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Am J Physiol Endocrinol Metab 284: E399-E406, 2003; doi:10.1152/ajpendo.00259.2002
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Vol. 284, Issue 2, E399-E406, February 2003

Creatine transporter activity and content in the rat heart supplemented by and depleted of creatine

Ernest Boehm, Sharon Chan, Mina Monfared, Theo Wallimann, Kieran Clarke, and Stefan Neubauer

Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN; Department of Biochemistry, University of Oxford, Oxford OX1 3BN, United Kingdom; and Eidgenoessisch-technische Hochschule (ETH)-Zürich, Institute of Cell Biology, ETH-Hönggerberg, CH-8093 Zürich, Switzerland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The intracellular creatine concentration is an important bioenergetic parameter in cardiac muscle. Although creatine uptake is known to be via a NaCl-dependent creatine transporter (CrT), its localization and regulation are poorly understood. We investigated CrT kinetics in isolated perfused hearts and, by using cardiomyocytes, measured CrT content at the plasma membrane or in total lysates. Rats were fed control diet or diet supplemented with creatine or the creatine analog beta -guanidinopropionic acid (beta -GPA). Creatine transport in control hearts followed saturation kinetics with a Km of 70 ± 13 mM and a Vmax of 3.7 ± 0.07 nmol · min-1 · g wet wt-1. Creatine supplementation significantly decreased the Vmax of the CrT (2.7 ± 0.17 nmol · min-1 · g wet wt-1). This was matched by an ~35% decrease in the plasma membrane CrT; the total CrT pool was unchanged. Rats fed beta -GPA exhibited a >80% decrease in tissue creatine and increase in beta -GPAtotal. The Vmax of the CrT was increased (6.0 ± 0.25 nmol · min-1 · g wet wt-1) and the Km decreased (39.8 ± 3.0 mM). The plasma membrane CrT increased about fivefold, whereas the total CrT pool remained unchanged. We conclude that, in heart, creatine transport is determined by the content of a plasma membrane isoform of the CrT but not by the total cellular CrT pool.

creatine transport(er); creatine feeding; creatine depletion; isolated perfused heart; cell-surface biotinylation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CONCEPT of the creatine kinase/phosphocreatine (CK/PCr) shuttle describes the functional association of CK isoenzymes with discrete intracellular compartments of ATP production and hydrolysis and the suitability of PCr and creatine to serve as cytosolic energy transducers (2, 3, 27). In this view, the functions of PCr and creatine are to form essential links between sites of ATP synthesis and utilization.

Both PCr and creatine can be spontaneously converted into creatinine, which leaves the cardiomyocyte and is excreted by the kidney at a rate of ~2% per day (1). This loss of creatine must be replenished, either from food or by endogenous synthesis. Creatine is not synthesized by cardiomyocytes, however (10), but rather is taken up by the action of a highly specific Na+Cl--dependent creatine cotransporter (CrT) (for review see Ref. 25). It has been suggested that CrT content and/or activity may be a major determinant of the myocardial creatine and, hence, PCr concentrations (19). For example, we have reported that decreased CrT content correlates well with decreased myocardial creatine (and PCr) content in human, rat (19), and dog (5) failing heart, suggesting that a decreased energy reserve in the failing heart might be a direct consequence of loss of CrT protein. Furthermore, the failure of the myocardium and other tissues to substantially increase intracellular creatine content in response to elevated plasma creatine levels caused by chronic creatine feeding has been proposed to be due to either an inactivation of the CrT or a decrease in CrT content (12, 13).

Many unanswered questions with regard to the biology of the CrT remain, however: it has yet to be shown how CrT activity and creatine content are related, and the precise mechanism by which the subcellular distribution and activity of the CrT are regulated remains unclear. We have recently shown that two distinct pools of the CrT protein exist in the cardiomyocyte (28), one associated with the mitochondrial compartment and a second with the plasma membrane. Studies (25) quantifying what we now know as a predominantly mitochondrial protein must be reassessed to take into account the existence of this discreet membrane-bound CrT isoform. The plasma membrane CrT is likely to be the only CrT pool relevant for the regulation of creatine content in the cardiomyocyte, yet there are currently no studies on this specific CrT pool in the heart. The purpose of this work, therefore, was to study the relationship between intracellular creatine content, subcellular CrT pools, and CrT uptake kinetics under conditions of creatine supplementation or depletion.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental animals. Male Wistar rats were obtained at ~250 g from Harlan UK (Bicester, UK). These were housed (3 animals to a cage) in a standard experimental animal laboratory (12:12-h light-dark schedule, room temperature 22°C), with free access to drinking water and a creatine-free powdered diet (RM1 diet; Special Diet Services, Witham, Essex, UK). Rats were randomly assigned for 6 wk to one of three feeding regimens: 1) Control diet; 2) diet + 3% creatine (Apin Chemicals, Abingdon, UK); and 3) diet + the creatine analog beta -guanidinopropionic acid (beta -GPA 1.5%). Rats were subsequently anesthetized with an intraperitoneal injection of sodium pentobarbitone (75 mg/kg body wt). Blood samples were removed from the femoral vein, and hearts were rapidly excised and arrested in ice-cold buffer (see Radiolabel studies). Hearts were cannulated via the ascending aorta for retrograde perfusion for radiolabeled [14C]creatine uptake studies, myocyte isolation, or metabolite assays. In addition to these feeding regimens, follow-up experiments were performed to investigate both the time course and dose dependence of the creatine feeding effect. In these studies, animals were fed 3% creatine for 2, 4, and 12 wk and 0.6 or 7% creatine for 6 wk.

Radiolabel studies. Hearts were perfused with Krebs-Henseleit buffer containing (in mmol/l) 110 NaCl, 4.7 KCl, 1.2 MgSO4 7 H2O, 1.75 CaCl2 · 2 H2O, 11 glucose, 2 KH2PO4, 25 NaHCO3, 0.5 Na2EDTA, 0.5 Na-lactate, and 4.5 Na-pyruvate. The buffer, aerated with 95% O2-5% CO2 to give a pH of 7.4 at 37°C, was perfused through the hearts in a recirculating mode (recirculating volume of ~400 ml) at a constant pressure of 100 mmHg. Recirculating buffer was supplemented with creatine (final [creatine] = 25, 100, and 500 µM); this was shown previously to have no effect on cardiac function, and hearts were stable for the duration of the protocol (<= 1 h). Cardiac contractile function was monitored throughout the protocol as previously described (12).

In an initial set of experiments, hearts were perfused in the presence of 100 µM creatine for 15 min, 30 min, and 1 h. Because the accumulation of [14C]creatine by the heart was linear with time, 30 min was taken as the optimal perfusion time. Hearts were allowed to equilibrate for 10 min, after which [14C]creatine (American Radiochemicals) was added in trace amounts to the recirculating buffer. Hearts were perfused for 30 min, after which time perfusion was switched for 15 min to a noncirculating mode with nonradioactive creatine-free buffer. The absence of 14C label in the effluent between 10 and 15 min showed that the extracellular space had been cleared and that leakage of creatine from the heart was negligible. At the end of perfusion, hearts were weighed and dissected into left ventricle, septum, and right ventricle. Tissue was digested in 1 ml of 1 M KOH at 60°C before scintillation counting for 14C. Creatine accumulation was used to estimate creatine uptake (29), with the assumption that creatine efflux was negligible (4). Because we observed no difference in creatine transport rates between left ventricle, right ventricle, and septum, data from each heart were calculated to yield the mean of [14C]creatine accumulation in these three sections.

Isolation of cardiac myocytes and cell surface biotinylation. Cardiac myocytes were isolated according to the method of Powell et al. (22). Briefly, after excision, hearts were perfused for 5 min by retrograde coronary perfusion with an isolation Tyrode buffer, containing (in mM) 130 NaCl, 5.4 KCl, 0.4 NaH2PO4 · 2 H2O, 3.5 MgCl2, 10 glucose, 5.0 HEPES, and 20 taurine, pH 7.4, at 37°C. This was followed by 10 min with isolation buffer supplemented with collagenase (1 mg/ml), protease (0.1 mg/ml), and CaCl2 (100 mM). The atria were removed, and the ventricles were placed in a stirred incubation solution (isolation buffer containing 1 mg/ml collagenase and 100 mM CaCl2) for 10 min at 37°C. The supernatant was filtered through a gauze mesh and made up to 10 ml with wash solution (10 mg/ml albumin and 500 µM CaCl2). Cell samples were centrifuged for 1 min at 20-30 g and then filtered through the gauze mesh, made up to 10 ml with wash solution, and centrifuged again for 1 min at 20-30 g. Cells were stored in storage solution containing DMEM (20 g/ml), NaH2CO3 (1 mM), and UltraSer G (2 mg/ml) at pH 7.35 until required.

Cell surface expression of the CrT was determined by using the membrane-impermeable biotinylation reagent Sulpho-NHS-LC-biotin (Pierce, Rockford, IL) (6, 26). Isolated myocytes were washed three times with ice-cold PBS and then incubated with PBS containing 1.0 mg/ml Sulfo-NHS-LC-biotin for 45 min with constant shaking (4°C). After labeling, the cells were washed three times in ice-cold PBS containing glycine (15 mM) to quench the residual Sulpho-NHS-LC-biotin. Subsequently, cells were solubilized by resuspension in solubilization buffer [containing (in mM) 150 NaCl, 20 HEPES, 2 phenylmethylsulfonyl fluoride, 1 EDTA, 0.1% Triton X-100, and anti-protease cocktail (Sigma, 1:100 dilution)] and gentle shaking for 1 h. Extracts were clarified by centrifugation at 13,000 g for 5 min (4°C), and a portion of the lysate was taken for incubation with streptavidin beads (Sigma Chemical). The remaining lysate was kept for protein determination and immunoblotting of the total cell extract.

Streptavidin beads (150 µl) were washed three times in wash buffer [containing (in mM) 150 NaCl and 10 Tris, pH 7.0] by rapid centrifugation. Lysate (2,000 µg protein) was added to the beads, incubated on ice, and rotated/shaken overnight. Samples were then washed six times by rapid centrifugation with wash buffer plus anti-proteases. To elute the biotinylated protein from the beads, 80 µl of urea buffer (containing 9 M urea, 2% SDS, and 100 mM Tris, pH 6.8) plus anti-proteases were added and incubated (with constant shaking) for 45 min at 95°C. Finally, tubes were centrifuged at 13,000 g, and the supernatant was collected.

Blood preparation. Blood samples were centrifuged at 12,000 rpm for 10 min. The pellet was washed with isosmotic buffer (MOPS-buffered saline) three times by rapid centrifugation. Approximately 250 µl were resuspended in solubilization buffer (see above) and incubated with shaking for 30 min at 4°C. The supernatant was used for the determination of plasma concentration of creatine or beta -GPA by HPLC, as has been previously described (18). The pellet was used for immunoblotting to observe the erythrocyte CrT (data not shown).

Western blotting. Total cell extracts (myocyte and erythrocyte at 20 and 100 µg protein/lane, respectively) and captured biotinylated proteins (100 µg protein/lane) were separated on a 10% SDS-polyacrylamide gel and subsequently transferred onto a nitrocellulose membrane (Gelman Laboratories). The membrane was blocked with 5% fat-free milk powder in PBS-T buffer (Tween, 0.1%) for 1 h at room temperature followed by 1 h in PBS supplemented with avidin (Sigma) at a concentration of 20 µg/ml. After washing for 1 h, membranes were incubated with a 1:3,000 dilution of the anti-CrT antibody overnight at 4°C. After washing with PBS-T buffer, the blot was incubated with a 1:5,000 dilution of the HRP-conjugated anti-rabbit antibody (Amersham Pharmacia Biotech). The immunoreactive bands were visualized using the ECL-Plus chemiluminescence detection kit (Amersham Pharmacia Biotech) and quantified by scanning densitometry. The intensity values for both CrT bands were added together for calculation of absolute CrT content.

Biochemical analysis. Hearts were perfused for 10 min before being rapidly freeze-clamped using Wollenberger tongs. For HPLC analysis, powdered tissue was homogenized in 0.4 N perchloric acid at 0°C, and aliquots of homogenates were removed for protein determination. The homogenate was neutralized and centrifuged for 5 min. The supernatant was used for measuring beta -GPA (where appropriate), PCr, free Cr, and ATP, as previously described (18). Noncollagen protein was measured by the method of Lowry et al. (14). Tissue concentrations were expressed as nanomoles per milligram protein. To investigate any possible coregulation of creatine kinase (CK) with the CrT, hearts were analyzed for total CK activity and isoenzyme specific activity (17, 18).

Statistical analysis. The data are expressed as means ± SD. To determine statistical significance between feeding groups, a one-way analysis of variance was used followed by a Bonferroni post hoc test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Heart weights, body weights, and cardiac function. Table 1 shows heart weight (HW) and body weight (BW) data from the three main feeding groups. Creatine feeding had no effect on BW. HWs and HW-to-BW ratios (HW/BW) were similarly unaffected. beta -GPA-fed rats displayed a small decrease in BW (P < 0.01) and a small increase in both HW and HW/BW (P < 0.01), suggesting the development of mild myocardial hypertrophy. Table 1 also shows that there was no difference in cardiac function between control and fed animals.

                              
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  		Table 1.   BW, HW, plasma creatine or beta -GPA level, and cardiac function after 6 wk of creatine supplementation (3% creatine) or depletion (1.5% beta -GPA)

Plasma levels of creatine and beta -GPA. The mean plasma creatine and beta -GPA concentrations from the main feeding groups are shown in Table 1. Creatine supplementation led to an about sevenfold significant increase in plasma creatine concentration. Feeding beta -GPA for 6 wk resulted in a plasma beta -GPA concentration of 1.5 ± 0.9 mM. There was a small but significant increase in plasma creatine in beta -GPA-fed rats (P < 0.01).

CrT kinetics in perfused heart. Figure 1 shows creatine uptake rates in the three main feeding groups. Uptake of creatine in control hearts followed first-order saturation kinetics. Rates of [14C]creatine uptake were dependent on the extracellular creatine concentration and reached saturation by 500 µM. The apparent Km and Vmax for [14C]creatine uptake in isolated perfused control hearts were calculated as 69.6 ± 13.1 µM and 3.7 ± 0.07 nmol · min-1 · g wet wt-1, respectively. Feeding 3% creatine for 6 wk led to an ~30% significant decrease in the Vmax (P < 0.001), despite no change in the calculated Km. Feeding 1.5% beta -GPA for 6 wk induced an ~70% significant increase in the Vmax (P < 0.001), as well as a significant decrease in the calculated Km (P < 0.05).


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  		Fig. 1.   Effects of extracellular creatine concentration on creatine uptake in the isolated perfused heart. Hearts were perfused with Krebs-Henseleit buffer containing [14C]creatine. Extracellular creatine was 25, 100, and 500 µM. The Km and Vmax are shown for hearts from control rats (), rats fed creatine (3% for 6 wk; ), and rats fed beta -guanidinopropionic acid (beta -GPA, 1.5% for 6 wk; black-down-triangle ). Each point represents the mean of 4 animals. Vertical arrows, actual situation in creatine-fed and control animals in which actual Vmax in control rats and creatine-fed rats is equivalent. Data are presented as means ± SD. * P < 0.001 vs. controls.

Quantification of total and membrane-bound CrT. Two major protein bands with an apparent mobility of 72 and 52 kDa were consistently recognized in the whole lysate preparations from all three experimental groups (Fig. 2). There was no significant difference in the total content of CrT between rats fed control, creatine (Fig. 2, A and B), or beta -GPA diets (Fig. 2, C and D).


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  		Fig. 2.   Immunoblot analysis (10 µg protein loaded/lane) of total creatine cotransporter (CrT) content in cardiomyocytes isolated from control rats vs. rats fed creatine (A and B) or beta -GPA (C and D). Note that the CrT exists as 2 bands, with molecular masses of ~52 and 72 kDa, respectively. There is no difference in the calculated total CrT content. Data are presented as means ± SD. * P < 0.05 vs. controls.

The membrane-bound protein also occurred as two bands. However, these were at slightly higher molecular masses (~60 and 75 kDa) than the total CrT protein (Figs. 3, A and C).


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  		Fig. 3.   Immunoblot analysis (100 µg protein loaded/lane) of plasma membrane-bound CrT in cardiomyocytes isolated from control rats vs. animals fed creatine (A and C) or beta -GPA (C and D). Note that the CrT exists as 2 bands, with molecular masses of ~60 and 75 kDa, respectively. There is an ~30% significant decrease in membrane CrT content in cardiomyocytes isolated from creatine-fed animals, whereas there is an about fivefold increase in the plasma membrane CrT content in cardiomyocytes isolated from beta -GPA-fed animals. Data are presented as means ± SD. * P < 0.01 vs. controls.

The content of the plasma membrane CrT was much (>10 times) lower than the total CrT. There was an ~30% significant decrease in CrT protein content in the creatine-fed group (Fig. 3, A and B), whereas beta -GPA-fed rats demonstrated a very large and significant increase, about fivefold, in membrane-bound CrT content compared with controls (Fig. 3, C and D).

Intracellular high-energy phosphates and CK. Table 2 shows high-energy phosphate and creatine concentrations, as well as CK activity, for the three main experimental groups. PCr, Cr, and ATP levels did not change significantly in the creatine-fed group. Total creatine content (PCr + creatinefree), however, showed a significant 11% increase in creatine-fed rats.

                              
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  		Table 2.   Metabolite concentrations and CK isoenzyme activities in hearts from rats fed either control diet or diet supplemented with creatine or beta -GPA for 6 wk

beta -GPA-fed rats exhibited a significant >80% decrease in total creatine compared with controls (Table 2) but no significant change in ATP. Because of the difficulty of separating beta -GPA and creatine with the HPLC method, only total creatine could be quantified. Total beta -GPA was 72.9 ± 5.7 nmol/mg protein.

Total CK activity and isoenzyme distribution were not different between groups.

Time and concentration dependence of creatine. To investigate whether the effect of feeding 3% creatine for 6 wk was maximal, a further series of animals was studied at different time points and with different doses of creatine. Plasma creatine was measured together with the maximal rate of creatine transport. Data from these experiments are presented in Fig. 4. Feeding 0.6% creatine for 6 wk, or 3% for 2 wk, had small effects on both plasma creatine and creatine transport compared with control animals. After 4 wk, the effect of 3% creatine was not significantly different from that after 6 wk. Feeding 3% creatine for 12 wk led to a decreasing trend in the plasma creatine concentration compared with 6 wk, although the Vmax was unchanged. Feeding creatine at a higher dose (7%) for 6 wk failed to have any additional effect on plasma creatine or the Vmax of the CrT compared with 3%. Heart weights, body weights, and cardiac function were unaffected in these additional feeding protocols (data not shown). Thus, by feeding 3% creatine for 6 wk, we observed a maximal effect on creatine absorption, retention, and transport.


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  		Fig. 4.   Plasma creatine concentration and Vmax of the CrT in 5 additional groups fed creatine. Data from control animals and those fed 3% creatine for 6 wk are included for comparison. Data are presented as means ± SD. * P < 0.01 vs. animals fed 3% for 6 wk.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results presented in this study show that creatine supplementation leads to a decrease in the Vmax of creatine transport in the heart and to a concomitant decrease in the content of the plasma membrane CrT pool. Creatine depletion by beta -GPA feeding, on the other hand, leads to an increase in the Vmax of creatine transport and a concomitant increase in the plasma membrane CrT pool. To the best of our knowledge, these are the first data to show directly a chronic coregulation of CrT activity and plasma CrT content in the intact perfused tissue.

Creatine and beta -GPA metabolism in rats. The creatine doses were chosen on the basis of the recommended daily maximum in humans of 20 g/day (0.6%) and our previous work (3 and 7%) (12). HWs and HW/BW ratios in all creatine-fed groups were not different from those of controls. Animals appeared to tolerate the feeding regimens well, with no signs of distress even at the highest creatine dose, which was equivalent to ~200 g/day in humans.

beta -GPA is a creatine analog and an efficient substrate for the CrT (8). It was given at a dose of 1.5% to deplete intracellular creatine stores (20, 24, 31). After 6 wk of feeding, beta -GPA-fed animals exhibited a small but significant reduction in BW and a small increase in HW and HW/BW compared with control animals. This is in agreement with a previous study showing the development of slight hypertrophy in rats fed 1.5% beta -GPA (15).

The plasma creatine concentration in control animals was found to be ~130 µM, which agrees with published data (25). As the control diet is creatine free, this level of creatine must be endogenously synthesized by the liver and kidneys (for review see Ref. 30), which would explain its consistency. Significant differences in the plasma creatine and beta -GPA concentrations in fed animals were probably due to variations in the feeding habits and times when experiments were performed. Nevertheless, there was an about sevenfold increase in plasma creatine in 3% creatine-supplemented rats at 4-6 wk, although this was less pronounced at 12 wk. A downregulation of the CrT in the kidney (9) leading to increased creatine excretion might explain this observation. It could also explain why the plasma creatine concentration was not further increased by feeding 7% creatine. We did not measure the concentration of creatine in the urine, although it has been shown previously that there is a dose-dependent increase in creatine excretion during creatine supplementation in humans (11).

Despite feeding beta -GPA at a dose of just 1.5%, plasma levels were increased substantially. Increased reabsorption by the kidneys due to an increase in CrT protein (opposite from that seen with creatine) might explain this observation. Plasma creatine levels were marginally increased by beta -GPA feeding. Although increased renal reabsorption might explain this observation, the effects of creatine supplementation/depletion on the kidney CrT or creatine synthesis have yet to be determined.

Creatine transporter kinetics. Our data are in close agreement with the one previous study investigating creatine transport kinetics in the rat heart (23). The uptake data for control mice fit well to a Michaelis-Menten plot with a calculated Km of ~70 µM. As this is lower than the plasma creatine concentration, the CrT is working at ~70% of its calculated Vmax.

The maximal rate of creatine transport was significantly decreased after feeding 3% creatine for 6 wk; there was no significant change in Km. This can classically be explained by a decrease in the CrT protein content without kinetic regulation. Although the ~30% decrease in Vmax is small, under these fed conditions the actual calculated rate of creatine transport is ~2.7 nmol · min-1 · g wet wt-1. This is the same as the actual calculated rate of creatine transport under control conditions (also ~2.7 nmol · min-1 · g wet wt-1; see arrows on Fig. 1). Therefore, it appears that any form of creatine supplementation leading to a plasma creatine concentration that is greater than that required for the Vmax of the CrT (i.e., >500 µM) would be expected to have an equivalent effect on CrT kinetics. This is what we find here (Fig. 4). Because the CrT operates close to saturation, only small decreases in CrT activity are necessary to maintain creatine homeostasis in the tissue.

Six weeks of beta -GPA feeding led to a highly significant increase in the Vmax of the CrT by ~70%, as well as a significant decrease in the calculated Kmax. The increased CrT in combination with high plasma beta -GPA leads to a rapid and significant increase in beta -GPA uptake and subsequent creatine depletion. As creatine depletion could increase the Vmax further, this might result in a positive feedback loop, whereby more beta -GPA was transported as more creatine was depleted.

CrT content. The plasma CrT and total mitochondrial CrT appeared as two bands. This is different from what was previously observed in biotinylated mouse cardiomyocytes, where only one band was observed in the membrane fraction (28). The two plasma membrane bands were at a higher molecular mass than those observed in whole myocyte preparations. We do not believe that this was a result of the biotinylation process, because biotinylation of whole cell lysates followed by immunoblotting did not result in any change in the apparent molecular mass of the CrT proteins (data not shown). In addition, isolated rat erythrocytes that possess only a plasma membrane CrT exhibit bands identical to those found in membrane preparations here (data not shown). It is possible that the membrane-bound form of the CrT is glycosylated, as has been previously suggested (25). However, the question as to why the plasma membrane CrTs from rat and mouse differ warrants further study.

Feeding 3% creatine for 6 wk consistently resulted in an ~30% decrease in the content of the plasma CrT compared with controls. As this decrease was similar to that found in CrT activity, we may conclude that the regulation of creatine transport during creatine supplementation is due to a decrease in the number of active CrTs rather than any kinetic regulation, such as phosphorylation (25). We found no difference in the total CrT pool, which agrees with the understanding that creatine feeding has no effect on mitochondrial content or function.

Feeding beta -GPA led to a dramatic fivefold increase in the content of the plasma membrane CrT. This is surprising when we consider that creatine transport rates were increased at most twofold. One can speculate either that the CrT is, under these conditions, subject to kinetic inhibition or that some of the protein overexpressed might be dysfunctional, for example, due to aggregation. However, there was no evidence of this on immunoblots, and although we have clearly demonstrated that CrT activity and content are related, the exact relationship between CrT kinetics and CrT content is unclear.

There was no change in the mitochondrial isoform of the CrT after beta -GPA feeding. This is in agreement with the literature, in which no changes in mitochondrial activity or content (per mg protein) were shown in the heart after beta -GPA feeding (20, 24, 31). Thus, importantly, it appears clear from our data that the plasma membrane CrT and mitochondrial CrT do not appear to be subject to the same mechanism of regulation.

Regulation of intracellular creatine content. The total creatine content of a cardiomyocyte is dependent on the rates of creatine uptake, creatine retention, and creatine loss via creatinine. Production of creatinine is spontaneous and nonenzymatic and occurs at a rate of ~0.2 µmol · g wet wt-1 · day-1 (2% of the total pool). The calculated creatine uptake rate measured in this study in control hearts is equivalent to ~4.0 µmol · g wet wt-1 · day-1. Because intracellular creatine is normally constant, a steady state must be attained by the simultaneous transport of creatine into the cell and efflux of creatine from the cell. As the rate of creatine loss as creatinine is relatively low, creatine efflux from the cell must be by a second mechanism at a rate approaching that of creatine influx. The mechanism by which creatine can simultaneously be transported into and out of the cell is unclear. Dodd et al. (7) demonstrated that cells with a preloaded pool of radiolabeled creatine did not lose label to the medium unless creatine was provided extracellularly. They showed that, in the presence of extracellular creatine, creatine efflux was catalyzed at a rate equal to that of creatine influx, suggesting that the creatine concentration is maintained at a steady state under normal conditions by a simultaneous transport into the cell and efflux from the cell, both processes catalyzed by the CrT.

If creatine efflux is first order, an increase in the rate of creatine influx (by increasing the plasma creatine concentration, for example) will result in a corresponding increase in the rate of efflux. Under these conditions, a new steady state will be established whereby the intracellular creatine concentration is increased by an amount that is proportional to the increase in plasma creatine. The intracellular concentration of creatine would appear to be set by a combination of CrT activity and thermodynamic driving force into the cell. For a cell to maintain its original creatine concentration in the face of an increasing driving force, the net rate of creatine transport must be decreased. This can be explained by decreasing the total number or activity of CrTs in the plasma membrane, as is shown in this study. Alternatively, the rate of creatine efflux could increase through a selective change in the Km or Vmax of the efflux pathway.

The rate of creatine transport in the rat heart is such that the total creatine pool could be turned over in ~2 days. It is possible, therefore, that intracellular creatine might be regulated by changes in CrT content on a daily basis, which supports the theory that decreases in total creatine content seen in the failing myocardium (5, 19) are due to decreases in CrT content or activity at an unchanged plasma creatine concentration.

Previous work has suggested that creatine transport is Na+Cl- dependent, with a stoichiometry of either 1 or 2 Na+ (9, 16). By equating the free energies of the sodium, creatine, and chloride gradients, the maximum possible accumulation of creatine in the rat heart, if a stoichiometry of 2 Na+ is used, is ~1,000/1 (inside/outside). In vitro and in vivo, the actual accumulation ratio is at least an order of magnitude less than this, implying that the CrT is not near equilibrium and is therefore a potential site for the control of intracellular creatine content. If 1 Na+/creatine is used, then the transport process is at equilibrium and is unlikely to be a control site. This difference is important with regard to how CrT activity might be regulated. A transporter working at equilibrium could not be regulated solely by varying the amount of transporter protein; a transporter far from equilibrium, however, could be regulated either by changing the amount of the protein or by additional allosteric regulation.

The intracellular signals for regulation of the CrT are unclear. In addition, although we show here that there are significant changes in CrT content, this does not rule out the possibility of allosteric regulation by, e.g., phosphorylation. Because creatine supplementation leads to an increase in total creatine, whereas beta -GPA leads to a depletion of total creatine, either creatine or PCr may regulate the expression/turnover of the CrT or allosterically control its activity. Previous studies have suggested that it is intracellular creatine and not PCr that regulates the CrT (7, 13). These authors found that when cultured cells were incubated in high extracellular creatine concentrations, they displayed a marked reduction in creatine transport over time, the major pool of total creatine being creatine and not PCr. This was not related to a decrease in the affinity of the transporter for creatine, but by a two- to threefold loss in maximal transporter activity. Loike et al. (13) also showed that it was not extracellular creatine that was controlling, as incubating cells in a sodium-free/creatine solution had no effect on creatine transport. It appears that high extracellular creatine leads to an initial increase in intracellular creatine concentration, which over time may feed back to limit Cr uptake. The data presented here support this notion but cannot distinguish between creatine or PCr as regulatory molecules. The signals triggering changes in total CrT protein expression/activity are unclear. One possibility is the AMP-activated protein kinase (AMPK), which has been shown to be sensitive to the energetic status of the cell and whose activity has been linked to the regulation of expression of many proteins (21).

Conclusions. Using new techniques for measuring both the plasma membrane content of the CrT and its activity under physiological conditions, we show a coregulation of the plasma membrane CrT content and activity under conditions of creatine supplementation and depletion. A predominantly mitochondrial pool of CrT remains constant. This has important implications for studies in heart and other tissues that have reported changes in the total CrT content only (25). To further understand the biology of the CrT, future studies must endeavor to combine measurements of CrT activity and CrT isoform localization in healthy and diseased myocardium.


    ACKNOWLEDGEMENTS

beta -Guanidinopropionic acid was provided as a gift from Dr. Ralf Jäger, Degussa Health and Nutrition, Freiburg, Germany.


    FOOTNOTES

This study was supported by the British Heart Foundation.

Address for reprint requests and other correspondence: E. Boehm, Wellcome Trust Centre for Human Genetics, Univ. of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom (E-mail: ernie.boehm{at}bioch.ox.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00259.2002

Received 11 June 2002; accepted in final form 31 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 284(2):E399-E406
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