Civelek V N, Deeney J T, Kubik K, Schultz V, Tornheim K, Corkey B E
Diabetes and Metabolism Unit, Evans Department of Medicine, Boston University Medical Center, MA 02118, USA.
Biochem J. 1996 May 1;315 ( Pt 3)(Pt 3):1015-9. doi: 10.1042/bj3151015.
Stimulation of insulin release by glucose requires increased metabolism of glucose and a rise in cytosolic free Ca2+ in the pancreatic beta-cell. It is accompanied by increases in respiratory rate, pyridine and flavin nucleotide reduction state, intracellular pH and the ATP/ADP ratio. To test alternative proposals of the regulatory relationships among free Ca2+, mitochondrial metabolism and cellular energy state, we determined the temporal sequence of these metabolic and ionic changes following addition of glucose to clonal pancreatic beta-cells (HIT). Combined measurements of the native fluorescence of reduced pyridine nucleotides and oxidized flavin, intracellular pH, and free Ca2+ were performed together with simultaneous measurement of O2 tension or removal of samples for assay of the ATP/ADP ratio. The initial changes were detected in three phases. First, decreases occurred in the ATP/ADP ratio (<3 s) and increases in pyridine (2 +/- 1 s) and flavin (2 +/- 1 s) nucleotide reduction. Next, increases in the O2 consumption rate (20 +/- 5 s), the ATP/ADP ratio (29 +/- 12 s) and internal pH (48 +/- 5 s) were observed. Finally, cytosolic free Ca2+ rose (114 +/- 10 s). Maximal changes in the ATP/ADP ratio, O2 consumption and pyridine and flavin nucleotide fluorescence preceded the beginning of the Ca2+ change. These relationships are consistent with a model in which phosphorylation of glucose is the initial event which generates the signals that lead to an increase in respiration, a rise in the ATP/ADP ratio and finally influx of Ca2+. Our results indicate that Ca2+ does not function as the initiator of increased mitochondrial respiration.
葡萄糖刺激胰岛素释放需要葡萄糖代谢增加以及胰腺β细胞胞质游离Ca2+升高。同时伴有呼吸速率、吡啶和黄素核苷酸还原状态、细胞内pH值以及ATP/ADP比值的增加。为了检验游离Ca2+、线粒体代谢和细胞能量状态之间调节关系的其他提议，我们测定了向克隆胰腺β细胞（HIT）中添加葡萄糖后这些代谢和离子变化的时间顺序。结合测量还原型吡啶核苷酸和氧化型黄素的天然荧光、细胞内pH值以及游离Ca2+，同时测量O2张力或取出样品测定ATP/ADP比值。最初的变化在三个阶段被检测到。首先，ATP/ADP比值下降（<3秒），吡啶（2±1秒）和黄素（2±1秒）核苷酸还原增加。接下来，观察到O2消耗速率增加（20±5秒）、ATP/ADP比值增加（29±12秒）和细胞内pH值增加（48±5秒）。最后，胞质游离Ca2+升高（114±10秒）。ATP/ADP比值、O2消耗以及吡啶和黄素核苷酸荧光的最大变化先于Ca2+变化开始。这些关系与一个模型一致，在该模型中葡萄糖磷酸化是引发信号的初始事件，这些信号导致呼吸增加、ATP/ADP比值升高，最终Ca2+内流。我们的结果表明Ca²⁺并非线粒体呼吸增加的启动因素。
