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

Abstract

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-[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 ϳ65% reduction in both insulininduced T-tubular PIP3 production and GLUT4-GFP translocation was seen. Sarcolemma was less affected, with reductions of ϳ40% in PIP3 production and ϳ15% 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. Diabetes 57: 13-23, 2008 S keletal muscle plays an important role in glucose homeostasis, and defects in glucose uptake in muscle are involved in states of insulin resistance (e.g., type 2 diabetes). Upon stimulation with insulin, the GLUT4 glucose transporters in skeletal muscle fibers are translocated from intracellular compartments to the plasma membrane and T-tubules, and, in turn, glucose uptake is increased (1,2). In type 2 diabetes, the number of GLUT4 transporters in muscle is normal (3,4), and in other states of insulin resistance as well, reductions in GLUT4 content cannot fully explain the diminished glucose transport in muscle (5-7). Accordingly, the insulin signaling and/or the trafficking of GLUT4 must be impaired (4). However, the nature of these cellular defects is poorly understood. By imaging the dynamic changes in localization of green fluorescent protein (GFP)-tagged proteins in skeletal muscle fibers in situ in normal living mice (8), we have revealed previously unnoticed signaling functions of the T-tubule system ...

The cholesterol-lowering agent methyl-β-cyclodextrin promotes glucose uptake via GLUT4 in adult muscle fibers and reduces insulin resistance in obese mice

“…The high plasma membrane cholesterol content of 3T3-L1 adipocytes (37) and L6 myotubes cultured in the presence of fatty acids (19) decreases insulin-promoted glucose transport in these cells. Additionally, a recent report indicates that a highfat diet reduces insulin signaling, abolishing phosphatidylinositol 3,4,5-phosphate production and GLUT4 translocation to the T-tubules of live mouse skeletal muscle (35). Moreover, treatment of 3T3-L1 adipocytes with chromium picolinate, a compound that removes membrane cholesterol, activates GLUT4 trafficking and enhances insulin-stimulated glucose transport via a cholesterol-dependent mechanism (10).…”

Increased subsarcolemmal lipids in type 2 diabetes: effect of training on localization of lipids, mitochondria, and glycogen in sedentary human skeletal muscle

“…Moreover, morphological analyses have shown that the content of SS mitochondria is increased more than IMF mitochondria following endurance training (13). Advances in confocal imaging techniques have revealed compartmentalized GLUT4 translocation, where GLUT4 vesicle movement of longer distances only took place in the SS region (21), and a high-fat-fed diet reduced GLUT4 translocation in the t-system to a greater extent than in the sarcolemma (22). Furthermore, in carbohydrate metabolism, localization dependency has been found regarding glycogen synthase activity (34), rate of glycogen resyntheses after exercise (25), and role of glycogen in single fiber contractility (29).…”

A common trafficking route for GLUT4 in cardiomyocytes in response to insulin, contraction and energy-status signalling

“…The notion of differential regulation of GLUT4 recruitment to the sarcolemma and transverse-tubules is further supported by microscopy studies on skeletal muscle from animal models of insulin resistance. In denervated muscle and muscle from fat-fed animals, reductions in GFP-GLUT4 translocation are reported to occur mainly at the transverse-tubule system (Lauritzen et al, 2008b).…”