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1 Department of Physiological Sciences, University of Stellenbosch, Private Bag X1, Stellenbosch 7602, South Africa
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Abstract |
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/ß/
heterotrimer to preserve ATP levels and so cell viability during stressful conditions. However, its role in aiding survival of adult skeletal muscle precursor cells is unclear. Using the differentiating mouse C2C12 postnatal skeletal muscle myoblast cell line, we have determined that proteins for the AMPK subunit isoforms 2 and 2 are constitutively expressed, while those for 1, ß1 and ß2 are undetectable in undifferentiated myoblasts but increasingly expressed with differentiation to myotubes. Although the 3 subunit is expressed at a low level in myoblasts, it too is expressed increasingly with differentiation to myotubes. The p50 but not the p72 isoform of the embryonic subunit homologue MELK is expressed only in proliferating myoblasts, while the ARK5 subunit homologue is increasingly expressed with differentiation. Myotubes displayed higher basal and stimulated 1/2 AMPK activation than myoblasts. Furthermore, serum starvation resulted in less 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 AMPK activity with 6-[4-(2-piperidin-1-ylethoxy)-phenyl]-3-pyridin-4-ylpyrazolo[1,5-] pyrimidine (Compound C) exacerbated the apoptosis resulting from serum withdrawal. If these in vitro events can also occur in vivo, they could have implications for pathologies such as muscle wasting, in which undifferentiated satellite stem cells may be easier apoptotic targets than their differentiated counterparts. Furthermore, these results suggest that when interpreting results from in vitro or in vivo experiments on AMPK, the subunit expression profile should be taken into 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 Cellular Pathology, University of Louvain Medical School, ICP-UCL 7529, Avenue Hippocrate 75, B-1200 Brussels, Belgium. Email: frances.moore{at}horm.ucl.ac.be
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Introduction |
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AMP-activated protein kinase (AMPK) is a highly conserved cellular serine/threonine protein kinase (Hardie et al. 1998) that functions heterotrimerically with a catalytic subunit and two regulatory subunits, ß and . Multiple isoforms of the subunits exist, namely 1 and 2, ß1 and ß2, and 1, 2 and 3 (Beri et al. 1994; Gao et al. 1996; Stapleton et al. 1996, 1997). Subunit isoforms that make up a functional /ß/ AMPK heterotrimer are variously expressed in different tissues and locations (Stapleton et al. 1996; Salt et al. 1998). For example, the 3-subunit is mainly expressed in skeletal muscle (Cheung et al. 2000), and mutations in this isoform have been linked to skeletal muscle glycogen storage disease (Milan et al. 2000). AMPK acutely regulates energy homeostasis by phosphorylating key enzymes, thereby altering their activities to preserve ATP and increase ATP resynthesis (Moore et al. 1991; Hardie et al. 1998). It also phosphorylates transcriptional factors to adjust gene expression under conditions of chronic stress (Leff, 2003). Mammalian AMPK can be activated during times when the cellular AMP:ATP ratio is elevated owing to stresses such as substrate deprivation for ATP synthesis, trophic signal deprivation for cell survival, or excessive ATP consumption (Moore et al. 1991; Hardie et al. 1998; Barnes et al. 2001; Winder, 2001; Leff, 2003; Rutter et al. 2003; Myburgh, 2004). AMP allosterically activates AMPK, which then allows further covalent activation by upstream AMPK kinases (AMPKK) that phosphorylate threonine 172 on the subunit (Moore et al. 1991; Lizcano et al. 2004). Recently, three homologues of the AMPK catalytic subunit have been described. They are maternal embryonic leucine zipper kinase (MELK; Heyer et 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 phosphorylate the consensus SAMS (HMRSAMSGLHLVKRR) peptide AMPK phosphorylation substrate in vitro, and their expression is correlated with suppression of tumour cell apoptosis (Suzuki et al. 2003a, 2004). Under conditions of acute stress, the canonical 1/2 AMPK can also protect non-tumour cells by inhibiting apoptosis (Stefanelli et al. 1998; Blazquez et al. 2001). Chronically activated AMPK has 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 or differentiation depends on a complex set of input signals that interpret the balance between positive survival signals and negative pro-apoptotic signals (Bellamy et al. 1995; Allen et al. 1998; Kiess & Gallagher, 1998). Serum contains numerous anti-apoptotic and growth-promoting factors, 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 factors in survival, in addition to their proliferative and metabolic effects, by phosphorylating a number of cellular substrates. Those that inhibit apoptotic cell death include the Forkhead transcription factors, the Bcl-2 family member Bad and the AMPK subunit homologue ARK5 (Dudeck et al. 1997; Parrizas et al. 1997; Chan et al. 1999; Sen et al. 2003; Suzuki et al. 2003a). ARK5, in turn, has been shown to inhibit apoptosis by phosphorylating fas-associated death domain-like interleukin-1ß-converting enzyme-inhibitory protein (FLIP), caspase-8 and -6 in glucose-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 lowering phosphorylation at two inhibitory sites on the AMPK subunit (Horman et al. 2006). This suggests that serum withdrawal may not necessarily result in apoptosis if the cell expresses sufficient functional AMPK heterotrimer that can protect cell viability as shown in several studies (Kazuyoshi et al. 2002; Suzuki et al. 2003a,b, 2004; Russell et al. 2004). Nevertheless, withdrawal of serum is used, in many cell culture models, to induce apoptosis via the deprivation of these survival factors (Dudeck et al. 1997; Niesler et al. 2000).
Apoptosis induced by serum deprivation is generally accepted to be dependent on the caspase cascade (Allen et al. 1998; Shi, 2004). In C2C12 myoblasts, however, dropping the serum concentration from 10 to 1% results in cell cycle withdrawal and differentiation into myotubes (Yaffe & Saxel, 1977; Walsh & Perlman, 1997). Certain caspases are activated during this differentiation and indirectly they promote necessary protein degradation, migration and fusion via activation of calcium-activated proteases (calpains) that are themselves required for both apoptosis and myoblast differentiation. In this respect, it is known that calpains can be transiently activated by proteolytic degradation of the calpastatin inhibitor by caspase-1 in differentiating myoblasts. Thus, transient activation of both calpains and caspases is needed for the myoblast differentiation programme (Fernando et al. 2002; Barnoy & Kosower, 2003; Goll et al. 2003; Abraham & Shaham, 2004; Dedieu et al. 2004). These apoptotic events may not progress to cell death because some inhibitors of apoptotic cascade amplification may still be operating, as recently shown in Drosophila (Dotto & Silke, 2004).
In the present study, we have used the differentiating mouse C2C12 skeletal muscle myoblast cell line as an in vitro cell culture model of in vivo differentiating myoblasts because they provide a well-established and reproducible model of myogenesis (Yaffe & Saxel, 1977; Walsh & Perlman, 1997). Furthermore, except for AMPK, many skeletal muscle-specific genes and proteins have been studied in these cells (Tomczak et al. 2004), thus allowing validation of our study to investigate the possible comparative role AMPK might play in facilitating the survival of both the undifferentiated myoblast and the differentiated myotube. In doing so, we have found that myoblasts are more vulnerable to apoptosis than myotubes, which we believe is due, in part, to insufficient expression of functional AMPK /ß/ heterotrimer and ARK5.
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Methods |
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Mouse C2C12 satellite cells (Yaffe & Saxel, 1977) were a gift from Dr Rob Smith, Stellenbosch University, South Africa. Dulbecco's modified Eagle's medium (DMEM) and penicillin–streptomycin were from Highveld Biological (Pty) Ltd, Johannesburgh, South Africa. Compound C (6-[4-(2-piperidin-1-ylethoxy)-phenyl]-3-pyridin-4-ylpyrazolo[1,5-] pyrimidine; CC; Zhou et al. 2001) was a gift from Dr Zhou, Merck, Rahway, NJ, USA. Polyvinylidene fluoride (PVDF) membrane (0.2 µm) and protein kaleidoscope molecular weight markers were from Bio-Rad Laboratories, Ltd, Johannesburgh, South Africa. Primary antibodies that recognize mouse proteins were either purchased [anti-threonine 172 phospho-AMPK 1/2; anti-serine 79-phosphorylated acetyl CoA carboxylase (ACC/ß); antitotal ACC/ß and immunoprecipitating anti-AMPK ß1 (Cell Signaling, Danvers, MA, USA); anti-ICAD (an inhibitor of caspase-3-activated DNAase; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); MELK (Serotec, Kidlington, Oxford, UK); and anti-ARK5 (Abgent, San Diego, CA, USA)] or donated [anti-AMPK 1, anti-2, anti-ß1/ß2, anti-1, anti-2 and anti-3 (Professor David Carling, MRC Cellular Stress Laboratory, Imperial College, London, UK)]. DAKO HRP-linked secondary antibodies were purchased through Diagnostech, Kyalami, Gauteng, South Africa. The horse radish peroxidase (HRP) chemiluminescent substrate ECLplus and Hyperfilm were from AEC GE Healthcare (Pty) Ltd, Cape Town, South Africa. 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's medium (DMEM) containing 0.45% glucose, 2 mM L-glutamine, 100 i.u. penicillin, 100 µg ml–1 streptomycin and 10% FBS (proliferation medium) in a 37°C humidified incubator with a 95% air-5% CO2 atmosphere. Differentiation of 70% confluent C2C12 cells was induced by replacing the 10% FBS with 1% HS (differentiation medium). Media were changed every other day. In differentiation medium, multinucleated, fused myotubes formed from proliferating mononucleated day 0 myoblasts by differentiation day 7. When required, proliferating cells were plated onto glass coverslips (22 x 22 mm) in six-well plates before differentiation. Apoptosis was induced either on day 0 (undifferentiated cells) or on day 7 (differentiated cells) by incubation in complete serum-free DMEM for 24 h. Where indicated, inhibition of AMPK was achieved by adding a 0.1% dilution of a 20 mM Compound C (Zhou et al. 2001) stock solution in DMSO to the cells during the whole 24 h serum starvation period.
Morphological assessment and quantification of apoptotic C2C12 cells
The C2C12 cells, plated on the glass coverslips, were stained with Hoechst 33342, a fluorescent nuclear binding dye, which allows clear distinction between apoptotic and normal cells on the basis of nuclear morphology (chromatin condensation and fragmentation). Hoechst 33342 [prepared in phosphate-buffered saline (PBS)] was added to the culture medium to a final concentration of 50 µg ml–1. Cells were evaluated by fluorescence microscopy according to the following grading system: normal nuclei (blue chromatin with organized structure) and apoptotic nuclei (bright fluorescent chromatin which is highly condensed or fragmented; Niesler et al. 2000). For this study, three separate cell populations were prepared. Six randomly selected fields per coverslip were captured using a fluorescence microscope (Nikon ECLIPSE E400) and digital camera (Nikon DXM1200). The number of apoptotic nuclei, as well as the total number of nuclei in each field of view (total, 1000–1200 nuclei per slide), were determined using Simple PCI version 4.0 (Compix Inc., Imaging Systems, Sewickley, Pennsylvania, USA). The scoring was performed blind. The apoptotic index (AI; percentage of apoptotic nuclei per field) was calculated as the number of apoptotic nuclei divided by the 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's t 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 mM EDTA; 1 mM EGTA; 250 mM mannitol; 1 mM DTT) buffer containing phosphatase and protease inhibitors (0.1 mM PMSF; 0.1 mM leupeptin; 1 mM benzamidine; 4 µg/ml soya bean trypsin inhibitor; 50 mM NaF; 50 mM Na pyrophosphate) (RIPA++). Protein cell lysate (20 µg) was separated by SDS-PAGE and transferred to PVDF membrane 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 saline pH 7.4) before Western immunoblotting with one of the specific primary antibodies and species-compatible HRP-linked secondary antibody, as described in the Materials section. Detection was by ECLplus.
Densitometry
Comparative band densities on Western blots were determined using the UN-SCAN-IT automated digitization system (Silk Scientific Inc., Orem, UT, USA).
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Results |
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Using SDS-PAGE and Western immunoblotting, we have made a number of observations regarding the protein expression of AMPK subunit isoforms in C2C12 cells during differentiation. Firstly, there is no detectable protein expression of the 1, ARK5, ß1 or ß2 subunit isoforms and only low expression of the 3 subunit isoform in proliferating, undifferentiated day 0 C2C12 cells. As differentiation progresses, however, these subunit isoforms are increasingly expressed, reaching a maximum by differentiation day 7. This level of expression is sustained, at least until differentiation day 11. Secondly, neither undifferentiated nor differentiated C2C12 cells express detectable levels of 1 subunit isoform protein. Thirdly, the proteins for both 2 and 2 subunit isoforms are constitutively expressed at the same level in C2C12 cells at all stages investigated in this study. Finally, the p50 but not the p72 isoform of the embryonic subunit homologue MELK (Gil et al. 1997) was expressed only in the undifferentiated, proliferating myoblasts (Fig. 1A, B and C). Although we were unable to detect either ß subunit isoform in the undifferentiated, proliferating myoblasts using standard Western immunoblotting of a 20 µg protein cell lysate, we were able to detect the ß subunit in 1 mg protein by Western immunoblotting after immunoprecipitation with a specific anti-ß1 antibody (not shown). Thus, the level of ß subunit expression in day 0 myoblasts was about 50 times lower than that seen in the differentiated myotubes. We were unable to determine the expression level of the ß2 subunit isoform in myoblasts using immunoprecipitation because we could not source a commercially available ß2 immunoprecipitating antibody.
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Undifferentiated and differentiated C2C12 cells differ in their capacity to activate AMPK
We next investigated whether acute exposure of C2C12 cells to the AMPK activator and mitochondrial uncoupler oligomycin (1 µM; Hawley et al. 2002) would result in increased AMPK activation as measured by the increase in threonine 172 phosphorylation on 1/2 subunits of AMPK, using standard SDS-PAGE and Western immunoblotting techniques. We found that exposure to oligomycin for up to 45 min resulted in higher AMPK activation in differentiated day 7 myotubes than in undifferentiated day 0 myoblasts (Fig. 2).
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2 AMPK expression), it was found that the basal AMPK phosphorylation was higher in day 7 cells (0.45) compared with day 0 cells (0.09). In the presence of oligomycin (45 min), the AMPK phosphorylation in undifferentiated day 0 myoblast cells increased from 0.09 to 0.21 (a 2.33-fold increase) compared with an increase from 0.45 to 1.00 (a 2.22-fold increase) in differentiated day 7 myotube cells. Thus, because the basal AMPK phosphorylation in day 7 myotubes was five times higher than that in day 0 myoblasts, the maximum AMPK activation achieved with stimulation reached 11-fold in myotubes compared with 2.3-fold in myoblasts (Table 1).
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Twenty-four hours of serum starvation of undifferentiated day 0 C2C12 and differentiated day 7 C2C12 cells resulted in an increase in their apoptotic indices by 30 and 20%, respectively, as determined by Hoechst 33342 staining of apoptotic 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 AMPK inhibitor; Zhou et al. 2001) to the serum-starved cells (but not to the cells in serum-containing medium) resulted in the apoptotic index of undifferentiated day 0 C2C12 cells rising to 90%, whereas addition of Compound C to the serum-starved day 7 differentiated C2C12 cells resulted in their apoptotic index rising to only 40% (Fig. 3Ac).
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rather than CAD is involved in C2C12 differentiation (Shiokawa et al. 2002). By Western blotting protein lysates, from cells treated as above, we found that decreases in full-length ICAD (Fig. 3B) were similarily associated with the apoptotic index increases (Fig. 3A). Serum deprivation increases AMPK-mediated phosphorylation of its acetyl CoA carboxylase (ACC) substrate in differentiated but not in undifferentiated C2C12 cells
Undifferentiated (day 0) and differentiated (day 7) C2C12 cells, in either serum-containing or serum-free medium, were exposed for 24 h to either 0.1% DMSO or to 0.1% DMSO containing 20 µM CC, the specific AMPK inhibitor. The cells were then lysed with RIPA++ buffer and 20 µg of the protein cell lysates were subjected to Western immunoblotting using specific antibodies to detect levels of: (1) AMPK-mediated phosphorylation of serine 79 on ACC; (2) phosphorylated threonine 172 on the 1/2 AMPK catalytic subunits; and (3) total protein expression of both ACC and ACCß isoforms. Serum withdrawal from day 7 but not from day 0 cells resulted in increased AMPK activity towards isoform ACC (but not isoform ACCß) as demonstrated by its increased serine 79 phosphorylation. This AMPK-mediated ACC phosphorylation was completely blocked by the specific AMPK inhibitor, CC. Regardless of AMPK inhibition, day 7 cells but not day 0 cells displayed 
20% basal serine 79 phosphorylation of ACC in the presence of serum (Fig. 4A). Day 7 cells also showed higher basal and serum withdrawal-stimulated threonine 172 phosphorylation of 1/2 AMPK than day 0 cells. In the presence of CC, however, under both serum-free and serum-containing conditions, threonine 172 phosphorylation of 1/2 AMPK in day 7 cells increased to that obtained with serum withdrawal alone (Fig. 4B). Thus, it appears that in the presence of CC, AMPK catalytic activity and threonine 172 AMPK phosphorylation are dissociated. In C2C12 cells, the and ß isoforms of ACC are equally expressed, and the levels do not change during differentiation. They were therefore used as markers of equal protein loading (Fig. 4C).
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Discussion |
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1/2/ß/ heterotrimeric AMP-activated protein kinases are known to act as sensors of metabolic stress in skeletal muscle and have been implicated in promoting survival not of only skeletal muscle but also of other tissues during AMP elevation in response to ATP 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 skeletal muscle myoblast cell line as an in vitro cell culture model of the in vivo differentiating myoblast to investigate what role AMPK might play in protecting adult skeletal muscle progenitor satellite cells from apoptosis during their differentiation.
We demonstrate that differentiated C2C12 myotubes are less vulnerable to apoptosis than their undifferentiated counterparts (Fig. 3). We believe this is partly due not only to their increased protein expression of the AMPK /ß/ subunits (Fig. 1) but also to their higher basal and stimulated 1/2 AMPK activity (Fig. 2). Although the 2 and 2 subunit isoforms are expressed in undifferentiated myoblasts, their level of ß1 (and probably ß2) subunit expression is about 50 times lower than that in the differentiated myotubes. Therefore, we hypothesize that, owing to low ß subunit expression, the undifferentiated myoblasts are more vulnerable to apoptosis, since they are unable to assemble sufficient functional 1/2/ß/ AMPK heterotrimer compared to their differentiated counterparts. Heterotrimeric assembly of AMPK is required before AMP, the allosteric activator of AMPK, can bind between the and ß subunits to cause the conformational change in the subunit that exposes the higher activating threonine 172 phosphorylation site (Hardie et al. 1998). Whether the phosphorylating upstream AMPKK(s) (Lizcano et al. 2004) is also expressed at a higher level in differentiated myotubes has not been determined. However, we did determine that the AMPK subunit homologue ARK5 is more highly expressed in differentiated C2C12 myotubes than in the undifferentiated myoblasts, thereby providing an alternative or additional anti-apoptotic mechanism when the Akt/PKB pathway is active. However, it is not yet known whether ARK5 also acts as a heterotrimer. Furthermore, we show for the first time that the embryonic AMPK subunit homologue MELK is expressed only in undifferentiated, proliferating C2C12 cells and that its expression is rapidly downregulated after cell cycle withdrawal when ARK5 expression starts to increase. However, we only detected the p50 and not the p72 isoform of MELK, which may reflect an alternative MELK translation product as suggested by Gil et al. (1997). That MELK is only expressed in the undifferentiated, proliferating C2C12 myoblasts could imply that it has a role in promoting proliferation 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 preimplantation embryonic development (Suzuki et al. 2003b). Both the zinc-finger-like protein ZPR9 and the transcriptional repressor nuclear protein NIPP1 are phosphorylated by MELK during this time, and phosphorylated ZPR9 accumulates in the nucleus while phosphorylated NIPP1 blocks spliceosome assembly (Seong et al. 2002; Vulsteke et al. 2004). Expression of MELK in this proliferating skeletal muscle adult progenitor-like C2C12 myoblast cell line suggests that MELK may also have a role in adult stem cell renewal in vivo. Perhaps MELK could be used as a molecular marker whereby activated, proliferating undifferentiated myoblasts could be distinguished from quiescent myoblasts and from differentiated myotubes that coexpress ARK5 and the ß and 3 AMPK subunits but not MELK.
Although we have clearly demonstrated a changing expression profile of AMPK subunit isoforms and homologues in differentiating C2C12 cells, the question still remains as to what regulates their expression. Is expression of all the subunits under the control of the myogenic differentiation programme (Kim et al. 2003) or are some subunits or homologues under the control of stress-activated pathways?
While we were able to determine that higher basal and stimulated levels of 1/2 AMPK activity in differentiated myotubes (Figs 2 and 4) were associated with phosphorylation of its substrate, ACC (Fig. 4), and protection from apoptosis (Fig. 3), we were unable to directly determine whether MELK or ARK5 were active. However, MELK does not appear to increase phosphorylation of ACC, either in the presence or absence of serum (see Fig. 4). This could result either from ACC not being an in vivo MELK substrate, even though MELK can phosphorylate the SAMS synthetic peptide (containing a phosphorylation site based on the serine 79 AMPK phosphorylation site of ACC) in vitro (Heyer et al. 1997; Beullens et al. 2005), or because it is inactive. It would be surprising if the p50MELK isoform were inactive, since recently Beullens et al. (2005) determined that the p72MELK isoform is activated by autophosphorylation on threonine 167 and serine 171 when the COOH-terminus is displaced after its phosphorylation. This auto-inhibitory domain may be missing in the p50MELK isoform, thereby perhaps making it constitutively active. If so, it would argue against ACC being a p50MELK substrate. In contrast, ARK5 is activated when growth factors, such as insulin or insulin growth factor-1 (in the serum-containing medium), bind to their highly homologous receptors to activate cell signalling by phosphorylating insulin receptor substrates (Suzuki et al. 2003b). Downstream signalling is highly dependent on the phosphatidylinositol 3-kinase (PI3K) pathway and Akt/PKB. Phosphatidylinositol 3-kinase is responsible for phosphorylating phosphatidylinositol 4,5-bisphosphate (PIP2), to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3). Generation of PIP3 results in the activation of the protein kinase protein kinase C effector protein kinase D1 (PDK1) which in turn phosphorylates the serine/threonine protein kinase Akt/PKB on threonine 308. Another protein kinase (anticipated to be PKB2) also phosphorylates Akt/PKB on serine 473. These two phosphorylations additively activate Akt/PKB (Lawlor & Alessi, 2001; Datta et al. 2006), which can then inhibit apoptosis by phosphorylating downstream substrates that include Forkhead transcription factors, the Bcl-2 family member Bad 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, another serine/threonine protein kinase, called the nuclear Dbf-related kinase 2 (NDR2), has also been found to phosphorylate and activate ARK5 during IGF-1 signalling (Suzuki et al. 2006). Active ARK5 phosphorylates the caspase cascade components FLIP, caspase-8 and caspase-6 (Suzuki et al. 2003a,b, 2004), thereby helping to inhibit apoptosis. In these differentiated C2C12 cells, when ARK5 is expressed, it is presumed to be active in the presence of the growth factor-containing serum because there is basal phosphorylation of ACC that is not inhibited by CC (see Fig. 4). Whether CC affects other components of the PI3K–Akt/PKB pathway is not known.
The ACCß isoform is accepted as being the predominantly expressed isoform in adult skeletal and heart muscle tissue (Winder et al. 1997; Barnes et al. 2001; Kim et al. 2003). In these C2C12 cells, however, both ACC and ACCß isoforms appear to be constitutively and equally expressed and, in our hands, AMPK and ARK5 seem only to phosphorylate the ACC isoform (Fig. 4). One could question which ACC isoform is the in vivo target of AMPK and ARK5, and under what conditions. Maybe inhibitory phosphorylation of the ACCß isoform is mediated solely by cyclic AMP-dependent kinase events (Haystead et al. 1990), or perhaps alternative types of cellular stress other than serum withdrawal cause AMPK-mediated phosphorylation of ACCß. Serum provides a wide variety of macromolecular proteins, low molecular weight nutrients, carrier proteins for water-insoluble components, and other compounds such as cytokines, hormones and attachment factors necessary for in vitro growth of cells. Serum also adds buffering capacity to the medium and binds or neutralizes toxic components. Thus, serum withdrawal results in loss of many survival factors that in our system activates hetereotrimeric AMPK, when present. Whether this is due to a rise in cytosolic AMP activating AMPK in the classical way (Moore et al. 1991; Lizcano et al. 2004) or whether there is a rise in cytosolic calcium, associated with apoptotic events (Rizzuto et al. 2003), that alternatively activates AMPK by calcium–calmodulin-dependent protein kinase-mediated phosphorylation as has recently been found (Birbaum, 2005), is not known. What contribution residual Akt/PKB or ARK5 activity may have made to the survival of the C2C12 myotubes under serum-free conditions was not determined. However, Fujio et al. (2001) found that Akt2 expression was upregulated during C2C12 cell differentiation, while Stewart & Rotwein (1996) found that during this time IGF-2 was produced by the cells and acted as an autocrine survival factor, presumably via the PI3K–Akt/PKB pathway.
Finally, we found that the addition of the specific AMPK inhibitor, CC, to serum-deprived cells causes further and far greater apoptosis of undifferentiated C2C12 myoblasts (90%) compared with differentiated C2C12 myotubes (40%), thereby further suggesting that AMPK plays a protective anti-apoptotic role in these cells. However, additional interpretations can be made. Firstly, the dose of CC used may be supranormal for the undifferentiated myoblasts, in which AMPK binding maybe saturating, leading to additional targets being affected. Secondly, AMPK inhibition may have more effect on dividing myoblasts because they have fewer alternative protective mechanisms, such as ARK5, in place. Thirdly, proliferating myoblasts may normally encounter stressful times during the cell cycle when any available AMPK may be directed towards maintaining their viability (Jones et al. 2005). Any additional external cellular stresses during these times may be too much for the cells to withstand. We could not determine 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 that ARK5 is not inhibited by CC, since in its presence there was no decrease in basal ACC phosphorylation. Therefore we suggest that under serum-containing conditions, ARK5 is active and maintains basal ACC phosphorylation, whereas following serum deprivation, when ARK5 is inactivated, AMPK is activated (Horman et al. 2005) to protect the viability of the stressed myotubes but not the myoblasts.
The many well-known targets of AMPK act primarily to preserve and generate ATP levels in the stressed cell (Hardie, 2004). Unlike ARK5, however, few AMPK substrates that directly inhibit components of the apoptotic cascades have been found. One might suppose that, since ARK5 phosphorylates sites with similar surrounding consensus sequences to those phosphorylated by AMPK (Suzuki et al. 2003b), at least some of the anti-apoptotic targets of ARK5 would also be phosphorylated by AMPK, but this has not been determined experimentally. Apart from these putative anti-apoptotic AMPK substrates, additional possible substrates that contain AMPK consensus phosphorylation sequences are the voltage-dependent anion channel 2 (Van Goethem E & Manuel Lopéz J, unpublished observations), which regulates cytochrome c release from the mitochondria and ICAD (Moore F, personal observation), which inhibits DNA fragmentation.
In summary, our results demonstrate that differentiated C2C12 skeletal myotubes are able to withstand more cellular stress to inhibit apoptosis than can undifferentiated C2C12 skeletal myoblasts because, in part, they express more functional heterotrimeric 1/2 AMPK and ARK5. If these in vitro observations were confirmed in primary skeletal muscle cells, they may have implications for in vivo pathologies, such as muscle wasting, where undifferentiated satellite stem cells may be easier apoptotic targets than their differentiated counterparts. Furthermore, it might be advisable to take the AMPK subunit isoform expression profile into account when interpreting results from in vitro or in vivo experiments on AMPK.
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Acknowledgements |
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Author's present address
F. Moore: Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology, University of Louvain Medical School, ICP-UCL 7529, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
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) or serum-free media (
) with no further additions (Aa) 0.1% DMSO (Ab) or 20 µM Compound C in 0.1% DMSO (Ac), for 24 h. The experiment was repeated three times. ***P < 0.0001. B, molecular marker of apoptosis. Protein cell lysates (20 µg) were subjected to Western immunoblotting to determine the level of full-length ICAD. Rho guanine nucleotide dissociation inhibitor p28 (GDI) was used as a loading control ([LC]).