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Increased Adenosine Monophosphate–Activated Protein Kinase Activity in Rat Hearts With Pressure-Overload Hypertrophy

Originally publishedhttps://doi.org/10.1161/hc4001.097183Circulation. 2001;104:1664–1669

    Abstract

    Background Recent reports suggest that activation of adenosine monophosphate (AMP)–activated protein kinase (AMPK), in response to acute changes in cellular energy status in cardiac and skeletal muscles, results in altered substrate utilization. We hypothesized that chronic alterations in myocardial energetics in hypertrophied hearts (left ventricular hypertrophy, LVH) will lead to elevated AMPK activity, which in turn regulates substrate utilization.

    Methods and Results Using 31P NMR spectroscopy and biochemical assays, we found that in LVH hearts, adenosine triphosphate (ATP) concentration decreased by 10%, phosphocreatine concentration decreased by 30%, and total creatine concentration was unchanged. Thus, the ratio of phosphocreatine/creatine decreased to one third of controls, and the ratio of AMP/ATP increased to 5 times above controls. These changes were associated with increased α1 and α2 AMPK activity (3.5- and 4.8-fold above controls, respectively). The increase in AMPK α1 activity was accompanied by a 2-fold increase in α1 expression, whereas α2 expression was decreased by 30% in LVH. The basal rate of 2-deoxyglucose uptake increased by 3-fold in LVH, which was associated with an increased amount of glucose transporters present on the plasma membrane.

    Conclusions These results demonstrate for the first time that chronic changes in myocardial energetics in hypertrophied hearts are accompanied by significant elevations in AMPK activity and isoform-specific alterations in AMPK expression. It also raises the possibility that AMPK signaling plays an important role in regulating substrate utilization in hypertrophied hearts.

    Adenosine monophosphate (AMP)–activated protein kinase (AMPK) is a heterotrimeric protein consisting of 1 catalytic subunit (α) and 2 noncatalytic subunits (β and γ).1–3 Two isoforms of the α-subunit have been identified (α1 and α2), and both are expressed in the heart.3 Recently, there has been emerging evidence suggesting that AMPK regulates fatty acid oxidation and glucose uptake in the heart and skeletal muscle in response to altered energy supply and/or demand (summarized in Figure 1).4–7 These studies suggest that an increased ratio of AMP to ATP (AMP/ATP), caused by depletion of high-energy phosphates, activates AMPK and increases energy production by stimulating substrate utilization (Figure 1). Regulation of AMPK activity by AMP/ATP is mediated by both allosteric and covalent mechanisms. Besides a direct allosteric activation, binding of AMP to AMPK also facilitates its phosphorylation by an upstream kinase, AMPK kinase (AMPKK), and inhibits its dephosphorylation by protein phosphatases.1,8 Moreover, increased [AMP] allosterically activates AMPKK.1,8 In striated muscle, AMPK activity is also regulated by the ratio of phosphocreatine (PCr) to creatine (PCr/Cr).1,2,4 Decreased PCr/Cr can activate AMPK by two mechanisms: either direct activation or altering the AMP/ATP ratio by increasing AMP concentration through the creatine kinase reaction and the adenylate kinase reaction.2,4,9

    Hearts with hypertrophy caused by pressure overload are characterized by chronic depletion of [PCr] but near normal [ATP]. It is not known whether these changes result in altered AMPK activity. Furthermore, in hypertrophied hearts of animal models and patients, glucose uptake is increased,10–13 but the mechanism responsible for this increase remains largely unclear. Interestingly, in rat hearts with pressure overload–induced left ventricular hypertrophy (LVH), we have observed a close relation between increased glucose uptake and decreased [PCr], a key regulator of AMPK.12 Taken together, these studies raise the possibility that glucose utilization in cardiac hypertrophy is regulated by myocardial energetics, with AMPK as a key signaling intermediary. In the current study, we found increased activity and altered protein levels of AMPK in LVH hearts. Furthermore, these changes were associated with increased fractional distribution of glucose transporters on the plasma membrane and higher basal glucose uptake in LVH, supporting the hypothesis that AMPK may be a signal linking altered energy status and glucose uptake in hypertrophied hearts.

    Figure 1. Summary of proposed mechanisms for acute AMPK activation and its function in regulating glucose and fatty acid utilization in muscle. ACC indicates acetyl-CoA carboxylase.

    Methods

    Animal Model of LVH

    Weanling male Wistar rats weighing 50 to 75 g were obtained from Charles River Breeding Laboratories. LVH was induced by placing a titanium clip on the ascending aorta to generate pressure overload for the LV, as previously described.9 Although the clip did not cause immediate constriction in weanling rats, pressure overload of the LV gradually developed as the animal grew in size. Sham-operated animals were used as controls. Animals were studied 17 to 19 weeks after the surgery. Compared with the control hearts, LVH hearts showed a 35% increase in LV weight (1.28±0.04 versus 1.73±0.03 g, P<0.0001) and a 31% increase in atrial weight (0.13±0.01 versus 0.17±0.01 g, P<0.005), whereas right ventricular weight was unchanged (0.34±0.00 and 0.36±0.02 g, P=NS). All experimental procedures were performed according to the guidelines of the American Physiological Society and were approved by the institutional animal care and use committee.

    Isolated Perfused Hearts

    Rats were anesthetized with sodium pentobarbital (50 mg/kg IP). A blood sample was drawn and used to measure plasma insulin concentrations with the use of a radioimmunoassay kit (Linco Research). The heart was perfused at constant flow with modified Krebs-Henseleit buffer, as previously described.9 Since the in vivo coronary perfusion pressure is higher for LVH, the perfusion flow was titrated to achieve the mean perfusion pressures of 80 and 110 mm Hg for control and LVH, respectively. Prior experience showed that this approach would achieve comparable myocardial flow rates per gram of LV weight for the two groups.9 The LV end-diastolic pressure was set to 5 to 10 mm Hg. Compared with the controls, LV systolic pressure was higher in LVH (199±5 versus 122±5 mm Hg, P<0.05), whereas the heart rate was lower (216±7 versus 243±7 bpm, P<0.05). LV function, estimated by the product of heart rate and LV developed pressure (RPP), was 29±2 versus 41±1 103mm Hg/min for control and LVH hearts, respectively (P<0.0001). When normalized for LV mass, RPP/LV was not different for control and LVH hearts (23±1 versus 23±1 103 mm Hg/min/g). Thus, the biochemical measurements in isolated perfused hearts were obtained under conditions of comparable mechanical load.

    31P NMR Measurements

    31P NMR spectra of isolated perfused hearts were collected as previously described.9 Baseline spectra were obtained after the stabilization period and were used for the quantification of high-energy phosphate content. After baseline measurements, hearts were either freeze-clamped for biochemical assays (see below) or switched to a glucose-free buffer containing 5 mmol/L 2-deoxyglucose (2DG) and 5 mmol/L pyruvate for the measurement of 2DG uptake rate, as previously described.12,14 The transport rate of 2DG was measured both before and after adding insulin (2 U/L) to the perfusate. The amount of ATP in the isolated perfused hearts at the end of the stabilization period, determined by high-performance liquid chromatography, was used to calibrate the [β-P]ATP peak area of the baseline spectra. Concentrations of PCr, inorganic phosphate (Pi), and 2DG-P were calculated by using the ratios of their peak areas to ATP peak area and were corrected for saturation as previously described.9 Intracellular pH was calculated by comparing the chemical shift between the Pi and PCr peaks to the values from a standard curve. Cytosolic free [ADP] and [AMP] were calculated by using the creatine kinase reaction equilibrium expression15 and the adenylate kinase reaction equilibrium expression,16 respectively.

    Biochemical Assays

    In a separate group of animals, hearts were perfused and freeze-clamped for biochemical assays. Isoform-specific activities of AMPK were measured as previously described.5 Tissue lysates were immunoprecipitated with antibodies against the α1 or α2 catalytic subunit and protein A/G beads (SantaCruz Biotechnologies). The kinase activity of the respective immunoprecipitates was measured in the presence of 0.2 mmol/L AMP, with a synthetic peptide (HMRSAMSGLHLVKRR) used as substrate. Myocardial ATP content was measured by high-performance liquid chromatography with freeze-clamped tissue. Total Cr content in the heart was measured by a fluorometric assay.17

    Membrane Fractionation, Immunobloting, and Northern Blotting

    Freeze-clamped tissue was pulverized and homogenized in a buffer containing 250 mmol/L sucrose and 20 mmol/L HEPES, pH 7.4, and plasma membranes (PM) were isolated by means of sucrose gradient centrifugation, as previously described.18 Activities of the plasma membrane marker potassium stimulated p-nitrophenol phosphatase (Kpnppase) were measured in the homogenate and the PM fraction to determine the purity and recovery of plasma membranes.19 A high degree of enrichment in Kpnppase activity was achieved in the PM fractions from both control and LVH groups (Table 1). Immunoblots of homogenate and PM proteins were performed with antisera to GLUT1 and GLUT4 (Chemicon). The percentage of GLUT1 and GLUT4 present in PM fractions were estimated by comparing the total homogenate GLUT1 and GLUT4 content and the PM GLUT1 and GLUT4 contents, corrected for percentage of recovery of the plasma membrane marker Kpnppase.

    Table 1097183. Plasma Membrane Fraction Enrichments

    Kpnppase activity, nmol · mg−1 · h−1Enrichment, foldRecovery, %
    HomogenatePlasma Membrane
    *P<0.05 vs control.
    Control (n=8)363±265678±37016±216±2
    LVH (n=8)287±303431±281*13±216±3

    For immunoblotting of AMPK α1 and α2 isoforms, 40 μg protein from crude muscle lysates were separated by 8% SDS-PAGE and transferred to nitrocellulose membranes. The two isoforms of AMPK were identified by immunoblotting with the specific antibodies against the α1 and α2 catalytic subunits, as previously described.5 Northern blotting was performed with cDNA probes (kind gift from Dr Kelly at Washington University at St Louis) for carnitine palmitoyl transferase I (CPT-I) and medium-chain acyl-CoA dehydrogenase (MCAD) with 10 μg RNA isolated from LV of control and LVH hearts.

    All data are shown as mean±SEM. Comparisons between control and LVH were performed by means of an unpaired Student’s t test.

    Results

    High-Energy Phosphate Content

    As shown in Table 2, [PCr] decreased by 30%, whereas the total Cr was unchanged in LVH, resulting in a marked decrease in the PCr/free-Cr ratio. Consequently, free [ADP] and [AMP], calculated by creatine kinase and adenylate kinase reaction equilibrium expression, were increased by severalfold. Thus, despite a small decrease in [ATP], the ratio of AMP/ATP increased by nearly 5 times in LVH.

    Table 2097183. Concentrations of High-Energy Phosphate Compounds

    Control (n=7)LVH (n=6)P
    PCr, mmol/L22.8±1.016.5±0.4<0.0001
    Total Cr, mmol/L27.2±3.725.0±1.5NS
    PCr/Free Cr5.21.9
    ATP, mmol/L10.1±0.29.1±0.20.01
    ADP, μmol/L15.0±2.631.3±1.70.0004
    AMP, nmol/L27.6±9.0115.8±12.80.0001
    AMP/ATP, nmol/mmol2.8±0.912.9±1.5<0.0001

    AMPK Activities

    To determine whether altered myocardial energetics was associated with changes in AMPK activity in LVH, we measured the activities of the α1 and α2 isoforms of AMPK (Figure 2A). In LVH, α1 and α2 AMPK activities were 3.5- and 4.8-fold above controls, respectively (P<0.05 for both). The increase in AMPK α1 activity was accompanied by a 2-fold increase in α1 protein (Figure 2B). In contrast, the protein level of AMPK α2 was decreased by 30% in LVH (P<0.05, Figure 2B). Thus, both altered expression and posttranslational modification contribute to the activation of AMPK in LVH.

    Figure 2. Activities (A) and protein levels (B) of the α1 and α2 isoforms of AMPK in control (n=6) and LVH (n=8) hearts. Insert in B shows representative immunoblots for α1 and α2 isoforms of AMPK. *P<0.05 vs control.

    Glucose Transport and Glucose Transporters

    Figure 3 shows the rates of 2DG uptake in isolated, perfused control and LVH hearts. The 2DG uptake rate during insulin-free perfusion (baseline) was increased by 3-fold in LVH compared with the controls (P<0.05). During maximal insulin stimulation, 2DG uptake rate was not different for the two groups. Thus, the response to insulin was reduced by 70% in LVH compared with the controls. To determine whether increased basal glucose uptake in LVH was due to increased glucose transporter proteins, we measured the total amount as well as the plasma membrane distribution of GLUT1 and GLUT4 in the LVH of control and LVH hearts (Figure 4). The total GLUT1 content was increased by 28% in LVH (Figure 4A), with a trend for a higher relative distribution of GLUT1 on the PM (Figure 4C, P=0.08). This resulted in a >50% increase in the total amount of GLUT1 on the PM in LVH (63±7 versus 39±4 AU, P<0.05). Although total GLUT4 content decreased by 25% in LVH (Figure 4B), the relative distribution of GLUT4 on the PM increased significantly (Figure 4D). This resulted in a comparable amount of GLUT4 on the PM for the LVH and the controls (64±8 versus 54±5 AU, P=NS). Increases in the relative distribution of glucose transporters on the PM in the LVH could not be explained by preexisting effects of insulin. Plasma insulin levels were not different for control and LVH at the time when hearts were removed for perfusion (18.7±1.6 versus 20.9±1.1 mIU/L), respectively (P=NS).

    Figure 3. Rates of 2DG uptake in control and LVH during baseline (insulin-free) and insulin stimulation. *P<0.05 vs control.

    Figure 4. Quantification of GLUT1 (A) and GLUT4 (B) from immunoblots of tissue homogenates and relative percentages of GLUT1 (C) and GLUT4 (D) in PM fractions for control and LVH hearts. See Methods for calculations. *P<0.05 vs control.

    Expressions of CPT-I and MCAD

    To assess the capacity for fatty acid oxidation in LVH, we measured the mRNA levels of CPT-I and MCAD, rate-limiting enzymes for long- and medium-chain fatty acid oxidation. Compared with the controls, the mRNA levels for CPT-I and MCAD in LVH hearts were decreased by 63% and 72%, respectively (P<0.05 for both, Figure 5).

    Figure 5. mRNA levels of MCAD and CPT-I in control (n=5) and LVH (n=7) hearts. Insert shows representative Northern blots for MCAD and CPT-I. *P<0.05 vs control.

    Discussion

    The major finding of this study is that AMPK activity is increased in LVH hearts. We believe this is the first report showing changes in isoform-specific AMPK activities and expression in a disease model with chronic changes in high-energy phosphate content. Furthermore, the activation of AMPK in LVH occurs concomitantly with elevated rates of glucose uptake and increased relative distribution of glucose transporters on the plasma membrane, suggesting that increased AMPK activity in LVH may partially contribute to enhanced glucose uptake in these hearts.

    AMPK Activity and Expression

    Acute depletion of high-energy phosphate content has been shown to activate AMPK during metabolic stress in the heart and skeletal muscle.5,6 Under these conditions, substantial depletion of ATP leads to a marked increase in cytosolic [AMP]. In LVH hearts, [ATP] is nearly normal, whereas [PCr], the energy reserve compound, is decreased, resulting in modest increases in [ADP] and [AMP]. Our study shows that AMPK is also activated in hypertrophied hearts, with milder but chronic decreases in myocardial high-energy phosphate content. Because the method used in this study to assay AMPK activity does not preserve the allosteric changes of the enzyme, the increased activity of AMPK in LVH reported here probably is caused by enhanced phosphorylation. Although it has been shown that increases in [AMP] or AMP/ATP cause phosphorylation of AMPK by its upstream kinase,1,8 results from the present study do not allow conclusions on whether chronic decreases in high-energy phosphate content in vivo is the exclusive source of increased AMPK activity in LVH. Nevertheless, these data do provide important observations in a chronic model that are consistent with previously reported activation of AMPK by increased AMP/ATP in acute experiments.

    The decreased [PCr] in LVH is unlikely to be caused by hypoxia or ischemia of these hearts. This is based on several lines of evidence obtained by us and others in animal models of ascending aortic banding. First, in recognition of the difference between the in vivo coronary perfusion pressure in control and LVH rats, the perfusion pressure in the isolated, perfused heart preparation was adjusted to 80 and 110 mm Hg for control and LVH hearts, respectively. It has been shown that this approach achieves comparable myocardial flow rates per gram of LV weight for the two groups.9 Second, by simultaneous measurement of high-energy phosphate content and intracellular pH in LVH hearts, we observed no signs of decreased pH or heterogeneity of pH (manifests as split Pi peak, see Reference 20) in these hearts compared with controls.20 Intracellular pH was 7.08±0.01 and 7.09±0.01 for sham and LVH hearts, respectively. Finally, previous studies have shown that decreased PCr/ATP in hypertrophied and failing hearts is not caused by insufficient oxygen delivery.21

    We also found altered protein level of the α1 and α2 isoforms of AMPK in LVH. Increased activity of the α1 isoform was associated with an upregulation of the α1 protein. In contrast, the α2 protein was downregulated in LVH despite significant increases in kinase activity. These observations demonstrate that both expression and posttranslational modification contribute to the chronic regulation of AMPK activity in LVH hearts. Although the increased amount of protein may have contributed to the higher activity of α1-AMPK, phosphorylation probably is the primary mechanism responsible for the elevation of α2-AMPK activity in LVH. The greater degree of α2-AMPK activation is consistent with previous reports showing higher sensitivity of α2- than α1-AMPK for phosphorylation in response to increases in AMP/ATP.4,22 Although the mechanisms responsible for altered expression of AMPK in LVH hearts are unclear, our results suggest that expression of these two isoforms is differentially regulated in cardiac hypertrophy. It is possible that multiple mechanisms, dependent and/or independent of myocardial energy status, may be involved in the regulation of AMPK expression in hypertrophied hearts. Future studies will be needed to elucidate these mechanisms.

    AMPK and Glucose Uptake

    Increased glucose uptake in hypertrophied hearts is a well-established observation, but its underlying mechanisms are poorly understood. Previous studies have shown that GLUT4 expression is decreased in hypertrophied hearts, whereas GLUT1 expression is either unchanged or increased.13,23 In this study, we found a 25% decrease in total GLUT4 protein in LVH, consistent with previous reports of decreased GLUT4 expression. Interestingly, despite reduced total content, the relative percentage of GLUT4 on the plasma membrane increased significantly in LVH, suggesting enhanced translocation of GLUT4 under basal conditions. These results are consistent with previous observations showing translocation of GLUT4 in hearts and skeletal muscles in which AMPK is activated acutely by 5-aminoimidazole-4-carboxyamide-1-β-d-ribofuranoside (AICAR).18,24 However, because the total GLUT4 content is lower in LVH, increases in relative distribution of GLUT4 on the plasma membrane appear to be insufficient to account for the increases in glucose uptake in these hearts. Our results suggest that increases in GLUT1 content (28%) and in the amount of GLUT1 present on the plasma membrane (>50%) may also contribute to increased basal glucose uptake in LVH. In addition, increased glycolysis has been demonstrated in hypertrophied hearts,25 and we have previously shown that glycolytic rates increase by 2-fold in LVH.26 A recent study suggests that activation of AMPK results in phosphorylation of 6-phosphofructo-2-kinase in the heart, leading to increased synthesis of fructose 2,6-bisphosphate, a potent stimulator of glycolysis.27 Thus, it is likely that activation of AMPK in LVH can upregulate glucose uptake through multiple mechanisms, including increasing the amount of glucose transporters on the cell surface membrane and enhancing rates of glucose disposal.

    It has been proposed that AMPK causes translocation of glucose transporters through a distinct mechanism from insulin stimulation because the former is independent of phosphatidylinositol-3-kinase.7,24,28 This is supported by the observation that there is an additive effect of AMPK activation or ischemia to insulin-stimulated glucose uptake.7,28,29 In the present study, however, glucose uptake during insulin stimulation was similar for hearts with low AMPK activity (control) and hearts with high AMPK activity (LVH). This may result from decreased total GLUT4 content in LVH, which limits the capacity for GLUT4-mediated glucose transport. This is of clinical significance because increased basal glucose uptake in the face of decreased transporter capacity in hypertrophied hearts will decrease the reserve for further recruitment of GLUT4-mediated glucose transport during stress, such as hypoxia or ischemia.

    AMPK and Fatty Acid Oxidation

    It has been shown that AMPK also regulates fatty acid metabolism.6,30 Acute activation of AMPK by the pharmacological activator AICAR increases fatty acid oxidation in rat skeletal muscle,30 presumably through a decrease in acetyl-CoA carboxylase activity and subsequent decrease in the inhibition of CPT-I by malonyl-CoA (Figure 1). In contrast, we found increased AMPK activity in hypertrophied hearts caused by chronic pressure overload, in which decreased fatty acid oxidation has been shown.25 Consistent with the findings of diminished fatty acid oxidation in hypertrophied hearts, we found that the capacity for fatty acid oxidation pathway is downregulated in LVH, as indicated by the severe reductions in the expression of CPT-I and MCAD, the rate-limiting enzymes for this pathway. Taken together, these observations suggest that whereas acute AMPK stimulation enhances fatty acid oxidation, long-term AMPK activation in cardiac hypertrophy, associated with decreased expression of the enzymes critical to fatty acid oxidation, is unable to increase the flux through this pathway. These results exemplify the complexity of cardiac hypertrophy within which various signaling pathways are activated and interact, all of which could potentially contribute to altered substrate utilization. It also emphasizes the importance of dissecting the role of AMPK signaling in a pathophysiologically relevant model. In this regard, identification of hearts with pressure overload as a chronic disease model with increased AMPK activity provides the basis for future studies on the functional role of this protein under pathological conditions.

    This work was supported by National Institutes of Health grants HL-59246 and AG-00837 (Dr Tian) and AR-45670 and AR-42238 (Dr Goodyear).

    We thank Dr Daniel P. Kelly for providing the cDNA probes for CPT-I and MCAD and Dr Joanne S. Ingwall for critical reading of the manuscript.

    Footnotes

    Correspondence to Rong Tian, MD, PhD, NMR Laboratory for Physiological Chemistry, 221 Longwood Ave, Room 229, Boston, MA 02115. E-mail

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