Live cell GLUT4 translocation assay reveals Per3 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 in its pathology due to its role in postprandial glucose disposal. Insulin resistance in skeletal muscle often precedes T2D diagnosis and is linked to impaired GLUT4 trafficking. Additionally, circadian disruptions are increasingly recognized as contributors to metabolic dysfunction. We have shown that skeletal muscle cells from T2D donors exhibit disrupted CLOCK/BMAL1 rhythms, impairing mitochondrial oxidative function. To investigate circadian insulin sensitivity, we developed a high-throughput, live-cell assay for GLUT4 translocation, allowing us to explore novel regulators of metabolic rhythmicity in skeletal muscle.

In this study, we analysed circadian insulin sensitivity in skeletal muscle using transcriptomics, PheWAS, and GLUT4-HiBiT assays. L6 and human myocytes were cultured, transfected, and assessed via our novel live-cell, circadian GLUT4 translocation assay. Circadian rhythms were analysed (JTK_CYCLE), and siRNA knockdowns targeted key genes, with glucose uptake measured using the Uptake-Glo assay in human myocytes.

Skeletal muscle cells from individuals with T2D exhibited disrupted circadian rhythms, with altered rhythmicity in ARNTL, HOXB5, PER3 , and TSSK6 ( p <0.05). Public database analysis revealed significant associations between these genes and T2D ( p =0.0001–0.03). Our high-sensitivity, live-cell assay demonstrated a 120% increase in GLUT4 translocation at 40 nM insulin ( p <0.001). Circadian rhythmicity in GLUT4 translocation was evident at physiological insulin levels (5 & 30 nM). PER3/Per3 knockdown significantly reduced GLUT4 translocation ( p <0.001) and glucose uptake in human muscle cells ( p <0.01), while Arntl knockdown caused a moderate reduction ( p <0.01) in GLUT4 translocation. Per3 silencing also abolished circadian rhythmicity in GLUT4 translocation ( p <0.01).

These findings establish PER3/Per3 as a key regulator of circadian GLUT4 translocation and insulin sensitivity, linking its dysregulation to metabolic impairments in T2D. Additionally, this study presents a novel live-cell GLUT4 translocation assay that is highly sensitive and capable of performing time-course or circadian experiments in the same group of cells. The sensitivity of this assay, coupled with reduced variation that is possible from using live-cells in time-course assays make it a powerful new tool for the discovery of GLUT4 translocation regulators. In this study, we have identified Per3 as a novel regulator of GLUT4 translocation in skeletal muscle cells.