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

¹ Research Division, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts
² Copenhagen Muscle Research Centre, Department of Medical Physiology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark
³ Department of Rheumatology, Bispebjerg Hospital, Copenhagen, Denmark

Address correspondence and reprint requests to H.P.M. Mortensen Lauritzen, PhD, Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215. E-mail: hans.lauritzen{at}joslin.harvard.edu

Key Words: [³H]2-DG, 2-deoxy-D-[³H]-glucose • 2-DG, 2-deoxy-glucose • ARNO, ADP-ribosylation factor nucleotide-binding site opener • EDL, extensor digitorum longus • GFP, green fluorescent protein • PI3-K, phosphatidylinositide 3-kinase • PIP3, phosphatidylinositol 3,4,5 P₃ • ROI, region of interest

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-ribosylation 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 P₃ (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.

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