Abstract. Antibodies specific for the insulin-regulatable glucose transporter (GLUT 4) were used to immunolocalize this protein in brown adipose tissue from basal-and insulin-treated rats. Cryosections of fixed tissue were incubated with antibodies, which were subsequently labeled with Protein A/gold and examined by EM. Antibodies against albumin and cathepsin D were also used with gold particles of different sizes to identify early and late endosomes, respectively. Under basal conditions 99 % of the GLUT 4 labeling was located within the cell. Labeling was predominantly in the trans-Golgi reticulum and tubulo-vesicular structures elsewhere in the cytoplasm. In insulin-stimulated cells ,040% of the GLUT 4 labeling was at the cell surface, where it was randomly distributed, except for occasional clustering in coated pits. Moreover, after insulin treatment, GLUT 4 was also enriched in early endosomes. We conclude that translocation of GLUT 4 to the cell surface is the major mechanism by which insulin increases glucose transport. In addition, these results suggest that in the presence of insulin GLUT 4 recycles from the cell surface, probably via the coated pit-endosome pathway that has been characterized for cell surface receptors, and also that insulin causes the redistribution of GLUT 4 by stimulating exocytosis from GLUT 4-containing tubulo-vesicular structures, rather than by slowing endocytosis of GLUT 4.

The insulin-regulated glucose transporter GLUT4 was immunolocalized in rat cardiac muscle under conditions of basal and stimulated glucose uptake, achieved by fasting and a combined exercise/insulin stimulus, respectively. In basal myocytes there was very little (<1%) GLUT4 in the different domains of the plasma membrane (sarcolemma, intercalated disk, and transverse tubular system). GLUT4 was localized in small tubulo-vesicular elements that occur predominantly near the sarcolemma and the transverse tubular system and in the trans-Golgi region. Upon stimulation -42% of GLUT4 was found in the plasma membrane. Each domain of the plasma membrane contributed equally to this effect. GLUT4-positive, clathrin-coated pits were also present at each cell surface domain. The remainder of the labeling was in tubulo-vesicular elements at the same sites as in basal cells and in the intercalated disk areas. The localization of GLUT4 in cardiac myocytes is essentially the same as in brown adipocytes, skeletal muscle, and white adipocytes. We conclude that increased glucose transport in muscle and fat is accounted for by translocation of GLUT4 from the intracellular tubulo-vesicular elements to the plasma membrane. The labeling of coated pits indicates that in stimulated myocytes, as in adipocytes, GLUT4 recycles constantly between the endosomal compartment and the plasma membrane and that stimulation of the exocytotic rate constant is likely the major mechanism for GLUT4 translocation.Glucose transport in muscle and fat is acutely regulated by factors such as insulin and exercise (1, 2). These tissues express a unique insulin-regulatable glucose transporter, GLUT4, that is not found in other tissues. In the postabsorptive state, muscle is the primary site for insulinstimulated glucose disposal (3). Thus the regulation of muscle glucose transport by insulin is a critical determinant of whole body glucose homeostasis. Most research concerning insulin regulation ofglucose transport has focused on adipocytes. By using various techniques it has been shown that insulin stimulates glucose transport by means of translocation of GLUT4 from an internal pool to the plasma membrane (4-6). Due to a number of limitations it has been difficult to perform similar analyses in muscle. First, it is difficult to perform subcellular fractionation in muscle due in part to the presence of multiple surface membranes (7). These include the sarcolemma, the lateral membrane of the cell; the transverse tubular system (T system), the membrane that protrudes transversely (and axially in heart) through the muscle cells; and (only in heart) the intercalated disk (ID), the membrane adjacent to the sarcolemma that crosses the fibrillar texture of the tissue and that is occupied by many junctional specialties. Second, there is a lack of suitable muscle cell culture lines that exhibit insulin-stimulated glucose transport. Third, metabolic control is closely linked to innervation and/or contractility in muscle. Thus, glucose transport may be activated dur...