通道的活性，膜电位和 细胞内游离Ca2+导致胰岛素释放。 模型可以 考虑了观察到的胰岛素在体内和在体内的脉动释放 分离的胰岛，以及II型糖尿病中这种脉动的丧失。 它也可以解释为什么葡萄糖刺激胰岛素分泌 需要葡萄糖的代谢，但没有特异性的代谢物是 确定为效应器，因为振荡过程本身 提供耦合。 该模型的最新观察支持了该模型 （a）单个大鼠胰岛中细胞内游离Ca2+的振荡，（b） 胰岛中的NADH振荡（指示糖酵解振荡） 提取物，（c）通过振荡的ATP/ADP比诱导 透化克隆悬浮液的自由Ca2+振荡 胰腺B细胞和对ATP敏感的K通道的活性。 这 拟议的研究将测试并进一步完善该模型。 培养的大鼠 将使用荧光显微镜检查胰岛。 细胞内 自由Ca2+和pH将通过荧光探针进行监测，而NADH将通过 内源性荧光。 在CA2+振荡的不同阶段 循环，将对糖酵解中间体和腺嘌呤进行测定 核苷酸。 振幅和/或频率的依赖性 这些在葡萄糖浓度上的振荡参数将是 检查。 代谢振荡调节的机制 将从代谢物变化的详细分析确定 胰岛提取物中的剖面以及动力学的研究 胰岛磷酸激酶和丙酮酸激酶的特性。 效果 显示肌肉提取物中糖酵解振荡的因素 （例如，将检查果糖-2,6-P2，葡萄糖1,6-P2，柠檬酸盐）。 最后，研究。将扩展到非葡萄糖的影响 秘密的甘油醛，甲丙基胆碱和磺酰脲， 在胰岛中的糖酵解振荡上。