A new fractionation procedure for the simultaneous isolation of plasma membranes, transverse tubules, and intracellular membranes from skeletal muscle: characterization and effects of insulin on glut4. Dombrowski, Luce, Denis Roy, Bruno Marcotte, and Andr[acute]e Marette. Department of Physiology & Lipid Research Unit, Laval University Hospital Research Center, Qu[acute]ebec, Canada, G1V 4G2
APStracts 2:0233E, 1995.
A new subcellular fractionation procedure for the simultaneous isolation of plasma membranes and transverse tubule membranes from rat skeletal muscle was developed. This new technique allows the isolation and separation of plasma membranes and transverse 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 GLUT4 glucose transporters, thus strongly suggesting that it represents an intracellular pool of GLUT4. Using this new procedure, it was found that acute in vivo insulin administration (30 min) increased GLUT4 protein content in the plasma membrane and a transverse tubule fraction (by 80%) whereas a smaller elevation (35%) was observed in another fraction enriched with transverse tubules. Insulin induced a concomitant reduction (40%) in GLUT4 abundance in the intracellular fraction. These results further support the hypothesis that transverse 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 GLUT4 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. 

Received 12 July 1995; accepted in final form 9 November 1995.
APS Manuscript Number E324-5.
Article publication pending Am. J. Physiol. (Endocrinol. Metab.).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 8 December 95