Oscillations in oxygen consumption by permeabilized clonal pancreatic beta-cells (HIT) incubated in an oscillatory glycolyzing muscle extract - Roles of free Ca2+, substrates, and the ATP/ADP ratio
Oscillations in oxygen consumption by permeabilized clonal pancreatic beta-cells (HIT) incubated in an oscillatory glycolyzing muscle extract - Roles of free Ca2+, substrates, and the ATP/ADP ratio
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DOI：
10.2337/diabetes.46.1.51
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发表时间：
1997-01-01
期刊：
影响因子：
7.7
通讯作者：
Tornheim, K
中科院分区：
文献类型：
--
作者：
Civelek, VN;Deeney, JT;Tornheim, K
为了确定糖酵解中的振荡是否可能是完整胰岛中观察到的氧耗振荡的基础，我们评估了胰岛提取物在糖酵解中表现自发振荡的能力，当从类似于1000个胰岛获得的无细胞提取物中供应葡萄糖和糖酵解辅助因子时，获得了NADH荧光振荡。在证明了胰岛提取物中的自发振荡之后，我们用更丰富的自发振荡的糖酵解肌肉提取物来沐浴渗透的克隆性β细胞，这种肌肉提取物会产生α-甘油磷酸和丙酮酸的脉冲，并诱导游离钙离子和ATP/ADP比率的振荡。该制剂被用来研究钙离子和可能的α-甘油磷酸或丙酮酸供应的变化是否可以作为观察到的氧消耗振荡的基础，并解释细胞质和线粒体代谢之间的协调。我们发现，O-2消耗和Ca~(2+)也有类似周期的振荡。用EGTA去除中钙离子并不能阻止O2消耗的振荡，也不会受到thapsigargin抑制内质网隔离时中钙离子大幅增加的严重影响。加入较高浓度的丙酮酸或α-甘油磷酸也不能消除O-2振荡。然而，当添加浓度为2，6-P-2的果糖，以防止糖酵解振荡和ATP/ADP比率时，它们就消失了。高浓度ADP的加入增加了O2消耗，也阻止了O2振荡。这些结果表明，反映在O-2振荡中的呼吸变化是对ATP/ADP比率或ADP浓度波动的响应，该参数是胰岛β细胞消耗O-2的主要调节因素。
To determine whether oscillations in glycolysis could underlie the oscillations in O-2 consumption observed in intact islets, we evaluated the capacity of an islet extract to exhibit spontaneous oscillations in glycolysis, When a cell-free extract obtained from similar to 1,000 islets was supplied with glucose and glycolytic cofactors, oscillations in NADH fluorescence were obtained. After this demonstration of spontaneous oscillations in islet extracts, we bathed permeabilized clonal beta-cells in the more plentiful spontaneously oscillating glycolytic muscle extract that generates pulses of alpha-glycerophosphate and pyruvate and induces oscillations in free Ca2+ and the ATP/ADP ratio. This preparation was used to investigate whether changes in Ca2+ and possibly alpha-glycerophosphate or pyruvate supply could underlie observed oscillations in O-2 consumption and explain coordination between cytosolic and mitochondrial metabolism. We found that oscillations of O-2 consumption and Ca2+ of a similar period were induced. Removal of medium Ca2+ with EGTA did not prevent the oscillations in O-2 consumption nor were they greatly affected by the substantial rise in medium Ca on treatment with thapsigargin to inhibit sequestration into the endoplasmic reticulum. The O-2 oscillations were also not eliminated by the addition of relatively high concentrations of pyruvate or alpha-glycerophosphate. However, they were lost on addition of fructose-2,6-P-2 at concentrations that prevent oscillations of glycolysis and the ATP/ADP ratio. Addition of a high concentration of ADP increased O-2 consumption and also prevented O-2 oscillations. These results suggest that the changes in respiration reflected in the O-2 oscillations occur in response to the oscillations in the ATP/ADP ratio or ADP concentration and that this parameter is a primary regulator of O-2 consumption in the pancreatic beta-cell.