Grimaldi Mariagrazia, Karaca Melis, Latini Livia, Brioudes Estelle, Schalch Thomas, Maechler Pierre
Department of Cell Physiology and Metabolism.
Faculty Diabetes Center, University of Geneva Medical Center, 1206 Geneva, Switzerland.
Hum Mol Genet. 2017 Sep 15;26(18):3453-3465. doi: 10.1093/hmg/ddx213.
Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome gives rise to unregulated protein-induced insulin secretion from pancreatic beta-cells, fasting hypoglycemia and elevated plasma ammonia levels. Mutations associated with HI/HA were identified in the Glud1 gene, encoding for glutamate dehydrogenase (GDH). We aimed at identifying the molecular causes of dysregulation in insulin secretion and ammonia production conferred by the most frequent HI/HA mutation Ser445Leu. Following transduction with adenoviruses carrying the human GDH-wild type or GDH-S445L-mutant gene, immunoblotting showed efficient expression of the transgenes in all the investigated cell types. Enzymatic activity tested in INS-1E beta-cells revealed that the mutant was much more sensitive to the allosteric activator ADP, rendering it highly responsive to substrates. INS-1E cells expressing either the wild type or mutant GDH responded similarly to glucose stimulation regarding mitochondrial activation and insulin secretion. However, at basal glucose glutamine stimulation increased mitochondrial activity and insulin release only in the mutant cells. In mouse and human islets, expression of mutant GDH resulted in robust elevation of insulin secretion upon glutamine stimulation, not observed in control islets. Hepatocytes expressing either the wild type or mutant GDH produced similar levels of ammonia when exposed to glutamine, although alanine response was strongly elevated with the mutant form. In conclusion, the GDH-S445L mutation confers hyperactivity to this enzyme due to higher sensitivity to ADP allosteric activation. This renders beta-cells responsive to amino acid stimulation, explaining protein-induced hypoglycemia secondary to non-physiological insulin release. Hepatocytes carrying mutant GDH produced more ammonia upon alanine exposure, which underscores hyperammonemia developed by the patients.
先天性高胰岛素血症/高氨血症（HI/HA）综合征会导致胰腺β细胞中蛋白质诱导的胰岛素分泌失控、空腹低血糖以及血浆氨水平升高。在编码谷氨酸脱氢酶（GDH）的Glud1基因中鉴定出了与HI/HA相关的突变。我们旨在确定最常见的HI/HA突变Ser445Leu导致胰岛素分泌失调和氨生成的分子原因。在用携带人GDH野生型或GDH-S445L突变基因的腺病毒转导后，免疫印迹显示转基因在所有研究的细胞类型中均有效表达。在INS-1Eβ细胞中测试的酶活性表明，该突变体对变构激活剂ADP更为敏感，使其对底物具有高度反应性。表达野生型或突变型GDH的INS-1E细胞在对葡萄糖刺激的线粒体激活和胰岛素分泌方面反应相似。然而，在基础葡萄糖水平下，谷氨酰胺刺激仅在突变细胞中增加了线粒体活性和胰岛素释放。在小鼠和人类胰岛中，突变型GDH的表达导致谷氨酰胺刺激后胰岛素分泌显著升高，而对照胰岛中未观察到这种情况。表达野生型或突变型GDH的肝细胞在暴露于谷氨酰胺时产生的氨水平相似，尽管突变型对丙氨酸的反应强烈升高。总之，由于对ADP变构激活的更高敏感性，GDH-S445L突变赋予该酶高活性。这使得β细胞对氨基酸刺激有反应，解释了非生理性胰岛素释放继发的蛋白质诱导的低血糖。携带突变型GDH的肝细胞在暴露于丙氨酸时产生更多的氨，这突出了患者发生的高氨血症。
