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 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 similar to 80%), whereas a smaller elevation (35%) was observed in another fraction enriched with T tubules. Insulin induced a concomitant reduction (similar to 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.