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

in Journal of Molecular Endocrinology
Authors:
Selina MäkinenS Mäkinen, Group of Metabolism, Minerva Foundation Institute for Medical Research, Helsinki, Finland

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Neeta DattaN Datta, Group of Metabolism, Minerva Foundation Institute for Medical Research , Helsinki, Finland

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Savithri RangarajanS Rangarajan, Pam Gene, PamGene International BV, 's-Hertogenbosch, Netherlands

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Yen NguyenY Nguyen, Group of Metabolism, Minerva Foundation Institute for Medical Research, Helsinki, Finland

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Vesa OlkkonenV Olkkonen, Group of Lipid Signaling, Minerva Foundation Institute for Medical Research, Helsinki, Finland

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Aino Latva-RaskuA Latva-Rasku, University of Turku and Turku University Hospital, Turku PET Centre, Turku, Finland

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Pirjo NuutilaP Nuutila, University of Turku and Turku University Hospital, Turku PET Centre, Turku, Finland

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Markku LaaksoM Laakso, Internal Medicine, University of Eastern Finland Institute of Clinical Medicine, Kuopio, Finland

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Heikki A KoistinenH Koistinen, Department of Medicine, University of Helsinki Faculty of Medicine, Helsinki, Finland

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Correspondence: Heikki Koistinen, Email: heikki.koistinen@helsinki.fi
Open access

Finnish-specific gene variant p.P50T/AKT2 (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, that 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. Binding of variant and control AKT2-PH domains to phosphatidylinositol (3,4,5)-trisphosphate ((PI(3,4,5)P3)) was assayed using PIP Strips™ Membranes. Tyrosine (PTK) and serine-threonine (STK) kinase assay was 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, 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. Binding of variant AKT2-PH domain to PI(3,4,5)P3 was reduced as compared to 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.

 

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