Finnish-specific AKT2 gene variant leads to impaired insulin signalling in myotubes

Abstract

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)P ₃) 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-Thr ³⁰⁸, AS160-Thr ⁶⁴² and GSK3β-Ser ⁹ was reduced in variant myotubes compared to controls. The binding of variant AKT2-PH domain to PI(3,4,5)P ₃ 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.