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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

AMPK activation with AICAR provokes an acute fall in plasma [K⁺]

¹Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, ²Department of Anatomy-Physiology and Lipid Research Unit, Laval University Hospital Research Centre, Quebec, Canada

Submitted 4 October 2007 ; accepted in final form 12 November 2007

	ABSTRACT

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AMP-activated protein kinase (AMPK), activated by an increase in intracellular AMP-to-ATP ratio, stimulates pathways that can restore ATP levels. We tested the hypothesis that AMPK activation influences extracellular fluid (ECF) K⁺ homeostasis. In conscious rats, AMPK was activated with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) infusion: 38.4 mg/kg bolus then 4 mg·kg^–1·min^–1 infusion. Plasma [K⁺] and [glucose] both dropped at 1 h of AICAR infusion and [K⁺] dropped to 3.3 ± 0.04 mM by 3 h, linearly related to the increase in muscle AMPK phosphorylation. AICAR treatment did not increase urinary K⁺ excretion. AICAR lowered [K⁺] whether plasma [K⁺] was chronically elevated or lowered. The K⁺ infusion rate needed to maintain baseline plasma [K⁺] reached 15.7 ± 1.3 µmol K⁺·kg^–1·min^–1 between 120 and 180 min AICAR infusion. In mice expressing a dominant inhibitory form of AMPK in the muscle (Tg-KD1), baseline [K⁺] was not different from controls (4.2 ± 0.1 mM), but the fall in plasma [K⁺] in response to AICAR (0.25 g/kg) was blunted: [K⁺] fell to 3.6 ± 0.1 in controls and to 3.9 ± 0.1 mM in Tg-KD1, suggesting that ECF K⁺ redistributes, at least in part, to muscle ICF. In summary, these findings illustrate that activation of AMPK activity with AICAR provokes a significant fall in plasma [K⁺] and suggest a novel mechanism for redistributing K⁺ from ECF to ICF.

potassium homeostasis; sodium-potassium-ATPase; muscle; urine

During exercise and ischemia, potassium homeostasis is significantly altered. Interstitial fluid [K⁺] is significantly elevated in ischemia as the sodium pumps are unable to actively clear K⁺ from the extracellular fluid (ECF) at a rate to match passive leaks. [K⁺] is also elevated around contracting muscles as the rate of K⁺ leaving the cell during membrane depolarization (via channels) rises to a higher rate than the sodium pump can actively pump K⁺ back into the cell (12, 41). If ECF [K⁺] climbs above the normal range (3.5 to 5 mM), a number of changes ensue including a membrane depolarization, increased muscle excitability, and then weakness. At 6.0 mM [K⁺], marked changes in the electrocardiogram are evident and at higher K⁺ concentrations lethal arrythmias may develop (4, 44). Homeostasis is restored in both cases when the net K⁺ influx rate rises above the K⁺ efflux rate. The mediators of this response have not been clearly identified (41). Cellular AMPK is a good candidate mediator of net cell K⁺ uptake in exercise, analogous to the action of AMPK to stimulate cellular glucose transport in exercising muscles (20, 46). In addition, mice deficient in muscle and heart AMPK activity exhibit depressed exercise-induced glucose uptake and, notably, significant muscle weakness and impaired recovery from fatigue (33). Interestingly, mutations in AMPK -subunits are associated with abnormalities in cardiac electrical conductance (7, 15), and mouse hearts deficient in AMPK (due to a dominant negative -subunit) are more susceptible to damage during ischemia and reperfusion (40). These findings suggested to us that there might be unidentified electrolyte abnormalities in exercising muscles deficient in AMPK activity.

This study tested the hypothesis that AMPK activation could play a role in clearing K⁺ from the ECF. This hypothesis was tested in vivo in conscious rats, using real time plasma [K⁺] and [glucose] measurements and clamps as implemented in our previous studies (8, 10), and in mice deficient in muscle AMPK activity (34). AMPK was activated by infusion of 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), an adenosine analog that is converted to AICAR monophosphate (ZMP) after cellular uptake. ZMP mimics AMP to activate AMPK by both direct allosteric activation and, more importantly, by promoting phosphorylation of the -subunit mediated by an upstream kinase complex (46, 48). The effects of AICAR on glucose uptake have been shown to be mediated by AMPK and not by some other AMP-dependent reaction since the response is abolished in AMPK ₂-subunit knockout mice (33, 34).

In support of our hypothesis, AICAR infusion provoked a significant fall in plasma [K⁺] with a time course indistinguishable from the parallel stimulation of glucose uptake and AMPK phosphorylation, suggesting that both glucose and K⁺ net transport are increased by AMPK activation. These provocative findings demonstrate that regulation of AMPK activity is important for mediating electrolyte as well as metabolic homeostasis and suggest that some of the reported effects of AMPK activation may be secondary to changes in plasma [K⁺].

	METHODS

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Animal protocols. All animal experiments were approved by the University of Southern California Keck School of Medicine IACUC and conducted in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Infusion protocols in rats. Sprague-Dawley rats, 10–12 wk of age (Simonsen Laboratories, Gilroy, CA), were maintained at constant temperature (21°C) under a 12 h:12 h artificial light cycle on standard chow. As previously described (10, 39), 3 days before experiments, animals were placed in individual cages with wire floors, and the distal one-third of each rat's tail was drawn through a hole placed low on the side of the cage and secured with a rubber stopper to protect tail blood vessel catheters. Animals were free to move about and allowed unrestricted access to food and water. One day before the experiment, two tail vein infusion catheters were inserted; 6 h before the start of experiment a tail artery blood sampling catheter was inserted. Patency of the arterial catheter was maintained by a slow infusion of heparinized (10 U/ml) saline (1 ml/h). During the 6-h period before the experiment, rats were fasted but had free access to water. At the end of the 6-h equilibration period, blood samples were taken from the tail artery for measurement of baseline [K⁺] and [glucose].

At time 0, conscious rats were infused with AICAR (Toronto Research Chemical), as previously described (3), with a bolus of 38.4 mg/kg then continuously at 4 mg·kg^–1·min^–1, or saline at a matched volume flow rate through a tail vein catheter for 3 h. During infusion of AICAR or saline, plasma glucose was clamped at the basal [glucose] and potassium was either allowed to change (K-unclamped protocol) or was clamped at basal [K⁺] (K-clamped protocol). Specifically, 60-µl blood samples were collected at 10-min intervals from the tail arterial cannula, spun, and plasma analyzed immediately for [glucose] with a Beckman glucose analyzer II and [K⁺] with a Radiometer FLM3 flame photometer. Solutions of dextrose (20%) and KCl (150 mM, in K-clamped protocol only) were separately infused (via a Y connector) through a tail vein catheter at variable rates to clamp plasma [glucose] and [K⁺] at basal levels. At the end of the clamp, animals were anesthetized with pentobarbital sodium, and gastrocnemius muscles were rapidly removed, frozen using liquid N₂-cooled aluminum blocks, and stored at –70°C for later analysis of AMPK abundance and phosphorylation.

In one series, rats (n = 4 pairs) were fed a gelled high [K⁺] diet that also contained the mineralocorticoid receptor antagonist spironolactone for 8–10 days to raise plasma [K⁺]. The control diet was prepared from 97 g powdered rat chow (Dyets, Bethlehem, PA) that contained 0.8% NaCl and 1% KCl plus 5 g agar and 167 ml H₂O. The high K⁺ diet was prepared from this mixture with the addition of 3 g KCl (4% KCl final) and 300 mg spironolactone (3 mg/g diet, Sigma Chemicals).

In another series, rats (n = 3 pairs) were fed a nominally K⁺-deficient diet (KD) to lower plasma [K⁺] or a control 1% K⁺ diet prepared commercially (Harlan Teklad, Madison, WI) for 5–7 days. Rats were subsequently infused with AICAR as described above. In this series, urine was collected, as described below, for measurement of [K⁺].

Measurement of urinary K⁺ excretion in rats. To determine K⁺ excretion before and during AICAR infusion (K-unclamp protocol only), urine was collected for 3 h from a plastic sheet placed below the wire floor of the cage before AICAR or saline infusion and during the 3-h infusions. A mesh screen was placed under the wire floor above the plastic sheet to prevent feces from contaminating the urine passed. In addition, with the rat under pentobarbital anesthesia, urine was collected from the urinary bladder at the end of the clamp and combined with the urine from the bottom of the cage during infusions. K⁺ and Na⁺ excretion were calculated as the product of urine volume, calculated gravimetrically, and [K⁺] or [Na⁺] measured by flame photometry as described above.

AICAR study in muscle-specific ₂-kinase dead AMPK transgenic mouse (Tg-KD1). Transgenic mice (C57BL/6, KD1 line) that express a dominant negative kinase dead (KD) rat ₂-isoform (K45R mutation) driven in heart and skeletal muscle by the muscle creatine kinase promoter (34), were obtained from M. Birnbaum (University of Pennsylvania) and bred in the USC animal facility to C57BL/6 mice (Simonsen Laboratories, Gilroy, CA). Offspring were genotyped by PCR as previously detailed (34). Tg-KD1 mice and wild-type littermates (NTg) were studied at 18–24 wk of age. Both males and females were studied. The protocol of Mu et al. (34), developed to examine the effect of AICAR on blood glucose levels, was modified for collection of nonhemolyzed plasma, requisite for accurate [K⁺] analysis. Mice were acclimated to being handled, to being anesthetized, and to having blood draws for a week before the experiment since stress is known to provoke a significant fall in plasma [K⁺]. After fasting for 4 h with free access to water, mice were briefly anesthetized (<2 min) with isoflurane, and a baseline blood sample of 60–80 µl was obtained from the submandibular vascular bundle using a Goldenrod lancet (Medipoint, Mineola, NY), collected through a heparinized glass capillary tube into a 0.5-ml microfuge tube and spun for isolation of plasma, which was analyzed for [K⁺] and [glucose]. If the baseline [K⁺] was above 4.9 mM, indicative of hemolysis, or below 3.4 mM, indicative of stress, the mouse was not further studied that day. Only two blood samples could be collected per experiment per animal because the blood removed (2 x 60 µl = 120 µl) represented near 10% of the total mouse blood volume (assuming 1.5 ml/25 g mouse). Preliminary experiments established a reproducible and significant AICAR-stimulated fall in plasma [glucose] in NTg mice at 30 min, as previously reported (34). If the baseline [K⁺] was between 3.4 and 4.9 mM, the mouse was injected intraperitoneally with 0.25 g/kg AICAR and at 30 min after AICAR injection the mouse was again briefly anesthetized with isoflurane (for <2 min) and a second blood sample was collected, as described above. Plasma glucose was measured with a Free Style blood glucose monitor (ThermaSense, Alameda, CA) and plasma potassium by flame photometry as described above.

Immunoblot analysis. AMPK activation was verified in a series by immunoblot analysis of rat muscle extracts for the phosphorylated form of AMPK as well as immunoblot of total AMPK probed as a control. Gastrocnemius muscles were quickly frozen after dissection with paddles cooled in liquid N₂ and stored at –80°C until analysis. Lysates of muscle were prepared for immunoblot as described previously (2, 36). In brief, muscles were homogenized in ice-cold lysis buffer containing phosphatase and protease inhibitors and 1% Triton X-100 and then centrifuged for isolation of supernatant. Each sample of 40 µg was resolved by SDS-PAGE, transferred to polyvinyldifluoride membranes, blocked overnight in Licor Blocking buffer (LI-COR Bioscience, Lincoln, NE), and then incubated with antibodies against phosphorylated AMPK (pThr172) or total AMPK (both from Cell Signaling, Danvers, MA). Antibody binding was detected with goat anti-rabbit IgG (H+L) Alexa Fluor 680 (Molecular Probes, Eugene, OR) and analyzed by Odyssey Infrared Imaging System and accompanying software (LI-COR Bioscience). Linearity of the detection system with this amount of protein loading was verified in preliminary experiments (not shown).

Muscle Na⁺-K⁺-ATPase activity. Na⁺-K⁺-ATPase activity was measured in triplicate in total membrane preparations (3,000 g supernatant) of whole gastrocnemius muscle of rat collected after 3 h of AICAR infusion during which both plasma [glucose] and [K⁺] were clamped at baseline. Na⁺-K⁺-ATPase activity was measured as previously described in detail as ouabain-sensitive µmol inorganic phosphate (P_i) liberated from ATP per milligram of protein per hour (10). Total Na⁺-K⁺-ATPase activity was calculated as the rate of P_i generated during a 15-min incubation period in the absence of ouabain minus P_i generated in the presence of either 2 mM ouabain (inhibits both ₁- and ₂-isoforms of Na⁺-K⁺-ATPase) or 10^–5 M ouabain (inhibits ₂-isoform). Na⁺-K⁺-ATPase ₁ activity was calculated as total activity minus ₂ specific activity.

Muscle Na⁺-K⁺-ATPase subcellular distribution. To determine whether AICAR stimulated trafficking of muscle Na⁺-K⁺-ATPase ₂ from intracellular pools to the plasma membrane, a sample set that was used to establish AICAR-stimulated muscle glucose uptake and GLUT4 translocation from intracellular membranes to plasma membranes (29) was reprobed for Na⁺-K⁺-ATPase ₂ translocation, as well as reprobed to verify GLUT4 translocation. In brief, after AICAR infusion (3), plasma membranes, T-tubules, and intracellular membranes were isolated from 7 to 8 g of muscles (tibialis, gastrocnemius, and quadriceps) using a procedure established by the Marette laboratory (14). Membrane fractions (10 µg each) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis as described above using a polyclonal anti-Na⁺-K⁺-ATPase ₂ antiserum (McDonough lab) at 1:500 (39) and anti-GLUT 4 polyclonal antiserum (Biogenesis, Kingston, NH) at 1:1,000. Immunoblots were quantitated as described above and abundance in plasma membranes (PM), intracellular membranes (IM), and T-tubules (TT) expressed as fraction of the total where abundance in PM+IM+TT is defined as 1.0 within each animal.

Quantitation and statistical analyses. Data are expressed as means ± SE except where standard deviation is indicated. The significance of the differences in mean values between groups was evaluated using paired two-tailed t-test when comparing data before and after treatment in the same animal and unpaired two-tailed t-test when comparing different animals. Differences were considered significant at P < 0.05.

	RESULTS

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AICAR activates AMPK and produces a decrease in plasma K⁺ levels in eukalemic rats. To test the hypothesis that AMPK activation could play a role in clearing K⁺ from the ECF, conscious rats were infused with AICAR (bolus of 38.4 mg/kg then continuously at 4 mg·kg^–1·min^–1 in saline) through a tail vein while real time plasma [K⁺] and [glucose] were measured every 10 min and [glucose] clamped at baseline as described (8, 10). Controls, assayed in parallel on the same day and time, were infused with a matched volume of saline (n = 6 pairs). Plasma [K⁺] remained at baseline (4.2 ± 0.1 mM), began to drop after 60 min of AICAR infusion, and fell to 3.2 ± 0.1 mM between 140 and 180 min (Fig. 1A), a clearance of 24% of the ECF K⁺. In the saline-infused group, [K⁺] fell slightly to 4.0 ± 0.5 mM, likely due to fasting.

View larger version (30K): [in this window] [in a new window]   Fig. 1. 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuraside (AICAR) infusion stimulates clearance of potassium from the plasma as a function of increased AMP-activated protein kinase (AMPK) phosphorylation. AICAR was infused at time 0 (bolus of 38.4 mg/kg then continuously at 4 mg·kg–1·min–1 into tail vein). Controls were infused with paired volume of saline. A: plasma [K+] during euglycemic clamp. Values are means ± SE; n = 6 pairs. B: time course of activation of AMPK was measured in AICAR-infused animals euthanized at 20, 60, 120, and 180 min, and vehicle-infused animals were euthanized at 180 min (n = 3 each group). AMPK activity was assessed by measuring abundance of AMPK phosphorylated at Thr172 (pAMPK ) in a constant amount of lysate protein isolated from gastrocnemius muscle using a phosphor-specific antibody; AMPK total abundance was measured in the same samples as a control; values are means ± SE. C: relationship between decrease in plasma [K+] and increase in pAMPK measured in the same rats before euthanasia (n = set of 15 rats from B). Regression analysis results: y = –0.9561x + 4.9407, R2 = 0.7642, P = 2.03E – 05.

AICAR maintains its K⁺-lowering effect in rats when baseline [K⁺] is elevated or lowered. A new strategy to decrease plasma [K⁺] during hyperkalemia could be therapeutically useful (24). To determine whether AICAR could lower elevated plasma K⁺, rats were fed a gelled high-K⁺ diet, which also contained the mineralocorticoid antagonist spironolactone to inhibit renal K⁺ secretion. After 8–10 days, plasma [K⁺] rose significantly to 4.5 ± 0.1 mM. AICAR infusion (Fig. 2A) caused plasma [K⁺] to fall to 3.8 ± 0.15 mM, a 17% loss of plasma K⁺. In paired controls, plasma K⁺ fell from a baseline of 3.9 ± 0.09 to 3.0 ± 0.19 mM, a 23% loss of plasma K⁺. These results suggest that AMPK activation may be a useful adjunct strategy to lower K⁺ in hyperkalemia.

View larger version (19K): [in this window] [in a new window]   Fig. 2. AICAR infusion stimulates clearance of potassium from the plasma in rats with elevated or lowered plasma [K+]. AICAR was infused at time 0 (bolus of 38.4 mg/kg then continuously at 4 mg·kg–1·min–1 into tail vein). Controls were infused with paired volume of saline. Values are means ± SE. A: rats (n = 4 pairs) fed a 4% K+ diet containing 3 mg/g spironolactone had significantly elevated baseline plasma [K+], P < 0.001, which was significantly lowered with AICAR infusion, P < 0.001. B: rats fed a K+-free diet (n = 3 pairs) had significantly lower baseline plasma [K+], P < 0.002, which was significantly lowered with AICAR infusion, P < 0.004.

AICAR does not increase renal K⁺ excretion. A fall in plasma [K⁺] can be explained by: 1) active K⁺ uptake from ECF to intracellular fluid (ICF), analogous to insulin stimulation of muscle Na⁺-K⁺-ATPase, 2) net transfer of K⁺ from the ECF to the ICF by inhibition of a passive K⁺ efflux route, or 3) increased urinary or fecal K⁺ excretion. The AICAR-stimulated drop in [K⁺] (Figs. 1A and 2, A and B) represents a shift of about 23% of ECF K⁺ (60 µmol) out of the ECF compartment occurring over 90 min. It would be very difficult to detect a shift of this magnitude into the much larger ICF K⁺ pool [which is 14,400 µmol assuming ICF K⁺ is 120 µmol/ml (45) and ICF volume is 40% of a 300-g body wt = 120 ml]. For this reason, we did not try to measure an increase in ICF [K⁺] but measured urinary K⁺ excretion during AICAR infusion. In the experiments described in Fig. 1A, urine was collected from the bottom of the cage for 3 h before AICAR and for the 3 h during AICAR infusion (bladder urine was pooled with the AICAR infusion samples). Rats (n = 6 pairs) had free access to water, but no food, during these collections. As summarized in Fig. 3, urinary K⁺ values were quite variable between individual rats but did not increase consistently with AICAR infusion. Urinary K⁺ (in mmol·kg^–1·3 h^–1, means ± SD) in the control group was 0.60 ± 0.49 before infusion and 0.96 ± 0.60 during paired saline infusion, whereas in the AICAR group, urinary K⁺ was 1.13 ± 1.18 before infusion and 1.20 ± 0.51 during AICAR infusion. These results do not support the hypothesis that the AICAR-stimulated fall in plasma K⁺ is due to increased urinary K⁺ secretion during AICAR infusion. Related to the question of K⁺ clearance by urinary excretion, KD diet also lowers urinary K⁺ to conserve K⁺ stores. Urinary [K⁺] (in mM, means ± SD) was measured in urine collected from bladders at the end of the experiments in Fig. 2B. In the rats fed the KD diet, urine [K⁺] was 1.7 ± 1.0 mM, significantly lower than the 30.3 ± 8.5 mM [K⁺] measured in urine of control K⁺ fed rats. The very low [K⁺] in the AICAR-infused rats fed the KD diet suggests that K⁺ is not cleared from the ECF to the urine.

View larger version (16K): [in this window] [in a new window]   Fig. 3. AICAR infusion did not significantly increase urinary K+ excretion. Urinary K+ (mmol·kg–1·3 h–1) collected from the bottom of the cage 3 h before and 3 h during AICAR infusion, the latter pooled with urine collected from the bladder collected after the experiment. Same rats as in Fig. 1A, n = 6 pairs. Lines connect values from the same rat. Bar indicates mean.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Quantifying AICAR stimulated K+ and glucose clearance during AICAR infusion with euglycemic and eukalemic clamps. AICAR was infused at time 0 (bolus of 38.4 mg/kg then continuously at 4 mg·kg–1·min–1 into tail vein). Controls were infused with paired volume of saline. A: plasma [K+] clamped at baseline with KCl infusion. B: K+ infusion rate (Kinf) necessary to maintain plasma K+ at baseline, which reflects rate of K+ clearance from the plasma. C: plasma [glucose] clamped at baseline with glucose infusion. D: glucose infusion rate (Ginf) necessary to maintain plasma glucose at baseline, which reflects rate of glucose clearance from plasma.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Effect of AICAR on GLUT4, but not Na+-K+-ATPase (NKA) 2, translocation in rat skeletal muscle. Membrane samples (from Ref. 29) were rerun and probed for GLUT4 and NKA 2. Top and middle, effect of AICAR on NKA 2 and GLUT4 distribution, quantitation of n = 3–4 experiments, respectively. Values are means ± SE. *P < 0.05. Bottom, representative immunoblots showing the subcellular distribution of NKAse 2 and GLUT4 in plasma membranes (PM), intracellular membranes (IM), and transverse tubules (TT). Membrane protein (10 µg) from each sample was subjected to immunoblotting as described in METHODS.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Inactivation of AMPK blunts AICAR-stimulated clearance of glucose and potassium. AICAR (0.25 g/kg body wt) in saline injected into the peritoneal cavity of Tg-KD1 mice or their wild-type littermates (NTg). Blood was collected before injection and at 30 min after injection from the submandibular vascular bundle. Plasma isolated and analyzed for [glucose] and [K+]. Results are expressed as means ± SE of 7 (NTg) to 9 (Tg-KD1). *P < 0.05 compared with basal collection; +P < 0.05 compared with NTg after AICAR.

	DISCUSSION

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The phenotypic effects of AMPK activation in the muscle are currently assigned to changes in metabolism, without attention to electrolyte shifts. The results of this study predict that plasma [K⁺] fell in all of the AICAR infusion studies reported to date. We suggest that some of the phenotypic changes that have been reported to occur with AMPK stimulation or inactivation may be secondary to changes in the transmembrane potassium gradient and membrane potential. For example, mice deficient in muscle and heart AMPK activity exhibit significant muscle weakness and impaired recovery from fatigue (33); mutations in AMPK -subunits are associated with cardiac hypertrophy and abnormalities in cardiac electrical conductance (7); and mouse hearts deficient in AMPK are more susceptible to damage during ischemia and reperfusion (40). A critical question that remains to be examined is whether a lack of AMPK activity, as seen in these models, blunts K⁺ clearance during recovery from exercise or ischemia, which could provoke muscle fatigue and pain and weakness (41).

Is the AICAR-mediated clearance of plasma potassium K⁺ due to AMPK activation? The alternative explanation would be that the ZMP produced from AICAR could mimic other effects of AMP. The finding that the AICAR-stimulated clearance of glucose from the plasma is dependent on muscle AMPK activity was established in a mouse model expressing a kinase dead ₂-catalytic subunit (Tg-KD1), which has a dominant negative effect on AMPK activity (33, 34). If the AICAR clearance of K⁺ is indeed mediated by muscle AMPK activation, then the drop in plasma [K⁺] as well as [glucose] should be blocked or blunted in the Tg-KD1 mice. We did see a significant blunting in the AICAR-induced K⁺ clearance in the Tg-KD1 mice compared with the NTg controls, which supports the notion that the K⁺ clearance is secondary to AICAR activation of AMPK activity. Other evidence in this study supporting the hypothesis that the drop in plasma [K⁺] is secondary to the activation in AMPK include: a significant temporal relationship between the increase in AMPK activity, measured as AMPK-P abundance and the decrease in plasma [K⁺] (Fig. 1C), and the very similar temporal relationship between AICAR-induced G_inf [which has been established to be due to AMPK activation (3, 35)] and AICAR-induced K_inf (Fig. 4). The fact that significant decreases in glucose and K⁺ persist in the Tg-KD1 mice infused with AICAR suggest that that ZMP exerts unidentified AMP-like effects that stimulate glucose and K⁺ cellular uptake or, more likely, that other nonmuscle tissues may participate in AICAR-stimulated glucose and potassium clearance. Related to this second point, when DeFronzo and colleagues (13) reported that insulin caused a decrease in plasma [K⁺], they also determined that 70% of the drop in [K⁺] observed during the first hour of insulin infusion could be accounted for by net uptake by the liver (13).

The magnitude of the AICAR-provoked fall in plasma potassium suggests that this drug is a potent stimulator of K⁺ clearance. Interestingly, there are similarities between signals that control K⁺ and glucose homeostasis: insulin stimulates cellular uptake of both glucose and K⁺ after a meal (31), exercise stimulates insulin-independent cellular uptake of both glucose and potassium uptake in muscle, and in this study we establish that AICAR stimulates the clearance of both glucose and potassium from the ECF. In a recent study from this laboratory using the same rat vendor and tail vein infusion and blood sampling protocols (8), we measured K_inf and G_inf stimulated by infusion of insulin at 5 mU·kg^–1·min^–1 for 150 min in control rats. During the last 60 min of insulin infusion K_inf stayed at 9.7 ± 1.48 µmol·kg^–1·min^–1 and G_inf at 172 ± 16 µmol·kg^–1·min^–1. In comparison, in this study, AICAR increased K_inf to a new steady state of 15.7 ± 1.3 µmol·kg^–1·min^–1 and G_inf to 62 ± 6 µmol·kg^–1·min^–1 during the last 60 min. These two sets of findings can be compared in order to provide a relative measure of the strength of AICAR to insulin on K⁺ and glucose clearance. The comparison reveals that, at the doses studied, insulin stimulates nearly threefold more glucose clearance than AICAR, whereas AICAR stimulates 1.6-fold more K⁺ clearance than insulin, demonstrating that AICAR is a powerful stimulator of K⁺ clearance. In the study of Chen et al. (8) insulin infusion had to be raised to the pharmacological level of 50 mU kg^–1·min^–1 for 1 h to decrease plasma [K⁺] to 3.2 mM, which is in the range of the decrease seen with AICAR.

What is the destination of the potassium that leaves the plasma? Potential mechanisms to clear K⁺ include: 1) urinary or fecal excretion, 2) increase in rate of active K⁺ transport from ECF to ICF, and 3) decrease K⁺ leak from ICF to ECF with unchanged active K⁺ uptake. The mechanism to clear glucose from the plasma is restricted to shift from ECF to ICF because there is normally no glucose excretion. Evidence against a mechanism of increased urinary excretion of K⁺ in response to AICAR include the observation that there was not a significant increase in urinary K⁺ excretion during AICAR infusion (Fig. 3). In addition, we found that AICAR provoked a drop in plasma K⁺ even in rats fed spironolactone or KD diet, which are two interventions that are well known to depress K⁺ secretion and excretion (25). Since the ICF K⁺ pool is 14,400 µmol, it was not feasible to attempt to detect a transfer of 42 µmol from ECF to ICF. Along the same vein, since dietary intake of K⁺ is so high, detecting an increase in colonic excretion was not feasible. For these reasons, we cannot conclude that K⁺ is transferred from the ECF to the ICF during AICAR stimulation, but this is our working hypothesis that has the experimental support of the urinary excretion studies.

There is an extensive literature that demonstrates that insulin infusion, contractility, and catecholamine stimulation activate muscle Na⁺-K⁺-ATPase activity and increase active cellular uptake of K⁺ (12, 32, 43). There is also literature that demonstrates AICAR stimulates net translocation of GLUT4 to plasma membrane in muscle, which contributes to the AICAR-induced clearance of glucose (22, 27, 29, 50). For these reasons we aimed to examine whether there was evidence for sodium pump activation or translocation to the plasma membrane during AICAR infusion. In short, our analysis of total Na⁺-K⁺-ATPase V_max activity and reanalysis of a set of membranes that were used to demonstrate GLUT4 translocation during AICAR infusion (29) did not support the hypothesis that AICAR provokes an increase in sodium pump-mediated active K⁺ uptake that could account for the fall in plasma K⁺. It is possible that more direct in vivo assays of sodium pump activity or ouabain-sensitive oxygen consumption would detect a change in Na⁺-K⁺-ATPase. Whereas these studies are not informative regarding a molecular mechanism for K⁺ clearance from the plasma, the lack of stimulation Na⁺-K⁺-ATPase activity by AICAR is consistent with the overall role of AMPK as a cellular fuel gauge that decreases ATP utilization and increases ATP production as a stimulation of active K⁺ uptake via the Na⁺-K⁺-ATPase would certainly increase ATP consumption.

Whereas exercise was not a variable in this study, we hypothesize that AMPK activation could play a role in clearing the K⁺ from interstitial fluid during exercise. Interstitial [K⁺] climbs around an exercising muscle as the rate of K⁺ efflux rises above the rate of active K⁺ uptake via Na⁺-K⁺-ATPase (41). As the rates of K⁺ efflux and active K⁺ uptake are increasing, the rate of ATP utilization (by sodium pump and contractile apparatus) is increasing as well. We predict a tipping point will be reached, depending on severity and length of the exercise, at which the cell AMP-to-ATP ratio will climb sufficiently to increase AMPK activity. We hypothesize that this increased AMPK activity may contribute to normalizing the rate of K⁺ efflux to influx, which will ultimately restore baseline [K⁺] when the exercise stops. A corollary hypothesis is that chronic metformin treatment, an AMPK activator prescribed for Type 2 diabetes (37) may increase the rate of restoration baseline K⁺ following exercise.

There is a literature on the effects of AICAR and AMPK stimulation on ion transporters in epithelia that can shed light on potential molecular mechanisms of the potassium clearance observed in this study. AMPK was identified as a binding partner of the cystic fibrosis transmembrane conductance regulator (CFTR) in a yeast two-hybrid screen, and its co-expression with CFTR in an oocyte significantly inhibited cAMP-activated CFTR Cl^– conductance (18). This inhibition was also observed when AMPK activity was stimulated in cultured lung cells (17). AMPK activation similarly inhibited epithelial sodium channel (ENaC) currents expressed in oocytes and polarized mouse collecting duct cells. In this case the interaction is not direct but involves AMPK-mediated phosphorylation of a protein (Nedd4-2) that regulates ENaC retrieval from the plasma membrane (6). Taken together, these studies indicate that AMPK may, directly or indirectly, limit the dissipation of transmembrane ion gradients, which would conserve the ATP necessary to maintain the gradients (16). As discussed above, K⁺ could be translocated from the ECF to the ICF either by increasing active K⁺ uptake mediated by the Na⁺-K⁺-ATPase or by decreasing K⁺ efflux via a passive efflux route; that is, decreasing the ratio of K⁺ efflux to K⁺ influx. One hypothesis that reconciles the role of AMPK as a cellular fuel gauge with the observed clearance of K⁺ from the ECF is that AMPK, acting directly or indirectly, may decrease the K⁺ efflux rate, which would promote net cellular K⁺ uptake by decreasing the ratio of K⁺ efflux (via channels/leaks) to K⁺ influx (via ATPases). Such a change in K⁺ flux ratio would clear K⁺ that accumulates in the ECF during exercise or ischemia without increasing ATP consumption.

Kristensen et al. (26) have analyzed membrane proteins involved in potassium shifts in muscle using specific inhibitors and come to the conclusion that ATP-sensitive K⁺ channels (K_ATP) and large-conductance Ca²⁺-dependent K⁺ channels (BK_Ca) are responsible for K⁺ release during muscle contraction, whereas the strong inward rectifier 2.1 K⁺ channel (Kir2.1) and the NKCC (Na⁺-K⁺-2Cl^–) cotransporter participate in K⁺ reuptake. Wyatt et al. (49) have recently reported that AICAR treatment of carotid body type 1 cells inhibits oxygen-sensitive K⁺ currents carried by BK_Ca, indicating this is a candidate that could be pursued in analysis of the molecular basis for the plasma K⁺-lowering effect of AMPK activation._. Nielsen et al. (38) have shown that K_ATP channels are located mainly in the plasma membrane of muscle, found in all muscle fiber types, are active in resting muscle where they contribute to baseline interstitial [K⁺] and that the K_ATP channel inhibitor glibenclamide acutely lowers interstitial [K⁺], suggesting that the K_ATP channel would be another good candidate. This K⁺ efflux pathway is activated by decreasing concentrations of ATP and has been shown to be inhibited by AICAR in pancreatic β cells (47). In contrast, bouts of hypoxia (which would presumably activate AMPK) have been reported to traffic K_ATP channels to the plasma membrane and activate channel activity in cardiomyocytes (42). In summary, the molecular mechanism(s) responsible for the clearance of K⁺ from the ECF during AMPK stimulation with AICAR remain to be determined, as does the exact tissues involved in the response.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was supported by National Institutes of Health Grants DK-57678 and DK-34316 (to A. McDonough), DK-065998 (to J. H. Youn), and by a grant from the Canadian Diabetes Association (to A. Marette). K. Lemieux was supported by a studentship from the Canadian Institutes of Health Research.

	ACKNOWLEDGMENTS

 
We are very grateful for the muscle-specific ₂ kinase dead AMPK transgenic mouse breeders provided by the Birnbaum lab (University of Pennsylvania).

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. A. McDonough, Dept. of Physiology and Biophysics, Univ. of Southern California Keck School of Medicine, 1333 San Pablo St., Los Angeles, CA 90089-9142

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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