GLUT4 translocation by insulin in intact muscle cells: detection by a fast and quantitative assay

Wang, Qinghua; Khayat, Zayna A.; Kishi, Kazuhiro; Ebina, Yasuhiko; Klip, Amira

doi:10.1016/s0014-5793(98)00423-2

Cited by 209 publications

(226 citation statements)

References 32 publications

(32 reference statements)

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“…Cell culture and troglitazone treatment L6GLUT4myc myoblasts were differentiated into myotubes by growing them in α-MEM supplemented with 2% fetal bovine serum as described previously [30]. At a seeding density of 2× 10 4 cells/ml (1 and 2 ml per well in 24-or 12-well plates, respectively) L6 cells fully differentiate into myotubes by day 7.…”

Section: Methodsmentioning

confidence: 99%

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Troglitazone causes acute mitochondrial membrane depolarisation and an AMPK-mediated increase in glucose phosphorylation in muscle cells

et al. 2005

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Aims/hypothesis: Troglitazone was the first thiazolidinedione (TZD) approved for clinical use, exerting hypoglycaemic effects related to its action as a ligand of the peroxisome proliferator-activated receptor γ receptor in adipocytes. However, emerging evidence suggests that mitochondrial function may be affected by troglitazone, and that skeletal muscle cells acutely respond to troglitazone by enhancing glucose uptake. The aim of the present study was to determine the cellular mechanisms by which troglitazone acutely stimulates glucose utilisation in skeletal muscle cells. Methods: L6 cells overexpressing GLUT4myc were incubated with troglitazone. Glucose uptake, transport and phosphorylation as well as AMP-activated protein kinase (AMPK) signalling and insulin signalling were examined. Changes in mitochondrial membrane potential were measured using the J-aggregate-forming dye JC-1. AMPK signalling was interfered with using AMPK α1/α2 siRNA. Results: Troglitazone acutely (in 10 min) reduced the mitochondrial membrane potential in L6GLUT4myc myotubes and robustly stimulated AMPK activity. Following 30 min of incubation with troglitazone or insulin, 2-deoxyglucose uptake was stimulated 1.5-and 2.1-fold respectively, and in cells treated with troglitazone, a 1.8-fold increase in the 2-deoxyglucose-6-phosphate:2-deoxyglucose ratio was observed. Moreover, contrary to insulin, troglitazone did not significantly stimulate 3-O-methylglucose uptake. Unlike insulin, troglitazone did not increase surface GLUT4myc content and did not increase IRS1-associated phosphatidylinositol 3-kinase activity or Akt phosphorylation on T308 and S473. Interestingly, interfering with troglitazone-induced activation of AMPK by decreasing the expression of the enzyme using siRNA inhibited the stimulation of 2-deoxyglucose uptake by the TZD. Conclusions/interpretation: We propose that troglitazone acutely increases glucose flux in muscle via an AMPK-mediated increase in glucose phosphorylation.

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Section: Methodsmentioning

confidence: 99%

“…Detection of surface GLUT4myc or GLUT1myc was performed on intact myotubes as previously described [30]. Briefly, L6-GLUT4myc myotubes or L6-GLUT1myc myotubes grown in 24-well tissue culture plates were treated with vehicle (0.1% DMSO), troglitazone (5 μg/ml) or insulin (100 nmol/l) for 30 min.…”

Section: Immunodetection Of Cell Surface Glut4myc or Glut1mycmentioning

confidence: 99%

Troglitazone causes acute mitochondrial membrane depolarisation and an AMPK-mediated increase in glucose phosphorylation in muscle cells

et al. 2005

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“…This disparity between the magnitude of GLUT4 translocation and the stimulation of glucose uptake has been observed in mature skeletal muscle (2)(3)(4)(5), primary adipocytes (6 -8), and muscle and fat cell lines (9,10). Recently, we developed a muscle cell line overexpressing GLUT4 fused to a Myc epitope that becomes exposed at the cell surface, allowing for the detection of GLUT4 translocation in intact cells (11,12). Using this system, we reported that GLUT4 translocation precedes the stimulation of glucose uptake by at least 2 min (13).…”

mentioning

confidence: 99%

“…Detection of Cell Surface GLUT4myc-GLUT4myc levels at the cell surface was measured by an antibody-coupled colorimetric assay as described (11). L6-GLUT4myc myotubes were washed once with PBS, fixed with 3% paraformaldehyde (v/v) for 3 min at room temperature, and then neutralized with 1% (w/v) glycine in PBS at 4°C for 10 min.…”

mentioning

confidence: 99%

Differential Effects of Phosphatidylinositol 3-Kinase Inhibition on Intracellular Signals Regulating GLUT4 Translocation and Glucose Transport

Somwar

Niu

Kim

et al. 2001

Journal of Biological Chemistry

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Phosphatidylinositol (PI) 3-kinase is required for insulin-stimulated translocation of GLUT4 to the surface of muscle and fat cells. Recent evidence suggests that the full stimulation of glucose uptake by insulin also requires activation of GLUT4, possibly via a p38 mitogen-activated protein kinase (p38 MAPK)-dependent pathway. Here we used L6 myotubes expressing Myctagged GLUT4 to examine at what level the signals regulating GLUT4 translocation and activation bifurcate. We compared the sensitivity of each process, as well as of signals leading to GLUT4 translocation (Akt and atypical protein kinase C) to PI 3-kinase inhibition. Wortmannin inhibited insulin-stimulated glucose uptake with an IC 50 of 3 nM. In contrast, GLUT4myc appearance at the cell surface was less sensitive to inhibition (IC 50 ‫؍‬ 43 nM). This dissociation between insulin-stimulated glucose uptake and GLUT4myc translocation was not observed with LY294002 (IC 50 ‫؍‬ 8 and 10 M, respectively). The sensitivity of insulin-stimulated activation of PKC/, Akt1, Akt2, and Akt3 to wortmannin (IC 50 ‫؍‬ 24, 30, 35, and 60 nM, respectively) correlated closely with inhibition of GLUT4 translocation. In contrast, insulindependent p38 MAPK phosphorylation was efficiently reduced in cells pretreated with wortmannin, with an IC 50 of 7 nM. Insulin-dependent p38␣ and p38␤ MAPK activities were also markedly reduced by wortmannin (IC 50 ‫؍‬ 6 and 2 nM, respectively). LY294002 or transient expression of a dominant inhibitory PI 3-kinase construct (⌬p85), however, did not affect p38 MAPK phosphorylation. These results uncover a striking correlation between PI 3-kinase, Akt, PKC/, and GLUT4 translocation on one hand and their segregation from glucose uptake and p38 MAPK activation on the other, based on their wortmannin sensitivity. We propose that a distinct, high affinity target of wortmannin, other than PI 3-kinase, may be necessary for activation of p38 MAPK and GLUT4 in response to insulin.

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“…Plasmid transfection The L6 muscle cell line and hamster kidney fibroblast cell line BHK were maintained in culture [21,22]. Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada) was used for plasmid transfections.…”

Section: Methodsmentioning

confidence: 99%

Ectopic expression of glucagon receptor in skeletal muscles improves glucose homeostasis in a mouse model of diabetes

et al. 2012

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Aims/hypothesis Excessive secretion of glucagon partially contributes to the development of diabetic hyperglycaemia. However, complete blocking of glucagon action will lead to adverse effects, since glucagon exerts certain beneficial effects via its receptor in many organs. We aimed to study the effects of a 'decoy receptor' for circulating glucagon on modulating beta cell function and glucose homeostasis in mice by over-producing the glucagon receptor (GCGR) in skeletal muscles. Methods We generated transgenic mice in which the expression of Gcgr is driven by the muscle specific creatine kinase (Mck) promoter, and assessed the effects of glucagon on the modulation of glucose homeostasis under conditions of extremes of glucose influx or efflux.Results Mck/Gcgr mice showed increased circulating levels of glucagon and insulin, resulting in an unchanged ratio of glucagon-to-insulin. The levels of hepatic glucose-6-phosphatase (G6PC) and fructose-1,6-bisphosphatase (F1,6P2ase) were significantly decreased, whereas the phosphorylation level of pancreatic cAMP-response-element-binding-protein (CREB) was significantly increased in these transgenic mice. Under basal conditions, the mice displayed normal blood glucose levels and unchanged glucose tolerance and insulin sensitivity when compared with their age-matched wild-type (WT) littermates. However, following multiple lowdose streptozotocin injections, Mck/Gcgr mice exhibited a delay in the onset of hyperglycaemia compared with the WT controls. This was associated with preserved beta cell mass and beta cell secretory capacity in response to glucose challenge.A. Maharaj and L. Zhu contributed equally to this study. Electronic supplementary material The online version of this article

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GLUT4 translocation by insulin in intact muscle cells: detection by a fast and quantitative assay

Cited by 209 publications

References 32 publications

Troglitazone causes acute mitochondrial membrane depolarisation and an AMPK-mediated increase in glucose phosphorylation in muscle cells

Troglitazone causes acute mitochondrial membrane depolarisation and an AMPK-mediated increase in glucose phosphorylation in muscle cells

Differential Effects of Phosphatidylinositol 3-Kinase Inhibition on Intracellular Signals Regulating GLUT4 Translocation and Glucose Transport

Ectopic expression of glucagon receptor in skeletal muscles improves glucose homeostasis in a mouse model of diabetes

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