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.

scholarly journals Regulation of GLUT1-mediated glucose uptake by PKCλ–PKCβII interactions in 3T3-L1 adipocytes

2004 ◽  
Vol 384 (2) ◽  
pp. 349-355 ◽  
Author(s):  
Remko R. BOSCH ◽  
Merlijn BAZUINE ◽  
Paul N. SPAN ◽  
Peter H. G. M. WILLEMS ◽  
André J. OLTHAAR ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Mitogen Activated Protein Kinase ◽  
Glucose Transporter 1 ◽  
Protein Levels ◽  
Mitogen Activated Protein ◽  
Pkc Protein Kinase C

Members of the PKC (protein kinase C) superfamily play key regulatory roles in glucose transport. How the different PKC isotypes are involved in the regulation of glucose transport is still poorly defined. PMA is a potent activator of conventional and novel PKCs and PMA increases the rate of glucose uptake in many different cell systems. In the present study, we show that PMA treatment increases glucose uptake in 3T3-L1 adipocytes by two mechanisms: a mitogen-activated protein kinase kinase-dependent increase in GLUT1 (glucose transporter 1) expression levels and a PKCλ-dependent translocation of GLUT1 towards the plasma membrane. Intriguingly, PKCλ co-immunoprecipitated with PKCβII and did not with PKCβI. Previously, we have described that down-regulation of PKCβII protein levels or inhibiting PKCβII by means of the myristoylated PKCβC2–4 peptide inhibitor induced GLUT1 translocation towards the plasma membrane in 3T3-L1 adipocytes. Combined with the present findings, these results suggest that the liberation of PKCλ from PKCβII is an important factor in the regulation of GLUT1 distribution in 3T3-L1 adipocytes.

Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp

1999 ◽  
Vol 277 (6) ◽  
pp. G1165-G1172 ◽  
Author(s):  
Cynthia R. L. Webster ◽  
M. Sawkat Anwer
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Signaling Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Pi3k Inhibitors ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Pi3k Signaling Pathway ◽  
Stimulation Of

cAMP stimulates Na+-taurocholate (TC) cotransport by translocating the Na+-TC-cotransporting peptide (Ntcp) to the plasma membrane. The present study was undertaken to determine whether the phosphatidylinositol-3-kinase (PI3K)-signaling pathway is involved in cAMP-mediated translocation of Ntcp. The ability of cAMP to stimulate TC uptake declined significantly when hepatocytes were pretreated with PI3K inhibitors wortmannin or LY-294002. Wortmannin inhibited cAMP-mediated translocation of Ntcp to the plasma membrane. cAMP stimulated protein kinase B (PKB) activity by twofold within 5 min, an effect inhibited by wortmannin. Neither basal mitogen-activated protein kinase (MAPK) activity nor cAMP-mediated inhibition of MAPK activity was affected by wortmannin. cAMP also stimulated p70S6K activity. However, rapamycin, an inhibitor of p70S6K, failed to inhibit cAMP-mediated stimulation of TC uptake, indicating that the effect of cAMP is not mediated via p70S6K. Cytochalasin D, an inhibitor of actin filament formation, inhibited the ability of cAMP to stimulate TC uptake and Ntcp translocation. Together, these results suggest that the stimulation of TC uptake and Ntcp translocation by cAMP may be mediated via the PI3K/PKB signaling pathway and requires intact actin filaments.

MEK inhibitors impair insulin-stimulated glucose uptake in 3T3-L1 adipocytes

2004 ◽  
Vol 287 (4) ◽  
pp. E758-E766 ◽  
Author(s):  
Anne W. Harmon ◽  
David S. Paul ◽  
Yashomati M. Patel
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Pi3k Pathway ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Mek Inhibitors ◽  
Transport Assay

In 3T3-L1 adipocytes, insulin activates three major signaling cascades, the phosphoinositide 3-kinase (PI3K) pathway, the Cbl pathway, and the mitogen-activated protein kinase (MAPK) pathway. Although PI3K and Cbl mediate insulin-stimulated glucose uptake by promoting the translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane, the MAPK pathway does not have an established role in insulin-stimulated glucose uptake. We demonstrate in this report that PI3K inhibitors also inhibit the MAPK pathway. To investigate the role of the MAPK pathway separately from that of the PI3K pathway in insulin-stimulated glucose uptake, we used two specific inhibitors of MAPK kinase (MEK) activity, PD-98059 and U-0126, which reduced insulin-stimulated glucose uptake by ∼33 and 50%, respectively. Neither MEK inhibitor affected the activation of Akt or PKCζ/λ, downstream signaling molecules in the PI3K pathway. Inhibition of MEK with U-0126 did not prevent GLUT4 from translocating to the plasma membrane, nor did it inhibit the subsequent docking and fusion of GLUT4- myc with the plasma membrane. MEK inhibitors affected glucose transport mediated by GLUT4 but not GLUT1. Importantly, the presence of MEK inhibitors only at the time of the transport assay markedly impaired both insulin-stimulated glucose uptake and MAPK signaling. Conversely, removal of MEK inhibitors before the transport assay restored glucose uptake and MAPK signaling. Collectively, our studies suggest a possible role for MEK in the activation of GLUT4.

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.

Rapid stimulation of glucose transport by mitochondrial uncoupling depends in part on cytosolic Ca2+ and cPKC

1998 ◽  
Vol 275 (6) ◽  
pp. C1487-C1497 ◽  
Author(s):  
Zayna A. Khayat ◽  
Theodoros Tsakiridis ◽  
Atsunori Ueyama ◽  
Romel Somwar ◽  
Yousuke Ebina ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Energy Demand ◽  
Plasma Membranes ◽  
Muscle Cells ◽  
Atp Production ◽  
Pkc Β ◽  
Stimulation Of

2,4-Dinitrophenol (DNP) uncouples the mitochondrial oxidative chain from ATP production, preventing oxidative metabolism. The consequent increase in energy demand is, however, contested by cells increasing glucose uptake to produce ATP via glycolysis. In L6 skeletal muscle cells, DNP rapidly doubles glucose transport, reminiscent of the effect of insulin. However, glucose transport stimulation by DNP does not require insulin receptor substrate-1 phosphorylation and is wortmannin insensitive. We report here that, unlike insulin, DNP does not activate phosphatidylinositol 3-kinase, protein kinase B/Akt, or p70 S6 kinase. However, chelation of intra- and extracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM in conjunction with EGTA inhibited DNP-stimulated glucose uptake by 78.9 ± 3.5%. Because Ca2+-sensitive, conventional protein kinase C (cPKC) can activate glucose transport in L6 muscle cells, we examined whether cPKC may be translocated and activated in response to DNP in L6 myotubes. Acute DNP treatment led to translocation of cPKCs to plasma membrane. cPKC immunoprecipitated from plasma membranes exhibited a twofold increase in kinase activity in response to DNP. Overnight treatment with 4-phorbol 12-myristate 13-acetate downregulated cPKC isoforms α, β, and γ and partially inhibited (45.0 ± 3.6%) DNP- but not insulin-stimulated glucose uptake. Consistent with this, the PKC inhibitor bisindolylmaleimide I blocked PKC enzyme activity at the plasma membrane (100%) and inhibited DNP-stimulated 2-[3H]deoxyglucose uptake (61.2 ± 2.4%) with no effect on the stimulation of glucose transport by insulin. Finally, the selective PKC-β inhibitor LY-379196 partially inhibited DNP effects on glucose uptake (66.7 ± 1.6%). The results suggest interfering with mitochondrial ATP production acts on a signal transduction pathway independent from that of insulin and partly mediated by Ca2+ and cPKCs, of which PKC-β likely plays a significant role.

scholarly journals Requirement of Atypical Protein Kinase Cλ for Insulin Stimulation of Glucose Uptake but Not for Akt Activation in 3T3-L1 Adipocytes

1998 ◽  
Vol 18 (12) ◽  
pp. 6971-6982 ◽  
Author(s):  
Ko Kotani ◽  
Wataru Ogawa ◽  
Michihiro Matsumoto ◽  
Tadahiro Kitamura ◽  
Hiroshi Sakaue ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Pleckstrin Homology Domain ◽  
Dependent Manner ◽  
Insulin Stimulation ◽  
Negative Effects ◽  
Stimulation Of

ABSTRACT Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCζ and PKCλ) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKCλ in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKCλ (λKD or λΔNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKCλ, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by λKD or λΔNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKCλ was ∼50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKCλ mutant that lacks the pseudosubstrate domain (λΔPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of λΔPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKCλ. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKCλ pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.

scholarly journals Adenosine 5′-Monophosphate-Activated Protein Kinase and p38 Mitogen-Activated Protein Kinase Participate in the Stimulation of Glucose Uptake by Dinitrophenol in Adult Cardiomyocytes

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2285-2294 ◽  
Author(s):  
Amélie Pelletier ◽  
Érik Joly ◽  
Marc Prentki ◽  
Lise Coderre
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
P38 Mapk ◽  
Molecular Mechanisms ◽  
Primary Cultures ◽  
Metabolic Stress ◽  
Mapk Signaling ◽  
Mapk Phosphorylation ◽  
Adult Cardiomyocytes ◽  
Stimulation Of

Abstract During metabolic stress, such as ischemia or hypoxia, glucose becomes the principal energy source for the heart. It has been shown that increased cardiac glucose uptake during metabolic stress has a protective effect on cell survival and heart function. Despite its physiological importance, only limited data are available on the molecular mechanisms regulating glucose uptake under these conditions. We used 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, as a model to mimic hypoxia and gain insight into the signaling pathway underlying metabolic stress-induced glucose uptake in primary cultures of rat adult cardiomyocytes. The results demonstrate that 0.1 mm DNP induces 2.2- and 9-fold increases in AMP-activated protein kinase (AMPK) and p38 MAPK phosphorylation, respectively. This is associated with a 2.3-fold increase in glucose uptake in these cells. To further delineate the role of AMPK in the regulation of glucose uptake, we used two complementary approaches: pharmacological inhibition of the enzyme with adenine 9-β-D arabinofuranoside and adenoviral infection with a dominant-negative AMPK (DN-AMPK) mutant. Our results show that overexpression of DN-AMPK completely suppressed DNP-mediated phosphorylation of acetyl coenzyme A carboxylase, a downstream target of AMPK. Inhibition of AMPK with either 9-β-D arabinofuranoside or DN-AMPK also abolished DNP-mediated p38 MAPK phosphorylation. Importantly, AMPK inhibition only partially decreased DNP-stimulated glucose uptake in cardiomyocytes. Inhibition of p38 MAPK with the pharmacological agent PD169316 also partially reduced (70%) glucose uptake in response to DNP. In conclusion, our results indicate that p38 MAPK acts downstream of AMPK in cardiomyocytes and that activation of the AMPK/p38 MAPK signaling cascade is essential for maximal stimulation of glucose uptake in response to DNP in adult cardiomyocytes.

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.

Sign in / Sign up

Export Citation Format

Share Document