Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat.

Abstract

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.

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

“…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].…”

“…It is now well established that these membrane proteins recycle between an intracellular pool and the plasma membrane, and that this recycling involves the endosomal system (for a review, see [9]). Immunoelectron microscopy studies of GLUT4 in adipocytes have demonstrated that GLUT4 is also located, at least in part, within the endocytic system as it undergoes constitutive recycling through clathrin-coated pits [4,10]. Using membraneimpermeant, glucose transporter-specific photolabels, it has been clearly demonstrated that GLUT4 constantly recycles between the plasma membrane and an intracellular site, both in the presence and absence of insulin [11,12].…”

Selective release of circRNAs in platelet‐derived extracellular vesicles

“…For whole-mount analysis, EV suspensions (5 µL each) were incubated for 1 min on formvar and carbon-coated glow-discharged copper grids and subsequently stained with 2% uranyl acetate. Immunogold labelling was performed as described [22–24]. After blocking with 1% BSA in phosphate-buffered saline, grids were incubated with a 1:50 dilution of primary mouse anti-CD63 antibody (Thermo Fisher Scientific; TS63) in PBS containing 0.1% BSA for 1 h at room temperature.…”