Chromatographic resolution of insulin receptor from insulin-sensitive D-glucose transporter of adipocyte plasma membranes

Biochemistry ◽  
1981 ◽  
Vol 20 (1) ◽  
pp. 216-221 ◽  
Author(s):  
Christin Carter-Su ◽  
Paul F. Pilch ◽  
Michael P. Czech
Keyword(s):  
Insulin Receptor ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Chromatographic Resolution ◽  
Adipocyte Plasma Membranes

scholarly journals Insulin Stimulates Membrane Fusion and GLUT4 Accumulation in Clathrin Coats on Adipocyte Plasma Membranes

2007 ◽  
Vol 27 (9) ◽  
pp. 3456-3469 ◽  
Author(s):  
Shaohui Huang ◽  
Larry M. Lifshitz ◽  
Christine Jones ◽  
Karl D. Bellve ◽  
Clive Standley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Insulin Signaling ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Tirf Microscopy ◽  
Adipocyte Plasma Membranes ◽  
Kinetics Of

ABSTRACT Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by ∼4-fold, increasing GLUT4 content in the plasma membrane. Remarkably, insulin signaling modulates the kinetics of the fusion process, decreasing the vesicle tethering/docking duration prior to membrane fusion. In contrast, the kinetics of GLUT4 molecules spreading out in the plasma membrane from exocytic fusion sites is unchanged by insulin. As GLUT4 accumulates in the plasma membrane, it is also immobilized in punctate structures on the cell surface. A previous report suggested these structures are exocytic fusion sites (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF microscopy using fluorescent proteins fused to clathrin light chain or GLUT4 reveals these structures are clathrin-coated patches. Taken together, these data show that insulin signaling accelerates the transition from docking of GLUT4-containing vesicles to their fusion with the plasma membrane and promotes GLUT4 accumulation in clathrin-based endocytic structures on the plasma membrane.

scholarly journals Identification of the glucose transporter in mammalian cell membranes with a125I-forskolin photoaffinity label

1988 ◽  
Vol 255 (3) ◽  
pp. 983-990 ◽  
Author(s):  
B E Wadzinski ◽  
M F Shanahan ◽  
R B Clark ◽  
A E Ruoho
Keyword(s):  
Smooth Muscle ◽  
Molecular Mass ◽  
Mammalian Cell ◽  
Cell Membranes ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Apparent Molecular Mass ◽  
Synaptic Membranes ◽  
Placental Membranes ◽  
Adipocyte Plasma Membranes

The glucose transporter has been identified in a variety of mammalian cell membranes using a photoactivatable carrier-free radioiodinated derivative of forskolin, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n ([125I]IAPS-forskolin) at 1-3 nM. The membranes that were photolabelled with [125I]IAPS-forskolin were human placental membranes, rat cortical and cerebellar synaptic membranes, rat cardiac sarcolemmal membranes, rat adipocyte plasma membranes, smooth-muscle membranes, and S49 wild-type (WT) lymphoma-cell membranes. The glucose transporter in plasma membranes prepared from the insulin-responsive rat cardiac sarcolemmal cells, rat adipocytes and smooth-muscle cells were determined to be approx. 45 kDa by SDS/polyacrylamide-gel electrophoresis (PAGE). Photolysis of human placental membranes, rat cortical and cerebellar synaptic membranes, and WT lymphoma membranes with [125I]-IAPS-forskolin, followed by SDS/PAGE, indicated specific derivatization of a broad band (43-55 kDa) in placental membranes and a narrower band (approx. 45 kDa) in synaptic membranes and WT lymphoma membranes. Digestion of the [125I]IAPS-forskolin-labelled placental and WT lymphoma membranes with endo-beta-galactosidase showed a reduction in the apparent molecular mass of the radiolabelled band to approx. 40 kDa. The membranes that were photolabelled with [125I]IAPS-forskolin and trypsin-treated produced a radiolabelled proteolytic fragment with an apparent molecular mass of 18 kDa. [125I]IAPS-forskolin is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.

scholarly journals Cycloheximide decreases glucose transporters in rat adipocyte plasma membranes without affecting insulin-stimulated glucose transport

1988 ◽  
Vol 251 (2) ◽  
pp. 491-497 ◽  
Author(s):  
S Matthaei ◽  
J M Olefsky ◽  
E Karnieli
Keyword(s):  
Protein Synthesis ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Glucose Transporters ◽  
Electrophoretic Analysis ◽  
Adipocyte Plasma Membranes ◽  
Inhibitor Of Protein Synthesis ◽  
Adipose Cells

This study examines the relationship between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters in isolated rat adipocytes. Adipose cells were incubated with or without cycloheximide, a potent inhibitor of protein synthesis, for 60 min and then for an additional 30 min with or without insulin. After the incubation we measured 3-O-methylglucose transport in the adipose cells, and subcellular membrane fractions were prepared. The numbers of glucose transporters in the various membrane fractions were determined by the cytochalasin B binding assay. Basal and insulin-stimulated 3-O-methylglucose uptakes were not affected by cycloheximide. Furthermore, cycloheximide affected neither Vmax. nor Km of insulin-stimulated 3-O-methylglucose transport. In contrast, the number of glucose transporters in plasma membranes derived from cells preincubated with cycloheximide and insulin was markedly decreased compared with those from cells incubated with insulin alone (10.5 +/- 0.8 and 22.2 +/- 1.8 pmol/mg of protein respectively; P less than 0.005). The number of glucose transporters in cells incubated with cycloheximide alone was not significantly different compared with control cells. SDS/polyacrylamide-gel-electrophoretic analysis of [3H]cytochalasin-B-photolabelled plasma-membrane fractions revealed that cycloheximide decreases the amount of labelled glucose transporters in insulin-stimulated membranes. However, the apparent molecular mass of the protein was not changed by cycloheximide treatment. The effect of cycloheximide on the two-dimensional electrophoretic profile of the glucose transporter in insulin-stimulated low-density microsomal membranes revealed a decrease in the pI-6.4 glucose-transporter isoform, whereas the insulin-translocatable isoform (pI 5.6) was decreased. Thus the observed discrepancy between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters strongly suggests that a still unknown protein-synthesis-dependent mechanism is involved in insulin activation of glucose transport.

Quantity of Na/K-ATPase and glucose transporters in the plasma membrane of rat adipocytes is reduced by in vivo triiodothyronine

1995 ◽  
Vol 133 (5) ◽  
pp. 626-634 ◽  
Author(s):  
Marianne Voldstedlund ◽  
Jørgen Tranum-Jensen ◽  
Aase Handberg ◽  
Jørgen Vinten
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Insulin Stimulation ◽  
Rat Adipocytes ◽  
Adipocyte Plasma Membranes ◽  
Glucose Transporter Glut4

Voldstedlund M. Tranum-Jensen J, Handberg A, Vinten J. Quantity of Na/K-ATPase and glucose transporters in the plasma membrane of rat adipocytes is reduced by in vivo triiodothyronine. Eur J Endocrinol 1995:133:626–34. ISSN 0804–4643 The expression of sodium-potassium pumps and glucose transporters in pure adipocyte plasma membranes from a hyperthyroid animal model was studied. Hyperthyroidism was induced by enteral administration of five doses of 90 μg of triiodothyronine every second day to 8-week-old rats. Following isolation of epididymal adipocytes, 3-O-methylglucose transport was measured and the number of Na/K-ATPase-(α1- and α2-isoforms) and glucose transporter (GLUT1 and GLUT4) molecules in sheets of adipocyte plasma membrane were determined by quantitative immunoelectron microscopy, using gold labelling. Maximal in vitro insulin stimulation of adipocytes increased the glucose transport rate and the amount of GLUT4 in the plasma membrane 15-fold, whereas the amount of α2 was unaffected, In adipocytes from hyperthyroid rats, mean adipocyte volume was decreased by 18% and the quantities of GLUT4 per unit area of plasma membrane (maximal insulin stimulation) and of α2 were decreased by 19% and 15% respectively. Thus, hypotrophia of fat tissue in the hyperthyroid state is associated with a decreased expression in the plasma membrane of the glucose transporter GLUT4 and the α2 -isoform of Na/K-ATPase. Marianne Voldstedlund, Department of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark

Adipocyte insulin-binding species: the 40 Å Stoke's radius protein

1984 ◽  
Vol 62 (7) ◽  
pp. 566-570
Author(s):  
C. Elliott ◽  
H. Joseph Goren
Keyword(s):  
Insulin Receptor ◽  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Relative Mass ◽  
Single Polypeptide Chain ◽  
Dodecyl Sulfate ◽  
Single Polypeptide ◽  
Adipocyte Plasma Membranes

Several laboratories have demonstrated the presence of large (70 Å) (1 Å = 0.1 nm) and small (40 Å) insulin receptors. This report provides evidence that the 40 Å insulin receptor migrates on dodecyl sulfate – acrylamide gel electrophoresis as a 90 000 dalton protein and that this protein is a single polypeptide chain. 125I-labeled insulin was bound to plasma membranes from isolated rat adipocytes. Following removal of unbound 125I-labeled insulin, the mixture was exposed to disuccinimidyl suberate. Proteins tagged with 125I-labeled insulin were separated by dodecyl sulfate gel electrophoresis or Sepharose 6B chromatography. Autoradiography of the gels demonstrated several large (relative mass (Mr) > 300 000) and one small (Mr ~ 90 000) labeled protein in nonreduced membrane proteins. Dithiothreitol reduction decreased the large insulin-binding species to its known subunits, but the 90 000 dalton protein did not decrease in size. Triton X-100 solubilized plasma membranes were separated by Sepharose 6B chromatography. One labeled protein, with Kav = 0.57 elution position, on dodecyl sulfate gel electrophoresis migrated as a 90 000 dalton protein. Thus, rat adipocyte plasma membranes contain both an oligomeric insulin-binding species and a monomeric insulin-binding species. The relationship of the monomeric to the oligomeric insulin receptor is discussed.

scholarly journals Recycling of the glucose transporter, the insulin receptor, and insulin in rat adipocytes. Effect of acidtropic agents.

1986 ◽  
Vol 261 (7) ◽  
pp. 3295-3305
Author(s):  
O Ezaki ◽  
M Kasuga ◽  
Y Akanuma ◽  
K Takata ◽  
H Hirano ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Glucose Transporter ◽  
Rat Adipocytes

scholarly journals Quantitative Aspects of the Insulin-Receptor Interaction in Liver Plasma Membranes

1974 ◽  
Vol 249 (7) ◽  
pp. 2249-2257 ◽  
Author(s):  
C. Ronald Kahn ◽  
Pierre Freychet ◽  
Jesse Roth ◽  
David M. Neville
Keyword(s):  
Insulin Receptor ◽  
Plasma Membranes ◽  
Receptor Interaction ◽  
Liver Plasma Membranes
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