Activation of AMP-activated Protein Kinase Stimulates Na⁺,K⁺-ATPase Activity in Skeletal Muscle Cells

Background: Contractions activate the sodium pump, Na⁺,K⁺-ATPase, and the energy sensor, AMP-activated protein kinase (AMPK), in skeletal muscle.

Results: AMPK activation increases Na⁺,K⁺-ATPase activity in skeletal muscle cells.

Conclusion: Activation of Na⁺,K⁺-ATPase in skeletal muscle cells is AMPK-dependent.

Significance: AMPK activation of the sodium pump may be crucial during exercise to maintain ion homeostasis and delay muscle fatigue.

Abstract

Contraction stimulates Na⁺,K⁺-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na⁺,K⁺-ATPase activity in skeletal muscle remains to be determined. Short term stimulation of rat L6 myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), activates AMPK and promotes translocation of the Na⁺,K⁺-ATPase α₁-subunit to the plasma membrane and increases Na⁺,K⁺-ATPase activity as assessed by ouabain-sensitive ⁸⁶Rb⁺ uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increase AMPK phosphorylation and Na⁺,K⁺-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na⁺,K⁺-ATPase activity. The effect of AICAR on Na⁺,K⁺-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na⁺,K⁺-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na⁺,K⁺-ATPase α₁-subunit dephosphorylation at Ser¹⁸, which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na⁺,K⁺-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A·PME-1 complex in AMPK-mediated regulation of Na⁺,K⁺-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.

Introduction

Na⁺,K⁺-ATPase is a transmembrane heterodimer ion pump that exchanges intracellular Na⁺ for extracellular K⁺ (1). The minimal functional pump consists of a catalytic α-subunit and a β-subunit, which controls the assembly of the heterodimer and the insertion into the membrane. Several isoforms of these subunits exist, with four α- and three β-subunits identified (2). Skeletal muscle expresses α₁, α₂, β₁, β₂, and β₃ isoforms (3–5). Na⁺,K⁺-ATPase is critically involved in the maintenance of the intracellular Na⁺ and K⁺ concentrations and participates in the maintenance of cell volume and electrochemical gradients. Na⁺,K⁺-ATPase also promotes membrane repolarization and re-uptake of extracellular K⁺ in excitable cells, including skeletal muscle (6, 7). During exercise, K⁺ and Na⁺ are displaced across the skeletal muscle sarcolemma, which is counteracted by an increase in intrinsic activity of Na⁺,K⁺-ATPase and its abundance at the plasma membrane (7, 8). Contraction-induced up-regulation of Na⁺,K⁺-ATPase activity in skeletal muscle is therefore important for the prevention of fatigue during exercise (6, 7).

The AMP-activated protein kinase (AMPK)³ is a major metabolic sensor, which maintains cellular energy homeostasis under energy-deprived conditions (9, 10). AMPK, activated by an increase in the intracellular AMP/ATP ratio, facilitates a switch in cellular metabolism from an anabolic state, where nutrients are taken up and stored, to a catabolic state, where they are oxidized, which promotes resynthesis (11, 12).

A number of pharmacological agents activate AMPK. The most commonly used AMPK activator is the adenosine analog 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). AICAR is metabolized to the monophosphorylated nucleotide, ZMP by adenosine kinase, and mimics the effect of AMP on AMPK activation (13). The A-769662 compound is another AMPK activator (14). Using L6 muscle cells, we have previously shown that A-769662 promotes AMPK phosphorylation, whereas simultaneously directly inhibiting the activity of the purified Na⁺,K⁺-ATPase in a manner analogous to ouabain treatment (15).

AMPK acts as a metabolic sensor and regulates ion transport by inhibiting several transport proteins including the epithelial Na⁺ channel (16–18), the cystic fibrosis transmembrane conductance regulator Cl⁻ channel (19, 20), and the renal-specific Na⁺,K⁺-2Cl⁻ co-transporter 2 (21). AMPK inhibits Na⁺,K⁺-ATPase in lung epithelial cells (H441) (22, 23). Moreover, AMPK activation decreases pump activity and stimulates endocytosis in primary alveolar epithelial cells (24). Collectively, these studies provide evidence to suggest that most of the energy consuming processes in the cell, including Na⁺,K⁺-ATPase activity (25), are switched-off in response to AMPK activation.

Contrary to the common view that AMPK activation inhibits all energy consuming processes, in MDCK cultured renal epithelial cells, Na⁺,K⁺-ATPase is activated via an AMPK-dependent mechanism (26). Moreover, in lung epithelium of mice exposed to ozone, AMPK phosphorylation is increased, concomitant with appearance of the sodium pump at the cell surface (27). Additionally AICAR infusion in rodents increases AMPK phosphorylation, concomitant with an acute decrease in plasma [K⁺], without increasing urinary excretion of [K⁺] (28). The effect of AICAR on AMPK activation and plasma [K⁺] is blunted in mice overexpressing a kinase-dead form of AMPK (28). Thus, AICAR may increase either Na⁺,K⁺-ATPase or potassium channel activity in skeletal muscle. In parallel with Na⁺,K⁺-ATPase activation, exercise increases AMPK phosphorylation and activity. However, the direct effect of AMPK on Na⁺,K⁺-ATPase activity in skeletal muscle remains to be determined. Thus, the aim of the present study was to determine the effect of AMPK activation on Na⁺,K⁺-ATPase activity in skeletal muscle cells.

EXPERIMENTAL PROCEDURES

Antibodies and Reagents

Specific anti-Na⁺,K⁺-ATPase α₁-subunit monoclonal antibodies were obtained from Dr. M. Caplan (Yale University, New Haven, CT). The McK1 antibody was a generous gift by Dr. K. Sweadner (Massachusetts Central Hospital, MA). Immunoprecipitation of total Na⁺,K⁺-ATPase α-subunit was performed using the polyclonal antibody, anti-NK1, raised against purified rat kidney holoenzyme (29). The antibodies against phospho-Akt Ser(P)⁴⁷³, phospho-ERK1/2 Thr(P)²⁰²/Tyr(P)²⁰⁴, phospho-AMPK Thr(P)¹⁷², phospho-PKCζ/λ Thr(P)^410/403, phospho(Ser/Thr)-Akt substrate (PAS) and phospho-ACC Ser(P)⁷⁹ were from Cell Signaling (Beverly, MA). The antibodies against methyl-PP2Aα Leu³⁰⁹ and phospho-PP2Aα Tyr(P)³⁰⁷ were from Abcam (Cambridge, UK). Antibody against PME-1 was from Millipore (Billerica, MA). Cyanide, kinase inhibitors Compound C, GF109203X, and the PKC activator, phorbol 12-myristate 13-acetate (PMA), were from Calbiochem (La Jolla, CA). Streptavidin-agarose beads and EZ-link Sulfo-NHS-SS-biotin were from Pierce Chemical Co. (Rockford, IL). Cell culture media and reagents were from Invitrogen. Human insulin (Actrapid) was from Novo Nordisk AS (Copenhagen, Denmark). Dimethyl sulfoxide (Calbiochem) was used as a solvent for the protein kinase and phosphatase inhibitors. A-769662 was a generous gift of Dr. Kei Sakomoto (University of Dundee, Dundee, UK). All other reagents were of analytical grade (Sigma).

L6 Muscle Cells

Rat L6 muscle cells (provided by Dr. Amira Klip, The Hospital for Sick Children, Toronto, Canada) were grown in αMEM supplemented with 10% FBS, 1% penicillin-streptomycin (100 units/ml of penicillin and 100 μg/ml of streptomycin), and 1% Fungizone (Amphotericin B) in 5% CO₂, 95% O₂ humidified air at 37 °C. L6 cells were differentiated into myotubes for 6 days in αMEM supplemented with 2% FBS. Myotubes were serum-starved 18 h prior to the experiment. Differentiated L6 myotubes express only the α₁-subunit isoform of Na⁺,K⁺-ATPase (30).

AMPKα Knock-out Cells

Primary muscle cell cultures were derived from gastrocnemius and tibialis anterior muscles of 4-week-old wild-type and AMPKα1^−/−; AMPKα2^fl/fl mice as previously described (31). Targeted disruption of the AMPKα2 loci was performed by infection of myoblasts with an adenovirus expressing the Cre recombinase (32). Cells were grown with DMEM/F-12 containing 1% penicillin-streptomycin, 0.2% Fungizone, 1% glutamine (Invitrogen), 10% FBS, and 2% Ultroser G (Pall Life Sciences) in 5% CO₂, 95% O₂ humidified air at 37 °C. The cells were grown on plates coated with 0.02% gelatin. Cells were differentiated into myotubes for 4 days in DMEM/F-12, 1% penicillin-streptomycin, 0.2% Fungizone (Amphotericin B), 1% glutamine, and 2% horse serum (Invitrogen) on plates coated with Matrigel (BD Biosciences). On the day of the experiment, cells were serum starved for 4–5 h.

siRNA Transfection of Myotubes

The siRNA sequences used to silence rat AMPKα1, AMPKα2, and PME-1 were: AMPKα1, GCA UAU GCU GCA GGU AGA UdTdT; AMPKα2, CGU CAU UGA UGA UGA GGC UdTdT (Qiagen, Hilden, Germany) (33); and PME-1, CAA GGA UAC UUU UCG AGU UdTdT (Ambion, Carlsbad, CA). A scrambled siRNA was used as a control in all experiments. L6 cells (seeded and grown as described above) were transfected with 100 nm siRNA using calcium phosphate (CellPhect Transfection kit; GE Healthcare) on days 2 and 4 of differentiation. siRNA-calcium phosphate precipitates were removed 16–18 h after addition of siRNA. Experiments were performed 48 h after the last transfection.

Cell Incubation

For the protein phosphorylation analysis, myotubes, differentiated for 6–7 days, were incubated for 40 min with 0.1% dimethyl sulfoxide, AICAR (1 mm), or cyanide (100 μm). L6 cells were incubated for 1 h with 100 μm A-769662. Thereafter the medium was removed and the cells were incubated with starvation medium (serum-free αMEM) for an additional 1, 2, 3, or 4 h. For treatment with Compound C, cells were preincubated in serum-free αMEM with 0.1% dimethyl sulfoxide or 40 μm AMPK inhibitor Compound C for 10 min at 37 °C. Cells were then stimulated with AICAR (1 mm for 40 min) in the presence of Compound C for 40 min. Cells were washed twice with ice-cold PBS, and harvested by scraping cells into ice-cold lysis buffer A (20 mm Tris, pH 8.0, 135 mm NaCl, 1 mm MgCl₂, 2.7 mm KCl, 10 mm Na₄P₂O₇, 0.5 mm Na₃VO₄, 10 mm NaF, 1 μm okadaic acid, 1% Triton X-100, 10% (v/v) glycerol, 0.2 mm PMSF, 10 μg/ml of leupeptin, and 10 μg/ml of aprotinin). Cells were lysed by repeated pipetting, and lysates were agitated for 60 min at 4 °C and subjected to centrifugation (12,000 × g for 10 min at 4 °C). Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce Chemical Co.). Lysates were stored at −80 °C before subsequent Western blot analysis.

Cell Surface Biotinylation

Cells were washed 3 times with ice-cold PBS, and thereafter exposed to EZ-link Sulfo-NHS-SS-biotin (Pierce Chemical Co.) at a final concentration of 1.5 mg/ml in PBS at 4 °C for 60 min with gentle shaking. Cell surface biotinylation was performed as described (34). Cells were then harvested and lysed in ice-cold buffer A as described above, and cell lysates were subjected to streptavidin precipitation. Samples were analyzed by SDS-PAGE with subsequent Western blot.

Measurement of Ouabain-sensitive ⁸⁶Rb⁺ Uptake

Na⁺,K⁺-ATPase transport activity was measured as ouabain-sensitive uptake of ⁸⁶Rb⁺, under conditions of the initial rate, as previously described (34). Myotubes were incubated in the presence or absence of ouabain (2 mm) and AMPK activators and/or inhibitor for 15 min, as indicated. Na⁺,K⁺-ATPase transport activity was determined after the addition of 50 μl of medium containing tracer amounts of ⁸⁶RbCl (100 nCi/sample; GE Healthcare) for 10 min. Reactions were stopped by cooling on ice and cells were washed three times with an ice-cold washing solution containing 150 mm choline chloride, 1.2 mm MgSO₄, 1.2 mm CaCl₂, 2 mm BaCl₂, and 5 mm HEPES, pH 7.4. Cells were lysed in 500 μl of 0.4 m NaOH and the radioactivity was measured by liquid scintillation. Protein content was determined in parallel using the BCA protein assay. Ouabain-sensitive ⁸⁶Rb⁺ uptake was calculated as the difference between the mean values, measured in triplicate samples, incubated without or with 2 mm ouabain. Basal ouabain-sensitive ⁸⁶Rb⁺ uptake in L6 myotubes was 7.8 ± 0.2 pmol of Rb/μg of protein per minute.

Metabolic Labeling of Myotubes with ³²P_i

³²P_i metabolic labeling was performed (34) to investigate in vitro phosphorylation of α-subunits of Na⁺,K⁺-ATPase. Myotubes (day 6 of differentiation) grown on 60-mm dishes were incubated for 3 h at 37 °C in serum-free αMEM containing ³²P_i (1 mCi/ml). AICAR (1 mm) or PKC activator PMA (500 nm) were added during the last 40 min of the incubation time. The incubation was terminated by cooling the culture dishes on ice and washing the cells with ice-cold PBS. Myotubes were lysed in 0.5 ml of ice-cold lysis buffer A. Insoluble material was removed by centrifugation (12,000 × g for 10 min at 4 °C). Aliquots of supernatant (300 μg of protein) were immunoprecipitated overnight at 4 °C with 50 μl of polyclonal anti-NK1 rabbit antibodies. Immunoprecipitates were collected on protein G-agarose beads (Invitrogen; Dynal). Beads were washed four times in lysis buffer A and twice in PBS. Pellets were resuspended in Laemmli sample buffer (60 μl) (62.5 mm Tris-HCl, 2% SDS, 10% glycerol, and 10 mm DTT), separated by SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Phosphoproteins were analyzed using Bio-Imaging Analyzer BAS-1800II (Fuji Photo Film Co., Ltd., Japan), and quantification was performed using the Image Gauge software, version 3.4 (Fuji Photo Film Co., Ltd., Japan). In each experiment, the amount of radioactivity incorporated into the α-subunit was corrected for the α-subunit protein content detected by Western blot analysis. The quantitative data are reported as arbitrary units.

Western Blot Analysis

Aliquots of cell lysate (30 μg of protein) or immunoprecipitates were resuspended in Laemmli sample buffer. Proteins were then separated by SDS-PAGE, transferred to PVDF membranes, blocked with 7.5% nonfat milk, washed with TBST (10 mm Tris-HCl, 100 mm NaCl, 0.02% Tween 20), and incubated with the appropriate primary antibodies overnight at 4 °C. Membranes were washed with TBST and incubated with an appropriate secondary antibody. Proteins were visualized by enhanced chemiluminescence and quantified by densitometry.

Statistics

Data are presented as mean ± S.E. Comparisons between groups were performed using one-way analysis of variance or Student's t test when only two groups were compared. Significance was established at p < 0.05.

RESULTS

AMPK Activation Increases Transport Activity and Cell Surface Abundance of Na⁺,K⁺-ATPase in L6 Muscle Cells

Ouabain-sensitive ⁸⁶Rb⁺ uptake and cell surface biotinylation was measured to assess the activity and abundance of the sodium pump in the plasma membrane in the presence or absence of AMPK activators (AICAR or cyanide). Both AICAR and cyanide increased AMPK phosphorylation at Thr¹⁷² in L6 cells (Fig. 1A), concomitant with increased cell surface expression of α₁-subunit of the sodium pump (Fig. 1B) and pump activity (Fig. 1C). These data provide evidence to suggest that AMPK activation increases expression of the α₁-subunit Na⁺,K⁺-ATPaseat the plasma membrane, as well as the activity of the pump in L6 cells.

FIGURE 1.

AICAR and cyanide increase the cell surface abundance and transport activity of Na⁺,K⁺-ATPase in L6 cells. L6 cells were incubated with either AICAR (1 mm for 40 min) or cyanide (NaCN; 100 μm for 40 min). Cells were biotinylated on the cell surface. Biotinylated proteins were recovered from cell extracts by precipitation with streptavidin-agarose, and Na⁺,K⁺-ATPase α₁-subunit on the cell surface was detected by Western blot analysis using Na⁺,K⁺-ATPase α₁-subunit antibody (B). An aliquot of the total cell extract from each sample was also processed by a parallel SDS-PAGE and subjected to immunoblot analysis to determine AMPK Thr¹⁷² phosphorylation (A). Results are mean ± S.E. n = 3–5. *, p < 0.05 versus basal. C, ⁸⁶Rb⁺ uptake assay was performed as described under “Experimental Procedures.” Oubain (2 mm) was used to inhibit Na⁺,K⁺-ATPase. Data are mean ± S.E. n = 5. *, p < 0.05 versus basal.

A-769662 Increases the Activity and Cell Surface Expression of Na⁺,K⁺-ATPase α₁-Subunit after Washout

A-769662 is a potent direct AMPK activator (14). However, A-769662 directly inhibits the purified Na⁺,K⁺-ATPase α₁-isoform isolated from kidney cells (15). A washout experiment was performed to eliminate the direct effect of A-769662 on the sodium pump. L6 cells were incubated for 1 h with A-769662 (Fig. 2). Thereafter, the medium was removed, cells were washed and incubated in A-769662-free medium for an additional 1, 2, 3, or 4 h. A 1-h incubation with A-769662 transiently increased AMPK phosphorylation at Thr¹⁷², which returned to the basal level during the washout period (1–4 h; Fig. 2A). ACC phosphorylation at Ser⁷⁹ was increased after incubation with A-769662 and remained elevated for 3 h after removal of A-769662 (Fig. 2B). Consistent with our previous observation (15), the cell surface expression of the Na⁺,K⁺-ATPase α₁-subunit in L6 myotubes was decreased after a 1-h exposure to A-769662 (Fig. 2C). However, cell surface expression of the Na⁺,K⁺-ATPase α₁-subunit was quickly normalized during the washout and was markedly increased after a 2-h incubation with starvation medium, which was followed by return to the basal level (Fig. 2C). Sodium pump activity decreased after a 1-h incubation with A-769662, but then increased 2 h following removal of A-769662 (Fig. 2D), concomitant with an increase in the Na⁺,K⁺-ATPase α₁-subunit cell surface expression.

FIGURE 2.

AMPK activation by A-769662 after the compound washout increases the transport activity and cell surface abundance of Na⁺,K⁺-ATPase in L6 cells. Differentiated L6 cells were preincubated in the presence or absence of 100 μm A-769662 for 1 h. L6 cells were lysed or the medium containing A-769662 was removed and replaced by starvation medium and incubated for 1, 2, 3, or 4 h and then lysed. Cell lysates were processed as described under “Experimental Procedures.” Samples were separated on a 7.5–20% SDS-PAGE, and subjected to immunoblot analysis for pAMPK at Thr¹⁷² (A) and pACC at Ser⁷⁹ (B). A representative Western blot is shown in the upper panel. Densitometric analysis of different proteins is expressed in arbitrary units. Results are mean ± S.E. for n = 8. *, p < 0.05 versus basal. C, after incubation, the cell surface was biotinylated as specified in the legend to Fig. 1A. Cell surface Na⁺,K⁺-ATPase α₁-subunit was detected by Western blot analysis. Densitometric analysis of the protein is shown in arbitrary units. Results are mean ± S.E. for n = 3–6. *, p < 0.05 versus basal. D, ouabain-sensitive ⁸⁶Rb⁺ uptake assay was performed and results represent mean ± S.E. n = 3–5. *, p < 0.05 versus basal.

The AMPK Inhibitor Compound C Abolishes the Effect of AICAR on Na⁺,K⁺-ATPase in L6 Muscle Cells

Exposure of L6 myotubes to a chemical AMPK inhibitor, Compound C, was without effect on basal AMPK and ACC phosphorylation (Fig. 3, A and B). Furthermore, basal cell surface expression of the Na⁺,K⁺-ATPase α₁-subunit (Fig. 3C) and pump activity (Fig. 3D) were unaltered by Compound C. However, Compound C abolished the effect of AICAR on AMPK and ACC phosphorylation (Fig. 3, A and B). Similarly, Compound C blocked the effect of AICAR on expression of the Na⁺,K⁺-ATPase α₁-subunit at the plasma membrane, as well as pump activity (Fig. 3, C and D). These data further support a role for AMPK in activation of the sodium pump.

FIGURE 3.

Compound C blocks the effect of AICAR on cell surface abundance and transport activity of Na⁺,K⁺-ATPase in L6 cells. L6 cells were incubated with AICAR (1 mm for 40 min) and Compound C (40 μm for 50 min) or with AICAR or Compound C alone, and a cell surface biotinylation assay was performed. An aliquot of the total cell extract was processed by a parallel SDS-PAGE. Representative blots and densitometric analysis of: pAMPK at Thr¹⁷² (A), pACC at Ser⁷⁹ (B), and cell surface α₁ Na⁺,K⁺-ATPase (C). Densitometric analysis of different proteins is shown in arbitrary units. Results are mean ± S.E. for n = 3–6. *, p < 0.05 versus basal. D, ⁸⁶Rb⁺ uptake assay was performed as described under “Experimental Procedures.” The values are the mean ± S.E., n = 3–5. *, p < 0.05 versus basal. Ouabain (2 mm) was used to inhibit Na⁺,K⁺-ATPase.

The AICAR Effect on the Na⁺,K⁺-ATPase Transport Activity and Cell Surface Abundance Requires AMPK α-Subunits

To determine whether AMPK α-subunits are required for the AICAR-stimulated increase in Na⁺,K⁺-ATPase transport activity and cell surface abundance, we silenced AMPK using siRNA against AMPKα1 and AMPKα2. A 50% decrease in AMPK α-subunit expression (Fig. 4A) substantially reduced the AICAR-stimulated AMPK phosphorylation at Thr¹⁷² (Fig. 4B) and ACC phosphorylation at Ser⁷⁹ (Fig. 4C). The effect of AICAR on cell surface expression and activity of the sodium pump was prevented in cells following a 50% decrease in AMPK α-subunit expression (Fig. 4, D and E). Because we were unable to completely knock-down AMPK α-subunit expression in L6 cells by siRNA, we further tested our hypothesis in primary mouse muscle cells deficient in the α₁- and α₂-subunits of AMPK. In wild-type cells, where both α-subunits of AMPK are endogenously expressed, AICAR increased the phosphorylation of AMPK at Thr¹⁷² and ACC at Ser⁷⁹ (Fig. 5, A–C). Conversely, in AMPK α-subunit-deficient cells basal and AICAR-stimulated phosphorylation of AMPK and ACC could not be detected (Fig. 5, A–C). Wild-type and AMPKα knock-out cells expressed a similar amount of the α₁-subunit of Na⁺,K⁺-ATPase (Fig. 5D). Thus, we next assessed the effects of AICAR on Na⁺,K⁺-ATPase in AMPKα knock-out cells. AICAR was without effect on either transport activity or cell surface abundance of Na⁺,K⁺-ATPase in AMPKα knock-out cells (Fig. 6, A and B). These data provide evidence that the AMPK α-subunits are required for AICAR-induced effects on Na⁺,K⁺-ATPase.

FIGURE 4.

AMPKα siRNA abolishes the AICAR-induced increase in transport activity and cell surface abundance of Na⁺,K⁺-ATPase in L6 cells. L6 cells were transfected with siRNA against AMPKα or a scramble sequence as described under “Experimental Procedures.” Transfected cells were incubated in the presence or absence of 1 mm AICAR for 30 min. L6 cells were biotinylated with the cleavable biotinylation reagent sulfo-NHS-SS-biotin. An aliquot of the total cell extract was processed by a parallel SDS-PAGE. Biotinylated or total proteins were subjected to immunoblot analysis of: total AMPKα (A), pAMPK at Thr¹⁷² (B), pACC at Ser⁷⁹ (C), and cell surface α₁ Na⁺,K⁺-ATPase (D). Results are mean ± S.E. in arbitrary units, n = 7–10. *, p < 0.05 versus basal scrambled. E, ouabain-sensitive ⁸⁶Rb⁺ uptake assay was performed as described under “Experimental Procedures.” Results are mean ± S.E. n = 9–15. *, p < 0.05 versus basal. 2 mm ouabain was used to totally inhibit Na⁺,K⁺-ATPase.

FIGURE 5.

AMPKα knock-out cells express Na⁺,K⁺-ATPase but not the AMPK α-subunits. Wild-type and AMPKα knock-out (KO) cells were incubated with AICAR (1 mm for 20 min). Cell lysates were subjected to immunoblot analysis of total AMPKα (A), pAMPK Thr¹⁷² (B), pACC at Ser⁷⁹ (C), and Na⁺,K⁺-ATPase α₁-subunit (D). Densitometric analysis of different proteins is shown as fold of basal. Results are mean ± S.E., n = 3–6. *, p < 0.05 versus basal.

FIGURE 6.

The increase in the transport activity and cell surface abundance of Na⁺,K⁺-ATPase is blocked in AMPKα knock-out cells. Wild-type and AMPKα knock-out (KO) cells were incubated with AICAR (1 mm for 20 min). A, the ouabain-sensitive ⁸⁶Rb⁺ uptake assay was performed as described under “Experimental Procedures.” Results are mean ± S.E. n = 9–15. *, p < 0.05 versus wild-type basal. B, biotinylated proteins were recovered from cell extracts by precipitation on streptavidin-agarose. Na⁺,K⁺-ATPase α₁-subunit abundance on the cell surface was detected by Western blot analysis using an antibody against the Na⁺,K⁺-ATPase α₁-subunit. Representative blots and densitometric analysis of cell surface Na⁺,K⁺-ATPase α₁-subunit are shown. Results are mean ± S.E., n = 7–10. *, p < 0.05 versus wild-type basal.

Phosphorylation of Na⁺,K⁺-ATPase α-Subunit in L6 Muscle Cells in Response to AICAR

Phosphorylation of the α-subunit of Na⁺,K⁺-ATPase triggers signals controlling Na⁺,K⁺-ATPase endocytosis and exocytosis (34–36). To determine whether AICAR promotes phosphorylation of Na⁺,K⁺-ATPase, myotubes were metabolically labeled with ³²P_i for 3 h. Thereafter, cells were incubated for 40 min with AICAR, or the PKC activator PMA, as a positive control. AICAR was without effect on the pattern of total protein phosphorylation, whereas PMA notably increased protein phosphorylation (Fig. 7A). Next, we precipitated the α₁-subunit of Na⁺,K⁺-ATPase with the NK1 antibody. Total phosphorylation of the Na⁺,K⁺-ATPase α₁-subunit was decreased by AICAR exposure, whereas PMA increased the total phosphorylation of the Na⁺,K⁺-ATPase α₁-subunit (Fig. 7B). To further explore the potential role of AICAR in Na⁺,K⁺-ATPase α₁-subunit phosphorylation, we utilized the McK1 antibody that specifically recognizes the nonphosphorylated form of the Na⁺,K⁺-ATPase on Ser¹⁸, a residue that is a target for phosphorylation by protein kinase C (PKC) (35–37). Phosphorylation of the α₁-subunit on Ser¹⁸ is essential for Na⁺,K⁺-ATPase endocytosis (35–38). The use of McK1 as a tool to investigate phosphorylation of the α₁-subunit has been validated earlier (39, 40). Incubation with AICAR decreased phosphorylation of the Na⁺,K⁺-ATPase α₁-subunit on Ser¹⁸, whereas PMA decreased McK1 immunoreactivity (Fig. 7C), indicating PKC-dependent phosphorylation on Ser¹⁸. In alveolar epithelial cells, the protein phosphatase 2A catalytic subunit (PP2A) directly dephosphorylates the Na⁺,K⁺-ATPase α₁-subunit, thereby promoting translocation to the plasma membrane (41). We assessed whether AICAR altered PP2A methylation and phosphorylation. AICAR stimulation resulted in dephosphorylation (Fig. 7D) and methylation (Fig. 7E) of PP2A in L6 myotubes, which promotes activation of the phosphatase (42, 43) and may subsequently cause dephosphorylation and activation of the Na⁺,K⁺-ATPase. These data suggest that the increase in Na⁺,K⁺-ATPase abundance in response to AICAR is dependent on phosphorylation of the α-subunit of the sodium pump on Ser¹⁸.

FIGURE 7.

AICAR decreases the phosphorylation of Na⁺,K⁺-ATPase. Cells were pretreated with dimethyl sulfoxide (Basal), AICAR (1 mm for 30 min), or PMA (500 nm for 40 min). A, an autoradiogram of ³²P-labeled total proteins after SDS-PAGE and electrophoretic transfer is shown. B, Na⁺,K⁺-ATPase α₁-subunits were immunoprecipitated using Na⁺,K⁺-ATPase α₁-subunit antibody as described under “Experimental Procedures.” Immunoprecipitates (IP) were then processed by SDS-PAGE and subjected to immunoblot analysis using a Na⁺,K⁺-ATPase α₁-antibody. C, total proteins were subjected to immunoblot analysis with the McK1 antibody. The McK1 antibody recognizes the unphosphorylated α₁-subunits (on Ser¹⁸). Activation of PP2A was assessed by measurement of phosphorylation at Tyr³⁰⁷ (D) and methylation at Leu³⁰⁹ (E). Representative blots and densitometric analysis are shown. Results are mean ± S.E., n = 4–5. *, p < 0.05 versus basal. WB, Western blot.

PME-1 Is Required for AICAR-stimulated Na⁺,K⁺-ATPase α₁-Subunit Dephosphorylation and Increased Cell Surface Abundance

PP2A and a PP2A-specific protein phosphatase methylesterase-1 (PME-1) constitutively associate with Na⁺,K⁺-ATPase α-subunit (44). PME-1 is a negative regulator of PP2A and its dissociation from the complex triggers Na⁺,K⁺-ATPase dephosphorylation in kidney cells (44). To determine whether PME-1 is involved in AMPK-mediated Na⁺,K⁺-ATPase regulation in skeletal muscle, L6 muscle cells were transfected with siRNA against PME-1, which resulted in a markedly reduced expression of PME-1 protein (Fig. 8A). As expected, methylation of PP2A at Leu³⁰⁹ was increased in PME-1-deficient muscle cells, but incubation with AICAR had no additional effect (Fig. 8B). Next, the role of PME-1 in regulation of Na⁺,K⁺-ATPase phosphorylation and cell surface expression was assessed. PME-1 silencing was without effect under basal conditions, but completely abolished AICAR-stimulated dephosphorylation of the Na⁺,K⁺-ATPase α₁-subunit (Fig. 8C) and translocation to the cell membrane (Fig. 8D). These data indicate that PME-1 is required for AMPK-mediated effects on Na⁺,K⁺-ATPase.

FIGURE 8.

PME-1 siRNA prevents AICAR-stimulated Na⁺,K⁺-ATPase dephosphorylation and increased cell surface abundance in L6 cells. L6 cells were transfected with siRNA against PME-1 or a scramble sequence as described under “Experimental Procedures.” Transfected cells were incubated in the presence or absence of 1 mm AICAR for 30 min. L6 cells were biotinylated with the cleavable biotinylation reagent sulfo-NHS-SS-biotin. An aliquot of the total cell extract was processed by a parallel SDS-PAGE. Biotinylated or total proteins were subjected to immunoblot analysis of total PME-1 (A), methylated PP2A at Leu³⁰⁹ (B), unphosphorylated Na⁺,K⁺-ATPase α₁-subunit (on Ser¹⁸), as assessed by McK1 antibody (C) and cell surface α₁ Na⁺,K⁺-ATPase (D). Data are mean ± S.E. in arbitrary units, n = 6. *, p < 0.05 versus basal. WB, Western blot.

Phosphorylation of PKCζ and AS160 in Response to AICAR in L6 Muscle Cells

In rat alveolar epithelial cells, activation of AMPK by hypoxia phosphorylates PKCζ and promotes endocytosis of Na⁺,K⁺-ATPase (45). We tested the hypothesis that atypical PKC could be activated by AMPK concomitant with an increased Na⁺,K⁺-ATPase α₁-subunit translocation and pump activity in L6 muscle cells. PKCζ/λ Thr^410/403 phosphorylation, a marker of PKC activity, was unaltered after AICAR or A-769662 exposure in L6 cells (Fig. 9A), but insulin increased PKCζ/λ Thr^410/403 phosphorylation (Fig. 9B). Recently, the Rab-GTPase activating protein AS160 (TBC1D4) has been suggested to play a role in Na⁺,K⁺-ATPase translocation (26, 46). In MDCK-cultured renal epithelial cells, inhibition of AMPK resulted in an AS160-dependent intracellular accumulation of Na⁺,K⁺-ATPase (26). We determined AS160 phosphorylation in the presence or absence of AICAR (Fig. 9, C and D). As expected, insulin increased AS160 phosphorylation in L6 cells (Fig. 9D). However, exposure to either of the AMPK activators (AICAR or A-769662) was without effect on AS160 phosphorylation in L6 cells (Fig. 9C). Our data provide evidence to suggest that neither atypical PKCs nor AS160 are involved in the AMPK-dependent activation on Na⁺,K⁺-ATPase in L6 muscle cells.

FIGURE 9.

AICAR has no effect on PKCζ and AS160 activation in L6 cells. L6 cells were incubated with insulin (20 min at 120 nm), AICAR (40 min at 1 mm), A-769662 (40 min at 100 μm), or both AICAR and insulin. Cell lysates were separated on 7.5–20% SDS-PAGE, and subjected to immunoblot analysis for pPKCζ/λ Thr^410/403 (A and B) and PAS (C and D). Representative blots and densitometric analysis are shown. Results are mean ± S.E., n = 8. *, p < 0.05 versus basal.

DISCUSSION

The Na⁺,K⁺-ATPase plays a critical role in regulating fluid and electrolyte transport in a variety of tissues and different physiological stimuli can promote endocytosis or translocation of the pump to the plasma membrane (35, 47, 48). Contractile activity stimulates Na⁺,K⁺-ATPase activity in skeletal muscle, at least partially via translocation of the pump to the plasma membrane from intracellular stores (8). The molecular mechanism underpinning this phenomenon is incompletely resolved.

In skeletal muscle, contraction and metabolic stress are potent activators of AMPK (49). AMPK regulates Na⁺,K⁺-ATPase cell surface expression and activity in various cell lines (22–24). However, the effect of AMPK on Na⁺,K⁺-ATPase activity in skeletal muscle cells and the molecular mechanism behind this effect are unknown. Here we provide evidence that the AMPK activator AICAR increases Na⁺,K⁺-ATPase activity and abundance at the cell surface in L6 skeletal muscle cells with a magnitude similar to that of insulin. This effect is likely to be due to the dephosphorylation of the Na⁺,K⁺-ATPase on Ser¹⁸ in response to activation of the Ser/Thr protein phosphatase (PP) 2A. Our data provide evidence to suggest that AMPK activation modifies Na⁺,K⁺-ATPase activity, partly by increasing cell surface abundance of Na⁺,K⁺-ATPase. Collectively, these findings suggest that AICAR-induced activation of Na⁺,K⁺-ATPase is dependent on AMPK activation.

The Na⁺,K⁺-ATPase is a highly energy-dependent pump and it seems counterintuitive that AMPK, which generally turns-off ATP-consuming processes (49), would sustain or even increase Na⁺,K⁺-ATPase activity by prohibiting its internalization. AMPK activation promotes internalization of Na⁺,K⁺-ATPase in a lung epithelial cell line (H441) (22) and alveolar epithelial cells (24). Conversely, in rat L6 muscle cells, AICAR-induced AMPK activation did not promote Na⁺,K⁺-ATPase internalization. This observation is consistent with studies in MDCK epithelial cells, where AMPK inhibition by Compound C induces Na⁺,K⁺-ATPase internalization (26). Furthermore, in ATP-depleted MDCK cells, metformin prevents redistribution of Na⁺,K⁺-ATPase from the basolateral membrane to the intracellular vesicular compartment (50). These data indicate that AMPK is not a simple energy-sensor, which automatically turns-off energy-consuming processes regardless of specific tissue and cellular functions. There are substantial differences in energy requirements and demands of major energy-consuming tissues (51). Under standard resting conditions, 19–28% of whole body ATP is consumed by Na⁺,K⁺-ATPase, 25–30% by protein synthesis, 4–8% by Ca²⁺-ATPase, 2–8% by actinomyosin ATPase, 7–10% by gluconeogenesis, 3% by ureagenesis, and the remaining percentage by RNA synthesis and substrate cycling (51). However, Na⁺,K⁺-ATPase contributes to only 5–10% of oxygen consumption in skeletal muscle and even less in heart (1–5%), compared with 40–70% in kidney, 60% in the gastrointestinal tract, and 50–60% in brain (7, 51–53). Even during muscle contraction, the fractional energy consumption by Na⁺,K⁺-ATPase appears to remain constant (5–10%), whereas actinomyosin ATPase (up to 80%) and Ca²⁺-ATPase (10–50%) are by far the major consumers of ATP (7, 51, 54). Inconsistent results regarding AMPK-mediated effects on Na⁺,K⁺-ATPase activity and/or abundance obtained in different cells and tissues may therefore reflect these fundamental differences in the fractional energy requirements of Na⁺,K⁺-ATPase.

AICAR infusion and concomitant AMPK phosphorylation, is associated with an acute fall in plasma [K⁺], without increasing K⁺ urinary excretion. This effect of AICAR on plasma [K⁺] was blunted in mice overexpressing a kinase-dead form of AMPK in skeletal muscle (28). Moreover, Na⁺,K⁺-ATPase expression at the cell surface was unaltered, but only the Na⁺,K⁺-ATPase α₂-subunit was measured in the plasma membrane fraction. In mice exposed to ozone, AMPK phosphorylation and Na⁺,K⁺-ATPase expression at the plasma membrane of lung epithelium is increased. The effect of ozone on Na⁺,K⁺-ATPase cell surface abundance was not observed in AMPKα₁-deficient mice (27). Thus, in skeletal muscle, the Na⁺,K⁺-ATPase may not be a primary target of energy-conserving measures. Activation of the energy sensing AMPK may be a potential mechanism for contraction-induced activation of the sodium pump.

To dissect the molecular mechanism behind the increase of Na⁺,K⁺-ATPase activity following AMPK activation, we determined whether Na⁺,K⁺-ATPase α₁-subunit phosphorylation is altered in response to AMPK activation by AICAR in L6 cells. Total phosphorylation of the Na⁺,K⁺-ATPase α₁-subunit, as well as phosphorylation on Ser¹⁸ was decreased following AICAR treatment, possibly explaining the increased cell surface Na⁺,K⁺-ATPase abundance. Indeed, endocytosis of rodent Na⁺,K⁺-ATPase requires Ser¹⁸ phosphorylation of the α₁-subunit (35–38). Thus, AMPK activation decreases Na⁺,K⁺-ATPase phosphorylation on Ser¹⁸ and prevents endocytosis of Na⁺,K⁺-ATPase. PKCζ phosphorylates the Na⁺,K⁺-ATPase α₁-subunit on Ser¹⁸ and triggers Na⁺,K⁺-ATPase endocytosis in polarized epithelial cells from lung (24, 37). Here we report that PKCζ phosphorylation, a marker of PKCζ activity was unchanged after AICAR stimulation, consistent with the decrease of Na⁺,K⁺-ATPase α₁-subunit Ser¹⁸ phosphorylation after AICAR stimulation. This result provides evidence that AMPK stimulation of the sodium pump occurs via a mechanism independent of PKCζ activation.

The Rab-GTPase activating protein AS160 (TBC1D4) plays a role in regulating the sodium pump. In alveolar epithelial cells, AS160 phosphorylation by Akt is required for Na⁺,K⁺-ATPase trafficking to the cell surface (46). AS160 interacts directly with the α-subunit of the Na⁺,K⁺-ATPase in MDCK-cultured renal epithelial cells. Inhibition of AMPK by Compound C decreased cell surface expression of the Na⁺,K⁺-ATPase and enhanced the interaction of the pump with AS160 (26). Here we investigated the role of AS160 in the regulation of Na⁺,K⁺-ATPase in L6 myotubes, however, AS160 phosphorylation in response to AICAR was unaltered. Of note, the antibody used in this study recognizes phosphorylation of AS160 and another closely related Rab-GTPase activating protein TBC1D1, thus the integrated phosphorylation signal from these two proteins was measured. Therefore, we cannot exclude the possibility that TBC1D1 phosphorylation was modified. Additional experiments are required to further explore this question.

The FXYD1 protein (phospholemman) is one of the major substrates for protein kinases in the sarcolemma and is an important regulator of Na⁺,K⁺-ATPase activity (55). Therefore, we speculated that FXYD1 may be a candidate protein for the regulation of Na⁺,K⁺-ATPase by AMPK. However, in L6 muscle cells, the FXYD1 protein was undetectable and the level of FXYD1 mRNA expression was at the limit of detection (data not shown). Moreover, an acute exposure of isolated mouse muscles to AICAR was without effect on FXYD1 phosphorylation,⁴ which therefore excludes FXYD1 as a regulator of the sodium pump in L6 myotubes.

The observed decrease in Na⁺,K⁺-ATPase phosphorylation on Ser¹⁸ following AMPK activation appears to involve Ser/Thr phosphatase PP2A. The catalytic subunit PP2Ac regulates Na⁺,K⁺-ATPase activity (56, 57). In alveolar epithelial cells, Na⁺,K⁺-ATPase recruitment to the plasma membrane is regulated by dephosphorylation of the Na⁺,K⁺-ATPase α₁-subunit at the Ser¹⁸ residue by PP2Ac and it is mediated by a direct interaction between the proteins (41). However, PP2Ac directly dephosphorylates and inactivates AMPK phosphorylation in mammals (58, 59). Here we observed that AMPK activation increased pump activity and Na⁺,K⁺-ATPase α₁-subunit expression at the cell surface concomitant with dephosphorylation and increased methylation of PP2Ac. Salt-inducible kinase 1 (SIK1), a member of the sucrose-nonfermenting 1 protein kinase (SNF1)/AMPK family of the Ser/Thr protein kinases, activates PP2Ac, thereby promoting the dephosphorylation of the Na⁺,K⁺-ATPase α-subunits and increasing its catalytic activity (44). Therefore, increased PP2Ac activity is not always coincident with AMPK inhibition.

AICAR-stimulated dephosphorylation of Na⁺,K⁺-ATPase α₁-subunit at Ser¹⁸ in skeletal muscle cells was dependent on the PP2A-specific methylesterase PME-1. Upon activation, PP2A directly interacts with the Na⁺,K⁺-ATPase α₁-subunit, dephosphorylates Ser¹⁸, and stimulates its recruitment to the plasma membrane (41). Because PME-1 acts as a negative regulator of PP2A (60), increased activity at Ser¹⁸ could therefore be expected in PME-1-deficient muscle cells. However, PP2A and PME-1 constitutively form a complex, which associates with the Na⁺,K⁺-ATPase α-subunit and regulates its phosphorylation and activity (44). Disruption of the PP2A·PME-1 complex in PME-1-deficient cells may therefore affect PP2A targeting to the Na⁺,K⁺-ATPase α₁-subunit and prevent dephosphorylation of Ser¹⁸ upon activation of PP2A. Our result is consistent with an earlier observation showing that PP2A Thr phosphatase activity is reduced in PME-1^−/− mice despite increased methylation of PP2Ac (61). Thus, PME-1 seems to be particularly important for PP2A Ser/Thr phosphatase activity. Indeed, deletion of yeast methylesterase PPE1 was associated with increased methylation of PPH21 (PP2A homolog), but decreased phosphatase activity (62). Our results therefore provide evidence to suggest that AMPK may act on PP2Ac to regulate Na⁺,K⁺-ATPase in a PME-1-dependent manner.

In conclusion, AMPK activation increases activity and cell surface abundance of the sodium pump in skeletal muscle cells. This effect appears to be dependent on the activation of the phosphatase PP2Ac. Based on these findings, we propose the following mechanism: AMPK activation directly or indirectly stimulates phosphatase PP2Ac, which subsequently dephosphorylates the Na⁺,K⁺-ATPase α₁-subunit on Ser¹⁸, thereby preventing endocytosis and increasing cell surface abundance and activity of the sodium pump. The increase in sodium pump activity by AMPK activation may be crucial under physiological conditions, such as physical exercise, to maintain ion homeostasis and transmembrane currents and thereby delay muscle fatigue.