Contraction-Induced Fatty Acid Translocase/CD36 Translocation in Rat Cardiac Myocytes Is Mediated Through AMP-Activated Protein Kinase Signaling

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.

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.

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.

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).

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.

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.

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.

[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).

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.

AICAR has been frequently used to activate AMP kinase (11–13). 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).

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).

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).

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).

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).

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.

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.

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.

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.

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.

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.