Reduction of Insulin-Stimulated Glucose Uptake in L6 Myotubes by the Protein Kinase Inhibitor SB203580 Is Independent of p38MAPK Activity
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《内分泌学杂志》
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
Department of Pharmacology (K.A.H.), University of Colorado Health Sciences Center and Denver Veterans Affairs Medical Center, Denver, Colorado 80262
Abstract
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 and p38MAPK (e.g. SB203580) reduce insulin-stimulated glucose uptake without affecting GLUT4 translocation. This observation suggested that insulin may increase GLUT4 activity via p38 and/or p38. 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 (drug-resistant p38) but barely affected phosphorylation of the p38 substrate MAPK-activated protein kinase-2. Expression of dominant-negative p38 or p38 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 or p38 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
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 (19) and p38 (20) are ubiquitously expressed, whereas p38 (21) is highly expressed in skeletal muscle and p38 in the lung and kidney (22). Full activation of p38MAPK requires dual phosphorylation on residues corresponding to threonine-180 and tyrosine-182 on p38 (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 and p38 (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 and p38 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 or p38 (34) or drug-resistant (DR) p38 (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 or p38 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
Materials
SB203580 was obtained from Calbiochem (San Diego, CA). Protease inhibitor cocktail, cytochalasin B, O-phenylenediamine dihydrochloride, 2-deoxyglucose, and -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). -Phospho-p38 MAPK (recognizes all p38MAPK isoforms only when phosphorylated on residues corresponding to T180 and Y182 on p38), -phospho-MAPK-activated protein kinase-2 (MK-2; recognizes MK-2 when phosphorylated on T334), and -p38 MAPK antibodies were obtained from Cell Signaling Technologies (Beverly, MA). Polyclonal -myc antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat -rabbit and sheep -mouse antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). Adenovirus particles encoding cDNAs for green fluorescent protein (GFP), DN-p38 (T180A, Y182F), DN-p38 (T180A, Y182F), and wild-type (WT)-p38 were prepared as previously described (36). DR-p38 (T180M, H107P, L108F) has been previously characterized (32). Synthetic siRNAs specific for rat p38MAPK (AAG CAC GAG AAU GUG AUU GGU) and p38MAPK (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 were constructed using the AdEasy I system (Stratagene, La Jolla, CA) (42). Briefly, cDNA encoding DR-p38 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 MEM and 5 mM glucose for 4 h (in serum-free medium). Infection proceeded for 10–12 h (WT-p38 and DR-p38 adenovirus) or 18–24 h (DN-p38, DN-p38, and GFP adenovirus) before serum deprivation and experimentation.
siRNA transfection
Cells were incubated in antibiotic-free 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, p38, or p38 and p38 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 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
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. 1A), whereas insulin-stimulated GLUT4 translocation was not affected by SB203580 at either insulin concentration (Fig. 1B). 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.
Effect of DR mutant of p38 on SB203580-mediated reduction in insulin-stimulated glucose uptake
SB203580 inhibits p38 and p38 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 or p38. To explore this possibility, WT- or DR-p38 was overexpressed in L6-GLUT4myc myotubes via adenoviral delivery. We determined that infection of L6 myotubes with adenoviruses encoding either WT-p38 or DR-p38 resulted in a similar insulin-responsive increase in total phospho-p38MAPK (Fig. 2A). 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 and DR-p38. 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, 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 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 (Fig. 2, 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 activity in cells overexpressing DR-p38. Yet insulin-stimulated glucose uptake was inhibited by 10 μM SB203580 to a similar extent in cells overexpressing WT- and DR-p38 (Fig. 3).
Effect of DN mutants of p38 or - on insulin-stimulated glucose uptake
To confirm the unexpected result obtained with the DR-p38 mutant, we sought to achieve inhibition of p38 and p38 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 or DN-p38 mutant relative to endogenous WT-p38 or p38, 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 or DN-p38 reduced insulin-stimulated phosphorylation of p38MAPK by approximately 70% in whole-cell lysates (Fig. 4) 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 (Fig. 5A) nor DN-p38 (Fig. 5B) 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, or DN-p38 (34, 30, and 40%, respectively; data not shown). Hence, despite a marked reduction in insulin-stimulated p38MAPK phosphorylation by expression of DN-p38MAPK/, there was no concomitant reduction in insulin-stimulated glucose uptake.
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 and/or p38 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 protein was achieved, as determined by immunoblotting whole-cell lysates (Fig. 6A). Reduction of p38 by siRNA did not appreciably affect expression of p38 (Fig. 6B). Because currently available antibodies specific to p38 are poor at detecting endogenous p38 protein in L6 myotubes, we used L6 cells stably expressing a FLAG-tagged DN-p38 and measured FLAG signal after siRNA transfection (Fig. 6B). p38 siRNA gene silencing achieved a knockdown of 63–74% of overexpressed p38 protein (Fig. 6B). We surmise that the endogenous p38 of L6-GLUT4myc myotubes was reduced to a similar extent by p38 siRNA. The reduction in p38 by siRNA did not appreciably affect expression of p38 (Fig. 6A), indicating specificity of each siRNA for its cognate sequence. In L6 myotubes transfected with siRNAs to both p38 and p38, each isoform was reduced to the extents achieved when each siRNA was used separately (Fig. 6, A and B). In addition, insulin-stimulated p38 phosphorylation was markedly inhibited by siRNA gene silencing of p38 alone or by combined siRNA to p38 and p38, whereas siRNA gene silencing of p38 was without effect on p38 phosphorylation (Fig. 6C). 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, p38, or p38 and p38 together (Fig. 7) were not accompanied by a drop in insulin-stimulated glucose uptake.
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. 8). 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. 8). 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. 1B), 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.
Discussion
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 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 (DR-p38) resistant to SB203580; 2) dominant-inhibitory mutants of p38 and p38; and 3) siRNA against p38 and p38.
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. 2A). Expression of DR-p38 in L6-GLUT4myc myotubes reversed the SB203580-mediated inhibition of hyperosmolarity-induced MK-2 phosphorylation by 65% (Fig. 2). However, it did not prevent SB203580-mediated inhibition of insulin-stimulated glucose uptake (Fig. 3). The most obvious interpretation of these results is that SB203580-mediated inhibition of glucose uptake may not be due to inhibition of p38 and/or p38 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 did not fully replace the endogenous p38 or p38 involved in regulating glucose uptake, even though DR-p38 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 or p38 (DN-p38 or DN-p38). Overexpression of either DN-p38 or DN-p38 reduced insulin-stimulated p38MAPK phosphorylation by 70–80% (Fig. 4) but did not lower glucose uptake stimulated by either 10 or 100 nM insulin (Fig. 5). 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 or p38. Reducing the expression of endogenous p38 by 65% dropped insulin-stimulated p38MAPK phosphorylation by 70%. In contrast, reducing the expression of p38 by 60% did not affect insulin-stimulated p38MAPK phosphorylation (Fig. 6). These results suggest that in L6-GLUT4myc myotubes, p38 is responsible for the majority of insulin-stimulated p38MAPK phosphorylation, even though both p38 and p38 are activated by insulin (15, 18). It is noteworthy that expression of DN-p38 resulted in a significant (70%) inhibition of insulin-stimulated p38MAPK phosphorylation. Therefore, it is possible that this p38 mutant was able to also inhibit the activation of endogenous p38, perhaps by sequestering an upstream kinase common to both p38MAPK isoforms. In agreement with such an interpretation, overexpression of DN-p38 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 or p38 by siRNA gene silencing had no effect on insulin-stimulated glucose uptake (Fig. 7). 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 and p38 (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 and p38 (31, 53), whereas p38 (21) and p38 (22) are insensitive to these inhibitors. In addition, the in vivo specificity of SB203580 has been validated in several systems using the DR-p38 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 N, 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. 8). 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. 1), 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 did not rescue the SB203580-mediated inhibition of glucose uptake, and reduction of endogenous p38 and p38 activities by either dominant-negative mutants of p38 and p38 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/-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 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.
1 C.N.A. and C.H. contributed equally to this work.
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.
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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
Department of Pharmacology (K.A.H.), University of Colorado Health Sciences Center and Denver Veterans Affairs Medical Center, Denver, Colorado 80262
Abstract
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 and p38MAPK (e.g. SB203580) reduce insulin-stimulated glucose uptake without affecting GLUT4 translocation. This observation suggested that insulin may increase GLUT4 activity via p38 and/or p38. 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 (drug-resistant p38) but barely affected phosphorylation of the p38 substrate MAPK-activated protein kinase-2. Expression of dominant-negative p38 or p38 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 or p38 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
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 (19) and p38 (20) are ubiquitously expressed, whereas p38 (21) is highly expressed in skeletal muscle and p38 in the lung and kidney (22). Full activation of p38MAPK requires dual phosphorylation on residues corresponding to threonine-180 and tyrosine-182 on p38 (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 and p38 (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 and p38 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 or p38 (34) or drug-resistant (DR) p38 (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 or p38 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
Materials
SB203580 was obtained from Calbiochem (San Diego, CA). Protease inhibitor cocktail, cytochalasin B, O-phenylenediamine dihydrochloride, 2-deoxyglucose, and -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). -Phospho-p38 MAPK (recognizes all p38MAPK isoforms only when phosphorylated on residues corresponding to T180 and Y182 on p38), -phospho-MAPK-activated protein kinase-2 (MK-2; recognizes MK-2 when phosphorylated on T334), and -p38 MAPK antibodies were obtained from Cell Signaling Technologies (Beverly, MA). Polyclonal -myc antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat -rabbit and sheep -mouse antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). Adenovirus particles encoding cDNAs for green fluorescent protein (GFP), DN-p38 (T180A, Y182F), DN-p38 (T180A, Y182F), and wild-type (WT)-p38 were prepared as previously described (36). DR-p38 (T180M, H107P, L108F) has been previously characterized (32). Synthetic siRNAs specific for rat p38MAPK (AAG CAC GAG AAU GUG AUU GGU) and p38MAPK (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 were constructed using the AdEasy I system (Stratagene, La Jolla, CA) (42). Briefly, cDNA encoding DR-p38 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 MEM and 5 mM glucose for 4 h (in serum-free medium). Infection proceeded for 10–12 h (WT-p38 and DR-p38 adenovirus) or 18–24 h (DN-p38, DN-p38, and GFP adenovirus) before serum deprivation and experimentation.
siRNA transfection
Cells were incubated in antibiotic-free 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, p38, or p38 and p38 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 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
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. 1A), whereas insulin-stimulated GLUT4 translocation was not affected by SB203580 at either insulin concentration (Fig. 1B). 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.
Effect of DR mutant of p38 on SB203580-mediated reduction in insulin-stimulated glucose uptake
SB203580 inhibits p38 and p38 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 or p38. To explore this possibility, WT- or DR-p38 was overexpressed in L6-GLUT4myc myotubes via adenoviral delivery. We determined that infection of L6 myotubes with adenoviruses encoding either WT-p38 or DR-p38 resulted in a similar insulin-responsive increase in total phospho-p38MAPK (Fig. 2A). 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 and DR-p38. 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, 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 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 (Fig. 2, 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 activity in cells overexpressing DR-p38. Yet insulin-stimulated glucose uptake was inhibited by 10 μM SB203580 to a similar extent in cells overexpressing WT- and DR-p38 (Fig. 3).
Effect of DN mutants of p38 or - on insulin-stimulated glucose uptake
To confirm the unexpected result obtained with the DR-p38 mutant, we sought to achieve inhibition of p38 and p38 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 or DN-p38 mutant relative to endogenous WT-p38 or p38, 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 or DN-p38 reduced insulin-stimulated phosphorylation of p38MAPK by approximately 70% in whole-cell lysates (Fig. 4) 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 (Fig. 5A) nor DN-p38 (Fig. 5B) 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, or DN-p38 (34, 30, and 40%, respectively; data not shown). Hence, despite a marked reduction in insulin-stimulated p38MAPK phosphorylation by expression of DN-p38MAPK/, there was no concomitant reduction in insulin-stimulated glucose uptake.
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 and/or p38 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 protein was achieved, as determined by immunoblotting whole-cell lysates (Fig. 6A). Reduction of p38 by siRNA did not appreciably affect expression of p38 (Fig. 6B). Because currently available antibodies specific to p38 are poor at detecting endogenous p38 protein in L6 myotubes, we used L6 cells stably expressing a FLAG-tagged DN-p38 and measured FLAG signal after siRNA transfection (Fig. 6B). p38 siRNA gene silencing achieved a knockdown of 63–74% of overexpressed p38 protein (Fig. 6B). We surmise that the endogenous p38 of L6-GLUT4myc myotubes was reduced to a similar extent by p38 siRNA. The reduction in p38 by siRNA did not appreciably affect expression of p38 (Fig. 6A), indicating specificity of each siRNA for its cognate sequence. In L6 myotubes transfected with siRNAs to both p38 and p38, each isoform was reduced to the extents achieved when each siRNA was used separately (Fig. 6, A and B). In addition, insulin-stimulated p38 phosphorylation was markedly inhibited by siRNA gene silencing of p38 alone or by combined siRNA to p38 and p38, whereas siRNA gene silencing of p38 was without effect on p38 phosphorylation (Fig. 6C). 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, p38, or p38 and p38 together (Fig. 7) were not accompanied by a drop in insulin-stimulated glucose uptake.
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. 8). 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. 8). 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. 1B), 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.
Discussion
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 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 (DR-p38) resistant to SB203580; 2) dominant-inhibitory mutants of p38 and p38; and 3) siRNA against p38 and p38.
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. 2A). Expression of DR-p38 in L6-GLUT4myc myotubes reversed the SB203580-mediated inhibition of hyperosmolarity-induced MK-2 phosphorylation by 65% (Fig. 2). However, it did not prevent SB203580-mediated inhibition of insulin-stimulated glucose uptake (Fig. 3). The most obvious interpretation of these results is that SB203580-mediated inhibition of glucose uptake may not be due to inhibition of p38 and/or p38 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 did not fully replace the endogenous p38 or p38 involved in regulating glucose uptake, even though DR-p38 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 or p38 (DN-p38 or DN-p38). Overexpression of either DN-p38 or DN-p38 reduced insulin-stimulated p38MAPK phosphorylation by 70–80% (Fig. 4) but did not lower glucose uptake stimulated by either 10 or 100 nM insulin (Fig. 5). 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 or p38. Reducing the expression of endogenous p38 by 65% dropped insulin-stimulated p38MAPK phosphorylation by 70%. In contrast, reducing the expression of p38 by 60% did not affect insulin-stimulated p38MAPK phosphorylation (Fig. 6). These results suggest that in L6-GLUT4myc myotubes, p38 is responsible for the majority of insulin-stimulated p38MAPK phosphorylation, even though both p38 and p38 are activated by insulin (15, 18). It is noteworthy that expression of DN-p38 resulted in a significant (70%) inhibition of insulin-stimulated p38MAPK phosphorylation. Therefore, it is possible that this p38 mutant was able to also inhibit the activation of endogenous p38, perhaps by sequestering an upstream kinase common to both p38MAPK isoforms. In agreement with such an interpretation, overexpression of DN-p38 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 or p38 by siRNA gene silencing had no effect on insulin-stimulated glucose uptake (Fig. 7). 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 and p38 (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 and p38 (31, 53), whereas p38 (21) and p38 (22) are insensitive to these inhibitors. In addition, the in vivo specificity of SB203580 has been validated in several systems using the DR-p38 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 N, 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. 8). 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. 1), 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 did not rescue the SB203580-mediated inhibition of glucose uptake, and reduction of endogenous p38 and p38 activities by either dominant-negative mutants of p38 and p38 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/-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 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.
1 C.N.A. and C.H. contributed equally to this work.
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.
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