Diabetologia:与高胰岛素症相关的新型GCK变体p.Val455Leu易感变构激活,促进体重增加和糖尿病的发展。

2021-10-13 从医路漫漫 MedSci原创

哺乳动物葡萄糖激酶(GK)主要在肝脏和胰腺表达,在碳水化合物代谢中起重要作用。单基因GK紊乱强调GK在确定血糖设定点中的作用。

目的:哺乳动物葡萄糖激酶(GK)主要在肝脏和胰腺表达,在碳水化合物代谢中起重要作用。单基因GK紊乱强调GK在确定血糖设定点中的作用。

方法:采用Sanger测序法对一个先天性高胰岛素血症(CHI)家系进行了GCK基因变异检测。采用动力学分析(也使用GK激活剂和抑制剂)、细胞内易位分析、胰岛素分泌测量和结构建模的综合方法,与已知的变异体进行比较。

结果:我们报道了一个新的功能增益基因GCCK变异体p.Val455Leu(V455L),在一个伴有CHI和伴随肥胖(空腹血糖2.1 mmol/l,BMI 45.0 kg/m2,H O M A-I R1)的德国家系中,遗传为常染色体显性性状。1例男性家族成员至35岁前发生2型糖尿病(空腹血糖2.8~3.7 mmol/l,BMI 38.9 kg/m2,HOMA-IR 4.6)。动力学分析表明,V455L变异体的葡萄糖亲和力显著增强(葡萄糖浓度为其最大反应速率的一半[S0.5]:突变体2.4±0.3 mmol/l,野生型7.6±1.0 mmol/l),并对内源激活剂果糖2,6-二磷酸酶和合成变构激活剂RO-28-1675有明显的相加敏感性。与已知的GK变异体V455M和V455E相比,RO-28-1675的作用更为明显。V455L和V455E与抑制剂葡糖激酶调节蛋白的结合未受影响,而V455M则减少,而甘露七糖则抑制所有GK变异体和野生型酶。结构分析表明,残基455在GK的非活性构象和活性构象之间的重排以及变构激活中起作用。与V455M和V455E的比较,以及激活GK变异体的概述,从单一氨基酸改变引起的GK酶特性的改变为新的序列异常提供了背景。

图1 NovelGCK错义突变p.Val455Leu在一个高胰岛素血症家族中。(A)受影响家庭的家系:黑色符号表示被诊断为婴儿期高胰岛素低血糖的个人。男性家庭成员用正方形表示,女性家庭成员用圆圈表示。罗马数字表示一代内的世代,阿拉伯数字表示一代内的个体,代表兄弟姐妹的出生顺序。(B)GCK测序证实了新变异NM_000162.3的杂合性:C.1363G>C(p.Val455Leu)。(C)个人II-2、II-4和II-5的OGTT结果。在本试验中,口服葡萄糖75g,每30min测量血糖(白色符号,虚线)和胰岛素(填充符号,实线)。(D)个人II-2和II-5的IVGTT结果。给予300 mg/kg的葡萄糖,每隔一段时间测量血糖(白色符号,虚线)和胰岛素(填充符号,实线)。(E)在没有任何激活物的情况下,或与3μg的FBPase-2(F)或10μμ/l RO-28-1675(G)孵育25min后,测定了1 mol g重组野生型(V455)或L455变异的Dendra2-GK酶的葡萄糖磷酸化活性。图表显示了5个(野生型)或3个(L455)独立实验的拟合平均值±SEM。采用非线性回归方法比较变构Sigmoid模型和Michaelis-Menten模型的拟合效果。RO-28-1675处理的野生型酶的数据符合Michaelis-Menten模型,而对于所有其他曲线,包括突变体的曲线,则采用S型拟合。根据曲线计算出的动力学参数如表1所示

图2 重组Dendra2-GK野生型(V455)、-M455、-E455和-L455酶对内源和合成激活剂以及竞争性抑制剂甘露七糖的响应。分别在2(A)、5(B)、10(C)、25μ/l葡萄糖(D)、3μg重组FBPase-2、10μ/l RO-28-1675、2(A)、5(B)、10(C)或25μ/l葡萄糖(D)条件下,分别测定重组Dendra2-GK突变体(1 mmol g或被甘露七糖抑制时为2 mmol g)的葡萄糖磷酸化活性,以及联合使用2(A)、5(B)、10(C)或25 mmol/l葡萄糖(D)时的葡萄糖磷酸化活性。四个独立实验的数据以均值±SEM表示。*p<0.05,**p<0.01和***p<0.001,与野生型GK比较,或在括号所示的一个GK变异体内,††p<0.0。与激活突变GK-L455相比,0.001 1an d†††p<0.0 5;甘露七糖对所有被分析的GK变异体均有显著抑制作用(p<0.0 5),但对于清晰度符号被省略(ANOVA/Bonferroni或,在GK-E455数据集中)

图3 Dendra2-GK变异体的细胞内定位和胰岛素分泌。(A)瞬时转染Dendra2-GK野生型(V455)和突变体GKM455、-E455和-L455的分泌胰岛素的MIN6细胞的代表性图像显示Dendra2荧光在细胞质中均匀分布。Scale bar,10μm.(B,c)MIN6细胞瞬时过表达Dendra2-GK野生型和-M455、-E455或-L455突变体的静态胰岛素分泌。细胞在无糖条件下孵育1h,然后分别用3mmoll(白条)或10mmoll(填条)葡萄糖刺激1h,不加或不加激活剂RO-28-1675(10mmolmol/l)刺激1h。胰岛素分泌先归一化为胰岛素含量,然后归一化为总蛋白含量。五个独立实验的数据以均值±SEM表示。*p<0.05和**p<0.001与野生型酶相比,或者,当用括号表示时,与3 mmol/l葡萄糖刺激(ANOVA/Bonferroni)相比

图4 在超开放的、酶失活的GK构象的结构模型中,预测Val-455被Met、Glu或Leu取代的效果。(A)在超开放的GK构象(PDB ID 1v4t[29])中,酶的大结构域显示为深灰色,小结构域显示为浅灰色,相互连接区I-III显示为淡绿色,C-末端螺旋α13显示为紫罗兰色,螺旋α5显示为深蓝色。具有高度柔性的结构(His-156,ASN-180)两侧的残基被染成浅蓝色,以区别于酶的末端。残留物Val-455显示为棒状模型,并以红色突出显示。(b-e)中细节的观察方向由红色箭头指示。(B)变构位点的扩大部分包括455位残基(虚线)的极性接触,以及与邻近氨基酸的次要(绿盘)或主要(红盘)空间重叠。与Val-455接触的相邻氨基酸呈橙色。那些相互作用超过α-螺旋主干氢键的接触残基称为(也称为Inc-e)。在以棒状模型表示的氨基酸中,氧原子被染成红色,氮原子被染成蓝色。(C)Val-455突变为蛋氨酸,突变残基显示为绿色,硫原子显示为黄色。出于说明的目的,提出了两种可能的旋转体。极性和空间接触与以前一样,氨基酸与突变的(但不与野生型的)残基相互作用,呈黄色。(D)Val-455突变为谷氨酸(配色方案与以前相同)。(E)Val-455突变为亮氨酸(配色方案同前)

结论: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,潜在地为打破CHI和2型糖尿病患者血糖水平波动和随之而来的长期代谢变化风险的恶性循环提供了新的战略见解。

原文出处:

Langer S,  Waterstradt R,  Hillebrand G,  Santer R,  Baltrusch S,The novel GCK variant p.Val455Leu associated with hyperinsulinism is susceptible to allosteric activation and is conducive to weight gain and the development of diabetes.Diabetologia 2021 Sep 16

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