Stimulation of protein degradation by low pH in L6G8C5 skeletal muscle cells is independent of apoptosis but dependent on differentiation state
Warren Pickering,
Mai-Kim Cheng,
Jeremy Brown,
Heather Butler,
John Walls
and
Alan Bevington
Department of Nephrology, Leicester General Hospital, Leicester, UK
Correspondence and offprint requests to: Alan Bevington, Department of Nephrology, Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW, UK. Email: ab74{at}leicester.ac.uk
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Abstract
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Background. In chronic renal failure, metabolic acidosis increases protein degradation (PD) in skeletal muscle, an effect which in vivo requires glucocorticoid (GC). This disorder is poorly understood, but can be studied in vitro using L6G8C5 rat skeletal muscle cells. Two potential confounding factors in studies of PD in culture are apoptosis and dedifferentiation, both of which resemble catabolic states. The aim of this study was to determine the extent to which these factors contribute to the observed effects of acid and GC on PD.
Methods. PD was measured in intact cells by pre-labelling cell protein with [14C]phenylalanine. Apoptosis was assessed morphologically by staining DNA with Hoechst 33342, by terminal deoxynucleotide transferase-mediated nick-end labelling and by cell-surface binding of Annexin V. Differentiation was assessed morphologically from myotube fusion and from activity of the marker enzyme creatine phosphokinase (CPK).
Results. In undifferentiated myoblasts, pH had no detectable effect on apoptosis provided that serum was present and GC (dexamethasone; 5 nmol/l) decreased apoptosis. In spontaneously fused cultures in 2% serum, inhibition of apoptosis with caspase-3 inhibitor (C3I; Ac-Asp-Met-Gln-Asp-CHO; 50 µmol/l) only decreased PD by 9% at pH 7.4. In contrast, the proteasome inhibitor MG132 decreased PD by 79%. Acid (pH 7.1) increased PD, with no requirement for GC, and this effect was blocked by MG132, but not by C3I. Differentiation was unaffected by 14 days of exposure to acid or GC. However, differentiation to myotubes led to decreased sensitivity of PD to acid. This effect of acid was lost completely in highly fused myotubes, but was partly restored by 500 nmol/l dexamethasone.
Conclusions. Stimulation of PD in these cells by acid and GC is not an artefact of apoptosis or dedifferentiation, but differentiation state does determine whether PD responds spontaneously to acid or (as in vivo) only does so in the presence of GC.
Keywords: apoptosis; differentiation; glucocorticoid; L6 cells; pH; protein degradation
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Introduction
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The malnutrition and catabolic state observed in chronic renal failure (CRF) is regarded as a major cause of morbidity and mortality, particularly if it occurs in conjunction with inflammation and atherosclerosis [1]. There is considerable loss of lean body mass, especially in skeletal muscle, and this is attributed to increased protein degradation (PD) in the myocytes [2]. Much has been learned of the pathways of PD involved [2] from studies in vivo and from ex vivo incubations of freshly isolated skeletal muscle. However, the mechanism(s) triggering the catabolic state are still obscure and much remains to be learned about the fundamental cell biology of this process.
This study considers the possible role in muscle catabolism of two key processes: apoptosis and cell differentiation. Apoptosis is clearly a catabolic process involving rapid proteolysis and cell shrinkage. Until recently, clinically important catabolic states in vivo were regarded as entirely distinct from apoptosis, but recent work in cancer cachexia suggests that at least some of the enzymes (caspases) involved in apoptosis may also have a role in this wasting illness [3]. Also, muscle wasting following burns has been shown to involve apoptosis of skeletal muscle [4]. Changes in the proportion of the different muscle fibre types have also been reported in skeletal muscle in CRF [5] and selective death of some of the fibres or changes in myocyte differentiation may contribute to such effects.
An important practical problem in the study of these processes is that isolated skeletal muscle remains viable for only a few hours in vitro, which restricts the studies that can be performed. Organ culture or cell culture methods are therefore needed for the long-term cell biology and the molecular basis of catabolic states to be investigated. Such cultured-cell models of muscle catabolism also require an accurate knowledge of the contribution of apoptosis and differentiation, both for the theoretical reasons outlined above and for the following technical reasons. The rate of turnover of confluent cultures of skeletal muscle cells (cell division balanced by apoptosis [6]) is much higher than for skeletal muscle cells in vivo, in which the slow turnover rate has made muscle a favoured target for gene transfer therapy. In principle, therefore, the proteolysis in apoptosis might make a disproportionate contribution to the total PD rate in vitro and, in studies of PD rate in intact cells, accelerated apoptosis might be misinterpreted as a major contributor to the catabolic response. Differentiation of skeletal muscle cells from myoblasts to mature skeletal muscle also involves dramatic changes in total cell protein content with the protein/DNA ratio rising from 10 µg protein/µg DNA in myoblasts [7] to 300 in mature skeletal muscle [8]. For this reason, dedifferentiation of cultures in vitro might make an abnormally large contribution to apparent catabolic effects and changes in differentiation state might also alter the cells response to catabolic stimuli.
In CRF, increased PD in muscle arises partly from metabolic acidosis and, in vivo, this requires the presence of glucocorticoid (GC) [9]. The basis of this catabolic effect of acid and the permissive effect of GC is not understood [10]. Studies with cultured L6G8C5 rat skeletal muscle cells suggest that acid stimulates PD by inhibiting the pH-sensitive System A amino-acid transporter in the plasma membrane [11], but these cells differ from muscle in vivo in that they respond spontaneously to low pH without the need for GC [11]; indeed, the GC requirement has been difficult to model in vitro. A possible explanation for the discrepancy between muscle in vivo and L6G8C5 cells in vitro is that confounding factors in vitro affect PD. Clearly, two potential confounding factors are apoptosis and cell differentiation.
The aim of this study was therefore to examine the effects of the two related catabolic stimuli (low pH and GC) on L6G8C5 cells under culture conditions varying from simple unfused myoblasts to highly differentiated myotubes, to determine the extent to which changes in apoptosis and differentiation could be contributing to the observed effects on PD.
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Subjects and methods
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Spontaneously fused cultures
L6 rat skeletal muscle cells sub-clone G8C5 (European Collection of Animal Cell Cultures, ref. 92121114) were seeded at passages 1323 at 4.5 x 104 cells/cm2 on plastic wells in growth medium comprising Dulbeccos modified Eagles medium (DMEM; Life Technologies 11880) supplemented with 2 mmol/l glutamine, penicillin (105 IU/l), streptomycin (100 mg/l), phenol red (10 mg/l) and 10% v/v fetal bovine serum (FBS). Serum was heat-inactivated for 30 min at 56°C before use. By day 4 (i.e. after 3 days), the myoblasts were confluent and aligned (Figure 1A) and were switched to Eagles minimum essential medium (MEM; Life Technologies 21090) with 2% v/v FBS and antibiotics and glutamine as above. Fresh MEM/2% FBS was added on day 6. By day 8, a network of multinucleated myotubes had formed, interspersed with dense patches of small myotubes and unfused cells (Figure 1B).
Test medium at pH 7.17.5 comprising MEM as above, but with dialysed FBS (Life Technologies 10110), was added from day 8 onwards. The pH was adjusted by addition of HCl or NaHCO3, with extra NaCl at low pH to maintain a constant Na concentration.
Highly fused myotubes
L6G8C5 cells cultured as above give slow, incomplete fusion (Figure 1B). Rapid fusion is obtained with cells at lower passage number (less than 13), but these myotubes show poor adherence. Highly differentiated L6G8C5 myotubes were therefore prepared as described by Elsner et al. [12]. Cells were seeded (day 1) at passages 812 at 4 x 103 cells/cm2 in growth medium. Fresh medium was added on days 3 and 5. On day 7, a differentiation mixture was added comprising growth medium as above, but with only 2% v/v FBS without heat inactivation, 5 nmol/l recombinant human insulin-like growth factor-I (IGF-I; Sigma I-3769), 107 mol/l all-trans retinoic acid, 10 mmol/l creatine and 105 mol/l cytosine ß-D-arabinofuranoside. By day 10, densely packed, tightly adherent myotubes had formed [12].
Differentiation
Fusion was routinely monitored by estimating the percentage of the culture covered by large myotubes (Figure 1B). For studies relating fusion to the pH sensitivity of PD, all estimates of fusion were made blind, i.e. before measurement of PD in the same cultures. For experiments requiring more precise quantification of differentiation, the muscle marker enzyme creatine phosphokinase (CPK) was assayed using a commercially available kit (Sigma 45-1).
Measurements
Total PD rate (release of amino acids from protein, irrespective of the PD pathway involved) was measured as described in detail elsewhere [7], by monitoring release of acid-soluble radioactivity from cultures in which protein had been pre-labelled with L-[U-14C]phenylalanine from days 4 to 8 (in spontaneously fused cultures) or from days 7 to 10 (in highly fused cultures). Test medium was supplemented with an additional 2 mmol/l L-phenylalanine to minimize reincorporation of labelled phenylalanine into cellular protein. Medium was sampled at t = 7, 21, 31 and 45 h. PD rates are expressed as the rate of decline of log10 of the percentage of the total 14C (acid-soluble 14C released into the medium + total 14C remaining in the cells) recovered in each culture well. Rates were calculated as the linear regression slope through the five time points (including time zero) in 45 h experiments or (for initial rate measurements) as the slopes of lines interpolated between the 0 and 7 h time points. Total protein and DNA were determined as described previously [11].
Quantification of apoptosis
Cells (4 x 105) were plated in growth medium on 9 cm Petris containing sterile glass microscope slides. On day 4, the cultures were rinsed with Hanks balanced salt solution (HBSS) to remove serum and serum-free test medium [MEM at pH 7.1 or 7.5 with or without 5 nmol/l dexamethasone (DEX)] was added. After incubation for 24 h, fresh test medium containing 10 µg/ml Hoechst 33342 fluorescent dye (Sigma B2261) was added. After a further 2 h incubation in the culture incubator, cultures were rinsed with HBSS, followed by fixing in phosphate-buffered saline (PBS) with 1% w/v paraformaldehyde for 15 min. The slides were then dried in air, covered with Immu-mount (Shandon 9990402) and examined on a fluorescence microscope with a Nikon UV-2A filter block (Figure 1E). Apoptotic cells (showing cytoplasmic shrinkage and nuclei with condensed fragmented chromatin) and total cells were counted over six randomly selected fields on each of three to four replicate slides. In one further experiment, apoptotic DNA strand breaks were labelled with fluorescein-tagged nucleotides using a terminal deoxynucleotidyl transferase (TUNEL) labelling kit (Roche Molecular Biochemicals 1684795). In separate cultures, cells showing primary necrosis (cell swelling, nuclear swelling, chromatin flocculation and cell membrane lysis) were assessed as described previously [13].
In the presence of serum, dividing cells with retracted cytoplasm and condensed chromatin made counting apoptotic cells difficult. Annexin V was therefore used as an assay for phosphatidylserine exposure on the surface of apoptotic cells. Starting on day 4, cells were incubated in test media (MEM + 10% FBS at pH 7.1 or 7.5 with or without 5 nmol/l DEX). After 24 h, the cells were rinsed with HB buffer comprising 10 mmol/l HEPES/NaOH pH 7.4, 150 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l MgCl2 and 1.8 mmol/l CaCl2. Fluorescein isothiocyanate (FITC)-labelled Annexin V (Bio-Whittaker BMS-306F1) diluted 1:1000 in HB was added and incubated for 8 min at room temperature. The cells were rinsed three times with HB and fixed for 10 min at +4°C in 4% w/v paraformaldehyde in PBS, followed by three rinses with PBS. Non-specific binding sites were blocked for 30 min at room temperature with 3% w/v bovine serum albumin (BSA) followed by 30 min with alkaline phosphatase-conjugated anti-FITC Fab antibody fragment (Roche Diagnostics 1426338) diluted 1:1000 in 3% BSA. Wells were washed three times with 0.02% Tween-20 in PBS followed by incubation with alkaline phosphatase substrate (1 mg/ml Sigma 104-105) in 1 mol/l diethylamine titrated to pH 9.8 with HCl. Absorbance in arbitrary units was read at 405 and 660 nm after 2 h and the difference taken as an index of Annexin V binding.
Proteolysis inhibitors used in this study were the caspase-3 inhibitor (C3I; Ac-Asp-Met-Gln-Asp-CHO; Calbiochem 235421), the 26S proteasome inhibitor MG132 (Z-Leu-Leu-Leu-CHO; Calbiochem 474790), the lysosomal proteolysis inhibitor Leupeptin (Ac-Leu-Leu-arginal hemisulphate; Calbiochem 108975), the Ca2+-dependent proteolysis (calpain) inhibitor E64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; Sigma E3132] and the autophagy inhibitor 3-methyl-adenine (3-MA; Sigma M9281).
Statistical analysis
Values are expressed as means ± SEM, with n-values denoting the number of independent experiments. Statistical significance of changes was assessed by analysis of variance with post hoc testing by Duncans multiple range test (or Tamhanes test for data sets with widely differing variance). Skewed apoptosis data in Figure 2 were analysed by Friedmans analysis of variance with paired analysis of test and control data by the Wilcoxon signed ranks test. Correlation is expressed as the Spearman rank correlation coefficient. Changes were taken to be significant if P < 0.05.

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Fig. 2. Effect of DEX and pH on the percentage of cells showing apoptotic morphology in L6G8C5 cultures stained with Hoechst 33342 on day 5 after 24 h of incubation in serum-free MEM at the specified pH. *P < 0.05 relative to corresponding values with DEX. Data from five independent experiments are shown. Similar results were obtained when apoptotic cells were detected using a TUNEL protocol.
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Results
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Unfused cells
In serum-free medium, unfused myoblasts rapidly underwent apoptosis (Figure 1C and E), which readily allowed the direction of effects of pH and GC on apoptosis to be determined. No primary necrosis [13] was detected. Apoptosis was strongly inhibited by low dose GC (5 nmol/l DEX) (Figures 1D and 2). Increasing pH also suppressed apoptosis, but this was very variable and did not reach statistical significance (Figure 2). Even when 10% serum was added (which strongly suppresses apoptosis), apoptosis was still detectable (Figure 3) and this residual apoptosis was again suppressed by DEX, although more weakly than in serum-free medium (Figures 3 vs 2). In the presence of serum, pH had no detectable effect on apoptosis (Figure 3).

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Fig. 3. Effect of DEX and pH on apoptosis assessed using Annexin V in L6G8C5 cultures on day 5 after 24 h of incubation in MEM + 10% FBS at the specified pH. *P < 0.05 relative to corresponding values with DEX.
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Cells fused spontaneously in 2% serum
Apoptosis was also suppressed by 2% serum (Figure 1A vs C) and, unlike 10% serum, low serum medium promoted fusion and differentiation of the cells to myotubes (Figure 1B). In these spontaneously fused cultures (Figure 1B), blockade of apoptosis with the specific C3I at 50 µmol/l (a dose that abolishes apoptosis in intact cells [14]) only decreased total PD rate at pH 7.4 by 9% (Figure 4A). In contrast, MG132 (an inhibitor of the ubiquitinproteasome pathway) caused strong inhibition of PD and almost abolished the increment in PD induced by acid (Table 1), suggesting that, as in skeletal muscle in vivo [2], the catabolic effect of acid occurs largely through this pathway. Leupeptin (a lysosomal proteolysis inhibitor), E64 (a calpain inhibitor) and 3-MA (an autophagy inhibitor) had much weaker inhibitory effects on PD, although 3-MA partly blunted the acid-induced increment in PD (Table 1). This and the small effect of C3I suggests that the relatively small effects of pH and GC on residual apoptotic rate (Figure 3) will have little confounding effect on total PD, particularly as not all caspase proteolytic activity in muscle cells is coupled directly to apoptosis [3].

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Fig. 4. The effect of pH, C3I and DEX on PD rate. Open bars, medium at pH 7.1; hatched bars, medium at pH 7.4. All rates were determined by linear regression over a time course 045 h after the simultaneous addition of the pH-adjusted media and C3I or DEX to the cells. Data in (A) and (B) (n = 4) are from spontaneously fused cultures to which these media were added after 8 days in culture. Data in (C) (n = 3) are from highly fused myotubes to which the media were added after 10 days in culture. *P < 0.05 vs the corresponding cultures at pH 7.4. **P < 0.05 vs cultures at pH 7.4 without C3I.
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Table 1. Influence of inhibitors of PD pathways on PD rate in spontaneously fused L6G8C5 cells in 2% v/ v serum on day 8
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Effect of pH and GC on differentiation
Acute exposure of unfused serum-free cultures (Figure 1C) or spontaneously fused cultures with 2% serum (Figure 1B) to low pH or DEX for 24 h had no detectable effect on differentiation of the cells either assessed from the degree of fusion or from measurements of CPK activity (Figure 5, main figure). More prolonged exposure of fused cultures to a pH of 7.1 (for 4 days, the longest period routinely used for PD and acidosis studies) had no significant effect on CPK activity compared with controls at pH 7.4 (Table 2). Only prolonged severe blockade of the action of endogenous GC in the serum for the whole of the growth and fusion period of the cultures, by using the GC receptor antagonist RU38486, had any significant impact on CPK activity (Figure 5, inset), reducing CPK in the cultures and impairing myotube formation. Blockade of mineralocorticoid receptors with spironolactone had no additional effect (Figure 5, inset).

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Fig. 5. (Main figure) Effect of pH and DEX (5 nmol/l) on CPK activity in L6 cells (n = 4) during 24-h incubations in medium (MEM) with or without 2% dialysed serum. Each data point denotes a separate culture well. For fused cultures, incubations commenced after 8 days on the culture plate. For unfused cultures, incubations commenced after 4 days. Open symbols, cultures with DEX; horizontal bars, means. (Inset) Effect of glucocorticoid receptor antagonist RU38486 and mineralocorticoid receptor antagonist spironolactone on CPK activity in L6 cells. Cells were cultured for 3 days in growth medium (DMEM + 10% serum), followed by 5 days in fusion medium (MEM + 2% serum) and then 3 days in test medium (MEM + 2% dialysed serum at pH 7.4). A, cultures without receptor antagonists; B, with 5 µmol/l RU38486 on days 911; C, with 5 µmol/l RU38486 throughout; D, with 5 µmol/l RU38486 throughout +5 µmol/l spironolactone on days 911. *P < 0.05 vs A.
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Table 2. Total protein and DNA contents and expression of CPK in highly fused L6G8C5 cells obtained by incubating with differentiation promoters or in cells spontaneously fused in low serum medium
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Effect of apoptosis and differentiation state on the response of PD to pH and GC
In spontaneously fused cultures with 2% serum, low pH gave a clear stimulation of PD rate (Figure 4B) and this effect was not blocked when apoptosis was inhibited with C3I (Figure 4A). Even though stimulation of PD by metabolic acidosis in vivo requires GC [9], stimulation occurred here in the absence of added GC (Figure 4A and B), indeed, as shown previously [15], addition of DEX (<500 nmol/l) caused no sustained enhancement of the rise in PD induced by acid (Figure 4B).
For generation of spontaneously fused cultures, L6G8C5 cells are routinely used in this laboratory at passages 1323. At higher passage number, fusion of the cultures is impaired (Figure 6A) and, unexpectedly under these conditions, the responsiveness of the PD rate to low pH increased. Consequently, a significant inverse correlation was observed between the magnitude of the response of PD to low pH and the degree of fusion (differentiation) of the cells (Figure 6B).

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Fig. 6. (A) Impairment of differentiation (fusion to form myotubes) in L6G8C5 cells with increasing passage number. The approximate percentage of the area of the confluent culture covered with large myotubes was estimated after 8 days in culture. Each data point denotes a separate batch of cells. Rs = 0.65, P < 0.001. (B) Blunting of the acid-induced increase in PD by increasing cell fusion to form myotubes. The initial PD rate (t = 07 h after addition of the pH-adjusted media) was measured after 8 days on the culture plate in the same cultures shown in (A). PD was measured in MEM + 2% dialysed serum at pH 7.1 or 7.4 and the percentage increase was determined. Rs = 0.51, P < 0.001.
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Highly fused myotubes
To confirm this apparent impairment in myotubes of the catabolic response to low pH, highly fused cultures expressing high levels of CPK and elevated levels of total protein (Table 2) were prepared by the protocol of Elsner et al. [12]. These were morphologically identical to those described in the original study [12], but the myotubes showed a decline in total protein and a small decline in DNA between days 10 and 14 (Table 2), i.e. after the differentiation mixture containing strongly anabolic IGF-I had been removed from the cultures (see Subjects and methods). Cell viability, however, was unaffected and differentiation was maintained, as shown by high activity of CPK on day 14 (Table 2) and occasional spontaneous tetanic contractions of the myotubes on days 1214, as reported previously [12].
The impaired response to acid noted above (Figure 6B) was confirmed in these cells. No stimulation of PD whatever was detected with acid alone (Figure 4C). In part this might have arisen because basal PD rate had already been elevated (Figure 4C vs 4B) by withdrawal of the anabolic differentiation mixture. However, the response to acid was partly restored in the presence of GC (Figure 4C), in agreement with the effect of GC on acid-induced PD in the skeletal muscle of adrenalectomized rats in vivo [2].
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Discussion
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The effect of acid on PD in vitro is not an artefact of apoptosis or dedifferentiation
The results of the present study and earlier work [6] show that, in addition to their well-described effects on protein turnover in skeletal muscle, GC [6] and, to a lesser extent, pH exert effects on apoptosis and differentiation of cultured skeletal muscle cells. These observations do not preclude the use of these cells for studies of catabolic states, however. Culture conditions were found under which apoptosis and differentiation are not major confounding factors in measurements of PD. In the presence of serum, pH has no significant effect on apoptosis (Figure 3) or differentiation (Figure 5 and Table 2) and GC promotes rather than prevents differentiation (Figure 5, inset) and suppresses rather than increases apoptosis (Figures 2 and 3; [6]), in contrast with the stimulation of apoptosis by GC observed in other cell types [16,17]. The stimulation of total PD by acid and GC observed in L6G8C5 cells cannot, therefore, be attributed to PD secondary to increased apoptosis or dedifferentiation. Viewed in conjunction with earlier work [7,11], the effects of acid and GC on PD in these cells seem therefore to be a reasonable model for the analogous catabolic effects in vivo. At least for acid, these occur through the same PD pathway (the ubiquitin-proteasome pathway) (Table 1), which is activated in uraemic metabolic acidosis in vivo [2].
It should be emphasized, however, that these findings in vitro do not completely rule out a role for muscle differentiation or apoptosis in the catabolic state or in the changes in muscle fibre composition [5] during CRF in vivo. Apoptotic and differentiation effects do occur in mature skeletal muscle in vivo. In addition to the caspase activation in cancer cachexia [3] and apoptosis during muscle wasting following burns [4], impaired differentiation like that observed on prolonged GC receptor blockade in Figure 5 has also been observed in the muscles of adrenalectomized rats in vivo and is reversed by GC supplements [18].
Why does the catabolic response to acid decline in fused cultures?
A potentially important observation in this study was that the response of PD to low pH was strongly suppressed in fused cells (Figures 4C and 6B). Like adrenalectomized rat skeletal muscle in vivo [9], myotubes were insensitive to acid alone, but the effect was restored in vivo [9] and weakly restored in myotubes (Figure 4C) when GC was added. This restoration of pH sensitivity by GC is reminiscent of the observation that pH sensitivity of PD in L6G8C5 cells re-emerges on exposure to GC after acid-induced PD has been suppressed by a low dose of IGF-I [10]. A possible explanation for the present results (Figures 4C and 6B), therefore, is that the pH sensitivity of PD does not depend on differentiation per se. It may depend instead on the changes in the ambient concentration of insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs), which are known to accompany fusion and differentiation [19]. In view of the observation that the metabolic effects of acid on these cells resemble those of insulin withdrawal [11], the role of IGFs and IGFBPs in acid-induced protein wasting clearly merits further investigation.
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Acknowledgments
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The authors gratefully acknowledge support for this work through project grants from the National Kidney Research Fund (ref. R29/1/97), the Wellcome Trust (ref. 059828/Z/99/Z) and the Renal Care and Research Association (ref. 452001). M.-K.C. thanks the Rank Prize Funds for a research studentship.
Conflict of interest statement. None declared.
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Notes
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Deceased. 
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Received for publication: 30.10.02
Accepted in revised form: 6. 3.03