Adenosine 5'-Monophosphate-Activated Protein Kinase and p38 Mitogen-Activated Protein Kinase Participate in the Stimulation of Glucose Uptak
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内分泌学杂志 2005年第5期
Montreal Diabetes Research Centre, Centre Hospitalier de l’Université de Montréal and the Departments of Medicine (A.P., L.C.) and Nutrition (E.J., M.P.), Université de Montréal, Montréal, Québec, Canada H2W 1T7
Address all correspondence and requests for reprints to: Lise Coderre, Ph.D., Research Centre, Centre Hospitalier de l’Université de Montréal-H?tel-Dieu, 3850 rue Saint-Urbain, Montréal (Québec) Canada H2W 1T7. E-mail: lise.coderre@umontreal.ca.
Abstract
During metabolic stress, such as ischemia or hypoxia, glucose becomes the principal energy source for the heart. It has been shown that increased cardiac glucose uptake during metabolic stress has a protective effect on cell survival and heart function. Despite its physiological importance, only limited data are available on the molecular mechanisms regulating glucose uptake under these conditions. We used 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, as a model to mimic hypoxia and gain insight into the signaling pathway underlying metabolic stress-induced glucose uptake in primary cultures of rat adult cardiomyocytes. The results demonstrate that 0.1 mM DNP induces 2.2- and 9-fold increases in AMP-activated protein kinase (AMPK) and p38 MAPK phosphorylation, respectively. This is associated with a 2.3-fold increase in glucose uptake in these cells. To further delineate the role of AMPK in the regulation of glucose uptake, we used two complementary approaches: pharmacological inhibition of the enzyme with adenine 9-?-D arabinofuranoside and adenoviral infection with a dominant-negative AMPK (DN-AMPK) mutant. Our results show that overexpression of DN-AMPK completely suppressed DNP-mediated phosphorylation of acetyl coenzyme A carboxylase, a downstream target of AMPK. Inhibition of AMPK with either 9-?-D arabinofuranoside or DN-AMPK also abolished DNP-mediated p38 MAPK phosphorylation. Importantly, AMPK inhibition only partially decreased DNP-stimulated glucose uptake in cardiomyocytes. Inhibition of p38 MAPK with the pharmacological agent PD169316 also partially reduced (70%) glucose uptake in response to DNP. In conclusion, our results indicate that p38 MAPK acts downstream of AMPK in cardiomyocytes and that activation of the AMPK/p38 MAPK signaling cascade is essential for maximal stimulation of glucose uptake in response to DNP in adult cardiomyocytes.
Introduction
THE HEART USES a variety of substrates, such as fatty acids, ketone bodies, and glucose for ATP production. Although fatty acids account for 60–70% of total energy production during aerobic conditions (1), carbohydrates become the major substrate during anaerobic periods and metabolic stress (2, 3, 4). It is well established that metabolic stressors, such as contractions (5), ischemia (6), and hypoxia (7), stimulate cardiac glucose uptake. It has been demonstrated that the ability of the heart to increase glucose uptake during ischemia is essential in preserving contractile function (3). Conversely, a decrease in glucose uptake and failure to maintain ATP levels during hypoxia increase cardiomyocyte death (8). Thus, the ability of the heart to up-regulate glucose uptake during metabolic stress is crucial for the maintenance of cardiac energy homeostasis and function.
Several laboratories have reported that AMP-activated protein kinase (AMPK) plays a major role in the regulation of metabolic stress-induced glucose uptake. AMPK is activated by increased AMP to ATP or creatine to phosphocreatine ratios (9, 10) and Thr172 phosphorylation by one or more upstream AMPK kinases (11, 12, 13). In skeletal muscle, activation of AMPK is observed in response to the adenosine analog 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (14, 15, 16) as well as various metabolic stimuli, such as muscle contractions (17), hypoxia (18, 19), and 2,4-dinitrophenol (DNP) (18, 20). Adenovirus-mediated expression of a constitutively active AMPK mutant (21) or activation of this enzyme by AICAR (22) increases glucose uptake in skeletal muscles. Conversely, overexpression of a dominant-negative AMPK (DN-AMPK) mutant in skeletal muscle cells or a kinase-dead AMPK2 (KD-AMPK) isoform in mice skeletal muscle completely suppresses both hypoxia- and AICAR-stimulated glucose uptake (19, 23). In contrast, expression of the KD-AMPK mutant only partially reduces contraction-mediated glucose uptake, suggesting that activation of an additional signaling pathway is required for this stimulus.
AMPK activation has been associated with the activation of numerous kinases, including p38 MAPK (24, 25, 26). As with AMPK, p38 MAPK activation is observed in response to ischemia (27), hypoxia (28), and DNP (29). It has been suggested that, in skeletal muscle, p38 MAPK activation is essential for maximal stimulation of glucose uptake in response to insulin (30) and contractions (31). Furthermore, it has been shown that in liver-derived Clone 9 cells, p38 MAPK is downstream of AMPK (32), and inhibition of p38 MAPK suppresses AICAR-stimulated glucose uptake in these cells.
Most of the studies cited above were performed in skeletal muscle. However, it is unclear whether the signaling pathway identified in skeletal muscle also operates in the heart. In skeletal muscle, stimulation of glucose uptake by exercise is independent of phosphatidylinositol 3-kinase (PI3-K). In contrast, inhibition of this enzyme diminishes glucose uptake in contracting cardiomyocytes (33, 34). Furthermore, hypoxia-induced glucose uptake is completely abolished in skeletal muscle overexpressing a KD-AMPK mutant (19), whereas ischemia-induced glucose uptake is only partially suppressed in transgenic mice overexpressing a DN-AMPK2 mutant in the heart (6). Together, these results suggest that significant differences exist in the regulation of glucose uptake between skeletal and cardiac muscles.
The aim of this project was to examine the signaling cascade activated in response to metabolic stress that leads to increased glucose uptake in primary cultures of adult cardiomyocytes. We used DNP, a weak base that dissipates the H+ gradient and uncouples the mitochondrial oxidative chain, as a model to mimic hypoxia in cardiomyocytes and study the molecular effectors that participate in the regulation of glucose uptake under these conditions. We chose to examine the contribution of AMPK and p38 MAPK, two enzymes involved in metabolic stress-mediated glucose uptake in skeletal muscle but whose role has not been clearly defined in the heart. Our results indicate that, in adult cardiomyocytes, p38 MAPK acts downstream of AMPK. Furthermore, and in contrast to skeletal muscle, inhibition of the AMPK/p38 MAPK signaling pathway only partially abolishes the stimulation of glucose uptake in response to DNP.
Materials and Methods
Chemicals
All cell culture solutions, fatty acid-free BSA (FAF BSA), water, supplements, wortmannin, DNP, adenine 9-?-D arabinofuranoside (AraA), potassium ferricyanide, potassium ferrocyanide, and Dnase I were purchased from Sigma-Aldrich Canada (Oakville, Ontario, Canada), and X-gal was from Roche (Laval, Québec, Canada). Collagenase was obtained from Worthington Biochemical Corp. (Lakewood, NJ). Human insulin (Humulin R) was procured from Eli Lilly Canada Inc. (Toronto, Ontario, Canada). Phospho-p38 MAPK (Thr 180/Tyr 182), p38 MAPK, phospho-AMPK (Thr 172), and AMPK polyclonal antibodies were from Cell Signaling Technology (Beverly, MA), whereas phospho-acetyl coenzyme A carboxylase (Ser 79) (ACC) was from Upstate Cell Signaling Solutions (Lake Placid, NY). DuPont NEN Life Science Products Research Products (Boston, MA) supplied [3H] 2-deoxyglucose. Polyvinylidene difluoride membranes were purchased from Immobilon Millipore (Bedford, MA). The enhanced chemiluminescence detection system was bought from Amersham Pharmacia Biotech (Baie d’Urfé, Québec, Canada). PD169316 was from Calbiochem (La Jolla, CA). The Bradford protein assay kit was from Bio-Rad (Hercules, CA). All electrophoresis reagents were obtained from Roche Molecular Biochemicals (Laval, Québec, Canada).
Isolation of adult rat cardiomyocytes
All experiments conformed to guidelines of the Canadian Council on Animal Care and were approved by the Animal Care Committee of the Centre Hospitalier de l’Université de Montréal. Male Sprague Dawley rats weighing 175–200 g were injected ip with 500 U heparin sulfate 15 min before anesthesia with sodium pentobarbital (60 mg/kg, ip). The heart was excised, and calcium-tolerant cardiomyocytes were isolated by the Langendorff method as described previously (35). During the whole procedure, the cells were maintained at 37 C. Briefly, all hearts were rinsed (4 ml/min) for 5 min in Krebs-Ringer (KR) buffer containing (in millimoles) 119 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 11 dextrose, and 25 HEPES (pH 7.4). They were then perfused with a calcium-free KR solution for 5 min to stop spontaneous cardiac contractions. This was followed by perfusion with KR buffer supplemented with 0.05% collagenase, 15 mM 2–3 butanedione monoxime, and 0.1% FAF BSA for 15 min. For the last 5 min of perfusion, the KR buffer was supplemented with 0.05 mM CaCl2, after which the ventricles were separated from the atria. The ventricles were minced in KR supplemented with 0.05% collagenase, 15 mM 2–3 butanedione monoxime, 0.2 mg/ml Dnase I, 0.1 mM CaCl2, and 0.1% FAF BSA. The resulting cell suspension was filtered through a nylon mesh and centrifuged at 1000 x g for 45 sec. The cells were washed twice and diluted in medium 199 supplemented with 11 mM glucose, 0.2% FAF BSA, 5 mM creatine, 2 mM L-carnitine, 5 mM taurine, 10–7 M insulin, 0.1 M ascorbic acid, 100 IU/ml penicillin, 25 μg/ml gentamicin, and 100 mg/ml streptomycin and then plated onto laminin-coated dishes. After 4 h, they were washed to remove damaged cells and debris before incubation at 37 C for 16 h in media containing 10–11 M insulin and 5.5 mM glucose.
Cardiomyocyte infection
The Ad5/cytomegalovirus (CMV)/nuclear localization signal (NLS)-adenovirus expressing ?-galactosidase (LacZ), which encodes the simian virus 40 large T-antigen nuclear-localization signal fused to the Escherichia coli LacZ reporter gene (36), served as a control to assess the percentage of infected cardiomyocytes at different multiplicities of infection (MOI). Dr. Lee A. Witters (Dartmouth Medical School, Hanover, NH) kindly provided the adenovirus containing the DN-AMPK mutant under the CMV promoter. This construct has a point mutation of the phosphorylation site (Thr 172) within the AMPK1 subunit and inhibits both AMPK1 and -2 heterotrimers (Witters, L. A., personal communication). Isolated cardiomyocytes were infected with 100, 200, or 500 MOI of either Ad5/CMV/NLS-LacZ or DN-AMPK in media containing 50 μg/ml of laminin. After 16 h, they were washed and incubated for an additional 8 h before glucose uptake measurements or enzyme activation studies.
In situ ?-galactosidase staining with X-gal
Cells were washed twice with KR buffer and fixed with ice-cold methanol for 10 min at –20 C. They were rewashed with KR buffer before ?-galactosidase staining was performed (37). Briefly, the cells were incubated for 2 h at 37 C with stain solution that contained 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, and 1 mg/ml X-gal. The cells were then rinsed twice with KR buffer and fixed with 10% formalin for 10 min at room temperature. ?-Galactosidase-positive cells with blue nuclei were then counted.
Glucose uptake in primary cultures of cardiomyocytes
The cells were washed twice with 1 ml KR buffer. They were then incubated for 30 min in 1 ml KR buffer containing 5 mM glucose and 0.2% FAF BSA. For the adenovirus experiments, glucose assays were conducted in media containing 10–11 M insulin to prevent DNP-associated cell death in DN-AMPK-infected cardiomyocytes. Inhibitors of PI3-K (100 nM wortmannin), AMPK (0.1, 0.5, and 1 mM AraA), and p38 MAPK (0.1, 0.2, and 0.5 μM PD169316) were added during the preincubation step as indicated in the figure legends. The glucose uptake assay was started by the addition of 1 μCi/ml [3H] 2-deoxyglucose. Vehicle (basal), DNP, and insulin were also added at this step as indicated in the figure legends and measured as described previously (35). Glucose uptake measurement was terminated by three rapid washes with 1 ml ice-cold KR buffer. The cells were disrupted with 0.5 ml of 0.5 M NaOH for 60 min at 37 C, and cell-associated radioactivity was quantified by scintillation counting. Glucose uptake was normalized to total protein, as measured by Bio-Rad assay.
AMPK, ACC, and p38 MAPK activation
The cells were washed twice with media containing no insulin and incubated for 1 h before stimulation with 0.1 mM DNP for 5 or 10 min. Inhibitors of AMPK and p38 MAPK were added 30 min before stimulation with DNP. As in the transport experiments, 10–11 M insulin was added to the media when the cardiomyocytes were infected with adenovirus. The reaction was stopped by three rapid washes with ice-cold KR buffer. The cells were then lysed in buffer containing 25 mM Tris-HCl (pH 7.4), 25 mM NaCl, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM EGTA, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 10 μg/ml aprotinin, 0.5 μg/ml leupeptin, 1% Triton X-100, and 0.1% sodium dodecyl sulfate. The lysate was centrifuged for 5 min at 12,000 x g at 4 C to remove insoluble material, and the resulting supernatant was taken for immunoblotting.
Gel electrophoresis and immunoblotting
Samples were electrophoresed on 6 or 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene difluoride membranes for Western blotting. The membranes were blocked for 1 h with 5% (wt/vol) milk in PBS solution (pH 7.4) containing 137 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, 0.05% Tween 20, and 0.02% sodium azide. They were then incubated with the primary antibody, followed by incubation with the appropriate secondary antibody conjugated to horseradish peroxidase. Antigen-antibody complexes were detected by the enhanced chemiluminescence method. Quantitative analysis was performed with a scanning densitometer.
Statistical analysis
Two-way ANOVA was applied for multiple comparisons, followed by the Fisher post hoc test. All data are reported as means ± SE. Values of P < 0.05 were considered to be significant.
Results
DNP and insulin stimulate glucose uptake through the activation of distinct signaling pathways in cardiomyocytes
In skeletal muscle, exercise and hypoxia stimulate glucose uptake in a PI3-K-independent manner. However, in the heart, PI3-K is activated during contraction, ischemia/reperfusion, and preconditioning (34, 38, 39). Thus, we investigated the role of PI3-K in DNP-mediated activation of glucose uptake in adult cardiomyocytes. We first established the concentration of DNP necessary to stimulate glucose uptake in these cells. The addition of 0.05 mM and 0.1 mM DNP increased cardiac glucose uptake by 1.4- and 2.3-fold, respectively (P < 0.02). At concentrations of 0.2 mM and above, we noted that a subset of cells lost their characteristic rod-shaped form, suggesting a cytotoxic effect of DNP at higher concentrations (data not shown). Therefore, all subsequent experiments were performed at 0.1 mM DNP. We next examined whether PI3-K played a role in DNP-mediated glucose uptake in adult cardiomyocytes. Our results showed that, in adult cardiomyocytes, the stimulatory effect of insulin and DNP on glucose uptake was additive (P < 0.02), indicating that the two stimuli used distinct signaling cascades (Fig. 1A). Inhibition of PI3-K by 100 nM wortmannin completely suppressed the increase of glucose uptake induced by insulin (P < 0.02) (Fig. 1B). In contrast, the inhibitor was without any effect on DNP-mediated glucose uptake, suggesting that PI3-K does not play a role in this process. Thus, in isolated adult cardiomyocytes, DNP and insulin stimulated glucose uptake through the activation of distinct signaling cascades.
FIG. 1. Insulin and DNP induce glucose uptake through activation of independent signaling pathways in cardiomyocytes. A, Cardiomyocytes were stimulated with 10–8 M insulin, 0.1 mM DNP, or a combination of both agents before measurement of glucose uptake as described in Materials and Methods. Means ± SE of three independent experiments. *, P < 0.05 compared with basal; , P < 0.02 different from DNP- and insulin-stimulated controls. B, Cardiomyocytes were preincubated for 30 min with 100 nM wortmannin before insulin or DNP stimulation. Means ± SE of four independent experiments. *, P < 0.006 vs. basal control; **, P < 0.009 vs. basal wortmannin; , P < 0.02 different from insulin-stimulated controls.
Pharmacological inhibition of AMPK reduces DNP-mediated p38 MAPK activation and glucose uptake in cardiomyocytes
AMPK plays a major role in stress-induced glucose uptake in skeletal muscle (25). The primary site responsible for AMPK activation is Thr 172, and phosphorylation of this site increases enzyme activity by 50- to 100-fold (40). As a first step to delineate the contribution of AMPK to DNP-mediated glucose uptake, we inhibited the enzyme with the pharmacological agent, AraA. Adult cardiomyocytes were pretreated with increasing concentrations of AraA (0.1, 0.5, and 1 mM) for 30 min, after which the cells were stimulated with DNP before measurement of AMPK activation with an antibody that recognizes its phosphorylated form. As shown in Fig. 2A, DNP induced a 1.8-fold increase in AMPK phosphorylation (P < 0.007). Incubation of cardiomyocytes with increasing concentrations of AraA provoked a dose-dependent decrease of DNP-mediated AMPK phosphorylation, resulting in 60 (P < 0.02) and 80% (P < 0.007) reductions of DNP-induced enzyme phosphorylation at 0.5 and 1 mM AraA, respectively. Because AraA primarily inhibits the AMPK2 isoform (17), the residual AMPK activity observed in cardiomyocytes is probably due to the AMPK1 isoform.
FIG. 2. AraA inhibits the activation of AMPK, p38 MAPK, and glucose uptake in response to DNP. Cardiomyocytes were preincubated for 30 min with 0.1, 0.5, or 1 mM AraA before stimulation with 0.1 mM DNP for either 5 (A) or 10 min (B). A, Top, Representative immunoblot of phospho-AMPK. Bottom, Densitometric analysis of immunoblots. Means ± SE of three independent experiments. *, P < 0.005 basal control vs. DNP-treated cells; , P < 0.02 different from DNP-stimulated controls; , P < 0.007 different from DNP-stimulated controls. B, Top, Representative immunoblot of phospho-p38 MAPK. Bottom, Densitometric analysis of immunoblots. Means ± SE of four independent experiments. *, P < 0.01 basal control vs. DNP-treated cells; , P < 0.05 different from DNP-stimulated controls.
Xi et al (32) demonstrated that p38 MAPK is localized downstream of the AMPK signaling cascade in liver-derived Clone 9 cells. We, therefore, examined whether AMPK inhibition suppresses DNP-mediated p38 MAPK activation in cardiomyocytes. p38 MAPK activation was assessed with an antibody that recognizes the active and phosphorylated form of the enzyme. Maximal activation of this enzyme was achieved after 10 min of stimulation (data not shown). This time point was used in subsequent experiments. As shown in Fig. 2B, DNP is a powerful activator of p38 MAPK, inducing a 9.4-fold increase of enzyme phosphorylation in control cardiomyocytes (P < 0.002). Inhibition of AMPK with 1 mM AraA was associated with a 62% reduction of p38 MAPK phosphorylation in response to DNP (P < 0.05).
We next studied the contribution of AMPK to DNP-stimulated glucose uptake in adult cardiomyocytes. As illustrated in Fig. 3, DNP increased glucose uptake by 2.4-fold in adult cardiomyocytes (P < 0.001). Incubation with increasing concentrations of AraA inhibited DNP action in a dose-dependent manner in these cells. At 0.5 and 1 mM AraA, we observed 41 (P < 0.02) and 51% (P < 0.002) reductions of glucose uptake in response to DNP. Incubation with a higher concentration of AraA (2 mM) did not further inhibit DNP-mediated glucose uptake in cardiomyocytes (data not shown).
FIG. 3. AraA inhibits glucose uptake in response to DNP in a dose-dependent manner. Cardiomyocytes were preincubated for 30 min with 0.1, 0.5, or 1 mM AraA before stimulation with 0.1 mM DNP for 20 min. Glucose uptake was evaluated as described in Materials and Methods. Means ± SE of three independent experiments. *, P < 0.006 basal control vs. DNP-treated cells; , P < 0.01 basal vs. DNP-treated cells (0.5 mM AraA) and different from DNP-stimulated controls; , P < 0.03 basal vs. DNP-treated cells (1 mM AraA) and different from DNP-stimulated controls.
DN-AMPK inhibits AMPK, ACC, and p38 MAPK phosphorylation and reduces glucose uptake in response to DNP
Pharmacological inhibitors are useful tools to examine the role of an enzyme in signaling pathways. However, most inhibitors also demonstrated nonspecific effects toward other enzymes. To further ascertain the role of AMPK in p38 MAPK activation and glucose uptake, we overexpressed a DN-AMPK mutant by adenoviral infection. Cardiomyocytes express very low levels of coxsackievirus and adenovirus receptors and show low infectivity, compared with other cells (41). We, therefore, used a nuclear-engineered ?-galactosidase reporter gene to titer virus infectivity in cardiomyocytes. Infection with 100, 200, and 500 MOI resulted in 68, 72, and 81% nuclear staining, respectively (data not shown). Based on these results, we infected cardiomyocytes with 500 MOI of DN-AMPK adenovirus in subsequent experiments. We monitored DN-AMPK expression by Western blotting with an anti-AMPK antibody. As shown in Fig. 4A, infection with 500 MOI of DN-AMPK increased AMPK content by 9.5-fold (P < 0.003). We next determined whether DN-AMPK could inhibit endogenous AMPK and prevent DNP-mediated phosphorylation of AMPK. As shown in Fig. 4A, stimulation with DNP produced a 2.2-fold increase in AMPK phosphorylation (P < 0.01). Overexpression of 500 MOI of DN-AMPK completely suppressed DNP-mediated phosphorylation of AMPK1 and -2 isoforms (P < 0.01). These results suggest that DN-AMPK acts as a dominant-negative inhibitor of endogenous AMPK.
FIG. 4. Overexpression of DN-AMPK inhibits DNP-induced AMPK, ACC, and p38 MAPK phosphorylation and activation of glucose uptake in cardiomyocytes. Cells were exposed for 24 h to 500 MOI of control or DN-AMPK adenovirus as described in Materials and Methods before stimulation with 0.1 mM DNP for either 5 (A and B) or 10 min (C). A, Top, Representative immunoblot of phospho-AMPK. Bottom, Densitometric analysis of immunoblots. Means ± SE of four independent experiments. *, P < 0.01 basal control vs. DNP-treated cells; , P < 0.01 different from DNP-stimulated controls. B, Top, Representative immunoblot of phospho-ACC, Bottom, Densitometric analysis of immunoblots. Means ± SE of four independent experiments. *, P < 0.03 basal control vs. DNP-treated cells; , P < 0.04, different from DNP-stimulated controls. C, Top, Representative immunoblot of phospho-p38 MAPK. Bottom, Densitometric analysis of immunoblots. Means ± SE of four independent experiments. *, P < 0.05 basal control vs. DNP-treated cells; , P < 0.05, different from DNP-stimulated controls.
To further ascertain AMPK inhibition by DN-AMPK, we examined the phosphorylation of a downstream target of AMPK, ACC, in response to DNP. Phosphorylation of ACC on ser79 reflects AMPK activation by phosphorylation and allosteric effectors. As illustrated in Fig. 4B, DNP induced a 3-fold-increase of ACC phosphorylation in control cells (P < 0.03), which was completely abolished upon infection with DN-AMPK (P < 0.04). The parallel reduction in phosphorylated AMPK and ACC demonstrated that overexpression of DN-AMPK inhibited the activation of the endogenous enzyme in response to DNP.
We next established the effect of DN-AMPK on p38 MAPK activation in cardiomyocytes. As shown in Fig. 4C, infection with DN-AMPK did not affect the concentration of p38 MAPK. Stimulation with DNP provoked a 2-fold increase in p38 MAPK activation in cardiomyocytes (P < 0.05). Infection with 500 MOI of DN-AMPK completely inhibited p38 MAPK phosphorylation in response to DNP (P < 0.05). These results confirmed the role of AMPK in DNP-mediated activation of p38 MAPK.
To examine the contribution of AMPK to DNP-stimulated glucose uptake in adult cardiomyocytes, we infected cardiomyocytes with 500 MOI of DN-AMPK adenovirus. Stimulation with DNP produced 1.6-fold stimulation of glucose uptake in cardiomyocytes (P < 0.05). In concordance with what was observed with AraA, DN-AMPK overexpression only partially reduced (39%) the increase of glucose uptake after DNP stimulation (Fig. 5A). In contrast, insulin-stimulated glucose uptake (P < 0.05) was not affected by DN-AMPK overexpression, and a 2.1- to 2.5-fold increase in glucose uptake was obtained in control and DN-AMPK-infected cells, respectively (Fig. 5B).
FIG. 5. Overexpression of DN-AMPK inhibits DNP-induced glucose uptake in cardiomyocytes. Cells were exposed for 24 h to 500 MOI of control or DN-AMPK adenovirus as described in Materials and Methods before stimulation with 0.1 mM DNP for 20 min (A) or with 10–7 M insulin for 30 min (B). Glucose uptake was measured as described in Materials and Methods. Means ± SE of four independent experiments. *, P < 0.05 basal control vs. DNP- and insulin-treated cells; , P < 0.03, different from DNP-stimulated controls.
DN-AMPK does not modulate glucose transporter (GLUT)1 and GLUT4 content in cardiomyocytes
GLUT1 and GLUT4 are the primary glucose transporters expressed in the heart. Because an alteration in the concentration of either GLUT1 and GLUT4 levels could modulate DNP-stimulated glucose uptake, we evaluated their protein content by Western blotting in adenoviral infected cardiomyocytes. Infection with 500 MOI DN-AMPK did not affect the concentration of either GLUT1 or GLUT4 proteins in cardiomyocytes (data not shown).
PD169316 inhibits DNP-induced p38 MAPK phosphorylation and glucose uptake in cardiomyocytes
Our results suggested that AMPK participates in DNP-induced glucose uptake and that p38 MAPK acts downstream of AMPK in this signaling pathway. To determine the contribution of p38 MAPK to DNP-mediated glucose uptake, we inhibited the enzyme with PD169316. This pharmacological compound binds to the ATP-binding pocket of the enzyme and inhibits p38 MAPK activity with an IC50 of 0.89 nM. Inactivation of p38 MAPK is associated with a reduction in Tyr182 and/or Thr180 phosphorylation, which is detectable by Western blotting. Cardiomyocytes were pretreated with increasing concentration of PD169316 for 30 min before evaluation of DNP-mediated glucose uptake in these cells. As shown in Fig. 6A, PD169316 inhibited DNP-stimulated glucose uptake in a dose-dependent manner in cardiomyocytes. At 0.5 μM, PD169316 provoked a 69% decrease in DNP-stimulated glucose uptake in cardiomyocytes (P < 0.003). Pretreatment with 1 μM PD169316 did not further inhibit DNP-mediated glucose uptake (73% reduction; data not shown). Together, these results suggested that maximal inhibition of glucose uptake is achieved at 0.5 μM PD169316. We next evaluated the ability of this compound to inhibit p38 MAPK activation in cardiomyocytes. As shown in Fig. 6B, DNP increased p38 MAPK phosphorylation by 4.5-fold in control cells (P < 0.004), and preincubation with 0.5 μM PD169316 provoked a 40% reduction of DNP-mediated p38 MAPK phosphorylation in cardiomyocytes (P < 0.04).
FIG. 6. PD169316 partially inhibits glucose uptake and p38 MAPK activation in response to DNP in cardiomyocytes. A, Cells were preincubated for 30 min with 0.1, 0.2, or 0.5 μM PD169316 before stimulation with 0.1 mM DNP for 20 min. Glucose uptake was measured as described in Materials and Methods. Means ± SE of five independent experiments. *, P < 0.003 basal vs. DNP-treated cells; **, P < 0.02 basal vs. DNP-treated cells; , P < 0.003, different from DNP-stimulated controls. B, Cells were preincubated for 30 min with 0.5 μM PD169316 before stimulation with 0.1 mM DNP for 10 min. Top, Representative immunoblot of phospho-p38 MAPK. Bottom, Densitometric analysis of immunoblots. Means ± SE of three independent experiments. *, P < 0.004 basal vs. DNP-treated cells; , P < 0.04, different from DNP-stimulated controls.
Discussion
DNP was used as a model to mimic hypoxia in cardiomyocytes and study the molecular mechanisms underlying the stimulation of glucose uptake in response to stress in these cells. Our data demonstrated that PI3-K activation is not required for DNP-mediated glucose uptake in cardiomyocytes. Our results also show that AMPK inhibition suppressed p38 MAPK activation in response to DNP in adult cardiomyocytes. More importantly, inhibition of the AMPK/p38 MAPK signaling cascade only partially abolished DNP-stimulated glucose uptake in these cells. Collectively, these findings demonstrated that p38 MAPK is downstream of the AMPK signaling cascade in cardiomyocytes and that activation of AMPK and p38 MAPK is required for maximal stimulation of glucose uptake during metabolic stress.
In skeletal muscle, insulin and contractions stimulate glucose uptake through activation of distinct signaling cascades, as demonstrated by the additive effect of these stimuli on this process. Furthermore, whereas activation of PI3-K is required for insulin action, the enzyme does not participate in the regulation of glucose uptake by contractions. In comparison, in isolated adult cardiomyocytes, the effects of insulin and contractions on glucose uptake are not additive (34). In addition, in cardiomyocytes, contractions activate PI3-K, and inhibition of the enzyme reduces contraction-mediated glucose uptake in these cells (34). These results suggest that PI3-K participates in the regulation of cardiac glucose uptake during contractions. In contrast to what has been observed for contractions, the effects of DNP and insulin are additive in cardiomyocytes. Furthermore, inhibition of PI3-K by wortmannin does not alter DNP-mediated glucose uptake, whereas it completely suppresses insulin’s stimulation of this process. Together, these results demonstrated that PI3-K does not contribute to DNP-stimulated glucose uptake in cardiomyocytes. This is in agreement with what has been reported for ischemia and preconditioning, both of which activate glucose uptake in a PI3-K-independent manner (39, 42). It should be noted that in skeletal muscle, the contribution of AMPK to the stimulation of glucose uptake differs between hypoxia and contractions (19). Potentially similar differences in the signaling pathway activated in response to contractions and DNP may also exist in the myocardium.
Several enzymes have been identified as potent mediators of glucose uptake in response to metabolic stress (25). AMPK, which regulates both glycolysis and fatty oxidation, is considered an important metabolic fuel gauge. The pharmacological activation of AMPK by AICAR stimulates glucose uptake in cardiac papillary (43) and skeletal muscles (44). However, stimulation with AICAR did not increase glucose uptake in cardiomyocytes (Pelletier, A., and L. Coderre, unpublished data). Studies have demonstrated that AICAR does not activate AMPK in these cells (45, 46, 47) probably because they lack adenylate cyclase, the enzyme responsible for its conversion to 5-aminoimidazole-4-carboxamide-1-?-D-ribofuranotide (47). In skeletal muscles, there is a good correlation between AMPK activation and enhanced glucose uptake in response to various metabolic stressors (25, 48). On the other hand, the role of AMPK in the regulation of cardiac glucose uptake has been largely unexplored. Therefore, our objective was to delineate the contribution of AMPK to p38 MAPK activation and the stimulation of glucose uptake in response to DNP in adult cardiomyocytes. The role of AMPK was examined using two complementary approaches: pharmacological inhibition of the enzyme with AraA, and adenoviral-mediated infection of cardiomyocytes with a DN-AMPK mutant. Our results showed that inhibition with AraA reduced DNP-mediated AMPK phosphorylation in a dose-dependent manner in adult cardiomyocytes. Overexpression of the DN-AMPK mutant also prevented AMPK and ACC phosphorylation in response to DNP. Inhibition of DNP-mediated ACC phosphorylation by DN-AMPK suggests that the mutant acts as a dominant negative of endogenous enzyme.
Activation of p38 MAPK has been observed in response to various stimuli including hyperosmolarity, hypoxia, ischemia, preconditioning, and DNP, stimuli that are also associated with increased AMPK activity (49). Furthermore, in skeletal muscles, pharmacological activation of AMPK by AICAR stimulated p38 MAPK (24). Our results show that stimulation with DNP also activates AMPK and p38 MAPK. Importantly, inhibition of AMPK with AraA greatly decreased DNP-mediated p38 MAPK phosphorylation in cardiomyocytes. Furthermore, overexpression of DN-AMPK completely inhibited DNP-mediated p38 MAPK phosphorylation in cardiomyocytes. Whereas we cannot exclude that DN-AMPK action is mediated by its binding to a component of the p38 MAPK signaling cascade, the fact that we observed a reduction of p38 MAPK phosphorylation after AMPK inhibition by a pharmacological agent or by the molecular approach strongly suggests that p38 MAPK is a downstream target of AMPK. Similar findings were also reported by Xi et al. (32), who demonstrated that overexpressing a DN-AMPK abolished p38 MAPK activation in liver-derived Clone 9 cells. In contrast, in 3T3-L1 adipocytes, activation of AMPK impaired insulin-mediated glucose uptake (50), whereas activation of p38 MAPK participates in the stimulatory effect of insulin on glucose uptake through modulation of GLUT4 intrinsic activity (30). This latter result suggests that the coupling between AMPK and p38 MAPK may be tissue specific. Our results demonstrated that, in the heart, p38 MAPK is downstream of AMPK. Additional work will be needed to further define the relationship between AMPK and p38 MAPK in different tissues.
Studies have suggested that activation of AMPK is required for AICAR- and hypoxia-stimulated glucose uptake in skeletal muscle. However, whether this enzyme plays a similar role in the heart is still not resolved. We thus examined AMPK’s contribution to the regulation of glucose uptake in adult cardiomyocytes. Inhibition of AMPK by AraA or DN-AMPK reduces the stimulatory effect of DNP on glucose uptake by 51 and 39%, respectively. Thus, in adult cardiomyocytes, inhibition of AMPK provokes only a partial reduction of glucose uptake in response to DNP. Xing et al. (6) also reported similar results in which no-flow ischemia-induced glucose uptake was inhibited by 62% in transgenic mice hearts overexpressing a DN-AMPK2 mutant. On the other hand, overexpression of a KD-AMPK mutant completely abolished the stimulation of cardiac glucose uptake during low-flow ischemia (51). These differences in the contribution of AMPK to glucose uptake may be related to the severity of the metabolic stress generated. Alternatively, they may be compensatory mechanisms activated in response to inhibition of both 1- and 2-AMPK isoforms in the heart. Our studies suggest that in cardiomyocytes, inhibition of AMPK only partially decreased glucose uptake in response to DNP. Studies have demonstrated that glucose is an important substrate during metabolic stresses such as ischemia or hypoxia. Thus, the partial reduction of glucose uptake in response to AMPK inhibition may be a protective mechanism to prevent complete suppression of cardiac glucose uptake, which could be detrimental for the heart. Thus, the contribution of multiple pathways to the stimulation of glucose uptake during metabolic stress may allow for the maintenance of glucose supply and thus cardiac function. Further studies exploring the role of AMPK in various models of ischemia will be needed to clarify the contribution of this enzyme to the stimulation of glucose uptake in the heart.
AMPK also phosphorylates and activates phosphofructokinase-2 (PFK-2) in the heart. PFK-2 is the enzyme responsible for the synthesis of fructose 2, 6-phosphate, a powerful activator of the glycolytic pathway (46). Thus, inhibition of AMPK should be paralleled by a concomitant reduction of PKF-2 activity, glycolysis and potentially glucose uptake. Studies have shown, however, that stimulation of glycolysis by increased cardiac workload does not require AMPK activation (52). Furthermore, in transgenic mice overexpressing DN-AMPK or KD-AMPK, cardiac glycogen breakdown occurs normally, suggesting that glycolysis was not impaired in these animals (6, 51). In addition to AMPK, PFK-2 can be phosphorylated and activated by other kinases, including protein kinase A. Cardiac glycolysis is also regulated by local factors such as adenine nucleotide concentration and intracellular pH (53, 54). Thus, the regulation of PFK-2 activity involves multiple and overlapping mechanisms, reflecting the importance of glycolysis in maintaining energy supplies during stressful conditions. Whether inhibition of AMPK impairs glycolysis in DNP-stimulated cardiomyocytes remains to be investigated.
Several studies have demonstrated that p38 MAPK participates in the regulation of glucose uptake in response to various stimuli probably through the modulation of GLUT1 (55) or GLUT4 intrinsic activity (30, 56). We have shown that p38 MAPK is downstream of the AMPK signaling cascade and that inhibition of AMPK decreases glucose uptake in cardiomyocytes. We thus sought to determine whether inhibition of p38 MAPK would also diminish glucose uptake in response to DNP in these cells. Pharmacological inhibition of p38 MAPK with PD169316 only partially decreased DNP-mediated glucose uptake in cardiomyocytes. Whereas we cannot rule out that the decrease in glucose uptake by PD169316 is independent of p38 MAPK inhibition, this result is consistent with the partial reduction of glucose uptake observed in response to AMPK inhibition in cardiomyocytes. On the other hand, these data contrast with the study of Xi et al. (32), in which they reported that inhibition of p38 MAPK completely suppresses AICAR-mediated glucose uptake in Clone 9 cells. Similar results were also obtained for skeletal muscle, in which pharmacological inhibition of p38 MAPK completely suppressed AICAR-mediated glucose uptake (24). On the other hand, inhibition of the enzyme decreased only partially contraction-mediated glucose uptake (31). As mentioned above, the contribution of the AMPK/p38 MAPK signaling pathway to glucose uptake is both tissue and stimuli specific. Our data in adult cardiomyocytes strongly suggest that in this tissue, activation of an additional pathway is required for maximal stimulation of glucose uptake in response to DNP.
In skeletal muscle, overexpression of KD-AMPK mutant or selective ablation of AMPK2 completely blocked the stimulatory effect of either AICAR or hypoxia in this tissue (57). Similar results have been reported by Fryer et al. (21), who demonstrated that AICAR-induced glucose uptake was completely inhibited by DN-AMPK overexpression in H-2K muscle cells. Thus, in skeletal muscle, activation of AMPK is required for both AICAR- and hypoxia-stimulated glucose uptake. On the other hand, inhibition of AMPK with AraA or DN-AMPK provokes only a partial reduction of DNP-mediated glucose uptake. This latter result suggests that the contribution of AMPK to the regulation of glucose uptake is tissue specific. Other differences between cardiac and skeletal muscles have also been reported. Eckel and colleagues (34) demonstrated that stimulation of glucose uptake by contraction required PI3-K activation in cardiomyocytes. Furthermore, in the heart, AMPK phosphorylates and activates PFK-2, leading to enhanced glycolysis, whereas this does not occur in muscle because, in this tissue, the PFK-2 isoform does not contain an AMPK phosphorylation site (58). Together, these studies indicate that important mechanistic differences may exist between the heart and skeletal muscle regarding the regulation of glucose metabolism. Because the concentrations and activities of signaling molecules can be modulated, primary cultures of adult cardiomyocytes may represent a good model to explore the activation of signaling pathways in the heart.
What could be the additional signaling pathway contributing to increased glucose uptake in cardiomyocytes? Stimulation with DNP increases intracellular Ca2+ concentration in adipocytes (59) and cardiomyocytes (60). In L6 myotubes, calcium chelation decreases glucose uptake in response to DNP, suggesting that Ca2+ contributes to this process (61). Other potential candidates include calcium/calmodulin-dependent protein kinase and classical protein kinase C, both of which can be activated by calcium and are involved in glucose uptake in skeletal muscles (61, 62, 63). Interestingly, pharmacological inhibition of calmodulin-dependent protein kinase reduces the stimulatory effect of hypoxia on glucose uptake in skeletal muscles (62, 63) and the heart (Pelletier, A., and L. Coderre, unpublished data), suggesting that the enzyme plays an important role in this process. However, confirmation of its role in the regulation of glucose uptake in the heart awaits further investigation.
In conclusion, our results demonstrate that p38 MAPK acts downstream of AMPK in adult cardiomyocytes. Importantly, inhibition of the AMPK/p38 MAPK signaling cascade decreased DNP-mediated glucose uptake, suggesting that activation of these enzymes is required for maximal stimulation of glucose uptake in cardiomyocytes.
Acknowledgments
We thank Dr. Pierre Paradis for providing the Ad5/CMV/NLS-LacZ adenovirus and for helpful discussions. We also thank Dr. James C. Engert for helpful discussions. The editorial assistance of Mr. Ovid Da Silva (editor, Research Support Office, Research Centre, Centre Hospitalier de l’Université de Montréal) is acknowledged.
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Address all correspondence and requests for reprints to: Lise Coderre, Ph.D., Research Centre, Centre Hospitalier de l’Université de Montréal-H?tel-Dieu, 3850 rue Saint-Urbain, Montréal (Québec) Canada H2W 1T7. E-mail: lise.coderre@umontreal.ca.
Abstract
During metabolic stress, such as ischemia or hypoxia, glucose becomes the principal energy source for the heart. It has been shown that increased cardiac glucose uptake during metabolic stress has a protective effect on cell survival and heart function. Despite its physiological importance, only limited data are available on the molecular mechanisms regulating glucose uptake under these conditions. We used 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, as a model to mimic hypoxia and gain insight into the signaling pathway underlying metabolic stress-induced glucose uptake in primary cultures of rat adult cardiomyocytes. The results demonstrate that 0.1 mM DNP induces 2.2- and 9-fold increases in AMP-activated protein kinase (AMPK) and p38 MAPK phosphorylation, respectively. This is associated with a 2.3-fold increase in glucose uptake in these cells. To further delineate the role of AMPK in the regulation of glucose uptake, we used two complementary approaches: pharmacological inhibition of the enzyme with adenine 9-?-D arabinofuranoside and adenoviral infection with a dominant-negative AMPK (DN-AMPK) mutant. Our results show that overexpression of DN-AMPK completely suppressed DNP-mediated phosphorylation of acetyl coenzyme A carboxylase, a downstream target of AMPK. Inhibition of AMPK with either 9-?-D arabinofuranoside or DN-AMPK also abolished DNP-mediated p38 MAPK phosphorylation. Importantly, AMPK inhibition only partially decreased DNP-stimulated glucose uptake in cardiomyocytes. Inhibition of p38 MAPK with the pharmacological agent PD169316 also partially reduced (70%) glucose uptake in response to DNP. In conclusion, our results indicate that p38 MAPK acts downstream of AMPK in cardiomyocytes and that activation of the AMPK/p38 MAPK signaling cascade is essential for maximal stimulation of glucose uptake in response to DNP in adult cardiomyocytes.
Introduction
THE HEART USES a variety of substrates, such as fatty acids, ketone bodies, and glucose for ATP production. Although fatty acids account for 60–70% of total energy production during aerobic conditions (1), carbohydrates become the major substrate during anaerobic periods and metabolic stress (2, 3, 4). It is well established that metabolic stressors, such as contractions (5), ischemia (6), and hypoxia (7), stimulate cardiac glucose uptake. It has been demonstrated that the ability of the heart to increase glucose uptake during ischemia is essential in preserving contractile function (3). Conversely, a decrease in glucose uptake and failure to maintain ATP levels during hypoxia increase cardiomyocyte death (8). Thus, the ability of the heart to up-regulate glucose uptake during metabolic stress is crucial for the maintenance of cardiac energy homeostasis and function.
Several laboratories have reported that AMP-activated protein kinase (AMPK) plays a major role in the regulation of metabolic stress-induced glucose uptake. AMPK is activated by increased AMP to ATP or creatine to phosphocreatine ratios (9, 10) and Thr172 phosphorylation by one or more upstream AMPK kinases (11, 12, 13). In skeletal muscle, activation of AMPK is observed in response to the adenosine analog 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (14, 15, 16) as well as various metabolic stimuli, such as muscle contractions (17), hypoxia (18, 19), and 2,4-dinitrophenol (DNP) (18, 20). Adenovirus-mediated expression of a constitutively active AMPK mutant (21) or activation of this enzyme by AICAR (22) increases glucose uptake in skeletal muscles. Conversely, overexpression of a dominant-negative AMPK (DN-AMPK) mutant in skeletal muscle cells or a kinase-dead AMPK2 (KD-AMPK) isoform in mice skeletal muscle completely suppresses both hypoxia- and AICAR-stimulated glucose uptake (19, 23). In contrast, expression of the KD-AMPK mutant only partially reduces contraction-mediated glucose uptake, suggesting that activation of an additional signaling pathway is required for this stimulus.
AMPK activation has been associated with the activation of numerous kinases, including p38 MAPK (24, 25, 26). As with AMPK, p38 MAPK activation is observed in response to ischemia (27), hypoxia (28), and DNP (29). It has been suggested that, in skeletal muscle, p38 MAPK activation is essential for maximal stimulation of glucose uptake in response to insulin (30) and contractions (31). Furthermore, it has been shown that in liver-derived Clone 9 cells, p38 MAPK is downstream of AMPK (32), and inhibition of p38 MAPK suppresses AICAR-stimulated glucose uptake in these cells.
Most of the studies cited above were performed in skeletal muscle. However, it is unclear whether the signaling pathway identified in skeletal muscle also operates in the heart. In skeletal muscle, stimulation of glucose uptake by exercise is independent of phosphatidylinositol 3-kinase (PI3-K). In contrast, inhibition of this enzyme diminishes glucose uptake in contracting cardiomyocytes (33, 34). Furthermore, hypoxia-induced glucose uptake is completely abolished in skeletal muscle overexpressing a KD-AMPK mutant (19), whereas ischemia-induced glucose uptake is only partially suppressed in transgenic mice overexpressing a DN-AMPK2 mutant in the heart (6). Together, these results suggest that significant differences exist in the regulation of glucose uptake between skeletal and cardiac muscles.
The aim of this project was to examine the signaling cascade activated in response to metabolic stress that leads to increased glucose uptake in primary cultures of adult cardiomyocytes. We used DNP, a weak base that dissipates the H+ gradient and uncouples the mitochondrial oxidative chain, as a model to mimic hypoxia in cardiomyocytes and study the molecular effectors that participate in the regulation of glucose uptake under these conditions. We chose to examine the contribution of AMPK and p38 MAPK, two enzymes involved in metabolic stress-mediated glucose uptake in skeletal muscle but whose role has not been clearly defined in the heart. Our results indicate that, in adult cardiomyocytes, p38 MAPK acts downstream of AMPK. Furthermore, and in contrast to skeletal muscle, inhibition of the AMPK/p38 MAPK signaling pathway only partially abolishes the stimulation of glucose uptake in response to DNP.
Materials and Methods
Chemicals
All cell culture solutions, fatty acid-free BSA (FAF BSA), water, supplements, wortmannin, DNP, adenine 9-?-D arabinofuranoside (AraA), potassium ferricyanide, potassium ferrocyanide, and Dnase I were purchased from Sigma-Aldrich Canada (Oakville, Ontario, Canada), and X-gal was from Roche (Laval, Québec, Canada). Collagenase was obtained from Worthington Biochemical Corp. (Lakewood, NJ). Human insulin (Humulin R) was procured from Eli Lilly Canada Inc. (Toronto, Ontario, Canada). Phospho-p38 MAPK (Thr 180/Tyr 182), p38 MAPK, phospho-AMPK (Thr 172), and AMPK polyclonal antibodies were from Cell Signaling Technology (Beverly, MA), whereas phospho-acetyl coenzyme A carboxylase (Ser 79) (ACC) was from Upstate Cell Signaling Solutions (Lake Placid, NY). DuPont NEN Life Science Products Research Products (Boston, MA) supplied [3H] 2-deoxyglucose. Polyvinylidene difluoride membranes were purchased from Immobilon Millipore (Bedford, MA). The enhanced chemiluminescence detection system was bought from Amersham Pharmacia Biotech (Baie d’Urfé, Québec, Canada). PD169316 was from Calbiochem (La Jolla, CA). The Bradford protein assay kit was from Bio-Rad (Hercules, CA). All electrophoresis reagents were obtained from Roche Molecular Biochemicals (Laval, Québec, Canada).
Isolation of adult rat cardiomyocytes
All experiments conformed to guidelines of the Canadian Council on Animal Care and were approved by the Animal Care Committee of the Centre Hospitalier de l’Université de Montréal. Male Sprague Dawley rats weighing 175–200 g were injected ip with 500 U heparin sulfate 15 min before anesthesia with sodium pentobarbital (60 mg/kg, ip). The heart was excised, and calcium-tolerant cardiomyocytes were isolated by the Langendorff method as described previously (35). During the whole procedure, the cells were maintained at 37 C. Briefly, all hearts were rinsed (4 ml/min) for 5 min in Krebs-Ringer (KR) buffer containing (in millimoles) 119 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 11 dextrose, and 25 HEPES (pH 7.4). They were then perfused with a calcium-free KR solution for 5 min to stop spontaneous cardiac contractions. This was followed by perfusion with KR buffer supplemented with 0.05% collagenase, 15 mM 2–3 butanedione monoxime, and 0.1% FAF BSA for 15 min. For the last 5 min of perfusion, the KR buffer was supplemented with 0.05 mM CaCl2, after which the ventricles were separated from the atria. The ventricles were minced in KR supplemented with 0.05% collagenase, 15 mM 2–3 butanedione monoxime, 0.2 mg/ml Dnase I, 0.1 mM CaCl2, and 0.1% FAF BSA. The resulting cell suspension was filtered through a nylon mesh and centrifuged at 1000 x g for 45 sec. The cells were washed twice and diluted in medium 199 supplemented with 11 mM glucose, 0.2% FAF BSA, 5 mM creatine, 2 mM L-carnitine, 5 mM taurine, 10–7 M insulin, 0.1 M ascorbic acid, 100 IU/ml penicillin, 25 μg/ml gentamicin, and 100 mg/ml streptomycin and then plated onto laminin-coated dishes. After 4 h, they were washed to remove damaged cells and debris before incubation at 37 C for 16 h in media containing 10–11 M insulin and 5.5 mM glucose.
Cardiomyocyte infection
The Ad5/cytomegalovirus (CMV)/nuclear localization signal (NLS)-adenovirus expressing ?-galactosidase (LacZ), which encodes the simian virus 40 large T-antigen nuclear-localization signal fused to the Escherichia coli LacZ reporter gene (36), served as a control to assess the percentage of infected cardiomyocytes at different multiplicities of infection (MOI). Dr. Lee A. Witters (Dartmouth Medical School, Hanover, NH) kindly provided the adenovirus containing the DN-AMPK mutant under the CMV promoter. This construct has a point mutation of the phosphorylation site (Thr 172) within the AMPK1 subunit and inhibits both AMPK1 and -2 heterotrimers (Witters, L. A., personal communication). Isolated cardiomyocytes were infected with 100, 200, or 500 MOI of either Ad5/CMV/NLS-LacZ or DN-AMPK in media containing 50 μg/ml of laminin. After 16 h, they were washed and incubated for an additional 8 h before glucose uptake measurements or enzyme activation studies.
In situ ?-galactosidase staining with X-gal
Cells were washed twice with KR buffer and fixed with ice-cold methanol for 10 min at –20 C. They were rewashed with KR buffer before ?-galactosidase staining was performed (37). Briefly, the cells were incubated for 2 h at 37 C with stain solution that contained 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, and 1 mg/ml X-gal. The cells were then rinsed twice with KR buffer and fixed with 10% formalin for 10 min at room temperature. ?-Galactosidase-positive cells with blue nuclei were then counted.
Glucose uptake in primary cultures of cardiomyocytes
The cells were washed twice with 1 ml KR buffer. They were then incubated for 30 min in 1 ml KR buffer containing 5 mM glucose and 0.2% FAF BSA. For the adenovirus experiments, glucose assays were conducted in media containing 10–11 M insulin to prevent DNP-associated cell death in DN-AMPK-infected cardiomyocytes. Inhibitors of PI3-K (100 nM wortmannin), AMPK (0.1, 0.5, and 1 mM AraA), and p38 MAPK (0.1, 0.2, and 0.5 μM PD169316) were added during the preincubation step as indicated in the figure legends. The glucose uptake assay was started by the addition of 1 μCi/ml [3H] 2-deoxyglucose. Vehicle (basal), DNP, and insulin were also added at this step as indicated in the figure legends and measured as described previously (35). Glucose uptake measurement was terminated by three rapid washes with 1 ml ice-cold KR buffer. The cells were disrupted with 0.5 ml of 0.5 M NaOH for 60 min at 37 C, and cell-associated radioactivity was quantified by scintillation counting. Glucose uptake was normalized to total protein, as measured by Bio-Rad assay.
AMPK, ACC, and p38 MAPK activation
The cells were washed twice with media containing no insulin and incubated for 1 h before stimulation with 0.1 mM DNP for 5 or 10 min. Inhibitors of AMPK and p38 MAPK were added 30 min before stimulation with DNP. As in the transport experiments, 10–11 M insulin was added to the media when the cardiomyocytes were infected with adenovirus. The reaction was stopped by three rapid washes with ice-cold KR buffer. The cells were then lysed in buffer containing 25 mM Tris-HCl (pH 7.4), 25 mM NaCl, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM EGTA, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 10 μg/ml aprotinin, 0.5 μg/ml leupeptin, 1% Triton X-100, and 0.1% sodium dodecyl sulfate. The lysate was centrifuged for 5 min at 12,000 x g at 4 C to remove insoluble material, and the resulting supernatant was taken for immunoblotting.
Gel electrophoresis and immunoblotting
Samples were electrophoresed on 6 or 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene difluoride membranes for Western blotting. The membranes were blocked for 1 h with 5% (wt/vol) milk in PBS solution (pH 7.4) containing 137 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, 0.05% Tween 20, and 0.02% sodium azide. They were then incubated with the primary antibody, followed by incubation with the appropriate secondary antibody conjugated to horseradish peroxidase. Antigen-antibody complexes were detected by the enhanced chemiluminescence method. Quantitative analysis was performed with a scanning densitometer.
Statistical analysis
Two-way ANOVA was applied for multiple comparisons, followed by the Fisher post hoc test. All data are reported as means ± SE. Values of P < 0.05 were considered to be significant.
Results
DNP and insulin stimulate glucose uptake through the activation of distinct signaling pathways in cardiomyocytes
In skeletal muscle, exercise and hypoxia stimulate glucose uptake in a PI3-K-independent manner. However, in the heart, PI3-K is activated during contraction, ischemia/reperfusion, and preconditioning (34, 38, 39). Thus, we investigated the role of PI3-K in DNP-mediated activation of glucose uptake in adult cardiomyocytes. We first established the concentration of DNP necessary to stimulate glucose uptake in these cells. The addition of 0.05 mM and 0.1 mM DNP increased cardiac glucose uptake by 1.4- and 2.3-fold, respectively (P < 0.02). At concentrations of 0.2 mM and above, we noted that a subset of cells lost their characteristic rod-shaped form, suggesting a cytotoxic effect of DNP at higher concentrations (data not shown). Therefore, all subsequent experiments were performed at 0.1 mM DNP. We next examined whether PI3-K played a role in DNP-mediated glucose uptake in adult cardiomyocytes. Our results showed that, in adult cardiomyocytes, the stimulatory effect of insulin and DNP on glucose uptake was additive (P < 0.02), indicating that the two stimuli used distinct signaling cascades (Fig. 1A). Inhibition of PI3-K by 100 nM wortmannin completely suppressed the increase of glucose uptake induced by insulin (P < 0.02) (Fig. 1B). In contrast, the inhibitor was without any effect on DNP-mediated glucose uptake, suggesting that PI3-K does not play a role in this process. Thus, in isolated adult cardiomyocytes, DNP and insulin stimulated glucose uptake through the activation of distinct signaling cascades.
FIG. 1. Insulin and DNP induce glucose uptake through activation of independent signaling pathways in cardiomyocytes. A, Cardiomyocytes were stimulated with 10–8 M insulin, 0.1 mM DNP, or a combination of both agents before measurement of glucose uptake as described in Materials and Methods. Means ± SE of three independent experiments. *, P < 0.05 compared with basal; , P < 0.02 different from DNP- and insulin-stimulated controls. B, Cardiomyocytes were preincubated for 30 min with 100 nM wortmannin before insulin or DNP stimulation. Means ± SE of four independent experiments. *, P < 0.006 vs. basal control; **, P < 0.009 vs. basal wortmannin; , P < 0.02 different from insulin-stimulated controls.
Pharmacological inhibition of AMPK reduces DNP-mediated p38 MAPK activation and glucose uptake in cardiomyocytes
AMPK plays a major role in stress-induced glucose uptake in skeletal muscle (25). The primary site responsible for AMPK activation is Thr 172, and phosphorylation of this site increases enzyme activity by 50- to 100-fold (40). As a first step to delineate the contribution of AMPK to DNP-mediated glucose uptake, we inhibited the enzyme with the pharmacological agent, AraA. Adult cardiomyocytes were pretreated with increasing concentrations of AraA (0.1, 0.5, and 1 mM) for 30 min, after which the cells were stimulated with DNP before measurement of AMPK activation with an antibody that recognizes its phosphorylated form. As shown in Fig. 2A, DNP induced a 1.8-fold increase in AMPK phosphorylation (P < 0.007). Incubation of cardiomyocytes with increasing concentrations of AraA provoked a dose-dependent decrease of DNP-mediated AMPK phosphorylation, resulting in 60 (P < 0.02) and 80% (P < 0.007) reductions of DNP-induced enzyme phosphorylation at 0.5 and 1 mM AraA, respectively. Because AraA primarily inhibits the AMPK2 isoform (17), the residual AMPK activity observed in cardiomyocytes is probably due to the AMPK1 isoform.
FIG. 2. AraA inhibits the activation of AMPK, p38 MAPK, and glucose uptake in response to DNP. Cardiomyocytes were preincubated for 30 min with 0.1, 0.5, or 1 mM AraA before stimulation with 0.1 mM DNP for either 5 (A) or 10 min (B). A, Top, Representative immunoblot of phospho-AMPK. Bottom, Densitometric analysis of immunoblots. Means ± SE of three independent experiments. *, P < 0.005 basal control vs. DNP-treated cells; , P < 0.02 different from DNP-stimulated controls; , P < 0.007 different from DNP-stimulated controls. B, Top, Representative immunoblot of phospho-p38 MAPK. Bottom, Densitometric analysis of immunoblots. Means ± SE of four independent experiments. *, P < 0.01 basal control vs. DNP-treated cells; , P < 0.05 different from DNP-stimulated controls.
Xi et al (32) demonstrated that p38 MAPK is localized downstream of the AMPK signaling cascade in liver-derived Clone 9 cells. We, therefore, examined whether AMPK inhibition suppresses DNP-mediated p38 MAPK activation in cardiomyocytes. p38 MAPK activation was assessed with an antibody that recognizes the active and phosphorylated form of the enzyme. Maximal activation of this enzyme was achieved after 10 min of stimulation (data not shown). This time point was used in subsequent experiments. As shown in Fig. 2B, DNP is a powerful activator of p38 MAPK, inducing a 9.4-fold increase of enzyme phosphorylation in control cardiomyocytes (P < 0.002). Inhibition of AMPK with 1 mM AraA was associated with a 62% reduction of p38 MAPK phosphorylation in response to DNP (P < 0.05).
We next studied the contribution of AMPK to DNP-stimulated glucose uptake in adult cardiomyocytes. As illustrated in Fig. 3, DNP increased glucose uptake by 2.4-fold in adult cardiomyocytes (P < 0.001). Incubation with increasing concentrations of AraA inhibited DNP action in a dose-dependent manner in these cells. At 0.5 and 1 mM AraA, we observed 41 (P < 0.02) and 51% (P < 0.002) reductions of glucose uptake in response to DNP. Incubation with a higher concentration of AraA (2 mM) did not further inhibit DNP-mediated glucose uptake in cardiomyocytes (data not shown).
FIG. 3. AraA inhibits glucose uptake in response to DNP in a dose-dependent manner. Cardiomyocytes were preincubated for 30 min with 0.1, 0.5, or 1 mM AraA before stimulation with 0.1 mM DNP for 20 min. Glucose uptake was evaluated as described in Materials and Methods. Means ± SE of three independent experiments. *, P < 0.006 basal control vs. DNP-treated cells; , P < 0.01 basal vs. DNP-treated cells (0.5 mM AraA) and different from DNP-stimulated controls; , P < 0.03 basal vs. DNP-treated cells (1 mM AraA) and different from DNP-stimulated controls.
DN-AMPK inhibits AMPK, ACC, and p38 MAPK phosphorylation and reduces glucose uptake in response to DNP
Pharmacological inhibitors are useful tools to examine the role of an enzyme in signaling pathways. However, most inhibitors also demonstrated nonspecific effects toward other enzymes. To further ascertain the role of AMPK in p38 MAPK activation and glucose uptake, we overexpressed a DN-AMPK mutant by adenoviral infection. Cardiomyocytes express very low levels of coxsackievirus and adenovirus receptors and show low infectivity, compared with other cells (41). We, therefore, used a nuclear-engineered ?-galactosidase reporter gene to titer virus infectivity in cardiomyocytes. Infection with 100, 200, and 500 MOI resulted in 68, 72, and 81% nuclear staining, respectively (data not shown). Based on these results, we infected cardiomyocytes with 500 MOI of DN-AMPK adenovirus in subsequent experiments. We monitored DN-AMPK expression by Western blotting with an anti-AMPK antibody. As shown in Fig. 4A, infection with 500 MOI of DN-AMPK increased AMPK content by 9.5-fold (P < 0.003). We next determined whether DN-AMPK could inhibit endogenous AMPK and prevent DNP-mediated phosphorylation of AMPK. As shown in Fig. 4A, stimulation with DNP produced a 2.2-fold increase in AMPK phosphorylation (P < 0.01). Overexpression of 500 MOI of DN-AMPK completely suppressed DNP-mediated phosphorylation of AMPK1 and -2 isoforms (P < 0.01). These results suggest that DN-AMPK acts as a dominant-negative inhibitor of endogenous AMPK.
FIG. 4. Overexpression of DN-AMPK inhibits DNP-induced AMPK, ACC, and p38 MAPK phosphorylation and activation of glucose uptake in cardiomyocytes. Cells were exposed for 24 h to 500 MOI of control or DN-AMPK adenovirus as described in Materials and Methods before stimulation with 0.1 mM DNP for either 5 (A and B) or 10 min (C). A, Top, Representative immunoblot of phospho-AMPK. Bottom, Densitometric analysis of immunoblots. Means ± SE of four independent experiments. *, P < 0.01 basal control vs. DNP-treated cells; , P < 0.01 different from DNP-stimulated controls. B, Top, Representative immunoblot of phospho-ACC, Bottom, Densitometric analysis of immunoblots. Means ± SE of four independent experiments. *, P < 0.03 basal control vs. DNP-treated cells; , P < 0.04, different from DNP-stimulated controls. C, Top, Representative immunoblot of phospho-p38 MAPK. Bottom, Densitometric analysis of immunoblots. Means ± SE of four independent experiments. *, P < 0.05 basal control vs. DNP-treated cells; , P < 0.05, different from DNP-stimulated controls.
To further ascertain AMPK inhibition by DN-AMPK, we examined the phosphorylation of a downstream target of AMPK, ACC, in response to DNP. Phosphorylation of ACC on ser79 reflects AMPK activation by phosphorylation and allosteric effectors. As illustrated in Fig. 4B, DNP induced a 3-fold-increase of ACC phosphorylation in control cells (P < 0.03), which was completely abolished upon infection with DN-AMPK (P < 0.04). The parallel reduction in phosphorylated AMPK and ACC demonstrated that overexpression of DN-AMPK inhibited the activation of the endogenous enzyme in response to DNP.
We next established the effect of DN-AMPK on p38 MAPK activation in cardiomyocytes. As shown in Fig. 4C, infection with DN-AMPK did not affect the concentration of p38 MAPK. Stimulation with DNP provoked a 2-fold increase in p38 MAPK activation in cardiomyocytes (P < 0.05). Infection with 500 MOI of DN-AMPK completely inhibited p38 MAPK phosphorylation in response to DNP (P < 0.05). These results confirmed the role of AMPK in DNP-mediated activation of p38 MAPK.
To examine the contribution of AMPK to DNP-stimulated glucose uptake in adult cardiomyocytes, we infected cardiomyocytes with 500 MOI of DN-AMPK adenovirus. Stimulation with DNP produced 1.6-fold stimulation of glucose uptake in cardiomyocytes (P < 0.05). In concordance with what was observed with AraA, DN-AMPK overexpression only partially reduced (39%) the increase of glucose uptake after DNP stimulation (Fig. 5A). In contrast, insulin-stimulated glucose uptake (P < 0.05) was not affected by DN-AMPK overexpression, and a 2.1- to 2.5-fold increase in glucose uptake was obtained in control and DN-AMPK-infected cells, respectively (Fig. 5B).
FIG. 5. Overexpression of DN-AMPK inhibits DNP-induced glucose uptake in cardiomyocytes. Cells were exposed for 24 h to 500 MOI of control or DN-AMPK adenovirus as described in Materials and Methods before stimulation with 0.1 mM DNP for 20 min (A) or with 10–7 M insulin for 30 min (B). Glucose uptake was measured as described in Materials and Methods. Means ± SE of four independent experiments. *, P < 0.05 basal control vs. DNP- and insulin-treated cells; , P < 0.03, different from DNP-stimulated controls.
DN-AMPK does not modulate glucose transporter (GLUT)1 and GLUT4 content in cardiomyocytes
GLUT1 and GLUT4 are the primary glucose transporters expressed in the heart. Because an alteration in the concentration of either GLUT1 and GLUT4 levels could modulate DNP-stimulated glucose uptake, we evaluated their protein content by Western blotting in adenoviral infected cardiomyocytes. Infection with 500 MOI DN-AMPK did not affect the concentration of either GLUT1 or GLUT4 proteins in cardiomyocytes (data not shown).
PD169316 inhibits DNP-induced p38 MAPK phosphorylation and glucose uptake in cardiomyocytes
Our results suggested that AMPK participates in DNP-induced glucose uptake and that p38 MAPK acts downstream of AMPK in this signaling pathway. To determine the contribution of p38 MAPK to DNP-mediated glucose uptake, we inhibited the enzyme with PD169316. This pharmacological compound binds to the ATP-binding pocket of the enzyme and inhibits p38 MAPK activity with an IC50 of 0.89 nM. Inactivation of p38 MAPK is associated with a reduction in Tyr182 and/or Thr180 phosphorylation, which is detectable by Western blotting. Cardiomyocytes were pretreated with increasing concentration of PD169316 for 30 min before evaluation of DNP-mediated glucose uptake in these cells. As shown in Fig. 6A, PD169316 inhibited DNP-stimulated glucose uptake in a dose-dependent manner in cardiomyocytes. At 0.5 μM, PD169316 provoked a 69% decrease in DNP-stimulated glucose uptake in cardiomyocytes (P < 0.003). Pretreatment with 1 μM PD169316 did not further inhibit DNP-mediated glucose uptake (73% reduction; data not shown). Together, these results suggested that maximal inhibition of glucose uptake is achieved at 0.5 μM PD169316. We next evaluated the ability of this compound to inhibit p38 MAPK activation in cardiomyocytes. As shown in Fig. 6B, DNP increased p38 MAPK phosphorylation by 4.5-fold in control cells (P < 0.004), and preincubation with 0.5 μM PD169316 provoked a 40% reduction of DNP-mediated p38 MAPK phosphorylation in cardiomyocytes (P < 0.04).
FIG. 6. PD169316 partially inhibits glucose uptake and p38 MAPK activation in response to DNP in cardiomyocytes. A, Cells were preincubated for 30 min with 0.1, 0.2, or 0.5 μM PD169316 before stimulation with 0.1 mM DNP for 20 min. Glucose uptake was measured as described in Materials and Methods. Means ± SE of five independent experiments. *, P < 0.003 basal vs. DNP-treated cells; **, P < 0.02 basal vs. DNP-treated cells; , P < 0.003, different from DNP-stimulated controls. B, Cells were preincubated for 30 min with 0.5 μM PD169316 before stimulation with 0.1 mM DNP for 10 min. Top, Representative immunoblot of phospho-p38 MAPK. Bottom, Densitometric analysis of immunoblots. Means ± SE of three independent experiments. *, P < 0.004 basal vs. DNP-treated cells; , P < 0.04, different from DNP-stimulated controls.
Discussion
DNP was used as a model to mimic hypoxia in cardiomyocytes and study the molecular mechanisms underlying the stimulation of glucose uptake in response to stress in these cells. Our data demonstrated that PI3-K activation is not required for DNP-mediated glucose uptake in cardiomyocytes. Our results also show that AMPK inhibition suppressed p38 MAPK activation in response to DNP in adult cardiomyocytes. More importantly, inhibition of the AMPK/p38 MAPK signaling cascade only partially abolished DNP-stimulated glucose uptake in these cells. Collectively, these findings demonstrated that p38 MAPK is downstream of the AMPK signaling cascade in cardiomyocytes and that activation of AMPK and p38 MAPK is required for maximal stimulation of glucose uptake during metabolic stress.
In skeletal muscle, insulin and contractions stimulate glucose uptake through activation of distinct signaling cascades, as demonstrated by the additive effect of these stimuli on this process. Furthermore, whereas activation of PI3-K is required for insulin action, the enzyme does not participate in the regulation of glucose uptake by contractions. In comparison, in isolated adult cardiomyocytes, the effects of insulin and contractions on glucose uptake are not additive (34). In addition, in cardiomyocytes, contractions activate PI3-K, and inhibition of the enzyme reduces contraction-mediated glucose uptake in these cells (34). These results suggest that PI3-K participates in the regulation of cardiac glucose uptake during contractions. In contrast to what has been observed for contractions, the effects of DNP and insulin are additive in cardiomyocytes. Furthermore, inhibition of PI3-K by wortmannin does not alter DNP-mediated glucose uptake, whereas it completely suppresses insulin’s stimulation of this process. Together, these results demonstrated that PI3-K does not contribute to DNP-stimulated glucose uptake in cardiomyocytes. This is in agreement with what has been reported for ischemia and preconditioning, both of which activate glucose uptake in a PI3-K-independent manner (39, 42). It should be noted that in skeletal muscle, the contribution of AMPK to the stimulation of glucose uptake differs between hypoxia and contractions (19). Potentially similar differences in the signaling pathway activated in response to contractions and DNP may also exist in the myocardium.
Several enzymes have been identified as potent mediators of glucose uptake in response to metabolic stress (25). AMPK, which regulates both glycolysis and fatty oxidation, is considered an important metabolic fuel gauge. The pharmacological activation of AMPK by AICAR stimulates glucose uptake in cardiac papillary (43) and skeletal muscles (44). However, stimulation with AICAR did not increase glucose uptake in cardiomyocytes (Pelletier, A., and L. Coderre, unpublished data). Studies have demonstrated that AICAR does not activate AMPK in these cells (45, 46, 47) probably because they lack adenylate cyclase, the enzyme responsible for its conversion to 5-aminoimidazole-4-carboxamide-1-?-D-ribofuranotide (47). In skeletal muscles, there is a good correlation between AMPK activation and enhanced glucose uptake in response to various metabolic stressors (25, 48). On the other hand, the role of AMPK in the regulation of cardiac glucose uptake has been largely unexplored. Therefore, our objective was to delineate the contribution of AMPK to p38 MAPK activation and the stimulation of glucose uptake in response to DNP in adult cardiomyocytes. The role of AMPK was examined using two complementary approaches: pharmacological inhibition of the enzyme with AraA, and adenoviral-mediated infection of cardiomyocytes with a DN-AMPK mutant. Our results showed that inhibition with AraA reduced DNP-mediated AMPK phosphorylation in a dose-dependent manner in adult cardiomyocytes. Overexpression of the DN-AMPK mutant also prevented AMPK and ACC phosphorylation in response to DNP. Inhibition of DNP-mediated ACC phosphorylation by DN-AMPK suggests that the mutant acts as a dominant negative of endogenous enzyme.
Activation of p38 MAPK has been observed in response to various stimuli including hyperosmolarity, hypoxia, ischemia, preconditioning, and DNP, stimuli that are also associated with increased AMPK activity (49). Furthermore, in skeletal muscles, pharmacological activation of AMPK by AICAR stimulated p38 MAPK (24). Our results show that stimulation with DNP also activates AMPK and p38 MAPK. Importantly, inhibition of AMPK with AraA greatly decreased DNP-mediated p38 MAPK phosphorylation in cardiomyocytes. Furthermore, overexpression of DN-AMPK completely inhibited DNP-mediated p38 MAPK phosphorylation in cardiomyocytes. Whereas we cannot exclude that DN-AMPK action is mediated by its binding to a component of the p38 MAPK signaling cascade, the fact that we observed a reduction of p38 MAPK phosphorylation after AMPK inhibition by a pharmacological agent or by the molecular approach strongly suggests that p38 MAPK is a downstream target of AMPK. Similar findings were also reported by Xi et al. (32), who demonstrated that overexpressing a DN-AMPK abolished p38 MAPK activation in liver-derived Clone 9 cells. In contrast, in 3T3-L1 adipocytes, activation of AMPK impaired insulin-mediated glucose uptake (50), whereas activation of p38 MAPK participates in the stimulatory effect of insulin on glucose uptake through modulation of GLUT4 intrinsic activity (30). This latter result suggests that the coupling between AMPK and p38 MAPK may be tissue specific. Our results demonstrated that, in the heart, p38 MAPK is downstream of AMPK. Additional work will be needed to further define the relationship between AMPK and p38 MAPK in different tissues.
Studies have suggested that activation of AMPK is required for AICAR- and hypoxia-stimulated glucose uptake in skeletal muscle. However, whether this enzyme plays a similar role in the heart is still not resolved. We thus examined AMPK’s contribution to the regulation of glucose uptake in adult cardiomyocytes. Inhibition of AMPK by AraA or DN-AMPK reduces the stimulatory effect of DNP on glucose uptake by 51 and 39%, respectively. Thus, in adult cardiomyocytes, inhibition of AMPK provokes only a partial reduction of glucose uptake in response to DNP. Xing et al. (6) also reported similar results in which no-flow ischemia-induced glucose uptake was inhibited by 62% in transgenic mice hearts overexpressing a DN-AMPK2 mutant. On the other hand, overexpression of a KD-AMPK mutant completely abolished the stimulation of cardiac glucose uptake during low-flow ischemia (51). These differences in the contribution of AMPK to glucose uptake may be related to the severity of the metabolic stress generated. Alternatively, they may be compensatory mechanisms activated in response to inhibition of both 1- and 2-AMPK isoforms in the heart. Our studies suggest that in cardiomyocytes, inhibition of AMPK only partially decreased glucose uptake in response to DNP. Studies have demonstrated that glucose is an important substrate during metabolic stresses such as ischemia or hypoxia. Thus, the partial reduction of glucose uptake in response to AMPK inhibition may be a protective mechanism to prevent complete suppression of cardiac glucose uptake, which could be detrimental for the heart. Thus, the contribution of multiple pathways to the stimulation of glucose uptake during metabolic stress may allow for the maintenance of glucose supply and thus cardiac function. Further studies exploring the role of AMPK in various models of ischemia will be needed to clarify the contribution of this enzyme to the stimulation of glucose uptake in the heart.
AMPK also phosphorylates and activates phosphofructokinase-2 (PFK-2) in the heart. PFK-2 is the enzyme responsible for the synthesis of fructose 2, 6-phosphate, a powerful activator of the glycolytic pathway (46). Thus, inhibition of AMPK should be paralleled by a concomitant reduction of PKF-2 activity, glycolysis and potentially glucose uptake. Studies have shown, however, that stimulation of glycolysis by increased cardiac workload does not require AMPK activation (52). Furthermore, in transgenic mice overexpressing DN-AMPK or KD-AMPK, cardiac glycogen breakdown occurs normally, suggesting that glycolysis was not impaired in these animals (6, 51). In addition to AMPK, PFK-2 can be phosphorylated and activated by other kinases, including protein kinase A. Cardiac glycolysis is also regulated by local factors such as adenine nucleotide concentration and intracellular pH (53, 54). Thus, the regulation of PFK-2 activity involves multiple and overlapping mechanisms, reflecting the importance of glycolysis in maintaining energy supplies during stressful conditions. Whether inhibition of AMPK impairs glycolysis in DNP-stimulated cardiomyocytes remains to be investigated.
Several studies have demonstrated that p38 MAPK participates in the regulation of glucose uptake in response to various stimuli probably through the modulation of GLUT1 (55) or GLUT4 intrinsic activity (30, 56). We have shown that p38 MAPK is downstream of the AMPK signaling cascade and that inhibition of AMPK decreases glucose uptake in cardiomyocytes. We thus sought to determine whether inhibition of p38 MAPK would also diminish glucose uptake in response to DNP in these cells. Pharmacological inhibition of p38 MAPK with PD169316 only partially decreased DNP-mediated glucose uptake in cardiomyocytes. Whereas we cannot rule out that the decrease in glucose uptake by PD169316 is independent of p38 MAPK inhibition, this result is consistent with the partial reduction of glucose uptake observed in response to AMPK inhibition in cardiomyocytes. On the other hand, these data contrast with the study of Xi et al. (32), in which they reported that inhibition of p38 MAPK completely suppresses AICAR-mediated glucose uptake in Clone 9 cells. Similar results were also obtained for skeletal muscle, in which pharmacological inhibition of p38 MAPK completely suppressed AICAR-mediated glucose uptake (24). On the other hand, inhibition of the enzyme decreased only partially contraction-mediated glucose uptake (31). As mentioned above, the contribution of the AMPK/p38 MAPK signaling pathway to glucose uptake is both tissue and stimuli specific. Our data in adult cardiomyocytes strongly suggest that in this tissue, activation of an additional pathway is required for maximal stimulation of glucose uptake in response to DNP.
In skeletal muscle, overexpression of KD-AMPK mutant or selective ablation of AMPK2 completely blocked the stimulatory effect of either AICAR or hypoxia in this tissue (57). Similar results have been reported by Fryer et al. (21), who demonstrated that AICAR-induced glucose uptake was completely inhibited by DN-AMPK overexpression in H-2K muscle cells. Thus, in skeletal muscle, activation of AMPK is required for both AICAR- and hypoxia-stimulated glucose uptake. On the other hand, inhibition of AMPK with AraA or DN-AMPK provokes only a partial reduction of DNP-mediated glucose uptake. This latter result suggests that the contribution of AMPK to the regulation of glucose uptake is tissue specific. Other differences between cardiac and skeletal muscles have also been reported. Eckel and colleagues (34) demonstrated that stimulation of glucose uptake by contraction required PI3-K activation in cardiomyocytes. Furthermore, in the heart, AMPK phosphorylates and activates PFK-2, leading to enhanced glycolysis, whereas this does not occur in muscle because, in this tissue, the PFK-2 isoform does not contain an AMPK phosphorylation site (58). Together, these studies indicate that important mechanistic differences may exist between the heart and skeletal muscle regarding the regulation of glucose metabolism. Because the concentrations and activities of signaling molecules can be modulated, primary cultures of adult cardiomyocytes may represent a good model to explore the activation of signaling pathways in the heart.
What could be the additional signaling pathway contributing to increased glucose uptake in cardiomyocytes? Stimulation with DNP increases intracellular Ca2+ concentration in adipocytes (59) and cardiomyocytes (60). In L6 myotubes, calcium chelation decreases glucose uptake in response to DNP, suggesting that Ca2+ contributes to this process (61). Other potential candidates include calcium/calmodulin-dependent protein kinase and classical protein kinase C, both of which can be activated by calcium and are involved in glucose uptake in skeletal muscles (61, 62, 63). Interestingly, pharmacological inhibition of calmodulin-dependent protein kinase reduces the stimulatory effect of hypoxia on glucose uptake in skeletal muscles (62, 63) and the heart (Pelletier, A., and L. Coderre, unpublished data), suggesting that the enzyme plays an important role in this process. However, confirmation of its role in the regulation of glucose uptake in the heart awaits further investigation.
In conclusion, our results demonstrate that p38 MAPK acts downstream of AMPK in adult cardiomyocytes. Importantly, inhibition of the AMPK/p38 MAPK signaling cascade decreased DNP-mediated glucose uptake, suggesting that activation of these enzymes is required for maximal stimulation of glucose uptake in cardiomyocytes.
Acknowledgments
We thank Dr. Pierre Paradis for providing the Ad5/CMV/NLS-LacZ adenovirus and for helpful discussions. We also thank Dr. James C. Engert for helpful discussions. The editorial assistance of Mr. Ovid Da Silva (editor, Research Support Office, Research Centre, Centre Hospitalier de l’Université de Montréal) is acknowledged.
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