Insulin-resistant human and rodent muscle exhibit impaired insulin-stimulated GLUT4 translocation ( Zierath et al., 1996 King et al., 1992 Etgen et al., 1997 Garvey et al., 1998) and muscle-specific deletion of GLUT4 is sufficient to cause systemic insulin resistance and glucose intolerance ( Zisman et al., 2000). Skeletal muscle is quantitatively the largest site of glucose disposal, a process facilitated by insulin and contraction-responsive translocation and insertion of glucose transporter 4 (GLUT4) into the surface membrane of muscle fibers ( Jaldin-Fincati et al., 2017 Klip et al., 2019). Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell surface recruitable GLUT4 pool via kinesin-1-mediated trafficking. Transient knockdown of the microtubule motor protein kinesin-1 protein KIF5B in L6 muscle cells reduced insulin-stimulated GLUT4 translocation while pharmacological kinesin-1 inhibition in incubated mouse muscles strongly impaired insulin-stimulated glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. In contrast, a 2-hr Noco treatment markedly decreased insulin responsiveness of glucose uptake. Using a perifused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. We found GLUT4 localized on the microtubules in mouse and human muscle fibers. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Microsystems Laboratory 2, Institute of Electrical and Micro Engineering, École Polytechnique Fédérale de Lausanne, Switzerland.Laboratory of Integrative Systems Physiology, Institute of Bioengineering, École Polytechnique Fédérale de Lausanne, Switzerland.Department of Electrical Engineering, Polytechnique Montréal, Canada.Clinical Drug Development, Novo Nordisk, Denmark.Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.College of Physical Education, Chongqing University, China.Exercise Science Laboratory, Faculty of Medicine, Universidad Finis Terrae, Chile.Heart and Skeletal Muscle Biology, Global Drug Discovery, Novo Nordisk, Denmark.August Krogh Section for Molecular Physiology, Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Denmark.
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