Metabolic consideration on PFK isozymes with reference to the first and the second phase of insulin secretory response from the pancreatic beta cells.

在胰腺细胞中，胰岛素分泌对葡萄糖的反应模式被称为双相。在胰岛素分泌的第一阶段，葡萄糖激酶（GK）作为葡萄糖传感器，调节葡萄糖通量和ATP合成速率。但是，如果我们试图通过代谢的考虑来解释胰岛素分泌的双相特征，那么只有非变构的GK成分不足以解释双相反应模式。磷酸果糖激酶（PFK）是肝脏中糖酵解的另一个关键调节酶，在细胞中也有三种不同的同工酶。我们已经表明，在胰腺组织中可以看到L（肝脏形式），M（肌肉形式）和P（血小板，脑或成纤维细胞形式）的所有三种同工酶的表达。其中，β细胞中的同工酶主要为L型和M型。PFK的两种同工酶都是众所周知的典型变构酶。值得注意的是，ATP和柠檬酸盐对PFK-M的变构抑制作用比PFK-L更明显。经AD活化后，PFK-M中P/AMP含量增加。葡萄糖进入β细胞的第一阶段以低ATP/ADP比率和低柠檬酸盐水平为特征。然后PFK-M主要参与中间的糖酵解步骤。当稳态葡萄糖摄取和磷酸化建立，细胞内ATP和柠檬酸盐含量增加时，变构抑制对PFK-M的影响更大，糖酵解主要由PFK-L进行。从代谢的角度来看，后一种现象可能是胰岛素分泌反应第二阶段的特征。为了评估和检验这一假设，我们正试图敲除细胞中特定的PFK同工酶。在本研究过程中，我们建立了适用于活细胞的细胞内钙离子、质子和ATP浓度的精确分析方法，分别采用实时细胞成像分析仪、共聚焦激光显微镜和荧光素酶过表达系统。目前，我们正在通过反义RNA表达系统的稳定转化，在MIN6细胞中建立PFK同工酶的细胞敲除。腺病毒系统也制备了反义载体的瞬时过表达。后者的目的是利用离体胰岛和胰腺器官灌注来评估这一假设。在此研究期间，为特异性敲除系统的建立和细胞分析系统的建立做了准备。这些将用于接下来的研究阶段，即将到来的结果将为建立同工酶特异性敲除小鼠提供进一步的理论依据，以确定β细胞PFK同工酶的全身效应。少

In the pancreatic beta cells, the mode of insulin secretion in response to glucose is known as biphasic. In the first phase of insulin secretion, glucokinase (GK) acts as a glucose sensor, regulating the glucose flux and ATP synthesis rate. But if we try to explain the biphasic profile of insulin secretion through the metabolic consideration, only the nonallosteric GK component is not sufficient for a biphasic mode of reaction.There are three distinct isozymes in phosphofructokinase (PFK), the other key regulatory enzyme of glycolysis in the liver, as well as in the beta cell. We have shown that expression of all three isozymes of L (liver form), M (muscle form) and P (platelet, brain or fibroblast form) is seen in the pancreatic tissue. Among these, the major isozymes in the beta cells are L and M forms. Both isozymes of PFK are well-known typical allosteric enzymes. To note, the attitue of allosteric inhibition by ATP and citrate is more steep in PFK-M than in PFK-L. Activation by AD … More P/AMP is stronger in PFK-M. The first stage of glucose entry to beta cells would be characterized by low ATP/ADP ratio and low citrate levels. Then PFK-M dominantly works the intermediate glycolytic step. When the steady state glucose uptake and phosphorylation are established and intracellular contents of ATP and citrate are increased, allosteric inhibition affects more on PFK-M and glycolysis will go on mainly by PFK-L. The latter phenomenon may responsible for characterizing the second phase of insulin secretory response in view from the metabolic consideration.To evaluate and examine this hypothesis, we are trying to knockout the specific PFK isozyme in the beta cells.We have established in this research process, the precise analytical methods applicable to living cells for the determination of intracellular calcium ion, proton and ATP concentrations by using the real-time cell-imaging analyzer, confocal laser microscopy, and luciferase overexpression system, respectively.We are now establishing the cellular knockout of PFK isozymes in MIN6 cells by the stable transformation of antisense RNA expression system. Transient overexpression of antisense vector is also prepared by the adenoviral system. The latter is aimed to evaluate the hypothesis using the isolated islets and pancreas organ perfusion.During this research period, establishment of the specific knockout system and the cytoanalytical system have been prepared. These will be used in the following study periods and the forthcoming results will give further rationale for establishing isozyme specific knockout mice to determine the whole body effect of beta cell PFK isozymes. Less