help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2005-0404
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Antonescu, C. N.
Right arrow Articles by Klip, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Antonescu, C. N.
Right arrow Articles by Klip, A.
Endocrinology Vol. 146, No. 9 3773-3781
Copyright © 2005 by The Endocrine Society

Reduction of Insulin-Stimulated Glucose Uptake in L6 Myotubes by the Protein Kinase Inhibitor SB203580 Is Independent of p38MAPK Activity

C. N. Antonescu1, C. Huang1, W. Niu, Z. Liu, P. A. Eyers, K. A. Heidenreich, P. J. Bilan and A. Klip

Programme in Cell Biology (C.N.A., C.H., W.N., Z.L., P.J.B., A.K.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; Department of Biochemistry (C.N.A., A.K.) and Institute of Medical Sciences (C.H.), University of Toronto, Ontario, Canada M5S 1A8; School of Life Sciences (P.A.E.), University of Manchester, Manchester M13 9PT, United Kingdom; and Department of Pharmacology (K.A.H.), University of Colorado Health Sciences Center and Denver Veterans Affairs Medical Center, Denver, Colorado 80262

Address all correspondence and requests for reprints to: A. Klip, Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8. E-mail: amira{at}sickkids.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin increases glucose uptake through translocation of the glucose transporter GLUT4 to the plasma membrane. We previously showed that insulin activates p38MAPK, and inhibitors of p38MAPK{alpha} and p38MAPKß (e.g. SB203580) reduce insulin-stimulated glucose uptake without affecting GLUT4 translocation. This observation suggested that insulin may increase GLUT4 activity via p38{alpha} and/or p38ß. Here we further explore the possible participation of p38MAPK through a combination of molecular strategies. SB203580 reduced insulin stimulation of glucose uptake in L6 myotubes overexpressing an SB203580-resistant p38{alpha} (drug-resistant p38{alpha}) but barely affected phosphorylation of the p38 substrate MAPK-activated protein kinase-2. Expression of dominant-negative p38{alpha} or p38ß reduced p38MAPK phosphorylation by 70% but had no effect on insulin-stimulated glucose uptake. Gene silencing via isoform-specific small interfering RNAs reduced expression of p38{alpha} or p38ß by 60–70% without diminishing insulin-stimulated glucose uptake. SB203580 reduced photoaffinity labeling of GLUT4 by bio-LC-ATB-BMPA only in the insulin-stimulated state. Unless low levels of p38MAPK suffice to regulate glucose uptake, these results suggest that the inhibition of insulin-stimulated glucose transport by SB203580 is likely not mediated by p38MAPK. Instead, changes experienced by insulin-stimulated GLUT4 make it susceptible to inhibition by SB203580.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUT4 IS THE main insulin-responsive glucose transporter (1, 2), and upon insulin stimulation, it is recruited from intracellular compartments to the plasma membrane (3, 4, 5). It is widely accepted that GLUT4 translocation is required for insulin-stimulated glucose uptake, but it has been debated whether translocation is the sole mechanism responsible for this outcome (6). Several studies have demonstrated that insulin-stimulated glucose uptake can be segregated from GLUT4 translocation based on differential sensitivities to insulin concentrations (7) and inhibition by the phosphatidylinositol 3-kinase inhibitor wortmannin (8, 9). These results suggested that GLUT4 translocation and the intrinsic activity of GLUT4 are differentially regulated (10).

p38MAPKs are serine/threonine kinases activated by environmental stressors (11), inflammatory cytokines (12), and protein synthesis inhibitors (13, 14) as well as growth factors (e.g. insulin, platelet-derived growth factor) (15, 16) and muscle contraction (17, 18). Four isoforms of p38MAPK have been identified: p38{alpha} (19) and p38ß (20) are ubiquitously expressed, whereas p38{gamma} (21) is highly expressed in skeletal muscle and p38{delta} in the lung and kidney (22). Full activation of p38MAPK requires dual phosphorylation on residues corresponding to threonine-180 and tyrosine-182 on p38{alpha} (12) catalyzed by dual-specificity MAPK kinases 3 and 6 (MKK3 and 6). Insulin-stimulated activation of p38MAPK has been observed in rat skeletal muscles (18), mouse brown adipocytes (23), 3T3-L1 adipocytes (15, 24), L6 muscle cells (7, 25), and human skeletal muscles (26) and adipocytes (27). Notably, p38MAPK inhibitors such as pyridinyl imidazole derivatives (SB203580 and SB202190) or azaazulene pharmacophores (A291077 and A304000) inhibit insulin-mediated glucose uptake (15, 18, 23, 24, 25, 28, 29) with no effect on GLUT4 translocation in 3T3-L1 adipocytes (15, 24), L6 myotubes (25), mouse brown adipocytes (23), and rat skeletal muscles (18). Collectively, these results have raised the hypothesis that insulin increases the intrinsic activity of GLUT4 via p38MAPK.

Although they have been shown to be highly potent and specific inhibitor of p38{alpha} and p38ß (30, 31), recent studies have revealed some previously unknown targets of these drugs (32). When this manuscript was in progress, Holman and colleagues (33) suggested that SB203580 directly inhibits the endofacial glucose binding site of the transporter, based on the competitive effect of the drug on glucose uptake under equilibrium exchange but not zero-trans conditions. Of note, such inhibition of glucose uptake was reported only for the insulin-stimulated state. Moreover, in that and other studies, an analog of SB203580 that does not inhibit p38MAPK (SB202474) failed to inhibit glucose transport, thereby maintaining the correlation between inhibition of p38MAPK and inhibition of glucose uptake. Thus, whether p38MAPK regulates the availability of glucose-binding sites and thus transport activity of GLUT4 deserves direct testing. The aim of this study was to use small interfering (si)RNA and p38MAPK mutants to examine the role of p38{alpha} and p38ß in insulin-stimulated glucose uptake and GLUT4 translocation in muscle cells. We used adenovirus-mediated gene transfer to introduce either dominant-negative (DN) mutants of p38{alpha} or p38ß (34) or drug-resistant (DR) p38{alpha} (32) into L6 myotubes to explore whether the SB203580-mediated reduction in insulin-stimulated glucose uptake occurs via inhibition of p38MAPK activity. In addition, we reduced p38{alpha} or p38ß expression via siRNA gene silencing in an effort to determine which isoform is involved in insulin signaling. Finally, we examined the effect of SB203580 on Bio-LC-ATB-BMPA photolabeling (35). The photolabel binds to the exofacial glucose binding site of GLUT4, which becomes unavailable when the transporter is locked in the inner-facing configuration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
SB203580 was obtained from Calbiochem (San Diego, CA). Protease inhibitor cocktail, cytochalasin B, O-phenylenediamine dihydrochloride, 2-deoxyglucose, and {alpha}-FLAG antibodies were obtained from Sigma (St. Louis, MO). 2-Deoxy-D-[3H]glucose was purchased from PerkinElmer Life Sciences (St. Laurent, Quebec, Canada). Streptavidin-immobilized agarose beads were obtained from Pierce Biotechnology (Rockford, Il). Bio-LC-ATB-BMPA was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). {alpha}-Phospho-p38 MAPK (recognizes all p38MAPK isoforms only when phosphorylated on residues corresponding to T180 and Y182 on p38{alpha}), {alpha}-phospho-MAPK-activated protein kinase-2 (MK-2; recognizes MK-2 when phosphorylated on T334), and {alpha}-p38 MAPK{alpha} antibodies were obtained from Cell Signaling Technologies (Beverly, MA). Polyclonal {alpha}-myc antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat {alpha}-rabbit and sheep {alpha}-mouse antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). Adenovirus particles encoding cDNAs for green fluorescent protein (GFP), DN-p38{alpha} (T180A, Y182F), DN-p38ß (T180A, Y182F), and wild-type (WT)-p38{alpha} were prepared as previously described (36). DR-p38{alpha} (T180M, H107P, L108F) has been previously characterized (32). Synthetic siRNAs specific for rat p38MAPK{alpha} (AAG CAC GAG AAU GUG AUU GGU) and p38MAPKß (AAG CAC GAG AAC GUC AUA GGA) isoforms were obtained from Dharmacon (Lafayette, CO).

Cell culture, lysates, and immunoblotting
GLUT4myc cDNA was constructed by inserting the human c-myc epitope into the first ectodomain of GLUT4 and subcloned into the pCXN2 vector (37), stably transfected into L6 myoblasts, and characterized (38, 39, 40). L6-GLUT4myc myoblasts were grown and differentiated into myotubes as previously described (41) and were deprived of serum for 3–5 h before all experimental procedures. Cell lysates and immunoblots were performed as previously described (7).

2-Deoxyglucose uptake and GLUT4myc translocation
2-Deoxyglucose uptake was measured for 5 min at room temperature using 10 µM D-2-deoxyglucose (0.5 µCi/ml 2-deoxy-D-[3H]glucose) as previously described (7). Uptake was terminated by rinsing the cells three times with 1 ml ice-cold 0.9% NaCl (wt/vol). GLUT4myc translocation was assayed by a colorimetric method as previously described (15).

Construction and isolation of adenovirus particles and adenovirus infection
Adenovirus particles containing cDNAs encoding DR-p38{alpha} were constructed using the AdEasy I system (Stratagene, La Jolla, CA) (42). Briefly, cDNA encoding DR-p38{alpha} was subcloned into adenovirus shuttle vector (pAdTrack-CMV) and subsequently inserted into a plasmid (pAdEasy-I) encoding modified adenoviral genome by homologous recombination in Escherichia coli strain BJ5183. Subsequently recombined pAdEasy-I was transfected into HEK293AD cells. Adenovirus particles encoding all cDNAs used were harvested and purified by discontinuous cesium chloride gradient. The infectivity of adenovirus particles was determined using tissue culture infectious dose 50 criteria and then converted to plaque-forming units according to the AdEasy I system manual. Differentiating L6 myotubes were incubated with 2.5 x 105 PFU/well (24-well plate) adenovirus particles in antibiotic/antimycotic-free {alpha}MEM and 5 mM glucose for 4 h (in serum-free medium). Infection proceeded for 10–12 h (WT-p38{alpha} and DR-p38{alpha} adenovirus) or 18–24 h (DN-p38{alpha}, DN-p38ß, and GFP adenovirus) before serum deprivation and experimentation.

siRNA transfection
Cells were incubated in antibiotic-free {alpha}MEM and 5 mM glucose, supplemented with 2% fetal bovine serum for 72 h, and then transfected with 500 nM siRNA of nonrelevant (NR) sequence or siRNA specific for p38{alpha}, p38ß, or p38{alpha} and p38ß together, using oligofectamine transfection reagent (Life Technologies, Carlsbad CA), 48 h and again 24 h before experimentation. Transfection involved incubation of L6 cells for 4 h in serum-free antibiotic/antimycotic-free {alpha}MEM containing 5 mM glucose for 4 h and the siRNA-oligofectamine mixture. Subsequently fetal bovine serum was added to each well to a final concentration of 2% and the cells were incubated until the next transfection or experiment.

Bio-LC-ATB-BMPA photolabeling of GLUT4myc
The method of the bio-LC-ATB-BMPA photolabel (43) was adapted as follows: after experimental treatments, L6-GLUT4myc myotubes were washed in 18 degree 10 mM HEPES buffer (pH 7.4), containing 140 mM NaCl, 4.7 mM KCl, 1.25 mM MgSO4, 2.5 mM CaCl2, and 2.5 mM NaH2PO4 and then exposed to 0.2 mM bio-LC-ATB-BMPA and irradiated for 1 min in a Rayonet photochemical reactor under 350 nm light. After washing in ice-cold HEPES buffer, cells were lysed in PBS containing 2% Thesit (C12E9) and centrifuged at 20,000 x g for 20 min at 4 degrees. Supernatants were incubated with immobilized streptavidin on 6% beaded agarose for 20 h at 4 degrees. Agarose beads were pelleted by centrifugation at 12,000 x g for 5 min, washed twice in PBS containing 1% (wt/vol) Thesit and twice in PBS containing 0.1% (wt/vol) Thesit. Precipitated proteins were solubilized in Laemmli sample buffer containing 8 M urea and 1 mM EDTA, heated at 98 C for 30 min, resolved by SDS-PAGE, and GLUT4myc detected by immunoblotting with anti-myc antibodies.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SB203580 inhibits insulin-stimulated glucose uptake in L6 muscle cells
In agreement with previous results (15), glucose uptake into L6 myotubes stimulated by either submaximal (10 nM) or maximal (100 nM) insulin doses was reduced by preincubation with SB203580 (Fig. 1AGo), whereas insulin-stimulated GLUT4 translocation was not affected by SB203580 at either insulin concentration (Fig. 1BGo). Notice that SB203580 is removed before all assays. Together the differential sensitivity of insulin-stimulated glucose uptake vs. GLUT4 translocation to SB203580 suggests that glucose uptake may require the participation of SB203580 sensitive target(s) independently of GLUT4 translocation.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 1. SB203580 inhibits insulin-stimulated glucose uptake without affecting the amount of cell surface GLUT4. L6-GLUT4myc myotubes were incubated with vehicle (open bars) or 10 µM SB203580 (closed bars) for 40 min in the absence or presence of insulin during the last 20 min of the incubation. A, Cells were washed three times in HEPES buffer to remove the inhibitor and 2-deoxyglucose uptake was measured as described in Materials and Methods. Results are expressed relative to the vehicle-treated basal condition and represent the mean ± SE of three independent experiments. Each condition was assayed in triplicate. B, GLUT4myc translocation was performed as described in Materials and Methods. Values were normalized to the surface GLUT4myc level of the vehicle-treated basal condition. Results shown are the mean ± SE of three independent experiments. Each condition was assayed in triplicate. *, P < 0.05

 
Effect of DR mutant of p38{alpha} on SB203580-mediated reduction in insulin-stimulated glucose uptake
SB203580 inhibits p38{alpha} and p38ß with IC50 of 50 nM and 500 nM in vitro (31) and IC50 of 1.4 µM in intact cells including L6 myotubes (Ref.9 and references within). Maximal inhibition of insulin-stimulated glucose uptake occurs at 10 µM SB203580; therefore, it is plausible that the effect might be mediated by a target other than p38{alpha} or p38ß. To explore this possibility, WT- or DR-p38{alpha} was overexpressed in L6-GLUT4myc myotubes via adenoviral delivery. We determined that infection of L6 myotubes with adenoviruses encoding either WT-p38{alpha} or DR-p38{alpha} resulted in a similar insulin-responsive increase in total phospho-p38MAPK (Fig. 2AGo). The gels show the slower migration of the FLAG-tagged transfected proteins, compared with the endogenous p38 MAPK. In these experiments the SDS-PAGE conditions were optimized to facilitate detection of the two bands, and this may explain why a doublet is now visible in the control, untransfected cells, potentially reflecting more than one isoform of the endogenous enzyme. Similar levels of p38MAPK phosphorylation were observed in cells overexpressing WT-p38{alpha} and DR-p38{alpha}. Determination of p38 MAPK action in vivo requires analysis of the phosphorylation of a known target, typically MK-2. Although MK-2 is phosphorylated in response to insulin in L6 myotubes, this largely involves the MEK1-Erk pathway (data not shown). Hence, as readout of the effectiveness of the WT- and DR-p38{alpha}, we examined the phosphorylation of MK-2 in response to another activator of p38MAPK-MK-2, namely hyperosmolar stress. In the presence of 10 µM SB203580, the hyperosmolarity-induced MK-2 phosphorylation was reduced by 89% in cells overexpressing WT-p38{alpha} and to a similar degree as in nontransfected cells. In contrast, 10 µM SB203580 reduced MK-2 phosphorylation by only 35% in cells overexpressing DR-p38{alpha} (Fig. 2Go, B and C). Because adenovirus infection efficiency was consistently around 80% (assessed by bicistronic expression of enhanced GFP AdEasy vector, data not shown), these results suggest that SB203580 is largely unable to inhibit cellular p38{alpha} activity in cells overexpressing DR-p38{alpha}. Yet insulin-stimulated glucose uptake was inhibited by 10 µM SB203580 to a similar extent in cells overexpressing WT- and DR-p38{alpha} (Fig. 3Go).



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 2. DR-p38{alpha} in L6 myotubes protects hyperosmolarity-stimulated MK-2 phosphorylation from the effect of SB203580. L6 myotubes were infected with 2.5 x 105 PFU/well (24-well plate) of WT-p38{alpha} or DR-p38{alpha} adenovirus for 10–12 h, as described in Materials and Methods. L6-GLUT4myc myotubes were preincubated with vehicle (open bars) or 10 µM SB203580 (closed bars) for 20 min. Then in the continued absence or presence of the drug, cells were stimulated with insulin (100 nM for 10 min) or hyperosmolar stress (0.5 M NaCl for 20 min) as indicated. Total cellular lysates were prepared in Laemmli sample buffer. Fifty micrograms of total protein were resolved on SDS-PAGE and immunoblotted for p38{alpha}, phospho-p38MAPK (T180/Y182) or phospho-MK-2 (T334), as indicated. Representative blots are shown in A and B. Seven immunoblots were scanned within the linear range to quantify MK-2 phosphorylation, and the results, expressed relative to the respective NaCl-treated condition are shown in C. *, P < 0.05, compared with the WT-p38{alpha} transfected, NaCl-, and SB203580-treated condition.

 


View larger version (25K):
[in this window]
[in a new window]
  		FIG. 3. DR-p38{alpha} does not prevent inhibition of insulin-stimulated glucose uptake by SB203580. L6-GLUT4myc myotubes were infected with 2.5 x 105 PFU/well (24-well plate) of WT-p38{alpha} or DR-p38{alpha} adenovirus for 10–12 h and then incubated with 10 µM SB203580 (striped bars) or without the agent (open bars) for 40 min. During the last 20 min, myotubes were stimulated with 100 nM insulin for 20 min in the continued presence (gray bars) or absence (closed bars) of the inhibitor. 2-Deoxyglucose uptake was measured as described in Materials and Methods. Results are expressed relative to the WT-p38{alpha}-infected unstimulated condition and represent the mean ± SE of seven independent experiments. Each condition was assayed in triplicate. *, P < 0.05, compared with the corresponding WT-p38{alpha}-infected conditions.

 
Effect of DN mutants of p38{alpha} or -ß on insulin-stimulated glucose uptake
To confirm the unexpected result obtained with the DR-p38{alpha} mutant, we sought to achieve inhibition of p38{alpha} and p38ß activity by expressing DN mutants of each isoform. The DN-p38MAPK mutants are mutated at both of the phosphorylation sites (T180A, Y182F); therefore, they cannot be phosphorylated or activated by their upstream kinases, MKK3 and MKK6. Adenovirus-mediated infection of L6-GLUT4myc myotubes resulted in a 50-fold overexpression of the DN-p38{alpha} or DN-p38ß mutant relative to endogenous WT-p38{alpha} or p38ß, as determined by immunoblotting of whole-cell lysates. In separate experiments, adenovirus infection efficiency was consistently 80% or higher, assessed by immunofluorescence microscopy (data not shown). Expression of either DN-p38{alpha} or DN-p38ß reduced insulin-stimulated phosphorylation of p38MAPK by approximately 70% in whole-cell lysates (Fig. 4Go) and did not affect total GLUT4 protein amount in L6-GLUT4myc myotubes (data not shown). Therefore, it appears that these mutants inhibit activation of p38MAPK by functionally sequestering MKK3 and/or MKK6, thereby preventing activation of downstream targets. Neither DN-p38{alpha} (Fig. 5AGo) nor DN-p38ß (Fig. 5BGo) expression reduced glucose uptake stimulated by submaximal (10 nM) or maximal (100 nM) insulin doses. In addition, the inhibition of insulin-stimulated glucose uptake seen by pretreatment with 10 µM SB203580 occurred to similar extents in L6-GLUT4myc myotubes expressing GFP, DN-p38{alpha}, or DN-p38ß (34, 30, and 40%, respectively; data not shown). Hence, despite a marked reduction in insulin-stimulated p38MAPK phosphorylation by expression of DN-p38MAPK{alpha}/ß, there was no concomitant reduction in insulin-stimulated glucose uptake.



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 4. DN-p38{alpha} or DN-p38ß inhibit insulin-stimulated p38MAPK phosphorylation. L6-GLUT4myc myotubes were infected with 2.5 x 105 PFU/well (24-well plate) of GFP, FLAG-DN-p38{alpha}, or FLAG-DN-p38ß adenovirus for 18–24 h. Myotubes were left unstimulated (open bars) or stimulated with 10 (gray bars) or 100 nM insulin (closed bars) for 10 min. Total cellular lysates were prepared in Laemmli sample buffer, and 50 µg of total protein were resolved by SDS-PAGE and immunoblotted for phospho-p38MAPK. Immunoblots were scanned within the linear range to quantify protein phosphorylation, and the results of at least six independent experiments are expressed relative to the GFP-infected basal condition, as the mean ± SE. A representative immunoblot of phosphorylated p38MAPK is shown in the top panel, and a representative immunoblot (i.b.) showing expression of FLAG-DN-p38{alpha} and FLAG-DN-p38ß in each condition, detected using FLAG-specific antibodies, is shown in the bottom panel. *, P < 0.001; **, P < 0.01, relative to the respective condition in GFP virus-infected cells.

 


View larger version (20K):
[in this window]
[in a new window]
  		FIG. 5. Expression of DN-p38{alpha} or DN-p38ß does not affect insulin-stimulated glucose uptake. L6-GLUT4myc myotubes were infected with 2.5 x 105 PFU/well (24-well plate) of GFP and either FLAG-DN-p38{alpha} (A) or FLAG-DN-p38ß (B) adenovirus for 18–24 h and then were left unstimulated (open bars) or stimulated with 10 (gray bars) or 100 nM (closed bars) insulin for 20 min. 2-Deoxyglucose uptake was measured as described in Materials and Methods. Results are expressed relative to the GFP-infected basal condition and represent the mean ± SE of six to seven independent experiments. Each condition was assayed in triplicate.

 
Effect of siRNAs to p38 isoforms on insulin-stimulated glucose uptake
To complement the above analysis, we examined the effect of siRNA gene silencing to reduce p38{alpha} and/or p38ß proteins on insulin-stimulated glucose uptake. Sequence-specific siRNAs were transfected into L6 myotubes on d 5, and experiments were performed on d 7 of myotube differentiation. A 65–70% reduction of p38{alpha} protein was achieved, as determined by immunoblotting whole-cell lysates (Fig. 6AGo). Reduction of p38{alpha} by siRNA did not appreciably affect expression of p38ß (Fig. 6BGo). Because currently available antibodies specific to p38ß are poor at detecting endogenous p38ß protein in L6 myotubes, we used L6 cells stably expressing a FLAG-tagged DN-p38ß and measured FLAG signal after siRNA transfection (Fig. 6BGo). p38ß siRNA gene silencing achieved a knockdown of 63–74% of overexpressed p38ß protein (Fig. 6BGo). We surmise that the endogenous p38ß of L6-GLUT4myc myotubes was reduced to a similar extent by p38ß siRNA. The reduction in p38ß by siRNA did not appreciably affect expression of p38{alpha} (Fig. 6AGo), indicating specificity of each siRNA for its cognate sequence. In L6 myotubes transfected with siRNAs to both p38{alpha} and p38ß, each isoform was reduced to the extents achieved when each siRNA was used separately (Fig. 6Go, A and B). In addition, insulin-stimulated p38 phosphorylation was markedly inhibited by siRNA gene silencing of p38{alpha} alone or by combined siRNA to p38{alpha} and p38ß, whereas siRNA gene silencing of p38ß was without effect on p38 phosphorylation (Fig. 6CGo). The total amount of GLUT4 in L6-GLUT4myc myotubes was unaffected by siRNA gene silencing of p38 isoforms (data not shown). Importantly, the reductions in p38{alpha}, p38ß, or p38{alpha} and p38ß together (Fig. 7Go) were not accompanied by a drop in insulin-stimulated glucose uptake.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 6. p38MAPK isoform-specific siRNA reduces p38MAPK protein and insulin-stimulated p38MAPK phosphorylation. L6-GLUT4myc myotubes were transfected with 500 nM of NR siRNA or p38{alpha}- or p38ß-specific siRNA or both p38{alpha}- and p38ß-specific siRNA together as described in Materials and Methods. Total cellular lysates were prepared in Laemmli sample buffer, and 50 µg of total protein were resolved by SDS-PAGE and immunoblotted as indicated. Each panel shows the averaged results of seven independent experiments and illustrates a representative gel. A, Immunodetection of p38{alpha}. B, Immunodetection of FLAG from L6-FLAG-DN-p38ß. C, After transfection, L6-GLUT4myc myotubes were either left unstimulated (open bars) or stimulated with 100 nM insulin (closed bars). Phospho-p38MAPK was detected by immunoblotting (i.b.). *, P < 0.001; **, P < 0.01 relative to insulin-treated NR-siRNA transfected cells.

 


View larger version (21K):
[in this window]
[in a new window]
  		FIG. 7. p38-Isoform-specific siRNA has no effect on insulin-stimulated glucose uptake. L6-GLUT4myc myotubes were transfected with 500 nM of NR siRNA, p38{alpha}- or p38ß-specific siRNA, or both p38{alpha}- and p38ß-specific siRNA together as described in Materials and Methods. After transfection, cells were left unstimulated (open bars) or stimulated with 100 nM insulin (closed bars) for 20 min as indicated, and 2-deoxyglucose uptake was measured as described in Materials and Methods. Results are expressed relative to the NR-siRNA transfected basal condition and represent the mean ± SE of seven independent experiments. Each condition was assayed in triplicate.

 
Effect of SB203580 on GLUT4myc photolabeling
Bio-LC-ATB-BMPA contains a sugar-like moiety recognized by GLUTs and a reactive group that undergoes covalent insertion into neighboring atoms of the transporter on photoactivation (35). Thus, it is expected that the amount of photolabeled GLUT4 is an indication of the accessibility of the extracellular glucose binding site of GLUT4. In agreement with previous results in adipose cells (44, 45, 46), insulin caused an increase in the amount of bio-LC-ATB-BMPA photolabeled GLUT4myc in L6 myotubes (Fig. 8Go). Pretreatment of cells with SB203580, followed by washout of the drug before bio-LC-ATB-BMPA incubation significantly reduced the amount of photolabeled transporter in the insulin-stimulated condition but was without effect in the basal state (Fig. 8Go). This results suggests that pretreatment of cells with SB203580 reduces the accessibility of the extracellular glucose binding site of insulin-stimulated GLUT4myc. Because availability of the myc epitope to the extracellular medium was not affected by SB203580 (Fig. 1BGo), these results suggest that it is the extracellular availability of the glucose binding site, rather than of the entire transporter, that is reduced by the drug.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 8. SB203580 reduces insulin-stimulated but not basal ATB-BMPA photolabeling of cell surface GLUT4myc. L6-GLUT4myc myotubes were pretreated with 10 µM SB203580 for 20 min and then stimulated with insulin, as indicated, for 20 min in the continued presence of the inhibitor. SB203580 was then washed out before photolabeling of GLUT4 with biotin-conjugated ATB-BMPA and precipitation of the photolabeled transporters via immobilized streptavidin, as described in Materials and Methods. The amount of precipitated (photolabeled) GLUT4myc was detected by immunoblotting with anti-myc antibody. A representative immunoblot is shown, and the results of n = 3 experiments are quantified. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been repeatedly observed that in muscle cells and adipocytes pretreated with specific inhibitors of p38MAPK such as pyridinyl imidazoles (SB203580 and SB202190) or azaazulene inhibitors (27) have reduced insulin-stimulated glucose uptake (30–40%) yet undiminished GLUT4 translocation (15, 23, 47). These results raised the hypothesis that insulin-stimulated glucose uptake requires both GLUT4 translocation and GLUT4 activation and that insulin-stimulated GLUT4 activation may involve a p38MAPK-dependent pathway (48). However, the hypothesis has largely relied on pharmacological inhibitors of p38MAPK.

Surprisingly, two studies employing DN mutants of p38MAPK to explore the regulation of glucose uptake have yielded contradicting results. Fujishiro et al. (28) showed that adenovirus-driven expression of DN-p38{alpha} in 3T3-L1 adipocytes elevated insulin-stimulated glucose uptake and that expression of DN mutants of upstream activators of p38MAPK (MKK3 and MKK6) elevated GLUT1 and diminished GLUT4 levels. DN-p38 MAPK may have had a similar effect, likely as a result of transcriptional input from the p38MAPK pathway on GLUT4 expression (27). In a related study from our group using 3T3-L1 adipocytes, induction of DN-p38 MAPK expression did not change GLUT1 or GLUT4 levels yet lowered insulin-stimulated glucose uptake (47). The amount of plasma membrane lawn-associated GLUT4 was also unchanged, but this approach does not distinguish GLUT4 vesicles docked/adhered from those fully inserted in the membrane. These considerations prompted us to reexamine the role of DN-p38 MAPK mutants in L6-GLUT4myc cells, a stably transfected cell line in which GLUT4myc insertion into the membrane can be accurately determined and in which GLUT4myc biosynthesis is unlikely to be prone to regulation because it is driven by a heterologous promoter (37). Therefore, in these cells, GLUT4myc expression cannot be regulated by p38MAPK input, and GLUT4myc largely surpasses the expression and function of endogenous glucose transporters (2, 25). This cellular system was therefore selected to determine whether p38MAPK is required for insulin-stimulated glucose uptake in L6-GLUT4myc myotubes using: 1) a mutant of p38{alpha} (DR-p38{alpha}) resistant to SB203580; 2) dominant-inhibitory mutants of p38{alpha} and p38ß; and 3) siRNA against p38{alpha} and p38ß.

The DR-p38MAPK has been previously used to determine whether SB203580-mediated effects occur via p38MAPK or other unknown SB203580 targets (32, 49, 50). Here we delivered WT- and DR-p38MAPK to L6 muscle cells, in which they were viably responsive to insulin, as judged from their phosphorylation in response to the hormone (Fig. 2AGo). Expression of DR-p38{alpha} in L6-GLUT4myc myotubes reversed the SB203580-mediated inhibition of hyperosmolarity-induced MK-2 phosphorylation by 65% (Fig. 2Go). However, it did not prevent SB203580-mediated inhibition of insulin-stimulated glucose uptake (Fig. 3Go). The most obvious interpretation of these results is that SB203580-mediated inhibition of glucose uptake may not be due to inhibition of p38{alpha} and/or p38ß by SB203580. Still it is also possible that p38MAPK is the target of SB203580, but an approximately 35% reduction in p38MAPK activity suffices to produce maximal inhibition of the p38MAPK-dependent component of insulin-stimulated glucose uptake. Alternatively, it is possible that the DR-p38{alpha} did not fully replace the endogenous p38{alpha} or p38ß involved in regulating glucose uptake, even though DR-p38{alpha} was able to functionally replace the endogenous p38MAPK activity required for MK-2 phosphorylation.

To distinguish among these interpretations, we overexpressed DN mutants of p38{alpha} or p38ß (DN-p38{alpha} or DN-p38ß). Overexpression of either DN-p38{alpha} or DN-p38ß reduced insulin-stimulated p38MAPK phosphorylation by 70–80% (Fig. 4Go) but did not lower glucose uptake stimulated by either 10 or 100 nM insulin (Fig. 5Go). Because each DN mutant reduced the total insulin-stimulated p38MAPK phosphorylation by 70–80%, these mutants likely inhibited signaling by both their cognate p38MAPK and other isoforms of p38MAPK. It is possible that the remaining 20–30% of p38MAPK activity suffices to stimulate GLUT4 activation. However, it is more likely that the remaining 20% p38MAPK phosphorylation arises from uninfected cells (see Results) and that, in infected cells, p38MAPK may not regulate GLUT4 activity. Instead, a different target may be responsible for the effects of SB203580.

To corroborate results obtained with the DN-p38 mutants, the expression of endogenous p38MAPK was diminished using siRNA against either p38{alpha} or p38ß. Reducing the expression of endogenous p38{alpha} by 65% dropped insulin-stimulated p38MAPK phosphorylation by 70%. In contrast, reducing the expression of p38ß by 60% did not affect insulin-stimulated p38MAPK phosphorylation (Fig. 6Go). These results suggest that in L6-GLUT4myc myotubes, p38{alpha} is responsible for the majority of insulin-stimulated p38MAPK phosphorylation, even though both p38{alpha} and p38ß are activated by insulin (15, 18). It is noteworthy that expression of DN-p38ß resulted in a significant (~70%) inhibition of insulin-stimulated p38MAPK phosphorylation. Therefore, it is possible that this p38ß mutant was able to also inhibit the activation of endogenous p38{alpha}, perhaps by sequestering an upstream kinase common to both p38MAPK isoforms. In agreement with such an interpretation, overexpression of DN-p38{alpha} was shown to sequester the upstream kinase MKK3, thereby preventing the activation of another MKK3 substrate, Mirk (51). Consistent with the results obtained with DN-p38, reducing endogenous p38{alpha} or p38ß by siRNA gene silencing had no effect on insulin-stimulated glucose uptake (Fig. 7Go). As in the case of L6 myotubes expressing DN-p38 mutants, siRNA gene silencing reduced only approximately 70% of p38MAPK activity, making it possible that the remaining small amount of active p38MAPK activity was sufficient to mediate maximal stimulation of GLUT4 activation. However, this argument is countered by the observation that in cells expressing DR-p38MAPK, glucose uptake was still reduced by SB203580, even though in the presence of the drug, the majority (65%) of p38MAPK was preserved. This result argues against a low level of p38MAPK sufficing to regulate glucose uptake.

The results obtained by three independent molecular methods to interfere with p38MAPK signaling did not interfere with submaximally or maximally stimulated glucose uptake. It may be possible that each of the DR-p38 and DN-p38 mutants and siRNA gene silencing of p38MAPK was unable to affect a small pool of p38MAPK that is responsible for SB203580-sensitive stimulation of GLUT4 activity in response to insulin. However, we recently observed that another pyridinyl imidazole, SB220025, inhibited p38MAPK as robustly as SB203580 (measured by hyperosmolarity-stimulated MK-2 phosphorylation) yet had no effect on insulin-stimulated glucose uptake (data not shown). Thus, the most likely interpretation of the collective results is that p38MAPK is not involved in insulin-stimulated glucose uptake and that the SB203580-mediated reduction in glucose uptake is due to an effect of SB203580 on another protein.

SB203580 binds to the ATP pocket of p38{alpha} and p38ß (52) with a 1:1 stoichiometry. The specificity of several pyridinyl imidazoles has been tested in many studies, and the in vitro IC50 values range from 50 to 500 nM for p38{alpha} and p38ß (31, 53), whereas p38{gamma} (21) and p38{delta} (22) are insensitive to these inhibitors. In addition, the in vivo specificity of SB203580 has been validated in several systems using the DR-p38{alpha} mutant used in this study (11, 32, 49, 50). Notably, however, selective outcomes were shown to be independent of participation of p38MAPK (50). Moreover, very recently, Godl et al. (54) detected several previously unknown targets of SB203580 in HeLa cells. Cell extracts from these cells were screened for proteins binding to SB203580 using affinity chromatography coupled to mass spectrometry. Several proteins not previously known to be targets of SB203580 were identified, some with IC50 values similar to or lower than p38MAPK, including rip-like interacting caspase-like apoptosis-regulatory protein kinase, CK1, cyclin G-associated kinase, protein kinase Nß, Janus kinase 1, and c-Jun N-terminal kinase (JNK)1/2. Indeed, under certain in vitro reaction conditions, the IC50 of JNK1 and JNK2 can be 13 and 0.7 µM, respectively, making SB203580 a much more potent inhibitor of JNK1/2 than previously reported. However, the JNK inhibitor SP600125 had virtually no effect on insulin-stimulated glucose uptake in L6 myotubes (Niu, W., and A. Klip, unpublished observations), rat neonatal myotubes (55), or primary rat adipocytes (56). Therefore, the possibility that SB203580 inhibited insulin-stimulated glucose uptake and GLUT4 activation via protein(s) other than p38MAPK and JNK seems possible, and future work seeking to identify such protein(s) would be of great interest. In support of this interpretation, Kayali et al. (29) observed discrepancies between the dose of SB203580 required to reduce insulin-stimulated glucose uptake in 3T3-L1 adipocytes and the EC50 of inhibition of activating transcription factor-2 activity recovered in vitro.

Alternatively, it is also plausible that the SB203580-mediated inhibition of insulin-stimulated glucose uptake is due to direct inhibition of GLUT4 by the compound. However, this is unlikely because SB203580 has no effect on basal glucose uptake (15, 23), which is mediated by GLUT4 in L6-GLUT4myc myotubes (2). Moreover, in our experimental protocol, SB203580 is washed away before the assay of glucose uptake. However, difficulties in removing the compound from cells has been described (33), raising the possibility that some quantity of SB203580 is still present in the cells at the time of measurement of glucose transport. Holman and colleagues (33) recently suggested that SB203580 may interact with insulin-stimulated GLUT4 based on its competitive action on equilibrium-exchange glucose uptake in insulin-stimulated rat adipocytes. Such an effect required loading of the cells with SB203580 and presence of the compound during the transport assay. Those results are compatible with either SB203580 acting on a target that reduces GLUT4 affinity of the intracellular glucose-binding site (such as an endogenous inhibitory protein) or SB203580 selectively binding to and directly inhibiting the insulin-stimulated configuration of GLUT4. In either case, SB203580 would diminish glucose uptake through GLUT4 in the insulin-stimulated but not the basal state.

To better understand the nature of the SB203580-mediated reduction in glucose uptake, we examined the effect of SB203580 on photoaffinity labeling of GLUT4myc by bio-LC-ATB-BMPA (Fig. 8Go). As for glucose uptake, SB203580 reduced ATB-BMPA photolabeling of GLUT4 in insulin-stimulated but not basal conditions. Because SB203580 does not reduce the amount of cell surface transporters detected via the myc epitope (Fig. 1Go), the drug appears to reduce the accessibility of the extracellular glucose-binding site of cell surface GLUT4myc. During the preparation of this manuscript, Bazuine et al. (57) observed a similar effect of SB203580 on ATB-BMPA photolabeling of GLUT4myc in insulin-stimulated 3T3-L1 adipocytes. This reduction is likely not due to SB203580 binding to the extracellular hexose-binding site of GLUT4 because the drug inhibits hexose influx noncompetitively (58). Instead, the SB203580-dependent reduction in GLUT4myc photolabeling at the extracellular site could be envisaged to occur concomitantly with the decreased affinity of the intracellular glucose binding site, potentially even as a consequence of it. Because insulin does not affect the Michaelis constant of GLUT4 for hexose entry, these results also suggest that stimulation with insulin may alter the rate of turnover of GLUT4 between outward- and inward-facing conformations.

In summary, pretreatment of L6-GLUT4myc myotubes with the pyridinyl imidazole SB203580 inhibited insulin-stimulated glucose uptake without decreasing GLUT4 translocation. The expression of DR-p38{alpha} did not rescue the SB203580-mediated inhibition of glucose uptake, and reduction of endogenous p38{alpha} and p38ß activities by either dominant-negative mutants of p38{alpha} and p38ß or siRNA-mediated gene silencing had no effect on insulin-stimulated glucose uptake. These results support the hypothesis that insulin-mediated glucose uptake and GLUT4 translocation can be segregated and that a p38{alpha}/ß-independent, SB203580-sensitive target or GLUT4 conformation may be required for insulin-stimulated activation of GLUT4 but not for GLUT4 translocation.


   Acknowledgments
 
We gratefully acknowledge Dr. Phil Cohen (University of Dundee, Dundee, Scotland, UK) for the gift of the cDNA encoding DR-p38MAPK{alpha} and valuable comments on this manuscript and Dr. Geoff Holman (University of Bath, Bath, UK) for providing initial samples of bio-LC-ATB-BMPA.


   Footnotes
 
This work was supported by Grant MT-12601 from the Canadian Institutes of Health Research (to A.K.). A Novartis Studentship from the Banting and Best Diabetes Centre and an Ontario Graduate Scholarship supported C.N.A. A Canadian Institute of Health Research fellowship supported C.H.

First Published Online June 9, 2005

1 C.N.A. and C.H. contributed equally to this work. Back

Abbreviations: DN, Dominant negative; DR, drug resistant; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; MK-2, MAPK-activated protein kinase-2; MKK, MAPK-kinase; NR, nonrelevant; si, small interfering; WT, wild type.

Received April 7, 2005.

Accepted for publication May 31, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Douen AG, Ramlal T, Rastogi S, Bilan PJ, Cartee GD, Vranic M, Holloszy JO, Klip A 1990 Exercise induces recruitment of the "insulin-responsive glucose transporter." Evidence for distinct intracellular insulin- and exercise-recruitable transporter pools in skeletal muscle. J Biol Chem 265:13427–13430[Abstract/Free Full Text]
  2. Rudich A, Konrad D, Torok D, Ben-Romano R, Huang C, Niu W, Garg RR, Wijesekara N, Germinario RJ, Bilan PJ, Klip A 2003 Indinavir uncovers different contributions of GLUT4 and GLUT1 toward glucose uptake in muscle and fat cells and tissues. Diabetologia 46:649–658[Medline]
  3. Cushman S, Wardzala L 1980 Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J Biol Chem 255:4758–4762[Free Full Text]
  4. Klip A, Ramlal T, Young DA, Holloszy JO 1987 Insulin-induced translocation of glucose transporters in rat hindlimb muscles. FEBS Lett 224:224–230[CrossRef][Medline]
  5. Marette A, Burdett E, Douen A, Vranic M, Klip A 1992 Insulin induces the translocation of GLUT4 from a unique intracellular organelle to transverse tubules in rat skeletal muscle. Diabetes 41:1562–1569[Abstract]
  6. Zierler K 1998 Does insulin-induced increase in the amount of plasma membrane GLUTs quantitatively account for insulin-induced increase in glucose uptake? Diabetologia 41:724–730[CrossRef][Medline]
  7. Niu W, Huang C, Nawaz Z, Levy M, Somwar R, Li D, Bilan PJ, Klip A 2003 Maturation of the regulation of GLUT4 activity by p38 MAPK during L6 cell myogenesis. J Biol Chem 278:17953–17962[Abstract/Free Full Text]
  8. Hausdorff SF, Fingar DC, Morioka K, Garza LA, Whiteman EL, Summers SA, Birnbaum MJ 1999 Identification of wortmannin-sensitive targets in 3T3–L1 adipocytes. Dissociation of insulin-stimulated glucose uptake and glut4 translocation. J Biol Chem 274:24677–24684[Abstract/Free Full Text]
  9. Somwar R, Niu W, Kim DY, Sweeney G, Randhawa VK, Huang C, Ramlal T, Klip A 2001 Differential effects of phosphatidylinositol 3-kinase inhibition on intracellular signals regulating GLUT4 translocation and glucose transport. J Biol Chem 276:46079–46087[Abstract/Free Full Text]
  10. Furtado LM, Somwar R, Sweeney G, Niu W, Klip A 2002 Activation of the glucose transporter GLUT4 by insulin. Biochem Cell Biol 80:569–578[CrossRef][Medline]
  11. Kovarik P, Stoiber D, Eyers PA, Menghini R, Neininger A, Gaestel M, Cohen P, Decker T 1999 Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-{gamma} uses a different signaling pathway. Proc Natl Acad Sci USA 96:13956–13961[Abstract/Free Full Text]
  12. Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, Davis RJ 1995 Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 270:7420–7426[Abstract/Free Full Text]
  13. Barros LF, Young M, Saklatvala J, Baldwin SA 1997 Evidence of two mechanisms for the activation of the glucose transporter GLUT1 by anisomycin: p38(MAP kinase) activation and protein synthesis inhibition in mammalian cells. J Physiol 504(Pt 3):517–525
  14. Kyriakis JM, Avruch J 2001 Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81:807–869[Abstract/Free Full Text]
  15. Sweeney G, Somwar R, Ramlal T, Volchuk A, Ueyama A, Klip A 1999 An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3–L1 adipocytes and L6 myotubes. J Biol Chem 274:10071–10078[Abstract/Free Full Text]
  16. Pyne NJ, Pyne S 1997 Platelet-derived growth factor activates a mammalian Ste20 coupled mitogen-activated protein kinase in airway smooth muscle. Cell Signal 9:311–317[CrossRef][Medline]
  17. Ryder JW, Fahlman R, Wallberg-Henriksson H, Alessi DR, Krook A, Zierath JR 2000 Effect of contraction on mitogen-activated protein kinase signal transduction in skeletal muscle. Involvement of the mitogen- and stress-activated protein kinase 1. J Biol Chem 275:1457–1462[Abstract/Free Full Text]
  18. Somwar R, Perreault M, Kapur S, Taha C, Sweeney G, Ramlal T, Kim DY, Keen J, Cote CH, Klip A, Marette A 2000 Activation of p38 mitogen-activated protein kinase {alpha} and ß by insulin and contraction in rat skeletal muscle: potential role in the stimulation of glucose transport. Diabetes 49:1794–1800[Abstract]
  19. Han J, Lee JD, Bibbs L, Ulevitch RJ 1994 A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265:808–811[Abstract/Free Full Text]
  20. Jiang Y, Chen C, Li Z, Guo W, Gegner JA, Lin S, Han J 1996 Characterization of the structure and function of a new mitogen-activated protein kinase (p38ß). J Biol Chem 271:17920–17926[Abstract/Free Full Text]
  21. Cuenda A, Cohen P, Buee-Scherrer V, Goedert M 1997 Activation of stress-activated protein kinase-3 (SAPK3) by cytokines and cellular stresses is mediated via SAPKK3 (MKK6); comparison of the specificities of SAPK3 and SAPK2 (RK/p38). EMBO J 16:295–305[CrossRef][Medline]
  22. Jiang Y, Gram H, Zhao M, New L, Gu J, Feng L, Di Padova F, Ulevitch RJ, Han J 1997 Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38{Delta}. J Biol Chem 272:30122–30128[Abstract/Free Full Text]
  23. Konrad D, Bilan PJ, Nawaz Z, Sweeney G, Niu W, Liu Z, Antonescu CN, Rudich A, Klip A 2002 Need for GLUT4 activation to reach maximum effect of insulin-mediated glucose uptake in brown adipocytes isolated from GLUT4myc-expressing mice. Diabetes 51:2719–2726[Abstract/Free Full Text]
  24. Bazuine M, Ouwens DM, Gomes de Mesquita DS, Maassen JA 2003 Arsenite stimulated glucose transport in 3T3–L1 adipocytes involves both Glut4 translocation and p38 MAPK activity. Eur J Biochem 270:3891–3903[Medline]
  25. Huang C, Somwar R, Patel N, Niu W, Torok D, Klip A 2002 Sustained exposure of L6 myotubes to high glucose and insulin decreases insulin-stimulated GLUT4 translocation but upregulates GLUT4 activity. Diabetes 51:2090–2098[Abstract/Free Full Text]
  26. Koistinen HA, Chibalin AV, Zierath JR 2003 Aberrant p38 mitogen-activated protein kinase signalling in skeletal muscle from type 2 diabetic patients. Diabetologia 46:1324–1328[CrossRef][Medline]
  27. Carlson CJ, Koterski S, Sciotti RJ, Poccard GB, Rondinone CM 2003 Enhanced basal activation of mitogen-activated protein kinases in adipocytes from type 2 diabetes: potential role of p38 in the downregulation of GLUT4 expression. Diabetes 52:634–641[Abstract/Free Full Text]
  28. Fujishiro M, Gotoh Y, Katagiri H, Sakoda H, Ogihara T, Anai M, Onishi Y, Ono H, Funaki M, Inukai K, Fukushima Y, Kikuchi M, Oka Y, Asano T 2001 MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-induced glucose uptake but regulates glucose transporter expression. J Biol Chem 276:19800–19806[Abstract/Free Full Text]
  29. Kayali AG, Austin DA, Webster NJ 2000 Stimulation of MAPK cascades by insulin and osmotic shock: lack of an involvement of p38 mitogen-activated protein kinase in glucose transport in 3T3–L1 adipocytes. Diabetes 49:1783–1793[Abstract]
  30. Goedert M, Cuenda A, Craxton M, Jakes R, Cohen P 1997 Activation of the novel stress-activated protein kinase SAPK4 by cytokines and cellular stresses is mediated by SKK3 (MKK6); comparison of its substrate specificity with that of other SAP kinases. EMBO J 16:3563–3571[CrossRef][Medline]
  31. Davies SP, Reddy H, Caivano M, Cohen P 2000 Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351:95–105[CrossRef][Medline]
  32. Eyers PA, van den Ijessel P, Quinlan RA, Goedert M, Cohen P 1999 Use of a drug-resistant mutant of stress-activated protein kinase 2a/p38 to validate the in vivo specificity of SB 203580. FEBS Lett 451:191–196[CrossRef][Medline]
  33. Ribe D, Yang J, Patel S, Koumanov F, Cushman SW, Holman GD 2005 Endofacial competitive inhibition of glucose transporter-4 intrinsic activity by the mitogen-activated protein kinase inhibitor SB203580. Endocrinology 146:1713–1717[Abstract/Free Full Text]
  34. Engelman JA, Lisanti MP, Scherer PE 1998 Specific inhibitors of p38 mitogen-activated protein kinase block 3T3–L1 adipogenesis. J Biol Chem 273:32111–32120[Abstract/Free Full Text]
  35. Holman GD, Kozka IJ, Clark AE, Flower CJ, Saltis J, Habberfield AD, Simpson IA, Cushman SW 1990 Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester. J Biol Chem 265:18172–18179[Abstract/Free Full Text]
  36. Allen MP, Linseman DA, Udo H, Xu M, Schaack JB, Varnum B, Kandel ER, Heidenreich KA, Wierman ME 2002 Novel mechanism for gonadotropin-releasing hormone neuronal migration involving Gas6/Ark signaling to p38 mitogen-activated protein kinase. Mol Cell Biol 22:599–613[Abstract/Free Full Text]
  37. Kanai F, Nishioka Y, Hayashi H, Kamohara S, Todaka M, Ebina Y 1993 Direct demonstration of insulin-induced GLUT4 translocation to the surface of intact cells by insertion of a c-myc epitope into an exofacial GLUT4 domain. J Biol Chem 268:14523–14526[Abstract/Free Full Text]
  38. Ueyama A, Yaworsky KL, Wang Q, Ebina Y, Klip A 1999 GLUT-4myc ectopic expression in L6 myoblasts generates a GLUT-4-specific pool conferring insulin sensitivity. Am J Physiol 277:E572–E578
  39. Foster LJ, Li D, Randhawa VK, Klip A 2001 Insulin accelerates inter-endosomal GLUT4 traffic via phosphatidylinositol 3-kinase and protein kinase B. J Biol Chem 276:44212–44221[Abstract/Free Full Text]
  40. Li D, Randhawa VK, Patel N, Hayashi M, Klip A 2001 Hyperosmolarity reduces GLUT4 endocytosis and increases its exocytosis from a VAMP2-independent pool in l6 muscle cells. J Biol Chem 276:22883–22891[Abstract/Free Full Text]
  41. Mitsumoto Y, Klip A 1992 Development regulation of the subcellular distribution and glycosylation of GLUT1 and GLUT4 glucose transporters during myogenesis of L6 muscle cells. J Biol Chem 267:4957–4962[Abstract/Free Full Text]
  42. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B 1998 A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95:2509–2514[Abstract/Free Full Text]
  43. Ryder JW, Yang J, Galuska D, Rincon J, Bjornholm M, Krook A, Lund S, Pedersen O, Wallberg-Henriksson H, Zierath JR, Holman GD 2000 Use of a novel impermeable biotinylated photolabeling reagent to assess insulin- and hypoxia-stimulated cell surface GLUT4 content in skeletal muscle from type 2 diabetic patients. Diabetes 49:647–654[Abstract]
  44. Wilson CM, Mitsumoto Y, Maher F, Klip A 1995 Regulation of cell surface GLUT1, GLUT3, and GLUT4 by insulin and IGF-I in L6 myotubes. FEBS Lett 368:19–22[CrossRef][Medline]
  45. Calderhead DM, Kitagawa K, Tanner LI, Holman GD, Lienhard GE 1990 Insulin regulation of the two glucose transporters in 3T3–L1 adipocytes. J Biol Chem 265:13801–13808
  46. Harrison SA, Clancy BM, Pessino A, Czech MP 1992 Activation of cell surface glucose transporters measured by photoaffinity labeling of insulin-sensitive 3T3–L1 adipocytes. J Biol Chem 267:3783–3788[Abstract/Free Full Text]
  47. Somwar R, Koterski S, Sweeney G, Sciotti R, Djuric S, Berg C, Trevillyan J, Scherer PE, Rondinone CM, Klip A 2002 A dominant-negative p38 MAPK mutant and novel selective inhibitors of p38 MAPK reduce insulin-stimulated glucose uptake in 3T3–L1 adipocytes without affecting GLUT4 translocation. J Biol Chem 277:50386–50395[Abstract/Free Full Text]
  48. Furtado L, Poon V, Klip A 2003 GLUT4 activation: thoughts on possible mechanisms. Acta Physiol Scand 178:287–296[CrossRef][Medline]
  49. Martin JL, Avkiran M, Quinlan RA, Cohen P, Marber MS 2001 Antiischemic effects of SB203580 are mediated through the inhibition of p38{alpha} mitogen-activated protein kinase: evidence from ectopic expression of an inhibition-resistant kinase. Circ Res 89:750–752[Abstract/Free Full Text]
  50. Guo X, Gerl RE, Schrader JW 2003 Defining the involvement of p38{alpha} MAPK in the production of anti- and proinflammatory cytokines using an SB 203580-resistant form of the kinase. J Biol Chem 278:22237–22242[Abstract/Free Full Text]
  51. Lim S, Zou Y, Friedman E 2002 The transcriptional activator Mirk/Dyrk1B is sequestered by p38{alpha}/ß MAP kinase. J Biol Chem 277:49438–49445[Abstract/Free Full Text]
  52. Young PR, McLaughlin MM, Kumar S, Kassis S, Doyle ML, McNulty D, Gallagher TF, Fisher S, McDonnell PC, Carr SA, Huddleston MJ, Seibel G, Porter TG, Livi GP, Adams JL, Lee JC 1997 Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site. J Biol Chem 272:12116–12121[Abstract/Free Full Text]
  53. Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, Lee JC 1995 SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 364:229–233[CrossRef][Medline]
  54. Godl K, Wissing J, Kurtenbach A, Habenberger P, Blencke S, Gutbrod H, Salassidis K, Stein-Gerlach M, Missio A, Cotten M, Daub H 2003 An efficient proteomics method to identify the cellular targets of protein kinase inhibitors. Proc Natl Acad Sci USA 100:15434–15439[Abstract/Free Full Text]
  55. de Alvaro C, Teruel T, Hernandez R, Lorenzo M 2004 Tumor necrosis factor {alpha} produces insulin resistance in skeletal muscle by activation of inhibitor {kappa}B kinase in a p38 MAPK-dependent manner. J Biol Chem 279:17070–17078[Abstract/Free Full Text]
  56. Goransson O, Ryden M, Nilsson R, Arner P, Degerman E 2004 Dimethylaminopurine inhibits metabolic effects of insulin in primary adipocytes. J Nutr Biochem 15:303–312[CrossRef][Medline]
  57. Bazuine M, Carlotti F, Rabelink MJ, Vellinga J, Hoeben RC, Maassen JA 2005 The p38 mitogen-activated protein kinase inhibitor SB203580 reduces glucose turnover by the glucose transporter-4 of 3T3–L1 adipocytes in the insulin-stimulated state. Endocrinology 146:1818–1824[Abstract/Free Full Text]
  58. Somwar R, Kim DY, Sweeney G, Huang C, Niu W, Lador C, Ramlal T, Klip A 2001 GLUT4 translocation precedes the stimulation of glucose uptake by insulin in muscle cells: potential activation of GLUT4 via p38 mitogen-activated protein kinase. Biochem J 359:639–649[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J ANIM SCIHome page
M. Lorenzo, S. Fernandez-Veledo, R. Vila-Bedmar, L. Garcia-Guerra, C. De Alvaro, and I. Nieto-Vazquez
Insulin resistance induced by tumor necrosis factor-{alpha} in myocytes and brown adipocytes
J Anim Sci, April 1, 2008; 86(14_suppl): E94 - E104.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
B. Emanuelli, D. Eberle, R. Suzuki, and C. R. Kahn
Overexpression of the dual-specificity phosphatase MKP-4/DUSP-9 protects against stress-induced insulin resistance
PNAS, March 4, 2008; 105(9): 3545 - 3550.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Diaz, C. N. Antonescu, E. Capilla, A. Klip, and J. V. Planas
Fish Glucose Transporter (GLUT)-4 Differs from Rat GLUT4 in Its Traffic Characteristics but Can Translocate to the Cell Surface in Response to Insulin in Skeletal Muscle Cells
Endocrinology, November 1, 2007; 148(11): 5248 - 5257.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
H. F. Kramer and L. J. Goodyear
Exercise, MAPK, and NF-{kappa}B signaling in skeletal muscle
J Appl Physiol, July 1, 2007; 103(1): 388 - 395.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
N. Konstantopoulos, S. Marcuccio, S. Kyi, V. Stoichevska, L. A. Castelli, C. W. Ward, and S. L. Macaulay
A Purine Analog Kinase Inhibitor, Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor 59, Reveals a Role for Calcium/Calmodulin-Dependent Protein Kinase II in Insulin-Stimulated Glucose Transport
Endocrinology, January 1, 2007; 148(1): 374 - 385.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
N. Fujii, N. Jessen, and L. J. Goodyear
AMP-activated protein kinase and the regulation of glucose transport
Am J Physiol Endocrinol Metab, November 1, 2006; 291(5): E867 - E877.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
Y. Tao, H. Maegawa, S. Ugi, K. Ikeda, Y. Nagai, K. Egawa, T. Nakamura, S. Tsukada, Y. Nishio, S. Maeda, et al.
The Transcription Factor AP-2{beta} Causes Cell Enlargement and Insulin Resistance in 3T3-L1 Adipocytes
Endocrinology, April 1, 2006; 147(4): 1685 - 1696.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
D. C. Wright, P. C. Geiger, D.-H. Han, and J. O. Holloszy
Are tyrosine kinases involved in mediating contraction-stimulated muscle glucose transport?
Am J Physiol Endocrinol Metab, January 1, 2006; 290(1): E123 - E128.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Antonescu, C. N.
Right arrow Articles by Klip, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Antonescu, C. N.
Right arrow Articles by Klip, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals