A new procedure for the isolation of plasma membranes, T tubules, and internal membranes from skeletal muscle.
A new subcellular fractionation procedure for the simultaneous isolation of plasma membranes and transverse (T) tubule membranes from a rat skeletal muscle was developed. This new technique allows the isolation and separation of plasma membranes and T tubules in distinct subcellular fractions, as revealed by the membrane distribution of enzymatic and immunologic markers of both cell surface compartments. The procedure also yields a novel membrane fraction that is devoid of markers of both surface domains but is markedly enriched with GLUT-4 glucose transporters, thus strongly suggesting that it represents an intracellular pool of GLUT-4. Using this new procedure, we found that acute in vivo insulin administration (30 min) increased GLUT-4 protein content in the plasma membrane and a T tubule fraction (by approximately 80%), whereas a smaller elevation (35%) was observed in another fraction enriched with T tubules. Insulin induced a concomitant reduction (approximately 40%) in GLUT-4 abundance in the intracellular fraction. These results further support the hypothesis that T tubules are involved in the regulation of glucose transport in skeletal muscle. This novel fractionation method will be useful in investigating the regulation of muscle GLUT-4 transporters in other physiological and disease states such as diabetes, where defective translocation of the transporter protein to either one or both cell surface domains is suspected to occur.
Dombrowski
L
L
Department of Physiology, Laval University Hospital Research Center, Quebec, Canada.
Roy
D
D
Marcotte
B
B
Marette
A
A
D000818:Animals; D002462:Cell Membrane; D005591:Chemical Fractionation; D051275:Glucose Transporter Type 4; D006652:Histological Techniques; D007328:Insulin / Q000378:metabolism; D007425:Intracellular Membranes; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D018482:Muscle, Skeletal / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000378:metabolism
D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat