Denervation and high-fat diet reduce insulin signaling in T-tubules in skeletal muscle of living mice.
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.


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-[(3)H]-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 approximately 65% reduction in both insulin-induced T-tubular PIP3 production and GLUT4-GFP translocation was seen. Sarcolemma was less affected, with reductions of approximately 40% in PIP3 production and approximately 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.
Lauritzen
Hans P M
HP
Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USA. hans.lauritzen@joslin.harvard.edu
Ploug
Thorkil
T
Ai
Hua
H
Donsmark
Morten
M
Prats
Clara
C
Galbo
Henrik
H
D000818:Animals; D004041:Dietary Fats / Q000494:pharmacology*; D005267:Femoral Nerve / Q000502:physiology; D051275:Glucose Transporter Type 4 / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D007328:Insulin / Q000502:physiology*; D008297:Male; D051379:Mice; D008870:Microtubules / Q000187:drug effects / Q000502:physiology*; D009121:Muscle Denervation; D018482:Muscle, Skeletal / Q000187:drug effects / Q000502:physiology*; D012584:Sciatic Nerve / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014162:Transfection
D004041:Dietary Fats; D051275:Glucose Transporter Type 4; D007328:Insulin; D049452:Green Fluorescent Proteins