<b>Analysis of multiple insulin actions in single muscle fibres from insulin resistant mice reveals selective defect in endogenous GLUT4 translocation</b>

<p dir="ltr">Accurate measurement of GLUT4 translocation is crucial for understanding insulin resistance in skeletal muscle, a key factor in the development of metabolic diseases. However, current methods rely on overexpressed epitope-tagged GLUT4 constructs or indirect measurements, limiting their physiological relevance and applicability. To overcome these challenges, we developed an innovative high-sensitivity imaging-based method that enables the direct assessment of endogenous GLUT4 translocation in primary skeletal muscle fibres. This approach utilises antibodies targeting exofacial epitopes on native GLUT4. Our method allows multiplexed analysis of multiple insulin-sensitive processes, including transferrin receptor trafficking and FOXO nuclear exclusion, alongside mitochondrial oxidative stress. This comprehensive approach provides a unique opportunity to simultaneously assess insulin action across different signalling branches within individual muscle fibres. We validated this method across multiple inbred mouse strains and models of insulin resistance, including chronic insulin exposure, palmitate treatment, and high-fat diet-induced obesity. Notably, we identified a selective defect in GLUT4 trafficking in insulin-resistant muscle fibres, while other insulin-dependent processes remained intact. By offering a high-fidelity model that maintains physiological relevance, this novel approach represents a significant advancement in the study of skeletal muscle insulin resistance and provides a powerful tool for dissecting gene-environment interactions that underlpin metabolic disease.</p>