Analysis of Multiple Insulin Actions in Single Muscle Fibers From Insulin-Resistant Mice Reveals Selective Defect in Endogenous GLUT4 Translocation

Insulin-stimulated GLUT4 translocation in skeletal muscle is crucial for whole-body glucose homeostasis, but direct methods to measure endogenous GLUT4 translocation in adult muscle are lacking. We developed a high-sensitivity, high-fidelity imaging method to directly assess endogenous GLUT4 translocation in primary skeletal muscle fibers without the need for overexpressed epitope-tagged constructs. The method enables simultaneous assessment of several makers of insulin sensitivity and mitochondrial oxidative stress within individual muscle fibers. The approach is versatile and was validated across genetically diverse mouse strains and under various insulin resistance conditions.