Live-cell GLUT4 translocation assay reveals <i>Per3</i> as a novel regulator of circadian insulin sensitivity in skeletal muscle cells

Type 2 diabetes (T2D) is a growing global health concern, with skeletal muscle playing a central role due to its contribution to postprandial glucose disposal. Insulin resistance in skeletal muscle often precedes the clinical onset of T2D and is characterised by impaired GLUT4 trafficking. Circadian disruption is increasingly recognised as a contributor to metabolic dysfunction, yet its impact on skeletal muscle insulin sensitivity remains poorly defined. We hypothesised that circadian regulators influence GLUT4 translocation and glucose uptake, contributing to the metabolic impairments observed in T2D. To investigate this, we developed a high-throughput, live-cell GLUT4 translocation assay capable of capturing circadian dynamics in skeletal muscle cells. Using publicly available transcriptomic data from primary human myotubes derived from individuals with and without T2D, our re-analysis identified altered rhythmic expression of several genes, including PER3, ARNTL, HOXB5, and TSSK6. Publicly available phenome-wide association study (PheWAS) data further supported associations between these genes and T2D-related traits. Functional validation using siRNA knockdown revealed that PER3 silencing significantly impaired GLUT4 translocation and glucose uptake in human skeletal muscle cells, while also abolishing rhythmic insulin responsiveness. ARNTL knockdown caused a moderate reduction in GLUT4 translocation, suggesting complementary roles in metabolic regulation. Our findings identify PER3 as a novel circadian regulator of GLUT4 translocation and insulin sensitivity in skeletal muscle. This work also introduces a sensitive, live-cell assay suitable for real-time assessment of GLUT4 dynamics and circadian regulation, offering a powerful platform for discovering new therapeutic targets in T2D.