Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat.

Abstract

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...

Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes

“…Under the same conditions, "40 % of the intracellular GLUT4 was ablated (Figures 4 and 5). This result is consistent with the immunoadsorption studies ( Figure 1) and also with previous electron microscopy studies [3,4]. Hence, in nonstimulated adipocytes as much as 40 % of the intracellular GLUT4 is in the recycling compartment.…”

“…The TfR is considered to be the best marker of early recycling endosomes and so this would tend to exclude this compartment as being a candidate. This is consistent with previous electron microscopy studies [3,4], and also supported by our observation that cellubrevin is quantitatively ablated using this methodology (S. Martin, C. Livingstone, G. W. Gould and D. E. James, unpublished work). This conclusion is further strengthened by our demonstration that the distribution of rab5 is markedly changed on sucrose gradients.…”

“…For instance, the most likely interpretation of the data in Figure 1 is that GLUT4 is distributed both in early endosomes containing the TfR and a distinct compartment. This interpretation is consistent with electron microscopy data [3,4]. In order to test this hypothesis more directly and to further characterize the intracellular GLUT4 compartment in adipocytes we have utilized a Tf-HRP conjugate to ablate the early endosomal\recycling system.…”

“…Despite considerable efforts to characterize the intracellular GLUT4 compartment in muscle and fat cells it is still unclear whether GLUT4 is targeted to a unique secretory compartment or to a subcompartment within the pre-existing endosomal system [3,4,9,37,38]. In the present study we have utilized the TfR as a prototypic marker of the endosomal\recycling system in an effort to distinguish between each of these possibilities.…”

“…GLUT4 is decreased by 40-50 % at each of these locations by insulin, suggesting that all of these compartments participate in the insulin-regulated recycling of GLUT4 [3,4].…”