Immunolocalization of Glucose Transporter GLUT4 Within Human Skeletal Muscle

Abstract

To investigate the cellular and subcellular distribution of glucose transporters in skeletal muscle, the glucose transporter isoform GLUT4 was localized in human muscle by electron microscopy via immunogold labeling with monoclonal (1F8) or COOH-terminal peptide polyclonal (ECU4) antibody and in isolated rat membranes by Western blot. There was no labeling of GLUT4 in endothelial cells of the capillaries. There also was no labeling of GLUT4 on the surface plasma membrane (sarcolemma) under either basal or insulinstimulated conditions. Specific labeling for GLUT4 was clearly observed in two compartments: within the triad (on terminal cisternae and transverse tubules) and on an intracellular compartment, possibly sarcoplasmic tubules. Isolated triad membranes from rat muscle also contained substantial quantities of GLUT4 transporter, but there was no detectable GLUT4 protein in isolated sarcolemmal membranes. These data suggest a possible mechanism that involves glucose transport across the muscle cell at the transverse tubule membrane, not the sarcolemma. Diabetes 40:150-54, 1991 V ilaro et al. (1) reported that the insulin-regulatable glucose transporter (GLUT4) is found predominantly in capillary endothelial cells of muscle and adipose tissue. This intriguing observation suggests that control of glucose transport by insulin may be primarily at the level of the capillary rather than the cell membrane as previously believed. Because of the importance of this question, we have further investigated the cellular and subcellular distribution of GLUT4 in human skeletal muscle via immunocytochemical localization. In addition, we have isolated triad and sarcolemmal membranes from rat skeletal muscle to determine the localization of the GLUT4 transporter. Our data clearly demonstrate that there is no GLUT4 in capillary endothelium in human muscle. Moreover, no GLUT4 transporters are detectable in the surface plasma membrane (sarcolemma) under either basal or insulin-stimulated conditions. Instead, the transporters are concentrated in the triad (transverse tubules and terminal cisternae) and in an intracellular compartment, possibly sarcoplasmic tubules. In agreement with this distribution pattern in situ, we found high concentrations of GLUT4 protein in isolated triad membranes from rat muscle but no detectable amounts in isolated sarcolemma. RESEARCH DESIGN AND METHODSFor this study, nondiabetic lean human volunteers underwent a muscle punch biopsy of the vastus lateralis muscle in the basal state after an overnight fast and during the final 30 min of a 2-h hyperinsulinemic-euglycemic clamp at 40 mil • nrr 2 • min" 1 . Informed consent was obtained before the biopsy after the nature and risks of the procedure had been fully explained. Muscle biopsies and membrane fractions were fixed in 4% paraformaldehyde/1 % glutaraldehyde at 4°C for 2 h, followed by 1% osmium tetroxide at 4°C for 1 h and embedding in LR white resin. Fixatives were made in 0.1 M phosphate buffer at pH 7.4. LR white resin (medium grade; Ted Pella, Redd...

Immunogold labeling study of the distribution of GLUT-1 and GLUT-4 in cardiac tissue following stimulation by insulin or ischemia

“…This addition of contraction stimulation could account for the higher GLUT-4 in sarcolemmal membranes seen in these studies compared with our insulin-only stimulated hearts. Our data support previous findings in skeletal muscle, where insulinmediated glucose uptake occurred at the T tubules, only with no increase in GLUT-4 seen at the sarcolemmal membrane (13). This observation is also in agreement with other immunofluorescence studies performed in skeletal muscle cells (1,4,36).…”

The molecular basis of insulin-stimulated glucose uptake: signalling, trafficking and potential drug targets

“…Early studies involving the isolation of GLUT4-containing vesicles identified numerous GLUT4 vesicle resident proteins that were known to localise to distinct intracellular compartments, suggesting that GLUT4 localises to several different membrane pools (Tanner & Lienhard 1987, Kandror & Pilch 1994a,b, 1996, Hanpeter & James 1995, Keller et al 1995. These observations were supported by immunofluorescence and electron microscopy studies in insulin-responsive tissues, which demonstrated that GLUT4 is localised within complex tubulovesicular structures at the perinuclear region of the cell, to small peripheral vesicles in the cytoplasm and to clathrincoated pits at the cell surface (Friedman et al 1991, Slot et al 1991, Smith et al 1991, Bornemann et al 1992. Colocalisation studies using known protein markers confirmed that GLUT4 localises to numerous intracellular organelles that include the Golgi complex, the trans-Golgi network, lysosomes, the late endosomes and the recycling endosomes (Ralston & Ploug 1996).…”

Immunocytochemical localization of fat/muscle-type glucose transporter (GLUT4) in the rat skeletal muscle: Effect of insulin treatment.

“…Based on immunoelectron microscopic observations, Friedman et al reported that GLUT4 in the human vastus lateralis muscle was localized mainly in the triad (transverse tubules and terminal cisternae) and sarcoplasmic tubules (7). In our present work, the labeling for GLUT4 in these intracellular compartments was weak, if anything, compared with the dense labeling in vesicles and tubules in the Golgi and near the cell surface.…”