Sergej Pirkmajer,1 Sameer S. Kulkarni,1 Robby Z. Tom,1 Fiona A. Ross,2 Simon A. Hawley,2

D. Grahame Hardie,2 Juleen R. Zierath,1,3 and Alexander V. Chibalin1

Methotrexate PromotesGlucose Uptake and LipidOxidation in Skeletal Muscle viaAMPK ActivationDiabetes 2015;64:360–369 | DOI: 10.2337/db14-0508

Methotrexate (MTX) is a widely used anticancer andantirheumatic drug that has been postulated to protectagainst metabolic risk factors associated with type 2diabetes, although the mechanism remains unknown.MTX inhibits 5-aminoimidazole-4-carboxamide ribonu-cleotide formyltransferase/inosine monophosphate cyclo-hydrolase (ATIC) and thereby slows the metabolism of5-aminoimidazole-4-carboxamide-1-b-D-ribofuranosyl-59-monophosphate (ZMP) and its precursor AICAR,which is a pharmacological AMPK activator. We ex-plored whether MTX promotes AMPK activation in cul-tured myotubes and isolated skeletal muscle. We foundMTX markedly reduced the threshold for AICAR-inducedAMPK activation and potentiated glucose uptake andlipid oxidation. Gene silencing of the MTX target ATICactivated AMPK and stimulated lipid oxidation in culturedmyotubes. Furthermore, MTX activated AMPK in wild-type HEK-293 cells. These effects were abolished in skel-etal muscle lacking the muscle-specific, ZMP-sensitiveAMPK-g3 subunit and in HEK-293 cells expressinga ZMP-insensitive mutant AMPK-g2 subunit. Collectively,our findings underscore a role for AMPK as a direct mo-lecular link between MTX and energy metabolism in skel-etal muscle. Cotherapy with AICAR and MTX couldrepresent a novel strategy to treat metabolic disordersand overcome current limitations of AICARmonotherapy.

Chronic therapy with methotrexate (MTX), a broadly usedanticancer and antirheumatic drug, may protect rheu-matic patients against metabolic risk factors associated

with cardiovascular disease, obesity (1), and type 2 diabe-tes (2). This notion is supported by recent observationsthat MTX alleviates hyperglycemia and insulin resistancein diabetic (db/db) mice (3) and obese mice fed a high-fatdiet (4). MTX is a folate antagonist and inhibits DNAreplication (5), which explains its anticancer action but thenot improvements in glucose homeostasis in type 2 diabetesor obesity. Suppression of chronic inflammation, thoughtto arise from MTX-stimulated adenosine release (6), mayimprove glucose homeostasis indirectly (7). Because anti-rheumatic drugs are not invariably associated with metabolicprotection (1,8), other mechanisms are likely. AlthoughMTX-induced adenosine release may have direct metaboliceffects (4), its exact role is ambiguous, because adenosinereceptor activation (9) and blockage (10) both improveinsulin sensitivity. Clearly, the molecular underpinningsof MTX action in relation to metabolic disease remainundefined.

MTX has several pharmacological targets, includ-ing 5-aminoimidazole-4-carboxamide ribonucleotideformyltransferase/inosine monophosphate cyclohydro-lase (ATIC) (11). ATIC is essential for the conversion of5-aminoimidazole-4-carboxamide-1-b-D-ribofuranosyl-59-monophosphate (ZMP) to inosine monophosphate inthe final two steps of the de novo purine synthesis path-way. Thus, congenital ATIC deficiency or MTX therapyelevates intracellular ZMP content and excretion of its metab-olites (6,12,13). The purine precursor ZMP is also an AMPmimetic and activates AMPK (14). AMPK is a heterotrimeric

1Department of Molecular Medicine and Surgery, Integrative Physiology, KarolinskaInstitutet, Stockholm, Sweden2Division of Cell Signalling & Immunology, College of Life Sciences, University ofDundee, Dundee, Scotland, U.K.3Department of Physiology and Pharmacology, Integrative Physiology, KarolinskaInstitutet, Stockholm, Sweden

Corresponding author: Alexander V. Chibalin, alexander.chibalin@ki.se.

Received 28 March 2014 and accepted 20 August 2014.

S.P. and S.S.K. contributed equally to this work.

© 2015 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

360 Diabetes Volume 64, February 2015

METABOLISM

serine-threonine kinase composed of the catalytic a- andnoncatalytic b- and g-subunits (15). ZMP binds to theAMP-binding sites on the g-subunits (16), and this is re-quired for its ability to activate AMPK (17). AMPK playsa role in maintaining energy homeostasis and is currentlya target for the treatment of type 2 diabetes (18–20).Thus, MTX might mitigate metabolic impairments by pro-moting ZMP-stimulated AMPK activation in key organscontrolling glucose homeostasis.

AMPK activation increases glucose uptake, fatty acidoxidation, and mitochondrial biogenesis, which helps toameliorate different aspects of metabolic dysregulation,including hyperglycemia and insulin resistance (19). MTXcould promote ZMP accumulation and AMPK activationby suppressing ATIC, which is expressed and active inskeletal muscle (21,22). ZMP is also the active metaboliteof the pharmacological AMPK activator AICAR (14). AICARtherapy promotes favorable metabolic reprogramming inskeletal muscle of sedentary (23) and diabetic (ob/ob)mice (24). However, a relatively high threshold for AMPKactivation (25), in conjunction with poor bioavailability(26), limits the usefulness of AICAR in the treatment oftype 2 diabetes (27). MTX may enhance AICAR-stimulatedAMPK activation in skeletal muscle by suppressing ATIC-mediated ZMP clearance and overcome this problem.

Here, we show that MTX markedly reduces the thresholdfor AICAR-stimulated AMPK activation and potentiatesglucose uptake and lipid oxidation in skeletal muscle. Thus,cotherapy with AICAR and MTX could represent a novelstrategy to treat metabolic disorders and overcome currentlimitations of AICAR monotherapy.

RESEARCH DESIGN AND METHODS

Antibodies and ReagentsAntibodies against phospho-AMPKa (Thr172) and phospho-ACC (Ser79) were from Cell Signaling Technology (Beverly,MA), the antibody against ATIC was from Sigma-Aldrich(Stockholm, Sweden), the GLUT4 antibody was fromMillipore (Temecula, CA), and the antibody against GAPDHwas from Santa Cruz Biotechnology (Santa Cruz, CA). TheECL reagent and the protein molecular weight marker werefrom GE Healthcare (Uppsala, Sweden). The BCA ProteinAssay kit was from Pierce (Rockford, IL), and the polyvinyl-idene fluoride Immobilon-P membrane was from Millipore(Bedford, MA). Cell culture materials were from Costar(Täby, Sweden), and [9-10(n)-3H]-palmitic acid and [1,2-3H]-2-deoxy-D-glucose were from Perkin-Elmer. All other reagents,unless otherwise specified, were of analytical grade andobtained from Sigma-Aldrich.

L6 Cells and Small Interfering RNA TransfectionL6 muscle cells were grown in a-minimum essential me-dium (a-MEM) supplemented with 10% FBS, 1% PeSt(100 units/mL penicillin and 100 mg/mL streptomycin),and 1% Fungizone at 37°C in humidified air and 5% CO2.They were differentiated into myotubes in a-MEM (2%FBS, 1% PeSt, and 1% Fungizone). L6 cells were transfected

with a pool of short interfering (si)RNAs (100 nmol/L)against ATIC mRNA or scrambled siRNA (Dharmacon, Chi-cago, IL) using the CellPhect Transfection Kit on days 2 and4 of differentiation. Experiments were performed 48–72 hafter the last transfection.

Primary Human Skeletal Muscle CellsPrimary human skeletal muscle myoblasts were from LonzaGroup Ltd. (Basel, Switzerland) and maintained in DMEM/F12 supplemented with 20% FBS, 1% PeSt, and 1%Fungizone at 37°C in humidified air and 7% CO2. Myoblastswere differentiated into myotubes for 5 days in DMEM (2%FBS, 1% PeSt, and 1% Fungizone). Serum-starved myotubeswere incubated for 4 h and subsequently with or without5 mmol/L MTX and/or 0.2 mmol/L AICAR for 5 h.

Metabolic Assays in L6 MyotubesL6 myotubes were incubated in a-MEM (without nucleo-sides) with or without 5 mmol/L MTX for 16 h before theexperiment. For fatty acid oxidation, myotubes were serum-starved for 4 h and then incubated with or without MTX(5 mmol/L) and/or AICAR (0.2 or 2 mmol/L) for 5 h ina-MEM, supplemented with 0.2% BSA, 20 mmol/L coldpalmitate, and 0.5 mCi/mL [3H]-palmitic acid. Palmitateoxidation was determined by measuring the amount of3H2O in cell culture media. Nonmetabolized palmitatewas adsorbed to charcoal and removed by centrifugationat 16,000 rpm for 15 min. For glucose uptake, L6 myo-tubes were incubated with or without MTX (5 mmol/L)and/or AICAR (0.2 or 2 mmol/L) in serum-free a-MEM(without nucleosides) for 5 h. Myotubes were thenwashed with HEPES-buffered saline (140 mmol/L NaCl,20 mmol/L HEPES, 5 mmol/L KCl, 2.5 mmol/L MgSO4,1 mmol/L CaCl2, pH 7.4) and subsequently incubated inHEPES-buffered saline supplemented with 10 mmol/L2-deoxy-D-glucose and 0.75–1 mCi/mL [1,2-3H]-2-deoxy-D-glucose for 10 min. Nonspecific glucose transport wasassessed in the presence of 10 mmol/L cytochalasinB. Myotubes were then washed with ice-cold stop solution(25 mmol/L glucose, 0.9% [w/v] NaCl) and lysed in 0.03%(w/v) SDS. Radioactivity was measured in cell lysates byliquid scintillation counting.

Experimental AnimalsThis study was approved by the Regional Animal EthicalCommittee (Stockholm, Sweden). AMPK-g3 knockout(AMPK-g32/2) mice were bred onto a C57BL/6 geneticbackground and have been thoroughly characterized (28).Before all experiments, AMPK-g32/2 and wild-type (WT)mice were fasted for 4 h before being anesthetized withAvertin (0.02 mL/g i.p.).

Metabolic Assays in Isolated Skeletal MuscleExtensor digitorum longus (EDL) and soleus muscle fromWT or AMPK-g32/2 mice were preincubated for 2 h at 30°Cin oxygenated (95% O2 and 5% CO2) Krebs-Henseleit bicar-bonate buffer (KHB) supplemented with 5 mmol/L glucose,15 mmol/L mannitol, 5 mmol/L HEPES, and 0.1% (w/v)BSA with or without 10 mmol/L MTX. Thereafter, muscles

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were incubated for 2 h with or without 10 mmol/L MTXand/or AICAR (0.2 mmol/L or 2 mmol/L). Glucose trans-port was assessed by measuring the uptake of [1,2-3H]-2-deoxy-D-glucose as described (28). Muscles were incubatedfor 20 min in KHB supplemented with 1 mmol/L 2-deoxy-D-glucose, 19 mmol/L mannitol, 2.5 mCi/mL [1,2-3H]-2-deoxy-D-glucose, and 0.7 mCi/mL [14C] mannitol. For fattyacid oxidation, KHB was supplemented with [H3]-palmiticacid (0.4 mCi/mL) and 0.3 mmol/L cold palmitate, andoxidation was determined by analyzing the [3H]-labeledwater content using liquid scintillation counting as de-scribed above.

Lysate Preparation and Immunoblot AnalysisMyotubes were washed with ice-cold PBS and lysed inhomogenization buffer (1% Protease Inhibitor Cocktail,137 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl2,5 mmol/L Na4P2O7, 0.5 mmol/L Na3VO4, 1% Triton X-100,10% glycerol, 20 mmol/L Tris, 10 mmol/L NaF, 1 mmol/LEDTA, and 0.2 mmol/L phenylmethylsulfonyl fluoride,pH 7.8). Lysates were centrifuged for 15 min at 12,000g(4°C). Muscle was homogenized in ice-cold buffer, fol-lowed by end-over-end rotation for 60 min (4°C) andcentrifugation at 12,000g for 10 min (4°C). Supernatantsof cell or muscle lysates were collected, and protein con-tent was measured (BCA Protein Assay). Proteins dis-solved in Laemmli buffer (25 mg per well) were resolvedon 4–12% Bis-Tris gel (Bio-Rad, Richmond, CA), trans-ferred to polyvinylidene fluoride membrane, blockedwith 7.5% nonfat milk, and probed overnight (4°C) withprimary antibodies. All primary antibodies were diluted1:1,000 in primary antibody buffer (Tris-buffered saline[TBS], 0.5% BSA, 0.5% NaN3, pH 7.6). After the overnightincubation, membranes were washed with washing buffer(TBS, 0.02% Tween-20) and incubated with appropriatehorseradish peroxidase–conjugated secondary antibodies(Bio-Rad) for 1 h at room temperature. The secondaryantibodies were diluted 1:25,000 in TBS supplementedwith 5% nonfat milk. Immunoreactive proteins were vi-sualized by enhanced chemiluminescence and quantifiedby the Quantity One 1-D Analysis Software (Bio-Rad).Data were normalized to GAPDH.

Plasmids and Generation of Stable WT and R531GAMPK-g2 HEK-293 Cell LinesFull-length human g2 was amplified with primersdesigned to encode a 59-BamHI site and a COOH-terminalFLAG tag, followed by an XhoI site. The resulting PCRproduct was cloned into the pcDNA5/FRT/TO plasmid(Invitrogen) to create pcDNA5/FRT/TO/g2. Mutagenesisof the construct was performed using the QuikChangeSite-Directed Mutagenesis system (Stratagene). The plas-mid (POG44) expressing Flp recombinase was from Invi-trogen. T-Rex HEK-293 cells containing a single FRT site(Invitrogen) were transfected with Fugene6 (Promega)using the plasmids POG44 and pcDNA5/FRT/TO/g2 ata ratio of 9:1. Fresh medium was added to the cells 24 hafter transfection, and media containing 200 mg/mL

hygromycin B and 15 mg/mL blasticidin was added 48 hafter transfection. The medium was replaced every 3 daysuntil foci could be identified, and individual foci were thenselected and expanded. The expression of FLAG-tagged g2was induced by adding 1 mg/mL tetracycline for 40 h, andexpression was detected using immunofluorescence micros-copy and Western blot using anti-FLAG antibodies.

Culture of WT and R531G AMPK-g2 HEK-293 Cells andCell TreatmentsHEK-293 cells expressing WT or R531G AMPK-g2 were gen-erated as described (17). T-Rex-Flp-In HEK-293 cells (Invi-trogen) containing a single FRT site were cultured in DMEMcontaining 4.5 g/L glucose, 10% (v/v) FBS, 100 IU/mLpenicillin, 100 mg/mL streptomycin, 15 mg/mL blasticidin,and 100 mg/mL zeocin. After transfection, T-Rex-Flp-InHEK-293 cells stably expressing WT or R531G AMPK-g2were cultured as above except that zeocin was replaced by200 mg/mL hygromycin B. Forty hours after the induction ofprotein, 3 mmol/L MTX/DMSO was added to cells for 16 h,followed by 1 h of AICAR in different concentrations.

AMPK Activity AssaysWT HEK-293 cells or mutant HEK-293 (RG) cells wereassayed for AMPK activity as described (17). Activity ofthe purified rat liver AMPK, which is approximately anequal mixture of the a1b1g1 and a2b1g1 heterotrimer(29), was measured in the presence or absence of AMP orprotein phosphatase 2C (PP2C) as described (30).

StatisticsResults are presented as means 6 SEM or means 6 SD.Comparisons among multiple groups were performed us-ing ANOVA, followed by the Bonferroni or Dunnett posthoc test. The Student t test was used when two groupswere compared. Statistical significance was established atP , 0.05.

RESULTS

MTX Promotes AMPK Activation in Cultured MyotubesTo determine whether MTX modulates AMPK activation,L6 myotubes and primary human myotubes were exposedto 5 mmol/L MTX and subthreshold (0.2 mmol/L) and/ormaximally effective (2 mmol/L) concentrations of AICAR(14,25). AMPK activation was determined by measuringAMPK Thr172 and acetyl-CoA carboxylase Ser79 (ACC) phos-phorylation. Exposure of cultured myotubes to 0.2 mmol/LAICAR alone did not elevate AMPK or ACC phosphorylation(Fig. 1A–D), demonstrating that 0.2 mmol/L AICAR is be-low the threshold for AMPK activation. Conversely, coincu-bation with MTX and 0.2 mmol/L AICAR robustly increasedphosphorylation of AMPK (Fig. 1A and B) and ACC (Fig. 1Cand D), demonstrating that MTX markedly reduces thethreshold for AMPK activation.

MTX Promotes Metabolic Effects in Skeletal Musclevia AMPK ActivationTo investigate whether the augmented AICAR-stimulatedACC phosphorylation in MTX-treated myotubes translates

362 Methotrexate Enhances AMPK Activation Diabetes Volume 64, February 2015

into increased lipid utilization, palmitate oxidationwas measured in L6 myotubes incubated with MTX(5 mmol/L) and/or AICAR (0.2 or 2 mmol/L). Although0.2 mmol/L AICAR alone failed to increase palmitateoxidation, treatment with 0.2 mmol/L AICAR and MTXtogether stimulated palmitate oxidation in L6 myotubes(Fig. 2A).

We next examined whether AICAR-stimulated glucoseuptake is augmented in L6 myotubes after exposure to5 mmol/L MTX (Fig. 2B). Although exposure of L6 myo-tubes to 0.2 mmol/L AICAR alone failed to stimulate glu-cose uptake, a combined treatment with 0.2 mmol/LAICAR and MTX markedly elevated glucose uptake.MTX enhanced glucose uptake in myotubes treated witha saturating concentration of AICAR (2 mmol/L), indicat-ing AICAR responsiveness was improved. Glucose trans-port was also determined in EDL and soleus muscletreated with 2 mmol/L AICAR and/or 10 mmol/L MTX(Fig. 2C). MTX robustly augmented AICAR-stimulated glu-cose uptake in EDL muscle, underscoring the effect ofMTX to potentiate AICAR responsiveness. Although so-leus muscle was less responsive to AICAR, glucose uptakewas elevated upon MTX and AICAR exposure. MTX treat-ment did not affect GLUT4 protein abundance or GLUT4mRNA expression (data not shown).

MTX Promotes AMPK Activation Through IndirectMechanismsTo confirm that MTX treatment inhibited ATIC, ZMPcontent was measured in L6 cells exposed to 0.2 mmol/LAICAR and/or 5 mmol/L MTX. AICAR alone did not ele-vate ZMP content, indicative of the high ZMP turnover(Fig. 3A). However, a combined treatment with MTX and0.2 mmol/L AICAR robustly increased the ZMP-to-ATPratio. MTX exposure suppresses de novo purine synthesis(31) and may provoke ATP depletion in some situations(32,33). ATP depletion decreases the adenylate energycharge (defined in Table 1), which stimulates AMPK acti-vation directly (34). Given that the AMP concentration incultured myotubes was too low for a reliable quantitativeestimation of energy charge, we determined the ADP-to-ATP ratio as a marker of cellular energy status (17). MTXexposure, in the absence or presence of AICAR, increasedthe ADP-to-ATP ratio 24–28% (Fig. 3B), indicating a mod-est degree of energy stress. The observed increases in theZMP content and the ADP-to-ATP ratio provide evidence

Figure 1—MTX promotes AMPK activation in cultured myotubes.Phosphorylation (p) of AMPK (pAMPK) at Thr172 (A and B) and its targetpACC at Ser79 (C and D) were determined in L6 myotubes and inmyotubes cultured from primary human skeletal muscle cells (HSMC)upon exposure to 5 mmol/L MTX and/or 0.2 mmol/L or 2 mmol/LAICAR (5 h). Myotubes were preincubated with 5 mmol/L MTX orvehicle for 16 h before the experiment. Results are means 6 SEM(n = 6). #P < 0.05 vs. basal, *P < 0.05 vs. AICAR (0.2 mmol/L).

Figure 2—MTX promotes metabolic effects in skeletal muscle viaAMPK activation. Palmitate oxidation (n = 7–8; except basal andMTX, where n = 15) (A) and glucose uptake (n = 24) (B) in L6 myo-tubes upon exposure to 5 mmol/L MTX and/or 0.2 mmol/L or2 mmol/L AICAR (5 h). L6 myotubes were preincubated with5 mmol/L MTX or vehicle for 16 h before the palmitate oxidationor glucose uptake assay. C: Glucose uptake in isolated EDL andsoleus muscle after exposure to 10 mmol/L MTX, 120 nmol/L in-sulin, and/or 2 mmol/L AICAR (n = 6–8). EDL and soleus musclewere preincubated with 10 mmol/L MTX or vehicle for 2 h before theglucose uptake assay. Results are means 6 SEM. #P < 0.05 vs.basal, *P < 0.05 vs. AICAR (0.2 mmol/L) or as indicated.

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that MTX promotes AMPK activation in cultured myo-tubes primarily by decreasing ZMP clearance, althoughATP depletion may play a secondary role.

To establish a causal link between MTX-induced alter-ations in cellular nucleotide content and AMPK activation,WT HEK-293 and RG cell lines were exposed to MTX(3 mmol/L) and different concentrations of AICAR (Fig.3C–F). RG cells harbor a single substitution (R531G) withinthe AMPK-g2 subunit that renders AMPK insensitive tostimulation by AMP and ZMP (17). ZMP content and theADP-to-ATP ratio were both increased in WT cells treatedwith MTX and AICAR (Fig. 3C and D). The elevated ZMPcontent during MTX and AICAR treatment was paralleledby an increase in AMPK activity (Fig. 3E). Notably, MTX

alone was sufficient to activate AMPK in WT cells. How-ever, the increase in AMPK activity was almost entirelyabrogated in RG cells during coincubation with MTX andAICAR (Fig. 3F), and MTX alone did not stimulate AMPKactivation in RG cells. Because AMPK complexes in RG cellsremain responsive to direct AMPK activators other thanAMP and ZMP, such as A-769662 (17), this suggests thatMTX is not a direct AMPK activator.

MTX Is Not a Direct AMPK ActivatorTo evaluate whether MTX activates AMPK allosterically,the activity of AMPK, purified from rat liver, was mea-sured during incubation with AMP and/or increasingconcentrations of MTX (Fig. 4A). AMPK activity was in-creased in the presence of 200 mmol/L AMP, but MTXalone or combined with AMP did not alter AMPK activity.We also determined whether MTX protects AMPK fromdephosphorylation by PP2C, which dephosphorylates andthereby inactivates AMPK (35). Purified AMPK was incu-bated with PP2C and 200 mmol/L AMP or different con-centrations of MTX (Fig. 4B). AMP almost completelyprevented the inactivation of AMPK by PP2C, but MTXdid not, showing that MTX does not inhibit PP2C-mediateddephosphorylation of AMPK.

MTX Depends on AMPK to Promote Lipid OxidationOur results have established that MTX enhances AICARaction in skeletal muscle. To determine whether MTXdepends on AMPK to promote metabolic alterations inskeletal muscle, we studied EDL and soleus muscles fromWT and AMPK-g32/2 mice. Isolated EDL and soleusmuscles were incubated in the absence or presence of10 mmol/L MTX and/or a subthreshold concentration ofAICAR (0.2 mmol/L). Treatment with 0.2 mmol/L AICARand MTX was sufficient to markedly stimulate AMPK ac-tivation and palmitate oxidation in WT EDL and soleusmuscles (Fig. 5). However, coincubation with MTX and0.2 mmol/L AICAR did not stimulate phosphorylation ofAMPK (Fig. 5A) and ACC (Fig. 5C) in AMPK-g32/2 EDLmuscle. MTX also failed to enhance AICAR-stimulated

Figure 3—MTX promotes AMPK activation through indirect mecha-nisms. ZMP content (A) and the ADP-to-ATP ratio (B) in L6 myotubesexposed to 5 mmol/L MTX and/or 0.2 mmol/L AICAR (5 h). L6 myo-tubes were preincubated with 5 mmol/L MTX or vehicle for 16 h beforethe experiment. Results are means 6 SEM (n = 3–5). ZMP content(C) and the ADP-to-ATP ratio (D) in WT cells exposed to 3 mmol/LMTX and/or different concentrations of AICAR. Results are means 6SD (n = 2). Activity of AMPK in WT cells (E) and RG cells (F) exposedto 3 mmol/L MTX and/or different concentrations of AICAR (1 h). WTand RG cells were preincubated with 3 mmol/L MTX (●) or vehicle (○)for 16 h before the AMPK activity assay. Results are means 6 SEM(n = 2–4). AMPK activity is expressed as a percentage of the activity incontrols without added treatments. Results are means 6 SEM (n =11–12). *P < 0.05 vs. without MTX or as indicated.

Table 1—Effect of MTX on ZMP content and adenylateenergy charge in AICAR-treated murine skeletal muscle

Skeletalmuscle Treatment

ZMP-to-ATPratio

Adenylateenergycharge

EDLAICAR 0.041 6 0.002 0.93 6 0.005

MTX + AICAR 0.039 6 0.002 0.92 6 0.01

SoleusAICAR 0.12 6 0.013 0.80 6 0.04

MTX + AICAR 0.12 6 0.016 0.80 6 0.05

ZMP content, expressed as the ZMP-to-ATP ratio, and adenylateenergy charge, calculated as ([ATP] + 0.5[ADP])/([ATP] + [ADP] +[AMP]), were determined in WT EDL and soleus muscle afterexposure to 0.2 mmol/L AICAR and 10 mmol/L MTX (2 h). EDLand soleus muscle were preincubated with 10 mmol/L MTX orvehicle for 2 h before the experiment. ZMP was below detectionlimit under basal conditions (data not shown). Results aremeans 6 SEM (n = 3–4).

364 Methotrexate Enhances AMPK Activation Diabetes Volume 64, February 2015

palmitate oxidation in AMPK-g32/2 EDL muscle (Fig. 5E),supporting the notion that MTX promotes these metabolicalterations through AMPK. Owing to low expression ofAMPK-g3 in WT soleus muscle (36), the response to a com-bined treatment with MTX and 0.2 mmol/L AICAR wassimilar between soleus muscles from WT and AMPK-g32/2

mice (Fig. 5B, D, and F).Nucleotide content was measured to establish whether

MTX promotes AMPK activation by decreasing ZMP clear-ance. ZMP content was markedly elevated upon addition ofAICAR in WT EDL and soleus muscle (Table 1). AlthoughMTX markedly augmented AICAR-stimulated AMPK acti-vation and palmitate utilization, it did not enhance ZMPaccumulation in EDL and soleus muscle. The adenylate en-ergy charge also remained unaltered upon MTX treatment(Table 1). These results suggest that diminished ZMP clear-ance and/or energy depletion are not the exclusive mecha-nisms underlying an MTX-induced reduction in the thresholdfor AICAR-stimulated AMPK activation in skeletal muscle.

Suppression of ATIC Directly Promotes AMPKActivation and Increases Lipid OxidationL6 myotubes were used to validate ATIC as an MTX target(Fig. 6A). Expression of ATIC was suppressed in L6

myotubes using siRNA (Fig. 6B). Exposure of myotubestransfected with scrambled siRNA to 0.2 mmol/L AICARdid not increase ZMP content (Fig. 6C). Conversely, stim-ulation with 0.2 mmol/L AICAR robustly elevated ZMPcontent in ATIC-depleted myotubes (Fig. 6C), indicatingdiminished ZMP clearance. The ADP-to-ATP ratio wasalso elevated 12–15% in ATIC-depleted cells (Fig. 6D),possibly reflecting purine depletion due to decreased denovo synthesis. The increased ZMP content in ATIC-depleted myotubes treated with AICAR increased AMPKand ACC phosphorylation and palmitate oxidation (Fig.6E–G), consistent with the hypothesis that decreased ZMPclearance promotes AMPK activation. Notably, ATIC deple-tion alone increased AMPK phosphorylation and palmitateoxidation (Fig. 6E and G), underscoring the link betweenATIC suppression and AMPK activation in skeletal muscle.

Gene silencing suppressed ATIC expression 70% (Fig.6B). To test whether inhibition of residual ATIC activityprovides an additional stimulus for AMPK activation, L6myotubes were transfected with siRNA, subsequentlyexposed to MTX, and incubated in the presence of0.2 mmol/L AICAR. In ATIC-depleted myotubes, MTX ro-bustly stimulated AMPK and ACC phosphorylation as wellas palmitate oxidation (Fig. 6H–J). ATIC depletion in con-junction with MTX exposure achieved maximal AMPKactivation, because incubation with 0.2 mmol/L AICARfailed to exert an additional effect. However, a combinedtreatment with 0.2 mmol/L AICAR and MTX triggereda similar response in control myotubes transfected withscrambled siRNA.

DISCUSSION

Epidemiological (1,2) and experimental (3,4) evidencesuggests that MTX may protect against type 2 diabetes,but the underlying mechanisms have been unclear. Weprovide evidence that MTX potentiates AICAR-stimulatedAMPK activation and glucose uptake as well as lipid oxi-dation in skeletal muscle. We also demonstrate that sup-pression of ATIC, a well-characterized MTX target (11),directly promotes AMPK activation in cultured myotubes.Finally, MTX alone is sufficient to stimulate AMPK acti-vation in HEK-293 cells. These results highlight a role forAMPK as a link between MTX and skeletal muscle energymetabolism.

Pharmacological activation of AMPK in skeletal musclerepresents a promising strategy to bypass insulin-resistant pathways to stimulate glucose uptake andimprove insulin sensitivity in type 2 diabetes (19). How-ever, the search for peripheral AMPK activators has beenchallenging. AMPK activators, such as A-769662, targetAMPK-b1 complexes that are poorly expressed in skeletalmuscle (37,38), whereas AICAR has clinical limitations dueto its poor bioavailability (18,26). Even with intravenousinfusion, the plasma concentration of AICAR (0.16–0.18mmol/L) remains below the threshold for AMPK activa-tion in skeletal muscle (27,39). Here we report coincuba-tion with 0.2 mmol/L AICAR and MTX activates AMPK

Figure 4—MTX is not a direct AMPK activator. A: Activity of purifiedrat liver AMPK was measured after incubation with increasing con-centrations of MTX in the absence (□) or presence (■) of 200mmol/L AMP. B: Activity of purified rat liver AMPK before (□) andafter incubation with PP2C for 10 min (■) in the presence or ab-sence of MTX (1–30 mmol/L) or 200 mmol/L AMP. AMPK activitiesare expressed as percentages of activity in the controls withoutadded treatments. Results are means 6 SD (n = 2).

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and stimulates glucose uptake and lipid oxidation in skel-etal muscle. These results are consistent with effects ofMTX on AICAR in cancer cells (32). Thus, combined ther-apy with MTX and AICAR might provide a novel pharma-cological strategy to overcome current limitations ofAICAR monotherapy.

AMPK-g3 is the muscle-specific isoform of the AMP- andZMP-sensitive AMPK-g subunit, which exists in three iso-forms (g1–g3) (40), and is a major isoform involved inexercise-induced AMPK activation (41). Although AMPK-g3 is expressed primarily in glycolytic muscles (28,36),a complete failure of MTX to enhance AICAR action inAMPK-g32/2 EDL muscle was unexpected because theAMPK-g1 and -g2 isoforms are also expressed (36). Fur-thermore, MTX enhanced AICAR-stimulated AMPKphosphorylation in WT and AMPK-g32/2 soleus muscle,despite low endogenous expression in WT soleus muscle(28,36). Thus, MTX preferentially promotes activation of

AMPK-g3–containing complexes in glycolytic skeletal mus-cle. The g3 subunit offers an entry point to tissue-specificregulation of AMPK function and a possible therapeutictarget for treatment of insulin resistance in type 2 diabetes(28). Activating mutations in the g3 isoform are associatedwith skeletal muscle remodeling analogous to exercise train-ing, including augmented mitochondrial biogenesis and fatoxidation as well as protection against insulin resistance(28,42). Cotherapy with MTX and AICAR may thereforepromote a plethora of beneficial metabolic alterations, in-cluding mimicking the effects of exercise training (23).

MTX promotes AMPK activation in cultured myotubesand WT cells primarily via inhibition of ATIC, whereasATP depletion may also have a secondary role. However,MTX did not inhibit ATIC or evoke energy depletion inmurine EDL and soleus muscles, although AICAR actionwas markedly enhanced. This failure to suppress ATICactivity may be explained by the fact that MTX is an

Figure 5—MTX is dependent on AMPK to promote lipid oxidation in isolated skeletal muscle. Phosphorylation (p) of AMPK (pAMPK) (A andB) and its target pACC (C and D) and palmitate oxidation (E and F ) were measured in WT or AMPK-g32/2 EDL and soleus muscle afterexposure to 10 mmol/L MTX and/or 0.2 mmol/L AICAR (2 h). EDL and soleus muscle were preincubated with 10 mmol/L MTX or vehicle for2 h before the experiment. Results are means 6 SEM (n = 12). *P < 0.05 vs. AICAR.

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efficient ATIC inhibitor only after MTX polyglutamatesare formed by the action of folylpolyglutamate synthe-tase, which is a slow process (11). Thus, the exposure timeduring the isolated skeletal muscle incubation may havebeen insufficient for MTX to depress ATIC activity. Al-though our data do not exclude a role for ATIC inhibitionduring chronic MTX therapy, they nevertheless suggestthat MTX promotes AMPK activation through additionalATIC-independent mechanisms. This ATIC-independentpathway appears to operate independently of any activa-tion of upstream kinases, such as LKB1, which phosphor-ylate AMPK at Thr172 (15). Indeed, MTX alone failed to

activate AMPK in RG cells, although they are normallyresponsive to AMPK activators other than ZMP andAMP (17). However, MTX decreases the concentrationof S-adenosylmethionine (43), a biological methyl groupdonor that promotes AMPK inactivation (44), which mayprovide one possible ATIC-independent pathway.

MTX has several targets aside from ATIC (11) that mayalso modulate AMPK activation. In particular, the activityof glycinamide ribonucleotide transformylase (GART),an enzyme upstream of ATIC in the de novo pathwayof purine synthesis (Fig. 7), may critically determine thelevel of endogenous ZMP. Concurrent inhibition of ATICand GART by MTX suppresses the entire de novo synthe-sis pathway and decreases endogenous ZMP formation(31). This provides a possible explanation why MTX alonedid not increase the content of endogenous ZMPin cultured myotubes, although the clearance of ZMPderived from AICAR was decreased. Similarly, MTXincreases ZMP content and enhances AMPK activationin AICAR-treated cancer cells, but not when used alone(32). Conversely, ATIC depletion elevated basal AMPKphosphorylation and palmitate oxidation in cultured myo-tubes, suggesting selective inhibition of ATIC in skeletalmuscle may introduce a metabolic block leading to ZMPaccumulation and AMPK activation.

MTX has a greater propensity to inhibit ATIC thanGART (11,31). Patients receiving low-dose MTX exhibitincreased urinary excretion of 5-aminoimidazole-4-carboxamide (13), a ZMP degradation product, whichis indicative of ZMP accumulation due to ATIC inhibi-tion. Conversely, high-dose MTX fails to increase uri-nary excretion of endogenous AICAR (45). Scaling downthe MTX dosage may create a therapeutic window formore selective ATIC inhibition with subsequent ZMPaccumulation and AMPK activation in skeletal muscleand other metabolic tissues. Consistent with this hy-pothesis, low-dose MTX therapy increased intracellularZMP content in mice (6). Furthermore, low-dose MTXmonotherapy increases skeletal muscle GLUT4 abun-dance in diabetic (db/db) mice (3) and suppresses adi-pose tissue lipolysis in obese mice (4), consistent withAMPK activation in skeletal muscle (24,46) and adiposetissue (14), respectively. Finally, we report MTX alone issufficient to induce AMPK activation in HEK-293 cells,thus providing proof-of-concept that MTX acts as AMPKactivator rather than just as an enhancer of effects ofAICAR.

We show that MTX enhances AICAR-stimulated AMPKactivation in skeletal muscle as well as in cells of kidneyorigin (HEK-293 cells). Taken together with studies indifferent types of cancer cells (31,32), these data suggestthat MTX potentiates effects of AICAR not only in skele-tal muscle but also in other organs. For instance, ATICis expressed and active in the heart (21,47), indicatingMTX could enhance AICAR action in cardiomyocytes.Also, liver expresses ATIC (47), and MTX-treated hepaticcarcinoma cells release adenosine (48), which results from

Figure 6—Suppression of ATIC promotes AMPK activation andincreases lipid oxidation in L6 myotubes by a direct mechanism.A: ATIC expression was determined by immunoblot in L6 myo-tubes, differentiated primary human skeletal muscle cells (HSMC),HEK-293 cells, and in murine EDL and soleus muscle. B: ATIC pro-tein expression in L6 myotubes after transfection with scrambledsiRNA (Scr) or siRNA against ATIC (siATIC). ZMP content (C ), theADP-to-ATP ratio (D), AMPK phosphorylation (pAMPK) (E ), ACCphosphorylation (pACC) (F), and palmitate oxidation (G) measuredin control or ATIC-depleted L6 myotubes upon exposure to0.2 mmol/L AICAR (5 h). Results for nucleotide measurements aremeans for basal (n = 2) and means6 SEM (n = 4) for AICAR. Resultsfor all others are means 6 SEM (n = 8). #P < 0.05 vs. basal (Scr),*P < 0.05 vs. AICAR (Scr). ATIC-depleted or control L6 myotubeswere preincubated with 5 mmol/L MTX for 16 h. AMPK phosphor-ylation (H), ACC phosphorylation (I), and palmitate oxidation (J) weredetermined upon exposure to 0.2 mmol/L AICAR (5 h). Results aremeans 6 SEM (n = 6). *P < 0.05 vs. MTX (Scr).

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MTX-induced ATIC inhibition (6). Consistent with theinvolvement of ATIC, we determined that MTX enhancesZMP accumulation and AMPK activation in AICAR-treated H4IIE cells of liver origin (data not shown). Asidefrom ATIC expression, differences in MTX uptake mightalso determine tissue responsiveness to cotherapy withAICAR and MTX. Indeed, cells lacking MTX transportersare resistant to MTX despite ATIC expression (32,49).Collectively, these studies indicate that MTX is likely toenhance AICAR action in all tissues that express ATIC andMTX uptake transporters.

In conclusion, MTX markedly reduces the threshold forAICAR-induced AMPK activation and increases glucoseuptake and lipid oxidation in skeletal muscle. The un-derlying mechanisms include reduced ZMP clearance dueto ATIC inhibition and ATP depletion as well as ATIC-independent effects. Collectively, our results highlighta role for AMPK as a direct link between MTX and energymetabolism. Cotherapy with AICAR and MTX or anotherMTX-based agent represents a novel strategy to treatmetabolic disorders and overcome current limitations ofAICAR monotherapy.

Acknowledgments. The authors thank Dr. Megan E. Osler (KarolinskaInstitutet) for critical reading of the manuscript and Dr. Amira Klip (The Hospitalfor Sick Children, Toronto, Ontario, Canada) for generously providing the L6muscle cells.Funding. This study was supported with funding from the European ResearchCouncil Ideas Program (ICEBERG, ERC-2008-AdG23285), the Swedish ResearchCouncil, the Swedish Diabetes Association, the Strategic Research Foundation,the Novo Nordisk Research Foundation, the Commission of the European Com-munities (Contract No. LSHM-CT-2004-005272 EXGENESIS), and the Strategic

Research Programme in Diabetes at Karolinska Institutet. Studies in the Hardielaboratory are supported by a Senior Investigator Award (097726) from theWellcome Trust and by a Programme Grant (C37030/A15101) from CancerResearch UK. S.P. was supported by the Slovenian Research Agency (grantP3-0043) and the Erasmus Lifelong Learning Programme.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. S.P., D.G.H., J.R.Z., and A.V.C. designed thestudy. S.P., S.S.K., R.Z.T., F.A.R., and S.A.H. performed experiments. S.P., J.R.Z.,and A.V.C. wrote the manuscript. D.G.H., J.R.Z., and A.V.C. revised the finalmanuscript. S.P., S.S.K., R.Z.T., F.A.R., D.G.H., J.R.Z., and A.V.C. approved thefinal manuscript. A.V.C. is the guarantor of this work and, as such, had fullaccess to all the data in the study and takes responsibility for the integrity ofthe data and the accuracy of the data analysis.

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Figure 7—MTX promotes AMPK activation in skeletal musclethrough different mechanisms. Clearance of AICAR through thede novo purine synthesis pathway limits effectiveness of AICARas an AMPK activator in skeletal muscle. Cotherapy with AICARand MTX may overcome these limitations by reducing the thresholdfor AMPK activation in skeletal muscle. Underlying mechanismsinclude diminished ZMP clearance and energy deprivation. How-ever, other ATIC-independent mechanisms are likely involved.IMP, inosine monophosphate; PRPP, 5-phosphoribosyl-1-pyrophosphate.

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