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）的“功能获得”引起的。已经发现了几个错义突变;然而，由过度GDH活性引发的细胞行为尚未得到充分证明。本研究旨在阐明GDH突变的胰腺β细胞胰岛素分泌失调的电生理机制。在过表达G446 D和L413 V的MIN 6细胞中测量了GDH动力学和胰岛素分泌。膜电位和通道活动记录下的穿孔补丁配置，保留细胞内环境。在突变MIN 6细胞中，GDH对三磷酸鸟苷（GTP）的敏感性降低，在低葡萄糖浓度下胰岛素分泌增强。在触角样结构发生突变的L413 V中，基础GDH活性升高。在没有葡萄糖的情况下，L413 V细胞发生去极化，通常伴有重复的Ca^<2+>放电。三磷酸腺苷（ATP）存在时，去极化得以维持，耗尽ATP后，去极化消失，表明去极化依赖于细胞内ATP。在L413 V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断剂或激动剂的敏感性未受损。这些数据表明，L413 V细胞增加细胞内ATP/二磷酸腺苷（ADP）的比例，这反过来又导致持续的去极化，不仅通过关闭KATP通道，但也通过开放的NSCC。由此产生的电压门控Ca^2+通道激活似乎可诱导高胰岛素血症。本研究提供的证据表明，在HI/HA综合征的胰腺β细胞中，多个通道在不受调节的胰岛素分泌中协同作用。

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.