scholarly journals Dissection of stress-activated glucose transport from insulin-induced glucose transport in mammalian cells using wortmannin and ML-9

1995 ◽  
Vol 309 (3) ◽  
pp. 731-736 ◽  
Author(s):  
L F Barros ◽  
R B Marchant ◽  
S A Baldwin
Keyword(s):  
Glucose Uptake ◽  
Glucose Transport ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Metabolic Stress ◽  
Cell Types ◽  
Mitogen Activated Protein Kinase ◽  
Uptake Capacity ◽  
Mitogen Activated Protein ◽  
Stimulation Of

The signaling pathways responsible for the activation of glucose transport by insulin and by metabolic stress in mammalian cells were studied in Clone 9 cells and 3T3-L1 adipocytes. Exposure of both cell types to azide or insulin markedly increased their glucose uptake capacity (Vmax.) without affecting their apparent affinity for glucose (Km). The effects of azide and insulin were not additive. Wortmannin, a selective inhibitor of phosphatidylinositol (PI) 3-kinase, did not affect stimulation of transport by azide but inhibited insulin-induced glucose transport with a Ki of < 10 nM. ML-9, a putative mitogen-activated protein kinase inhibitor, was equipotent in its inhibition of azide- and insulin-stimulated glucose transport. These findings suggest that multiple signalling cascades are involved in the stimulation of glucose transport in mammalian cells and that PI 3-kinase, an essential link in the pathway by which insulin stimulates glucose transport, is not necessary for the activation of glucose uptake by metabolic stress.

scholarly journals Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes.

1994 ◽  
Vol 14 (4) ◽  
pp. 2372-2377 ◽  
Author(s):  
N van den Berghe ◽  
D M Ouwens ◽  
J A Maassen ◽  
M G van Mackelenbergh ◽  
H C Sips ◽  
...  
Keyword(s):  
Map Kinase ◽  
Glucose Uptake ◽  
Signaling Pathway ◽  
Glucose Transport ◽  
Signal Transduction Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Signaling ◽  
Mitogen Activated Protein ◽  
Kinase Signaling Pathway ◽  
Stimulation Of

The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport.

Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes

1994 ◽  
Vol 14 (4) ◽  
pp. 2372-2377
Author(s):  
N van den Berghe ◽  
D M Ouwens ◽  
J A Maassen ◽  
M G van Mackelenbergh ◽  
H C Sips ◽  
...  
Keyword(s):  
Map Kinase ◽  
Glucose Uptake ◽  
Signaling Pathway ◽  
Glucose Transport ◽  
Signal Transduction Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Signaling ◽  
Mitogen Activated Protein ◽  
Kinase Signaling Pathway ◽  
Stimulation Of

The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport.

Role of ERK1/2 activation in microtubule stabilization and glucose transport in cardiomyocytes

2011 ◽  
Vol 301 (5) ◽  
pp. E836-E843 ◽  
Author(s):  
Mohamed Asrih ◽  
Corinne Pellieux ◽  
Irène Papageorgiou ◽  
René Lerch ◽  
Christophe Montessuit
Keyword(s):  
Glucose Uptake ◽  
Glucose Transport ◽  
Metabolic Stress ◽  
Mitogen Activated Protein Kinase ◽  
Rat Cardiomyocytes ◽  
Microtubule Stabilization ◽  
Positive Effects ◽  
Microtubule Disruption ◽  
Extracellular Signal ◽  
Stimulation Of

We previously demonstrated that microtubule disruption impairs stimulation of glucose uptake in cardiomyocytes and that 9-cis retinoic acid (9cRA) treatment preserved both microtubule integrity and stimulated glucose transport. Herein we investigated whether 1) activation of the extracellular signal-regulated kinases (ERK1/2) is responsible for microtubule destabilization and 2) ERK1/2 inactivation may explain the positive effects of 9cRA on glucose uptake and microtubule stabilization. Adult rat cardiomyocytes in primary culture showed increased basal ERK1/2 phosphorylation. Cardiomyocytes exposed to inhibitors of the ERK1/2 kinase mitogen/extracellular signal-regulated kinase (MEK) 1/2 had preserved microtubular scaffold, including microtubule-organizing centers (MTOC), together with increased insulin and metabolic stress-stimulated glucose transport as well as signaling, thus replicating the effects of 9cRA treatment. Although 9cRA treatment did not significantly reduce global ERK1/2 activation, it markedly reduced perinuclear-activated ERK1/2 at the location of MTOC. 9cRA also triggered relocation of the ERK1/2 phosphatase mitogen-activated protein kinase phosphatase-3 from the cytosol to the nucleus. These results indicate that, in cardiomyocytes, microtubule destabilization, leading to impaired stimulation of glucose transport, is mediated by ERK1/2 activation, impacting on the MTOC. 9cRA acid restores stimulated glucose transport indirectly through compartmentalized inactivation of ERK1/2.

scholarly journals Fc gamma R-dependent mitogen-activated protein kinase activation in leukocytes: a common signal transduction event necessary for expression of TNF-alpha and early activation genes.

1996 ◽  
Vol 184 (3) ◽  
pp. 1027-1035 ◽  
Author(s):  
R Trotta ◽  
P Kanakaraj ◽  
B Perussia
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Kinase Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mitogen Activated Protein Kinase ◽  
Monocytic Cell ◽  
Fast Kinetics ◽  
Mitogen Activated Protein ◽  
Stimulation Of

Cross-linking the receptors for the Fc domain of IgG (Fc gamma R) on leukocytes induces activation of protein tyrosine kinases. The intermediary molecules that transduce to the nucleus the signals leading to induction of the diverse biological responses mediated by these receptors are not clearly identified. We have investigated whether mitogen-activated protein kinases (MAPK) are involved in transmembrane signaling via the three Fc gamma R present on monocytic, polymorphonuclear, and natural killer (NK) cells. Our results indicate that occupancy of Fc gamma RI and Fc gamma RII on the monocytic cell line THP-I and on polymorphonuclear leukocytes (PMN) induces, transiently and with fast kinetics, MAPK phosphorylation, as indicated by decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and increased amounts of the proteins in antiphosphotyrosine antibody immunoprecipitates. This, associated with increased enzymatic activity, also occurs upon stimulation of the transmembrane isoform of CD16 (Fc gamma RIIIA) in NK cells and in a T cell line expressing transfected Fc gamma RIIIA alpha ligand-binding chain in association with zeta, but not upon stimulation of the glycosil-phosphatidylinositol-anchored Fc gamma RIIIB on PMN. Using the specific MAP kinase kinase inhibitor-PD 098059, we show that activation of MAPK is necessary for the Fc gamma R-dependent induction of c-fos and tumor necrosis factor alpha mRNA expression in monocytes and NK cells. These results underscore the role of MAPK as signal-transducing molecules controlling the expression of different genes relevant to leukocyte biology upon Fc gamma R stimulation.

GLUT4 translocation precedes the stimulation of glucose uptake by insulin in muscle cells: potential activation of GLUT4 via p38 mitogen-activated protein kinase

2001 ◽  
Vol 359 (3) ◽  
pp. 639-649 ◽  
Author(s):  
Romel SOMWAR ◽  
David Y. KIM ◽  
Gary SWEENEY ◽  
Carol HUANG ◽  
Wenyan NIU ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
P38 Mapk ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Assay ◽  
Glut4 Translocation ◽  
L6 Myotubes ◽  
Mitogen Activated Protein ◽  
Stimulation Of

We previously reported that SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), attenuates insulin-stimulated glucose uptake without altering GLUT4 translocation. These results suggested that insulin might activate GLUT4 via a p38 MAPK-dependent pathway. Here we explore this hypothesis by temporal and kinetic analyses of the stimulation of GLUT4 translocation, glucose uptake and activation of p38 MAPK isoforms by insulin. In L6 myotubes stably expressing GLUT4 with an exofacial Myc epitope, we found that GLUT4 translocation (t1/2 = 2.5min) preceded the stimulation of 2-deoxyglucose uptake (t1/2 = 6min). This segregation of glucose uptake from GLUT4 translocation became more apparent when the two parameters were measured at 22°C. Preincubation with the p38 MAPK inhibitors SB202190 and SB203580 reduced insulin-stimulated transport of either 2-deoxyglucose or 3-O-methylglucose by 40–60%. Pretreatment with SB203580 lowered the apparent transport Vmax of insulin-mediated 2-deoxyglucose and 3-O-methylglucose without any significant change in the apparent Km for either hexose. The IC50 values for the partial inhibition of 2-deoxyglucose uptake by SB202190 and SB203580 were 1 and 2μM respectively, and correlated with the IC50 for full inhibition of p38 MAPK by the two inhibitors in myotubes (2 and 1.4μM, respectively). Insulin caused a dose- (EC50 = 15nM) and time- (t1/2 = 3min) dependent increase in p38 MAPK phosphorylation, which peaked at 10min (2.3±0.3-fold). In vitro kinase assay of immunoprecipitates from insulin-stimulated myotubes showed activation of p38α (2.6±0.3-fold) and p38β (2.3±0.2-fold) MAPK. These results suggest that activation of GLUT4 follows GLUT4 translocation and that both mechanisms contribute to the full stimulation of glucose uptake by insulin. Furthermore, activation of GLUT4 may occur via an SB203580-sensitive pathway, possibly involving p38 MAPK.

scholarly journals 5-Aminoimidazole-4-carboxamide riboside (AICAR) enhances GLUT2-dependent jejunal glucose transport: a possible role for AMPK

2005 ◽  
Vol 385 (2) ◽  
pp. 485-491 ◽  
Author(s):  
John WALKER ◽  
Humberto B. JIJON ◽  
Hugo DIAZ ◽  
Payam SALEHI ◽  
Thomas CHURCHILL ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Western Blotting ◽  
Glucose Transporter ◽  
Mitogen Activated Protein Kinase ◽  
Mrna Levels ◽  
Energy Status ◽  
P38 Inhibitor ◽  
Mitogen Activated Protein

AMPK (AMP-activated protein kinase) is a key sensor of energy status within the cell. Activated by an increase in the AMP/ATP ratio, AMPK acts to limit cellular energy depletion by down-regulating selective ATP-dependent processes. The purpose of the present study was to determine the role of AMPK in regulating intestinal glucose transport. [3H]3-O-methyl glucose fluxes were measured in murine jejunum in the presence and absence of the AMPK activators AICAR (5-aminoimidazole-4-carboxamide riboside) and metformin and the p38 inhibitor, SB203580. To differentiate between a sodium-coupled (SGLT1) and diffusive (GLUT2) route of entry, fluxes were measured in the presence of the SGLT1 and GLUT2 inhibitors phloridzin and phloretin. Glucose transporter mRNA levels were measured by reverse transcriptase–PCR, and localization by Western blotting. Surface-expressed GLUT2 was assessed by luminal biotinylation. Activation of p38 mitogen-activated protein kinase was analysed by Western blotting. We found that treatment of jejunal tissue with AICAR resulted in enhanced net glucose uptake and was associated with phosphorylation of p38 mitogen-activated protein kinase. Inhibition of p38 abrogated the stimulation of AICAR-stimulated glucose uptake. Phloretin abolished the AICAR-mediated increase in glucose flux, whereas phloridzin had no effect, suggesting the involvement of GLUT2. In addition, AICAR decreased total protein levels of SGLT1, concurrently increasing levels of GLUT2 in the brush-border membrane. The anti-diabetic drug metformin, a known activator of AMPK, also induced the localization of GLUT2 to the luminal surface. We conclude that the activation of AMPK results in an up-regulation of non-energy requiring glucose uptake by GLUT2 and a concurrent down-regulation of sodium-dependent glucose transport.

scholarly journals Methyl-beta-cyclodextrin stimulates glucose uptake in Clone 9 cells: a possible role for lipid rafts

2004 ◽  
Vol 378 (2) ◽  
pp. 343-351 ◽  
Author(s):  
Kay BARNES ◽  
Jean C. INGRAM ◽  
Matthew D. M. BENNETT ◽  
Gordon W. STEWART ◽  
Stephen A. BALDWIN
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Gradient Centrifugation ◽  
Metabolic Stress ◽  
Mitogen Activated Protein Kinase ◽  
Cholesterol Depletion ◽  
Lipid Environment ◽  
Stimulation Of

An acute increase in the Vmax for glucose uptake occurs in many mammalian cell types after exposure to osmotic or metabolic stress. In the rat epithelial Clone 9 cell line, the glucose transporter isoform GLUT1 is responsible for this enhanced uptake. Although stimulation of transport in these cells is known to result from the unmasking of ‘cryptic’ exofacial permeant-binding sites in GLUT1 molecules resident in the plasma membrane, the mechanism of such unmasking remains unclear. One possibility involves changes in the lipid environment of the transporter: reconstitution experiments have shown that transport activity in vitro is acutely sensitive to the phospholipid and cholesterol composition of the membrane. In the current study we found that treatment of Clone 9 cells with methyl-β-cyclodextrin, which removed >80% of the cell cholesterol, led to a 3.5-fold increase in the Vmax for 3-O-methyl-d-glucose transport while having little effect on the Km. In contrast to the metabolic stress induced by inhibition of oxidative phosphorylation, cholesterol depletion led neither to depletion of cellular ATP nor stimulation of AMP-activated protein kinase. Similarly, it did not result in stimulation of members of the stress- and mitogen-activated protein kinase families. In unstressed, cholesterol-replete cells, a substantial proportion of GLUT1 in detergent lysates co-fractionated with the lipid-raft proteins caveolin and stomatin on density-gradient centrifugation. Immunocytochemistry also revealed the presence of GLUT1-enriched domains, some of which co-localized with stomatin, in the plasma membrane. Both techniques revealed that the abundance of such putative GLUT1-containing domains was decreased not only by cholesterol depletion but also in cells subjected to metabolic stress. Taken together, these data suggest that a change in the lipid environment of GLUT1, possibly associated with its re-distribution between different microdomains of the plasma membrane, could play a role in its activation in response to stress.

Role of tyrosine kinase and p44/42 MAPK in D2-like receptor-mediated stimulation of Na+, K+-ATPase in kidney

2002 ◽  
Vol 282 (4) ◽  
pp. F697-F702 ◽  
Author(s):  
Vihang Narkar ◽  
Tahir Hussain ◽  
Mustafa Lokhandwala
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Rat Kidney ◽  
Receptor Activation ◽  
Proximal Tubules ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Stimulation Of

Our laboratory has shown that dopamine D2-like receptor activation causes stimulation of Na+, K+-ATPase (NKA) activity in the proximal tubules of the rat kidney. The present study was designed to investigate the cellular signaling mechanisms mediating this response to D2-like receptor activation. We measured the stimulation of NKA activity by bromocriptine (D2-like receptor agonist) in the absence and presence of PD-98059 [p44/42 mitogen-activated protein kinase (MAPK) kinase inhibitor] and genistein (tyrosine kinase inhibitor) in renal proximal tubules. Both agents inhibited bromocriptine-mediated stimulation of NKA, suggesting the involvement of p44/42 MAPK and tyrosine kinase in this response. Additionally, we found that bromocriptine increased the phosphorylation of p44/42 MAPK in the proximal tubules, which was blocked by PD-98059 and genistein. These results show that D2-like receptor activation causes stimulation of NKA activity by means of a tyrosine kinase-p44/42 MAPK pathway in the proximal tubules of the kidney.

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