Oscillations in activities of enzymes in pancreatic islet subcellular fractions induced by physiological concentrations of effectors
Oscillations in activities of enzymes in pancreatic islet subcellular fractions induced by physiological concentrations of effectors
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DOI：
10.2337/diabetes.46.12.1996
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发表时间：
1997-12-01
期刊：
影响因子：
7.7
通讯作者：
Cruz, MO
中科院分区：
文献类型：
--
作者：
MacDonald, MJ;AlMasri, H;Cruz, MO
葡萄糖是最有效的胰岛素促分泌剂，它通过有氧糖酵解刺激胰岛素分泌，而其他促分泌剂仅通过线粒体代谢刺激胰岛素释放。众所周知，在完整的胰岛β细胞中，任何一种促分泌剂都可以引起代谢的振荡(如糖酵解、ATP/ADP、NAD(P)/NAD(P)H比率)，这种振荡发生的周期类似于膜电位和胰岛素分泌的振荡。在这项研究中，胰岛胞浆或线粒体部分在生理浓度的底物存在下孵育。重复添加生理效应器会导致所研究的三种酶的活性发生振荡。加入草酰乙酸酯(5 MU/L)抑制胰岛线粒体提取液中琥珀酸脱氢酶的活性，使该酶活性振荡。在柠檬酸合成酶反应中，加入乙酰辅酶A(3MU/L)，与草酰乙酸酯结合，降低草酰乙酸酯的浓度，使酶重新激活，从而开始另一次振荡。加入果糖二磷酸(10MU/L)，使丙酮酸激酶活性振荡。果糖二磷酸被相当快地降解成磷酸三糖，而且，当它被降解时，丙酮酸激酶的活性也有平行的下降。该酶被重新激活，并随着随后添加的二磷酸果糖而振荡。通过加入EGTA来螯合钙，使线粒体甘油磷酸脱氢酶振荡，从而激活该酶。当游离钙浓度增加到0.1mU/L时，酶活力增加。重复添加螯合剂和钙会引起酶活性的振荡。这三种酶和自然产生的效应物的生理浓度的结果增加了这样的可能性，即不仅这些酶的活性，而且许多酶的活性在体内振荡，以响应变构效应物和底物的水平。如果是这样的话，起搏器的活性可能是分布在胞浆和线粒体中多个调控部位的复杂效应的结果，而不是单一的酶作为主要起搏器的结果。
Glucose, the most potent insulin secretagogue, stimulates insulin secretion by aerobic glycolysis, but other secretagogues stimulate insulin release exclusively by mitochondrial metabolism. It is well known that in the intact pancreatic beta-cell, either kind of secretagogue can induce oscillations in metabolism (e.g., glycolysis, ATP/ADP, NAD(P)/NAD(P)H ratios) that occur with a periodicity similar to oscillations in membrane electrical potential and insulin secretion. In this study, pancreatic islet cytosol or mitochondrial fractions were incubated in the presence of physiological concentrations of substrates. Repeated additions of physiological effecters caused oscillations in the activities of the three enzymes studied. Succinate dehydrogenase activity in islet mitochondrial extracts was made to oscillate by adding oxaloacetate (5 mu mol/l) to inhibit the enzyme. The enzyme was reactivated by adding acetyl-CoA (3 mu mol/l), which combines with oxaloacetate in the citrate synthase reaction and lowers the concentration of oxaloacetate, thus beginning another oscillation. Pyruvate kinase activity was made to oscillate by adding fructose bisphosphate (10 mu mol/l). Fructose bisphosphate was degraded to triose phosphates fairly rapidly, and, as it was degraded, there was a parallel decrease in pyruvate kinase activity. The enzyme was reactivated and made to oscillate with subsequent additions of fructose bisphosphate. The mitochondrial glycerol phosphate dehydrogenase was made to oscillate by adding EGTA to chelate calcium, which activates the enzyme. When the concentration of free calcium was raised to >0.1 mu mol/l by adding more calcium, the activity of the enzyme increased. Repeated additions of chelator and calcium caused the enzyme activity to oscillate. The results with these three enzymes and physiological concentrations of naturally occurring effecters raise the possibility that the activities of not only these enzymes but of numerous enzymes oscillate in vivo in response to levels of allosteric effecters and substrates. If this is the case, pacemaker activity may result from complex effects distributed across multiple regulatory sites in both the cytosol and mitochondria, rather than from a single enzyme acting as a primary pacemaker.