Contraction of rat cardiac myocytes induces translocation of fatty acid translocase (FAT)/CD36 and GLUT4 from intracellular stores to the sarcolemma, leading to enhanced rates of long-chain fatty acid (FA) and glucose uptake, respectively. Because intracellular AMP/ATP is elevated in contracting cardiac myocytes, we investigated whether activation of AMP-activated protein kinase (AMP kinase) is involved in contraction-inducible FAT/CD36 translocation. The cell-permeable adenosine analog 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) and the mitochondrial inhibitor oligomycin, similar to 4-Hz electrostimulation, evoked a more than threefold activation of cardiomyocytic AMP kinase. Both AICAR and oligomycin stimulated FA uptake into noncontracting myocytes by 1.4- and 2.0-fold, respectively, but were ineffective in 4 Hz-contracting myocytes. These findings indicate that both agents stimulate FA uptake by a similar mechanism as electrostimulation, involving activation of AMP kinase, as evidenced from phosphorylation of acetyl-CoA carboxylase. Furthermore, the stimulating effects of both AICAR and oligomycin were antagonized by blocking FAT/CD36 with sulfo-N-succinimidylpalmitate, but not by inhibiting phosphatidylinositol 3-kinase with wortmannin, indicating the involvement of FAT/CD36, but excluding a role for insulin signaling. Subcellular fractionation showed that oligomycin was able to mobilize intracellularly stored FAT/CD36 to the sarcolemma. We conclude that AMP kinase regulates cardiac FA use through mobilization of FAT/CD36 from a contraction-inducible intracellular storage compartment.

Fatty acid translocase (FAT)/CD36 is increasingly becoming recognized as a physiologically important long-chain fatty acid (FA) transport facilitator within the sarcolemma of muscle tissues (13). We have gathered convincing evidence that FAT/CD36-mediated FA uptake is a rate-limiting step in FA use by heart and skeletal muscle (2). Moreover, FA uptake appears to be regulated by translocation of FAT/CD36 from intracellular, presumably endosomal, stores to the sarcolemma. Insulin and cellular contractions are two important physiological factors able to recruit FAT/CD36 to the sarcolemma (47). Interestingly, the stimulation of FA uptake by contractions was additive to that of insulin, indicating that both factors operate through independent mechanisms. Furthermore, wortmannin completely blocked insulin-inducible FA uptake but had no effect on contraction-inducible FA uptake. Altogether, these findings indicate that insulin-inducible and contraction-inducible FAT/CD36 pools are stored in two distinct intracellular sites (7). Strikingly, the effects of insulin and contractions on FA uptake by translocation of FAT/CD36 are remarkably similar to their effects on glucose uptake by translocation of the glucose transporter GLUT4 from insulin-inducible and contraction-inducible intracellular stores (7). Whereas the wortmannin sensitivity of insulin-inducible FA uptake indicates an involvement of phosphatidylinositol (PI) 3-kinase, the signaling pathway responsible for contraction-inducible FAT/CD36 translocation is largely unknown. Recently, we have established that cAMP-dependent protein kinase A is not involved in regulation of FA uptake (8). Another likely candidate-signaling enzyme could be AMP-activated protein kinase, which has been firmly established to be involved in translocation of GLUT4 during an increase in contractile activity (9,10).

During such an increase in mechanical performance, the high constitutive activity of adenylate kinase in the heart prevents a drastic fall in ATP via the conversion of two molecules of ADP into ATP and AMP (11,12). This process also renders the intracellular concentration of AMP a sensitive indicator of the metabolic state of the cell. The subsequent activation of AMP kinase has now been established to play an important role in the cellular response to an increase in mechanical activity. In resting muscle, AMP kinase can be activated pharmacologically by 5′-amino-4-imidazolecarboxamide ribonucleoside (AICAR), an adenosine analog (1315). After being readily taken up by the muscle cell, AICAR is phosphorylated by the catalytic action of adenosine kinase to form AICAR monophosphate (ZMP), which closely resembles the chemical structure of AMP. ZMP, in turn, is known to activate AMP kinase, and in this it mimics the effect of contractions on muscle energy metabolism.

It has been widely accepted that AMP kinase activation has a great impact on substrate metabolism in heart and skeletal muscle, perhaps most notably by stimulating glucose utilization via induction of GLUT4 translocation (9,10). Furthermore, AMP kinase has also been recognized to be involved in FA utilization by heart and muscle. In these tissues, activation of AMP kinase causes a drop in acetyl-CoA carboxylase activity concomitant with lowering intracellular levels of malonyl-CoA (13,1618). This results in an enhancement of mitochondrial FA oxidation, because the decline in the cytoplasmic malonyl-CoA concentration relieves the inhibition on carnitine palmitoyltransferase-I.

The present study explores the possibility that there is another level of regulation of FA utilization by AMP kinase (i.e., at the level of the sarcolemmal uptake process through modulating the subcellular distribution of FAT/CD36). To this end, we investigated whether activation of AMP is able to stimulate FA uptake. For this purpose, we applied the following two pharmacological manipulations: 1) the adenosine analog AICAR leading to activation of AMP kinase by ZMP and 2) the mitochondrial inhibitor oligomycin to elevate the intracellular level of AMP. Both AICAR and oligomycin were added to quiescent cardiac myocytes as well as cardiac myocytes electrically stimulated to contract at 4 Hz (5) to study the degree of convergence of their potential effects with contraction-inducible FA uptake. Subsequently, it was determined whether FAT/CD36 and its translocation to the sarcolemma were involved in stimulation of FA uptake by AMP kinase.

Isolation of cardiac myocytes.

Cardiac myocytes were isolated from male Lewis rats (200–250 g) using a Langendorff perfusion system and a Krebs-Henseleit bicarbonate medium supplemented with 11 mmol/l glucose and equilibrated with a 95% O2 and 5% CO2 gas phase (medium A) at 37°C, according to Fischer et al. (19) as previously described (20). After isolation, the cells were washed twice with medium A supplemented with 1.0 mmol/l CaCl2 and 2% (wt/vol) BSA (medium B) and then suspended in 15 ml medium B. The isolated cells were allowed to recover for ∼2 h at room temperature. At the end of the recovery period, cells were washed and suspended in medium B. Only when >80% of the cells had a rod-shaped appearance and excluded trypan blue were they used for subsequent tracer uptake studies.

Electrical stimulation of cardiac myocytes in suspension.

As described in detail elsewhere (5), cell suspensions were subjected to an electric field via two platinum electrodes that were connected to a pulse generator capable of generating biphasic pulses up to 250 V. The monophasic components of the pulses exhibited a block profile. The duration of a monophasic pulse was set at 100 μs, and the time interval between the monophasic components before reversal of the voltage was fixed at 10 μs. The voltage was set at 200 V and the frequency at 4 Hz.

Substrate utilization by cardiac myocytes.

Cells (1.8 ml; 5–8 mg wet mass/ml), suspended in medium B without glucose, were preincubated in capped 20-ml incubation vials for 15 min at 37°C under continuous shaking. To study palmitate uptake, 0.6 ml of the [1-14C]palmitate/BSA complex was added at the start of the incubations so that the final concentration of palmitate amounted to 100 μmol/l with a corresponding palmitate/BSA ratio of 0.3. This palmitate/BSA complex was prepared as previously described (20). To study deoxyglucose uptake, [3H]deoxyglucose was added at the start of the incubations in 0.6 ml medium B without glucose to a final concentration of 100 μmol/l. Cellular uptake of palmitate (3-min incubation) and deoxyglucose (3-min incubation) was determined upon washing the cells three times for 2 min at 100g in an ice-cold stop solution containing 0.2 mmol/l phloretin as previously described (20). The washing procedure did not affect cellular integrity as evaluated by microscopical inspection.

AICAR (1.0 mmol/l), oligomycin (30 μmol/l), insulin (10 nmol/l), wortmannin (200 nmol/l), and adenosine (≤1.0 mmol/l) were added to the cell incubations 15 min before addition of the radiolabeled substrate. 5-Iodotubercidin (20 μmol/l) was added 90 min before any other addition. Cell suspensions were incubated with sulfo-N-succinimidylpalmitate (SSP) (400 μmol/l) for 15 min, and subsequently washed to remove unbound SSP, and resuspended in medium B before addition of radiolabel. Stock solutions of oligomycin, wortmannin and SSP, and 5-iodotubercidin were prepared in DMSO, which never exceeded a final concentration of 0.5% in the cell suspensions. At this concentration, DMSO did not affect cellular substrate utilization. All agents were added at the minimal concentration, at which they exerted the maximal effect. None of these agents, alone or in combination and including SSP, were found to affect the percentage of cells that 1) were rod-shaped and 2) excluded trypan blue as parameters of cellular integrity.

Measurement of adenosine phosphates and ZMP in cardiac myocytes.

Cardiac myocytes (12–15 mg wet mass/ml) were incubated in medium B in the absence and presence of additions for 15 min. Then, they were centrifugated in a microcentrifuge at 2,000 rpm. Upon removal of the supernatant, the cell pellet was extracted with 250 μl 3 mol/l perchloric acid and neutralized with 1 mol/l KHCO3. Hereafter, the cellular content of adenosine phosphates and ZMP was determined by high-performance liquid chromatography according to a variation of the procedure of Wynants and Van Belle (21), as described earlier (22).

Measurement of activation of AMP kinase.

Cardiac myocytes (8–12 mg wet mass/ml) were incubated in medium B in the absence and presence of additions for 15 min. At the end of the incubation, an aliquot was quickly transferred to one-third volume of a sample buffer containing 62.5 mmol/l Tris-HCl (pH 6.8), 2 mmol/l EDTA, 20 mmol/l dithiothreitol, and 7.5% (wt/vol) SDS and used for SDS-PAGE. Subsequently, Western blotting was performed with an antiserum specific for the serine 79-phosphorylated acetyl-CoA carboxylase according to the manufacturer’s instructions.

Subcellular fractionation of cardiac myocytes.

Cardiac myocytes (2.25 ml; 20–25 mg wet mass/ml) were incubated in medium B without further additions or with 30 μmol/l oligomycin or 100 μmol/l dipyridamole for 15 min. At the end of the incubation, the total volume of cell incubations was quickly transferred to a tightly fitting 5-ml Potter-Elvejhem glass homogenizer on ice containing 1 ml cold H2O, after which NaN3 was added to a final concentration of 5 mmol/l to stop ATP-dependent vesicular trafficking events such as GLUT4 translocation (23). Immediately thereafter, cell suspensions were homogenized with 10 strokes. Subsequently, fractionation was carried out according to Fischer et al. (24), with minor modifications as described previously (8). For determination of the GLUT4 and FAT/CD36 content in the plasma membrane (PM) and low-density microsomal (LDM) fraction, aliquots of the membrane fractions were separated with SDS-PAGE and Western blotting, as we have described recently (4). To detect FAT/CD36, we used a monoclonal antibody (MO25) directed against human CD36, and for detection of GLUT4, a polyclonal IgG antiserum was applied. Signals obtained by Western blotting were quantified by densitometry.

Other procedures.

Cellular wet mass was obtained from cell samples taken during the incubation period and determined after centrifugation for 2–3 s at maximal speed in a microcentrifuge and subsequent removal of the supernatant. Protein was quantified with the Bicinchichonic acid protein assay (Pierce, Rockford, IL) according to the manufacturer’s instructions.

Materials.

[1-14C]palmitic acid was obtained from Amersham Life Science (Little Chalfont, U.K.). BSA (fraction V, essentially FA free) phloretin, AICAR, oligomycin, adenosine, insulin, and wortmannin were all obtained from Sigma (St. Louis, MO). 5-Iodotubercidin was purchased from Biomol (Plymouth Meeting, PA). Collagenase type 2 was purchased from Worthington (Lakewood, NJ). The BCA protein assay reagent kit was from Pierce. Antibody MO25 was a gift from Dr. N.N. Tandon, Thrombosis and Vascular Biology Laboratory, Otsuka America Pharmaceutical, Rockville, MD. Antibodies directed against GLUT4 were obtained from Sanver Tech (Heerhugowaard, the Netherlands). Anti-phospho-acetyl-CoA carboxylase was obtained from Brunschwig Chemie (Amsterdam, the Netherlands). SSP is routinely synthesized in our laboratory, as has been previously described (25). Purity of these compounds was confirmed with infrared spectroscopy (performed by Dr. van Genderen, Eindhoven Technical University).

Data presentation and statistics.

All data are presented as means ± SD for the indicated number of myocyte preparations. Statistical difference between groups of observations was tested with a paired Student’s t test. P values ≤0.05 were considered significant.

Dose dependence of effects of AICAR and oligomycin on FA uptake.

AICAR has been frequently used to activate AMP kinase (1113). Its phosphorylated product has been observed to gradually accumulate in hepatocyte incubations up to 60 min. However, ongoing accumulation of ZMP beyond an optimal level can be rather inhibitory to AMP kinase (26). Therefore, we tested in cardiac myocytes the optimal AICAR concentration that would, in combination with 15 min preincubation, exert the maximal effect on FA uptake, the process under study. Indeed, we found an optimal concentration of AICAR, which was determined to be 1.0 mmol/l (Fig. 1).

Inhibition of mitochondrial ATP production also will result in an increase in intracellular AMP and hence activation of AMP kinase. The drop in intracellular ATP acts synergistically, because the AMP-driven activation is inhibited by high ATP, making the AMP/ATP ratio the major determinant of AMP kinase activation (27,28). However, this study requires the inhibition of mitochondrial ATP production, while at the same time aerobic metabolism is not inhibited. Notably, FA uptake, the process under study, depends highly on aerobic metabolism, because we previously observed that a complete block in mitochondrial β-oxidation upon addition of the carnitine palmitoyltransferase-I inhibitor etomoxir was able to markedly inhibit FA uptake by cardiac myocytes (20). Oligomycin, a potent inhibitor of mitochondrial F1F0-ATPase, has long been used to inhibit state 3 respiration in isolated mitochondria (27). However, recent observations by Ylitalo et al. (28) showed that the concentration of oligomycin required for 50% inhibition of the mitochondrial F1F0-ATPase was fivefold lower than its concentration required for 50% inhibition of electron flux through the respiratory chain, and hence, oxygen consumption. This leaves open a window of oligomycin concentrations at which intracellular AMP is elevated while simultaneously oxygen consumption is not inhibited. Based on these observations, we studied FA uptake as a function of the oligomycin concentration and found it to be maximally stimulated at 30 μmol/l oligomycin (Fig. 1).

Effect of insulin, cellular contractions, AICAR, and oligomycin on the intracellular levels of adenosine phosphates and ZMP in cardiac myocytes.

We have compared the effects of 1.0 mmol/l AICAR and 30 μmol/l oligomycin with that of 4-Hz contractions on intracellular adenosine phosphates. A comparison with insulin is also relevant so as to explore a possible convergence between insulin signaling and contraction signaling at the level of AMP. In nonstimulated cells, the AMP/ATP ratio was 0.041. None of the selected manipulations significantly affected intracellular ATP levels, although electrical stimulation and oligomycin had a small lowering effect (Fig. 2). Insulin and AICAR did not affect the intracellular level of AMP (Fig. 2) nor the AMP/ATP ratio. In cells contracting at 4 Hz, the AMP/ATP ratio was increased by 1.9-fold (Fig. 2). Oligomycin enhanced the AMP/ATP ratio by 2.5-fold. Only in AICAR-treated cardiac myocytes was the formation of ZMP observed, which greatly exceeded the intracellular concentration of AMP (ZMP/AMP ratio: 20.9 ± 2.4 [n = 6]; Fig. 2).

Effects of insulin, contractions, AICAR, and oligomycin on activation of AMP kinase in cardiac myocytes.

Activation of AMP kinase was assessed by determining its ability to phosphorylate acetyl-CoA carboxylase, one of its major substrates. The heart expresses predominantly a 280-kDa isoform and to a lesser extent a 265-kDa isoform (16), both of which can be phosphorylated by AMP kinase (29). This phosphorylation decreases the activity of both isoforms of acetyl-CoA carboxylase and results in a drop in the intracellular malonyl-CoA concentration and a consequent de-inhibition of mitochondrial FA oxidation (30). The only other signaling enzyme known to be able to phosphorylate acetyl-CoA carboxylase (i.e., cAMP-dependent protein kinase A) (29), is likely not activated by contractions, AICAR, or oligomycin. Notably, 4-Hz contractions do not elevate intracellular levels of cAMP (8) nor does AICAR treatment (26,31). In addition, mitochondrial inhibitors, such as oligomycin, are expected to decrease rather than increase cAMP levels as a result of decreased availability of ATP, the substrate for adenylyl cyclase. Furthermore, recent studies in electrically stimulated skeletal muscle have convincingly shown that there is a direct link between AMP kinase activation and phosphorylation of the muscle-specific acetyl-CoA carboxylase-β isoform (32).

Under basal conditions, we observed a low basal level of phosphorylation of the predominant 280-kDa isoform of acetyl-CoA carboxylase. Insulin did not stimulate phosphorylation of acetyl-CoA carboxylase. In contrast, cellular contractions at 4 Hz and preincubation with oligomycin and AICAR increased this phosphorylation by 3.3-, 5.5-, and 8.7-fold, respectively (Fig. 3). In the case of AICAR and oligomycin-treated cardiac myocytes, a second lower band appears, which likely corresponds to phosphorylation of the 265-kDa isoform. The effect of AICAR on phosphorylation of acetyl-CoA carboxylase could be blocked in the presence of 5-iodotubercidin, a potent inhibitor of adenosine kinase (33).

Effects of AICAR and oligomycin on deoxyglucose by cardiac myocytes.

Uptake of deoxyglucose into cardiac myocytes was markedly elevated by 4-Hz stimulation (1.5-fold). In addition, AICAR (1.4-fold) and oligomycin (2.0-fold) substantially stimulated deoxyglucose uptake (Fig. 4, right panel). These findings verify that manipulations that activate AMP kinase exert an expected metabolic response (9).

Mechanism of the effects of AICAR and oligomycin on FA uptake by cardiac myocytes.

Electrical stimulation of cardiac myocytes at 4 Hz resulted in a 1.5-fold increased FA uptake rate, in agreement with our previous findings (5). FA uptake into quiescent cardiac myocytes was markedly enhanced by AICAR (1.4-fold) or oligomycin (2.1-fold) (Fig. 4). Electrical stimulation at 4 Hz had no significant additional effect on FA uptake by cardiac myocytes treated with either AICAR or oligomycin. Similarly, when AICAR was added together with oligomycin, FA uptake could not be stimulated further (Fig. 4), suggesting that these agents, as well as cellular contractions, exert their action through the same pathway.

To study the involvement of FAT/CD36, we applied its inhibitor SSP, for which we earlier collected evidence that it specifically inhibits FAT/CD36 without affecting 43-kDa plasma membrane FA binding protein (FABPpm) and 62-kDa FA transport proteins (FATPs) (discussed in detail in the study by Coort et al. [34]). In addition, mammalian cells are impermeable for the sulfo-N-succinimidyl moiety (35), excluding possible effects of SSP on intracellular FA metabolizing enzymes. Preincubation of cardiac myocytes with SSP reduced palmitate uptake by 47% (Fig. 5). In the presence of this FA transport inhibitor, cellular FA uptake is not enhanced by cellular contractions nor by AICAR or oligomycin (Fig. 5). Hence, inhibition of FAT/CD36 completely abolishes the stimulatory effects of all these manipulations on cardiomyocytic FA uptake.

Inclusion of the adenosine kinase inhibitor 5-iodotubercidin had no effect on basal FA uptake by cardiac myocytes but caused a complete blockade of the stimulatory effect of AICAR on cellular FA uptake (Fig. 5). However, 5-iodotubercidin did not affect stimulation of FA uptake by cellular contractions nor by oligomycin (Fig. 5).

The PI 3-kinase inhibitor wortmannin had no effect on palmitate uptake by quiescent cardiac myocytes and was also noneffective on FA uptake by cardiac myocytes stimulated at 4 Hz or treated with AICAR or oligomycin (Fig. 6). Insulin enhanced FA uptake by cardiac myocytes incubated under basal conditions by 1.4-fold. Insulin also significantly further enhanced FA uptake by cardiac myocytes stimulated at 4 Hz (1.5-fold) or treated with AICAR (1.3-fold) or oligomycin (1.4-fold) (Fig. 6).

Effects of insulin and oligomycin subcellular distribution of FAT/CD36.

Incubation of cardiac myocytes in the presence of insulin or oligomycin during 15 min before subcellular fractionation decreased the content of FAT/CD36 in the LDM fraction by 49 and 42%, respectively. Simultaneously, these agents increased the content of FAT/CD36 in the PM fraction to a similar magnitude of 1.5-fold (Fig. 7). Both insulin and oligomycin had comparable effects on subcellular distribution of GLUT4. Moreover, these effects of both agents were very similar in magnitude (i.e., a decrease in GLUT4 content in the LDM fraction by 57 and 56%, respectively, and a concomitant increase in the PM fraction by 1.8- and 1.7-fold, respectively).

The primary goal of this study was to delineate the signaling process involved in contraction-inducible FA uptake in heart. Using the cell-permeable AMP kinase activator AICAR and the mitochondrial inhibitor oligomycin, we found strong evidence that contraction-inducible FA uptake is mediated through elevated intracellular AMP and subsequent activation of AMP kinase resulting in the translocation of FAT/CD36 from intracellular stores to the sarcolemma of cardiac myocytes. These data reveal another level of regulation of cardiac substrate utilization by AMP kinase.

Central role for AMP kinase.

The ability of AICAR to induce FA uptake by cardiac myocytes is a completely novel observation and suggests an involvement of AMP kinase in the regulation of FA uptake. While the consensus is that AICAR acts mainly through activation of AMP kinase (9,10,26), it has been occasionally reported in cardiomyocyte studies that AICAR does not cause accumulation of ZMP (36) and does not activate AMP kinase (37). Therefore, it was important to verify whether AICAR in the present study indeed would exert these effects. Measurement of nucleoside phosphates in AICAR-treated cardiac myocytes demonstrated an accumulation of ZMP to a concentration of 1.38 ± 0.36 μmol/g wet mass (Fig. 2). This is a concentration very near to the optimal concentration of ZMP to stimulate AMP kinase in rat adipocytes (26). With respect to AMP kinase activation, AICAR treatment clearly resulted in a 5.5-fold increase in phosphorylation of acetyl-CoA carboxylase, which indicates that there is a marked increase in AMP kinase activity.

The adenosine kinase inhibitor 5-iodotubercidin inhibits the conversion of AICAR to ZMP and therefore blocks the effects of AICAR that are mediated through AMP kinase. The ability of 5-iodotubercidin to completely inhibit the AICAR-induced increase in phosphorylation of acetyl-CoA carboxylase as well as the AICAR-induced increase in FA uptake by cardiac myocytes indicates that ZMP formation is necessary for AICAR to stimulate both AMP kinase activity and FA uptake. It could however be argued that these effects of 5-iodotubercidin are mediated via high local endogenous adenosine concentrations, which are also a consequence of inhibition of adenosine kinase. To investigate this issue, cardiac myocytes were incubated in the presence of adenosine. It was found that adenosine in concentrations up to 1.0 mmol/l was without effect on FA uptake (data not shown), thus excluding that the effects of 5-iodotubercidin are mediated via adenosine. Therefore, the present findings strongly favor the notion that AICAR stimulates FA uptake by cardiac myocytes through its phosphorylated product, ZMP, and thereafter through activation of AMP kinase.

Besides direct activation of AMP kinase by AICAR, elevation of the intracellular AMP/ATP ratio by using oligomycin was also found to enhance cardiomyocytic FA uptake. The ability of oligomycin to strongly augment the phosphorylation of acetyl-CoA carboxylase is in favor of an important role of AMP kinase in oligomycin-induced FA uptake. The nonadditivity of the effects of oligomycin and AICAR further indicates that these agonists operate via a similar mechanism involving AMP kinase. In contrast to AICAR, the stimulatory effect of oligomycin was insensitive to inhibition by 5-iodotubercidin. This is in line with the notion that AMP formation in the presence of this mitochondrial inhibitor is due to hydrolysis of mitochondrially produced ATP rather than phosphorylation of adenosine.

Contractile activity appeared to markedly (1.9-fold) elevate the intracellular AMP/ATP ratio, which is, however, less effective than the stimulating effect of oligomycin on this ratio (2.5-fold stimulation). In line with this, 4-Hz contractions substantially (3.3-fold) increased the activity of AMP kinase, as evidenced by measurement of phosphorylation of acetyl-CoA carboxylase. This increase is also of a lesser magnitude than that of oligomycin (8.7-fold stimulation). Thus, when considering these two different stimuli, there appears to be a positive correlation between the AMP/ATP ratio and the degree of activation of AMP kinase. These findings also show that electrically stimulated cardiac myocytes are a suitable system to study the role of AMP kinase in cardiac substrate utilization. It is of interest to note that increasing workload in Langendorf-perfused hearts did not induce activation of AMP kinase (38,39). The discrepancy between these latter studies and the present study must be attributed to the different models used. In contrast to the stimulatory effect of AICAR and oligomycin on FA uptake in quiescent cardiac myocytes, neither agents were effective in stimulating FA uptake in electrically stimulated cardiac myocytes. This lack in additivity is regarded as evidence that both agonists increase cardiomyocytic FA uptake via a similar mechanism, including activation of AMP kinase. Similar to oligomycin, contractile activity maintains its ability to stimulate FA uptake in the presence of 5-iodotubercidin-imposed blockade in adenosine kinase activity. Hence, contraction-induced AMP accumulation occurs at the expense of intracellular ATP levels and does not depend on phosphorylation of adenosine.

Involvement of FAT/CD36 translocation.

Treatment of cardiac myocytes with SSP resulted in a complete blockade of the stimulation of FA uptake by oligomycin and AICAR, powerfully indicating that increased involvement of FAT/CD36 is causally linked to the increase in FA uptake in the presence of AMP kinase activation. Subsequent subcellular fractionation demonstrated that oligomycin was able to mobilize FAT/CD36 from intracellular membrane stores to the sarcolemma. Because both oligomycin and AICAR activate AMP kinase and because the effects of oligomycin and AICAR on FA uptake are nonadditive and sensitive to inhibition by SSP, it can also be deduced that AICAR induces translocation of FAT/CD36. Interestingly, the oligomycin-induced increase in sarcolemmal abundance of FAT/CD36, amounting to 1.5-fold, is in the same order of magnitude as the increase in cardiomyocytic FA uptake in the presence of this mitochondrial inhibitor. It can therefore be concluded that activation of AMP kinase stimulates FA uptake through translocation of FAT/CD36 to the sarcolemma.

The stimulatory effect of AMP kinase activation on FA uptake by cardiac myocytes was found to be independent of insulin’s stimulation of FA uptake, because insulin stimulates FA uptake to a similar extent whether in the absence or presence of contractions, oligomycin, or AICAR. Furthermore, when PI 3-kinase, a key enzyme in insulin-signaling events, is inhibited by wortmannin, the stimulatory effects of contraction, oligomycin, and AICAR on cardiomyocytic FA uptake are maintained. Taken together, these findings implicate that in the presence of an increase in mechanical activity, FAT/CD36 is translocated from its contraction-inducible intracellular store upon activation of AMP kinase and independent of insulin signaling. Vice versa, insulin is unable to mobilize FAT/CD36 from the contraction-inducible storage compartment because the intracellular AMP/ATP ratio is not altered by insulin, nor is AMP kinase activated.

FA and glucose uptake compared.

In addition to stimulating FA uptake and FAT/CD36 translocation, the present study indicates that AICAR and oligomycin also stimulate glucose uptake as well as GLUT4 translocation. These findings are in line with recent studies by Russell et al. (9) and Kurth-Kraczek et al. (40) showing that AMP kinase activation increases cardiac muscle glucose uptake through translocation of GLUT4 via a pathway that is independent of PI 3-kinase. The stimulating effect of AICAR, added at an optimal concentration of 1 mmol/l, on both deoxyglucose and FA uptake, however, is markedly smaller than that of oligomycin, which corresponds to an earlier report in which potassium cyanide was found to be more effective than AICAR in inducing glucose uptake (14). Furthermore, the confirmation of GLUT4 translocation in response to insulin- and contraction signaling validates the suitability of isolated cardiac myocytes as an experimental model to study signaling and vesicular trafficking processes.

Concluding remarks.

AMP kinase is known as a stress- and exercise-induced multisubstrate enzyme. It precludes ATP utilization for anabolic purposes, and it switches on catabolic processes for meeting increased energy demands (41,42). Among these latter processes are glucose transport and FA oxidation. Based on the present study, AMP kinase is now also shown to upregulate FAT/CD36-mediated sarcolemmal FA transport. In light of the accumulating evidence that FA uptake into myocytes of heart and skeletal muscle is a rate-governing step (2,5), the combined induction of FAT/CD36 mobilization and of carnitine palmitoyltransferase-I activity through AMP kinase activation is metabolically efficient, because this allows the extra incoming FA to be preferentially channeled into mitochondrial β-oxidation. It remains to be established whether AMP kinase activation is fully responsible for the increase in FA uptake in the presence of cardiomyocytic contractions or that AMP kinase-independent mechanisms are necessary to mobilize FAT/CD36 from contraction-sensitive stores.

FIG. 1.

Dose-dependent effects of AICAR and oligomycin on FA uptake by cardiac myocytes. Cardiac myocytes were incubated for 15 min with various concentrations of AICAR and oligomycin. Data are means ± SD of three experiments carried out with different cardiomyocyte preparations. *Significantly different from myocytes without additions (P < 0.05).

FIG. 1.

Dose-dependent effects of AICAR and oligomycin on FA uptake by cardiac myocytes. Cardiac myocytes were incubated for 15 min with various concentrations of AICAR and oligomycin. Data are means ± SD of three experiments carried out with different cardiomyocyte preparations. *Significantly different from myocytes without additions (P < 0.05).

Close modal
FIG. 2.

Influence of insulin, electrical stimulation, AICAR, and oligomycin on intracellular nucleoside phosphates in cardiac myocytes. Cardiac myocytes were incubated for 15 min in the absence (Ctrl) or presence of 10 nmol/l insulin (Ins), electrical stimulation at 200 V/4 Hz, 1.0 mmol/l AICAR, and 30 μmol/l oligomycin (Oli). Intracellular levels of AMP (A), ATP (B) and ZMP (C) were determined as described in research design and methods. Data are means ± SD of three to six experiments carried out with different cardiomyocyte preparations. *Significantly different from myocytes without additions (Ctrl) (P < 0.05).

FIG. 2.

Influence of insulin, electrical stimulation, AICAR, and oligomycin on intracellular nucleoside phosphates in cardiac myocytes. Cardiac myocytes were incubated for 15 min in the absence (Ctrl) or presence of 10 nmol/l insulin (Ins), electrical stimulation at 200 V/4 Hz, 1.0 mmol/l AICAR, and 30 μmol/l oligomycin (Oli). Intracellular levels of AMP (A), ATP (B) and ZMP (C) were determined as described in research design and methods. Data are means ± SD of three to six experiments carried out with different cardiomyocyte preparations. *Significantly different from myocytes without additions (Ctrl) (P < 0.05).

Close modal
FIG. 3.

Influence of insulin, electrical stimulation, AICAR, and oligomycin on activation of AMP kinase. Cardiac myocytes were incubated for 15 min in absence (Ctrl) or presence of 10 nmol/l insulin (Ins), electrical stimulation at 200 V/4 Hz, 1.0 mmol/l AICAR (in the absence and presence of 10 μmol/l 5-iodotubercidin [iTu], which was added 90 min before AICAR), and 30 μmol/l oligomycin (Oli). Phosphorylation of acetyl-CoA carboxylase was detected at 280 kDa (i.e., the major isoform of this enzyme in heart). However, in the AICAR- and oligomycin-treated cells, a second lower band is apparent, corresponding to the 265-kDa isoform. Data are means ± SD of three experiments carried out with different cardiomyocyte preparations. *Significantly different from myocytes without additions (Ctrl) (P < 0.05).

FIG. 3.

Influence of insulin, electrical stimulation, AICAR, and oligomycin on activation of AMP kinase. Cardiac myocytes were incubated for 15 min in absence (Ctrl) or presence of 10 nmol/l insulin (Ins), electrical stimulation at 200 V/4 Hz, 1.0 mmol/l AICAR (in the absence and presence of 10 μmol/l 5-iodotubercidin [iTu], which was added 90 min before AICAR), and 30 μmol/l oligomycin (Oli). Phosphorylation of acetyl-CoA carboxylase was detected at 280 kDa (i.e., the major isoform of this enzyme in heart). However, in the AICAR- and oligomycin-treated cells, a second lower band is apparent, corresponding to the 265-kDa isoform. Data are means ± SD of three experiments carried out with different cardiomyocyte preparations. *Significantly different from myocytes without additions (Ctrl) (P < 0.05).

Close modal
FIG. 4.

Effects of AICAR and oligomycin in the absence and presence of electrical stimulation on substrate uptake by cardiac myocytes. Cell suspensions were incubated in the absence of additions (Ctlr) or presence of 1.0 mmol/l AICAR or 30 μmol/l oligomycin (Oli) for 15 min before execution of palmitate uptake studies (3 min; left panel) or deoxyglucose uptake studies (3 min; right panel) without and with electrical field stimulation at 200 V and 4 Hz (200V/4Hz). Data are means ± SD of four to six experiments carried out with different cardiomyocyte preparations. *Significantly different from myocytes without additions (Ctrl) in the absence of electrical stimulation (P < 0.05). **Significantly different from corresponding quiescent myocytes (Quiescent) (P < 0.05).

FIG. 4.

Effects of AICAR and oligomycin in the absence and presence of electrical stimulation on substrate uptake by cardiac myocytes. Cell suspensions were incubated in the absence of additions (Ctlr) or presence of 1.0 mmol/l AICAR or 30 μmol/l oligomycin (Oli) for 15 min before execution of palmitate uptake studies (3 min; left panel) or deoxyglucose uptake studies (3 min; right panel) without and with electrical field stimulation at 200 V and 4 Hz (200V/4Hz). Data are means ± SD of four to six experiments carried out with different cardiomyocyte preparations. *Significantly different from myocytes without additions (Ctrl) in the absence of electrical stimulation (P < 0.05). **Significantly different from corresponding quiescent myocytes (Quiescent) (P < 0.05).

Close modal
FIG. 5.

Modulation of the stimulatory effects of electrical stimulation, AICAR, and oligomycin on FA uptake by SSP and 5-iodotubercidin. Cell suspensions were preincubated with 400 μmol/l SSP or with 20 μmol/l 5-iodotubercidin (iTu) as described in research design and methods. In the case of SSP, cells were centrifuged at the end of the incubation period, washed twice, and resuspended in medium B. The SSP-matched basal incubation did not display an FA uptake rate significantly different from the iTu-matched basal. Subsequently, cells were incubated in the absence of additions (Ctrl) or in the absence of additions but in the presence of electrical stimulation at 200 V/4 Hz or in the presence of 1.0 mmol/l AICAR or 30 μmol/l oligomycin (Oli) before determination of palmitate uptake (3 min). Data are means ± SD of four to eight experiments carried out with different cardiomyocyte preparations. *Significantly different from myocytes without additions (Ctrl) (P < 0.05). **Significantly different from corresponding basal myocytes (P < 0.05).

FIG. 5.

Modulation of the stimulatory effects of electrical stimulation, AICAR, and oligomycin on FA uptake by SSP and 5-iodotubercidin. Cell suspensions were preincubated with 400 μmol/l SSP or with 20 μmol/l 5-iodotubercidin (iTu) as described in research design and methods. In the case of SSP, cells were centrifuged at the end of the incubation period, washed twice, and resuspended in medium B. The SSP-matched basal incubation did not display an FA uptake rate significantly different from the iTu-matched basal. Subsequently, cells were incubated in the absence of additions (Ctrl) or in the absence of additions but in the presence of electrical stimulation at 200 V/4 Hz or in the presence of 1.0 mmol/l AICAR or 30 μmol/l oligomycin (Oli) before determination of palmitate uptake (3 min). Data are means ± SD of four to eight experiments carried out with different cardiomyocyte preparations. *Significantly different from myocytes without additions (Ctrl) (P < 0.05). **Significantly different from corresponding basal myocytes (P < 0.05).

Close modal
FIG. 6.

Effects of insulin and insulin signaling on stimulation of FA uptake by electrical stimulation, AICAR, and oligomycin. Cell suspensions were incubated for 15 min before execution of palmitate uptake studies (3 min) under basal conditions (Ctrl), in the presence of electrical field stimulation at 200 V/4 Hz, or in the presence of 1.0 mmol/l AICAR, 30 μmol/l oligomycin (Oli), 10 nmol/l insulin (Ins), or 200 nmol/l wortmannin (Wort). Data are means ± SD of four to six experiments carried out with different cardiomyocyte preparations. *Significantly different from myocytes without additions (Ctrl) (P < 0.05). **Significantly different from corresponding basal myocytes (P < 0.05).

FIG. 6.

Effects of insulin and insulin signaling on stimulation of FA uptake by electrical stimulation, AICAR, and oligomycin. Cell suspensions were incubated for 15 min before execution of palmitate uptake studies (3 min) under basal conditions (Ctrl), in the presence of electrical field stimulation at 200 V/4 Hz, or in the presence of 1.0 mmol/l AICAR, 30 μmol/l oligomycin (Oli), 10 nmol/l insulin (Ins), or 200 nmol/l wortmannin (Wort). Data are means ± SD of four to six experiments carried out with different cardiomyocyte preparations. *Significantly different from myocytes without additions (Ctrl) (P < 0.05). **Significantly different from corresponding basal myocytes (P < 0.05).

Close modal
FIG. 7.

Effect of insulin and oligomycin on subcellular distribution of FAT/CD36 in cardiac myocytes. Cardiac myocytes were incubated for 15 min in the absence (Basal) and presence of 10 nmol/l insulin (Ins) or 30 μmol/l oligomycin (Oli), after which NaN3 was added to a final concentration of 5 mmol/l to stop ATP-demanding processes. Immediately thereafter, cells were frozen in liquid nitrogen and, upon thawing, subjected to subcellular fractionation. The collected fractions were subjected to electrophoresis and Western blotting. Signals were quantified by densitometry. Transporter content was expressed as multiple of control (Basal) in the corresponding fraction. Data are means ± SD of four experiments carried out with different cardiomyocyte preparations. Representative Western blots are shown. GLUT4 was detected at 45 kDa and FAT/CD36 at 88 kDa. *Significantly different from myocytes in the absence of additions (Basal) (P < 0.05).

FIG. 7.

Effect of insulin and oligomycin on subcellular distribution of FAT/CD36 in cardiac myocytes. Cardiac myocytes were incubated for 15 min in the absence (Basal) and presence of 10 nmol/l insulin (Ins) or 30 μmol/l oligomycin (Oli), after which NaN3 was added to a final concentration of 5 mmol/l to stop ATP-demanding processes. Immediately thereafter, cells were frozen in liquid nitrogen and, upon thawing, subjected to subcellular fractionation. The collected fractions were subjected to electrophoresis and Western blotting. Signals were quantified by densitometry. Transporter content was expressed as multiple of control (Basal) in the corresponding fraction. Data are means ± SD of four experiments carried out with different cardiomyocyte preparations. Representative Western blots are shown. GLUT4 was detected at 45 kDa and FAT/CD36 at 88 kDa. *Significantly different from myocytes in the absence of additions (Basal) (P < 0.05).

Close modal

This study was supported by the Netherlands Heart Foundation, grants D98.012 and 2000.156, and by the Heart & Stroke Foundation of Ontario. J.J.F.P.L. was the recipient of a VIDI-Innovational Research Grant from the Netherlands Organization for Scientific Research (NWO-ZonMw grant number 016.036.305).

Antibody MO25 was provided by Dr. N.N. Tandon, Thrombosis and Vascular Biology Laboratory, Otsuka America Pharmaceutical, Rockville, MD.

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