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RECEPTORS AND SIGNAL TRANSDUCTION
1Department of Medicine, 2Department of Epidemiology & Biostatistics, and 3Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio
Submitted 28 January 2008 ; accepted in final form 28 July 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
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80%. Finally, we found no evidence for coimmunoprecipitation of Glut1 and p-AS160. We conclude that although azide, hypoxia, and AICAR all activate AMPK, the downstream signaling pathways are distinct, with azide and hypoxia stimulating ERK1/2 and AICAR stimulating the p38 pathway. AMP-activated protein kinase; glucose transporter 1; oxidative phosphorylation; small interfering RNA directed against AS160
The rate of glucose transport is regulated by a large number of agents and stimuli including insulin, hypoxia, inhibition of oxidative phosphorylation, cellular transformation, exposure to alkaline pH, serum and growth factors, and a rise in cell calcium concentration (1, 6, 8, 14, 15, 19, 20, 26, 28). In many of our previous studies we have examined the stimulation of glucose transport in response to exposure to azide, a chemical inhibitor of mitochondrial respiration, utilizing Clone 9 cells (a rat liver-derived cell line that near-exclusively expresses the Glut1 isoform) (20, 26, 33). Importantly, and in contrast to cells expressing Glut4, in which exposure to insulin, hypoxia, or chemical inhibitors of oxidative phosphorylation increases the abundance of the transporter at the cell surface (3, 27), stimulation of glucose transport in Glut1-expressing cells appears to be mediated predominantly by increased activity of Glut1 transporters preexisting in the plasma membrane (4, 19, 33).
It is well known that phosphorylation of AMP-activated protein kinase (AMPK) leads to stimulation of glucose transport in a variety of cells examined (1, 8, 13). This kinase, which appears to function as a "sensor" of the energy status of cells, becomes phosphorylated and activated in response to conditions associated with increased intracellular concentration of AMP (and decreased ATP), changes that are present during hypoxia or inhibition of oxidative phosphorylation (1, 4, 10, 20, 37). We and others have previously reported that AMPK phosphorylation and enzymatic activity are stimulated following exposure of Clone 9 cells to azide or 5'-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and that the activation of AMPK is associated with an increased rate of Glut1-mediated glucose transport (1, 4, 20). More recently, employing small interfering RNA (siRNA) technology and compound C as an inhibitor of AMPK, we have established that activation of AMPK following exposure to azide or AICAR constitutes a critical step in the glucose transport response (21). However, the steps subsequent to activation of AMPK and the signaling pathway(s) leading to stimulation of glucose transport remain largely unknown. The present study is focused on delineating some of the pathways mediating the glucose transport response.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
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1 plus AMPK2 knockout (AMPK KO) fibroblasts were a gift from the laboratory of Dr. Benoit Viollet (Paris, France). DMEM, trypsin-EDTA, calf serum, newborn bovine serum, bovine calf serum, and recombinant protein G-agarose were purchased from GIBCO (Grand Island, NY). Cell culture dishes were obtained from Corning Glass Works (Medfield, MA), and 3-O-methyl-D-[3H]glucose ([3H]3-OMG; 3.4 mCi/mmol) and an enhanced chemiluminescence Western blotting detection kit were obtained from Amersham Life Science (Arlington Heights, IL). Mouse anti-β-actin, peroxidase-conjugated goat anti-mouse and anti-rabbit IgG, phloretin, cytochalasin B (CB), PMSF, AICAR, U0126 ethanolate, and standard chemical reagents were obtained from Sigma (St. Louis, MO). Rabbit anti-AMPK--pan, rabbit monoclonal anti-phosphorylated (Thr172) AMPK (recognizing both phosphorylated 1- and 2-isoforms), mouse anti-phosphorylated p38 MAPK (Thr180/Tyr182), rabbit anti-total p38 MAPK, and rabbit anti-total AS160 were obtained from Cell Signaling Technology (Danvers, MA). Mouse anti-phosphorylated ERK1/2 (E-4) and rabbit anti-total ERK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and the p38/SAPK2 inhibitor SB 203580 and rabbit anti-phosphorylated AS160 (Thr642) were obtained from Upstate (Temecula, CA). siRNA directed against AS160 (siAS160) was obtained from Sigma (St. Louis, MO), and Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA). Block-iT fluorescent oligo and siRNA for nonspecific targeting of mRNAs were purchased from Dharmacon (Lafayette, CO). Cell culture. Clone 9 cells in 60-mm dishes were maintained in DMEM containing 5.6 mM D-glucose supplemented with 10% calf serum at 37°C in a 9% CO2-humidified chamber (pH 7.4), as described previously (20, 33). In experiments with hypoxia, cells were incubated in a sealed chamber gassed with 95% N2 and 5% CO2 (nominally <0.05% O2). HepG2 cells were maintained in MEM/F12-1 medium with 10% new born bovine serum in accordance with suggestions by the American Type Culture Collection. H9c2 cells were kept in DMEM containing 5.6 mM D-glucose with 10% bovine calf serum. AMPK WT and AMPK KO mouse fibroblasts were maintained in DMEM (5.6 mM glucose) supplemented with 10% newborn bovine serum at 37°C in a 5% CO2-humidified chamber (pH 7.4), as described previously (24).
AS160 knockdown by siRNA directed against AS160. Seventy percent confluent Clone 9 cells in 60-mm culture dishes were incubated with fresh DMEM with no antibiotics for 2 h before transfection. Cells were transfected with an siAS160-targeted sequence of (5'-3') UCAGGGUAAACUGUAGUAGTT (100 pmol/ml) or nonspecific scrambled siRNA as negative control by using Lipofectamine 2000 according to the manufacture's protocol. Forty-eight hours after transfection, the media were changed and the cells were treated with diluent, 10 mM azide, or 2 mM AICAR for 1 h, followed by CB-inhibitable [3H]3-OMG glucose uptake assay and Western blotting.
Transfection efficiency was monitored using the same quantity of Block-iT fluorescent oligo, and fluorescence was observed at 24–48 h after transfection by microscopy. A transfection efficiency of 90–95% was obtained at 24–48 h.
Measurement of CB-inhibitable [3H]3-OMG uptake. Cells in triplicate 60-mm dishes were used in uptake experiments, as described previously (21). The uptake medium consisted of 1.0 ml of DMEM with 1 µCi of [3H]3-OMG and 1 µl of DMSO or CB in DMSO at a final concentration of 50 µM. Uptake was conducted for 1.0 min (4, 21, 26). [3H]3-OMG uptake was calculated as the difference between uptake in the absence and presence of CB (21, 26).
SDS-PAGE and Western blotting.
Cells were lysed in Nonidet P-40 (NP-40)-containing buffer (5 mM NaCl, 25 mM Tris, 25 mM sodium fluoride, 10 mM sodium pyrophosphate, 0.5 mg/l leupeptin, 1 mg/l aprotinin, 1 mM sodium orthovanadate, 0.1 mM PMSF, and 0.5% NP-40; pH 7.5). In experiments on phosphorylation of AS160, cells were directly lysed in SDS sample buffer. Proteins were separated by 8 or 10% SDS-PAGE. Polyvinylidene difluoride membranes were blocked using 5% nonfat milk and incubated with rabbit anti-AMPK--pan (63 kDa) for measurement of total AMPK, rabbit anti-phosphorylated (Thr172) AMPK for measurement of 1- plus 2-isoform phosphorylation (p-AMPK; 63 kDa), mouse anti-phosphorylated p38 (p-p38; 43 kDa) or rabbit anti-total p38 (43 kDa), mouse anti-phosphorylated ERK1/2 (p-ERK1/2; 42 and 44 kDa) or rabbit anti-total ERK (42 and 44 kDa), or rabbit anti-phosphorylated AS160 for measurement of phosphorylated AS160 (p-AS160; 160 kDa). Mouse anti-β-actin (42 kDa) antibody was used to verify equal loading of the gels. The secondary antibody was a 1:1,000 to 1:4,000 (vol/vol) dilution of horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse in Tris-buffered saline-Tween 20 (50 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.5). Blots were developed using enhanced chemiluminescence reagents, and immunoreactive bands were visualized on Kodak X-Omat film; the intensity of the bands was determined by densitometry. Multiple exposures of blots were developed to ensure linearity of the signals. Each experiment was repeated at least three times. In the case of ERK1/2, the abundance of ERK1 and ERK2 bands was added together. In all experiments measuring the relative abundance of p-AMPK, p-ERK1/2, and p-p38, the density of the bands was normalized against the total abundance of each of the proteins.
Immunoprecipitation of p-p38. Clone 9 cells were treated with diluent, 10 mM azide, or 2 mM AICAR for 1 h and then lysed in the NP-40 buffer. Postnuclear cell lysates from each condition were immunoprecipitated overnight using mouse monoclonal anti-phospho-p38 MAPK antibody (1:100) with prewashed protein G-Sepharose. Samples were centrifuged at 3,000 rpm for 1 min at 4°C, and the pellets were washed three times with 10 volumes of ice-cold 0.5% NP-40 buffer. The final pellet was treated with SDS sample buffer. Membranes were probed with rabbit anti-total p38.
Immunoprecipitation of AS160 and p-AS160. Clone 9 cells and HepG2 cells were treated with diluent, 10 mM azide, or 2 mM AICAR for 1 h and then lysed in the NP-40 buffer. Postnuclear cell lysates from each condition were immunoprecipitated overnight using mouse monoclonal anti-Glut1 antibody (1:10) and prewashed protein G-Sepharose. Samples were washed three times with 0.5% NP-40 buffer and lysed with SDS sample buffer. Immunoprecipitated products were employed for Western blotting using rabbit polyclonal anti-phospho-AS160 or rabbit polyclonal anti-total AS160 antibody. HepG2 cell lysates (following the same treatments) were immunoprecipitated with rabbit anti-total AS160 antibody, and the immunoprecipitated products were employed for Western blotting using mouse anti-Glut1 antibody.
Statistical analysis. Values are means ± SE. Data are expressed in terms of natural logarithmic scale, and differences between each treatment and control were computed. To test for differences between each treatment and control, we conducted a Student's t-test for single mean. One-way analysis of variance was conducted to test for differences among the three treatments, followed by the Bonferroni adjustment factor for multiple comparison tests. All tests were conducted with a 0.05 level of significance. Analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
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1- and 2-isoforms of the enzyme are inactivated (24). Treatment of AMPK WT cells with azide or AICAR stimulated glucose transport (Fig. 1A). In contrast, AMPK KO fibroblasts manifested no change in the rate of glucose transport in response to either azide or AICAR. The rate of transport in AMPK KO cells under control conditions was similar to the rate in AMPK WT cells. Treatment with either azide or AICAR increased the phosphorylation of AMPK in WT fibroblasts; the abundance of p-AMPK increased 1.5 ± 0.1- and 2.3 ± 0.1-fold in cells treated with azide and AICAR, respectively, whereas p-AMPK was absent in AMPK KO cells (Fig. 1B).
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1- and 2-isoforms of AMPK, we found that the content of p-AMPK (normalized against total AMPK) increased in both azide- and AICAR-treated cells, and simultaneous expose to both agents resulted in a higher abundance of p-AMPK in a near-additive manner (Fig. 2, B and C). The content of total AMPK was unchanged.
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To verify the above results using an alternative approach, we immunoprecipitated p-p38 from Clone 9 cells treated with AICAR, azide, and their combination and developed the immunoblots using antibodies directed at total p-38. Consistent with the above observations, exposure to AICAR, but not azide, increased the abundance of p-p38 (data not shown).
Selective phosphorylation of ERK1/2 by azide and of p38 by AICAR in mouse fibroblasts. To explore for possible generality of the divergent signaling pathways observed in Clone 9 cells, we examined the response to azide and AICAR in the mouse AMPK WT and AMPK KO fibroblast cell lines. In WT cells, phosphorylation of ERK1/2 increased selectively in azide-treated cells (1.4 ± 0.1-fold), and phosphorylation of p38 increased 2.1 ± 0.1-fold only in AICAR-treated cells, whereas the abundances of total AMPK, ERK1/2, and p38 in the WT cells remained unchanged (Fig. 4, left 3 lanes). In KO cells, the abundance of p-p38 was increased, as was the content of total p38. However, the ratio of p-p38 to p38 in KO cells was the same as that in WT cells. In addition, the selective activation of p-ERK1/2 and p-p38 was totally absent in KO cells in response to azide or AICAR treatment (Fig. 4, right 3 lanes). It should be noted that the increased abundance of p-p38 and p-38 in KO cells under control conditions is not associated with an increase in the basal level of glucose transport (see Fig. 1). Quantitation of three separate experiments is shown in Fig. 4B.
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50%, whereas the AICAR-induced stimulation of glucose transport was unaffected (Fig. 6A). U0126 had no significant effect on phosphorylation of AMPK in response to AICAR or azide (Fig. 6, B and C). However, U0126 significantly reduced the abundance of p-ERK1/2 in cells treated with azide and had no effect on AICAR-induced increase in p-p38.
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Effect of downregulation of AS160 expression on the glucose transport response in Clone 9 cells. To further investigate the role of AS160 in azide- or AICAR-induced glucose transport, we decreased the expression of AS160 by application of siAS160. Forty-eight hours after transfection with 100 pmol/ml siAS160 or nonspecific scrambled siRNA, the acute effect of 10 mM azide and 2 mM AICAR on 3-OMG-inhibitable glucose transport was measured. Treatment with azide or AICAR for 1 h stimulated the rate of glucose transport in control-transfected cells by 2.9 ± 0.1- and 3.0 ± 0.2-fold, respectively, and the stimulation of transport by these agents was not affected in cells treated with siAS160 (2.7 ± 0.2- and 3.4 ± 0.2-fold, respectively; P > 0.05) (Fig. 9A). Samples of lysates assayed for p-AS160 content in two independent experiments showed an average decrease of 81, 80, and 75% in basal, azide-, and AICAR-treated cells, respectively (Fig. 9B). (Antibody against p-AS160 was employed in this experiment because of the poor quality of the blots using the anti-total AS160 antibody against the rat protein; nevertheless, the same degree of depression of AS160 was observed using the latter antibody.) The abundance of β-actin (used as a control) remained unchanged.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
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1- and 2-isoforms showed no stimulation of glucose transport in response to either azide or AICAR, verifying the essential role of AMPK in this response. 2) The stimulatory effect of azide and AICAR on glucose transport was not additive, whereas their effect on the abundance of p-AMPK was near-additive. 3) Although azide and AICAR both lead to increased phosphorylation of AMPK that is associated with stimulation of AMPK activity (1, 4, 32, 34, 35), the signaling pathways appear to be divergent (in Clone 9 cells, mouse WT fibroblasts, and H9C2 cells); azide selectively resulted in phosphorylation of ERK1/2, whereas exposure to AICAR led to phosphorylation of p38. 4) Similar to azide, hypoxia selectively phosphorylated ERK1/2 in both Clone 9 cells and H9c2 cells. 5) Exposure of Clone 9 cells to hypoxia, azide, or AICAR resulted in phosphorylation of AS160. However, Glut1 was not physically associated with AS160 (or p-AS160), and reduced expression of AS160 by siRNA directed against the protein did not diminish the glucose transport response to either azide or AICAR. Finally, 6) the results raise the possibility that cells contain different functional pools of AMPK, one responsive to azide and hypoxia and the other responsive to AICAR. It is worth emphasizing that the above results involve Glut1- and not Glut4-mediated glucose transport.
We took advantage of the availability of mouse fibroblasts in which both -isoforms of AMPK have been deleted (24). These cells afforded us the opportunity to rigorously test the hypothesis that stimulation of AMPK is critical for stimulation of glucose transport in response to azide and AICAR. The results indicated that in the absence of phosphorylation of AMPK, the rate of glucose transport was completely unaffected by the above stimuli. In contrast, WT fibroblasts showed a positive glucose transport response to both azide and AICAR.
Exposure to azide or AICAR alone stimulated the rate of glucose uptake, and simultaneous exposure to both agents was not additive and resulted in no further increase in the rate of glucose transport compared with AICAR alone. Interestingly, simultaneous exposure to both agents resulted in a higher abundance of p-AMPK than exposure to either agent alone in a near-additive manner. These findings raise the possibility of potential heterogeneity in cell AMPK distribution; alternatively, cells may contain different functional pools of AMPK, one responsive to azide and the other to AICAR. One source of this presumed heterogeneity could result from the possibility that 1- and 2-isoforms of AMPK respond differentially to the above two stimuli. However, the available evidence is not in keeping with this premise for two reasons: 1) we and others have reported that the 1-isoform of AMPK is by far the predominant isoform of AMPK in Clone 9 cells (4), and more importantly, 2) suppression of AMPK2 mRNA and protein content using siRNA technology did not alter the glucose transport response of these cells to either azide or AICAR, whereas siRNA directed toward the 1-isoform significantly inhibited the response to either agent (21).
We also made the novel observation that stimulation of AMPK by exposure to azide or hypoxia leads to a selective stimulation of the ERK1/2 pathway, whereas AMPK stimulation by AICAR leads to selective activation of the p38 pathway in three cell lines, namely, Clone 9, H9c2 cells, and fibroblasts. Neither agent stimulated the phosphorylation of JNK. It has been reported that exposure of L6 cells to AICAR increases the phosphorylation of ERK1/2 (9). In agreement with this report, we found an 1.7-fold increase in phosphorylation of ERK1/2 by AICAR in L6 cells; however, exposure to AICAR resulted in an 1.9-fold increase in p-p38 (data not shown). In further experiments employing inhibitors of ERK1/2 and p38 pathways, we made two critical observations, namely, 1) although U0126 and SB 203580 were observed to selectively inhibit the phosphorylation of these two mitogen-activated protein kinases (MAPKs), they had no effect on phosphorylation of AMPK in response to either azide or AICAR, suggesting that these mediators are "downstream" to activation of AMPK, and 2) phosphorylation of ERK1/2 and p38 in response to azide and AICAR, respectively, appeared to be responsible for the observed stimulation of glucose transport in response to the above stimuli. Finally, we found that the signaling pathway following the activation of AMPK in response to hypoxia was similar to the pathway that is stimulated in response to azide but not to AICAR.
An unexpected finding was that SB 203580 significantly inhibited the glucose transport response to azide as well as to AICAR. We also noted that exposure to SB 203580 alone stimulated the rate of glucose transport. The reasons for these findings are not clear and probably reflect well-known nonspecific actions of this inhibitor, a situation that limits analysis of results of experiments using this inhibitor (5, 25). Other investigators have noted the structural similarities between Glut1 and Glut4 and that SB 203580 can inhibit Glut4 function (2, 31). Although it is possible that this agent has a similar effect on Glut1, our finding that addition of SB 203580 alone to control cells stimulated transport makes this possibility less likely.
The nonadditive nature of the glucose transport response to azide plus AICAR raises the possibility of a common pathway in which the signals generated by azide and AICAR converge. We considered AS160 as a potential candidate. AS160 is a novel Rab GTPase-activating protein with two phosphotyrosine domains, a COOH-terminal Rab-GAP domain, and six phosphorylation motifs (RXRXXS*/T*) targeted by Akt and other kinases (7). Interestingly, in addition to insulin, AICAR increases the phosphorylation of AS160 in rat skeletal muscle (11, 22, 36). Furthermore, under basal conditions, AS160 in skeletal muscle is attached to intracellular Glut4-containing vesicles, and phosphorylation of AS160 results in its binding to 14-3-3 protein, leading to dissociation of p-AS160 from the vesicles (18). Finally, in a cell-free system, AS160 is phosphorylated by activated AMPK (36). On the basis of these findings, we examined whether AS160 becomes phosphorylated in response to azide and hypoxia similar to that reported for AICAR. The results indicated that this is indeed the case: phosphorylation of AS160 was significantly increased in response to azide, hypoxia, and AICAR. This finding raises the intriguing possibility that AS160 is either in the presumed final common pathway leading to increased glucose uptake or might itself serve as the point of convergence in the signaling pathways that are stimulated by azide, hypoxia, and AICAR. However, our results demonstrate that AS160 (or p-AS160) is not physically associated with Glut1 (similar to Glut4-expressing tissues) and that decreased expression of AS160 with the use of siRNA did not diminish the glucose transport response to the above stimuli. Hence, the phosphorylation of AS160 that occurs in response to AMPK activation may not play a mediating role in Glut1-mediated glucose transport response. This result is not totally unexpected, since in contrast to Glut4-expressing cells, the major mode of stimulation of glucose transport in cells expressing Glut1 is through activation of transporters preexisting in the plasma membrane (15). On the other hand, it should be noted that in the case of insulin signaling in 3T3-L1 adipocytes leading to Glut4 translocation, a minor amount (5–10%) of AS160 (Rab GAP TBC1D4/AS160) is sufficient for the glucose transport response (18). This raises the possibility that the 80% decrease in AS160 content was not sufficient to inhibit the glucose transport response in our experiments.
The present study has illuminated some of the signaling steps that occur following the inhibition of oxidative phosphorylation and activation of AMPK that lead to stimulation of Glut1-mediated glucose transport. In addition, it appears that activation of ERK1/2 and p38 pathways by azide and hypoxia on the one hand and AICAR on the other, respectively, is essential in the glucose transport response. Nevertheless, the absolute necessity of phosphorylation of these mediators, and the steps leading to the apparent activation of Glut transporters residing in the plasma membrane, is unclear at present and requires further study.
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NOTE ADDED IN PROOF |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
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