AMPK negatively regulates PXR activity

AMPK is the energy sensor in cells that functions to restore energy balance during periods of ATP depletion²¹. In an attempt to understand the link between glucose and PXR regulation, we examined the activation status of AMPK in HepG2 C3A cells under starvation conditions and in the presence of 50mM glucose. AMPK was activated, as detected by the phosphorylation of threonine 172, when cells were glucose starved but was inactive in the presence of glucose (Fig. 2a). This is consistent with the results of a previous study showing rapid activation of AMPK and PKA in glucose-deprived hepatocarcinoma cells³². Interestingly, we observed an inverse correlation of PXR activity: PXR activity was downregulated under glucose-starvation conditions but upregulated in the presence of glucose, as determined by the increase in the protein levels of CYP3A4 and MDR1 (both of which are PXR transcriptional targets) (Fig. 2a). This inverse correlation led us to postulate that AMPK negatively regulated PXR. To test this hypothesis, we treated HepG2 PXR CYP-luc cells with 50mM glucose and increasing concentrations of metformin—previously reported to be an AMPK activator^33,34. We observed that PXR activity was impeded by AMPK activation in a dose-dependent manner (Fig. 2b). To confirm this observation, HepG2 PXR CYP-luc cells were starved of glucose to minimize all PXR activity and then treated with increasing concentrations of dorsomorphin—a known AMPK inhibitor^35,36. PXR activity increased in a dose-dependent manner in response to increasing concentrations of dorsomorphin (Fig. 2c). Taken together, these observations suggest that the activation of AMPK represses the activity of PXR.

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Figure 2

AMPK activation is inversely correlated with PXR activation.

(a) HepG2 cells were deprived of serum and glucose for 24h. The cells were then treated with or without 50mM glucose. Whole-cell lysates were probed with antibodies as indicated (left panel). AMPK activity (pAMPK Thr 172) was normalized to total AMPK expression. Other protein expression quantifications were normalized to actin. HepG2 PXR CYP-luc cells were deprived as described in the legend for Fig. 1, and luciferase activities were measured 24h after the cells were treated with glucose and metformin (an AMPK activator) (b) or compound C (an AMPK inhibitor) (c). The P-values were determined using ANOVA with Tukey’s HSD test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3. Results are expressed as the mean±SE.

Identification of AMPK modulators

Dorsomorphin (6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine) is the only agent reported to be used as a cell-permeable AMPK inhibitor. Unfortunately, dorsomorphin (also known as compound C) is not specific for AMPK; it also inhibits the BMP pathway^37,38 and a number of kinases other than AMPK^39,40. The lack of a specific AMPK inhibitor prompted us to identify additional and more potent, cell-permeable AMPK inhibitors. To this end, we performed our primary compound screen with a library of 244 chemicals previously identified as kinase inhibitors. In an in vitro kinase assay coupled with an ADP-Glo assay (the latter assay measures the generation of ADP from the kinase reaction), we treated recombinant active AMPK with varying concentrations of the test compounds in 384-well plates and measured the ADP-mediated luminescence. Of the compounds tested, 32 that displayed more than 80% inhibition of AMPK at a concentration of 2 μM, as compared to DMSO without AMPK protein (Fig. 3a), were selected for analysis to determine their IC₅₀ values (Table 1). We selected three compounds, the indirubin derivative E804 (ID E804), Cdk1/2 inhibitor III, and UCN-01, for further validation based on their potent inhibition of AMPK (Fig. 3a and Table 1) and cell permeability^41,42,43. The ability of these three compounds to inhibit AMPK was assessed in a cell-based assay, and only ID E804 and Cdk1/2 inhibitor III inhibited AMPK activity effectively (see Supplementary Fig. S3). We also further characterized reported AMPK activators. A769662 and salicylate, both are known AMPK activators^44,45,46, displayed dose-responsive activation of AMPK in the biochemical ADP-Glo kinase assay. AICAR (5-aminoimidazole-4-carboxamide ribonucleotide), another well-known cellular AMPK activator⁴⁷, was included in our assay but did not activate AMPK in vitro (Fig. 3b). A similar observation was previously made by Hawley et al., who found that metformin did not activate AMPK in a cell-free system³⁴. These results suggest that A769662 and salicylate are allosteric activators of AMPK and that AICAR may activate AMPK indirectly by decreasing cellular ATP and increasing AMP. All three activators were assessed for AMPK activation in a cell-based assay, and all three effectively increased AMPK activity (see Supplementary Fig. S3).

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Figure 3

Screening of potent AMPK activators and inhibitors.

The Calbiochem Kinase Inhibitor Library I and Calbiochem Kinase Inhibitor Library III were screened for potent AMPK inhibitors by using an in vitro AMPK kinase assay coupled with an ADP-Glo assay. (a) A total of 244 compounds were screened, and 32 compounds displayed more than 80% inhibition at a concentration of 2 μM, as compared to DMSO. (b) A-769662 and salicylate were identified as potent AMPK activators in the ADP-Glo assay.

AMPK kinase activity is essential in the regulation of PXR

To confirm the inhibitory effects of AMPK on PXR (Fig. 2), we used the AMPK activators A769662, AICAR, and salicylate. We starved HepG2 PXR CYP-luc cells of glucose then treated them with 50mM glucose and increasing concentrations of the test compounds. Similar to our previous observation, AMPK activation inhibited PXR activity as assessed by CYP3A4 promoter inhibition (Fig. 4a–c). AMPK activation also inhibited glucose-induced CYP3A4 protein expression (Fig. 4d). To confirm that the observed effects of the activators were not the result of cytotoxicity, we performed a cell viability assay and observed that at the concentrations tested, the compounds had no significant effect on cell viability (see Supplementary Fig. S4a–c). Conversely, the inhibition of AMPK by ID E804 and Cdk 1/2 inhibitor III activated PXR (Fig. 4e–f). PXR binds many structurally diverse compounds⁴⁸. To determine if the effects of the AMPK inhibitors on PXR activation were the result of direct binding and activation of PXR, we tested the direct binding of the AMPK inhibitors to PXR by using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay^49,50. As shown in Supplementary Fig. S4d, all three inhibitors tested bound, but more weakly than the known potent PXR ligand T0901317⁵¹. It is important to note that although UCN-01 weakly bound PXR in our binding assay, UNC-01 did not induce PXR activity (Fig. 4g), which is consistent with its failure to activate AMPK in a cell-based assay (see Supplementary Fig. S3). This observation suggests that the activation of PXR by ID E804 and Cdk 1/2 inhibitor III was not mainly due to their binding to PXR where they may act as agonists, rather the activation of PXR observed is through the modulation of AMPK activity. Overexpression of WT AMPK, but not of the kinase-dead mutant of AMPK (AMPK KD), recapitulated the effects of the AMPK activators by causing a decrease in CYP3A4 promoter activity (Fig. 4h). These results suggest that the kinase activity of AMPK is essential for its regulation of PXR activity. An siRNA-mediated knockdown of AMPK increased PXR activity, supporting our observations with AMPK inhibitors (Fig. 4i).

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Figure 4

AMPK modulates PXR activity.

HepG2 PXR CYP-luc cells were deprived as described in the legend for Fig. 1. Cells were co-treated with increasing concentrations of AMPK activators and 50mM glucose. CYP3A4 promoter activity was assessed by luciferase assay (a–c), or PXR and CYP3A4 protein expression were assessed (d). (e–g) Deprived HepG2 PXR CYP-luc cells were treated with increasing concentrations of indicated AMPK inhibitors, and luciferase assays were performed. Wild-type AMPK (AMPK WT), kinase-dead AMPK (AMPK KD), or control vector (Vector) were expressed in HepG2 PXR CYP-luc cells (h), or AMPK was knocked down in cells using siRNA targeting AMPK (AMPK siRNA) or NT siRNA as control (i), and cells were deprived for 24h. Luciferase activities were measured 24h after treatment. The P-values were determined using ANOVA with Tukey’s HSD test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3. Results are expressed as the mean±SE.

PXR is not a direct target of AMPK

AMPK activation has previously been shown to promote phosphorylation and regulate the localization of constitutive androstane receptor (CAR) in cells^29,30,31. Therefore, we performed an in vitro kinase assay to determine if PXR was a direct target of AMPK. In our in vitro study, we used purified active AMPK with purified PXR-GST as a substrate and found that the PXR was not phosphorylated by the AMPK (Fig. 5, lane 7). We did observe AMPK autophosphorylation (Fig. 5, lane 4), and this was out-competed by the SAMS peptide (an AMPK synthetic substrate) (Fig. 5, lanes 9–11), indicating that the kinase assay worked as expected. This suggests that although AMPK plays an essential role in regulating PXR and the kinase activity of AMPK is required, PXR is not a direct substrate of AMPK.

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Figure 5

PXR is not a direct target of AMPK.

Active AMPK was subjected to in vitro kinase assay with PXR and SAM peptide as substrates in the presence of nonradioactive ATP. The incorporation of phosphate into the substrate was determined by Western blot analysis with anti-phosphorylated serine/threonine antibody (pSer/Thr) (upper two panels). The lower three panels show the same blot reprobed with the indicated antibodies to detect PXR-GST, GST, or total AMPK.

Cell culture

All cell lines were maintained in a humidified incubator at 37°C with 5% CO₂. A clone of HepG2 cells stably over-expressing FLAG-hPXR (in pCDNA3.1 vector) and CYP3A4-luciferase reporter (in pGL3 vector) (HepG2 PXR CYP-luc clone 1) (previously described as PXR clone 1 [ref. ⁶⁶]) was generated and maintained in double-selection medium containing 500μg/mL G418 and 200μg/mL hygromycin (Life Technologies). Primary human hepatocytes were maintained in Williams E medium containing Primary Hepatocyte Maintenance Supplement (Life Technologies) and medium was changed to DMEM for experimentation. Cells collected for RNA and protein extraction were detached by scraping. For all luminescence-based assays, the cells were plated using phenol-red–free DMEM medium (Life Technologies). Cells were routinely verified to be mycoplasma-free by using the MycoProbe Mycoplasma Detection Kit (R&D Systems).

Protein extraction and Western blot analysis

Protein was isolated from cells by incubating them in Pierce RIPA lysis buffer with added Halt Protease Inhibitor cocktail (Thermo Fisher Scientific) and Phosphatase Inhibitor Cocktail (Sigma) for 30min on ice then sonicating the lysate for 10s at 50% amplitude to shear the DNA. The protein concentration was measured using the Pierce BCA Protein Assay (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. Protein lysates were resolved on NuPAGE 4–12% SDS-PAGE gradient gels (Life Technologies). After electrophoresis was completed, the proteins were transferred from the gels to nitrocellulose membranes with an iBlot Dry Blotting System (Life Technologies). All membranes were blocked with Odyssey Blocking Buffer (LI-COR Biotechnology, Lincoln, NE). All antibodies were diluted in Odyssey Blocking Buffer. The secondary antibodies were diluted in TBS. All Western blot imaging was performed using a LI-COR Odyssey Infrared Imaging System.

RNA extraction and quantitative real-time PCR

RNA was extracted using Maxwell simplyRNA Kits and a Maxwell 16 Instrument (Promega). RNA concentrations were measured using a NanoDrop 8000UV-Vis Spectrophotometer (Thermo Fisher Scientific). All cDNA used in mRNA quantitative real-time PCR (qPCR) analyses was synthesized from extracted RNA by using the SuperScript VILO cDNA Synthesis Kit (Life Technologies) in accordance with the manufacturer’s protocol. The mRNA expression data were generated using Applied Biosystems TaqMan assays (20×) and Fast Advanced Master Mix (Life Technologies). Thermal cycling for qPCR was performed with an Applied Biosystems 7900HT Fast Real-Time PCR system (Life Technologies) in accordance with the TaqMan Fast protocol. Gene expression was normalized to the housekeeping gene 18S ribosomal RNA (18S), the expression of which did not vary in the different cell lines as a function of the glucose levels. Data are shown as the mRNA fold change (2^−ΔΔCT) relative to the mRNA level of the corresponding transcript in the control samples as indicated. Each experiment was performed at least three times, and all samples were analyzed in triplicate.

Luciferase assays

HepG2 PXR CYP-luc cells grown in flasks were trypsinized and plated in 96-well plates (CulturPlate-96, PerkinElmer) at 20,000 cells/well. Cells were treated as described above. PXR activity was determined as a measure of CYP3A4- luciferase reporter activity. The luciferase assay was performed in accordance with the manufacturer’s protocol. Briefly, a volume of the reagent equal to the volume of the medium on the cells was added and the plates were incubated at room temperature for 30min with gentle agitation. The luminescence signal was measured with an EnVision plate reader (PerkinElmer), and the fold change in PXR activation was calculated.

AMPK kinase assay

Recombinant active AMPK (400ng) and purified PXR-GST (200ng) were incubated in the presence of 300μM nonradioactive ATP (Sigma-Aldrich) in AMPK kinase buffer (400mM Tris, pH7.5, 20mM MgCl₂, 0.1mg/mL BSA, 50 μM DTT). The reaction mixture was incubated at 30°C for 1h with gentle agitation, then the reaction was stopped by adding sample loading buffer and boiling for 5min. The entire reaction volume was resolved on SDS-PAGE and immunoblotted with the indicated antibodies.

AMPK inhibitor and activator screen

In the primary screening to identify potent AMPK inhibitors, a library of 240 putative kinase inhibitors (EMD Millipore) at concentrations of 2μM, along with DMSO controls, was incubated with or without 10ng AMPK protein in 15μL reaction mixture (50μM ATP and 50μM SAMS peptide in the assay buffer of 40mM Tris, pH 7.5, 20mM MgCl₂, 0.1mg/mL BSA, 50μM DTT) at room temperature for 60min in 384-well white solid-bottom plates. An ADP-Glo assay was then performed. The luminescence signal for each well was measured with a PHERAstar FS plate reader (BMG Labtech, Durham, NC). The DMSO concentration was 0.2% for all assay wells. The signal values from DMSO without AMPK and with AMPK were used as positive (100% inhibition) and negative (0% inhibition) controls, respectively. The activity of each well was normalized to that of the positive and negative controls. Those 32 chemicals with %inhibition values of at least 80% were selected for further dose-response confirmation. In the dose-response characterization of potent AMPK inhibitors and activators, titrations of the 32 selected AMPK inhibitors from the primary screening and three selected putative AMPK activators (A-769662, salicylate, and AICAR), along with DMSO controls, were incubated with or without 10ng AMPK protein in 15μL reaction mixture (50μM ATP and 50μM SAMS peptide in the assay buffer of 40mM Tris, pH 7.5, 20mM MgCl₂, 0.1mg/mL BSA, 50μM DTT) at room temperature for 60min in 384-well white solid-bottom plates. An ADP-Glo assay was then performed. The luminescence signal for each well was measured with a PHERAstar FS plate reader. The DMSO concentration was 0.2% for all assay wells. The signal values for DMSO without AMPK and with AMPK were used as positive (100% inhibition) and negative (0% inhibition) controls, respectively. The activity of each well was normalized to that of the positive and negative controls. The results for each chemical were fitted into a one-site competitive binding equation with GraphPad Prism to derive the IC₅₀ values.

Cell proliferation assays

Real-time cell growth in response to the various treatments was measured as the degree of cell confluence in culture plates and was determined using an IncuCyte ZOOM live-cell imaging system (Essen BioScience, Ann Arbor, MI). In brief, 10,000 cells/well were seeded in 96-well plates 24h before the addition of the compounds. The cell density was assessed every 6h throughout the experiment. Treatment with vehicle (DMSO) alone was used as a negative control. Cell proliferation curves were plotted using confluence values at specified time points for each treatment.

Time-resolved fluorescence resonance energy transfer (TR-FRET) PXR ligand binding assay

PXR TR-FRET binding assays were performed as previously described⁴⁹ with only minor modifications. Briefly, in black 384-well low-volume assay plates, concentrations of chemicals, T0901317, DMSO, or 10μM T0901317 were incubated with 100nM BODIPY FL vindoline, 5nM GST-hPXR-LBD, and 5nM Tb-anti-GST for 30min in 20μL/well assay buffer (50mM Tris, pH 7.5, 20mM MgCl2, 0.1mg/mL BSA, 0.05mM DTT). The TR-FRET signals (10,000×520nm/490nm) were then measured with a PHERAstar FS plate reader (BMG Labtech, Durham, NC) using a 340-nm excitation filter, a 100-μs delay time, and a 200-μs integration time. The final DMSO concentration was 1.1% for all assay wells. Signal values from 10μM T0901317 and DMSO were used as positive (100% inhibition) and negative (0% inhibition) controls, respectively. The activity of each well was normalized to that of the positive and negative controls. The normalized results for each chemical were fitted into a one-site competitive binding equation with GraphPad Prism to derive the IC₅₀ values. In the test, the positive control T0901317 had an IC₅₀ value of 95.9±7.0nM, which is consistent with the value of 101.6nM previously recorded under similar assay conditions⁴⁹.

Plasmid and siRNA transient transfection

For plasmid transfection, HepG2 cells were grown to 80% confluency. The transfection reagent Lipofectamine 2000 (Invitrogen) and 3μg AMPK plasmids were pre-incubated at room temperature for 5min at a ratio of 3:1 in serum-free Opti-MEM medium (Gibco). The mixture was then added to the cells in culture medium. The culture flasks were incubated at 37°C for 24h, then the cells were trypsinized and replated in 96-well plates for further analysis. For siRNA transfection, cells were plated as described above. The transfection reagent RNAi max (Invitrogen) at a ratio of 3:1 with 100nmol/L nontargeting (ctrl siRNA) or targeting siRNA (AMPK and PXR), was pre-incubated at room temperature for 5min in Dharmacon ACCELL siRNA delivery medium (Thermo Scientific). The mixture was then added to the HepG2 cells in culture medium. The culture flasks were incubated at 37°C for 24h, then the cells were trypsinized and replated in 96-well plated for further analysis.

We thank Drs Yueming Wang and Jing Wu for their independent confirmation of our initial observations. We are grateful to Dr. Keith A. Laycock (St. Jude Department of Scientific Editing) for editing the manuscript and to other members of the Chen research laboratory for valuable discussions of the paper. This work was supported by ALSAC, St. Jude Children’s Research Hospital, and the National Institutes of Health [Grants RO1-GM086415, RO1-GM110034, R35-GM118041, and P30-CA21765]. Human primary hepatocytes were obtained through the Liver Tissue and Cell Distribution System (Pittsburgh, PA), which is funded by NIH Contract #N01-DK-7- 0004/HHSN267200700004C.