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Experimental Physiology

The changing AMPK expression profile in differentiatingmouse skeletal muscle myoblast cells helps conferincreasing resistance to apoptosis

Carola U. Niesler, Katherine H. Myburgh and Frances Moore

Department of Physiological Sciences, University of Stellenbosch, Private Bag X1, Stellenbosch 7602, South Africa

AMP-activated protein kinase (AMPK) functions as a α/β/γ heterotrimer to preserve ATPlevels and so cell viability during stressful conditions. However, its role in aiding survival ofadult skeletal muscle precursor cells is unclear. Using the differentiating mouse C2C12 postnatalskeletal muscle myoblast cell line, we have determined that proteins for the AMPK subunitisoformsα2 andγ2 are constitutively expressed, while those forα1,β1 andβ2 are undetectablein undifferentiated myoblasts but increasingly expressed with differentiation to myotubes.Although the γ3 subunit is expressed at a low level in myoblasts, it too is expressed increasinglywith differentiation to myotubes. The p50 but not the p72 isoform of the embryonic α subunithomologue MELK is expressed only in proliferating myoblasts, while the ARK5 α subunithomologue is increasingly expressed with differentiation. Myotubes displayed higher basal andstimulated α1/α2 AMPK activation than myoblasts. Furthermore, serum starvation resulted inless apoptosis of differentiated myotubes than of undifferentiated myoblasts. This reflects, in part,the increased expression of functional AMPK in the myotubes, since specific inhibition of AMPKactivity with 6-[4-(2-piperidin-1-ylethoxy)-phenyl]-3-pyridin-4-ylpyrazolo[1,5-α] pyrimidine(Compound C) exacerbated the apoptosis resulting from serum withdrawal. If these in vitroevents can also occur in vivo, they could have implications for pathologies such as musclewasting, in which undifferentiated satellite stem cells may be easier apoptotic targets than theirdifferentiated counterparts. Furthermore, these results suggest that when interpreting resultsfrom in vitro or in vivo experiments on AMPK, the subunit expression profile should be takeninto account.

(Received 20 June 2006; accepted after revision 29 August 2006; first published online 31 August 2006)Corresponding author F. Moore: Hormone and Metabolic Research Unit, Christian de Duve Institute of CellularPathology, University of Louvain Medical School, ICP-UCL 7529, Avenue Hippocrate 75, B-1200 Brussels, Belgium.Email: frances.moore@horm.ucl.ac.be

Satellite cells are mitotically quiescent mononucleatedadult skeletal muscle progenitor cells that lie between thebasal lamina and basement membrane of each skeletalmuscle fibre. Once activated into myoblasts, they facilitatemuscle growth and repair by proliferating and terminallydifferentiating into postmitotic fused multinucleatedmyotubes (Hughes & Blau, 1990; Morgan & Partridge,2003). In vivo, numerous stimuli, e.g. stretch, muscledamage and exercise, are capable of activating quiescentsatellite cells (Darr & Schultz, 1987; Crameri et al. 2004;Kadi et al. 2004).

AMP-activated protein kinase (AMPK) is a highlyconserved cellular serine/threonine protein kinase (Hardieet al. 1998) that functions heterotrimerically with a

catalytic α subunit and two regulatory subunits, β andγ . Multiple isoforms of the subunits exist, namely α1 andα2, β1 and β2, and γ 1, γ 2 and γ 3 (Beri et al. 1994; Gaoet al. 1996; Stapleton et al. 1996, 1997). Subunit isoformsthat make up a functional α/β/γ AMPK heterotrimerare variously expressed in different tissues and locations(Stapleton et al. 1996; Salt et al. 1998). For example,the γ 3-subunit is mainly expressed in skeletal muscle(Cheung et al. 2000), and mutations in this isoformhave been linked to skeletal muscle glycogen storagedisease (Milan et al. 2000). AMPK acutely regulates energyhomeostasis by phosphorylating key enzymes, therebyaltering their activities to preserve ATP and increaseATP resynthesis (Moore et al. 1991; Hardie et al. 1998).

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society DOI: 10.1113/expphysiol.2006.034736

208 C. U. Niesler and others Exp Physiol 92.1 pp 207–217

It also phosphorylates transcriptional factors to adjustgene expression under conditions of chronic stress (Leff,2003). Mammalian AMPK can be activated during timeswhen the cellular AMP:ATP ratio is elevated owing tostresses such as substrate deprivation for ATP synthesis,trophic signal deprivation for cell survival, or excessiveATP consumption (Moore et al. 1991; Hardie et al. 1998;Barnes et al. 2001; Winder, 2001; Leff, 2003; Rutteret al. 2003; Myburgh, 2004). AMP allosterically activatesAMPK, which then allows further covalent activation byupstream AMPK kinases (AMPKK) that phosphorylatethreonine 172 on the α subunit (Moore et al. 1991;Lizcano et al. 2004). Recently, three homologues of theAMPK catalytic α subunit have been described. They arematernal embryonic leucine zipper kinase (MELK; Heyeret al. 1997), Snf1/AMP kinase-related kinase (SNARK;Suzuki et al. 2003c) and AMPK-related kinase 5 (ARK5;Suzuki et al. 2003b). Each homologue can phosphorylatethe consensus SAMS (HMRSAMSGLHLVKRR) peptideAMPK phosphorylation substrate in vitro, and theirexpression is correlated with suppression of tumour cellapoptosis (Suzuki et al. 2003a, 2004). Under conditions ofacute stress, the canonical α1/α2 AMPK can also protectnon-tumour cells by inhibiting apoptosis (Stefanelli et al.1998; Blazquez et al. 2001). Chronically activated AMPKhas been shown either to act protectively (Russell et al.2004) or to target cells towards apoptosis (Meisse et al.2002; Campas et al. 2004).

Whether a normal cell commits to death ordifferentiation depends on a complex set of input signalsthat interpret the balance between positive survival signalsand negative pro-apoptotic signals (Bellamy et al. 1995;Allen et al. 1998; Kiess & Gallagher, 1998). Serumcontains numerous anti-apoptotic and growth-promotingfactors, including insulin, necessary for cell survival (Kiess& Gallagher, 1998). In many cases, protein kinase B(Akt/PKB) acts as a key mediator for these growth factorsin survival, in addition to their proliferative and metaboliceffects, by phosphorylating a number of cellular substrates.Those that inhibit apoptotic cell death include theForkhead transcription factors, the Bcl-2 family memberBad and the AMPK α subunit homologue ARK5 (Dudecket al. 1997; Parrizas et al. 1997; Chan et al. 1999; Senet al. 2003; Suzuki et al. 2003a). ARK5, in turn, hasbeen shown to inhibit apoptosis by phosphorylating fas-associated death domain-like interleukin-1β-convertingenzyme-inhibitory protein (FLIP), caspase-8 and -6 inglucose-deprived tumour cells (Suzuki et al. 2003a,b,2004). Recent work has shown that removal of insulin(in serum) can allow activation of AMPK by loweringphosphorylation at two inhibitory sites on the AMPKα subunit (Horman et al. 2006). This suggests that serumwithdrawal may not necessarily result in apoptosis if thecell expresses sufficient functional AMPK heterotrimerthat can protect cell viability as shown in several studies

(Kazuyoshi et al. 2002; Suzuki et al. 2003a,b, 2004; Russellet al. 2004). Nevertheless, withdrawal of serum is used,in many cell culture models, to induce apoptosis via thedeprivation of these survival factors (Dudeck et al. 1997;Niesler et al. 2000).

Apoptosis induced by serum deprivation is generallyaccepted to be dependent on the caspase cascade (Allenet al. 1998; Shi, 2004). In C2C12 myoblasts, however,dropping the serum concentration from 10 to 1% resultsin cell cycle withdrawal and differentiation into myotubes(Yaffe & Saxel, 1977; Walsh & Perlman, 1997). Certaincaspases are activated during this differentiation andindirectly they promote necessary protein degradation,migration and fusion via activation of calcium-activatedproteases (calpains) that are themselves required for bothapoptosis and myoblast differentiation. In this respect,it is known that calpains can be transiently activatedby proteolytic degradation of the calpastatin inhibitorby caspase-1 in differentiating myoblasts. Thus, transientactivation of both calpains and caspases is needed forthe myoblast differentiation programme (Fernando et al.2002; Barnoy & Kosower, 2003; Goll et al. 2003; Abraham &Shaham, 2004; Dedieu et al. 2004). These apoptotic eventsmay not progress to cell death because some inhibitors ofapoptotic cascade amplification may still be operating, asrecently shown in Drosophila (Dotto & Silke, 2004).

In the present study, we have used the differentiatingmouse C2C12 skeletal muscle myoblast cell line as anin vitro cell culture model of in vivo differentiatingmyoblasts because they provide a well-established andreproducible model of myogenesis (Yaffe & Saxel, 1977;Walsh & Perlman, 1997). Furthermore, except for AMPK,many skeletal muscle-specific genes and proteins havebeen studied in these cells (Tomczak et al. 2004), thusallowing validation of our study to investigate the possiblecomparative role AMPK might play in facilitating thesurvival of both the undifferentiated myoblast and thedifferentiated myotube. In doing so, we have foundthat myoblasts are more vulnerable to apoptosis thanmyotubes, which we believe is due, in part, to insufficientexpression of functional AMPK α/β/γ heterotrimer andARK5.

Methods

Materials

Mouse C2C12 satellite cells (Yaffe & Saxel, 1977) were agift from Dr Rob Smith, Stellenbosch University, SouthAfrica. Dulbecco’s modified Eagle’s medium (DMEM)and penicillin–streptomycin were from HighveldBiological (Pty) Ltd, Johannesburgh, South Africa.Compound C (6-[4-(2-piperidin-1-ylethoxy)-phenyl]-3-pyridin-4-ylpyrazolo[1,5-α] pyrimidine; CC; Zhou et al.2001) was a gift from Dr Zhou, Merck, Rahway, NJ, USA.

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Polyvinylidene fluoride (PVDF) membrane (0.2 µm)and protein kaleidoscope molecular weight markers werefrom Bio-Rad Laboratories, Ltd, Johannesburgh, SouthAfrica. Primary antibodies that recognize mouse proteinswere either purchased [anti-threonine 172 phospho-AMPK α1/2; anti-serine 79-phosphorylated acetylCoA carboxylase (ACCα/β); antitotal ACCα/β andimmunoprecipitating anti-AMPK β1 (Cell Signaling,Danvers, MA, USA); anti-ICAD (an inhibitor of caspase-3-activated DNAase; Santa Cruz Biotechnology Inc., SantaCruz, CA, USA); MELK (Serotec, Kidlington, Oxford,UK); and anti-ARK5 (Abgent, San Diego, CA, USA)] ordonated [anti-AMPK α1, anti-α2, anti-β1/β2, anti-γ 1,anti-γ 2 and anti-γ 3 (Professor David Carling, MRCCellular Stress Laboratory, Imperial College, London,UK)]. DAKO HRP-linked secondary antibodies werepurchased through Diagnostech, Kyalami, Gauteng,South Africa. The horse radish peroxidase (HRP)chemiluminescent substrate ECLplus and Hyperfilm werefrom AEC GE Healthcare (Pty) Ltd, Cape Town, SouthAfrica. Fetal bovine serum (FBS), donor herd horse serum(HS) and all other reagents were from Sigma-Aldrich,Aston Manor, Gauteng, South Africa.

Cell culture

C2C12 cells [ATCC CRL-1772] (Dedieu et al. 2004)were routinely cultured in Dulbecco’s modified Eagle’smedium (DMEM) containing 0.45% glucose, 2 mm l-glutamine, 100 i.u. penicillin, 100 µg ml−1 streptomycinand 10% FBS (proliferation medium) in a 37◦Chumidified incubator with a 95% air-5% CO2 atmosphere.Differentiation of 70% confluent C2C12 cells was inducedby replacing the 10% FBS with 1% HS (differentiationmedium). Media were changed every other day. Indifferentiation medium, multinucleated, fused myotubesformed from proliferating mononucleated day 0 myoblastsby differentiation day 7. When required, proliferating cellswere plated onto glass coverslips (22 × 22 mm) in six-wellplates before differentiation. Apoptosis was induced eitheron day 0 (undifferentiated cells) or on day 7 (differentiatedcells) by incubation in complete serum-free DMEM for24 h. Where indicated, inhibition of AMPK was achievedby adding a 0.1% dilution of a 20 mm Compound C (Zhouet al. 2001) stock solution in DMSO to the cells during thewhole 24 h serum starvation period.

Morphological assessment and quantification ofapoptotic C2C12 cells

The C2C12 cells, plated on the glass coverslips,were stained with Hoechst 33342, a fluorescentnuclear binding dye, which allows clear distinctionbetween apoptotic and normal cells on the basis ofnuclear morphology (chromatin condensation and

fragmentation). Hoechst 33342 [prepared in phosphate-buffered saline (PBS)] was added to the culture mediumto a final concentration of 50 µg ml−1. Cells wereevaluated by fluorescence microscopy according tothe following grading system: normal nuclei (bluechromatin with organized structure) and apoptoticnuclei (bright fluorescent chromatin which is highlycondensed or fragmented; Niesler et al. 2000). For thisstudy, three separate cell populations were prepared.Six randomly selected fields per coverslip were capturedusing a fluorescence microscope (Nikon ECLIPSE E400)and digital camera (Nikon DXM1200). The numberof apoptotic nuclei, as well as the total number ofnuclei in each field of view (total, 1000–1200 nuclei perslide), were determined using Simple PCI version 4.0(Compix Inc., Imaging Systems, Sewickley, Pennsylvania,USA). The scoring was performed blind. The apoptoticindex (AI; percentage of apoptotic nuclei per field) wascalculated as the number of apoptotic nuclei divided bythe total nuclei counted in one field multiplied by 100.The mean ± s.e.m. AI was calculated from the 18 fields(3 samples; 6 fields per sample) using unpaired Student’st test.

Western immunoblotting

Cells were lysed with RIPA/HBSS (1% NP4O; 0.1% SDS;0.5% Na deoxycholate; 2.5 mm Tris/HC1 pH 7.4; 1 mmEDTA; 1 mm EGTA; 250 mm mannitol; 1 mm DTT) buffercontaining phosphatase and protease inhibitors (0.1 mmPMSF; 0.1 mm leupeptin; 1 mm benzamidine; 4 µg/mlsoya bean trypsin inhibitor; 50 mm NaF; 50 mm Napyrophosphate) (RIPA++). Protein cell lysate (20 µg)was separated by SDS-PAGE and transferred to PVDFmembrane using a BioRad semidry blotting system.Proteins on the membrane were blocked in Blotto A (5%non fat dried milk; 0.05% Tween-20; Tris buffered salinepH 7.4) before Western immunoblotting with one of thespecific primary antibodies and species-compatible HRP-linked secondary antibody, as described in the Materialssection. Detection was by ECLplus.

Densitometry

Comparative band densities on Western blots weredetermined using the UN-SCAN-IT automateddigitization system (Silk Scientific Inc., Orem, UT,USA).

Results

Protein expression of AMPK subunit isoforms indifferentiating C2C12 cells

Using SDS-PAGE and Western immunoblotting, we havemade a number of observations regarding the proteinexpression of AMPK subunit isoforms in C2C12 cells

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during differentiation. Firstly, there is no detectableprotein expression of the α1, ARK5, β1 or β2 subunitisoforms and only low expression of the γ 3 subunitisoform in proliferating, undifferentiated day 0 C2C12cells. As differentiation progresses, however, these subunitisoforms are increasingly expressed, reaching a maximumby differentiation day 7. This level of expression issustained, at least until differentiation day 11. Secondly,neither undifferentiated nor differentiated C2C12 cellsexpress detectable levels of γ 1 subunit isoform protein.Thirdly, the proteins for both α2 and γ 2 subunit isoformsare constitutively expressed at the same level in C2C12cells at all stages investigated in this study. Finally, thep50 but not the p72 isoform of the embryonic α subunithomologue MELK (Gil et al. 1997) was expressed onlyin the undifferentiated, proliferating myoblasts (Fig. 1A, Band C). Although we were unable to detect eitherβ subunitisoform in the undifferentiated, proliferating myoblastsusing standard Western immunoblotting of a 20 µgprotein cell lysate, we were able to detect the β subunitin 1 mg protein by Western immunoblotting afterimmunoprecipitation with a specific anti-β1 antibody(not shown). Thus, the level of β subunit expression inday 0 myoblasts was about 50 times lower than that seen inthe differentiated myotubes. We were unable to determinethe expression level of the β2 subunit isoform in myoblastsusing immunoprecipitation because we could not source acommercially availableβ2 immunoprecipitating antibody.

As a marker of in vitro C2C12 myogenic differentiation,we determined that the secondary myogenic regulatory

Figure 1. Protein expression of AMPKsubunit isoforms in differentiatingC2C12 cellsProtein cell lysates (20 µg) fromdifferentiating day 0, 1, 3, 5, 7, 9 or 11C2C12 cells were subjected to Westernimmunoblotting using specific antibodiesthat recognize AMPK subunit isoforms andhomologues, as well as a transcriptionalfactor that is induced during myogenicdifferentiation. Detection was with ECLplus.A, expression of the AMPK α subunitisoforms and α subunit homologues.B, expression of the AMPK β subunitisoforms. C, expression of the AMPKγ subunit isoforms. D, expression of thesecondary regulatory factor of myogenicdifferentiation, myogenin. Either the AMPKα2 or γ 2 subunit isoform (in the same orparallel blot) was used as a loading control([LC]). The protein extracts used for therepresentative blots came from more thanfour differentiating cell cultures. Each blotwas repeated at least twice.

factor myogenin was transiently expressed between days 3and 7 of differentiation (Fig. 1D).

Undifferentiated and differentiated C2C12 cells differin their capacity to activate AMPK

We next investigated whether acute exposure of C2C12cells to the AMPK activator and mitochondrial uncoupleroligomycin (1 µm; Hawley et al. 2002) would result inincreased AMPK activation as measured by the increasein threonine 172 phosphorylation on α1/2 subunitsof AMPK, using standard SDS-PAGE and Westernimmunoblotting techniques. We found that exposureto oligomycin for up to 45 min resulted in higherAMPK activation in differentiated day 7 myotubes thanin undifferentiated day 0 myoblasts (Fig. 2).

Using densitometry, we quantified (in arbitrary units,a.u.) the basal and stimulated AMPK phosphorylation at 0and 45 min in both day 0 and day 7 cells and found that theapparent increase in AMPK activity over 45 min on day 0and day 7 rose from 2.00 to 4.16 (a 2.08-fold increase)and from 9.20 to 19.98 (a 2.17-fold increase), respectively.When the phosphorylations were normalized (AMPK-P/α2 AMPK expression), it was found that the basal AMPKphosphorylation was higher in day 7 cells (0.45) comparedwith day 0 cells (0.09). In the presence of oligomycin(45 min), the AMPK phosphorylation in undifferentiatedday 0 myoblast cells increased from 0.09 to 0.21 (a 2.33-fold increase) compared with an increase from 0.45 to1.00 (a 2.22-fold increase) in differentiated day 7 myotube

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Exp Physiol 92.1 pp 207–217 AMPK lowers apoptosis of C2C12 myotubes 211

Table 1. Degree of AMPK activation in C2C12 myoblasts and myotubes

AMPK-P α2 AMPK Normalized foldOligomycin (activation) band Fold expression, band AMPK-P/α2 activation from(1 µM) density (a.u.) activation density (a.u.) AMPK expression day 0 (0 min)

Day 0, 0 min 2.00 1.00 21.36 0.09 1.00Day 0, 45 min 4.16 2.08 20.15 0.21 2.33Day 7, 0 min 9.20 1.00 20.30 0.45 5.00Day 7, 45 min 19.98 2.17 19.90 1.00 11.11

cells. Thus, because the basal AMPK phosphorylation inday 7 myotubes was five times higher than that in day 0myoblasts, the maximum AMPK activation achieved withstimulation reached 11-fold in myotubes compared with2.3-fold in myoblasts (Table 1).

Inhibition of AMPK increases apoptosis more inundifferentiated than in differentiated C2C12 cells

Twenty-four hours of serum starvation of undifferentiatedday 0 C2C12 and differentiated day 7 C2C12 cells resultedin an increase in their apoptotic indices by 30 and 20%,respectively, as determined by Hoechst 33342 staining ofapoptotic nuclei (Fig. 3Aa). Concomitant addition of 0.1%DMSO did not increase the apoptotic indices further(Fig. 3Ab). However, concomitant addition of 0.1%DMSO containing 20 µm Compound C (a specific AMPKinhibitor; Zhou et al. 2001) to the serum-starved cells (butnot to the cells in serum-containing medium) resulted inthe apoptotic index of undifferentiated day 0 C2C12 cellsrising to 90%, whereas addition of Compound C to theserum-starved day 7 differentiated C2C12 cells resulted intheir apoptotic index rising to only 40% (Fig. 3Ac).

As a second assay for apoptosis induced by total serumwithdrawal rather than differentiation, we analysed theprotein expression level of full-length inhibitor of caspase-3-activated DNAase (ICAD) by Western immunoblotting.We chose ICAD because DNAase γ rather than CAD isinvolved in C2C12 differentiation (Shiokawa et al. 2002).By Western blotting protein lysates, from cells treatedas above, we found that decreases in full-length ICAD(Fig. 3B) were similarily associated with the apoptoticindex increases (Fig. 3A).

Serum deprivation increases AMPK-mediatedphosphorylation of its acetyl CoA carboxylase (ACC)substrate in differentiated but not in undifferentiatedC2C12 cells

Undifferentiated (day 0) and differentiated (day 7) C2C12cells, in either serum-containing or serum-free medium,were exposed for 24 h to either 0.1% DMSO or to0.1% DMSO containing 20 µm CC, the specific AMPKinhibitor. The cells were then lysed with RIPA++ bufferand 20 µg of the protein cell lysates were subjected to

Western immunoblotting using specific antibodies todetect levels of: (1) AMPK-mediated phosphorylation ofserine 79 on ACC; (2) phosphorylated threonine 172 onthe α1/2 AMPK catalytic subunits; and (3) total proteinexpression of both ACCα and ACCβ isoforms. Serumwithdrawal from day 7 but not from day 0 cells resultedin increased AMPK activity towards isoform ACCα (butnot isoform ACCβ) as demonstrated by its increasedserine 79 phosphorylation. This AMPK-mediated ACCphosphorylation was completely blocked by the specificAMPK inhibitor, CC. Regardless of AMPK inhibition,day 7 cells but not day 0 cells displayed ∼20% basalserine 79 phosphorylation of ACCα in the presenceof serum (Fig. 4A). Day 7 cells also showed higherbasal and serum withdrawal-stimulated threonine 172phosphorylation of α1/2 AMPK than day 0 cells. Inthe presence of CC, however, under both serum-free and serum-containing conditions, threonine 172phosphorylation of α1/2 AMPK in day 7 cells increasedto that obtained with serum withdrawal alone (Fig. 4B).Thus, it appears that in the presence of CC, AMPK catalyticactivity and threonine 172 AMPK phosphorylation are

α1/ α2 AMPK-P

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Figure 2. Undifferentiated and differentiated C2C12 cells differin their capacity to activate AMPKC2C12 cells were exposed to 1 µM oligomycin for up to 45 min 20 µgprotein cell lysates were then subjected to Western immunoblotting todetect threonine 172 phosphorylation on the AMPK α1/2 subunits(marker of AMPK activation). Detection was with ECLplus. Left upperpanel, AMPK activation in undifferentiated day 0 cells; right upperpanel, loading control; left lower panel, AMPK activation indifferentiated day 7 cells; and right lower panel, loading control. Thereis more basal AMPK activity in day 7 myotubes than in day 0myoblasts, and exposure to oligomycin for 45 min raises AMPK activityin day 7 cells further. Detection of total α2 AMPK subunit expressionwas used as the loading control, in parallel blots. The blots arerepresentative of two whole experiments.

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212 C. U. Niesler and others Exp Physiol 92.1 pp 207–217

dissociated. In C2C12 cells, the α and β isoforms of ACCare equally expressed, and the levels do not change duringdifferentiation. They were therefore used as markers ofequal protein loading (Fig. 4C).

Discussion

The canonical α1/α2/β/γ heterotrimeric AMP-activatedprotein kinases are known to act as sensors of metabolicstress in skeletal muscle and have been implicated inpromoting survival not of only skeletal muscle but alsoof other tissues during AMP elevation in response toATP depletion (Barnes et al. 2001; Culmsee et al. 2001;Russell et al. 2004; Horman et al. 2006). In this study,we have used the differentiating mouse C2C12 skeletalmuscle myoblast cell line as an in vitro cell culture modelof the in vivo differentiating myoblast to investigate whatrole AMPK might play in protecting adult skeletal muscleprogenitor satellite cells from apoptosis during theirdifferentiation.

We demonstrate that differentiated C2C12 myotubesare less vulnerable to apoptosis than their undifferentiatedcounterparts (Fig. 3). We believe this is partly due notonly to their increased protein expression of the AMPK

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Figure 3. Inhibition of AMPK increasesapoptosis more in day 0undifferentiated than in day 7differentiated C2C12 cellsA, apoptotic indices. Hoechst staining ofday 0 and day 7 C2C12 cells was used toidentify both normal and apoptotic nuclei.The apoptotic index (AI) was calculated asthe percentage of apoptotic nuclei per totalnuclei number per field. Themean ± S.E.M. percentage AI was calculatedusing unpaired Student’s t test from 6randomly selected fields per treatment. Cellswere exposed to standard serum-containingmedia (�) or serum-free media (�) with nofurther additions (Aa) 0.1% DMSO (Ab) or20 µM Compound C in 0.1% DMSO (Ac),for 24 h. The experiment was repeatedthree times. ∗∗∗P < 0.0001. B, molecularmarker of apoptosis. Protein cell lysates(20 µg) were subjected to Westernimmunoblotting to determine the level offull-length ICAD. Rho guanine nucleotidedissociation inhibitor p28 (GDI) was used asa loading control ([LC]).

α/β/γ subunits (Fig. 1) but also to their higher basal andstimulated α1/2 AMPK activity (Fig. 2). Although the α2and γ 2 subunit isoforms are expressed in undifferentiatedmyoblasts, their level of β1 (and probably β2) subunitexpression is about 50 times lower than that in thedifferentiated myotubes. Therefore, we hypothesize that,owing to low β subunit expression, the undifferentiatedmyoblasts are more vulnerable to apoptosis, since theyare unable to assemble sufficient functional α1/2/β/γAMPK heterotrimer compared to their differentiatedcounterparts. Heterotrimeric assembly of AMPK isrequired before AMP, the allosteric activator of AMPK,can bind between the α and β subunits to cause theconformational change in the α subunit that exposesthe higher activating threonine 172 phosphorylation site(Hardie et al. 1998). Whether the phosphorylatingupstream AMPKK(s) (Lizcano et al. 2004) is alsoexpressed at a higher level in differentiated myotubeshas not been determined. However, we did determinethat the AMPK α subunit homologue ARK5 is morehighly expressed in differentiated C2C12 myotubes thanin the undifferentiated myoblasts, thereby providing analternative or additional anti-apoptotic mechanism whenthe Akt/PKB pathway is active. However, it is not yet known

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Exp Physiol 92.1 pp 207–217 AMPK lowers apoptosis of C2C12 myotubes 213

whether ARK5 also acts as a heterotrimer. Furthermore, weshow for the first time that the embryonic AMPKα subunithomologue MELK is expressed only in undifferentiated,proliferating C2C12 cells and that its expression is rapidlydownregulated after cell cycle withdrawal when ARK5expression starts to increase. However, we only detectedthe p50 and not the p72 isoform of MELK, whichmay reflect an alternative MELK translation productas suggested by Gil et al. (1997). That MELK is onlyexpressed in the undifferentiated, proliferating C2C12myoblasts could imply that it has a role in promotingproliferation while inhibiting differentiation programmes,as in other cells (Heyer et al. 1997; Gil et al. 1998;Davezac et al. 2002; Nakano et al. 2005). For example,MELK plays an important role in preimplantationembryonic development (Suzuki et al. 2003b). Both thezinc-finger-like protein ZPR9 and the transcriptionalrepressor nuclear protein NIPP1 are phosphorylatedby MELK during this time, and phosphorylated ZPR9accumulates in the nucleus while phosphorylated NIPP1blocks spliceosome assembly (Seong et al. 2002; Vulstekeet al. 2004). Expression of MELK in this proliferatingskeletal muscle adult progenitor-like C2C12 myoblast cellline suggests that MELK may also have a role in adultstem cell renewal in vivo. Perhaps MELK could be usedas a molecular marker whereby activated, proliferatingundifferentiated myoblasts could be distinguished fromquiescent myoblasts and from differentiated myotubes thatcoexpress ARK5 and the β and γ 3 AMPK subunits but notMELK.

Although we have clearly demonstrated a changingexpression profile of AMPK subunit isoforms andhomologues in differentiating C2C12 cells, the questionstill remains as to what regulates their expression. Isexpression of all the subunits under the control of themyogenic differentiation programme (Kim et al. 2003) orare some subunits or homologues under the control ofstress-activated pathways?

While we were able to determine that higherbasal and stimulated levels of α1/2 AMPK activity indifferentiated myotubes (Figs 2 and 4) were associatedwith phosphorylation of its substrate, ACC (Fig. 4),and protection from apoptosis (Fig. 3), we were unableto directly determine whether MELK or ARK5 wereactive. However, MELK does not appear to increasephosphorylation of ACC, either in the presence orabsence of serum (see Fig. 4). This could result eitherfrom ACC not being an in vivo MELK substrate, eventhough MELK can phosphorylate the SAMS syntheticpeptide (containing a phosphorylation site based onthe serine 79 AMPK phosphorylation site of ACC)in vitro (Heyer et al. 1997; Beullens et al. 2005), orbecause it is inactive. It would be surprising if thep50MELK isoform were inactive, since recently Beullenset al. (2005) determined that the p72MELK isoform is

activated by autophosphorylation on threonine 167 andserine 171 when the COOH-terminus is displaced afterits phosphorylation. This auto-inhibitory domain maybe missing in the p50MELK isoform, thereby perhapsmaking it constitutively active. If so, it would argue againstACC being a p50MELK substrate. In contrast, ARK5 isactivated when growth factors, such as insulin or insulingrowth factor-1 (in the serum-containing medium), bindto their highly homologous receptors to activate cellsignalling by phosphorylating insulin receptor substrates(Suzuki et al. 2003b). Downstream signalling is highlydependent on the phosphatidylinositol 3-kinase (PI3K)pathway and Akt/PKB. Phosphatidylinositol 3-kinase isresponsible for phosphorylating phosphatidylinositol 4,5-bisphosphate (PIP2), to generate phosphatidylinositol3,4,5-trisphosphate (PIP3). Generation of PIP3 results

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DMSO+ CC

Figure 4. Serum deprivation increases AMPK-mediatedphosphorylation of its ACC substrate in differentiated but notin undifferentiated C2C12 cellsUndifferentiated (day 0) and differentiated (day 7) C2C12 cells wereexposed for 24 h to 0.1% DMSO or to 0.1% DMSO containing 20 µM

CC in the presence or absence of serum. Protein cell lysates (20 µg)were than subjected to Western immunoblotting to detectphosphorylated serine 79 ACC (A), phosphorylated threonine 172α1/2 AMPK (B), and total ACCα and ACCβ protein expression used asthe loading control (C). Serum withdrawal alone in day 7 but notday 0 cells resulted in increased AMPK activity, as demonstrated by theincrease in serine 79 phosphorylation on ACCα. AMPK-mediatedphosphorylation of ACC was completely blocked by the inclusion ofthe AMPK inhibitor CC. Regardless of the presence or absence of CC,day 7 cells displayed ∼20% more basal serine 79 phosphorylation ofACCα in the presence of serum than day 0 cells (A). Day 7 cellsshowed higher basal and stimulated threonine 172 phosphorylation ofα1/2 AMPK than day 0 cells. In the presence of CC, under bothserum-free and serum-containing conditions, threonine 172phosphorylation of α1/2 AMPK increased to that obtained with serumwithdrawal alone. Thus, in the presence of CC, AMPK catalytic activityand AMPK phosphorylation appears to be dissociated. Expression ofthe α and β ACC isoforms was used as equal protein loading controls.Detection was by ECLplus. The blots are representative of two wholeexperiments.

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214 C. U. Niesler and others Exp Physiol 92.1 pp 207–217

in the activation of the protein kinase proteinkinase C effector protein kinase D1 (PDK1) whichin turn phosphorylates the serine/threonine proteinkinase Akt/PKB on threonine 308. Another proteinkinase (anticipated to be PKB2) also phosphorylatesAkt/PKB on serine 473. These two phosphorylationsadditively activate Akt/PKB (Lawlor & Alessi, 2001;Datta et al. 2006), which can then inhibit apoptosisby phosphorylating downstream substrates that includeForkhead transcription factors, the Bcl-2 family memberBad and ARK5 (Dudeck et al. 1997; Parrizas et al.1997; Chan et al. 1999; Sen et al. 2003; Suzuki et al.2003a). Recently, downstream of Akt/PKB, anotherserine/threonine protein kinase, called the nuclear Dbf-related kinase 2 (NDR2), has also been found tophosphorylate and activate ARK5 during IGF-1 signalling(Suzuki et al. 2006). Active ARK5 phosphorylates thecaspase cascade components FLIP, caspase-8 and caspase-6(Suzuki et al. 2003a,b, 2004), thereby helping to inhibitapoptosis. In these differentiated C2C12 cells, when ARK5is expressed, it is presumed to be active in the presenceof the growth factor-containing serum because there isbasal phosphorylation of ACCα that is not inhibited byCC (see Fig. 4). Whether CC affects other components ofthe PI3K–Akt/PKB pathway is not known.

The ACCβ isoform is accepted as being thepredominantly expressed isoform in adult skeletal andheart muscle tissue (Winder et al. 1997; Barnes et al. 2001;Kim et al. 2003). In these C2C12 cells, however, bothACCα and ACCβ isoforms appear to be constitutively andequally expressed and, in our hands, AMPK and ARK5seem only to phosphorylate the ACCα isoform (Fig. 4).One could question which ACC isoform is the in vivotarget of AMPK and ARK5, and under what conditions.Maybe inhibitory phosphorylation of the ACCβ isoformis mediated solely by cyclic AMP-dependent kinase events(Haystead et al. 1990), or perhaps alternative types ofcellular stress other than serum withdrawal cause AMPK-mediated phosphorylation of ACCβ. Serum provides awide variety of macromolecular proteins, low molecularweight nutrients, carrier proteins for water-insolublecomponents, and other compounds such as cytokines,hormones and attachment factors necessary for in vitrogrowth of cells. Serum also adds buffering capacity tothe medium and binds or neutralizes toxic components.Thus, serum withdrawal results in loss of many survivalfactors that in our system activates hetereotrimeric AMPK,when present. Whether this is due to a rise in cytosolicAMP activating AMPK in the classical way (Moore et al.1991; Lizcano et al. 2004) or whether there is a risein cytosolic calcium, associated with apoptotic events(Rizzuto et al. 2003), that alternatively activates AMPK bycalcium–calmodulin-dependent protein kinase-mediatedphosphorylation as has recently been found (Birbaum,2005), is not known. What contribution residual Akt/PKB

or ARK5 activity may have made to the survival ofthe C2C12 myotubes under serum-free conditions wasnot determined. However, Fujio et al. (2001) foundthat Akt2 expression was upregulated during C2C12 celldifferentiation, while Stewart & Rotwein (1996) found thatduring this time IGF-2 was produced by the cells and actedas an autocrine survival factor, presumably via the PI3K–Akt/PKB pathway.

Finally, we found that the addition of the specific AMPKinhibitor, CC, to serum-deprived cells causes further andfar greater apoptosis of undifferentiated C2C12 myoblasts(∼90%) compared with differentiated C2C12 myotubes(∼40%), thereby further suggesting that AMPK playsa protective anti-apoptotic role in these cells. However,additional interpretations can be made. Firstly, the doseof CC used may be supranormal for the undifferentiatedmyoblasts, in which AMPK binding maybe saturating,leading to additional targets being affected. Secondly,AMPK inhibition may have more effect on dividingmyoblasts because they have fewer alternative protectivemechanisms, such as ARK5, in place. Thirdly, proliferatingmyoblasts may normally encounter stressful times duringthe cell cycle when any available AMPK may be directedtowards maintaining their viability (Jones et al. 2005).Any additional external cellular stresses during these timesmay be too much for the cells to withstand. We could notdetermine whether MELK was inhibited by CC. However,assuming that basal phosphorylation of ACC in serum-containing medium is mediated mainly by active ARK5(when AMPK is normally relatively inactive; Horman et al.2006; King et al. 2006; Bertrand et al. 2006), it appears thatARK5 is not inhibited by CC, since in its presence there wasno decrease in basal ACC phosphorylation. Therefore wesuggest that under serum-containing conditions, ARK5is active and maintains basal ACCα phosphorylation,whereas following serum deprivation, when ARK5 isinactivated, AMPK is activated (Horman et al. 2005) toprotect the viability of the stressed myotubes but not themyoblasts.

The many well-known targets of AMPK act primarilyto preserve and generate ATP levels in the stressed cell(Hardie, 2004). Unlike ARK5, however, few AMPKsubstrates that directly inhibit components of theapoptotic cascades have been found. One might supposethat, since ARK5 phosphorylates sites with similarsurrounding consensus sequences to those phosphorylatedby AMPK (Suzuki et al. 2003b), at least some of the anti-apoptotic targets of ARK5 would also be phosphorylatedby AMPK, but this has not been determinedexperimentally. Apart from these putative anti-apoptoticAMPK substrates, additional possible substrates thatcontain AMPK consensus phosphorylation sequences arethe voltage-dependent anion channel 2 (Van Goethem E& Manuel Lopez J, unpublished observations), whichregulates cytochrome c release from the mitochondria and

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Exp Physiol 92.1 pp 207–217 AMPK lowers apoptosis of C2C12 myotubes 215

ICAD (Moore F, personal observation), which inhibitsDNA fragmentation.

In summary, our results demonstrate that differentiatedC2C12 skeletal myotubes are able to withstandmore cellular stress to inhibit apoptosis than canundifferentiated C2C12 skeletal myoblasts because, inpart, they express more functional heterotrimeric α1/2AMPK and ARK5. If these in vitro observations wereconfirmed in primary skeletal muscle cells, they mayhave implications for in vivo pathologies, such as musclewasting, where undifferentiated satellite stem cells maybe easier apoptotic targets than their differentiatedcounterparts. Furthermore, it might be advisable totake the AMPK subunit isoform expression profile intoaccount when interpreting results from in vitro or in vivoexperiments on AMPK.

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Acknowledgements

We thank the National Research Foundation and the MedicalResearch Council, South Africa for funding this work; alsoMathilde van der Merwe for cell culture and Karen van Tubberghfor immunohistochemistry.

Author’s present address

F. Moore: Hormone and Metabolic Research Unit, Christiande Duve Institute of Cellular Pathology, University of LouvainMedical School, ICP-UCL 7529, Avenue Hippocrate 75, B-1200Brussels, Belgium.

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