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 XF^e96 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 Strips^TM 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.

芬兰特异性基因变异 p.P50T/ AKT2（次要等位基因频率 (MAF) = 1.1%）与胰岛素抵抗和 2 型糖尿病易感性增加有关。在这里，我们在体外研究了基因变异对人类初级骨骼肌细胞中葡萄糖代谢和细胞内信号传导的影响，这些细胞是从 14 名男性 p.P50T/ AKT2变异携带者和 14 名对照中建立的。^{分别用 2-[1,2- 3} H]-脱氧-D-葡萄糖和 D-[ ¹⁴ C]-葡萄糖检测胰岛素刺激的葡萄糖摄取和葡萄糖掺入糖原，并用海马XF ^e96分析仪。用蛋白质印迹研究胰岛素信号传导。使用 PIP Strips ^TM Membranes测定变体和对照 AKT2-PH 结构域与磷脂酰肌醇 (3,4,5)-三磷酸 (PI(3,4,5)P _{3 ) 的结合。}使用 PamGene® 激酶组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着影响。然而，胰岛素刺激的糖酵解速率在变体肌管中受损。蛋白质印迹分析显示胰岛素刺激的 AKT-Thr ³⁰⁸、AS160-Thr ⁶⁴²和 GSK3β-Ser ^9磷酸化与对照相比，变体肌管中减少。与对照蛋白相比，变体 AKT2-PH 结构域与 PI(3,4,5)P ₃的结合减少了。PamGene® 激酶组分析揭示了基因型之间的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的激酶活性会出现大规模损伤。总之，来自 p.P50T/ AKT2变体携带者的肌管显示出多种信号改变，这可能导致该信号变体携带者易患胰岛素抵抗和 T2D。