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Am J Physiol Heart Circ Physiol 288: H2412-H2421, 2005. First published January 6, 2005; doi:10.1152/ajpheart.00558.2004
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Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

Markus Frederich,* Li Zhang,* and James A. Balschi

Nuclear Magnetic Resonance Laboratory for Physiological Chemistry, Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts

Submitted 9 June 2004 ; accepted in final form 30 December 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 µM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol·min–1·mg protein–1 in normoxic hearts and from 5 to 55 pmol·min–1·mg protein–1 in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity (A0.5) was 3 ± 1 µM for hypoxic hearts and 28 ± 13 µM for normoxic hearts. The A0.5 for {alpha}2-isoform AMPK activity was 2 ± 1 µM for hypoxic hearts and 13 ± 8 µM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK {alpha}-subunit. In potassium-arrested hearts perfused with variable O2 content, {alpha}-subunit Thr172 phosphorylation increased at O2 ≤ 21% even though [AMP] was <0.3 µM. Thus hypoxia or O2 ≤ 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].

signal transduction; 31P-nuclear magnetic resonance spectroscopy; acetyl-CoA carboxylase

AMP-ACTIVATED PROTEIN KINASE (AMPK) and AMP-activated protein kinase kinase (AMPKK) comprise a protein kinase cascade that has been highly conserved throughout evolution (17, 18). The AMPK cascade acts as a cellular low-fuel warning system (17). Increases in AMP concentration ([AMP]) increase AMPK activity. AMPK functions to either conserve ATP or promote alternative methods of ATP generation. One way that AMPK stimulates ATP synthesis in heart is by phosphorylation of acetyl-CoA carboxylase (ACC). The phosphorylation inhibits ACC activity, which lowers malonyl-CoA formation. Lower malonyl-CoA in turn relieves the inhibition of carnitine palmitoyl transferase 1, the primary transporter and control point for fatty acyl-CoA entry into the mitochondrion. This results in acceleration of the {beta}-oxidation of fatty acids and the generation of ATP.

AMPK is a heterotrimeric protein that consists of one catalytic subunit ({alpha}) and two noncatalytic subunits ({beta} and {gamma}) (33). Phosphorylation of the {alpha}-subunit Thr172 increases AMPK activity (21, 34). Two isoforms of the {alpha}-subunit have been identified ({alpha}1 and {alpha}2). Both isoforms are expressed in rat heart. The {beta}-subunit, which has two isoforms, functions as a scaffold for the binding of the {alpha}- and {gamma}-subunits (37) and can affect activity and localization (36). The {gamma}-subunit, which has three isoforms, contains cystathionine {beta}-synthase (CBS) domains that are the binding sites for AMP and ATP (31).

Recently, we described the relationship between AMPK activity measured in vitro and cytosolic [AMP] determined in vivo for oxygenated perfused rat hearts (15). The maximal AMPK activity measured was, however, only about one-half of that measured from ischemic hearts (15). This led us to speculate that mechanisms in addition to activation by [AMP] alter AMPK activity in intact hearts.

The aim of the present study was to test whether varying O2 tension alters the activity of AMPK in heart. AMPK activity and phosphorylation were measured in vitro from hearts in which the cytosolic [AMP] had been determined in vivo during periods of different O2 tension. The protocol design created steady-state conditions that generated similar [AMP] but different O2 tensions. In the first series of hearts, cytosolic [AMP] was changed from 0.2 to 16 µM using metabolic inhibitors (4) during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). In a second series of hearts, [AMP] was held <0.3 µM by arresting the hearts with high potassium concentration during variable hypoxia, which was created by changing O2 gas content (95, 40, 21, 10, and 0%). The results demonstrate that AMPK activity correlates to cytosolic [AMP] in oxygenated heart, and the absence of O2 increases the phosphorylation of Thr172 {alpha}-subunits and AMPK activity by a mechanism that is independent of [AMP]. This amplification of activity is most evident in contracting hearts at cytosolic [AMP] between ~2 and ~10 µM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
Preparation of Isolated Perfused Rat Hearts

Hearts of male Sprague-Dawley rats (body wt, 280–320 g) were isolated and perfused in the isovolumic Langendorff model (4). Krebs-Henseleit buffer (KH) perfusate contained (in mM) 118 NaCl, 5.9 KCl, 1.2 MgSO4, 25 NaHCO3, 1.75 CaCl2, 0.5 sodium EDTA, and 10 D-glucose and was equilibrated with 95% O2-5% CO2 with a resultant pH of 7.4. In a second series of studies, KH perfusate was also prepared as described above but with the addition of 0.5 mM pyruvate (Pyr-KH). Specific metabolic inhibitors or exogenous substrates were added to this perfusate as noted. A Stratham P23dB pressure transducer connected to the left ventricle balloon measured the left ventricular pressure and heart rate (HR). Hearts were electrically paced using a Grass stimulator (Grass; Quincy, MA). Left ventricular pressures were recorded with a MacLab system (ADInstruments; Colorado Springs, CO).

The experimental protocol was approved by the Harvard Medical Area Standing Committee on Animals and followed the recommendations of National Institutes of Health and the American Physiological Society's guidelines for the use and care of laboratory animals.

Metabolic Inhibitors

Addition of 0.3 mM 2-bromooctanoic acid (BrO; Aldrich; Milwaukee, WI) to the KH perfusate for 25 min irreversibly inhibits 3-ketothiolase and, thereby, the {beta}-oxidation of long-chain fatty acids, which arise from endogenous triacylglycerol (30). Ten minutes of perfusion with 0.4 mM amino-oxyacetate (AOA; Sigma; St. Louis, MO), which is a reversible inhibitor of the malate-aspartate shuttle (29), results in nearly complete inhibition of glucose oxidation (4).

Study Design and Experimental Protocols to Vary [AMP] and O2

All hearts were paced at a HR of 300 beats/min unless otherwise noted. Each of four groups consisted of two subgroups as follows: the O2 subgroup was perfused with KH equilibrated with 95% O2-5% CO2 that contained inhibitors or KCl, and the N2 subgroup was perfused identically until the final 10 min, when the KH perfusate was switched to one equilibrated with 95% N2-5% CO2. The four groups included 1) the KCl group, in which hearts were perfused with KH during the baseline period and then by KH with 25 mM KCl (98.9 mM NaCl) for 20 min with no pacing (n = 4 each for O2 and N2); 2) the Glc group, in which hearts were perfused with KH for the 30 min (n = 8 for O2 and 5 for N2); 3) the GA group, in which hearts were perfused with KH during the baseline period and subsequently by KH plus AOA for 20 min (n = 11 for O2 and 10 for N2); and 4) the GBA group, in which hearts were perfused with KH that contained BrO during the baseline period and then by KH plus AOA for a 10-min period with a HR of 300 beats/min and subsequently for 10 min at a HR of 450 beats/min (n = 5 each for O2 and N2). Half of all GA group hearts were paced at 300 beats/min for the entire 20 min, and half were paced at 300 beats/min for 10 min and 450 beats/min for 10 min. The different pacing rates did not result in different cardiac energetics; therefore, the results were combined. Six additional hearts were perfused with KHO2 plus AOA for 6 min (n = 2), 10 min (n = 2), and 12 min (n = 2) while being paced at 300 beats/min. Equilibration of 95% N2-5% CO2 with KH perfusate results in a minimal O2 partial pressure of 20 mmHg. At the end of the protocol, hearts were freeze-clamped with Wollenberger tongs cooled in liquid N2 and stored at –80°C until additional measurements were made.

A second series of hearts was perfused with Pyr-KH equilibrated with 95% O2-5% CO2 for 20 min and then with Pyr-KH with 25 mM KCl (98.9 mM NaCl; KCl Pyr-KH) for 16 min equilibrated with 95% O2-5% CO2. The perfusate was then switched to KCl Pyr-KH equilibrated with one of the following gases for 16 min: 1) 95% O2-5% CO2 (95% O2), n = 6; 2) 40% O2-55% N2-5% CO2 (40% O2), n = 4; 3) 21% O2-74% N2-5% CO2 (21% O2), n = 6; 4) 10% O2-85% N2-5% CO2 (10% O2), n = 4; and 5) 95% N2-5% CO2 (0% O2), n = 4. The solutions with different O2 tension created a variable hypoxia. At the end of the perfusion period, hearts were freeze-clamped with Wollenberger tongs cooled in liquid N2 and stored at –80°C until additional measurements were made.

Spectroscopy of Isolated Perfused Hearts

The 31P NMR free-induction decays (FIDs) were acquired at 161.8 MHz using an Inova spectrometer (Varian; Palo Alto, CA). Typically, 48 FIDs were averaged over 2 min using 60° pulses and a recycle time of 2.5 s. The resonance areas of phosphocreatine (PCr) and ATP and the chemical shifts of the inorganic phosphate (Pi) were quantified using Bayesian analysis software (G. L. Bretthorst; Washington University; St. Louis, MO). The analysis software uses a direct statistical analysis of the time-domain data (the FID) that is based on Bayesian probability theory (8). We have found it to provide superior signal amplitude analysis of the data compared with discrete Fourier transformation and analysis of the spectra. Saturation factors for resonances were determined from fully relaxed spectra (recycle time, 15 s).

Heart 31P Metabolite Content and Calculation of AMP

The concentrations of the 31P NMR-visible metabolites of hearts were derived using ATP as an internal standard. The area of the {beta}-resonance of ATP at the beginning of each protocol was set to 10.8 mM (3). The areas of other resonances, corrected for variable saturation, were then calculated on this basis. Intracellular pH (pHi) was calculated from the chemical shift of intracellular Pi. Metabolite measurements for all hearts are an average of the final 4 min of 31P NMR data. The creatine kinase equilibrium expression and 31P NMR measurements of concentrations of PCr ([PCr]), ATP ([ATP]), and H+ ([H+]) were used to calculate the cytosolic ADP concentration ([ADP]) (26). Cytosolic AMP concentration ([AMP]) was calculated using the adenylate kinase equilibrium expression (15, 26).

Measurement of AMPK Activity

Total AMPK activity was measured using the method of Dagher et al. (12), except that 0.1 µM of P1,P5-diadenosine-5'-pentaphosphate (Ad2P5), which is an inhibitor of adenylate kinase, was added to the homogenization and resuspension buffer. Addition of Ad2P5 to the homogenization and assay yields significantly higher and more consistent AMPK activity measurements. This indicates that adenylate kinase is present and active in the extract. Protein was precipitated from the homogenate using (NH4)2SO4. AMPK activity was quantified in the resuspended pellet as the incorporation of 32P from [{gamma}-32P]ATP (10 GBq/mmol; New England Nuclear; Boston, MA) into a synthetic peptide with the specific target sequence for AMPK, the SAMS peptide, which has amino acid sequence HMRSAMSGLHLVKRR (American Peptides; Sunnyvale, CA). Radioactivity was measured using a liquid scintillation counter (Tri-Carb 2100TR; Packard BioSciences, Meriden, CT). Protein content in the solution that contained the resuspended (NH4)2SO4 pellet was determined using the Bradford method (7).

The isoform-specific activity of AMPK in heart tissue was measured after tissue was homogenized in a buffer of the following composition: 50 mM HEPES, 150 mM NaCl, 100 mM NaF, 10 mM Na4P2O7, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 0.1 µM Ad2P5, pH 7.5. After immunoprecipitation of the {alpha}1- and {alpha}2-subunits by anti-AMPK-{alpha}1 and -{alpha}2 antibodies (a gift from Dr. Neil Ruderman) coupled to protein A agarose beads (Upstate Biotechnology; Lake Placid, NY), activity was measured as the incorporation of 32P into the SAMS peptide. Protein content was determined in the homogenization buffer and was adjusted to a content of 0.5 mg/ml before immunoprecipitation.

Measurement of Phosphorylation of {alpha}-Thr172 of AMPK

Frozen rat heart tissue samples were homogenized in ice-cold lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM Na4P2O7, 1 mM {beta}-glycerol phosphate, 1 mM Na3VO4, and 1 µg/ml leupeptin] using a T25 Basic homogenizer (IKA Works; Wilmington, NC) and were incubated with 1% Triton X-100 at 4°C for 2 h. Lysates were generated by centrifugation at 20,000 g for 30 min at 4°C as total cellular protein. Protein concentration in lysates was measured by the modified Bradford method (Protein Assay Kit II; Bio-Rad; Hercules, CA) with bovine serum albumin as a standard. Protein samples (50 µg each) were electrophoresed under reducing denaturing conditions in 8% polyacrylamide-SDS gels and transferred by electroblotting onto a nitrocellulose membrane. Membranes were blocked in 5% nonfat milk (Bio-Rad) for 1 h at room temperature. Membranes were then incubated with an anti-phospho-AMPK-{alpha} polyclonal antibody (Thr172; Cell Signaling Technology; Beverly, MA), an anti-AMPK-{alpha} polyclonal antibody (Cell Signaling Technology), an anti-phospho-acetyl-CoA carboxylase polyclonal antibody (S-79; Upstate), or an anti-acetyl-CoA carboxylase polyclonal antibody (Upstate) overnight at 4°C and were subsequently incubated with horseradish peroxidase-conjugated secondary antibody (Southern Biotech; Birmingham, AL) for 2 h at room temperature. Signal detection was facilitated with enhanced chemiluminescence (ECL Kit; Amersham; Piscataway, NJ). The membrane immunosignals were quantitated using an image-scanning densitometer (model GS-700 imaging densitometer and Quantity One 4.2.1 software; Bio-Rad).

Statistical Analysis

The data are presented as means ± SE. Statistical computations were performed with StatView 5.0.1 (SAS Institute; Cary, NC). An ANOVA was used to compare measurements between matched O2 and N2 groups and among all groups. A post hoc Fishers protected least-significant difference was used for comparison of the means. Differences were declared statistically significant if P < 0.05. Prism 4.0 for Windows (GraphPad Software; San Diego, CA) was used for graphs and fitting of the measurements of AMPK activity and [AMP] to the appropriate equations.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANT
 REFERENCES
 
31P NMR-Measured Metabolite Content of Isolated Heart During Protocol

The isolated perfused rat heart, in which glucose is the only exogenous fuel supplied, also oxidizes endogenous fuels including glucose from glycogen and fatty acids from triacylglycerols. To increase the cytosolic [AMP] in these hearts, the heart's supply of acetyl-CoA was limited using the inhibitors AOA and BrO separately or together. AOA limits the oxidation of glucose and thus restricts oxidation to fatty acids. BrO inhibits the {beta}-oxidation of long-chain fatty acids. The combination of AOA and BrO limited the oxidation of pyruvate and fatty acids, which restricted the hearts to ATP derived from glycolytic activity. The use of these inhibitors decreases energy production (ATP synthesis) in aerobic hearts. This reduces high-energy phosphate contents, particularly PCr (4). Because of the equilibrium of the creatine kinase reaction, the reduction of [PCr] results in an increase in [ADP]. In turn, the near equilibrium of the adenylate kinase reaction translates the increase in [ADP] into an increase in [AMP]. Thus 31P NMR measures of PCr, ATP, and pHi in intact hearts provided an estimate of cytosolic [AMP] in vivo. To alter the O2 supply, heart perfusion was switched from KH equilibrated with 95% O2-5% CO2 (normoxia, O2 hearts) to KH equilibrated with 95% N2-5% CO2 (hypoxia, N2 hearts). The protocol design created steady-state conditions that generated similar ranges of [AMP] but different O2 tensions for the O2 and N2 hearts. The KCl Pyr-KH hearts were not treated with inhibitors. The low ATP demand of a K+-arrested heart allows glycolysis to maintain the ATP supply. Thus these hearts maintain relatively low, constant [AMP] during variable hypoxia.

Altering metabolic supply and demand in the four groups of hearts (KCl, Glc, GA, and GBA) resulted in a range of [PCr], which in turn generated a range of [AMP] (Table 1). Hearts perfused with GA KH that was equilibrated with 95% O2-5% CO2 or 95% N2-5% CO2 exhibited reduced PCr and increased [AMP] (Fig. 1).


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  		Table 1. Concentrations of heart metabolites and intracellular pH of KH-perfused hearts

 


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  		Fig. 1. 31P NMR spectra obtained from isolated rat hearts during experimental protocol. Perfusion conditions were as follows: GA O2, Krebs-Henseleit (KH) plus amino-oxyacetate (AOA) gassed with 95% O2-5% CO2 (top); and GA N2, KH plus AOA gassed with 95% N2-5% CO2 (bottom) 31P NMR resonances are identified as phenylphosphonic acid (PPA), the external standard; phosphomonoesters (PME); inorganic phosphate (Pi); phosphocreatine (PCr); the {gamma}-, {alpha}-, and {beta}-phosphates of ATP; and NAD+ and NADH (NAD). Total AMP-activated protein kinase (AMPK) activities measured in vitro for these hearts (left) and in vivo AMP concentration ([AMP]) calculated from each spectrum (right) are indicated.

 
Hardie and Carling (17) suggested that AMPK responds to the AMP/ATP ratio rather than just to AMP. Because [ATP] varies little in these hearts, the differences in the AMP/ATP ratios between the normoxic and hypoxic hearts were similar to the [AMP] values (Table 1). The AMP/ATP ratio does not explain the greater AMPK activity of the N2 hearts.

AMPK Activity

Total AMPK activity. For any pair of O2 and N2 heart perfusion groups, the total AMPK activity was higher in the N2 hearts (Table 2). This difference in activity occurred even though the [AMP] was lower in the GA N2 than the GA O2 group hearts and equal in the KCl and the GBA O2 and N2 groups. As expected, the Glc N2 hearts had higher [AMP] and AMPK activity than the Glc O2 hearts.


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  		Table 2. AMP-activated protein kinase activities of hearts

 
Relationship between total AMPK activity and cytosolic [AMP]. AMP is an allosteric activator of AMPK. Binding of AMP increases the phosphorylation of {alpha}-Thr172 by AMPKK, which increases AMPK activity (Fig. 2). Rapid freeze-clamping preserves the physiological phosphorylation status of AMPK. The in vitro activity measurements, therefore, reflect the in vivo phosphorylation state of the enzyme. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the individual measurements from O2 and N2 hearts to the equation

(1)
where {upsilon} is the AMPK activity, A0.5 is the [AMP] at 50% AMPK activation, and Vmax is the maximal activity. Parameters (means ± SE) obtained from the fitting were as follows: for the O2 hearts, A0.5 = 28 ± 13 µM and Vmax = 102 ± 36 pmol·min–1·mg protein–1; for the N2 hearts, A0.5 = 3 ± 1 µM and Vmax = 61 ± 11 pmol·min–1·mg protein–1. A0.5 for the O2 and N2 group hearts were different, but the Vmax for the O2 and N2 group hearts were similar.



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  		Fig. 2. Total AMPK activity as a function of [AMP]. A curve of the equation {upsilon} = Vmax x [AMP]/A0.5 + [AMP] where {upsilon} is AMPK activity, A0.5 is [AMP] at 50% AMPK activation, and Vmax is the maximal activation, was fit to the measurements of [AMP] and total AMPK activity from the individual O2 and N2 hearts. A0.5 values for the O2 and N2 group hearts were different (P = 0.002), whereas the Vmax values were the same (P = 0.31). Fitting of the total AMPK activity from the O2 hearts (n = 36) yielded A0.5 = 28 ± 13 µM and Vmax = 102 ± 36 pmol·min–1·mg protein–1 with 95% confidence intervals (A0.5 = 2–54 µM and Vmax 29–175 pmol·min–1·mg protein–1) and R2 = 0.88. Fitting of the total AMPK activity from the N2 hearts (n = 25) yielded A0.5 = 3.1 ± 1.5 µM and Vmax = 61 ± 11 pmol·min–1·mg protein–1 with 95% confidence intervals (A0.5 = 0.1–6.2 µM and Vmax = 37–84 pmol·min–1·mg protein–1) and R2 = 0.61.

 
In our previous study of AMPK activity and cytosolic [AMP] in heart, we used the same methods to increase [AMP] as were used with the O2 groups of the present study (15). The results reported here are different. The previous study reported the AMP A0.5 to be 1.8 ± 0.3 µM. This is considerably lower than the 28 ± 13 µM identified for the O2 hearts of the present study. The reasons for this discrepancy are not yet known. In the previous study, the NMR and AMPK activities were measured in separate groups of hearts. One possibility is that the hearts that underwent NMR measurements may have been perfused at a slightly lower temperature (1–2°C) than the hearts in which AMPK activity was measured. This and/or other differences between the two studies could account for the different values of A0.5. The determination of in vivo [AMP] and in vitro AMPK activity in the same heart, as reported here, is clearly optimal.

Isoform-specific AMPK activity. Both catalytic {alpha}-isoforms of AMPK are present in heart. Our measurements of {alpha}-isoform-specific activity for Glc O2 hearts are in agreement with reports for the well-perfused oxygenated rat heart, that the {alpha}2-isoform activity is ~10 times that of the {alpha}1-isoform activity (33, 35). For all pairs of O2 and N2 heart perfusion groups, the {alpha}1-isoform-specific AMPK activities were equal (Table 2). For the Glc and GA O2 and N2 heart perfusion groups, the {alpha}2-isoform-specific AMPK activity was higher in the N2 hearts. The {alpha}2-isoform-specific AMPK activities were equal among the Glc, GA, and GBA N2 heart groups.

To determine the dependence of {alpha}1-isoform-specific AMPK activity on cytosolic [AMP], measurements from O2 and N2 hearts were fit to Eq. 1. The curves for the two sets were not different; therefore, one curve was fit to both sets. The parameters for the combined O2 and N2 data were A0.5 = 3 ± 2 µM and Vmax = 2.8 ± 0.6 pmol·min–1·mg protein–1 (Fig. 3A).



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  		Fig. 3. Isoform-specific AMPK activity as a function of [AMP]. A curve of the equation used in Fig. 2 was fit to the measurements of [AMP] and isoform-specific AMPK activity from the individual O2 hearts and N2 hearts. A: fitting of the {alpha}1-isoform activity from the combined O2 hearts (n = 24) and N2 hearts (n = 25) yielded A0.5 = 3.3 ± 1.8 µM and Vmax = 29.8 ± 0.6 pmol·min–1·mg protein–1 with 95% confidence intervals (A0.5 = –0.5–7.0 µM and Vmax = 1.6–4.0 pmol·min–1·mg protein–1) and R2 = 0.30. B: fitting of the {alpha}2-isoform activity from the O2 hearts (n = 24) yielded A0.5 = 13.1 ± 7.6 µM and Vmax = 191 ± 67 pmol·min–1·mg protein–1 with 95% confidence intervals (A0.5 = –2.7 to 28.9 µM and Vmax = 53–329 pmol·min–1·mg protein–1) and R2 = 0.79. Fitting of the {alpha}2-isoform activity from the N2 hearts (n = 25) yielded A0.5 = 1.9 ± 0.8 µM and Vmax = 161 ± 21 pmol·min–1·mg protein–1 with 95% confidence intervals (A0.5 = 0.2–3.6 µM and Vmax = 117–205 pmol·min–1·mg protein–1) and R2 = 0.64. The {alpha}2-AMPK A0.5 values for the O2 and N2 hearts were different (P = 0.014), but the Vmax values were the same (P = 0.69).

 
The dependence of {alpha}2-isoform-specific AMPK activity on cytosolic [AMP] was determined using Eq. 1. The parameters obtained were as follows: for the O2 hearts, A0.5 = 13 ± 8 µM and Vmax = 191 ± 67 pmol·min–1·mg protein–1; for the N2 hearts, A0.5 = 1.9 ± 0.8 µM and Vmax = 161 ± 21 pmol·min–1·mg protein–1 (Fig. 3B). Similar to the results for total AMPK activity, the {alpha}2-AMPK A0.5 values for the O2 and N2 hearts were different, whereas the Vmax values were not different.

Because the protein determinations for total activity and isoform-specific activities are different, the sum of {alpha}1- and {alpha}2-activities does not equal the total activity. The total activity protein was measured after (NH4)2SO4 precipitation from the homogenization buffer. The isoform-specific activity protein was determined in homogenization buffer before immunoprecipitation.

Phosphorylation of AMPK-{alpha}-Thr172 and ACC Ser79 in Normoxic and Anoxic Hearts with Variable [AMP]

The phosphorylation state of Thr172 on the {alpha}-subunit of AMPK was determined by Western blot analysis for KCl, Glc, GA, and GBA O2 and N2 hearts (Fig. 4A). The phosphorylation of AMPK increased as cytosolic [AMP] increased in the O2 hearts. An increase in AMPK phosphorylation that is independent of [AMP] (see Table 1) was evident for the KCl N2 hearts (Fig. 4B).



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  		Fig. 4. Western blot analyses of the phosphorylation of AMPK {alpha}-Thr172 and acetyl-CoA carboxylase (ACC) Ser79 during normoxia and hypoxia (see Study Design and Experimental Protocols to Vary [AMP] and O2 for group descriptions). A: representative Western immunoblots were used to measure the phosphorylation of Thr172 on the {alpha}-subunit of AMPK in Glc and KCl O2 and N2 hearts (top) and Glc, GA, and GBA O2 and N2 hearts (bottom); 50 µg of protein was loaded in each lane. B: a plot of the relative phosphorylation of the AMPK {alpha}-subunit Thr172 from Western immunoblots of the O2 and N2 hearts of KCl, Glc, GA, and GBA groups. AMPK-P signal values were normalized as percentages of the optical density of one Glc-N2 sample on each blot. Values are means ± SE; *P < 0.05, measurement vs. relevant O2 group; {dagger}P < 0.05, measurement vs. Glc O2 group; {ddagger}P < 0.05, measurement vs. Glc N2 group. C: a representative Western immunoblot was used to measure the phosphorylation of Ser79 on ACC in Glc O2 and N2 hearts and KCl O2 and N2 hearts; 50 µg of protein was loaded in each lane. D: relative phosphorylation of ACC Ser79 from Western immunoblots plotted for hearts. ACC-P signal values were normalized as percentages of the density of one Glc-N2 sample on each blot. *P < 0.05, measurement vs. relevant O2 group; {dagger}P < 0.05, measurement vs. Glc O2 group.

 
Because AMPK plays a major role in the regulation of ACC activity, the phosphorylation state of Ser79 was determined by Western blot analysis (Fig. 4C). The results demonstrate an increase in ACC phosphorylation that is evident between the KCl O2 and N2 hearts as well as between the KCl O2 and Glc O2 hearts. This parallels the AMPK activity of these hearts (see Table 2). The relative phosphorylation of ACC Ser79 is, however, nearly complete in the Glc O2 hearts (Fig. 4D).

ACC phosphorylation was also studied in four groups of hearts in which the [AMP] was varied by alteration of oxygenated KH as follows: 1) Pyr-KH plus KCl (Pyr-KCl), 2) Pyr alone, 3) reduction of Glc group concentration to 5 mM (Glc 5 mM), and 4) no change in Glc group (Glc 10 mM). The in vivo [AMP] in these hearts (n = 18) varied from 0.08 to 1.3 µM, and the in vitro total AMPK activity varied from 1 to 4 pmol·min–1·mg protein–1. The phosphorylation of ACC Ser79 was determined by Western blot analysis (Fig. 5A). Relative ACC phosphorylation increased from 0.1 to 1.0 as AMPK activity rose from 1 to 2 pmol·min–1·mg protein–1 (Fig. 5B). The data were fit to the equation for a Boltzmann sigmoid function

(2)
where V50 is the center. The V50 value was found to be 1.6 ± 0.01 pmol·min–1·mg protein–1. This is the AMPK activity at which the ACC phosphorylation is at 50% of maximal activity (top).



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  		Fig. 5. Western blot analysis of the phosphorylation of ACC Ser79 during normoxia. A: a representative Western immunoblot was used to measure the phosphorylation of Ser79 on ACC in hearts from the following groups (from the left): lanes 1–3, Pyr-KCl; lanes 4 and 5, Pyr; lanes 6 and 7, 5 mM Glc; and lanes 8–10, 10 mM Glc; 50 µg of protein was loaded in each lane. B: relative phosphorylation of ACC Ser79 from two Western immunoblots plotted as a function of the AMPK activity (n = 18). ACC-P signal values were normalized as percentages of the density of one 10 mM glucose-KH sample or 5 mM glucose-KH sample on each blot. Solid line was fit to the data from the Pyr-KCl (n = 6), Pyr (n = 4), 5 mM Glc (n = 4), and 10 mM Glc (n = 4) hearts using a Boltzmann sigmoid function (Eq. 2), where V50 is the center. Bottom = 0.13 ± 0.06, top = 0.85 ± 0.06, V50 = 1.6 ± 0.1, slope = 0.08 ± 0.09; R2 = 0.86.

 
In KH-perfused hearts, the phosphorylation of ACC Ser79 correlates with in vivo [AMP] and AMPK activity over a very small range. The phosphorylation of ACC by AMPK will be complete as [AMP] approaches ~1 µM. This is consistent with the AMPK cascade being poised to maximize fatty acid oxidation within the range of [AMP] encountered in the oxygenated heart. It also suggests that phosphorylation of ACC is not a reliable indicator of AMPK activity during situations where [AMP] is greatly altered, i.e., during ischemia.

AMPK Activity and Phosphorylation of AMPK-{alpha}-Thr172 in Normoxic and Anoxic Hearts with Variable [AMP]

The {alpha}-subunit Thr172 has been identified as the major site of AMPK phosphorylation by AMPKK in vitro (34). The phosphorylation of this site has been shown to correlate with increased AMPK activity in gastrocnemius muscle in vivo (28). In the present study, the relative phosphorylation of the Thr172 residue of the AMPK {alpha}-subunit for heart increased as total activity measurements increased (Fig. 6). The maximal AMPK activity was observed with a relative phosphorylation of ≥0.75 (arbitrary scale). The minimal activity was with a relative phosphorylation of about ≤0.4. A straight (dashed) line was fit to the data. The data are, however, not randomly distributed around the line, as would be expected for a linear relationship. The data were also fit to the equation for a Boltzmann sigmoid function. To the eye, this sigmoid (solid line) provides a better description of the data. The V50 value (or the center) was found to be 0.49 ± 0.03.



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  		Fig. 6. Relationship between total AMPK activity and relative phosphorylation of the {alpha}-subunit Thr172 of AMPK. Total AMPK activity for hearts from the groups KCl O2 (n = 4), Glc O2 (n = 4), GA O2 (n = 10), and GBA O2 (n = 4), KCl (n = 4), Glc N2 (n = 5), GA N2 (n = 10), and GBA N2 (n = 5) plotted vs. the relative phosphorylation of the AMPK {alpha}-subunit Thr172 (see Fig. 4B) from the same hearts. Dashed line, which was fit using linear regression, yielded the following values: y-intercept = 6 ± 4; slope = 25 ± 5; R2 = 0.78; P = 0.003. Solid line was fit using a Boltzmann sigmoid function with the following values: bottom = 3.2 ± 3 pmol·min–1·mg protein–1, top = 39 ± 2 pmol·min–1·mg protein–1, V50 = 0.49 ± 0.03, slope = 0.098 ± 0.033; and R2 = 0.77.

 
Phosphorylation of AMPK-{alpha}-Thr172 and ACC Ser79 During Variable Hypoxia in Hearts with Constant [AMP]

To define the minimum O2 content for the activation of AMPK, another series of hearts was perfused with Pyr-KH that contained KCl to arrest contraction. The addition of pyruvate to the KH perfusate increases the [PCr] and, hence, minimizes [AMP] in these hearts. The Pyr-KH perfusate was equilibrated with five different gases (95, 40, 21, 10, and 0% O2) in five groups of hearts during the final 16 min of perfusion. During variable hypoxia, the [AMP] for each of the five groups was similar and was ≤0.3 µM (Table 3).


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  		Table 3. Concentrations of heart metabolites of KCl Pyr-KH-perfused hearts during variable hypoxia

 
At [AMP] below 0.3 µM, the phosphorylation of AMPK in the hearts increased with decreasing O2 content in the Pyr-KH group (Fig. 7A). The total {alpha}-AMPK remained constant. The O2 content threshold was 21% for a significant increase in the phosphorylation of AMPK (Fig. 7B). The phosphorylation of AMPK-{alpha}-Thr172 in the 21, 10, and 0% O2 hearts was greater than that of the 95% O2 hearts. The phosphorylation of ACC Ser79 was increased only for the 0% O2 hearts (Fig. 7, C and D). The total ACC remained constant.



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  		Fig. 7. Western blot analysis of the phosphorylation of AMPK {alpha}-Thr172 and ACC Ser79 during variable hypoxia. A: a representative Western immunoblot was used to measure the phosphorylation of Thr172 on the {alpha}-subunit of AMPK (top) and the {alpha}-subunit of AMPK (bottom) in Pyr-KH KCl hearts (n = 3/group) for the 95, 40, 21, and 0% O2 groups; 50 µg of protein were loaded in each lane. B: relative phosphorylation of the {alpha}-subunit Thr172 was obtained from Western blots for all Pyr-KH KCl hearts. AMPK-P signal values were normalized as percentages of the density of one 0% O2 sample on each blot. *P < 0.05 vs. 95% O2 group; #P < 0.05 vs. 95, 40, 21, and 10% O2 groups. C: a representative Western immunoblot was used to measure the phosphorylation of Ser79 on ACC (top) and ACC (bottom) in Pyr-KH KCl hearts (n = 3/group) from the 95, 40, 21, and 0% O2 groups; 50 µg of protein were loaded in each lane. D: relative phosphorylation of ACC Ser79 was obtained from Western blots for all Pyr-KH KCl hearts. ACC-P signal values were normalized as percentages of the density of one 0% O2 sample on each blot. #P < 0.05 vs. 95, 40, 21, and 10% O2 groups.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANT
 REFERENCES
 
Results reported here define the relationship between AMPK activity measured in vitro and the cytosolic [AMP] measured in vivo in isolated perfused rat hearts during two states of oxygenation, normoxia and hypoxia. The results demonstrate an increase in total AMPK and {alpha}2-AMPK activity during hypoxia that is independent of [AMP]. The increase is most evident when [AMP] is greater than ~2 µM but less than ~10 µM. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or by some other effect that reduces the A0.5 for [AMP].

The AMPKK-AMPK protein kinase cascade responds to [AMP] increases (17, 18). Increases in [AMP] activate the cascade via four mechanisms (11, 13, 23). First, AMP allosterically activates AMPKK. Second, AMP binds to AMPK, which makes AMPK a better substrate for AMPKK, which then phosphorylates Thr172 of the AMPK {alpha}-subunit. This phosphorylation is apparently necessary for AMPK activity. Third, AMP binds to AMPK, which makes it a poorer substrate for protein phosphatases. Fourth, AMP allosterically activates phosphorylated AMPK. The activating effects of AMP are antagonized by high concentrations of ATP (17). Because AMPK is activated when AMP is elevated and ATP is depressed, AMPK is hypothesized to act as a cellular "fuel gauge" (17).

The {gamma}-subunit of AMPK contains four tandem pairs of CBS domains that provide two binding sites for AMP and ATP (31). CBS domain pairs have been termed Bateman domains (25). In vitro studies of the phosphorylation of AMPK by AMPKK, using calmodulin kinase kinase as a surrogate for AMPKK, found that the AMP A0.5 was 4.3 ± 0.6 µM in the presence of 200 µM ATP (19). At an [ATP] of 2 mM, the AMP A0.5 increased to ~100 µM. Assays of the AMP allosteric effect on isolated phosphorylated AMPK activity revealed an A0.5 of ~3 µM in the presence of 200 µM ATP and an A0.5 for AMP of 30 µM at an [ATP] of 4 mM (11). Adams et al. (1) modeled the {gamma}-subunit Bateman domain and proposed that when occupied by ATP, the {beta}- and {gamma}-phosphates extend out of the pocket and bridge the {alpha}- and {gamma}-subunits. In this way, it was proposed that ATP acts as an intrasteric inhibitor of AMPK.

The ~90% reduction in the A0.5 of AMP for AMPK activity (total and {alpha}2) observed here in the hypoxic heart could result from a change in the binding affinity of either AMP or ATP to the {gamma}-subunit. Replacement of amino acid residues within the Bateman domains of the {gamma}-subunit alters the A0.5 for AMP (1). There are no data to suggest that covalent modifications such as phosphorylation of the {gamma}-subunit occur. The A0.5 (2–3 µM) observed in the hypoxic heart could result from an [ATP] of ~200 µM. Because the cytosolic [ATP] is 6–10 mM in those hearts, this would require that AMPK be sensing a discrete compartment where AMP increases and ATP drastically decreases. The action of adenylate kinase concomitantly produces AMP with ATP. Such a hypothetical compartment would have to exclude adenylate kinase, which is in the cytosol and the mitochondrial intermembrane space. The formation of fatty acid-coenzyme A results in the conversion of ATP to AMP, and thus could result in locally elevated AMP with reduced ATP. This reaction takes place, however, on the outer membrane of the mitochondrion. AMPK cellular localization has been studied in HEK-293 cells, where it was found to be associated with the endoplasmic reticulum and/or the mitochondria (36).

Another hypothetical possibility to explain the apparent change in A0.5 is that phosphorylation of a site (other than Thr172) on the {alpha}-subunit reduces the potential for intrasteric inhibition by ATP bound to the {gamma}-subunit. Two other phosphorylation sites on the {alpha}2 subunit, Thr258 and Ser491 (Ser485 on the {alpha}1-subunit), have been identified (38). These sites may be phosphorylated by an AMPKK that is insensitive to AMP and sensitive to low O2 levels. Phosphorylation of these two sites on {alpha}1-AMPK, however, did not lead to increased activity. In the present study, there was no difference in the {alpha}1-AMPK activity between normoxic and hypoxic hearts, but the {alpha}2-AMPK activity displayed an increase resulting from hypoxia. The NH2-terminal portion of the {alpha}-subunit, which includes Ser485, is involved in the binding of {alpha}- to the {beta}- and {gamma}-subunits.

There are reports of increases in AMPK activity that do not involve alterations in [AMP]. Osmotic stress and metformin, which is used in the treatment of Type 2 diabetes, are reported to increase AMPK activity and phosphorylation of AMPK-{alpha}-Thr172 without alterations in [AMP] or the AMP/ATP ratio (16, 22). The signals resulting from these two stimuli that lead to the activation of AMPK have not been identified. Beauloye et al. (5) reported that treatment of hearts with high dosages of insulin before hypoxia reduced AMPK activity during hypoxia with no difference in the AMP/ATP ratio. They also reported that the reduction by insulin of AMPK activity during hypoxia could be blocked with wortmannin, which suggests the involvement of phosphatidylinositol-3 kinase. We tested the hypothesis that insulin pretreatment reduces AMPK activity during hypoxia and have not observed reductions in AMPK activity (unpublished observations).

Complexes of LKB-1 tumor suppressor, STRAD-{alpha},{beta} and MO25-{alpha},{beta}, have recently been identified as upstream kinases in the AMPK cascade (20). Two AMPKK activities, AMPKK-1 and AMPKK-2, which contain LKB-1, STRAD-{alpha},{beta}, and MO25-{alpha},{beta}, have been purified from rat liver. AMPKK activities were not increased by addition of AMP, but these AMPKK complexes increased the phosphorylation of AMPK when AMP was bound to AMPK. Because there are reports that AMPKK activity increases with added AMP (23), this may indicate that LKB-1 is not the only AMPKK.

The cytosolic [AMP]-independent increase in AMPK activity and phosphorylation found in this study during hypoxia is consistent with an AMP-independent increase in AMPKK activity. A recent report (2) has found evidence for an AMP-independent increase in AMPKK activity in mildly ischemic rat hearts. This suggests the hypothesis that AMPKK is responding to an O2-sensing system. O2-sensing systems consist of a sensor that alters some mediator in response to changes in O2 tension. This mediator then functions to elicit a physiological response. The activity of hypoxia-inducible factor 1 (HIF-1), a transcription factor, is based upon cellular O2 concentration with low O2 increasing HIF-1 activity. HIF-1 targets genes that encode proteins that increase O2 delivery and mediate adaptive responses to O2 deprivation (32). The HIF-1 intracellular O2 sensor has been identified as a prolyl hydroxylase (14, 24). Ideally, an O2 sensor/mediator system will respond to mild hypoxia before ATP declines. The increase in AMPK activity with hypoxia between ~2 and ~10 µM [AMP] and the increase in AMPK phosphorylation with decreasing O2 tension are consistent with the actions of an O2-sensor/mediator system interacting with the AMPK cascade.

Oxidative stress caused by treating NIH-3T3 cells with H2O2 elevated the AMP/ATP ratio and increased AMPK activity (10). This may indicate that reactive oxygen species activate AMPK. Superoxide, a reactive oxygen species, has been shown to increase during hypoxia (9) and ischemia (6) in neonatal rat myocytes and during ischemia-reperfusion in isolated rat hearts (27). Peroxynitrite is formed by the reaction of nitric oxide with superoxide. Exposure of bovine aortic endothelial cells to peroxynitrite increased the phosphorylation of the {alpha}-subunit Thr172 of AMPK without an increase in [AMP] (39). Hypoxia and reoxygenation increased peroxynitrite and the phosphorylation of the {alpha}-subunit Thr172 (39). Thus it is possible that the [AMP]-independent increase in AMPK activity results from an elevation of reactive oxygen species that increase peroxynitrite.

In conclusion, 31P NMR measures of PCr, ATP, and pHi combined with the creatine kinase and adenylate kinase equilibrium expressions provide the most accurate estimates of cytosolic [AMP] available. As [AMP] increases in the presence of abundant O2, the activities of both the total AMPK and the AMPK {alpha}-isoforms increase. In hypoxic hearts, AMPK activity increased in an [AMP]-independent fashion. The reduction in the A0.5 for AMP in hypoxic hearts is consistent with one or more of the following hypothesis: 1) AMPK senses a compartment with an [ATP] much lower than that of the cytoplasm, 2) the inhibitory effect of ATP upon AMPK is modified possibly by phosphorylation of a site other than {alpha}-Thr172, or 3) the binding of AMP to {gamma}-subunit Bateman domain is altered by phosphorylation of a site other than {alpha}-Thr172. Alternatively, the increase in AMPK activity may result from direct phosphorylation of {alpha}-Thr172 AMPK by an upstream kinase that is responding directly or indirectly to low O2 and not to an increase in [AMP]. This would reduce the apparent A0.5 of AMP during hypoxia.

AMPK is hypothesized to function as a cellular fuel gauge or low-energy sensor. The response of AMPK activity to hypoxia provides the low-energy sensor with another means to sense and respond to energy status. Thus the AMPK cascade responds to externally supplied O2, which may be considered a fuel, and to the internal metabolite AMP, which directly reflects alterations in ATP, the cellular energy currency that is generated by carbon fuel metabolism. The threshold for response by AMPK cascade to increasing [AMP] is lower in hypoxia. The mechanism by which the low-O2 signal is transmitted to the AMPK cascade is undefined.


   GRANT
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
REFERENCES
 
National Institutes of Health Grant HL-46033 (to J. A. Balschi) supported this work.

Present address of M. Frederich: Department of Biological Sciences, University of New England, Biddleford, ME 04005.


   ACKNOWLEDGMENTS
 
The authors thank Prof. Joanne Ingwall for a critical reading of the manuscript, Prof. Neil Ruderman for providing the {alpha}1- and {alpha}2-specific antibodies, and Dr. G. Larry Bretthorst of Washington University (St. Louis, MO) for Bayes analysis software.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Balschi, 221 Longwood Ave., BLI 247, Boston, MA 02115 (E-mail: jbalschi{at}rics.bwh.harvard.edu)

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.

* L. Zhang and M. Frederich contributed equally to this work. Back


   REFERENCES
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 DISCUSSION
 GRANT
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