Imaging GLUT4 in isolated human skeletal muscle fibres (#222)
Glucose transporter 4 (GLUT4) is the primary insulin and contraction responsive glucose transporter expressed in skeletal muscle. The translocation of GLUT4 to the cell surface is a crucial step in stimulating muscle glucose uptake and defects in this process underlie obesity-related insulin resistance and the pathogenesis of type 2 diabetes. However, skeletal muscle is complex with the sarcolemma and the T-tubules both important sites of GLUT4 translocation and glucose uptake, which makes the process difficult to assess in human skeletal muscle. We used an immunofluorescence approach to visualise the subcellular localisation of GLUT4 in isolated single human muscle fibres.
Muscle biopsies were collected following an overnight fast from 5 middle-aged males. Single muscle fibres were isolated from fresh tissue, pinned at ~1.5x resting length, fixed and permeabilised. Muscle fibres were immunostained using antibodies targeting GLUT4 and the dihydropyridine receptor (DHRP, a marker of the T-tubular network). Images were viewed with confocal microscopy.
GLUT4 staining was highly prevalent in perinuclear regions with some non-continuous staining at the sarcolemma. DHPR staining presented continuous double bands which ran perpendicular to the muscle fibre axis representing the T-tubular network. Single GLUT4 striations also ran perpendicular to the muscle fibres axis and closely associated with the T-tubule marker DHPR. At greater resolution, and in line with previous rodent data, small and larger GLUT4 clusters were distributed throughout the muscle fibre.
Immunofluorescence staining and high resolution imaging of isolated human muscle fibres allows GLUT4 localisation to be determined in relation to both the sarcolemma and the T-tubules. This offers a viable approach to investigate GLUT4 translocation to the entire surface membrane in response to both insulin and contraction in human skeletal muscle and provide new insight into GLUT4 translocation defects underlying skeletal muscle insulin resistance.