Minerva Foundation Institute for Medical Research, Tukholmankatu, Helsinki, Finland.
Department of Medicine, University of Helsinki and Helsinki University Hospital, Haartmaninkatu, Helsinki, Finland.
J Mol Endocrinol. 2023 Jan 4;70(2). doi: 10.1530/JME-21-0285. Print 2023 Feb 1.
Finnish-specific gene variant p.P50T/AKT2 (minor allele frequency (MAF) = 1.1%) is associated with insulin resistance and increased predisposition to type 2 diabetes. Here, we have investigated in vitro the impact of the gene variant on glucose metabolism and intracellular signalling in human primary skeletal muscle cells, which were established from 14 male p.P50T/AKT2 variant carriers and 14 controls. Insulin-stimulated glucose uptake and glucose incorporation into glycogen were detected with 2-[1,2-3H]-deoxy-D-glucose and D-[14C]-glucose, respectively, and the rate of glycolysis was measured with a Seahorse XFe96 analyzer. Insulin signalling was investigated with Western blotting. The binding of variant and control AKT2-PH domains to phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) was assayed using PIP StripsTM Membranes. Protein tyrosine kinase and serine-threonine kinase assays were performed using the PamGene® kinome profiling system. Insulin-stimulated glucose uptake and glycogen synthesis in myotubes in vitro were not significantly affected by the genotype. However, the insulin-stimulated glycolytic rate was impaired in variant myotubes. Western blot analysis showed that insulin-stimulated phosphorylation of AKT-Thr308, AS160-Thr642 and GSK3β-Ser9 was reduced in variant myotubes compared to controls. The binding of variant AKT2-PH domain to PI(3,4,5)P3 was reduced as compared to the control protein. PamGene® kinome profiling revealed multiple differentially phosphorylated kinase substrates, e.g. calmodulin, between the genotypes. Further in silico upstream kinase analysis predicted a large-scale impairment in activities of kinases participating, for example, in intracellular signal transduction, protein translation and cell cycle events. In conclusion, myotubes from p.P50T/AKT2 variant carriers show multiple signalling alterations which may contribute to predisposition to insulin resistance and T2D in the carriers of this signalling variant.
芬兰特有的基因变异 p.P50T/AKT2（次要等位基因频率（MAF）=1.1%）与胰岛素抵抗和 2 型糖尿病的易感性增加有关。在这里，我们研究了该基因变异对来自 14 名男性 p.P50T/AKT2 变异携带者和 14 名对照者的人原代骨骼肌细胞中葡萄糖代谢和细胞内信号转导的体外影响。用 2-[1,2-3H]-脱氧-D-葡萄糖和 D-[14C]-葡萄糖分别检测胰岛素刺激的葡萄糖摄取和葡萄糖掺入糖原，并用 Seahorse XFe96 分析仪测量糖酵解率。用 Western blot 法研究胰岛素信号转导。用 PIP StripTM 膜测定变异和对照 AKT2-PH 域与磷脂酰肌醇（3,4,5）-三磷酸（PI(3,4,5)P3）的结合。使用 PamGene®激酶组蛋白分析系统进行蛋白酪氨酸激酶和丝氨酸-苏氨酸激酶测定。体外肌管中胰岛素刺激的葡萄糖摄取和糖原合成不受基因型的显著影响。然而，胰岛素刺激的糖酵解率在变异肌管中受损。Western blot 分析显示，与对照相比，变异肌管中胰岛素刺激的 AKT-Thr308、AS160-Thr642 和 GSK3β-Ser9 的磷酸化减少。与对照蛋白相比，变异 AKT2-PH 结构域与 PI(3,4,5)P3 的结合减少。PamGene®激酶组蛋白分析显示，两种基因型之间存在多种差异磷酸化激酶底物，例如钙调蛋白。进一步的计算机上游激酶分析预测，参与细胞内信号转导、蛋白质翻译和细胞周期事件等的激酶的活性会出现大规模损伤。总之，p.P50T/AKT2 变异携带者的肌管显示出多种信号改变，这可能导致该信号变异携带者易患胰岛素抵抗和 2 型糖尿病。
