Molecular characterization of congenital hyperinsulinism/hyperammonemia caused by glutamate dehydrogenase gene defects

高胰岛素血症/高氨血症(HI/HA)综合征是由谷氨酸脱氢酶(GDH)“功能增强”引起的。已经发现了几个错义突变；然而，由过度的GDH活性引发的细胞行为还没有完全被证明。本研究旨在阐明GDH突变的胰岛β细胞胰岛素分泌失调的电生理学机制。在过表达G446D和L413V的MIN6细胞中检测GDH动力学和胰岛素分泌。在保留细胞内环境的穿孔贴片结构下，记录了膜电位和通道活动。在突变的MIN6细胞中，GDH对三磷酸鸟苷(GTP)的敏感性降低，在低糖浓度下胰岛素分泌增加。在L413V中，具有天线样结构突变的基础GDH活性升高。L413V细胞在没有葡萄糖的情况下去极化，经常伴随着重复的钙离子激发。去极化在三磷酸腺苷(ATP)存在下维持，并在耗尽ATP后消失，这表明去极化依赖于细胞内的ATP。在L413V细胞中，ATP敏感性钾通道(KATP通道)被抑制，非选择性阳离子通道(NSCC)被增强，而通道对其特异性阻滞剂或激动剂的敏感性不受影响。这些数据表明，L413V细胞增加了细胞内ATP/二磷酸腺苷(ADP)的比率，这反过来不仅通过关闭KATP通道，而且通过开放NSCC而导致持续的去极化。由此产生的电压门控钙离子通道的激活似乎诱导了高胰岛素血症。本研究提供的证据表明，在HI/HA综合征的胰岛β细胞中，多个渠道协同作用导致胰岛素的无调控分泌。

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by "gain of function" of glutamate dehydrogenase (GDH). Several missense mutations have been found ; however, cell behaviors triggered by the excessive GDH activity have not been fully demonstrated. This study was aimed to clarify electrophysiological mechanisms underlying the dysregulated insulin secretion in pancreatic beta cells with GDH mutations. GDH kinetics and insulin secretion were measured in MIN6 cells overexpressing the G446D and L413V. Membrane potentials and channel activity were recorded under the perforated-patch configuration that preserved intracellular environments. In mutant MIN6 cells, sensitivity of GDH to guanosine triphosphate (GTP) was reduced and insulin secretion at low glucose concentrations was enhanced. The basal GDH activity was elevated in L413V bearing a mutation in the antenna-like structure. The L413V cells were depolarized without glucose, often accompanying by repetitive Ca^<2+> firings. The depolarization was maintained in the presence of adenosine triphosphate (ATP) and disappeared by depleting ATP, suggesting that the depolarization depended on intracellular ATP. In L413V cells, the ATP-sensitive potassium channel (KATP channel) was suppressed and the nonselective cation channel (NSCC) was potentiated, while sensitivity of the channels to their specific blockers or agonists was not impaired. These data suggest that the L413V cells increase the intracellular ATP/adenosine diphosphate (ADP) ratio, which in turn causes sustained depolarization not only by closure of the KATP channel, but also by opening of the NSCC. The resultant activation of the voltagegated Ca^<2+> channel appears to induce hyperinsulinism. The present study provides evidence that multiple channels cooperate in unregulated insulin secretion in pancreatic beta cells of the HI/HA syndrome.