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Am J Physiol Heart Circ Physiol 290: H1235-H1243, 2006. First published October 28, 2005; doi:10.1152/ajpheart.00906.2005
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Relationship between 5-aminoimidazole-4-carboxamide-ribotide and AMP-activated protein kinase activity in the perfused mouse heart

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

NMR Laboratory for Physiological Chemistry, Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts

Submitted 23 August 2005 ; accepted in final form 24 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
AMP-activated protein kinase (AMPK) is a cellular energy sensor whose activity responds to AMP concentration ([AMP]). An agent that activates AMPK in cells is 5-aminoimidazole-4-carboxamide-1-riboside (AICA-riboside). Phosphorylated AICA-riboside or AICA-ribotide (ZMP) is an AMP analog. It is generally assumed that ZMP accumulation does not alter [AMP]. Additionally, the effect of AICA-riboside on AMPK activity of the heart is uncertain. Two hypotheses were tested in the isolated mouse heart: 1) sufficient ZMP concentration ([ZMP]) forms to increase AMPK activity, and 2) [ZMP] accumulation increases [AMP]. Perfusion of isolated mouse hearts with Krebs-Henseleit buffer containing 0.15–2 mM AICA-riboside concentration resulted in [ZMP] of 2–8 mM. ZMP accumulation reduced phosphocreatine concentration, which increased cytosolic [AMP]. In hearts with [ZMP] less than ~3 mM, in vivo AMPK allosteric activity effects of ZMP were observed; AMPK phosphorylation and [AMP] were not increased. With [ZMP] between 3 and 5 mM, in vitro AMPK activity and phosphorylation increased with unchanged [AMP]. This occurred in hearts perfused with 0.25 mM AICA-riboside for 48 min and 0.5 mM AICA-riboside for 24 min. The [ZMP] resulting in 50% AMPK activity (covalent phosphorylation of AMPK) was 4.1 ± 0.6 mM. Hearts with [ZMP] >5 mM displayed increased [AMP] and AMPK activity that was not different from hearts with similar [AMP] with no [ZMP]; the half-maximal activity of AMP was 5.6 ± 1.6 µM. Thus, in mouse hearts, AICA-riboside was metabolized to [ZMP] adequately to increase AMPK activity. Higher [ZMP] also increased cytosolic [AMP], which affects AMPK activity.

signal transduction; acetyl-CoA carboxylase; 5'-aminoimidazole-4-carboxamide-1-beta-D-riboside

AMP-ACTIVATED PROTEIN KINASE (AMPK) and AMPK kinase (AMPKK) comprise a protein kinase cascade that has been highly conserved throughout evolution (10, 11). The AMPK cascade acts as a cellular low fuel warning system (10). Increases in AMP concentration ([AMP]) increase AMPK activity (7, 8). Once activated, AMPK functions either to conserve ATP or to promote alternative methods of ATP generation. One way that AMPK stimulates ATP synthesis in the heart is by the 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 an acceleration of the beta-oxidation of fatty acids and the generation of ATP.

Increased AMPK activity has been elicited by use of ischemia, hypoxia, or metabolic inhibitors to alter cellular energetics and, hence, increase [AMP]. A pharmacological agent, 5-aminoimidazole-4-carboxamide-riboside (AICA-riboside; Z-riboside), has been used to increase AMPK activity in a wide range of tissues and cell types, including adipocytes (4) and skeletal muscle (17, 22). AICA-riboside does not directly activate AMPK. AICA-riboside must be phosphorylated to the active agent 5-aminoimidazole-4-carboxamide-ribotide (AICA-ribotide; ZMP). ZMP accumulation in INS-1 cells did not alter total AMP and ATP content but did increase AMPK activity (12). Total AMP content was also unchanged in gastrocnemius muscles, with ZMP formation accompanied by increased AMPK activity (22). ZMP accumulation, however, decreased phosphocreatine (PCr) in rat white quadriceps muscle (30). This raises the possibility that AICA-riboside metabolism can reduce PCr concentration ([PCr]). We have demonstrated that reductions in [PCr] increased cytosolic [AMP] in the heart (7, 8).

ZMP, a structural analog of AMP, increases AMPK activity in vitro. The ZMP concentrations ([ZMP]) that affect AMPK activity are 50 times higher than the [AMP] required for increased AMPK activity. In vitro measurements of the concentrations required for half-maximal allosteric activity (A0.5) of AMPK have elucidated the following: 1) in the presence of 4 mM ATP, the A0.5 for AMP was 29 ± 14 µM, whereas the A0.5 for ZMP was 1.5 ± 0.6 mM (4); and 2) the A0.5 for ZMP was ~5 mM in the presence of 3 mM ATP (16). Two measurements of the A0.5 for ZMP for phosphorylation of AMPK in vivo have been reported. In hepatocytes, the A0.5 of ZMP for phosphorylation of AMPK was ~2 mM (4). A ZMP/ATP of ~0.2 was required for A0.5 of AMPK in INS-1 cells (12).

Reports of the effects of AICA-riboside treatment on the AMPK activity of the heart have been contradictory. Russell et al. (23) reported that an in vivo arterial AICA-riboside concentration ([AICA-riboside]) of 0.9 mM increased myocardial AMPK activity. However, Longnus et al. (20) reported that perfusion of isolated rat hearts with 0.8 and 1.2 mM AICA-riboside did not increase AMPK activity or phosphorylation. We have confirmed that AICA-riboside does not increase AMPK activity in isolated rat hearts (Frederich and Balschi, unpublished observations). Longnus et al. reported, however, that the phosphorylation of Ser79 of ACC, an AMPK target, did increase with AICA-riboside perfusion. Because AMPK phosphorylation was not increased, these results are consistent with an allosteric increase in AMPK activity in vivo.

The present study tests two hypotheses in the isolated mouse heart: 1) AICA-riboside metabolism forms sufficient [ZMP] to increase AMPK activity and phosphorylation; and 2) ZMP accumulation alters cellular energetics and increases cytosolic [AMP]. This study defines the relationship among [ZMP], [AMP], and AMPK activity in isolated mouse hearts perfused with a range of [AICA-riboside]. PCr, ATP, and ZMP were measured in vivo using 31P-NMR spectroscopy. HPLC also measured Z nucleotide content. AMPK activity and phosphorylation of the hearts were measured in vitro. Cytosolic [AMP] was determined in vivo using [PCr], [ATP], and intracellular pH measurements (7).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of isolated perfused hearts. Hearts of male C57BL/6 mice (24–26 g) were isolated and perfused in the isovolumic Langendorff model. The Krebs-Henseleit (KH) perfusate contained (in mM) 118 NaCl, 5.3 KCl, 1.2 MgSO4, 25 NaHCO3, 2.5 CaCl2, 0.5 Na+-EDTA, 10 D-glucose, and 0.5 pyruvate and was equilibrated with 95% O2-5% CO2, with a resultant pH of 7.4. Specific metabolic inhibitors and AICA-riboside were added to this perfusate as noted. A pressure transducer connected to the left ventricle balloon measured the left ventricle pressure and heart rate. Hearts were electrically paced using a Grass stimulator (Grass, Quincy, MA). Left ventricular pressures were recorded with a PowerLab system (ADInstruments, Colorado Springs, CO). The animal protocol was approved by the Harvard Medical Area Standing Committee on Animals and followed the recommendations of the National Institutes of Health and the American Physiological Society guidelines for the use and care of laboratory animals.

Metabolic inhibitors. Addition of 0.3 mM 2-bromo-octanoic acid (Aldrich, Milwaukee, WI) to the KH perfusate irreversibly inhibits the beta-oxidation of long-chain fatty acids, which arise from endogenous triacylglycerol (26). Perfusion with 0.4 mM amino-oxyacetate (Sigma, St. Louis, MO), a reversible inhibitor of the malate-aspartate shuttle (25), results in near complete inhibition of glucose oxidation (1).

Study design and experimental protocols to vary [AMP] and [ZMP]. Figure 1 diagrams and details the perfusion protocols used to vary [AMP] (Fig. 1A) and [ZMP] (Fig. 1B) in the mouse heart.


Figure 1
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  		Fig. 1. Heart perfusion protocols to vary 5-aminoimidazole-4-carboxamide-ribotide and AMP concentrations ([ZMP] and [AMP]). A: hearts perfused without 5-aminoimidazole-4-carboxamide-1-riboside (AICA-riboside) in Krebs-Henseleit (KH). All hearts were paced at a heart rate (HR) of 420 beats per min (bpm) unless noted. Five groups of hearts were studied: 1) hearts perfused with KH during the entire protocol (Glc group, n = 15); 2) hearts perfused with KH at a HR of 420 bpm for 8 min, followed by a HR of 600 bpm for 40 min (Glc 600 group, n = 8); 3) hearts perfused with KH for 8 min, followed by KH plus amino-oxyacetate (AOA) without pyruvate (Pyr) for 40 min (GA group; n = 5); 4) hearts perfused with KH for 16 min followed by KH containing 2-bromo-octanoic acid (BrO) without Pyr for 24 min (GB group, n = 6); 5) hearts perfused with KH for 16 min followed by KH containing BrO without Pyr for 24 min, followed by KH without Pyr plus AOA and dobutamine (Db) for 16 min at a HR of 600 bpm (GBA group, n = 5). These conditions created variable cytosolic [AMP]. At the end of the perfusion, hearts were freeze-clamped with Wollenberger tongs cooled in liquid nitrogen and stored at –80°C until further measurements. B: hearts perfused with KH containing AICA-riboside. All hearts were paced at a HR of 420 bpm. Nine groups of hearts were perfused with KH for 8–24 min. Perfusate was then switched to KH containing one of the following [AICA-riboside] for 48 min unless noted otherwise: 1) 0.15 mM, n = 7; 2) 0.25 mM for 24 min, n = 3; 3) 0.25 mM for 32 min, n = 4; 4) 0.25 mM for 48 min, n = 4; 5), 0.5 mM for 20 min, n = 7; 6) 0.5 mM for 48 min, n = 4; 7) 1 mM, n = 11; 8) 1.5 mM, n = 4; and 9) 2 mM, n = 5. Perfusion of hearts with KH solutions containing different [AICA-riboside] for varying times created variable [ZMP]. At the end of the perfusion, hearts were freeze-clamped with Wollenberger tongs cooled in liquid nitrogen and stored at –80°C until further measurements.

 
31P-NMR spectroscopy of isolated perfused hearts. 31P-NMR free induction decays were acquired at 161.8 MHz using a Varian Inova spectrometer (Varian, Palo Alto, CA). Typically, 208 free-induction decays (FIDs) were averaged over 8 min using 60° pulses and a recycle time of 2.4 s. The frequency of the Pi resonance was measured using Varian software (VNMR 6.1C). The resonance areas of PCr, ATP, and ZMP were quantified using Bayesian Analysis software (G. L. Bretthorst, Washington University, St. Louis, MO). Bayesian Analysis software uses a direct statistical analysis of the free induction decay amplitudes, which corresponds to the resonance area (3). Saturation factors for resonances were determined from fully relaxed spectra, recycle time 15 s.

Heart 31P metabolite content and calculation of AMP. We derived the concentrations of the 31P-NMR-visible metabolites of hearts using the gamma-resonance of ATP as an internal standard. The area of the {gamma}-resonance of ATP at the beginning of each protocol was set to 10.0 mM. The areas of other resonances, corrected for variable saturation, were then calculated on this basis. Intracellular pH was calculated from the frequency of intracellular Pi relative to the frequency of PCr. The creatine kinase equilibrium expression and 31P-NMR measurements of [PCr], [ATP], and H+ concentration were used to calculate cytosolic [ADP] (19). We calculated cytosolic [AMP] using the adenylate kinase equilibrium expression (7, 19). ZTP and ATP peaks overlap in the spectrum. To correct for loss of ATP and gain in ZTP, the final NMR-measured [ATP] of hearts perfused for 48 min with AICA-riboside were corrected as follows. The initial [ATP] of each heart in the [AICA-riboside] groups was scaled by multiplication with the following factors: 0.25 mM (0.96), 0.5 mM (0.92), 1.0 mM (0.83), 1.5 mM (0.71), and 2.0 mM (0.69). These factors were derived from a line fit to the HPLC measurements of ATP content of hearts perfused with AICA-riboside (Table 1). The corrected value was then used as the final [ATP] measurement.


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  		Table 1. HPLC measured content of Z nucleotides and adenine nucleotides of mouse hearts perfused with and without AICA riboside

 
HPLC analysis. We measured adenine nucleotides (ATP, ADP, AMP) and Z nucleotides (ZMP, ZDP, and ZTP) in perchloric acid extracts of freeze-clamped hearts using methods described by Sabina et al. (24) (with results expressed as nmol/mg protein). We determined protein levels using the method of Lowry et al. (21).

Measurement of AMPK activity. We measured total AMPK activity using the method of Dagher et al. (5), as modified by Frederich et al. (8). No AMP was added to the assay. AMPK activity was quantified as the incorporation of 32P from [{gamma}-32P]ATP (10 GBq/mmol; NEN, Boston, MA) into a synthetic peptide with the specific target sequence for AMPK, the SAMS-peptide, amino acid sequence HMRSAMSGLHLVKRR (American Peptides, Sunnyvale, CA). We measured radioactivity using a liquid scintillation counter (Tri-Carb 2100TR, Packard Biosciences, Meriden, CT). We determined protein content in the solution containing the resuspended (NH4)2SO4 pellet using the Bradford method (2).

The isoform-specific activity of AMPK in heart tissue was measured (8) after immunoprecipitation of the {alpha}2-subunits by anti-AMPK-{alpha}2 antibodies 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 the phosphorylation of {alpha}-Thr172 of AMPK and Ser79 of ACC. We measured phosphorylation using Western immunoblots, as described by Frederich et al. (8). The primary antibodies were anti-phospho-AMPK-{alpha}-polyclonal antibody (Thr172; Cell Signaling Technology, Beverly, MA) and anti-phospho-ACC polyclonal antibody (S-79; Upstate Biotechnology). Signal detection was facilitated with enhanced chemiluminescence (Amersham, Piscataway, NJ). The membrane signals were quantitated using an image scanning densitometer (Bio-Rad model GS-700 imaging densitometer and Bio-Rad Quantity One 4.2.1).

Statistical analysis. The data are presented as means ± SD unless otherwise indicated. Statistical computations were performed with Statistica (Version 6.1; StatSoft, Tulsa, OK). ANOVA was used to compare measurements among all groups. A post hoc Fisher’s protected least significant difference was used for comparison of the means. Differences were declared statistically significant if P < 0.05. GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA) was used for graphs and fitting of the measurements of AMPK activity, [AMP], and [ZMP] to the appropriate equation.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Functional consequences of AICA-riboside perfusion in mouse hearts. AICA-riboside has an inotropic effect on the isolated mouse heart. Left ventricular pressures were as follows: 1) during KH perfusion, end diastolic = 9 ± 2 mmHg and systolic = 90 ± 8 mmHg; 2) after 20 min of AICA-riboside perfusion, end diastolic = 12 ± 2 mmHg (P < 0.05 vs. KH perfusion) and systolic = 108 ± 18 mmHg (P < 0.05); and 3) after 40 min of AICA-riboside perfusion, end diastolic = 14 ± 3 mmHg (P < 0.05) and systolic = 121 ± 22 mmHg (P < 0.05). During KH perfusion, coronary flow was 1.7 ± 0.2 ml/min; it increased to 2.0 ± 0.2 ml/min (P < 0.05) after 20 min of AICA-riboside and 2.4 ± 0.3 ml/min at 40 min (P < 0.05).

HPLC measured nucleotide content of the heart after perfusion with AICA-riboside for 48 min. The adenine nucleotide (ATP, ADP, and AMP) and Z nucleotide (ZMP, ZDP, and ZTP) contents were measured by HPLC (Table 1). Substantial ZTP was formed. Perfusion with 1.5 and 2.0 mM AICA-riboside for 48 min decreased ATP content. Because the phosphorus nuclei of ZTP resonate at the same frequency as those of ATP, NMR was not able to detect this. Thus the NMR peak area at the ATP frequency increased during the protocol, reporting the sum of ATP and ZTP. Final NMR-determined [ATP] areas were corrected to reflect the HPLC-measured decline (see MATERIALS AND METHODS).

31P-NMR measured metabolite content of the heart during perfusion with AICA-riboside Perfusion of isolated mouse heart with KH containing AICA-riboside resulted in the appearance of a new peak at 6.2 ppm in the 31P-NMR spectrum, which was assigned to ZMP (Fig. 2A). To confirm the identity of this resonance as ZMP, extracts were combined from three hearts after perfusion with 1 mM AICA-riboside KH. The 31P-NMR spectrum of the extract exhibited a peak at 6.4 ppm. Addition of authentic ZMP to the extract increased the peak at 6.4 ppm (Fig. 2B). The slight difference in resonance frequencies of the ZMP peaks in the heart and in the heart extract probably resulted from a difference in pH.


Figure 2
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  		Fig. 2. A: 31P-NMR spectra obtained from isolated mouse heart. Bottom: mouse heart perfused with KH. Top: same mouse heart 20 min after the perfusion was switched to KH containing 1.5 mM AICA-riboside. 31P-NMR resonances are identified as follows, going from left to right: ZMP, phosphocreatine (PCr), {gamma}-phosphate of ATP, {alpha}-phosphate of ATP, NAD+ and NADH (NAD), and beta-phosphate of ATP. B: 31P-NMR spectra obtained the extracts of isolated mouse hearts perfused with AICA-riboside. Bottom (I): extract of 3 mouse hearts perfused with KH containing AICA-riboside. Middle (II): same extract after addition of 5 mM ZMP. Top (II-I): difference spectrum results from the subtraction of I from II. 31P-NMR resonances are identified as follows, going from left to right: ZMP, Pi, and PCr.

 
Hearts perfused with KH containing from 0.15 to 2.0 mM AICA-riboside exhibit increasing rate of [ZMP] growth (Fig. 3A). The maximum [ZMP] resulted from 48-min perfusion with KH containing from 0.5 to 2.0 mM AICA-riboside.


Figure 3
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  		Fig. 3. A: 31P-NMR measured [ZMP] of isolated mouse hearts. Perfusion began with KH medium; at time 0 (min), perfusion medium was switched to KH containing one of the following concentrations of AICA-riboside: 0.15 mM ({blacktriangledown}) (n = 7), 0.25 mM ({blacksquare}) (n = 15), 0.5 mM (bullet) (n = 10), 1.0 mM ({blacklozenge}) (n = 17), or 2.0 mM ({blacktriangleup}) (n = 5). Values are means ± SE. B: 31P-NMR measured concentrations of ZMP ({blacksquare}) and PCr ({circ}) for isolated perfused mouse hearts (n = 4). Perfusion began with KH medium. At time 0, perfusion was switched to KH medium containing 1 mM AICA-riboside. At 48 min, perfusion was switched back to KH. Values are means ± SD. C: 31P-NMR measured [ZMP] of isolated perfused mouse hearts (n = 6). Perfusion began with KH medium. At time 0, perfusion was switched to KH medium containing 1 mM AICA-riboside. At 48 min, perfusion for 5 hearts ({blacksquare}, mean ± SD) was switched to KH containing 1 mM AICA-riboside plus 50 µM iodotubercidin, an inhibitor of adenosine kinase. One heart ({square}) was perfused with KH + 1 mM AICA-riboside continuously from time 0.

 
The [ZMP] growth during AICA-riboside was accompanied by reduction of [PCr] (Fig. 3B). Removal of AICA-riboside from KH perfusion medium results in a reduction of [ZMP] and a restoration of [PCr]. In separate experiments, 48-min perfusion with [AICA-riboside] KH was followed by [AICA-riboside] KH with 50 µM 5-iodotubercidin, an inhibitor of adenosine kinase (15). This caused [ZMP] to decrease (Fig. 3C). This indicates that AICA-riboside is continuously phosphorylated to form ZMP by adenosine kinase, whereas ZMP is continuously dephosphorylated, probably by cytosolic 5'-nucleotidase, to form AICA-riboside (28).

AICA-riboside is phosphorylated by adenosine kinase in a reaction that requires ATP. As a result, ZMP sequesters phosphoryl groups. Because PCr functions, in part, as a reservoir of phosphoryl groups that maintains [ATP] via the creatine kinase reaction, the sequestration of phosphoryl groups by ZMP decreases PCr. Because of the near equilibrium of the creatine kinase reaction, reduction in [PCr] increases cytosolic [ADP]. In turn, the near equilibrium of the adenylate kinase reaction converts the increased cytosolic [ADP] into an increase in cytosolic [AMP]. Sufficient accumulation of [ZMP] resulted in increased cytosolic [AMP] (Table 2). Note that the HPLC-measured total AMP (and ADP) content did not change (Table 1). Most AMP and ADP are bound to macromolecules in the myocyte and are not detected by NMR experiments. If the total AMP content in the well-oxygenated heart were cytosolic, the [AMP] would be ~300 µM and not the ~1 µM cytosolic [AMP] that we calculated. 31P-NMR spectroscopy, in combination with the creatine kinase and adenylate kinase equilibrium expressions, provides the best estimates for the metabolically active cytosolic [AMP] and [ADP]. We believe that AMPK activity responds to the cytosolic [AMP] and not to total AMP.


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  		Table 2. Concentration of heart 31P NMR-measured metabolites and pHi in hearts perfused with KH containing AICA-riboside

 
A primary goal of this study was to characterize the effect of [ZMP] on AMPK activity in the heart. Because [ZMP] accumulation in the heart increases [AMP], control hearts were treated with metabolic inhibitors to increase [AMP] without [ZMP] (Table 3).


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  		Table 3. Concentration of heart 31P NMR-measured metabolites and pHi in KH-perfused hearts

 
AMPK activity: relationship between total AMPK activity and cytosolic [AMP]. Figure 4, A and B, shows the relationship between total AMPK activity and cytosolic [AMP]. Binding of AMP to the {gamma}-subunit of AMPK increases AMPKK-catalyzed phosphorylation of AMPK {alpha}-Thr172. Phosphorylation of {alpha}-Thr172 increases AMPK activity. Rapid freezing preserves the physiological phosphorylation status of AMPK. The in vitro activity measurements, therefore, reflect the in vivo phosphorylation state or the degree of covalent modification of AMPK. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the individual measurements from hearts to the equation

Formula 1(1)
where v = AMPK activity, A0.5 = [AMP] at 50% AMPK activation, and Vmax = maximal activity.


Figure 4
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  		Fig. 4. A: total AMP-activated protein kinase (AMPK) activity as a function of [AMP] (hyperbolic curve of Eq. 1). Variables were fit to the measurements of in vivo [AMP] and in vitro total AMPK activity from the individual hearts. Fitting the total AMPK activity from hearts without ZMP ({square}, n = 40) to Eq. 1 (dashed line) produced the following parameters (best fit ± SE): for half-maximal allosteric activity of AMPK (A0.5) = 5.5 ± 1.6 µM, maximal activity (Vmax) = 9.6 ± 1.6 pmol·min–1·mg protein–1, 95% confidence intervals for A0.5 = 2.3–8.6 µM and for Vmax = 7.6–11.6 pmol·min–1·mg protein–1, R2 = 0.70. Fitting the total AMPK activity from the hearts with ZMP ({blacksquare}, n = 48) to Eq. 1 (solid line) produced the following parameters: A0.5 = 1.9 ± 0.7 µM, Vmax = 7.5 ± 0.8 pmol·min–1·mg protein–1, 95% confidence intervals for A0.5 = 0.5–3.2 µM and for Vmax = 6.0–9.1 pmol·min–1·mg protein–1, R2 = 0.38. B: expansion of total AMPK activity as a function of [AMP] shown in A. The shift of the curve (solid line) to the left results from an apparent reduction in the A0.5 for AMP for the hearts with ZMP ({blacksquare}). This indicates that ZMP is increasing activity at lower [AMP]. As [AMP] increased, effect of ZMP on activity was reduced. C: [AMP] and [ZMP] in hearts (cytosolic [AMP] and [ZMP] measured from hearts perfused with AICA-riboside). [AMP] measurements at 0 mM ZMP are from the Glc group hearts, which were not exposed to AICA-riboside. The dashed line is meant to guide the eye and does not imply a defined relationship between [AMP] and [ZMP]. The increase in [AMP] becomes significant near [ZMP] of 5 mM. D: total AMPK activity as a function of [ZMP] (curve of the Hill equation, Eq. 2). Variables were fit to the combined AMPK activity measurements of the Glc group hearts (for basal activity at 0 mM ZMP) and AICA-riboside-perfused hearts (in vivo [ZMP]). Fitting the data to Eq. 2 yields the following (best fit ± SE): A0.5 = 4.1 ± 0.6 mM, Vmax = 5.2 ± 1.0 pmol·min–1·mg protein–1; basal activity = 1.65 ± 0.44 pmol·min–1·mg protein–1 and Hill coefficient = 4 ± 1.9. The 95% confidence intervals were basal activity = 1.0–2.5 pmol·min–1·mg protein–1, Vmax = 3.1–7.3 pmol·min–1·mg protein–1, A0.5 = 3.0–5.2 mM, and Hill coefficient = 0.1–4.0, with R2 = 0.57.

 
Fitting the measurements of all hearts with all [AICA-riboside] and without AICA-riboside separately to Eq. 1 (Fig. 4A) yields the following values (best fit ± SE): 1) for hearts perfused without AICA-riboside, A0.5 = 5.5 ± 1.6 µM and Vmax = 9.6 ± 1.0 pmol·min–1·mg protein–1; and 2) for hearts perfused with AICA-riboside, A0.5 = 1.9 ± 0.7 µM and Vmax = 7.5 ± 0.8 pmol·min–1·mg protein–1. The A0.5 for the two heart groups were different (P = 0.025); the Vmax were not different (P = 0.11). The reduction in apparent A0.5 for AMP of the AICA-riboside hearts signals that the presence of cytosolic ZMP is increasing AMPK activity (Fig. 4B). The lower apparent A0.5 does not reflect a change in the sensitivity of AMPK to AMP binding. The effects of [ZMP] are most prominent below 5.5 µM (the A0.5 for AMP in the absence of ZMP).

The measurements of AMPK activity from all hearts that contained cytosolic [AMP] < 3 µM were merged into two new groups, with and without AICA-riboside. The AICA-riboside hearts, which had [ZMP] = 3.3 ± 0.9 mM, displayed an AMPK activity 50% higher (P = 0.004; 3.3 ± 1.3 pmol·min–1·mg protein–1) than hearts (2.0 ± 1.2 pmol·min–1·mg protein–1) without ZMP. Thus ZMP of ~3.5 mM increased AMPK activity without increasing [AMP].

The increase of [ZMP] correlated with increased cytosolic [AMP] (Fig. 4C). The relationship among AMPK activity, [ZMP], and [AMP] was complex. Inspection of the group data from hearts perfused with low [AICA-riboside] (0.15 and 0.25 mM for up to 32 min), which had ZMP < 3 mM and [AMP] < 3 µM, shows that the AMPK activity was unchanged (Table 4). Hearts perfused with [AICA-riboside] (0.25 mM for 48 min and 0.5 mM for 20 min), which had [ZMP] < 5 mM and [AMP] < 4 µM, had increased AMPK activity. In hearts with [ZMP] greater than ~5 mM, [AMP] increased (approaching the A0.5 for AMP) and the increase in AMPK activity cannot be ascribed to increased [ZMP] alone. For example, the AMPK activity of the GB hearts was equal to that of the 2.0 mM AICA-riboside hearts, which had equal [AMP] (Table 4).


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  		Table 4. AMP-activated protein kinase Activity of mouse hearts perfused with and without AICA-riboside

 
There was a sixfold increase in {alpha}2-isoform-specific AMPK activity for the hearts perfused with 1 mM AICA-riboside (Table 4). This was consistent with the fivefold increase in total activity measured for the 1 mM AICA-riboside hearts.

Relationship between total AMPK activity and cytosolic [ZMP]. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [ZMP] (Fig. 4D) was determined by fitting the individual measurements from all hearts to the Hill equation

Formula 2(2)
where A0.5 = [ZMP] at 50% AMPK activation, Vbasal = basal activity, and h = Hill coefficient.

Fitting the data from Glc group hearts (hearts perfused with KH during the entire protocol; [ZMP] = 0) and all AICA-riboside-perfused hearts to Eq. 2 (Fig. 4D) yields the following (best fit ± SE): A0.5 = 4.1 ± 0.6 mM, Vmax = 5.2 ± 1.0 pmol·min–1·mg protein–1, Vbasal = 1.65 ± 0.44 pmol·min–1·mg protein–1, and h = 4 ± 1.9. The [AMP] varied in these hearts, especially when [ZMP] was >5 mM (see Fig. 4C).

Note that the ZMP and AMPK activity can best be fit with a sigmoid equation. Modeling of the AMPK cascade predicted that the AMP-to-AMPK activity relationship should also be described by a sigmoid equation (12). To date, our measurements of the in vivo [AMP] and AMPK activity relationship (Fig. 4A) have been best fit with a hyperbola (8).

Relative magnitude of AMPK activity resulting from [ZMP]. In the oxygenated mouse heart, the AMPK activity resulting from either increased [ZMP] or increased [AMP] is less than 35% of the AMPK activity resulting from 8 min of no-flow ischemia (Fig. 5). This difference in the AMPK activity in the oxygenated and hypoxic or ischemic heart is consistent with our recent observation in the hypoxic rat heart of a twofold increase in AMPK activity that is independent of the effect of increased [AMP] (8). The AMPK activity from the 1 mM AICA-riboside group was the highest of the groups with [ZMP]. These hearts had elevated [AMP], which undoubtedly contributes to the AMPK activity.


Figure 5
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  		Fig. 5. AMPK activity (pmol·min–1·mg protein–1) measured for mouse hearts from the Glc, 1 mM AICA-riboside (6.9 mM cytosolic [ZMP]), 8 min of no-flow ischemia (8 min Isch), and 8-min Isch groups with 5 mM ZMP added to the in vitro assay (8 min Isch + 5 mM ZMP). Values are means ± SD. *P < 0.05 vs. Glc group; #P < 0.05 vs. 1 mM AICA-riboside group; {ddagger}P < 0.05 vs. 8-min Isch group.

 
The increase in AMPK activity and phosphorylation reflects covalent modification of AMPK resulting from ZMP binding in vivo. ZMP may also allosterically increase in vivo AMPK activity, which will not be revealed in the in vitro activity or in the in vivo {alpha}-Thr172 phosphorylation. To emulate the in vivo allosteric effects of ZMP in the in vitro activity assay, 5 mM ZMP was added to the in vitro AMPK activity assay for the 8-min ischemic hearts. The added [ZMP] increased the in vitro activity twofold (Fig. 5). It is quite possible that the in vivo AMPK activity in hearts with ZMP is up to two times greater than the in vitro activity.

Phosphorylation of AMPK {alpha}-Thr172 and ACC Ser79 in mouse hearts with and without [ZMP]. The phosphorylation state of Thr172 on the {alpha}-subunit of AMPK was determined by Western blot analysis of Glc hearts and of hearts perfused with [AICA-riboside] of 0.5 mM and below (Fig. 6). The AMPK {alpha}-Thr172 phosphorylation for hearts perfused with 0.5 and 0.25 mM AICA-riboside for 48 min was greater than that of Glc hearts and 0.15 and 0.25 mM (32 min) hearts (Fig. 6B). The phosphorylation is consistent with the activity (Table 4). AMPK phosphorylates Ser79 on ACC to regulate its activity. The phosphorylation state of ACC Ser79 was also determined (Fig. 6C). ACC phosphorylation increased in the hearts with ZMP. The relative phosphorylation of Ser79 was nearly complete in the hearts exposed to 0.5 mM AICA-riboside for 48 min (Fig. 6D). The magnitudes of the increase in ACC phosphorylation of the 0.15 mM and the 32-min 0.25 mM AICA-riboside hearts are relatively greater than the AMPK phosphorylations of these hearts (Fig. 6B). This disproportionate increase in the phosphorylation of ACC indicates that ZMP allosterically increased AMPK activity in vivo. These hearts had cytosolic [ZMP] of ~3 mM (Table 4).


Figure 6
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  		Fig. 6. Western blot analysis of the phosphorylation of AMPK {alpha}-Thr172 and acetyl-CoA carboxylase (ACC) Ser79 of mouse heart after perfusion with and without AICA-riboside: A: representative Western immunoblot used to measure the phosphorylation of Thr172 on the {alpha}-subunit of AMPK from hearts perfused with the conditions Glc (KH only) and KH having the following [AICA-riboside] and exposure times (in parentheses): 0.5 mM (48 min), 0.25 mM (48 min), 0.15 mM (48 min), and 0.25 mM (32 min). Protein (50 µg) was loaded in each lane. B: plot of the relative phosphorylation of the AMPK {alpha}-subunit Thr172 from Western immunoblot shown in A. AMPK-P signal values were normalized as percentages of the optical density of one 0.5 mM AICA-riboside heart sample (means ± SD). *P < 0.05 vs. Glc group. C: representative Western immunoblot used to measure the phosphorylation of Ser79 on ACC from hearts perfused with the conditions Glc (KH only) and KH having the following [AICA-riboside] and exposure times (in parentheses): 0.5 mM (48 min), 0.25 mM (48 min), 0.15 mM (48 min), and 0.25 mM (32 min). Protein (50 µg) was loaded in each lane. D: relative phosphorylation of ACC Ser79 from Western immunoblot shown in C. ACC phosphorylation signal values were normalized as percentages of the density of one 0.5 mM [AICA-riboside] sample on the blot (means ± SD). *P < 0.05 vs. Glc group, {dagger}P < 0.05 vs. 0.15 and 0.25 mM 32-min groups.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The AMPK protein kinase cascade responds to [AMP] increases (10, 11). Increases in [AMP] activate the cascade by four mechanisms (4, 6, 14). First, AMP allosterically activates AMPKK (12). This mechanism is somewhat controversial. LKB1 has been identified as an AMPKK; however, it does not respond to AMP (13). Second, AMP binds to AMPK, which promotes phosphorylation of Thr172 of the AMPK {alpha}-subunit by AMPKK. This phosphorylation correlates with 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 (10). This study examines the effect of cytosolic [ZMP] on mechanisms 2 and 4 and cytosolic [AMP] on mechanism 2. This study cannot rule out an effect of ZMP on the upstream AMPKK (mechanism 1). If mechanism 1 is operative, its [ZMP] dependence will not occur at lower [ZMP] than the effect that we have assigned to mechanism 2.

ZMP, or AICA-ribotide, is a structural analog of AMP. ZMP has been used in numerous studies to pharmacologically activate AMPK. The present study tested two hypotheses using the isolated perfused mouse heart: 1) AICA-riboside metabolism forms sufficient [ZMP] to increase AMPK activity and phosphorylation; 2) ZMP accumulation alters cellular energetics and increases cytosolic [AMP]. This study defined the relationship among AMPK activity measured in vitro and cytosolic [ZMP] and cytosolic [AMP] measured in vivo of isolated mouse hearts.

In the mouse heart, we found a substantial increase in [ZMP] that was time and [AICA-riboside] dependent. The A0.5 of ZMP for the phosphorylation of AMPK was 4.1 ± 0.6 mM, whereas the A0.5 for AMP was 5.5 ± 1.6 µM. Cytosolic ZMP binding to AMPK was, therefore, 800-fold less effective than AMP in promoting AMPK phosphorylation by AMPKK. These results explain why AICA-riboside does not increase AMPK activity in the isolated rat heart. The rat heart ZMP contents reported were equivalent to a [ZMP] of 0.2–0.4 mM (20). This [ZMP] is too low relative to the A0.5 for ZMP to promote AMPK phosphorylation. The A0.5 of ZMP for phosphorylation of AMPK has been reported in two other cell types. In hepatocytes, the A0.5 of ZMP for phosphorylation of AMPK was ~2 mM (4). In addition a ZMP/ATP of ~0.2 was required for A0.5 in INS-1 cells (12). In the mouse heart, ZMP/ATP for A0.5 was 0.43 ± 0.08; ZMP/ATP+ZTP for A0.5 was 0.36 ± 0.05 (not shown). Thus it appears that the A0.5 of ZMP for AMPK phosphorylation (covalent modification) in the heart is about twice that reported in hepatocytes and INS-1 cells.

Because the state of in vivo AMPK allosteric activation of AMPK is not preserved during protein isolation, it can only be inferred from AMPK actions on its target proteins. Hearts with [ZMP] of ~3 mM exhibited increased ACC phosphorylation relative to their AMPK phosphorylation. This is consistent with a ZMP A0.5 for allosteric activation of 3 mM or lower. In the rat heart [ZMP] of 0.2–0.4 mM increased phosphorylation of ACC (20). This implies ZMP A0.5 of ~0.3 mM or lower. In vitro determinations of the allosteric activity using purified AMPK have yielded a range of values. Henin et al. (16) reported an A0.5 for ZMP of ~ 5 mM in the presence of 3 mM ATP; Corton et al. (4) reported an A0.5 for ZMP of 1.5 ± 0.6 mM at 4 mM ATP with an A0.5 for AMP of 29 ± 14 µM. Thus the in vivo results from the mouse heart with [ATP] of ~10 mM are generally consistent with reported in vitro measurements of the ZMP A0.5 for allosteric activation.

In the mouse heart as in some other tissues, AICA-riboside metabolism resulted in ZMP accumulation to millimolar concentrations (4, 16, 24, 29). The reduction in [ZMP] with removal of AICA-riboside or by addition of iodotubercidin, an inhibitor of adenosine kinase, indicates an active futile cycle of AICA-riboside phosphorylation and ZMP dephosphorylation (28). The dephosphorylation of ZMP to AICA-riboside in the heart is most likely catalyzed by cytosolic 5'-nucleotidase activity. The lack of appreciable formation of ZMP in the rat heart suggests the possibility that adenosine kinase activity may be too low relative to cytosolic 5'-nucleotidase activity. Alternatively, the transport of AICA-riboside, which occurs via the adenosine transporter (9), may be much lower in the rat heart. Low [ZMP] accumulation may not be limited to the rat heart. ZMP was not detectable in rabbit cardiomyoctes exposed to 500 µM AICA-riboside (18). In the heart, the extent of ZMP formation is species dependent.

ZMP can be metabolized in several ways (28). One possible fate of ZMP is conversion into ZDP and ZTP. Substantial [ZTP] formed in the mouse heart. We estimate that [ZTP] was 3–5 mM in hearts perfused with 0.5–2 mM [AICA-riboside] for 48 min. [ZMP], an intermediate in the de novo synthesis of purine nucleotides, can also be converted into inosine monophosphate (IMP). IMP can be converted into the nucleotides GMP and AMP (anabolic pathway) or uric acid (catabolic pathway). AICA-riboside increased IMP and ATP in postischemic dog myocardium even though [ZMP] was low (27). In normal dog myocardium treated with AICA-riboside when [ZMP] was < 70 µM, IMP was predominantly converted to adenine nucleotides (27); when [ZMP] was > 190 µM, IMP was predominantly converted to uric acid (24). The lack of ZMP metabolism to adenine nucleotides in our study of the mouse heart may result from the high [ZMP] achieved, which were well above 190 µM.

In the mouse heart, [ZMP] accumulation, which was time and [AICA-riboside] dependent, resulted in variable decreases of [PCr] and [ATP]. Some studies of AICA-riboside metabolism in hepatocytes (4) and skeletal muscle (22) have found no change in ATP with ZMP formation. Others have reported that hepatocytes exposed to 0.5 mM AICA-riboside had unchanged ATP yet exposure to 2 mM AICA-riboside decreased ATP 40% (29). It is evident that the AICA-riboside metabolism-induced decrease in myocardial [PCr] and [ATP] translated into increased cytosolic [AMP].

In conclusion, AICA-riboside formed sufficient [ZMP] to increase AMPK activity in the mouse heart. The [ZMP] was time and [AICA-riboside] dependant. The in vivo A0.5 of ZMP for AMPK activity (covalent modification of AMPK) was 4.1 mM. AICA-riboside metabolism decreased [ATP] and increased the [AMP].


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-46033 to J. A. Balschi.


   ACKNOWLEDGMENTS
 
The authors thank Professor Joanne Ingwall for critically reading the manuscript.

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


   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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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
 REFERENCES
 

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