Critical Role of 5 -AMP-activated Protein Kinase in the Stimulation of Glucose. Transport in Response to Inhibition of Oxidative Phosphorylation

Transcription

1 Page 1 of 56 Articles in PresS. Am J Physiol Cell Physiol (August 30, 2006). doi: /ajpcell Critical Role of 5 -AMP-activated Protein Kinase in the Stimulation of Glucose Transport in Response to Inhibition of Oxidative Phosphorylation Ming Jing 1 and Faramarz Ismail-Beigi 1,2 1 Department of Medicine, and 2 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, Running title: AMPK and glucose transport Address correspondence to: F. Ismail-Beigi, MD, PhD Clinical and Molecular Endocrinology Department of Medicine Case Western Reserve University Euclid Avenue Cleveland, OH Tel: (216) FAX: (216) fxi2@case.edu Copyright 2006 by the American Physiological Society.

2 Page 2 of 56 ABSTRACT 5 -AMP-activated protein kinase (AMPK) functions as an energy sensor to provide metabolic adaptation under conditions of ATP depletion such as hypoxia or inhibition of oxidative phosphorylation. Whether activation of AMPK is critical for the stimulation of glucose transport that occurs in response to inhibition of oxidative phosphorylation is unknown. Here we found that treatment of Clone 9 cells (expressing Glut1) with sodium azide (5 mm for 2 h) or with the AMPK activator, 5 - aminoimidazole-4carboxamide-1--d-ribofuranoside (AICAR) (2 mm for 2 hours), stimulated the rate of glucose transport by 2- to 4-fold. Employing small interference RNA (sirna) directed against AMPK1 isoform alone or against both 1 and 2 isoforms resulted in a significant inhibition of the glucose transport response and the content of P-AMPK1&2 and P-ACC in response to azide. Transfection with sirna directed against AMPK2 alone did not affect the glucose transport response. The efficacy of transfection with sirnas in reducing AMPK content was confirmed by Western blotting. Incubation of cells with compound C, an inhibitor of AMPK, abrogated the glucose transport response, and abolished the increase in P-AMPK1&2 in cells treated with azide or hypoxia. Simultaneous exposure to azide and AICAR did not augment the rate of transport in response to AICAR alone. There was no evidence of co-immunoprecipitation of P-AMPK1&2 with Glut1. However, LKB1 was found to be associated with P-AMPK1&2. We conclude that activation of AMPK plays a sufficient and necessary role in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation.

3 Page 3 of 56 KEY WORDS: sirna, siampk1, siampk2, compound C, hypoxia, oxidative phosphorylation. ABBREVIATIONS: 5 -aminoimidazole-4-carboxamide-1--d-ribofuranoside (AICAR); 5 -AMP-activated protein kinase (AMPK); sodium azide; compound C; small interference RNA (sirna); phosphorylated 1 and 2 subunits of AMPK assayed in combination is designated as P-AMPK1&2; total cell AMPK including both 1&2 isoforms is denoted as Total AMPK; 1 subunit is denoted as AMPK1; 2 subunit is denoted as AMPK2; sirna directed against both 1 and 2 subunits of AMPK is denoted as siampk1&2; sirna directed against the 1 subunit of AMPK is denoted as siampk1; sirna direct against the 2 subunit of AMPK is denoted as siampk2; phospho-acetyl-coa carboxylase (phospho-acc; P-ACC); c-jun NH(2)-terminal kinase (JNK); phosphoserine (P-serine).

4 Page 4 of 56 Exposure of cells and tissues to conditions associated with decreased energy production such as ischemia, hypoxia, or inhibition of oxidative phosphorylation lead to a stimulation of glucose transport and glycolytic ATP production (4, 8, 13). This response is of vital importance in cells in which the rate of glucose transport is rate-limiting for glucose metabolism. For example, we have found that inhibition of oxidative phosphorylation by hypoxia, or chemical agents such as cyanide or azide in Clone 9 cells, a rat liver cell line expressing the Glut1 isoform of facilitative glucose transporters, leads to a marked early fall in cell ATP and a rise in cell ADP and AMP that is associated with increases in ADP/ATP and AMP/ATP ratios (13, 14, 25). However, within minutes after the start of the exposure, cell ATP, ADP and AMP levels return to normal (or near-normal levels) and are maintained at these new levels for 24 h despite the continued presence of the inhibitors (13, 14, 25). This adaptive response is associated with a stimulation of glucose transport and lactate production (5, 14). More prolonged exposure to azide or CoCl 2 (a stimulator of the hypoxia-responsive pathway) results in an increase in Glut1 mrna expression and subsequent enhanced Glut1 expression; however, this effect only occurs after ~4 h of exposure (6, 11, 23, 24). More recently, we have documented that the above response is associated with stimulation of 5 -AMP-activated protein kinase (AMPK) brought about by increased phosphorylation of AMPK, a finding that has been confirmed by others (1, 4, 13). Although the stimulation of AMPK by AICAR, or use of constitutively active AMPK, leads to stimulation of glucose transport in Glut1-expressing cells (1, 31), the potential effect of inhibition of AMPK pathway on stimulation of glucose transport has not been examined previously. Moreover, the signals, sequence of events and underlying

5 Page 5 of 56 mechanisms by which the activation of glucose transport is initiated and maintained following stimulation of AMPK are incompletely understood. While the above data establish that stimulation of AMPK is sufficient for stimulation of Glut-1 mediated glucose transport, they do not define whether the simulation of AMPK activity is a necessary step in the enhancement of glucose transport in response to inhibition of oxidative phosphorylation, or, alternatively, that activation of AMPK simply occurs in parallel with the glucose transport response. AMPK is a hetero-trimeric protein complex whose subunit becomes phosphorylated leading to activation of the enzyme s kinase activity under conditions of metabolic stress, especially in response to increased cellular AMP/ATP ratio (1, 4, 8, 13, 27). Indeed, it has been proposed that AMPK serves as a universal energy sensor (a switch ) that controls the metabolic pathways regulating rates of energy production and energy consumption (8, 27). Stimulation of AMPK in cells that express Glut4 is associated with translocation of this glucose transporter from intracellular vesicles to the plasma membrane, thereby leading to a stimulation of glucose transport (2, 15). In cells that express the Glut1 isoform (such as Clone 9 cells), enhancement of AMPK activity also stimulates glucose transport, and this effect appears to be predominantly due to activation of Glut1 transporters pre-existing in the plasma membrane (4, 12, 25). In the present study we examined the possibility that stimulation of AMPK plays a critical role in the enhancement of glucose transport in response to inhibition of oxidative phosphorylation using sirna technology to suppress AMPK activity and then to test whether the glucose transport response is negatively affected. AICAR was used as a positive control throughout. In addition, we employed compound C as an inhibitor of

6 Page 6 of 56 AMPK activity to explore the same question in cells treated with azide or exposed to hypoxia. Suppression of AMPK activity and its isoforms was determined by western blotting of total AMPK and its phosphorylated forms, and by measurement of P-ACC content, in response to azide (and AICAR). Potential additive effect of azide and AICAR on glucose transport was determined. We also examined whether Glut1 associates with AMPK in response to azide, and whether Glut1 becomes phosphorylated upon stimulation of AMPK activity. Finally, we examined the potential role of LKB1 (10, 22) and c-jun NH(2)-terminal kinase (JNK) (30) as upstream kinases that could phosphorylate AMPK in response to inhibition of oxidative phosphorylation. MATERIALS AND METHODS Materials Clone 9 cells were obtained from American Type Culture Collection (ATCC, Rockville, MD). Dulbecco's modified Eagle's medium (DMEM), Opti-MEM, trypsin EDTA, calf serum, lipofectamine 2000 and recombinant protein G agarose were purchased from GIBCO (Grand Island, NY). Cell culture dishes were from Corning Glass Work (Medfield, MA). 3-O-methyl-D-[ 3 H] glucose ([ 3 H]3-OMG; 3.4 mci/mmol) and enhanced chemiluminescence (ECL) western blotting detection kit were from Amersham Life Science (Arlington Heights, IL). Peroxidase conjugated goat anti-mouse and anti-rabbit IgG, mouse anti--actin, phloretin, cytochalasin B, phenylmethyl sulfonyl fluoride (PMSF), 5-animoimidazole-4-carboxamide ribonucleoside (AICAR), anisomysin and standard chemical reagents were from Sigma (St. Louis, MI). Anti-AMP-activated protein kinase -pan, anti-ampk1, anti-ampk2, monoclonal anti-phospho-thr 172 -

7 Page 7 of 56 AMPK1&2 (recognizing both phospho-isoforms), anti-phospho-acetyl CoA carboxylase (Ser 79) (all rabbit) and mouse anti-phospho-jnk were from Upstate Cell Signaling Solutions (Charlotsville, VA). Mouse anti-glut1 monoclonal antibody (Mab 37-4) was described previously (31). Rabbit anti-glut1 antibody was from Abcam (Cambridge, MA). Goat anti-total LKB1, LKB1 positive control, peroxidase conjugated donkey anti-goat IgG and protein A/G PLUS-Agarose were from Santa Cruz (Santa Cruz, CA). Rabbit anti-phosphoserine was from Zymed Laboratories Inc (San Francisco, CA). Small interference RNA (sirna) directed against AMPK1 or AMPK2, Block-iT fluorescent oligo, si-control non-specific targeting sirna were purchased from Dharmacon (Lafayette, CO). Cell Culture Clone 9 cells in 60 mm dishes in triplicate were maintained in DMEM containing 5.6 mm D-glucose supplemented with 10% calf serum at 37 C in a 9% CO 2 -humidified chamber (ph 7.4). Cells were used between passages 30 and 50. Upon confluence (at 3-4 days), the medium was replaced with serum-free DMEM for 24 hours. In experiments employing compound C, cells were pre-incubated in the presence or absence of the reagent (20 µm dissolved in 20 µl DMSO) for 30 min before co-incubation with diluent, 5 mm azide, or 2 mm AICAR for 2 h; cells not treated with compound C received the diluent. Similarly, in experiments involving hypoxia and compound C, cells were preincubated in the presence and absence of compound C for 30 min before being incubated in the hypoxic chamber with a nominal >0.5% O 2 for 2 h. Because the hypoxic chamber was equilibrated with 95% N2/5% CO2, cells not exposed to hypoxia were incubated in 5% CO 2 -humidified chamber for 2 h prior to assay. To test for the additive effect of

8 Page 8 of 56 azide and AICAR, Clone 9 cells were treated with diluent, 5 mm azide, 2 mm AICAR, or both for 2 h before CB-inhibitable [ 3 H] 3-OMG uptake assay. Following the uptake assay, cells were harvested in 100 µl of NP-40 lysis buffer (5 mm NaCl, 25 mm Tris, 25 mm sodium fluoride, 5 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). After centrifugation, 70 µl of the post-nuclear lysate was subjected to radioactive counting and the rest was used for SDS-PAGE and Western blot analysis. Application of small interfering RNA (sirna) directed against AMPK Seventy percent confluent Clone 9 cells in 6-well dishes were incubated with fresh DMEM with no antibiotics for 2 h prior to transfection. Cells were transfected in duplicate with sirna smart pool targeting 4 different sections of the mrna encoding either AMPK1, AMPK2, or both (100 pmol/ml) using lipofectamine 2000, according to the manufacture s protocol. 48 hours after transfection, the media were changed and the cells were treated with diluent, 5 mm azide, or 2 mm AICAR for 2 h followed by CB-inhibitable [ 3 H]3-OMG glucose uptake assay and Western blotting (as noted above). Potential nonspecific effects transfection with sirna on AMPK content was monitored by transfection of non-specific (scrambled) sirna and immunoblotting for AMPK. In preliminary experiments, use of non-specific sirna resulted in no change in the cellular content of AMPK1 or in the amount of P-AMPK1&2 generated in response to AICAR. Transfection efficiency was monitored using by same quantity of Block-iT fluorescent oligo and fluorescence was observed at h after transfection by microscopy. A transfection efficiency of 90-95% was obtained at 48 to 72 h. In

9 Page 9 of 56 preliminary experiments, various concentrations of sirna (from 25 to 200 pmol/ml) were employed to determine the optimal condition for reducing AMPK expression without causing cell toxicity. A concentration of 100 pmol/ml for each isoform used individually or 50 pmol/ml of each used in combination was found to be ideal. Use of 150 to 200 pmol/ml of sirna was associated with toxicity and cell death. Measurement of CB inhibitable [ 3 H]3-OMG uptake Cells in duplicate 6-well dishes in experiments using sirna, or in triplicate 60 mm dishes in experiments using compound C, azide, hypoxia, or AICAR were incubated for 60 sec in glucose uptake medium containing either dimethylsulfoxide (DMSO) alone or DMSO containing CB at a final concentration of 50 µm. The uptake medium consisted of 1.0 ml DMEM with 1 µci of [ 3 H]3-OMG and 1 µl of cytochalasin B solution or DMSO. Uptake was stopped by ice-cold solution of 100 mm MgCl 2 and 100 µm phloretin (2, 4). Cells were harvested in the NP-40 lysis buffer and the radioactivity was determined by scintillation spectrometry. [ 3 H]3-OMG uptake was calculated as the difference in uptake in the absence and presence of cytochalasin B, assayed in parallel. Unless noted otherwise, experiments were repeated three times and the results were averaged. Uptakes in control and treated cells were performed in parallel. We have noted that the rate of glucose transport in control cells under basal conditions and the degree of stimulation of transport by any specific agent varies between experiments, possibly reflecting the state of the cells, their passage number, and the degree of confluence (4, 13, 24). Nevertheless, a 2 to more than 4-fold stimulation of glucose transport in response to azide is the constant founding. For this reason, control cells and treated cells are performed in parallel all experiments.

10 Page 10 of 56 SDS-PAGE and western blotting All the cell lysates were prepared using the NP-40 lysis buffer. In certain experiments, a portion of the lysate was used for radioactivity counting, as indicated above. Cell lysates from different treatment groups were centrifuged at 14,000 X g for 20 min to remove nuclei and insoluble materials. Protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked using 5% nonfat milk and incubated with either rabbit anti-ampk1 (63 kda), rabbit anti- AMPK2 (63 kda), rabbit anti-ampk -pan (63 kda) for measuring total AMPK1&2 subunit, rabbit anti-phospho- Thr 172 -AMPK1&2 for measuring P- AMPK1&2 (63 kda), rabbit anti-phospho-acc and rabbit anti-total-acc (265 kda). Rabbit anti-glut 1 (55 kda) was employed. Goat anti-lkb1 (52 kda) was employed in western blot and immunoprecipitation experiments. Mouse anti--actin (42 kda) antibody was used to verify equal loading of the gels. The secondary antibody was 1: dilution of horseradish peroxidase-conjugated goat anti-rabbit, anti-mouse, or donkey anti-goat antibody in Tris-buffered saline-tween 20 (TBST: 50 mm Tris, 150 mm NaCl, and 0.05% Tween 20, v/v; ph 7.5). Blots were developed using ECL reagents, and immunoreactive bands were visualized on Kodak X-Omat film and the intensity of the bands were determined by densitometry. Blots were reprobed with mouse anti--actin for control of protein loading using horseradish peroxidase-conjugated goatanti-mouse antibody as secondary antibody. Each experiment was repeated at least three times. In experiments on activation of JNK, confluent cells pre-incubated in serum-free DMEM for 24 h were treated with 10 µm anisomycin or 2 mm AICAR for 1 h. Post-

11 Page 11 of 56 nuclear cell lysates were subjected to 10% SDS-PAGE and probed with mouse antiphospho-jnk (46, 54 kda), rabbit anti-p-ampk1&2, P-ACC, and mouse anti-actin. Immunoprecipitation of Glut1, P-AMPK1&2 and LKB. Cells were treated with diluent, 5 mm azide, or 2 mm AICAR for 2 h before being lysed in the NP-40 buffer. 3 mg protein of post-nuclear cell lysate from each condition was immnoprecipitated using mouse monoclonal anti-glut1 antibody (1:100) using pre-washed protein G-sepharose overnight. Samples were centrifuged at 3000 rpm for 1 minute at 4 C and the pellets were washed three times with 10 volume of ice-cold 0.5% NP-40 buffer. The final pellet was treated with SDS sample buffer and divided into three equal parts and analyzed in separate blots. Membranes were probed with rabbit anti-glut1, rabbit anti-phospho-thr 172 -AMPK1&2, and rabbit anti-phosphoserine antibody. In other experiments, 1 mg protein of post-nuclear lysate from each of the above conditions was immunoprecipitated with rabbit anti-phospho-thr 172 AMPK1&2 and the product was probed for Glut1 and P-AMPK1&2 in separate blots. To explore the association between AMPK and LKB1, Clone 9 cells were treated with diluent, 5 mm azide, or 2 mm AICAR for 2 h before being lysated in the NP-40 buffer. 1 mg protein of post-nuclear cell lysate prepared from each condition was immunoprecipitated in 500 µl of NP-40 buffer overnight at 4 C with 2 µg rabbit antiphospho-thr 172 -AMPK1&2. Products were prepared in duplicates blots and probed for LKB1 and P-AMPK1&2. In additional experiments, 1 mg protein of post-nuclear lysate prepared from cells treated as above was immunoprecipitated with 2 µg goat anti-

12 Page 12 of 56 total-lkb1 antibody, immunobloted and probed with rabbit anti-phospho-thr AMPK1&2 and anti-total-lkb1 in separate blots. Statistical analysis All data are presented as means ± S.E. ANOVA was used for significance throughout. In certain analyses involving direct comparison between two groups, Student s t-test was employed. In all cases, a P < 0.05 was considered significant. RESULTS Effect of sirna directed against AMPK2 on azide-stimulated glucose transport Forty eight hours after transfection with 100 pmol/ml of siampk2 directed against four segments of AMPK2 or being treated with lipofectamine alone, the acute effect of 5 mm azide on 3-OMG inhibitable glucose transport was measured. AICAR (2 mm for 2 h) was used as a positive control. We chose a 2-h time point for these studies because Glut1 expression which is increased at later times does not occur prior to 4 hours (24, 25). Treatment with azide or AICAR for 2 h respectively stimulated the rate of glucose transport in mock-transfected cells by 2.8 ± 0.3 and 3.5 ± 0.4 fold, and the stimulation by these agents were not affected in cells treated with sirna directed against the 2 isoform (2.9 ± 0.9 and 3.5 ± 0.4 fold, respectively) (P >0.05) (Fig. 1A). Samples of lysates were assayed for the content of AMPK2 and P-AMPK1&2. Immunoblots of AMPK2 showed an average decrease of 94%, 88%, and 98% in the content of the 2 subunit in basal, azide-, and AICAR-treated groups, respectively. However,

13 Page 13 of 56 immunoblots using anti-phosphothr 172 -AMPK1&2, which recognizes phosphorylation of both 1 and 2 subunits, showed that the content of P-AMPK1&2 was not decreased in siampk2-treated cells (lane 2 vs lane 1), and that the content of P-AMPK1&2 increased significantly in response to azide or AICAR, irrespective of treatment with siampk2 (Fig. 2B). Total AMPK1&2 content was slightly (but not significantly) reduced in cells exposure to the sirna. In addition, P-ACC content was significantly increased in cells exposed to azide or AICAR, irrespective of the presence or absence of siampk2. Finally, the abundance of -actin remained unchanged. The above results suggest that the 2 isoform may be minor component of total AMPK1&2 in Clone 9 cells. Effect of sirna directed against AMPK1 on azide-stimulated glucose transport We next determined the importance of AMPK1 in the glucose transport response to azide. Again, AICAR was used as a positive control. The sirna employed was directed against 4 different sections of AMPK1 mrna. Control cells received lipofectamine alone. Transfection with siampk1 (100 pmol/ml) caused a slight, but significant fall in the rate of glucose transport in control (basal) cells. Treatment with siampk1 significantly suppressed azide- and AICAR-induced stimulation of glucose transport, although the rate of uptake was not decreased to the level in control cells (Fig. 2A). To determine whether the decrease in glucose transport in siampk1-treated cells was associated with a suppression of AMPK1 expression and phosphorylation,

14 Page 14 of 56 immunoblots were prepared from samples from the above transport experiments. Immunoblots using anti-ampk1 demonstrated an 89%, 83%, and 78% decrease in the content of AMPK1 in control, azide and AICAR groups, respectively (Fig. 2B). Immunoblots using anti-phospho-thr 172 -AMPK1&2 showed that the content of P- AMPK1&2 increased in azide- and AICAR-treated cells compared to cells that had received lipofectamine alone. In contrast, P-AMPK1&2 content was significantly suppressed in siampk1-transfected cells treated with azide or AICAR (lane 4 and lane 6) to 0.23 ± 0.1- and 0.25 ± 0.1 fold of control cells, respectively. Endogenous P- AMPK1 under basal conditions was also suppressed to 0.04 ± 0.02-fold of controls with use of siampk1 (lane 2). Total AMPK1&2 content was also measured. Exposure to siampk1 resulted in a significant (~70%) decrease in total AMPK1&2 content in cells under basal conditions, and the marked decrease in AMPK1&2 content was also evident in siampk1-exposed cells treated with azide or AICAR. The increase in the content of P-ACC was greatly suppressed in cells pre-exposed to siampk1. Finally, no significant change in the content of -actin was observed in control or treated cells. The incomplete suppression of total AMPK1&2 and P-AMPK1&2 in siampk1- treated cells noted above is in keeping with the less than complete suppression of glucose transport in response to azide and AICAR noted above in these same cells. These results are consistent with the reported finding that the 1 isoform predominates in Clone 9 cells (4). Effect of sirna directed against both AMPK1 and AMPK2 isoforms on azide-stimulated glucose transport

15 Page 15 of 56 Given the above observations, we reasoned that simultaneous administration of siampk1 and siampk2 should have effects that either mimic or slightly exceed the results observed with use of siampk1 alone. Combined transfection with sirna directed against AMPK1 and AMPK2 (each at sub-maximal level of 50 pmol/ml) resulted in a significant decrease in the rate of glucose transport in azide- and AICARtreated cells (compared to control cells under basal conditions) to approximately the rate observed under basal conditions (Fig 3A); the stimulation of glucose transport in response to azide or AICAR was significantly suppressed in cells pre-exposed to siampk1 (P <0.05 by t-test). Treatment of cells with both siampk1 and siampk2 also reduced the rate glucose transport in control cells, perhaps due to suppression of endogenous AMPK activity, or in part due to non-specific effects. Immunoblots of samples from the above experiments showed a highly significant suppression total AMPK1&2 expression by 86%, 85% and 89% in control, azide and AICAR groups, respectively (Fig. 3B). P-AMPK1&2 was inhibited by 93%, 81%, and 88% in the three groups, respectively. The content of P-ACC in response to azide (or AICAR) was markedly reduced in cells pre-exposed to the combination of the sirnas, while -actin used as a control for loading remained constant. Effect of compound C on AMPK activation and on the glucose transport response to azide Clone 9 cells pre-exposed to 20 µm compound C for 30 min were treated with diluent, azide, or AICAR for 2 h prior to measurement of glucose transport. Exposure to compound C resulted in a slight reduction in the rate of glucose transport in control cells. Glucose uptake was stimulated by azide and AICAR by 3.7 ± 0.3 and 3.8 ± 0.4-fold,

16 Page 16 of 56 respectively, as expected in cells not exposed to compound C. In contrast, pre-treatment with compound C completely abolished the glucose transport response to both azide and AICAR (Fig. 4A). Treatment with compound C profoundly suppressed the content of P- AMPK1&2 in azide- and AICAR-treated cells to well below the level in control cells (Fig. 4B). Effect of compound C on AMPK activation and on the glucose transport response to hypoxia The purpose of these experiments was two-fold: 1) to verify that the stimulation of glucose transport occurred in to response to hypoxia and is associated with stimulation of AMPK, and 2) to test the effect of compound C on both of the above responses. Following a 2 hour incubation of cells under hypoxic conditions (nominal > 0.5% O 2 ) the rate of glucose transport was augmented to a similar degree as cells exposed to azide in the presence of oxygen (Figure 5A). The stimulation of glucose transport in response to hypoxia was completely prevented by compound C. Similar to experiments with azide detailed above, compound C decreased the content of P-AMPK1&2 in cells exposed to hypoxia while the content of total AMPK1&2 remained constant (Fig. 5B). Effect of azide, AICAR, and their combination on glucose transport and P-AMPK1&2 The above results suggest that activation of AMPK plays an important role in the stimulation of glucose transport in response to azide. We hence examined whether the stimulation of glucose transport in response to azide is additive to the response to AIACR. Cells were treated with diluent, azide, AICAR, or both for 2 h prior to measurement of glucose transport (Fig. 6A). Exposure to azide and AICAR alone

17 Page 17 of 56 resulted in 2.6 ± 0.3- and 4.5 ± 0.4-fold increase in the rate of transport, respectively. Exposure to both agents resulted in no further stimulation of glucose transport compared cells treated with AICAR alone. Immunoblots prepared from the above transport experiments showed that the content of P-AMPK1&2 increased in azide- and AICAR-treated cells (Fig. 6B). The content of total AMPK1&2 was unchanged. Interestingly, exposure to both agents resulted in a higher abundance of P-AMPK1&2 than exposure to either agent alone, although the rate of transport was not further augmented. Potential association of P-AMPK1&2 with Glut1 and phosphorylation of Glut1 The mechanism by which AMPK activation leads to simulation of glucose transport is only partially understood. Studies in the past few years have shown that activation of AMPK by AICAR or other stimuli leads to translocation of the Glut4 glucose transporter isoform to the plasma membrane, and consequently to a stimulation of glucose transport in skeletal muscle, adipose tissue, and heart (2, 15). In case of cells such as Clone 9 which express only the Glut1 isoform of facilitative glucose transporters, the mechanism by which stimulation of AMPK augments glucose transport is not understood, and activation of transporters Glut1 pre-existing in the plasma membrane has been proposed (1, 4). Here we explored the possibility that P-AMPK associates with Glut1 and leads to its phosphorylation. Lysates prepared from cells treated with diluent, 5 mm azide, or 2 mm AICAR for 2 h were immunoprecipated using anti-glut1 antibody, and cell lysates as well as the immunoprecipitates were assayed for Glut1 and P- AMPK1&2 (Fig. 7A). The content of P-AMPK1&2 increased in lysates of cells

18 Page 18 of 56 exposed to azide or AICAR. However, no P-AMPK1&2 was detected in Glut1 immunoprecipitates (Panel A, top). Similar blots were developed using antiphosphoserine antibody (Panel A, bottom). Multiple phosphorylated bands were present in lysates from control and treated cells with no obvious difference being discernible (Panel A, bottom section, left 3 lanes). In samples of Glut1 immunoprecipitates, a few P- serine-containing proteins bands with molecular masses of ~250 kda, ~120 kda, and kda were present; none of these proteins showed a differential change in their phophorylation status in response to exposure to azide or AICAR. In addition, no P- serine was detected in the region of Glut1 itself in immunoprecipitates from control and stimulated cells (Panel A, bottom section, right 3 lanes). In addition, we immunoprecipitated P-AMPK1&2 from control cells and cells exposed to azide or AICAR (Fig. 7B). Treatment with azide and AICAR increased the content of P- AMPK1&2 in cell lysates and in P-AMPK1&2 immunoprecipitates. However, no Glut1 was detected in immunoprecipitates of P-AMPK1&2. Potential Role of LKB1 and JNK in activation of AMPK Results of recent studies indicate that increased binding of AMP to the regulatory subunit of AMPK enhances the phosphorylation of the catalytic subunit of the enzyme by an upstream kinase, LKB1 (22, 28). We hence examined whether LKB1 is expressed in Clone 9 cells, and whether it can be found in association with P- AMPK1&2 in response to exposure to azide; AICAR was employed as a positive control. Cells were treated as described above. LKB1 was found to be expressed in Clone 9 cells (Fig. 8). As expected, the content of P-AMPK1&2 increased in cells treated with azide or AICAR (Fig. 8, left 3 lanes). Immunoprecipitates of P-

19 Page 19 of 56 AMPK1&2 contained LKB1, and the abundance of LKB1 was higher in immunoprecipitate products from azide- and AICAR-treated cells (Fig. 8; middle 3 lanes). Likewise, immunoprecipitates of LKB1 assayed for the presence of P- AMPK1&2 showed significant amounts of P-AMPK1&2 in the IP products from azide- or AICAR-treated cells (Fig. 8, right 3 lanes). Interestingly, virtually no P- AMPK1&2 was present in immunoprecipitates of LKB1 in control non-stimulated cells. JNK has been identified to be an upstream component of AMPK signaling cascade in glucose-deprived DU145 prostate carcinoma cells (30). To determine whether activation of JNK leads to phosphorylation of AMPK in Clone 9 cells, we employed anisomycin to stimulate JNK phosphorylation and activity (3). Cells were treated with diluent, 10 µm anisomycin, or 2 mm AICAR for 1 h and lysates were assayed for P-JNK, P-AMPK1&2, P-ACC, and -actin (Fig. 9). AICAR stimulated the phosphorylation of AMPK and ACC, as expected. While anisomycin increased P-JNK (as expected), there was no discernible increase in P-AMPK1&2 or P-ACC. Moreover, treatment with AICAR did not result in increased phosphorylation of JNK. DISCUSSION Under conditions of metabolic stress, such as inhibition of oxidative phosphorylation with reduced energy production, glycolytic ATP synthesis becomes the principle energy producing pathway. This is especially the case in cells in which glucose transport is rate-limiting for glucose metabolism where stimulation of glucose transport becomes a critical component of the adaptive response (12, 14). Despite the

20 Page 20 of 56 physiological importance of this general response, the molecular mechanisms underlying the regulation of glucose transport under these conditions are poorly understood. Results of our previous studies have shown that exposure to hypoxia, cyanide, or azide causes a sharp decrease in cell ATP and an increase in ADP and AMP concentrations within 15 min of treatment, with the nucleotide concentrations and ratios returning to normal or near-normal values by one hour despite the continued presence of the inhibitory conditions (13, 14, 25). As predicted by the increase in AMP/ATP ratio, AMPK becomes phosphorylated and activated under above conditions (1, 13). However, whether the stimulation of AMPK activity that is observed in conjunction with the enhanced glucose transport plays a critical role, a permissive role, or plays no role in the stimulation of glucose transport is not known. In the present study, we determined whether stimulation of AMPK in response to inhibition of oxidative phosphorylation is both sufficient and necessary for the increase rate of glucose transport. Although previous studies by us and other have shown that stimulation of AMPK in associated with an increase in the rate of glucose transport (i.e., establishing sufficiency of the premise) (1, 4, 31), the effect of inhibition of AMPK on the glucose transport response to inhibition of oxidative phosphorylation has not been reported. Hence the necessityof the stimulation of AMPK in the glucose transport response to inhibition of oxidative phosphorylation has not been established. Here, we utilized two independent approaches to suppress AMPK activity to determine the effect of such inhibition on the glucose transport response to azide. In these studies we have employed AICAR as a positive control throughout. We also tested for potential additive effect of azide and AICAR on AMPK activation and on the rate of glucose transport.

21 Page 21 of 56 The results reported herein place stimulation of AMPK activity directly in the pathway of the glucose transport response to inhibition of oxidative phosphorylation, signifying that the observed stimulation of AMPK is a necessary step in the glucose transport response. Mammalian AMPK is a hetero-trimeric enzyme comprised of a catalytic and regulatory and subunits (21). The subunit is expressed as two isoforms (1 and 2), and both contain a Thr at position 172 that upon phosphorylation lead to a ~ fold increase in the enzyme s activity (29). The two isoforms are differentially expressed in various tissues. AMPK complexes containing the 2 isoform predominate in skeletal and cardiac muscle (26), while approximately equal levels of the two isoforms is expressed in liver (27). In contrast, -cells in pancreatic islets largely express the 1 isoform (20). In Clone 9 cells that were used in the present study, we had previously detected the presence of both isoforms (1). Further detailed studies by Barnes et al utilizing immunoprecipitation of each AMPK in Clone 9 cells followed by measurement of enzyme activity in the immunoprecipitates showed that the 1 isoform of AMPK is responsible for the bulk of the enzyme s activity in these cells (4). To investigate the potential mediating, and perhaps critical, role of AMPK in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation, we employed the following two independent strategies. The first strategy involved suppression of AMPK1 and AMPK2 subunit expression (alone and in combination) using sirna technology, while the second strategy employed compound C as an inhibitor of AMPK (19). Both strategies would be predicted to result in a significant suppression of the glucose transport response, if the stimulation of AMPK activity plays an important role in mediating the response. Use of sirna directed against the 2

22 Page 22 of 56 isoform of AMPK resulted in a large decrease in AMPK2 abundance. However, upon stimulation of these cells with AICAR, no significant decrease in P-AMPK1&2 was observed, suggesting that the 2 isoform may be a minor component of total AMPK expressed in these cells. This inference is consistent with the findings of Barnes et al noted above on the expression and function of AMPK isoforms in Clone 9 cells (4). We then examined whether the stimulation of glucose transport in response to azide was affected in cells with suppressed AMPK2 content. The results showed that neither the stimulation in response to azide nor to AICAR was affected by use of siampk2. In addition, total AMPK1&2 content and the content of P-ACC in response to azide (and AICAR) were not affected. In contrast were the results following the use of sirna directed against AMPK1. Use of the siampk1 in control cells decreased basal transport by a slight extent, perhaps due to the dependence of basal glucose transport on AMPK activity. Treatment with siampk1 resulted in a dramatic suppression of azide-stimulated glucose transport. Western blot analysis showed that the abundance of AMPK1 was significantly depressed in sirna-treated cells, as was the abundance of total AMPK1&2 as well as the content of P-AMPK1&2 in response to azide (and AICAR), verifying that the 1 isoform is the predominant form of the enzyme in these cells (4). The content of P-ACC in response to azide (and AICAR) was significantly depressed in cells exposed to siampk1. The partial suppression of glucose transport in response to azide could be interpreted to mean that AMPK plays a significant, but partial, role in the response, or alternatively, that there was incomplete suppression of AMPK in

23 Page 23 of 56 response to the sirna treatment. The finding that the stimulation of glucose transport by AICAR was also partially suppressed strengthens the latter possibility. We next used sirnas against both AMPK isoforms in combination. In these experiments 50 pmol/ml of each sirna was used to limit cell toxicity. The effect of the mixture of the sirnas on glucose transport in response to azide was similar to that seen with use of siampk1 alone, a finding that would be expected if the 2 isoform was a minor component of total AMPK activity. Western blot analysis showed a dramatic (but incomplete) suppression of total cellular AMPK1&2 content and of P-AMPK1&2 abundance in response to azide and AICAR. Importantly, the content of P-ACC was markedly depressed in cells exposed to azide (and AICAR), consistent with great suppression of AMPK1&2 activity. We also used a complementary strategy to define the mediating role of AMPK activation in the stimulation of glucose transport in response to azide and hypoxia. Compound C has been reported to be a selective inhibitor of AMPK by preventing the phosphorylation of the subunit by competing with AMP binding to the subunit of the enzyme (19). Use of this reagent resulted in a near-complete suppression in the content of P-AMPK1&2 in cells treated with AICAR used as a positive control. Similarly, the content of P-AMPK1&2 in azide-treated cells and in cells exposed to hypoxia was suppressed by compound C to low levels, while, importantly, the total cellular content of AMPK1&2 remained unaltered. Under these conditions, we observed a nearcomplete suppression of the stimulation of glucose transport in response to azide, hypoxia, and to the positive control, AICAR. It should be noted that unlike the glucose transport response following exposure to azide, cyanide, or hypoxia that occurs within

24 Page 24 of 56 ~10 min of exposure, stimulation of the hypoxia-responsive pathway per se by CoCl 2 (which also induces Glut1 mrna expression and subsequently results in increased Glut1 expression and stimulation of glucose transport) occurs after a delay of ~4 h (11, 14, 24). Hence, the stimulation of glucose transport in response to hypoxia at 2 h and its suppression by compound C reflects the inhibition of oxidative phosphorylation and stimulation of AMPK leading to stimulation of glucose transport with the constant cell Glut1 content. Based on the results derived from the sirna and compound C experiments, we conclude that stimulation of AMPK activity plays a critical role in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation. The above results strongly suggest that stimulation of AMPK is necessary for the stimulation of glucose transport in response to inhibition of oxidative phosphorylation. To extend the accepted premise that stimulation of AMPK is sufficient for the glucose transport response to azide, we measured the effect of simultaneous exposure to aizde and AICAR. If azide stimulates glucose transport by pathways that are in whole, or in part, independent of AMPK, then addition of azide to cells exposed to AICAR should further augment the rate of transport observed in response to AICAR alone. We found no evidence for any additive effect of azide to the stimulation of glucose transport in response to AICAR. It is of interest to note that the content of P-AMPK1&2 was highest in cells treated with both agents, while the transport response was not further enhanced. The reason for this finding is not known, and suggests that activation of AMPK stimulates the rate of glucose transport only to a certain limiting extent. Nevertheless, the above results strongly suggest that activation of AMPK is associated with a stimulation of glucose transport in response to azide.

25 Page 25 of 56 Having found that stimulation of AMPK is critical for the glucose transport response to azide (and AICAR), we explored the possibility that Glut1 becomes phosphorylated by the direct action of AMPK. Such a modification could itself mediate the presumed activation of Glut1 and stimulate glucose transport. This possibility gains credence because of the presence of two potential consensus phosphorylation sequences for AMPK in the intracellular segments of Glut1 at Ser 95 and Ser 226 that are conserved in rat, human, and mouse (7, 17, 18). We thus assayed for the presence of P-serine in Glut1 immunoprecipitates from control cells and cells treated with azide or AICAR. No P-serine was detected in Glut1 immunoprecipitates under any of the above conditions. However, although a number of P-serine-containing protein bands were present in Glut1 immunoprecipitates, their abundance showed no differential change in control versus treated cells. Identification of these proteins could be a subject of future studies. We also did not find any P-AMPK1&2 in Glut1 immunoprecipitates, nor was there any Glut1 in P-AMPK1&2 immunoprecipitates. Based on these findings, we conclude that AMPK probably does not directly phosphorylate Glut1, and that the action of AMPK to stimulate glucose transport is probably indirect and is mediated by unknown intermediary steps. It should also be noted that the increase in Glut1 expression and content in response to azide is not observed prior to 4 h of exposure to the inhibitor (24, 25). We also examined the potential role of LKB1 and JNK in the phosphorylation and activation of AMPK in response to azide and AICAR. LKB1 has been identified as an upstream kinase that phosphorylates the -subunit of AMPK at position Thr172, especially upon binding of 5 -AMP to the subunit of AMPK (29). LKB1 is one of the two described modes of activation of AMPK, the other being mediated by increased cell

26 Page 26 of 56 calcium concentration and activation of calmodulin-dependent protein kinase kinase (CaMKK) (22). It is possible that other means of activation of AMPK exist and these potential mechanisms are under investigation (9). LKB1 has been identified as the site of action of metformin, a medication used in the treatment of type 2 diabetes (22). We found that LKB1 was immunoprecipitated in association with P-AMPK1&2, and viceversa. Interestingly, the degree of association between the two proteins was augmented in cells treated with AICAR (as expected), as well as in cells treated with azide. The latter finding suggests, but does not prove, that the stimulation of AMPK and its phosphorylation in cells treated with azide is mediated by LKB1. For example, we have previously described that the intracellular free calcium concentration increases following inhibition of oxidative phosphorylation (16). We additionally examined the potential activation of AMPK in response to phosphorylation and activation of JNK. This latter possibility was examined because of a previous report that in DU145 prostate carcinoma cells deprived of glucose, JNK phosphorylation and activation was associated with phosphorylation of AMPK (30). We found that JNK phosphorylation in response to anisomycin did not result in phosphorylation of AMPK, and that treatment with AICAR was not associated with phosphorylation of JNK in Clone 9 cells. The divergent results probably reflect the usage of different cells and stimuli. Our results suggest that LKB1 might play a mediating role in the activation of AMPK in response to azide. Our previous results have shown that intracellular free calcium concentration rises in cells treated with azide (16). This raises the possibility that activation of CaMKK mentioned above might also play a role in the stimulation of AMPK in response to azide. Further

27 Page 27 of 56 work is necessary to better define the upstream pathway(s) leading to stimulation of AMPK in response to inhibition of oxidative phosphorylation. Finally, it is worth emphasis that the steps and sequence of events that mediate the stimulation of glucose transport in response to activation of AMPK remain unknown. Our finding that AMPK plays a critical role in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation implies the involvement of downstream phosphorylation events, of kinases, and perhaps of phosphatases, in the response. Our results also demonstrate that AMPK is not associated with Glut1, nor is Glut1 itself phosphorylated in response exposure to azide or following stimulation of AMPK activity. Further work is necessary to delineate the sequence of events to identify the mediating mechanisms underlying the stimulation of glucose transport in response to inhibition of oxidative phosphorylation.

28 Page 28 of 56 ACKNOWLEDGMENTS We thank Ms. Li Song for her excellent technical support. GRANTS This study was supported by National Institute of Diabetes and Digestive and Kidney Disease Grant RO1-DK

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34 Page 34 of 56 FIGURE LEGENDS Figure 1. Effect of sirna directed against AMPK2 on glucose transport in response to azide and AICAR, and on AMPK2, P-AMPK1&2, total- AMPK1&2, and P-ACC abundance. A. Glucose transport. 70% confluent Clone 9 cells were transfected in duplicate with 5 µl/ml lipofectamine 2000 with or without 100 pmol/ml siampk2. 48 h after transfection, cells were treated with 5 mm azide or 2 mm AICAR (as indicated) for 2 h prior to measurement of CB-inhibitable 3-OMG transport. Cells were lysed in 100 µl NP-40 buffer, after centrifugation, 60 µl of the postnuclear lysates were used for radioactive counting. The experiment was repeated three times and the results were averaged. * denotes P < 0.05 compared to control (lipofectamine alone) using ANOVA. The stimulation of transport in response to azide or AICAR was not decreased by exposure to the sirna (P > 0.05 by t-test). B. AMPK2 expression, P-AMPK1&2, total-ampk1&2, and P-ACC abundance. Following determination of protein, samples of the above lysates were subjected to immunoblotting to measure the relative content of cell P-AMPK1&2, AMPK2, total-ampk1&2, P-ACC, and -actin under the different treatment conditions. Results of three blots normalized against values in control cells without treatment of siampk2 are given as fold-change below each lane. * denotes P < 0.05 compared to control (lipofectamine alone) using ANOVA. Exposure to sirna directed against AMPK2 had no effect on the content of total AMPK1&2, P-AMPK1&2,

35 Page 35 of 56 or P-ACC compared to their respective control by t-test (P > 0.05). The content of - actin remained constant. Figure 2. Effect of sirna directed against AMPK1 on glucose transport in response to azide and AICAR, and on AMPK1, P-AMPK1&2, total- AMPK1&2, and P-ACC abundance. A. Glucose transport. Conditions were the same as those detailed in the legend Fig. 1A, except sirna directed against AMPK1 was employed. * denotes P < 0.05 compared to control (lipofectamine alone) using ANOVA. Exposure to sirna directed against AMPK1 had significant effect on the rate of glucose transport in response to azide or AICAR compared to their respective controls by t-test (P < 0.05). B. AMPK1 expression, P-AMPK1&2, total- AMPK1&2, and P-ACC abundance. Conditions were identical to those in Fig. 1B, except sirna directed against AMPK1 was used. Antibodies against P-AMPK1&2, AMPK1, total-ampk1&2, and P-ACC were employed. * denotes P < 0.05 compared to control (lipofectamine alone) using ANOVA. Using t-test, all cells preexposed to sirna direct against AMPK1 showed different abundance of P- AMPK1&2, AMPK1, total AMPK1&2 and P-ACC compared to their respective controls (P < 0.05). The content of -actin remained constant. Figure 3. Effect of sirna direct against both AMPK1 and AMPK2 on glucose transport in response to azide and AICAR and on total AMPK1&2, P- AMPK1&2, and P-ACC abundance. A. Glucose transport. Conditions were the same as those detailed in Fig. 1A, except that 50 pmol/ml of sirna directed against both

36 Page 36 of 56 1 and 2 was employed. * denotes P < 0.05 compared to control (lipofectamine alone) using ANOVA. Exposure to sirna directed against both AMPK1 and AMPK2 had significant effect on the rate of glucose transport in response to azide or AICAR compared to their respective control by t-test (P < 0.05). B. Total AMPK1&2 expression, P-AMPK1&2, and P-ACC abundance. Conditions were the same as those in Fig.1B, except that 50 pmol/ml of each sirna was used. Antibodies against P- AMPK1&2, and total AMPK1&2, and P-ACC were used. * denotes P < 0.05 compared to control (lipofectamine only) using ANOVA. Using t-test, all cells preexposed to sirna direct against both AMPK1 and AMPK2 showed different abundance of P-AMPK1&2, total AMPK1&2 and P-ACC compared to their respective controls (P < 0.05). The content of -actin remained constant. Figure 4. Effect of compound C on azide- or AICAR-stimulated glucose transport, P-AMPK1&2 and total AMPK1&2. A. Glucose transport. Confluent Clone 9 cells in triplicate 60 mm dishes were incubated in serum-free media for 24 h prior to experimentation. Cells were pre-treated with 20 µm compound C for 30 min prior to being treated with diluent, 5 mm azide, or 2 mm AICAR for 2 h prior to measurement of CB-inhibitable 3-OMG transport. Cells were lysed in 100 µl of NP-40 buffer, and 70 µl of each post-nuclear lysate was used for radioactive counting and the remainder was used for immunoblotting (see below). Uptakes in control and treated cells were performed in parallel. The experiment was repeated three times using triplicate dishes for each condition, and the results were averaged. Values are expressed as fold-increase ± SE compared to basal. * denotes P < 0.05 compared to control using ANOVA. Glucose

37 Page 37 of 56 transport was lower in all cells pretreated with compound C compared to their respective controls by t-test (P < 0.05). B. P-AMPK1&2 and total AMPK1&2 abundance. The above lysates were used in replicate blots. In each experiment, the densitometry value of P-AMPK1&2 for each condition was normalized against the value for total AMPK1&2 abundance. The experiment was repeated three times and the results averaged. * denotes P < 0.05 compared to control using ANOVA. Using t-test, cells preexposed to compound C had decreased abundance of P-AMPK1&2 compared to their respective controls (P < 0.05). Figure 5. Effect of compound C on hypoxia-induced stimulation of glucose transport, P-AMPK1&2 and total AMPK1&2. A. Glucose transport. Confluent Clone 9 cells in triplicate 60 mm dishes were serum-starved for 24 h prior to the experiment. Cells were pre-treated with 20 µm compound C for 30 min before being incubated under hypoxic conditions (nominal 0.5% O 2 ) for 2 h and subsequent measurement of CB-inhibitable 3-OMG transport. Cells treated for 2 h with 5 mm azide served as positive control. Cells were lysed in 300 µl of NP-40 buffer, and 250 µl of each post-nuclear lysate was used for radioactive counting and the remainder was used for immunoblotting (see below). Uptakes in control and treated cells were performed in parallel. The experiment was repeated two times using triplicate dishes for each condition, and the results were averaged. Values are expressed as fold-increase ± SE compared to basal. * denotes P < 0.05 compared to control using ANOVA. Glucose transport was lower in all cells pretreated with compound C compared to their respective controls by t-test (P < 0.05). B. P-AMPK1&2 and total AMPK1&2 abundance. The above lysates were used for immunoblotting. In each experiment, the densitometry

38 Page 38 of 56 value of P-AMPK1&2 for each condition was normalized against the value for total AMPK1&2 abundance in the same culture dish and the results averaged. * denotes P < 0.05 compared to control using ANOVA. Using t-test, cells pre-exposed to compound C had decreased abundance of P-AMPK1&2 compared to hypoxia itself (P < 0.05). Figure 6. The effect of azide, AICAR, or both on glucose transport and on the abundance of P-AMPK1&2 and total AMPK. A. Glucose transport. 100% confluent Clone 9 cells were serum starved for 24 h. Cells were treated with diluent, 5 mm azide, 2 mm AICAR, or both for 2 h prior to measurement of CB-inhibitable [ 3 H] 3- OMG transport in three independent experiments. Cells were lysed in 100 µl NP-40 buffer, and after centrifugation, 60 µl of the post-nuclear lysates were used for radioactive counting. *denotes P < 0.05 compared to control using ANOVA. Student s t-test showed no significant difference between the rate of transport in AICAR- verse AICAR plus azide-treated cells. B. Total AMPK expression and P-AMPK1&2 abundance. Following determination of protein, sample of each of the above lysates was subjected to immunoblotting to measure the relative content of cell P-AMPK1&2 and total AMPK under the different treatment conditions. Results of three blots normalized against values in control cells are given as fold-change below each lane. *denotes P < 0.05 compared to control using ANOVA. The content of total AMPK1&2 remained constant. Fig. 7. Glut1 does not associate with P-AMPK1&2 and is not serinephosphorylated in azide- or AICAR-treated cells. A. Immunoprecipitation of Glut1. Clone 9 cells were treated with diluent, 5 mm azide, or 2 mm AICAR for 2 h and

39 Page 39 of 56 lysates were prepared using NP-40 buffer. Post-nuclear lysates were immunoprecipitated overnight using mouse anti-glut1 antibody. Immunoprecipitates were dissolved in SDS sample buffer and utilized in three individual western blots for measurement of P- AMPK1&2 using rabbit anti-phospho-thr 172 -AMPK (top panel), for Glut1 using rabbit anti-glut1 (middle panel), and for P-serine using rabbit anti-phosphoserine antibody (bottom panel). The region of Glut1 is indicated on the right. There is no significant difference in the Glut1 content in cell lysates from control and treated cells, or in Glut1 immunopercipitates. B. Immunoprecipitation P-AMPK1&2. Post-nuclear lysates were immunoprecipitated with rabbit anti-phospho-thr 172 -AMPK, and immunoprecipitates were fractionated in two individual blots for measurement of P- AMPK1&2 and Glut1. There is no significant difference in the Glut1 content in cell lysates from control and treated cells, and there is no Glut1 detected in Glut1 immunopercipitates. Fig. 8. Association of LKB1 and P-AMPK1&2. Clone 9 cells were treated with diluent, 5 mm azide, 2 mm AICAR for 2 h and lysates were prepared using NP-40 buffer. Immunoblot of cell lysates is shown in the left 3 lanes. Protein from post-nuclear lysates was immunoprecipitated overnight using goat anti-lkb1. The suspension during the final wash step was divided into two equal parts and one half of the immunoprecipitate product was dissolved in SDS buffer devoid of -ME and blotted for LKB1, and the other half was dissolved in SDS buffer containing -ME and immunobloted with rabbit anti-phospho-thr 172 -AMPK1&2 (middle 3 lanes). In a different set of experiment, 1 mg protein from each post-nuclear lysate was

40 Page 40 of 56 immunoprecipitated with goat anti-lkb1 antibody, and immunobloted with rabbit antiphospho-thr 172 -AMPK1&2 and anti-lkb1 (right 3 lanes). Fig. 9. Effect of AMPK activation on JNK phosphorylation. Confluent Clone 9 cell were serum starved for 24 h and treated with 20 µm anisomycin, 2 mm AICAR, or diluent for 1 h. Cells were lysed in NP-40 buffer and post-nuclear lysates were subjected to 10% SDS-PAGE and probed with mouse anti-phospho-jnk, rabbit anti-phospho Thr 172 -AMPK, rabbit anti-phospho-acc. -actin was used to control for loading of the lanes.

41 Page 41 of 56 Fig. 1A P>0.05 P>0.05 P>0.05

42 Page 42 of 56 Fig. 1B Control Azide AICAR siampk P-AMPK 1&2 (fold-change) * * 1.71* AMPK 2 (fold-change) * * * Total-AMPK (fold-change) P-ACC (fold-change) * 3.40* 3.81* 3.69* -actin

43 Page 43 of 56 Fig. 2A P<0.05 P<0.05 P<0.05

44 Page 44 of 56 Fig. 2B Control Azide AICAR siampk P-AMPK1&2 (fold-change) * 1.63* 0.23* 1.46* 0.25* AMPK1 (fold-change) * * * Total AMPK (fold-change) * * * P-ACC (fold-change) 1.00 <0.05* 2.80* * <0.05* -actin

45 Page 45 of 56 Fig. 3A P<0.05 P<0.05 P<0.05

46 Page 46 of 56 Fig. 3B Control Azide AICAR siampk1& P-AMPK1&2 (fold-change) * 1.43* 0.19* 2.16* 0.12* total AMPK1&2 (fold-change) * * * P-ACC (fold-change) * 1.61* 0.01* 2.15* 0.01* -actin

47 Page 47 of 56 Fig. 4A P<0.05 P<0.05 P<0.05

48 Page 48 of 56 Fig. 4B Control Azide AICAR compound C P-AMPK1& 2 (fold-change) * 1.45* 0.05* 2.50* 0.02* total AMPK1&2

49 Page 49 of 56 Fig. 5A P<0.05 P<0.05

50 Page 50 of 56 Fig. 5B Control hypoxia azide compound C P-AMPK1& 2 (fold-change) * * total AMPK1&2

51 Page 51 of 56 Fig. 6A P>0.05 P<0.05

52 Page 52 of 56 Fig. 6B Con AZ AIC AZ+AIC P-AMPK1& 2 (fold-change) * 2.13* 3.71* total AMPK1&2

53 Page 53 of 56 Fig. 7A Cell lysate IP: Glut1 P-AMPK1&2 CON AZ AIC CON AZ AIC Glut1 P-serine Glut1 region 37-

54 Page 54 of 56 Fig. 7B P-AMPK1&2 Cell lysate IP: P-AMPK CON AZ AIC CON AZ AIC Glut1

55 Page 55 of 56 Fig. 8 P-AMPK1&2 Cell lysate IP: P-AMPK1&2 IP: LKB1 CON AZ AIC CON AZ AIC CON AZ AIC Total-LKB1

56 Page 56 of 56 Fig. 9 P-JNK CON ANISO AIC P-AMPK1&2 P-ACC -actin