Insulin-Regulated Trafficking of Dual-Labeled Glucose Transporter 4 in Primary Rat Adipose Cells

Dawson, Kevin; Aviles-Hernandez, Armando; Cushman, Samuel W.; Malide, Daniela

doi:10.1006/bbrc.2001.5620

Cited by 63 publications

(70 citation statements)

References 37 publications

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“…The hemagglutinin tag is inserted in the exofacial loop of GLUT4. Thus, in nonpermeabilized cells, anti-hemagglutinin will bind only to GLUT4 that is inserted into the plasma membrane (20,21). Imaging confirmed that bradykinin-enhanced GLUT4 translocation (mean fluorescence intensity representing cell surface hemagglutinin-GLUT4-GFP [arbitrary units]: control basal 1.0 Ϯ 0, control ϩ insulin 1.99 Ϯ 0.13, bradykinin basal 1.17 Ϯ 0.015, and bradykinin ϩ insulin 2.57 Ϯ 0.08; P Ͻ 0.04 vs. control ϩ insulin (n ϭ 3) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Cushman, Yale University, New Haven, CT). Using the Gene Pulser XCell System (BioRad), cells were electroporated in 0.4-cm gap-width cuvettes with two pulses of 25 F at 800 V followed by one pulse of 1,050 F at 200 V. Eighteen hours after transfection, the adipocytes were washed, treated or not treated with bradykinin (1 mol/l) for 1 h and/or insulin (100 nmol/l) for 30 min, and incubated with mouse monoclonal anti-hydroxylamine antibody (1:150) and donkey anti-mouse IgG antibody conjugated with rhodamine red (1:100), fixed in 4% paraformaldehyde in PBS, and mounted on glass slides using Vectashield mounting medium as described (21). Fluorescence was observed using the Leica TCS SP2 Confocal Laser Scanning Microscope and analyzed with Leica Confocal Software.…”

Section: -Deoxy-d-[mentioning

confidence: 99%

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Bradykinin Augments Insulin-Stimulated Glucose Transport in Rat Adipocytes via Endothelial Nitric Oxide Synthase–Mediated Inhibition of Jun NH2-Terminal Kinase

Beard

et al. 2006

Diabetes

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An increase in bradykinin has been suggested to contribute to the enhanced insulin sensitivity observed in the presence of ACE inhibitors. To investigate a potential direct, nonvascular effect on an insulin target tissue, the effect of bradykinin on glucose uptake and insulin signaling was studied in primary rat adipocytes. Whereas basal glucose uptake was not altered, bradykinin augmented insulinstimulated glucose uptake twofold, which was blocked by HOE-140, a bradykinin B 2 receptor antagonist. The bradykinin effect on glucose uptake was nitric oxide (NO) dependent, mimicked by NO donors and absent in adipocytes from endothelial NO synthase ؊/؊ mice. Investigation of insulin signaling revealed that bradykinin enhanced insulin receptor substrate-1 (IRS-1) Tyr phosphorylation, Akt/protein kinase B phosphorylation, and GLUT4 translocation. In contrast, insulin-stimulated extracellular signal-regulated kinase1/2 and Jun NH 2 -terminal kinase (JNK) activation were decreased in the presence of bradykinin, accompanied by decreased IRS-1 Ser 307 phosphorylation. Furthermore, bradykinin did not enhance insulin action in the presence of the JNK inhibitor, SP-600125, or in adipocytes from JNK1 ؊/؊ mice. These data indicate that bradykinin enhances insulin sensitivity in adipocytes via an NO-dependent pathway that acts by modulating the feedback inhibition of insulin signaling at the level of IRS-1. Diabetes

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Section: Resultsmentioning

confidence: 99%

Section: -Deoxy-d-[mentioning

confidence: 99%

Bradykinin Augments Insulin-Stimulated Glucose Transport in Rat Adipocytes via Endothelial Nitric Oxide Synthase–Mediated Inhibition of Jun NH2-Terminal Kinase

Beard

et al. 2006

Diabetes

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“…6. The CMV-10 plasmid encoding human AS160 with a triple FLAG tag at the amino terminus was as described in Ref.…”

Section: Methodsmentioning

confidence: 99%

Insulin-stimulated Phosphorylation of a Rab GTPase-activating Protein Regulates GLUT4 Translocation

Sano

Kane

Sano

et al. 2003

Journal of Biological Chemistry

832

886

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Insulin stimulates the rapid translocation of intracellular glucose transporters of the GLUT4 isotype to the plasma membrane in fat and muscle cells. The connections between known insulin signaling pathways and the protein machinery of this membrane-trafficking process have not been fully defined. Recently, we identified a 160-kDa protein in adipocytes, designated AS160, that is phosphorylated by the insulin-activated kinase Akt. This protein contains a GTPase-activating domain (GAP) for Rabs, which are small G proteins required for membrane trafficking. In the present study we have identified six sites of in vivo phosphorylation on AS160. These sites lie in the motif characteristic of Akt phosphorylation, and insulin treatment increased phosphorylation at five of the sites. Expression of AS160 with two or more of these sites mutated to alanine markedly inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Moreover, this inhibition did not occur when the GAP function in the phosphorylation site mutant was inactivated by a point mutation. These findings strongly indicate that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation and that this phosphorylation signals translocation through inactivation of the Rab GAP function.Insulin rapidly stimulates glucose transport into fat and muscle cells by causing the insertion of additional glucose transporters of the GLUT4 isotype into the plasma membrane, in a process referred to as GLUT4 translocation. The overall process consists of generation of the specialized vesicles containing GLUT4 from the endosomal system, the movement of these vesicles from the perinuclear region to the plasma membrane, and the fusion of the vesicles with the plasma membrane (1). The steps in this process that insulin accelerates, and the complete signaling pathways from the insulin receptor that lead to their acceleration, have not yet been fully defined. One partial insulin signaling pathway that has been established to be required for GLUT4 translocation is the pathway that proceeds from the receptor through tyrosine phosphorylation of the insulin receptor substrates to activation of phosphatidylinositol 3-kinase and generation of phosphatidylinositol 3,4,5-trisphosphate. The latter leads to the activation of the protein kinase Akt and also protein kinase C /, and there is evidence that GLUT4 translocation requires the activation of one or both of these kinases (reviewed in Refs. 2 and 3). However, although a substrate linking either kinase to GLUT4 translocation has been sought for several years, hitherto none has been clearly identified.Recently, we reported the discovery of a new substrate for insulin-activated Akt in 3T3-L1 adipocytes, which was designated AS160 for Akt subtrate of 160 kDa (4). The most prominent feature of AS160 is the presence of a GTPase activating domain for a Rab. Since Rabs are small G proteins that play critical roles in vesicle formation, movement, and fusion (5), we investigated the role of AS160 in GLUT4 translocatio...

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“…tagging does not affect the insulin-responsive trafficking of GLUT4 (21,22). C-terminally EGFP-tagged WT GLUT4 or its glycosylation mutant (N57Q) was therefore transfected into HeLa cells to provide insights into the roles of the N-glycan of GLUT4.…”

Section: N-glycosylation Of Glut4 Contributes To Its Stability-egfpmentioning

confidence: 99%

N-Glycosylation Is Critical for the Stability and Intracellular Trafficking of Glucose Transporter GLUT4

Haga

Ishii

Suzuki

2011

Journal of Biological Chemistry

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The facilitative glucose transporter GLUT4 plays a key role in regulating whole body glucose homeostasis. GLUT4 dramatically changes its distribution upon insulin stimulation, and insulin-resistant diabetes is often linked with compromised translocation of GLUT4 under insulin stimulation. To elucidate the functional significance of the sole N-glycan chain on GLUT4, wild-type GLUT4 and a GLUT4 glycosylation mutant conjugated with enhanced GFP were stably expressed in HeLa cells. The N-glycan contributed to the overall stability of newly synthesized GLUT4. Moreover, cell surface expression of wildtype GLUT4 in HeLa cells was elevated upon insulin treatment, whereas the glycosylation mutant lost the ability to respond to insulin. Subcellular distribution of the mutant was distinct from that of wild-type GLUT4, implying that the subcellular localization required for insulin-mediated translocation was impaired in the mutant protein. Interestingly, kifunensine-treated cells also lost sensitivity to insulin, suggesting the functional importance of the N-glycan structure for GLUT4 trafficking. The K m or turnover rates of wild-type and mutant GLUT4, however, were similar, suggesting that the N-glycan had little effect on transporter activity. These findings underscore the critical roles of the N-glycan chain in quality control as well as intracellular trafficking of GLUT4.

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Insulin-Regulated Trafficking of Dual-Labeled Glucose Transporter 4 in Primary Rat Adipose Cells

Cited by 63 publications

References 37 publications

Bradykinin Augments Insulin-Stimulated Glucose Transport in Rat Adipocytes via Endothelial Nitric Oxide Synthase–Mediated Inhibition of Jun NH2-Terminal Kinase

Bradykinin Augments Insulin-Stimulated Glucose Transport in Rat Adipocytes via Endothelial Nitric Oxide Synthase–Mediated Inhibition of Jun NH2-Terminal Kinase

Insulin-stimulated Phosphorylation of a Rab GTPase-activating Protein Regulates GLUT4 Translocation

N-Glycosylation Is Critical for the Stability and Intracellular Trafficking of Glucose Transporter GLUT4

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