Denervation and High-Fat Diet Reduce Insulin Signaling in T-Tubules in Skeletal Muscle of Living Mice

摘要

OBJECTIVE-Insulin stimulates muscle glucose transport by translocation of GLUT4 to sarcolemma and T-tubules. Despite muscle glucose uptake playing a major role in insulin resistance and type 2 diabetes, the temporal and spatial changes in insulin signaling and GLUT4 translocation during these conditions are not well described. RESEARCH DESIGN AND METHODS-We used time lapse confocal imaging of green fluorescent protein (GFP) ADP-ribo-sylation factor nucleotide-binding site opener (ARNO) (evaluation of phosphatidylinositide 3-kinase activation) and GLUT4-GFP-transfected quadriceps muscle in living, anesthetized mice either muscle denervated or high-fat fed. T-tubules were visualized with sulforhodamine B dye. In incubated muscle, glucose transport was measured by 2-deoxy-D-[~3H]-glucose uptake, and functional detubulation was carried out by osmotic shock. Muscle fibers were immunostained for insulin receptors. RESULTS-Denervation and high-fat diet reduced insulin-mediated glucose transport. In denervated muscle, insulin-stimulated phosphatidylinositol 3,4,5 P_3 (PIP3) production was abolished in T-tubules, while PIP3 production at sarcolemma was increased 2.6-fold. Correspondingly, GLUT4-GFP translocation to T-tubules was abolished, while translocation to sarcolemma was increased 2.3-fold. In high fat-fed mice, a ～ 65% reduction in both insulin-induced T-tubular PIP3 production and GLUT4-GFP translocation was seen. Sarcolemma was less affected, with reductions of ～ 40% in PIP3 production and ～ 15% in GLUT4-GFP translocation. Access to T-tubules was not compromised, and insulin receptor distribution in sarcolemma and T-tubules was unaffected by denervation or high-fat feeding. Detubulation of normal muscle reduced basal and abolished insulin-induced glucose transport. CONCLUSIONS-Our findings demonstrate, for the first time, that impaired insulin signaling and GLUT4 translocation is compartmentalized in muscle and primarily localized to T-tubules and not sarcolemma during insulin resistance.