Institute of Medical Biochemistry and Molecular Biology, University Medicine Rostock, Rostock, Germany.
Department of Pediatrics, University Medical Center Eppendorf, Hamburg, Germany.
Diabetologia. 2021 Dec;64(12):2687-2700. doi: 10.1007/s00125-021-05553-w. Epub 2021 Sep 16.
AIMS/HYPOTHESIS: The mammalian enzyme glucokinase (GK), expressed predominantly in liver and pancreas, plays an essential role in carbohydrate metabolism. Monogenic GK disorders emphasise the role of GK in determining the blood glucose set point.
A family with congenital hyperinsulinism (CHI) was examined for GCK gene variants by Sanger sequencing. A combined approach, involving kinetic analysis (also using GK activators and inhibitors), intracellular translocation assays, insulin secretion measurements and structural modelling, was used to investigate the novel variant compared with known variants.
We report on the novel gain-of-function GCK variant p.Val455Leu (V455L), inherited as an autosomal dominant trait in a German family with CHI and concomitant obesity (fasting blood glucose 2.1 mmol/l, BMI 45.0 kg/m, HOMA-IR 1.5 in an adult female family member); one male family member developed type 2 diabetes until age 35 years (with fasting glucose 2.8-3.7 mmol/l, BMI 38.9 kg/m, HOMA-IR 4.6). Kinetic characterisation of the V455L variant revealed a significant increase in glucose affinity (glucose concentration at which reaction rate is half its maximum rate [S]: mutant 2.4 ± 0.3 mmol/l vs wild-type 7.6 ± 1.0 mmol/l), accompanied by a distinct additive susceptibility to both the endogenous activator fructose 2,6-bisphosphatase and the synthetic allosteric activator RO-28-1675. The effect of RO-28-1675 was more pronounced when compared with the previously known GK variants V455M and V455E. Binding to the inhibitor glucokinase regulatory protein was unimpaired for V455L and V455E but was reduced for V455M, whereas mannoheptulose inhibited all GK variants and the wild-type enzyme. Structural analyses suggested a role for residue 455 in rearrangements between the inactive and active conformations of GK and also in allosteric activation. Comparison with V455M and V455E and an overview of activating GK variants provided a context for the novel sequence aberration in terms of altered GK enzyme characteristics caused by single amino acid changes.
CONCLUSION/INTERPRETATION: We provide new knowledge on the structure-function relationship of GK, with special emphasis on enzyme activation, potentially yielding fresh strategic insights into breaking the vicious circle of fluctuating blood glucose levels and the attendant risk of long-lasting metabolic changes in both CHI and type 2 diabetes.
目的/假设：哺乳动物酶葡萄糖激酶（GK）主要在肝脏和胰腺中表达，在碳水化合物代谢中发挥重要作用。单基因 GK 紊乱强调了 GK 在确定血糖设定点中的作用。
通过 Sanger 测序检查一个患有先天性高胰岛素血症（CHI）的家族的 GCK 基因突变。采用一种综合方法，包括动力学分析（还使用 GK 激活剂和抑制剂）、细胞内转位测定、胰岛素分泌测量和结构建模，对与已知变体相比的新型变体进行了研究。
我们报告了一种新的获得功能的 GCK 变体 p.Val455Leu（V455L），该变体在一个患有 CHI 和伴随肥胖的德国家族中作为常染色体显性遗传（成年女性家族成员空腹血糖 2.1mmol/l，BMI 45.0kg/m，HOMA-IR 1.5）；一名男性家族成员在 35 岁前发展为 2 型糖尿病（空腹血糖 2.8-3.7mmol/l，BMI 38.9kg/m，HOMA-IR 4.6）。V455L 变体的动力学特征显示葡萄糖亲和力显著增加（反应速率达到最大速率一半时的葡萄糖浓度[S]：突变体 2.4±0.3mmol/l 与野生型 7.6±1.0mmol/l），同时对内源性激活剂果糖 2,6-双磷酸酶和合成变构激活剂 RO-28-1675 具有明显的附加敏感性。与先前已知的 GK 变体 V455M 和 V455E 相比，RO-28-1675 的作用更为明显。V455L 和 V455E 对葡萄糖激酶调节蛋白的结合不受影响，但 V455M 受影响，而甘露庚酮糖抑制所有 GK 变体和野生型酶。结构分析表明残基 455 在 GK 的非活性和活性构象之间的重排以及变构激活中起作用。与 V455M 和 V455E 进行比较，并对激活 GK 变体进行概述，为由于单个氨基酸变化导致的 GK 酶特性改变提供了新的结构-功能关系知识。
结论/解释：我们提供了关于 GK 结构-功能关系的新知识，特别强调酶的激活，这可能为打破波动的血糖水平和随之而来的 CHI 和 2 型糖尿病中持久代谢变化的恶性循环提供新的战略见解。
