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Endocrinology, doi:10.1210/en.2007-1326
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Endocrinology Vol. 149, No. 4 1490-1498
Copyright © 2008 by The Endocrine Society

5-Aminoimidazole-4-Carboxamide-1-β-D-Ribofuranoside Reduces Glucose Uptake via the Inhibition of Na+/H+ Exchanger 1 in Isolated Rat Ventricular Cardiomyocytes

Coralie Ségalen, Sarah L. Longnus, Delphine Baetz, Laurent Counillon and Emmanuel Van Obberghen

Institut National de la Santé et de la Recherche Médicale (C.S., S.L.L., E.V.O.), Unité (U) 145 and U907, Institut Fédératif de Recherche 50, Faculté de Médecine, Nice F-06107, France; Centre National de la Recherche Scientifique (D.B., L.C.), Unité Mixte de Recherche 6548, Parc Valrose, Nice F-06108, France; and Université de Nice-Sophia Antipolis (C.S., S.L.L., D.B., L.C., E.V.O.), Nice F-06108, France

Address all correspondence and requests for reprints to: Emmanuel Van Obberghen, Faculté de Medecine, Institut National de la Santé et de la Recherche Médicale Unité 907, Avenue de Valombrose, Nice F-06107, France. E-mail: vanobbeg{at}unice.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated by an increased AMP/ATP ratio. AMPK is now well recognized to induce glucose uptake in skeletal muscle and heart. 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is phosphorylated to form the AMP analog ZMP, which activates AMPK. Its effects on glucose transport appear to be tissue specific. The purpose of our study was to examine the effect of AICAR on insulin-induced glucose uptake in adult rat ventricular cardiomyocytes. We studied isolated adult rat ventricular cardiomyocytes treated or not with the AMPK activators AICAR and metformin and, subsequently, with insulin or not. Insulin action was investigated by determining deoxyglucose uptake, insulin receptor substrate-1- or -2-associated phosphatidylinositol 3-kinase activity and protein kinase B (PKB) cascade using antibodies to PKB, glycogen synthase kinase-3, and Akt substrate of 160 kDa. Intracellular pH was evaluated using the fluorescent pH-sensitive dye 2',7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Na+/H+ exchanger 1 (NHE1) activity was assessed using the NH4+ prepulse method. Our key findings are as follows. AICAR and metformin enhance insulin signaling downstream of PKB. Metformin potentiates insulin-induced glucose uptake, but surprisingly, AICAR inhibits both basal and insulin-induced glucose uptake. Moreover, we found that AICAR decreases intracellular pH, via inhibition of NHE1. In conclusion, AMPK potentiates insulin signaling downstream of PKB in isolated cardiac myocytes, consistent with findings in the heart in vivo. Furthermore, AICAR inhibits basal and insulin-induced glucose uptake in isolated cardiac myocytes via the inhibition of NHE1 and the subsequent reduction of intracellular pH. Importantly, AICAR exerts these effects in a manner independent of AMPK activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
5'-AMP-ACTIVATED PROTEIN kinase (AMPK) is a serine/threonine kinase that plays a key role in energy homeostasis by acting as a cellular warning system for low fuel (1). During metabolic stress such as exercise or ischemia, AMPK is activated by an increase in the AMP/ATP ratio and promotes ATP-generating pathways like glucose transport, glycolysis, and fatty acid oxidation, while decreasing energy-consuming anabolic pathways. Due to its effects on glucose metabolism, AMPK has been proposed to be a therapeutic target for metabolic diseases such as type 2 diabetes and obesity, in which glucose uptake and use are impaired.

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) alters glucose uptake differently in various tissues and cells. First, it is recognized that AICAR, via the activation of AMPK, stimulates glucose uptake in skeletal muscle (2) and in isolated cardiac papillary muscle (3, 4, 5). However, AICAR also inhibits glucose uptake in 3T3-L1 adipocytes (6) and isolated adipocytes (7) and in soleus muscle (8, 9, 10). To the best of our knowledge, the effects of AICAR on glucose uptake in isolated ventricular cardiomyocytes have never been determined.

Recently, Moopanar et al. (11) demonstrated that AICAR inhibits Na+/H+ exchanger-1 (NHE1) in the rat heart. NHE isoforms protect cells against internal acidification by the regulation of intracellular pH (pHi) (12). Furthermore, it has been observed that increasing extracellular pH (pHe) from 7.0 to 8.0, which, in parallel, augments pHi, stimulates glucose transport (13, 14). Insulin has been reported to induce cytosol alkalinization, which facilitates optimal glucose uptake (15, 16). In addition, Yang et al. (16) showed that cariporide and bafilomycin, which inhibit, respectively, NHE1 and H+-ATPases, reduce pHi and insulin-induced glucose uptake.

We have previously found in the heart in vivo that the activation of AMPK with AICAR or metformin potentiates insulin signaling downstream of protein kinase B (PKB) (17). Here, we were interested in the effect of AMPK activation, with AICAR or metformin, on glucose uptake in isolated adult rat ventricular cardiomyocytes exposed or not to insulin.

We demonstrate that AICAR, by decreasing pHi through the inhibition of NHE1, inhibits basal and insulin-induced glucose uptake in isolated cardiomyocytes. This inhibitory effect is independent of AMPK, because metformin potentiated insulin-induced glucose uptake and did not alter NHE1 activity. AICAR and metformin, via the activation of AMPK, potentiate insulin-induced activation of PKB and its downstream targets in cardiac myocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
AICAR was obtained from Toronto Research Chemicals (North York, Canada), metformin from Calbiochem (Darmstadt, Germany), collagenase type 2 from Worthington Biochemical (Lakewood, NJ), laminin from Beckton Dickinson (Franklin Lakes, NJ), and HEPES modified medium 199 from Invitrogen (Cergy Pontoise, France). Insulin was purchased from Novo-Nordisk (Bagsvaerd, Denmark) and radioisotopes from PerkinElmer, Inc. (Wellesley, MA). Antibodies to phosphorylated acetyl coenzyme A carboxylase (ACC) (Ser79), phosphorylated Akt substrate of 160 kDA (phospho-AS160) (Thr642), AS160, insulin receptor substrate (IRS)-1, and IRS-2 were from Upstate Biotechnology (Lake Placid, NY). Antibodies to AMPK-{alpha}, ACC, phospho-PKB (Ser473), phospho-PKB (Thr308), PKB, and phosphorylated glycogen synthase kinase-3{alpha}/β (phospho-GSK3{alpha}/β) (Ser21/9) were from Cell Signaling Technology (Beverly, MA). Antibody to GSK3β was from Santa Cruz Biotechnology (Santa Cruz, CA). Unless stated otherwise, all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

Ventricular cardiomyocyte isolation
Male Sprague Dawley rats (250–300 g) were euthanized with sodium pentobarbital (100 mg/kg) and the heart rapidly excised and placed in ice-cold calcium-free Joklik’s medium (modified method from Ref. 18). The aorta was cannulated and the heart perfused in a retrograde manner with calcium-free Joklik’s medium at a flow rate of 5 ml/min·mg heart for 5 min. Thereafter, the heart was perfused with 0.09% (wt/vol) collagenase and 0.7% (wt/vol) BSA in Joklik’s medium for 30 min. At 15 and 20 min, calcium was added to achieve a final concentration of 200 µM. All perfusion buffers were maintained at 37 C and gassed with a mixture of 95%O2/5%CO2. At the end of the perfusion period, the atria and aorta were removed. The ventricles were carefully opened using forceps and then incubated in 20 ml incubation buffer [10 ml perfusate, 8 ml Joklik medium, and 2 ml 10% (wt/vol) BSA] for 10 min in a shaking water bath (37 C, 120 rpm). The heart was subsequently dissected with forceps, and cells were further dissociated using a 10-ml pipette. The suspension was then transferred to a polypropylene Erlenmeyer flask and placed in a shaking water bath (37 C, 120 rpm) for 5 min, whereas the Ca2+ concentration was increased in steps of 200 µM to 1 mM. Next, the cell suspension was filtered through gauze, and cardiomyocytes were pelleted by centrifugation (2 min, 17 x g). Cells were resuspended in sedimentation buffer [Joklik’s medium containing 10% (vol/vol) fetal calf serum and 1 mM CaCl2] and allowed to settle for 5 min. This procedure was repeated twice. Cells, in HEPES-modified medium 199 supplemented with 10% (vol/vol) fetal calf serum, L-carnitine (2 mM), creatine (5 mM), taurine (5 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamicin (5 µg/ml), were allowed to adhere for 1 h to laminin-coated plates. Cardiomyocytes were then depleted for 2 h in HEPES-modified medium 199 with penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamicin (5 µg/ml). Cardiomyocytes were then treated with AICAR (40 min, 2 mM) or metformin (85 min, 5 mM) or left untreated and subsequently incubated for 5 min without or with insulin (10–8 M). Our investigation conforms to the current Guidelines for the Care and Use of Laboratory Animals of the National Institute of Health and Medical Research of France (INSERM, France).

Deoxyglucose uptake
Isolated cardiomyocytes were treated as described above, except that 1 µCi/ml 2-[3H]deoxyglucose (DOG) was added with insulin and cardiomyocytes incubated for an additional period of 30 min. Cardiomyocytes were washed three times with ice-cold PBS, DOG content was quantified by standard counting procedures, and protein levels were measured.

Western blot analysis
Cardiomyocytes were homogenized in 1% (vol/vol) Nonidet P-40, 10% (wt/vol) glycerol, 137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 10 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na-pyrophosphate, 1 mM orthovanadate, 4 µg/ml aprotinin, 4 µg/ml leupeptin, 4 µg/ml pepstatin, and 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride. Lysates were then used for immunoprecipitation or were subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA) using standard procedures. Blots were probed with the appropriate horseradish peroxidase-conjugated antirabbit or antimouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and visualized by the enhanced chemiluminescence system from Pierce (Rockford, IL).

Phosphatidylinositol 3-kinase (PI3K) activity
PI3K activity was measured in cardiomyocyte lysates after immunoprecipitation with antibodies to IRS-1 or to IRS-2 using 0.5 or 0.8 mg protein, respectively. After incubation with the immunoprecipitating antibody, pellets were washed three times with lysis buffer and then three times with PI3K buffer [10 mM HEPES (pH 7.4), 0.1 mM EGTA, 0.015% (vol/vol) Nonidet P-40, 1 mM dithiothreitol]. Pellets were resuspended in PI3K buffer with 1 µg/µl phosphatidylinositol from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). The reaction was initiated by the addition of the ATP mix (5 µM MgCl2, 100 µM ATP, 0.2 µCi/µl [{gamma}-32P]ATP) and allowed to continue for 10 min at room temperature. The reaction was stopped by the addition of 2 M HCl, and the phosphoinositides were extracted with a methanol-chloroform (vol/vol) mix. The phosphoinositides were resolved by thin-layer chromatography, revealed by a Storm 840 phosphoimager (Molecular Dynamics; Amersham Biosciences, Piscataway, NJ) and quantified using ImageQuant 5.1 software (Amersham Biosciences).

pHi measurement
Cardiomyocytes were cultured in laminin-coated 35-mm plates in medium 199. At the end of treatment with AMPK activators (AICAR or metformin) and with or without insulin as described in Ventricular cardiomyocyte isolation above, cells were incubated with the fluorescent pH-sensitive dye BCECF-acetoxymethyl ester (BCECF/AM) for 5 min at a concentration of 5 µM. Then cardiomyocytes were rinsed with HEPES-buffered Tyrode solution (132.5 mM NaCl, 5.4 mM KCl, 1.2 mM MgCl2-6H20, 10 mM HEPES, 1 mM CaCl2, 5.5 mM glucose, buffered at pH 7.4). Throughout the experiment, the pHi was monitored using a Zeiss ICM 405 inverted microscope with a Zeiss x40 objective, coupled to a video camera. The cells were excited successively at 490 and 450 nm, and each image was digitalized and stored. Image treatment was performed using the AxonR Imaging Workbench (AIW 4.0) software. Under these conditions, three to seven rod-shaped, calcium-tolerant myocytes were usually present per microscopic field and used in each experiment. The emission ratio 490/450 (R), obtained from intracellular BCECF, was calculated using the pH = 6.8' + log(R– Rmin/Rmax– R) equation and converted to a linear pH scale using in situ data calibration, which was performed at the end of the experiment using the nigericin technique described elsewhere (19, 20). The buffering power βi was estimated as described previously (19) and found unchanged between control vs. AICAR treatment (data not shown).

Intracellular acidification and pHi recovery
Cardiomyocytes were cultured for 1 h in HEPES-buffered Tyrode solution. After treatment with drugs, the addition and subsequent removal of NH4Cl (20 mM in HEPES-buffered Tyrode solution for 8 min) was used to induce an acid load to activate the pHi regulatory mechanisms (21). Because HCO3–-free solutions were used, only Na+-H+ exchange was responsible for pHi recovery observed during the time course of the experiments. The rate of pHi recovery at pH 6.8 and pH 6.9 were calculated using the formula {Delta}pHi/{Delta}t.

Statistical analysis
Results are presented as the mean ± SEM, and n represents the number of experiments, which have been performed at least three times, each consisting of cardiomyocytes isolated from one heart, used in each measurement. Differences between the groups were compared with the two-tailed unpaired Student’s t test. The Bonferroni correction was applied to the P values obtained to correct for multiple comparisons. A corrected P value of <0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We have previously shown that activation of AMPK in the rat heart in vivo potentiates insulin signaling downstream of PKB (17). To investigate the mechanism by which AMPK affects insulin action specifically in the myocardium, we used isolated rat ventricular cardiomyocytes.

AICAR inhibited DOG uptake in cardiomyocytes
First, we measured DOG uptake (Fig. 1Go). As expected, insulin increases glucose transport in cardiomyocytes 3-fold. Metformin potentiates insulin-induced glucose transport by 20% but does not modify the basal level. Surprisingly, AICAR partially inhibits basal and insulin-induced glucose transport.


Figure 1
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  		FIG. 1. Deoxyglucose uptake. Deoxyglucose uptake was measured after incubation with AMPK activators AICAR (40 min at 2 mM) or metformin (85 min at 5 mM) and with or without insulin (ins) (10–8 M) as described in Materials and Methods. At the beginning of the insulin stimulation, [3H]DOG was added to the medium, and the experiment was stopped after 30 min. Values are mean ± SEM. *, Significantly different from corresponding value without insulin (P < 0.00005); #, significantly different from corresponding value without AMPK activators (P < 0.001).

 
Both AICAR and metformin activate AMPK in cardiomyocytes, as illustrated by an increase in AMPK{alpha} phosphorylation on threonine 172 and of its target ACC on serine 79 (Fig. 2Go). These phosphorylation sites are well-recognized to reflect AMPK activity (22, 23).


Figure 2
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  		FIG. 2. AICAR and metformin activate AMPK in cardiomyocytes. Representative Western blots of lysates from cardiomyocytes treated as described in Materials and Methods are shown. Blots were probed with an antibody to phospho-AMPK (Thr172) or phospho-ACC (Ser79), stripped, and reprobed with an antibody to AMPK{alpha} (A) or ACC (B). Results were quantified, normalized for total amount of protein, and plotted. Values are mean ± SEM. *, P < 0.05; **, P < 0.01, significantly different from corresponding value without AMPK activators. ins, Insulin.

 
Metformin and AICAR via AMPK potentiate insulin signaling downstream of PKB
Next, we studied insulin signaling upstream of glucose transporter 4 (GLUT4) translocation to investigate the mechanism by which AICAR decreases glucose uptake. The activity of PI3K associated with IRS-1 (Fig. 3AGo) or IRS-2 (Fig. 3BGo) is increased by insulin stimulation. AMPK activators, such as AICAR or metformin, do not alter the insulin response. Then, we looked at the PKB pathway. Similar to what we have previously shown in the whole heart (17), AMPK activation by AICAR or metformin potentiates insulin-induced PKB phosphorylation on threonine 308 and serine 473 in isolated cardiomyocytes (Fig. 4Go, A and B). The effect on PKB is also observed on its targets GSK3 and AS160 (Fig. 4Go, C and D).


Figure 3
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  		FIG. 3. IRS-1- and IRS-2-associated PI3K activity. IRS-1- or IRS-2-associated PI3K activity was measured in immunoprecipitates (IP) from lysates using antibodies to IRS-1 (A) or IRS-2 (B) from cardiomyocytes treated as described in Materials and Methods. Results were quantified and plotted. Values are mean ± SEM. *, P < 0.005, significantly different from corresponding value without insulin (ins); **, P < 0.0005, significantly different from corresponding value without insulin.

 

Figure 4
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  		FIG. 4. AMPK activators potentiate the insulin-stimulated PKB cascade. Representative Western blots of lysates from cardiomyocytes treated as described in Materials and Methods are shown. Blots were probed with an antibody to phospho-PKB (Thr308 or Ser473), phospho-GSK3{alpha}/β (Ser21/9), or phospho-AS160 (Thr642), stripped, and reprobed with antibody to PKB (A and B), to GSK3β (C), or to AS160 (D). Results were quantified, normalized for total amount of protein, and plotted. Values are mean ± SEM. *, Significantly different from corresponding value without insulin (ins) (P < 0.005); #, significantly different from corresponding value without AMPK activators (P < 0.05).

 
To summarize, at this point, we could not explain the inhibition induced by AICAR on glucose uptake because AICAR, in fact, increases the insulin-induced PKB pathway.

AICAR decreases pHi via the inhibition of NHE1
Unlike its effect on glucose transport, AICAR does not block the insulin signaling pathway. AICAR is known to exert biological effects independent of AMPK activation, as illustrated, for example, by Moopanar et al. (11), who reported that AICAR inhibits the Na+/H+ exchanger (NHE1) in the rat heart independent of AMPK activation. Therefore, we hypothesize that AICAR could decrease the intracellular pH level by inhibiting NHE1 and, by doing so, could prevent GLUT4 translocation and glucose uptake. To test this hypothesis, we measure pHi, using the pH-sensitive fluorescent dye BCECF, after AICAR or metformin treatment of cardiomyocytes, with or without insulin. Basal pHi in cardiomyocytes is about 7.23. Insulin significantly increases pHi to 7.41, whereas metformin does not alter the pHi either in basal conditions or in response to insulin (Fig. 5AGo). Importantly, AICAR significantly reduces basal and insulin pHi in cardiomyocytes (Fig. 5AGo). To measure NHE1 activity, we carried out pHi recovery after acidification by a NH4+ prepulse (Fig. 5BGo). AICAR strongly inhibits basal pHi recovery as shown by a decrease in {Delta}pH/{Delta}t at pH 6.8 and 6.9 (Fig. 5CGo), which implicates AICAR in the inhibition of NHE1. In contrast, metformin does not alter pHi recovery (Fig. 5Go, B and C).


Figure 5
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  		FIG. 5. AICAR decreases pHi via the inhibition of NHE1. A, Cardiomyocytes were treated with metformin (85 min, 5 mM) or AICAR (40 min, 2 mM) and subsequently with or without insulin (ins) (5 min, 10–8 M). At the end of the treatment, cardiomyocytes were incubated with the fluorescent pH-sensitive dye BCECF/AM for 5 min. pHi was then recorded from single cardiomyocytes. B, Cardiomyocytes treated with AICAR (dark gray) or metformin (light gray) or left untreated (black) as in A were incubated with NH4+ for 7 min and then rinsed with HEPES-buffered Tyrode solution to induce the acidification. The pHi recoveries after acid load from representative curves were superimposed to facilitate comparison. C, Rate of pHi recovery {Delta}pHi/{Delta}t was estimated at pH 6.8 and pH 6.9. The average of four to 10 experiments was quantified and plotted. Values are mean ± SEM. *, Significantly different from corresponding value without insulin (P < 0.05); #, significantly different from corresponding value without AICAR (P < 0.05); **, significantly different from corresponding value without AICAR (P < 0.000005).

 
The reduction of the pHi to approximately 7.0 mimics the effect of AICAR on glucose uptake in cardiomyocytes
To mimic the effect of AICAR on glucose transport, cardiomyocytes were exposed for 5 min to HEPES-buffered Tyrode solution at pH 6.0, which decreases the pHi to approximately 7.0, which is the pHi obtained with AICAR treatment. Thereafter, the medium is changed to HEPES-buffered Tyrode solution at pH 7.4 with cariporide (10 µM) to clamp the pHi at 7.0 and to bring the pHe to 7.4, which is necessary to allow insulin binding at a physiological pH (24, 25). During this treatment, the pHi of cardiomyocytes is monitored with the fluorescent dye BCECF/AM and remains stable at a value close to 7.0 (Fig. 6AGo). To ensure that the change in pHi, but not in pHe, does not alter insulin signaling, we monitored the phosphorylation of PKB on serine 473 and its targets GSK3{alpha}/β and AS160 (Fig. 6BGo). Insulin induces an increase in the phosphorylation of PKB, GSK3, and AS160, and pHi of about 7.0 does not modify insulin action on PKB, GSK3, and AS160 when pHe is at 7.4. Under these conditions, the decrease in pHi to approximately 7.0 significantly decreases basal and insulin-induced DOG uptake (Fig. 6CGo) as observed with the AICAR treatment.


Figure 6
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  		FIG. 6. A decrease in pHi mimics the effect of AICAR on glucose uptake. Cardiomyocytes were cultured in HEPES-buffered Tyrode’s solution at pH 7.4 for 2 h. Cells were then incubated or not with a HEPES-buffered Tyrode’s solution at pH 6.0 for 5 min, which decreases the pHi to around 7.0. Thereafter, cardiomyocytes were put into a HEPES-buffered Tyrode’s solution at pH 7.4 with cariporide (10 µM) to keep the pHi at 7.0 and to restore pHe to 7.4, thus allowing insulin to bind to its receptor at physiological pH. In parallel, cardiomyocytes were treated only with cariporide (10 µM) for 5 min and then without or with insulin (ins). A, pHi was monitored using the fluorescent pH-sensitive dye BCECF/AM, and the average of nine individual cells is presented. B, Lysates from cardiomyocytes treated as described above were subjected to SDS-PAGE. Representative blots of four to six experiments are presented. Membranes were probed with antibodies to phospho-PKB (Ser473), phospho-GSK3{alpha}/β (Ser21/9), or phospho-AS160 (Thr642), stripped, and reprobed with antibodies to PKB, GSK3, or AS160. C, DOG uptake was measured with or without insulin. Values are mean ± SEM. *, Significantly different from corresponding value without insulin (P < 0.01); #, significantly different from corresponding value without pHi at about 7.0 (P < 0.005); §, significantly different from corresponding value without cariporide (P < 0.05); **, significantly different from corresponding value without insulin (P < 0.005).

 
Furthermore, we show that a short treatment with cariporide also decreases insulin-induced glucose uptake (Fig. 6CGo), whereas this inhibitor alone does not decrease basal uptake. Noticeably, this inhibition of insulin-induced glucose uptake is independent of a defect in insulin signaling because cariporide does not modify insulin-induced phosphorylation of PKB and its targets GSK3 and AS160 (Fig. 6BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have shown, for the first time to the best of our knowledge, that AICAR, independent of AMPK activation, inhibits both basal and insulin-stimulated glucose uptake in isolated adult rat ventricular cardiomyocytes via the inhibition of NHE1 (Fig. 7Go).


Figure 7
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  		FIG. 7. Schematic representation of AICAR effects on glucose uptake in adult rat ventricular cardiomyocytes. Insulin induces glucose uptake in cardiomyocytes through the activation of PKB, which inhibits AS160 and allows GLUT4 translocation to the membrane. GSK3 is also inhibited by PKB. Metformin and AICAR activate AMPK in cardiomyocytes, which potentiates insulin-induced PKB activation. But, independently of AMPK, AICAR inhibits the Na+/H+ exchanger NHE1. This inhibition causes the reduction of basal and insulin-induced GLUT4 translocation and glucose uptake.

 
In our conditions, AICAR inhibits glucose uptake in isolated cardiomyocytes. It seems unlikely that this effect is due to AMPK activation because, in the same condition, metformin, another AMPK activator, potentiates insulin-induced glucose uptake. Moreover, other studies using adenovirus or transgenic animals implicate AMPK in glucose uptake activation in the myocardium (26, 27, 28). Previously, AICAR was proposed to exert effects independent of AMPK activation in the heart. Longnus et al. (29) have indeed shown that AICAR induces allosteric activation of glycogen phosphorylase independently of AMPK, leading to glycogenolysis in the intact heart. Furthermore, Moopanar et al. (11) have recently demonstrated that AICAR inhibits the Na+/H+ exchanger (NHE1) in rat hearts independently of AMPK activation and adenosine receptors. Therefore, we hypothesized that AICAR, via the inhibition of NHE1, could decrease pHi and inhibit GLUT4 translocation and hence glucose uptake. Using the fluorescent pH-sensitive dye BCECF, we find that AICAR decreases pHi in isolated cardiomyocytes. Moopanar et al. (11) did not induce a change in pHi in their conditions, maybe because they stimulated cells with AICAR for a much shorter period, i.e. only a few minutes before their analysis. Moreover, insulin stimulation did not overcome the effect of AICAR on glucose transport even though insulin is known to activate NHE1 (30). Importantly, applying the NH4Cl prepulse method, we confirmed the inhibitory effect of AICAR on NHE1 in our conditions. Furthermore, we provide evidence that the inhibition of NHE1 is independent of AMPK activation because metformin did not alter pHi or its recovery after an acid load.

Concerning metformin and pHi, previous studies have shown that this drug induces an inhibition of complex I in the mitochondrial respiratory chain (31), which can lead, in the most serious cases, to lactic acidosis. However, complex I inhibition (31) or a drop in pH in cardiomyocytes (32) is observed only when cells are treated for a long period with metformin (more than 16 h). This is probably due to the slow permeation of the drug across the mitochondrial inner membrane and its ensuing accumulation where it inhibits complex I directly. Because we have treated cardiomyocytes for a short period with metformin (90 min), we probably have no detectable or only a slight effect on complex I, which does not alter pHi, as measured in our conditions.

Although the mechanism of NHE1 inhibition by AICAR remains unknown, we would like to suggest the following not mutually exclusive possibilities. First, AICAR could directly inhibit NHE1 because it has an aminoimidazole group. Ahmad et al. (33) have presented aminoimidazoles as potent, selective, and orally bioavailable inhibitors of NHE1 that can be used to replace the acylguanidine group of the cyclopropyl series of NHE1 inhibitors. However, AICAR failed to inhibit NHE1-mediated pH recovery when added extemporaneously to the cardiomyocytes after NH4+ acid loading (data not shown). This suggests that the inhibitory mechanism might be more complex than simple binding on the extracellular side of the transporter. Second, AICAR could act from the intracellular side, either directly or by activating pathways independent of AMPK, which could regulate NHE1 activity. Indeed, multiple phosphorylation sites or regulation domains in the C terminus of the NHE1 protein, which modulate the activation of the exchange by intracellular H+ (34), have been shown to be controlled by multiple hormones, growth factors, and signaling pathways. Generally speaking, the regulation of NHE1 activity is complex and might also be dependent on the cell type (12).

Previously, AICAR, also called acadesine, has been shown to provide protection against injury during ischemia and reperfusion when administered before ischemia or with cardioplegia (35). However, the mechanism of this beneficial effect is still unclear. Some studies have proposed that it could exert a protective effect by enhancing adenosine levels in the myocardium or by activating AMPK (36). On the other hand, during ischemia, NHE1 is activated and enhances intracellular Na+ concentration, which leads to calcium overload during reperfusion and exacerbation of myocardial injury. Furthermore, in the Goto-Kakizaki rat model of type 2 diabetes, NHE1 activity is increased in cardiomyocytes accompanied by a phenotype of hypertrophy that can be prevented by chronic treatment with the NHE1 inhibitor cariporide (37). Considering these facts as a whole, it is tempting to suggest that the protective effect of AICAR in the myocardium, which has been observed during ischemia/reperfusion-induced injury, may involve the inhibition of the Na+/H+ exchanger NHE1.

Insulin-induced alkalinization is necessary for optimal GLUT4 translocation and glucose uptake in isolated cardiomyocytes (16) and in muscle cells (15). However, previous studies have decreased both pHi and pHe in their approach to monitor insulin-induced glucose uptake (16). Because the binding of insulin to its receptor is dependent on the extracellular pH, which should be not lower than 7.4 (24, 25), these results should be interpreted with caution. In our study, we have confirmed that a decrease in pHi, but not in pHe, inhibits basal and insulin-induced glucose uptake. Our data obtained with the NHE1 inhibitor cariporide, which, similar to AICAR, inhibits insulin-induced glucose uptake, further support our conclusion. In addition, Yang et al. (16) have previously shown that cariporide reduces glucose transport and the availability of GLUT4 at the cell surface of insulin-stimulated cardiomyocytes. Taken together, their data suggest that cytosolic acidification, either due to cariporide or to the H+-ATPase inhibitor bafilomycin A, antagonizes the final stages of GLUT4 insertion at the sarcolemmal membrane.

Here, we used AICAR and metformin (38, 39) to activate AMPK in cardiomyocytes. AICAR, after entering the cell, is phosphorylated to form ZMP, which is an analog of AMP. Inside the cell, the AMP/ATP ratio appears to be increased, and AMPK is activated (40, 41). Previous studies have shown that metformin alters the activity of complex I of the respiratory chain (31). This inhibition was first thought to induce AMPK activation, but other studies have demonstrated that AMPK activation by metformin is independent of an adenine nucleotide mechanism and is mediated through the indirect activation of an AMPK kinase (39, 42, 43, 44). To summarize, in our study, we have activated AMPK by two different mechanisms to assess AMPK’s role.

It is well established that the activation of AMPK increases glucose transport in skeletal muscle and heart (45). Concerning the heart, observations gathered with different agents that activate AMPK such as oligomycin and metformin (26, 46), adiponectin (47), dinitrophenol (48), and ischemia (28) support the idea that AMPK induces glucose uptake. However, it is important to stress that studies showing an AMPK-induced glucose uptake via AICAR in the heart have been performed in isolated papillary muscle (3, 4, 5) and not in ventricular cardiomyocytes. In our work, metformin alone did not alter basal glucose uptake, but metformin significantly potentiated insulin-induced glucose uptake, most likely via the activation of AMPK. Surprisingly, we find here that AICAR inhibits glucose uptake in adult ventricular cardiomyocytes. In muscle, it is well established that AICAR, via the activation of AMPK, stimulates glucose uptake (2). However, AICAR inhibits glucose uptake in other cell types such as 3T3-L1 adipocytes (6), isolated rat adipocytes (7), and human umbilical vein endothelial cells (49) and does not affect glucose transport in rat soleus muscle (8, 9, 10). These previous studies did not provide mechanisms explaining AICAR’s inhibitory action. Although NHE1 is ubiquitously expressed, it can be connected to different cell-specific signaling pathways that could completely change its response to AICAR, depending on the tissues and cell types. Thus, it would be important to know whether AICAR alters signaling pathways in a specific fashion in different muscle fibers or tissues, resulting in particular effects on NHE activation. Additionally, because soleus muscle is insensitive to AICAR, it would be interesting to investigate whether other NHE isoforms are expressed in soleus muscle compared with other muscle types. In fact, NHE2 mRNA has been detected by Northern blot in skeletal muscle (50), but to our knowledge, no data are available on NHE isoforms expression in the different skeletal muscle types.

To investigate the mechanism of AICAR action on glucose uptake, we first investigated the insulin-induced PKB pathway, which plays a key role in insulin-induced glucose uptake (51). AICAR and metformin do not change IRS-1- or IRS-2-associated PI3K but significantly enhance the insulin-induced PKB cascade. This effect of AMPK on insulin signaling has been previously observed in the heart in vivo (17) and in cardiomyocytes (26), although the mechanism of enhanced PKB activity by AMPK has not yet been characterized. Moreover, we demonstrate here, for the first time to the best of our knowledge, that AMPK activation potentiates insulin-induced phosphorylation of the PKB substrate AS160 in the myocardium. AS160 is a Rab GTPase-activating protein (GAP) protein, implicated in GLUT4 trafficking by inhibiting its exocytosis. Insulin, via PKB, induces the phosphorylation on five sites (52) and the inhibition of AS160, thus allowing GLUT4 exocytosis and glucose transport (53). Recently, Kramer et al. (54) have shown that AMPK activation induces phosphorylation of AS160 and enhances insulin-induced AS160 phosphorylation in skeletal muscle. In their study, the authors used two different antibodies to measure AS160 phosphorylation: the anti-PAS antibody [for phospho-(Ser/Thr) Akt substrate antibody] and the anti-phospho-Thr642AS160 antibody. They demonstrated that AICAR and muscle contraction increase AS160 phosphorylation only when measured with the PAS antibody. In our work, we used the anti-phospho-Thr642AS160 antibody, which reflects PKB activation.

In conclusion, we show, in isolated rat cardiomyocytes, that AMPK activation, induced by either AICAR or metformin, increases insulin signaling downstream of PKB. However, contrary to metformin, which improves insulin-induced glucose uptake, AICAR inhibits basal and insulin-induced glucose transport, at least in part by decreasing pHi via the inhibition of NHE1 (see schematized view in Fig. 7Go). Therefore, our results provide novel evidence of an AICAR effect independent of AMPK activation in the heart. These results should be taken into consideration in studies aiming at drug design for AMPK regulation in the heart.


   Footnotes
 
This project was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis, le Conseil Général des Alpes Maritimes et le Conseil Régional Provence-Alpes-Côte d’Azur (PACA), grants from the European Community [FP6-Eugene2 (LSHM-CT-2004-512013)] and from Fondation de France (Paris, France), Recherche sur les Maladies Cardiovasculaires. S.L.L. was supported by an EFSD Eli Lilly Research Fellowship. C.S. was supported by a doctoral fellowship from INSERM-PACA with Nichols Institute Diagnostics as industrial partner.

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online January 10, 2008

Abbreviations: ACC, Acetyl coenzyme A carboxylase; AICAR, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; AMPK, AMP-activated protein kinase; AS160, Akt substrate of 160 kDa; BCECF, 2',7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein; BCECF/AM, BCECF acetoxymethyl ester; DOG, deoxyglucose; GLUT4, glucose transporter 4; GSK glycogen synthase kinase; IRS, insulin receptor substrate; NHE1, Na+/H+ exchanger 1; pHe, extracellular pH; pHi, intracellular pH; PI3K, phosphatidylinositol 3-kinase.

Received September 27, 2007.

Accepted for publication January 2, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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