J Physiol 576.2 (2006) pp 391–401 391

Creatine as a compatible osmolyte in muscle cells exposedto hypertonic stress

Roberta R. Alfieri1, Mara A. Bonelli1, Andrea Cavazzoni1, Maurizio Brigotti4, Claudia Fumarola1,Piero Sestili5, Paola Mozzoni2, Giuseppe De Palma2, Antonio Mutti2, Domenica Carnicelli2,Federica Vacondio3, Claudia Silva3, Angelo F. Borghetti1, Kenneth P. Wheeler6 and Pier Giorgio Petronini1

Dipartimenti di 1Medicina Sperimentale, Sezione di Patologia Molecolare e Immunologia, 2Clinica Medica, Nefrologia e Scienze della Prevenzione,and 3Farmaceutico, Universita degli Studi di Parma, 43100 Parma, Italy4Dipartimento di Patologia Sperimentale, Universita degli Studi di Bologna, 40126 Bologna, Italy5Istituto di Farmacologia e Farmacognosia, Universita degli Studi di Urbino ‘Carlo Bo’, 61029 Urbino, Italy6 Department of Biochemistry, School of Life Sciences, JMS Building, University of Sussex, Brighton BN1 9QG, UK

Exposure of C2C12 muscle cells to hypertonic stress induced an increase in cell content ofcreatine transporter mRNA and of creatine transport activity, which peaked after about 24 hincubation at 0.45 osmol (kg H2O)−1. This induction of transport activity was prevented byaddition of either cycloheximide, to inhibit protein synthesis, or of actinomycin D, to inhibitRNA synthesis. Creatine uptake by these cells is largely Na+ dependent and kinetic analysisrevealed that its increase under hypertonic conditions resulted from an increase in V max of theNa+-dependent component, with no significant change in the K m value of about 75 µmol l−1.Quantitative real-time PCR revealed a more than threefold increase in the expression of creatinetransporter mRNA in cells exposed to hypertonicity. Creatine supplementation significantlyenhanced survival of C2C12 cells incubated under hypertonic conditions and its effect was similarto that obtained with the well known compatible osmolytes, betaine, taurine and myo-inositol.This effect seemed not to be linked to the energy status of the C2C12 cells because hypertonicincubation caused a decrease in their ATP content, with or without the addition of creatine at20 mmol l−1 to the medium. This induction of creatine transport activity by hypertonicity is notconfined to muscle cells: a similar induction was shown in porcine endothelial cells.

(Received 9 June 2006; accepted after revision 24 July 2006; first published online 27 July 2006)Corresponding author K. P. Wheeler: Department of Biochemistry, School of Life Sciences, JMS Building, Universityof Sussex, Brighton BN1 9QG, UK. Email: k.p.wheeler@sussex.ac.uk

Creatine plays a key role in buffering the concentrationof ATP in skeletal muscle cells, which contain 90% oftotal body creatine (Wyss & Kaddurah-Daouk, 2000).It is synthesized mainly in the liver and kidneys and istaken up by other tissues from the blood by a specifictransporter (CT1). This has been classified as a member ofthe (Na++Cl−)-dependent neurotransmitter transporterfamily (www.bioparadigms.org/slc/intro.asp) and showshigh homology with the betaine/γ -aminobutyric acid andtaurine transporters (Guimbal & Kilimann, 1993; Zorzanoet al. 2000; Speer et al. 2004). Creatine kinase catalyses thephosphorylation of cellular creatine and the creatine pool(creatine plus creatine phosphate) depends on the rate ofcreatine uptake from blood and on the rate of non-enzymicconversion to creatinine (Wyss & Kaddurah-Daouk, 2000).As only about 2% of the creatine pool per day is convertedinto creatinine, the most important process that controlsthe creatine pool is the uptake from the extracellularfluid (Murphy et al. 2003a). Normally a high creatine

concentration gradient is maintained across muscle cellmembranes, the cellular concentration of creatine pluscreatine phosphate being 500–1000 times higher thanthat in the plasma (Beis & Newsholme, 1975; Wyss &Kaddurah-Daouk, 2000).

Oral creatine supplementation is used widely by athletesto improve performance and muscle mass (Casey et al.1996; Volek & Rawson, 2004) and has been extendedto the medical field to treat a number of muscular,neurological and cardiovascular diseases such as gyrateatrophy (Sipila et al. 1981; Heinanen et al. 1999), McArdledisease (Vorgerd et al. 2000), Duchenne dystrophy (Felberet al. 2000; Tarnopolsky et al. 2004), myastenia gravis(Stout et al. 2001), amyotrophic lateral sclerosis (Mazziniet al. 2001) and Parkinson’s disease (Matthews et al.1999). The putative benefits of creatine supplementationgenerally have been attributed to an ergogenic effect: anincrease in the concentration of creatine phosphate actingas an ATP buffer. Recently, however, other possibilities

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392 R. F. Alfieri and others J Physiol 576.2

have been suggested. For example, creatine showsantioxidant properties (Lawler et al. 2002) andhad protective effects against oxidative stress incultured mammalian cells (Lenz et al. 2005; Sestiliet al. 2006). Creatine has also been shown to haveanti-inflammatory activity in endothelial cells (Nomuraet al. 2003) and creatine supplementation improvedneuronal differentiation and dopaminergic cell survivalunder adverse conditions (Andres et al. 2005). Now wereport that supplementation of growth medium withcreatine can enable cultured muscle cells to surviveexposure to hypertonicity.

Several types of mammalian cells can survive whenexposed to a hypertonic environment (up to about500 mosmol (kg H2O)−1) because of a specific adaptationprocess that eventually results in their accumulatingcompatible osmolytes (Burg, 1995; Burg et al. 1997).This adaptation involves changes of gene expression thatresult in an increased synthesis either of a compatibleosmolyte itself (e.g. sorbitol) or of transporters for theosmolytes, such as SNAT-2 for neutral amino acids (Alfieriet al. 2001, 2005), BGT1 for betaine (Petronini et al.2000), SMIT for myo-inositol and TAUT for taurine (Burget al. 1997). The usual explanation of this phenomenonis the need to replace the early cellular accumulation ofinorganic ions with small organic molecules (compatibleosmolytes) that do not perturb macromolecular structuresas the increased concentrations of inorganic ions do. Theseresponses were extensively characterized in kidney-derivedcells (Beck et al. 1998; Beck & Neuhofer, 2005) buthave also been detected in chondrocytes (De Angeliset al. 1999), macrophages (Denkert et al. 1998), endo-thelial cells (Petronini et al. 2000; Alfieri et al. 2002,2004) and mesothelial cells (Matsuoka et al. 1999).Relatively few studies of muscle cells have been reported,but hypertonicity was shown to induce amino acidtransport system A in vascular smooth muscle cells(Chen & Kempson, 1995), whilst hypotonicity activatedtaurine efflux from C2C12 myoblasts, in keeping withtaurine’s role as an organic osmolyte (Manolopoulos et al.1997). Hypertonicity also caused a stimulation of NKCC(Na+–K+–2Cl− cotransporter) activity in rat L6 skeletalmuscle cells (Zhao et al. 2004). Here we show not onlythat hypertonicity induces creatine transport in C2C12cells but also that creatine can act like the well-establishedcompatible osmolytes betaine, taurine and myo-inositol inprotecting the cells against hypertonic stress.

Methods

Materials

[14C]Creatine (55 Ci mol−1) was obtained from AmericanRadiolabeled Chemicals Inc. (Saint Louis, MO, USA), andl-[4,5-3H]leucine from Amersham plc (Little Chalfont,

UK). Media, fetal calf serum and antibiotics for culturingthe cells were purchased from Gibco (Grand Island, NY,USA). Disposable plastics were obtained from Costar(Broadway, Cambridge, MA, USA), and other reagentsfrom Sigma Chemical Co. (St Louis, MO, USA).

Cell Culture

C2C12 mouse myoblasts from the Istituto ZooprofilatticoSperimentale (Brescia, Italy) were grown in Dulbecco’smodified Eagle’s medium (DMEM) supplemented withglutamine (2 mmol l−1), fetal calf serum (10%), penicillin(100 U ml−1) and streptomycin (100 µg ml−1). Endo-thelial cells were obtained and cultured as previouslydescribed (Petronini et al. 2000). All cultures were keptin an incubator at 37◦C in a water-saturated atmosphereof 5% CO2 in air.

Experimental growth media

The media consisted of DMEM supplemented with bovineserum albumin (0.1%, w/v), betaine (0.1 mmol l−1) andmyo-inositol (0.1 mmol l−1) (Alfieri et al. 2002). Whenrequired, media were made hypertonic by addition ofsucrose. The osmolalities of the media were checkedwith a vapour-pressure osmometer (Wescor) and normalmedium was about 0.3 osmol (kg H2O)−1.

Cell viability

Cell density was assayed in terms of the protein contentper dish or cell number, determined by cell counting(Alfieri et al. 2002). To test cell viability under hypertonicconditions, cells were first seeded at a density of about104 cells cm−2 and cultured for 2 days in normal isotonicmedium before it was replaced with the appropriate hyper-tonic media.

Cell protein

Cell protein, precipitated by trichloroacetic acid, wasdissolved in 0.2 n NaOH and its concentration measuredby a dye-fixation method (Bio-Rad, Hercules, CA, USA)with bovine serum albumin as standard (Bradford, 1976).

Rate of protein synthesis

The rate of protein synthesis was measured as the rateof incorporation of labelled leucine (2.5 mCi mmol−1,2 µCi ml−1) during a 30 min incubation of the cellmonolayers. This procedure has been described in detailelsewhere (Petronini et al. 2000).

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J Physiol 576.2 Creatine as a compatible osmolyte 393

Transport measurements

Approximate initial rates of uptake of creatine by C2C12cells were measured after the latter had been incubatedin control (0.3 osmol (kg H2O)−1) or test (0.480 osmol(kg H2O)−1) medium for the desired time. The cell mono-layers were quickly washed with Earle’s balanced saltsolution containing 0.1% glucose and then incubated inthis solution for 15 min at 37◦C to diminish the cellularpool of osmolytes. The cells were washed again andimmediately incubated at 37◦C for the desired time in thepresence of 14C-labelled creatine (usually 2 µCi µmol−1).The incubations were stopped by removal of the mediumand the cells were quickly washed three times with freshcold medium. Trichloroacetic acid (5%, w/v) was added todenature the cells and the radioactivity in samples of theacid extracts was measured by scintillation counting.

The accumulation of creatine by the cells was monitoredwith the use of [14C]creatine added to the incubationmedia. The incubations were stopped after the desired timeby removal of the medium and the cell monolayers quicklywashed three times with Earle’s balanced salt solution(containing 0.1% glucose) before they were denatured andassayed as just described.

Cellular ATP content

This was determined by a luminescence assay system(ATPLiteTM-M, Packard) as previously described(Fumarola et al. 2005).

Cellular contents of creatine and creatine phosphate

These were measured by HPLC analysis of cell extracts,obtained as follows The incubation medium was rapidlyremoved and ice-cold perchloric acid (3.8%) immediatelyadded to the cell monolayer. After 30 min on ice,the acid extract was removed and neutralized withsaturated Na2CO3 solution. The HPLC system consistedof two Gilson 305 solvent delivery pumps (GilsonInc., Middleton, WI, USA), a 20 µl capacity sampleinjector (Rheodyne LLC, Rohnert Park, CA, USA) and aGilson UV 115 detector (Gilson Inc.). Chromatographicseparation was achieved at room temperature on aAlltima C-18 column (5 µm, 250 × 4.6 mm, AlltechInc., Columbia, MD, USA). The mobile phase consistedof 70 mmol l−1 phosphate buffer (pH 6.0) containing7 mmol l−1 tetrabutylammonium hydrogen sulphate.The flow rate was 0.75 ml min−1 and the eluant wasmonitored at 218 nm. Standard curves for creatine,creatine phosphate and creatinine, constructed by plottingpeak area against solute concentration, were linear over therange 5–500 µmol l−1.

RNA isolation, cDNA synthesis and quantitativeRT-PCR

Total RNA was extracted from about 2 × 106 cells bythe method of Chomczynski & Sacchi (1987) with theuse of TRIzol (Gibco BRL, Gaithersburg, MD, USA)and digested with DNAseI (DNA-free kit, Ambion Inc.,Austin, TX, USA) to remove any contaminating genomicDNA. RNA quality was checked by electrophoresis on1% TBE agarose gel, using a denaturing loading buffer(RNA Ladder, New England Biolabs Inc., Beverly, MA,USA). The concentration of RNA was measured with theRiboGreen probe on a fluorescence spectrophotometerCary Eclipse (Varian Inc., Palo Alto, CA, USA). cDNAwas synthesized with the use of 500 ng of RNA, 250 ng ofrandom hexamer primers, 200 U of SuperScript II reversetranscriptase (Invitrogen, Carlsbad, CA, USA) and 1 U ofSUPERase IN (Ambion Inc., Austin, TX, USA), followingInvitrogen’s recommended experimental conditions.

Gene expression was assessed by quantitative realtime PCR with the use of an iCycler iQ MulticolorReal-Time PCR detection system (Bio-Rad, Hercules,CA, USA). Reaction mixtures contained 2 µl of templatecDNA with 300 nm of forward and reverse primers and12.5 µl of 2xiQ SybrGreen Supermix (Bio-Rad, Hercules,CA, USA) in a total volume of 25 µl. Triplicate assayswere run for each sample and each included a standardcurve and a negative control. Specific oligonucleotideprimers were designed with the Probe Finder 2.04programme (Roche Applied Science, F. Hoffmann-LaRoche Ltd, Basel, Switzerland) to span the exon–exonjunctions of the Mus musculus genes encoding thecreatine transporter (GenBank sequence AB077327.1;sense 5′-CTCTCCATGGTGACTGATGGT-3′; antisense5′-TGCCACTAGCTGAGTAGTAGTCAAA-3′) andβ-actin (GenBank sequence NM˙007393.1; sense5′-TGACAGGATGCAGAAGGAGA-3′; antisense 5′-CGC-TCAGGAGGAGCAATG-3′) to produce 67 and 75 bpproducts, respectively. The amplification protocolconsisted of 3 min at 95◦C followed by 40 cycles at 95◦Cfor 30 s, 61◦C for 30 s and 72◦C for 30 s. Then a finalmelting step with a gradual increase in temperaturefrom 50◦C to 94◦C was used to ensure there wereno non-specific products. The relative quantitativeexpression of the creatine transporter, after normalizationwith β-actin as housekeeping gene (Murphy et al. 2003b),was calculated as suggested by Pfaffl (2001).

Translation of endogenous mRNA by rabbitreticulocyte lysate

Rabbit reticulocyte lysate was prepared as describedby Allen & Schweet (1962). Translation of endo-genous mRNA in vitro by the unfractionated rabbitreticulocyte lysate was performed in reaction mixtures

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394 R. F. Alfieri and others J Physiol 576.2

(125 µl) containing 30 mmol l−1 Hepes-KOH, pH 7.5,80 mmol l−1 KCl, 1.8 mmol l−1 magnesium acetate,50 µmol l−1 of each amino acid except leucine,2 mmol l−1 ATP, 0.25 mmol l−1 GTP, 0.5 mmol l−1

dithiothreitol, 0.4 mmol l−1 spermidine, 0.24 µmol l−1

(2 µCi) [3H]leucine and 50 µl of lysate. Where indicated,the osmolarity of the reaction mixture was increased byaddition of KCl, creatine or betaine. The complete mixturewas incubated for 5 min at 28◦C and then a 62.5 µl samplewas taken and added to 1 ml of 0.1 m KOH, the solutionwas decolourized with two drops of 35% (w/v) H2O2

and 1 ml of 20% (w/v) trichloroacetic acid added. Theprecipitate was collected on a Whatman GF/C filter andits radioactivity measured by scintillation counting. Theosmolality of the residual reaction mixture was measuredwith a vapour pressure osmometer (Wescor), the standardmixture being 0.373 osmol (kg H2O)−1. Under thesestandard conditions the [3H]leucine incorporated was67368 ± 4883 dpm (mean ± s.d., n = 12).

Statistical analysis

Unless noted otherwise, the results are expressed as meanvalues ± s.d. for the indicated number of measurements.The significance of differences between the mean valuesrecorded for different experimental conditions wascalculated by Student’s t test and P-values are indicatedwhere appropriate in the figures and their legends.Curves were fitted to experimental values with the use of‘Kaleidagraph’ (Synergy Software, Reading, PA, USA).

Figure 1. Effect of hypertonicity oncreatine uptake by C2C12 cellsCells were seeded and cultured for 48 h inisotonic (0.3 osmol (kg H2O)−1) medium.A, samples were incubated in isotonicmedium or hypertonic medium(0.36–0.50 osmol (kg H2O)−1) for 16 hbefore the initial rate of creatine uptake bythe cells was measured from a solutioncontaining 0.1 mmol l−1 creatine. B, sampleswere incubated in isotonic medium orhypertonic medium (0.48 osmol (kg H2O)−1)and creatine influx was measured, asdescribed above, at the indicated time points.Mean values (± S.D.) of three measurementsare given. ❡, isotonic; •, hypertonic.

Results

Hypertonic stimulation of creatine uptakeby C2C12 cells

Figure 1A shows the rate of uptake of creatine by C2C12cells that had been incubated for 16 h in media withosmolalities ranging from 0.3 to 0.5 osmol (kg H2O)−1. Therate clearly increased in parallel with the imposed hyper-tonicity, reaching a peak at about 0.45 osmol (kg H2O)−1.Figure 1B shows that although an increase in the rate ofcreatine influx was detectable only 4 h after exposing thecells to hypertonic medium, the rate continued to increasefor several hours and the maximum value was reachedmuch later, after about 24 h.

Effect of hypertonicity on the kinetic parametersof creatine influx

Approximate initial rates of uptake of creatine byC2C12 cells were measured from a range of creatineconcentrations, and in the presence and absence ofNa+, after the cells had been exposed for 24 hto isotonic (0.3 osmol (kg H2O)−1) or hypertonic(0.48 osmol (kg H2O)−1) conditions. After subtractionof the linear Na+-independent components of influx,the remaining saturable, Na+-dependent, componentscould be expressed as Michaelis-Menten equations(Fig. 2). The lines shown, from the ‘Kaleidagraph’curve-fitting program, are for V max values of 4.5 ± 0.4and 7.1 ± 0.6 nmol (15 min)−1 (mg protein)−1 with K m

values of 75 ± 22 and 77 ± 19 µmol l−1 for cells exposed to

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J Physiol 576.2 Creatine as a compatible osmolyte 395

Figure 2. Effect of hypertonicity on the kinetics of creatineuptakeC2C12 cells were incubated for 24 h in either isotonic (0.3 osmol(kg H2O)−1) medium (controls) or hypertonic (0.48 osmol (kg H2O)−1)medium (test cells) before their initial rate of creatine uptake wasmeasured in the presence and absence of Na+, with creatineconcentrations ranging from 0.01 to 0.5 mmol l−1. Mean values(± S.E.M.) for the Na+-dependent influx are given for 4 independentduplicate measurements. The curves, drawn with the use of‘Kaleidagraph’ (Synergy Software, Reading, PA, USA), fit‘Michaelis-Menton’ equations with kinetic parametersKm = 75 ± 22 µmol l−1, Vmax = 4.5 ± 0.4 nmol (15 min)−1

(mg protein)−1 for control cells ( ❡) and Km = 77 ± 19 µmol l−1,Vmax = 7.1 ± 0.6 nmol (15 min)−1 (mg protein)−1 for test cells (•).

Figure 3. Effects of actinomycin D and cycloheximide oncreatine uptakeCells were seeded and cultured for 48 h in isotonic (0.30 osmol(kg H2O)−1) medium and then incubated for 16 h in isotonic mediumor hypertonic medium (0.48 osmol (kg H2O)−1) with or without theaddition of 0.8 µmol l−1 actinomycin D (Act D) or 35 µmol l−1

cycloheximide (CHX). Then creatine influx was measured fromsolutions containing 0.1 mmol l−1 creatine. Mean values (± S.D.) ofthree measurements are given. Open bars, isotonic condition; filledbars, hypertonic conditions.

isotonic and hypertonic conditions, respectively. Thus theincrease in the rate of uptake was caused by an increasein V max, with no significant change in the K m value,which was in the micromolar range, as reported in theliterature for Na+-dependent creatine transport (Guimbal& Kilimann, 1993; Tran et al. 2000; Peral et al. 2002).

Taken together, the results in Figs 1 and 2 show thathypertonic incubation of C2C12 cells stimulates theiruptake of creatine via a Na+-dependent mechanism thatinvolves an increase in the capacity of the transporter.

Dependence on RNA and protein synthesis

Since this last conclusion is consistent with a mechanisminvolving an increase in the number of activetransporters in the cell membrane, the dependenceof the hypertonicity-induced changes in creatinetransport on RNA and protein synthesis was examined. Asshown in Fig. 3, induction of creatine transport activitywas prevented by the addition of either actinomycinD (0.8 µmol l−1) or cycloheximide (35 µmol l−1) to thehypertonic culture medium, indicating that it requiresboth RNA and protein synthesis. In keeping with thisfinding, quantitative real-time PCR revealed a more thanthreefold increase in the expression of creatine transportermRNA in cells exposed to hypertonicity (Fig. 4).

Figure 4. Expression of CT1 mRNA in C2C12 cells exposed tohypertonicityCells were seeded and cultured for 48 h in isotonic (0.3 osmol(kg H2O)−1) medium and then samples were incubated in isotonic orin hypertonic medium (0.48 osmol (kg H2O)−1) for 16 h and 24 h. Theamounts of CT1 mRNA and β-actin mRNA in cell extracts were thenmeasured with the use of ‘Real Time RT-PCR’. Values of the expressionof CT mRNA were normalized to the corresponding values for β-actinmRNA and the results for the cells exposed to hypertonicity are givenrelative to those from the control (isotonic) cells. Mean values (± S.D.)from three experiments are given. Open bar, isotonic; filled bar,hypertonic.

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396 R. F. Alfieri and others J Physiol 576.2

Figure 5. Effect of creatine on cell survival under hypertonic conditionsCells were seeded and cultured for 48 h in isotonic (0.3 osmol (kg H2O)−1) medium. A, samples were incubatedfor 24 h in hypertonic media (0.44–0.56 osmol (kg H2O)−1) in the presence or absence of 0.1 mmol l−1 creatinebefore cell survival was estimated by cell counting. Mean values (± S.D.) from three measurements are given.Open bars, without added creatine; filled bars, with added creatine. B, samples were incubated for 16 h in isotonicmedium in the presence of creatine at the indicated concentrations before being transferred to hypertonic medium(0.53 osmol (kg H2O)−1) containing the same creatine concentrations and incubated for a further 24 h. Cell survivalwas then estimated by cell counting. Mean values (± S.D.) from three measurements are given. Open bar, isotonicconditions; filled bars, hypertonic conditions.

Creatine as a compatible osmolyte

To see if creatine could behave as a compatible osmolyte,cell survival was monitored after the C2C12 cells had

Figure 6. Similar effects of creatine and established compatibleosmolytesC2C12 muscle cells were seeded and cultured for 48 h in isotonic(0.3 osmol (kg H2O)−1) medium and then incubated in hypertonicmedium (0.53 osmol (kg H2O)−1), with or without the addition of20 mmol l−1 betaine (Be), myo-inositol (In), taurine (Ta) or creatine(Cr). Culture was continued for 24 h and cell growth was estimated bycell counting. Mean values (± S.D.) from three measurements aregiven. Open bar, isotonic conditions; filled bars, hypertonic conditions.

been incubated in the presence or absence of creatine(0.1 mmol l−1), first in isotonic (0.3 osmol (kg H2O)−1)medium for 16 h, and then for 24 h in media rangingin osmolality from 0.30 to 0.56 osmol (kg H2O)−1. Asshown in Fig. 5A, cell survival in the absence of creatinedecreased with exposure to hypertonicity, with a drasticloss at osmolalities above about 0.55 osmol (kg H2O)−1.The addition of the creatine, however, largely prevented theloss of cells, changing a survival of only 28 ± 3% to oneof 68 ± 9% with cells exposed to 0.56 osmol (kg H2O)−1.In a similar experiment, cell survival was checked after24 h exposure to 0.55 osmol (kg H2O)−1 in the presenceof added creatine at concentrations ranging from 0.1 to20 mmol l−1, again after a preliminary 16 h incubation inisotonic medium with the same creatine concentrations(Fig. 5B). It is clear that cell survival increased as theconcentration of added creatine increased. Significantprotection of the cells occurred only after they hadbeen incubated for at least 4 h in creatine-supplementedisotonic medium before exposure to hypertonicity,suggesting that accumulation of creatine by the cells isimportant (results not shown). Finally, Fig. 6 shows thatcell survival in hypertonic medium supplemented withcreatine was comparable to that observed in the presenceof the well known compatible osmolytes betaine, taurineand myo-inositol (Petronini et al. 2000; Alfieri et al. 2002).

All the above observations show that creatine can behaveas a compatible osmolyte in C2C12 cells, a conclusionsupported by the following findings which parallel those

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J Physiol 576.2 Creatine as a compatible osmolyte 397

Figure 7. Effect of creatine on the rate of protein synthesisunder hypertonic conditionsThe rate of protein synthesis was measured as the incorporation ofradio-labelled L-leucine into C2C12 cell proteins during a 30-min pulseafter the cells had been incubated for the indicated times inhypertonic (0.48 osmol (kg H2O)−1) medium in the absence orpresence of creatine (20 mmol l−1). Mean values (± S.D.) from fourmeasurements are given. Open bars, without added creatine; filledbars, with added creatine.

reported previously for other cells and other compatibleosmolytes. First, the partial inhibition of total cell proteinsynthesis caused by hypertonicity was alleviated and morequickly reversed in the presence of creatine (Fig. 7).

Figure 8. Effects of increased osmolarity, produced by differentsolutes, on the translation of globin mRNA by rabbitreticulocyte lysateTranslation was measured as the rate of incorporation of radio-labelledleucine into proteins, as described in Methods, with one of KCl,betaine or creatine added to give the indicated osmolalities. Theresults are expressed relative to the value obtained with the standardreaction mixture (0.37 osmol (kg H2O) −1).

Second, cell-free protein synthesis in the unfractionatedrabbit reticulocyte lysate system was not affected whencreatine was added to increase the medium’s osmolarity, incontrast to the marked inhibition caused by the addition ofKCl (Fig. 8). For this experiment the original compositionof the translating mixture (Brigotti et al. 2003) wasslightly modified by raising the ATP concentration to2 mmol l−1 and by omitting the ATP-regenerating system(composed by creatine kinase and a small amount ofcreatine phosphate). Under these conditions the proteinsynthesis efficiency was similar to that of the originalsystem. The limited solubility of creatine prevented themeasurement of effects above 0.45 osmol (kg H2O)−1.

Cell content of creatine, phosphocreatine and ATP

The accumulation of [14C]creatine in C2C12 cellsamounted to 488 ± 30 nmol (mg protein)−1 after 16 hin isotonic (0.31 osmol (kg H2O)−1) medium containing20 mmol l−1 creatine, and increased to 836 ± 128 nmol(mg protein)−1 after a subsequent 24 h in hyper-tonic (0.53 osmol (kg H2O)−1) medium. To distinguishbetween creatine and phosphocreatine, this experimentwas repeated with unlabelled creatine and the cell contentswere analysed with the use of HPLC. This revealed thatalthough the cell content of both creatine and creatinephosphate increased after the 16 h isotonic incubation,only the amount of creatine increased further after thesubsequent hypertonic treatment (Fig. 9). These increases

Figure 9. Cellular accumulation of creatine and phosphocreatineCells were seeded and cultured for 48 h in isotonic (0.3 osmol(kg H2O)−1) medium and then samples incubated for 16 h in isotonicmedium containing 20 mmol l−1 creatine. Some of these cells werethen analysed for creatine and phosphocreatine whilst other sampleswere transferred to hypertonic medium (0.53 osmol (kg H2O)−1), alsocontaining 20 mmol l−1 creatine, and incubated for a further 24 h.Cell content of creatine and phosphocreatine was determined withthe use of HPLC as described in Methods. Mean values (± S.D.) fromthree measurements are given. Open bars, isotonic; light grey bars,isotonic plus creatine; cross-hatched bars, hypertonic plus creatine.

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398 R. F. Alfieri and others J Physiol 576.2

in content of creatine and creatine phosphate, however,were not accompanied by any increase in the amount ofATP in the C2C12 cells, under either isotonic or hyper-tonic conditions. On the contrary, hypertonic incubation,with or without added creatine, caused a marked decrease(about 40%) in cellular ATP (Fig. 10).

Lack of cell specificity

The stimulation of creatine uptake by hypertonicity is notrestricted to C2C12 cells: parallel induction of creatinetransport activity (not shown) and creatine accumulation(Fig. 11) occur in the porcine endothelial cells that we haveused extensively for previous studies of responses to hyper-tonicity (Petronini et al. 2000; Alfieri et al. 2001, 2002,2004).

Discussion

The results described above showing the increased uptakeof creatine by C2C12 cells exposed to hypertonicity, andthe ability of creatine to afford protection against thehypertonicity by acting as a compatible osmolyte, areremarkably similar to those obtained previously in studiesof other cells with established compatible osmolytes suchas betaine, taurine and myo-inositol (see Alfieri et al.2002 for references). Although neither the percentageincrease in transport activity (Figs 1 and 2), nor the

Figure 10. Intracellular ATP contentC2C12 cells were seeded and cultured in isotonic (0.3 osmol(kg H2O)−1) medium for 24 h and then incubated for 16 h in isotonicmedium in the presence or absence of creatine (20 mmol l−1). Sampleswere taken for the measurement of ATP content and the rest of thecells transferred to hypertonic medium (0.53 osmol (kg H2O)−1), againwith or without the addition of creatine (20 mmol l−1) for a further5 h incubation, after which cell ATP content was again measured.Mean values (± S.D.) from four independent experiments are given.Open bars, without added creatine; filled bars, with added creatine.

protection against apoptosis consistently afforded by aphysiological concentration of creatine (Fig. 5) foundhere is as high as previously reported for porcine endo-thelial cells with betaine, etc. (Alfieri et al. 2002), theoverall picture is the same. Both the time course ofincrease in transporter activity in response to hyper-tonicity (Fig. 1B) and the increase in amount of creatinetransporter mRNA (Fig. 4) parallel those observed in othercells with other osmolytes. The finding that creatine uptakeby porcine endothelial cells is similarly stimulated byhypertonicity (Fig. 11) also indicates that creatine mayperhaps be added to the list of established compatibleosmolytes. Assuming that the C2C12 cells contain 6–8 µlwater (mg protein)−1, the value of about 840 nmol(mg protein)−1 for the cell content of creatine plusphosphocreatine, after 24 h hypertonic incubation in thepresence of 20 mmol l−1 creatine, is equivalent to a cellularconcentration of about 120 mmol l−1. This is enoughto have compensated for about 50% of the imposed0.22 osmol (kg H2O)−1 increase in osmolality. Othercompatible osmolytes, and accumulated amino acids,presumably provided the remaining necessary solutes.Our values for the relative increase in concentrations ofcreatine and phosphocreatine in C2C12 cells incubatedwith creatine (Fig. 9 and text) are in accordance with thosereported by Ceddia & Sweeney (2004) for L6 rat skeletalmuscle cells.

The finding that the ATP concentration in C2C12cells was not affected by changes in the concentrationsof creatine and phosphocreatine (Fig. 10) parallel the

Figure 11. Hypertonic stimulation of creatine uptake byendothelial cellsEndothelial cells were incubated in isotonic (0.3 osmol (kg H2O)−1) orhypertonic medium (0.5 osmol (kg H2O)−1) containing 0.05 mmol l−1

radiolabelled creatine and the cellular content of creatine wasmeasured at the indicated times. Mean values (± S.D.) from threemeasurements are given. ❡, isotonic; •, hypertonic.

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J Physiol 576.2 Creatine as a compatible osmolyte 399

observations of Nomura et al. (2003) with endothelialcells, as well those of Ceddia & Sweeney (2004) with L6 ratskeletal muscle cells. The decrease in ATP concentrationunder hypertonic conditions (Fig. 10) could result froman increased ATP consumption due to the stimulation ofthe Na+,K+-ATPase during the first hours of hypertonictreatment (Ferrer-Martinez et al. 1996).

Although it seems clear that muscles cells, like endo-thelial cells, are not normally subjected to such drastichypertonic conditions as those used here, physiologicallysignificant changes in plasma osmolality can occur incertain circumstances. For example, the increase inblood glucose concentration caused by diabetes canincrease plasma osmolality to 0.33–0.38 osmol (kg H2O)−1

(Powers, 2005), within the range that gave a detectableeffect with C2C12 muscle cells (Fig. 1A). Similarly, a 4%increase in plasma osmolality was reported to accompanyan increase in plasma taurine content in human subjectsdoing vigorous exercise with no fluid intake (Cuisinieret al. 2002). Such a relatively small change in osmolality,although physiologically important, would not producesignificant effects in the kind of experiments describedhere, so it is not clear if the accumulation of compatibleosmolytes would become important for cell survivalunder such conditions. Similarly, it is not clear if theaccumulation of creatine in muscle tissue that occurs whencreatine is taken as a food supplement could be beneficialsimply as a compatible osmolyte under conditions suchas those described by Cuisinier et al. (2002). On theother hand, the apparent safety of the consumption ofrelatively high concentrations of compounds such ascreatine and betaine, in the form of dietary supplementsmight be explained by their being compatible osmolytes.This property may be relevant to the recent finding,in a study of a mouse model for stroke, that dietarycreatine supplementation improved cerebral blood flowand had a clear neuro-protective effect, with no detectablechange in the energy status of brain tissue (Prass et al.2006).

Another possibility is that these responses to hyper-tonicity by osmolyte transporters in non-renal tissuesare relics from earlier requirements. The accumulationof compatible osmolytes might well have been a veryearly property of cells that enabled them to surviveunder temporary hypertonic conditions. This requirementcould subsequently have become redundant in cells incontrolled tissue environments in multicelluar organisms,and these osmolytes gradually used for other morespecific purposes, such as neurotransmitters (glycine andGABA), components of second messengers (inositol) andmetabolic intermediates (betaine, creatine). If this is thecase, it seems possible that other compounds, such as thecatecholamines and l-carnitine, might fall into the samecategory.

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Acknowledgements

This investigation was supported by Universita degli Studi diParma and by MIUR (Ministero della Istruzione, della Universitae della Ricerca), Rome, Italy and COFIN 2004 (PS).

C© 2006 The Authors. Journal compilation C© 2006 The Physiological Society