Photocatalyzed labeling of adipocyte plasma membranes with an aryl azide derivative of glucose.

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.

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HDL Binding to Human Adipocyte Plasma Membranes: Regional Variation in Omental and Subcutaneous Depots

Lipoprotein Deficiency Syndromes ◽

10.1007/978-1-4684-1262-8_6 ◽

1986 ◽

pp. 61-66

Author(s):

Bessie Fong ◽

Andrew Salter ◽

Jose Jimenez ◽

Jean-Pierre Despres ◽

Aubie Angel

Keyword(s):

Regional Variation ◽

Plasma Membranes ◽

Human Adipocyte ◽

Adipocyte Plasma Membranes

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Desensitization of beta-adrenergic receptors in adipocytes causes increased insulin sensitivity of glucose transport

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1996.271.2.e271 ◽

1996 ◽

Vol 271 (2) ◽

pp. E271-E276 ◽

Cited By ~ 5

Author(s):

A. Green ◽

R. M. Carroll ◽

S. B. Dobias

Keyword(s):

Insulin Sensitivity ◽

Glucose Transport ◽

Adrenergic Receptors ◽

Plasma Membranes ◽

Insulin Receptors ◽

Insulin Binding ◽

High Dose ◽

Beta Adrenergic Receptors ◽

Beta Adrenergic ◽

Adipocyte Plasma Membranes

To determine the effect of desensitization of adipocyte beta-adrenergic receptors on insulin sensitivity, rats were continuously infused with isoproterenol (50 or 100 micrograms.kg-1.h-1) for 3 days by osmotic minipumps. Epididymal adipocytes were isolated. The cells from treated animals were desensitized to isoproterenol, as determined by response of lipolysis (glycerol release). Binding of [125I]iodocyanopindolol was decreased by approximately 80% in adipocyte plasma membranes isolated from treated rats, indicating that beta-adrenergic receptors were downregulated. Cellular concentrations of Gn alpha and Gi alpha were not altered. Insulin sensitivity was determined by measuring the effect of insulin on glucose transport (2-deoxy-[3H]glucose uptake). Cells from the isoproterenol-infused rats were markedly more sensitive to insulin than those from control rats. This was evidenced by an approximately 50% increase in maximal glucose transport rate in cells from the high-dose isoproterenol-treated rats and by an approximately 40% decrease in the half-maximal effective concentration of insulin in both groups. 125I-labeled insulin binding to adipocytes was not altered by the isoproterenol infusions, indicating that desensitization of beta-adrenergic receptors results in tighter coupling between insulin receptors and stimulation of glucose transport.

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Insulin, oxytocin, and vasopressin stimulate protein kinase C activity in adipocyte plasma membranes.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.87.3.1052 ◽

1990 ◽

Vol 87 (3) ◽

pp. 1052-1056 ◽

Cited By ~ 52

Author(s):

J. J. Egan ◽

J. Saltis ◽

S. A. Wek ◽

I. A. Simpson ◽

C. Londos

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Plasma Membranes ◽

Kinase C ◽

Protein Kinase C Activity ◽

Adipocyte Plasma Membranes

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Selective uptake of cholesteryl ester from high density lipoproteins by plasma membranes of adipose tissue

Biochemistry and Cell Biology ◽

10.1139/o90-129 ◽

1990 ◽

Vol 68 (5) ◽

pp. 870-879 ◽

Cited By ~ 11

Author(s):

Joel G. Parkes ◽

Aubie Angel

Keyword(s):

Adipose Tissue ◽

Cholesteryl Ester ◽

Plasma Membranes ◽

Divalent Cations ◽

High Density ◽

Metabolic Energy ◽

High Density Lipoproteins ◽

High Affinity ◽

Lipoprotein Binding ◽

Adipocyte Plasma Membranes

The interaction between high density lipoproteins (HDL) and adipose tissue is an important pathway for cholesterol and cholesteryl ester flux. In intact fat cells, a disproportionately greater net uptake of cholesteryl ester occurs subsequent to lipoprotein binding than would have been predicted from a consideration of holoparticle uptake alone. To characterize the early events in this process, cholesteryl hexadecyl ether, a nonmetabolizable, accumulative marker of cholesteryl ester, was incorporated into canine HDL2, and its uptake by omental adipocyte plasma membranes was measured in relation to the binding of HDL2, which in this animal species is enriched in apolipoprotein A-I and free of apolipoprotein E. The dose–response profile for HDL2 binding was consistent with a single lipoprotein binding site at all concentrations of HDL2, whereas uptake of cholesteryl ester from HDL2 was biphasic, suggesting a high affinity site at low HDL2 concentrations and a low affinity site at high lipoprotein concentrations. Pronase treatment stimulated binding twofold and this was accompanied by a parallel twofold stimulation of cholesteryl ester uptake. EDTA, on the other hand, reduced binding and uptake of cholesteryl ester by 20%, indicating partial dependence upon divalent cations. The proportion of HDL2 cholesteryl ester accumulated by plasma membranes relative to HDL2 protein bound was not altered by either pronase or EDTA, despite the fact that these agents had opposite effects upon binding. In dissociation studies, a portion of membrane-associated HDL2 did not equilibrate with exogenous HDL2 and a greater proportion of the cholesteryl ester failed to dissociate. A stepwise mechanism for cholesteryl ester uptake, involving (i) saturable, high affinity HDL2 binding to cell surface sites, (ii) vectoral, HDL2 concentration-dependent delivery of cholesteryl ester to the membrane, and (iii) cholesteryl ester sequestration into a nonexchangeable membrane compartment, appears to be independent of metabolic energy or cell processing.Key words: cholesteryl ester transport, high density lipoprotein receptor, cholesterol storage.

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

Biochemical Journal ◽

10.1042/bj2550983 ◽

1988 ◽

Vol 255 (3) ◽

pp. 983-990 ◽

Cited By ~ 9

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.

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