Glucocorticoid antagonist RU38486 fails to block acid-induced muscle wasting in vivo or in vitro
Warren P. Pickering,
Frease E. Baker,
Jeremy Brown,
Heather L. Butler,
Sheena Govindji,
Julie M. Parsons,
Izabella Z. A. Pawluczyk,
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. Increased protein degradation during metabolic acidosis contributes to muscle wasting in uraemia. Adrenalectomy experiments in severely acidotic rats (arterial pH
7.15) have shown that this is prevented in the absence of glucocorticoid. It should therefore be possible to block such muscle wasting with glucocorticoid receptor antagonist 11ß-(4-dimethylaminophenyl)-17ß-hydroxy,-17a-(prop-1-ynyl)-estra-4,9-dien-3-one (RU38486).
Methods. The effect of oral RU38486 (50 mg/kg body weight/day) was studied in vivo by administration to rats receiving dietary HCl supplements which yielded moderate acidosis (plasma HCO3 19.7 ± 1.2 mmol/l), comparable with that observed in uraemia. The effect of the glucocorticoid dexamethasone (DEX) (up to 500 nmol/l) and RU38486 (up to 5 µmol/l) was also studied in vitro in acidified cultures of L6-G8C5 rat skeletal muscle cells.
Results. In vivo 15 days of moderate acidosis slowed weight gain and induced muscle wasting (6% weight loss in gastrocnemius with a commensurate decline in muscle protein) but, at this level of acidosis, muscle protein degradation showed no detectable increase. Wasting was not inhibited by RU38486 in spite of blockade of 80% of the glucocorticoid receptors in gastrocnemius. Unexpectedly, weight gain was significantly slower in acidotic rats receiving RU38486 than in acidotic rats receiving vehicle. In vitro acid spontaneously stimulated protein degradation, but even under strongly acidic conditions (pH 7.1) this was only weakly and transiently stimulated by 5 nmol/l DEX and transiently blunted by 5 µmol/l RU38486. In contrast, as little as 1 nmol/l insulin-like growth factor I (IGF-I) almost abolished the effect of acid and this was partly restored by 5 nmol/l DEX.
Conclusions. IGF-I is a potent determinant of acid-induced protein degradation in vitro and is antagonized by glucocorticoid. If glucocorticoid acts in this indirect way in vivo this may explain why, in moderate metabolic acidosis with intact adrenal glands, the action of RU38486 via glucocorticoid is too weak to be of therapeutic value.
Keywords: glucocorticoid; insulin-like growth factor I; metabolic acidosis; protein degradation; RU38486; skeletal muscle
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Introduction
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Metabolic acidosis is a contributor to the increased protein degradation (PD) and skeletal muscle wasting observed in uraemia [1,2], but there are few treatments available apart from alkali supplements such as NaHCO3 which, in renal patients, carry the risk of Na loading and fluid overload. An alternative approach is suggested by the observation in rats that glucocorticoid secretion increases during metabolic acidosis [3] and that the accompanying catabolic state in skeletal muscle is abolished by adrenalectomy [3,4] and restored by administration of glucocorticoid [3,4]. It is possible therefore that glucocorticoid rather than acid itself is the main determinant of protein catabolism during acidosis. If so, the glucocorticoid receptor antagonist 11ß-(4-dimethylaminophenyl)-17ß-hydroxy,-17a-(prop-1-ynyl)-estra-4,9-dien-3-one (RU38486), which has been shown to block muscle wasting induced by externally administered glucocorticoid in rats [5], should be an effective treatment for muscle wasting in metabolic acidosis.
The initial aim of this study was therefore to attempt to block the catabolic effect of acid in vivo using RU38486 in rats rendered acidotic by administration of a dietary acid load. The failure of this in vivo experiment to show the expected anabolic effect of RU38486 then prompted us to re-examine the role of glucocorticoid in acid-induced muscle wasting, using the L6 skeletal muscle cell line from rat which has been shown previously to be a useful in vitro model of protein wasting in metabolic acidosis [6]. The transience of the effects of glucocorticoid in L6 cells then led us to investigate whether the presence of another factor [insulin-like growth factor I (IGF-I)] was also required for glucocorticoid to act on PD in this cell line.
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Subjects and methods
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Study of the effect of RU38486 during metabolic acidosis in vivo
Twenty adult male rats were randomly allocated to four groups designated C (control), RC (control receiving RU38486), A (acidotic) and RA (acidotic receiving RU38486). The animals were housed in conditions of constant temperature and humidity with a 12 h light/dark cycle. They were fed a diet containing 20 g of protein (as casein) per 100 g dry weight (ICN 960259) with free access to water. To make the diet easier to handle, its viscosity was increased with methyl cellulose (Sigma M0512, 4000 centipoises viscosity at 2% w/v at 20°C) by mixing 24.5 g of dry diet with 24.5 ml of water and 1 g of methyl cellulose. After a run-in period of 15 days, the diet of animals in Groups A and RA was made up in 0.25 mol/l HCl instead of water, a dose calculated to administer 35 mmol of HCl per day per kg body weight as in an earlier study [7]. As a positive control, to confirm the correlation between plasma [HCO3
] and muscle PD reported previously [3,4], a separate group of five rats (with three simultaneous non-acidotic controls) was given a dietary acid load of 70 mmol of HCl per day per kg body weight.
Animals in Groups RC and RA received RU38486 by gavage in a single daily dose of 50 mg per kg body weight in suspension in an aqueous vehicle [5] of 0.25% (w/v) carboxymethyl-cellulose and 0.2% (w/v) polysorbate 80. Animals in Groups C and A received vehicle alone. Pair-feeding was performed to ensure that food intake in the acidotic animals was matched to that in the corresponding non-acidotic controls. Animals were weighed on days 0, 5, 10 and 15. Urine was collected in the last 24 h before death and stored at 20°C. After 15 days (
24 h after the last dose of drug) the animals were killed under light anaesthesia and exsanguinated by aortic cannulation. Blood pH and bicarbonate were immediately determined on a Corning 238 blood gas analyzer. Skeletal muscle was then processed as follows.
Glucocorticoid receptor binding in soluble protein fraction. The left gastrocnemius muscle (
2 g) was weighed, and homogenized using a T8 motorized homogenizer (IKA Labortechnik, Germany) in 5 ml of ice-cold buffer [5] comprising 25 mmol/l Na2HPO4, 1.5 mmol/l Na2EDTA, 10% (v/v) glycerol, 2 mmol/l dithiothreitol, 10 mmol/l Na2MoO4 and 1 mmol/l phenylmethylsulfonyl fluoride, adjusted to pH 7.2 at room temperature with HCl. The homogenate was stored under liquid nitrogen. Homogenates were thawed on ice and centrifuged for 60 min at 110 000 g at +4°C in a Beckman L8-60M preparative ultracentrifuge. The supernatant (soluble protein fraction) was used immediately for determination of total protein (Figure 1C) [6] and specific glucocorticoid receptor binding with a single saturating dose (15 nmol/l) of 3H-triamcinolone acetonide (3H-TA) (New England Nuclear NET 470) exactly as described previously [5]. A similar procedure was used for whole homogenates of L6-G8C5 cells.

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Fig. 1. Effect of 15 days of acid loading (35 mmol/kg body weight/day) and RU38486 (50 mg/kg body weight/day) on tissue weight and muscle protein (five rats in each group). (A) Muscle mean wet weights: gastrocnemius (open circles), heart (filled circles), soleus (open squares), EDL (filled squares). Acidosis significantly decreased weight (P < 0.05) in all except soleus. (B) Kidney wet weights. Acidosis (A) significantly increased weight relative to control (C) (*P < 0.05). (C) Influence of plasma [HCO3] on soluble protein content of gastrocnemius in rats receiving vehicle only (correlation coefficient Rs = 0.80, P = 0.006).
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Ubiquitin mRNA determination. The right gastrocnemius muscle was immediately removed and frozen in liquid nitrogen. Total RNA was isolated using an acid phenol-guanidinium reagent (Trizol, Life Technologies 15596) and was quantified by measuring absorption at 260/280 nm. RNA (30 µg per sample) was separated by electrophoresis in a 1% agarose 1.9% formaldehyde gel, transferred to a nylon membrane (Hybond, Amersham RPN 203N) and cross-linked to the membrane by ultraviolet irradiation. Membranes were incubated at 42°C for 4 h in a pre-hybridization solution containing 5x SSPE (Sigma S2015), 5x Denhardts solution (1x = 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 50% deionized formamide, 1% sodium dodecyl sulphate (SDS) and 200 µg/ml salmon sperm DNA. Membranes were then hybridized sequentially with 32P-dCTP-labelled cDNA probes (Promega 'Prime-a-Gene) for human ubiquitin and human cyclophilin in a hybridization solution of the same composition as above at 42°C overnight. After hybridization, membranes were washed twice with 2x SSPE, 0.2% SDS, at room temperature for 10 min, followed by two washes with 0.2x SSPE, 0.2% SDS at 65°C for 30 min each. Autoradiographic signals were quantified in arbitrary units using a Bio-Rad GS700 Imaging Densitometer. Densitometric signals for ubiquitin were normalized by expressing them relative to the corresponding cyclophilin values to correct for variations in the amount of RNA loaded onto the gel.
Ex vivo determination of muscle PD rate
The total PD rate was measured as described in Hasselgren et al. [8] from the rate of tyrosine (Tyr) output from extensor digitorum longus (EDL) muscles incubated in vitro in the presence of cycloheximide to block Tyr re-incorporation by protein synthesis. Briefly, EDLs were excised, weighed, attached to steel supports to maintain them at resting length [8] and transferred to 3 ml of KrebsHenseleit bicarbonate buffer (K-H) with 5 mmol/l glucose and 20 mmol/l N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid] (HEPES). This was incubated at 37°C under 95%O2/5%CO2 at pH 7.4 for 30 min with frequent swirling of the buffer to maximize gas exchange between the muscles and the K-H. The buffer was then discarded and incubated as above for a further 2 h in fresh K-H without HEPES, and supplemented with 0.5 mmol/l cycloheximide. Tyr release into the buffer was determined as described previously [7] and expressed as nmol/g wet weight over the 2 h incubation.
Extraction and assay of corticosterone from urine
The procedure was based on that described previously [3]. Urine was centrifuged for 10 min at 2000 g. To 1 ml of supernatant, 10 nCi (370 Bq) of [1,2,6,7-3H]corticosterone (Amersham TRK 406) was added as internal standard in 20 µl of dimethyl sulphoxide (DMSO). Corticosterone was extracted by vortexing with 1 ml of dichloromethane followed by centrifugation at 3000 g for 10 min at +4°C. The yellow aqueous phase was discarded. The residue was dried down under oxygen-free nitrogen, dissolved in 200 µl of 0.1 mol/l tris[hydroxymethyl]aminomethane (Tris), titrated to pH 7.4 with HCl, and applied to a 360 mg C18 reverse-phase column (Waters Corporation, Sep-Pak ref. WAT051910) which had been wetted with 1 ml of acetonitrile at 1 ml/min, followed by 2 ml of 0.1 mol/l Tris, pH 7.4 at 1 ml/min. The corticosterone-loaded column was washed with 10 ml of H2O at 23 ml/min, followed by 2 ml of 20% (v/v) aqueous acetonitrile at 12 ml/min. Corticosterone was eluted with 1.5 ml of acetonitrile at 0.5 ml/min. The first 0.5 ml of eluate was discarded as it contained negligible corticosterone. The remainder was dried down as above and dissolved by vortexing for 10 min in 0.5 ml of 0.1 mol/l Tris, pH 7.4. Total corticosterone was determined in this extract by 125I radio-immunoassay (Amersham, Biotrak ref. RPA 548) and 3H-corticosterone recovery (65 ± 2%, n = 20) was assessed by liquid scintillation counting.
Cell culture
L6-G8C5 rat skeletal muscle cells [L6 sub-clone G8C5 from the European Collection of Animal Cell Cultures (ECACC ref. 92121114)] were seeded at passage 1331 at 4.5 x 104 cells/cm2 on plastic culture wells in growth medium comprising Dulbeccos modified Eagles medium (Life Technologies 11880) with 5 mmol/l glucose and 1 mmol/l pyruvate, 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). FBS was obtained from Life Technologies (10106) and contained less than 20 nmol of cortisol per litre. All sera were heat-inactivated for 30 min at 56°C before use. Cultures were incubated at 37°C under humidified 95% air/5% CO2. By day 4 (i.e. after 3 days), the myoblasts were confluent and aligned and were switched to Eagles minimum essential medium with Earles salts (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, fusion to form myotubes had occurred. Previous studies have shown, however, that fusion is not a prerequisite for acid-induced protein wasting to be observed in this cell line [6].
Test medium at pH 7.17.5 comprising MEM as above, but with dialysed FBS (Life Technologies 10110), was added from day 8 onwards. In previous studies, pH values of 7.1 and 7.5 were chosen because the relationship between extracellular pH and protein wasting is approximately linear over this pH range [6]. To demonstrate that alkalaemia is not a requirement for the effects described here, some experiments were performed at the more physiological control pH of 7.4 (see Results). The pH was adjusted by addition of HCl or NaHCO3, with extra NaCl at low pH to maintain a constant Na concentration. Dexamethasone (DEX) (Sigma D-1756) and RU38486 (Mifepristone from Roussel UCLAF) were added dissolved in DMSO. The same total concentration of DMSO (0.011% v/v final concentration) was added to control cultures. To obtain test media depleted of steroid, MEM was made up without phenol red and was supplemented with charcoal-stripped dialysed FBS. Activated charcoal (0.75 g of Merck 33032) was wetted with 2 ml of water and centrifuged at 900 g for 5 min at 20°C. The supernatant was discarded and the charcoal pellet was resuspended in 10 ml of dialysed FBS followed by incubation at +4°C for 30 min. The charcoal was removed by centrifugation at 3000 g for 10 min at +4°C and the supernatant was sterilized by filtration through a 0.8 µm filter followed by a 0.2 µm filter.
Measurements
For studies of PD, L-[U-14C]-phenylalanine (Amersham CFB 70) was added to L6-G8C5 cells in 35 mm diameter culture wells during the fusion period (days 48 inclusive) at a final concentration of 0.23 mCi/l (8.5 MBq/l) to label the cellular proteins. At the end of this labelling period, cultures were incubated in 2 ml of unlabelled MEM + 2% FBS for 2 h to eliminate radioactivity from rapidly degraded proteins [6]. The medium was then discarded, and at this point (designated time zero), 3 ml of test medium was added. From this point onwards, test media were supplemented with unlabelled L-phenylalanine (2 mmol/l) to minimize re-incorporation of labelled phenylalanine into cellular protein [6]. Rates of PD were measured from the rate of release of acid-soluble 14C into the culture medium [6] by sampling 0.3 ml aliquots after 7, 21, 31, 45 and 55 h. (For experiments with IGF-I, incubations were shortened to 31 h to avoid nutrient exhaustion through insulin-like effects.) The labelled medium was mixed with an equal volume of 20% (w/v) trichloroacetic acid and chilled at +4°C for at least 30 min to precipitate protein. The samples were then centrifuged at 3000 g for 10 min at +4°C and 14C-Phe activity determined in the supernatant by liquid scintillation counting as an index of PD [6]. 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 plus total 14C remaining in the cells) recovered in each culture well [6]. Rates were calculated as the linear regression slope through the time points (including time zero) in 3155 h experiments (Figures 2B, 4, 6 and 8) or as the slopes of lines interpolated between the 0 and 7 h time points (Figures 5 and 7). 14C-Phe activity was also determined in the protein pellet derived from the acid-treated medium, as an index of cell detachment and leakage of intact labelled proteins. None of the conditions tested had any significant effect on this activity nor on the total 14C recovered in each culture well.

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Fig. 2. Relationship between the PD rate in rat skeletal muscle cells and severity of metabolic acidosis. (A) Tyr output from rat extensor digitorum longus muscles incubated for 2 h ex vivo in K-H, pH 7.4 at 37°C with 0.5 mmol/l cycloheximide after 15 days with (n = 5) or without (n = 3) oral HCl supplements of 70 mmol/kg body weight/day (correlation coefficient Rs = 0.76, P = 0.03). Vertical dashed lines indicate the mean plasma HCO3 concentration for the acidotic and control groups of rats in Table 1 and Figure 1. (B) Rate of release of 14C-Phe from pre-labelled cell protein in cultures of L6-G8C5 cells incubated for 45 h in MEM + 2% (v/v) dialysed serum at the specified HCO3 concentration starting on day 8 (see Subjects and methods) (correlation coefficient Rs = 0.946, P < 0.01).
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Fig. 5. (A) Doseresponse curves (n = 3) of the effect of DEX on PD rate measured over the first 7 h. Solid line denotes pH 7.1; dashed line denotes pH 7.5. *P < 0.05 vs corresponding values without DEX. (B) Effect of DEX (5 nmol/l) (open squares) on the time course of the acid-induced rise in PD in L6-G8C5 cells, commencing on day 8. Each data point represents the mean PD rate (n = 10) measured over the preceding time interval. #P < 0.01 vs the simultaneous value without DEX (filled squares). (C) Effect of RU38486 (5 µmol/l) on the acid-induced rise in PD rate in L6-G8C5 cells (n = 4). PD rate was determined over the first 7 h, commencing on day 8. **P < 0.04 vs control.
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Table 1. Influence of 15 days of acid-loading (35 mmol/ kg body weight/ day) and RU38486 (50 mg/ kg body weight/ day) in rats (n = 5 for all groups)
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At the end of the incubations, the cultures were placed on ice and rinsed three times with 0.9% (w/v) NaCl to remove extracellular protein and 14C-Phe before storage at 20°C. Cultures were thawed at room temperature, scraped in 1.00 ml of 0.5 M NaOH, digested at 70°C for 30 min and residual 14C-Phe activity determined by liquid scintillation counting.
Statistical analysis
Values are expressed as means ± SEM. Statistical significance of changes was assessed by unpaired Students t-test or, for multiple comparisons, by analysis of variance and Duncans multiple range test. Correlation is expressed as the Spearman rank correlation coefficient Rs. Effects were regarded as significant if P < 0.05. In culture experiments the n value denotes the number of independent experiments. For the small transient catabolic effects of DEX it was necessary to run each experiment 310 times with four to six replicate culture wells in each experiment. Pooled data from all experiments are presented.
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Results
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Effects of glucocorticoid antagonist during metabolic acidosis in vivo
Control and acidotic rats were treated with oral glucocorticoid antagonist RU38486 or vehicle for a period of 15 days. For acid-treated animals receiving vehicle, sufficient dietary acid was administered to induce metabolic acidosis with a moderate reduction in plasma HCO3 concentration and no significant decline in arterial pH (Table 1), the situation usually encountered in uraemic metabolic acidosis [2]. In the rats receiving RU38486, despite ingestion of the same food and acid intake (Table 1), both a significant decline in plasma HCO3 concentration and a significant fall in arterial pH were observed in acid-loaded animals relative to the controls receiving drug alone (Table 1).
Body weight gain over the 15 day study was significantly blunted in the acidotic animals compared with the controls (Table 1) and a catabolic state was detected in the skeletal muscle (gastrocnemius) of the acidotic group with an increase in mRNA for the catabolic marker ubiquitin (Table 1) and a net decline in muscle protein as plasma [HCO3] declined (Figure 1C). However, with this degree of acid-loading, stimulation of the PD rate (Tyr output from the animals EDLs) was too small to detect (Table 1). In a separate group of rats the anticipated inverse correlation between Tyr output and plasma [HCO3] was demonstrable (Figure 2A) but, as in L6G8C5 cells in vitro (Figure 2B) a much wider range of HCO3 concentration (13.726.3 mM, arterial pH range
7.17.45) had to be imposed for the effect to be observed, and the correlation was strongly dependent on the extreme values at high and low HCO3 concentration.
Oral RU38486 strongly inhibited specific binding of 3H-TA to glucocorticoid receptors in both acidotic and control rats in a soluble protein fraction derived from gastrocnemius (Table 1), suggesting that
80% of the receptors had been blocked by the drug. The 3H-TA binding data were corrected for the decline in total soluble protein content that occurred in these muscles with declining plasma [HCO3] (Figure 1C).
In spite of this receptor blockade, RU38486 did not significantly blunt the catabolic effect of acidosis on body weight (Table 1), wet weight of individual muscles (Figure 1A), or ubiquitin mRNA (Table 1), and unexpectedly Tyr output from the EDLs of non-acidotic animals was increased by the drug (Table 1). Furthermore, the 15 day body weight gain in the acidotic group receiving RU38486 was less than that in the acidotic group receiving vehicle (Table 1), suggesting that the drug was worsening the catabolic state. In contrast, acidosis exerted a hypertrophic effect on the rats kidneys (Figure 1B) and, unlike the effects on muscle (Figure 1A), this effect was no longer detectable in the presence of RU38486 (Figure 1B).
In the acidotic rats, urinary corticosterone excretion was increased (Table 1) as reported previously [3], and treatment with RU38486 had no effect on this. The failure of RU38486 to block muscle wasting in the acidotic animals cannot therefore be attributed to a compensatory increase in corticosterone secretion in response to receptor blockade by the drug.
Effects of glucocorticoid and acid in vitro
Sensitivity of L6-G8C5 cells to glucocorticoid.
The presence of glucocorticoid receptors in L6-G8C5 myoblasts and in fused cultures was demonstrated by measuring specific binding of 3H-TA (Figure 3). In homogenates from both types of culture, binding of this ligand was displaced by DEX and RU38486 over a dose range of 5500 nmol/l (Figure 3), implying that both agents essentially saturate the glucocorticoid receptors at 500 nmol/l irrespective of the degree of cell differentiation.

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Fig. 3. Displacement by competing ligands of specific binding of 3H-TA (15 nmol/l) to glucocorticoid receptors in homogenates of L6-G8C5 cells. DEX (filled squares) and RU38486 (open squares) in myoblasts on day 4. DEX (filled triangles) and RU38486 (open triangles) in fused cultures on day 8.
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Effect of low-dose glucocorticoid on L6-G8C5 cells.
No catabolic effect of 5 nmol/l DEX was detected when the rate of PD was determined over a 55 h time course (Figure 4), indeed the rate was slightly decreased by DEX both at pH 7.1 and 7.5. As reported previously [6], lowering the pH of the culture medium to 7.1 was sufficient to increase PD even in the absence of added glucocorticoid, and this effect was not enhanced by DEX at 5 nmol/l nor was it blocked by addition of RU38486 at 50 nmol/l (Figure 4).

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Fig. 4. Effect of DEX (5 nmol/l) and RU38486 (50 nmol/l) on the PD rate of L6-G8C5 cells (n = 4). PD rate was determined over 55 h commencing on day 8. Open bars denote cultures at pH 7.1. Hatched bars denote cultures at pH 7.5. *P < 0.03 vs corresponding values at pH 7.5. #P < 0.03 vs control values at the same pH.
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Transient effects on PD rate.
Even though DEX did not stimulate the rate of PD averaged over a 55 h experiment, it was noted that the initial rate of PD (measured from t = 0 to 7 h) (Figure 5A) and the accompanying acid-induced rise in PD (Figure 5B) were both transiently increased by DEX. In further measurements at t = 2, 4 and 7 h (data not shown) the catabolic effect of DEX was detectable from 4 h onwards.
At pH 7.1 the stimulation of PD by DEX at t = 07 h was blocked by RU38486 (Table 2), suggesting that classical glucocorticoid receptors were involved. Even in the absence of added glucocorticoid, 5 µM RU38486 halved the acid-induced rise in PD at t = 07 h (Figure 5C), suggesting that the trace of glucocorticoid in the 2% serum in the culture medium was initially enhancing the effect of acid. However, RU38486 failed to blunt the effect of acid in longer incubations (Figure 4). Similar negative results were obtained in three further 55 h experiments in which glucocorticoid action was blocked by the alternative approach of stripping steroid-like molecules from the medium using charcoal-treated serum and incubation without phenol red (data not shown). It should be emphasized that this persistent stimulation of PD at pH 7.1 in the absence of added glucocorticoid was not an artifact of using an alkalaemic control pH (7.5). The same result was obtained when a control pH of 7.4 was used (Figures 6 and 8).

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Fig. 6. Suppression of acid-induced PD by IGF-I in prolonged incubations (n = 37). PD rate was determined over 31 h commencing on day 8. Open bars denote incubation at pH 7.1, hatched bars pH 7.4. All media contained 2% DFBS. *P < 0.05 vs corresponding value at pH 7.4. #P < 0.05 vs value without IGF-I at pH 7.4.
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Does glucocorticoid antagonize a transient anabolic factor?
A possible explanation for the transient (07 h) enhancement of acid-induced PD by DEX (Figure 5B) and the transient blunting of it by RU38486 (Figure 5C) is that glucocorticoid acts by antagonizing the effect of an anabolic factor which suppresses PD only in the first 7 h after fresh medium is added to the cultures. Serum factors known to be antagonized by glucocorticoid are insulin and the IGFs. IGF-I seemed a promising candidate (see Discussion) as it is gradually inactivated when added to muscle cell cultures by IGF-binding proteins (IGFBPs) secreted by the cells [9].
In L6-G8C5 cultures, large IGF-I supplements (10100 nmol/l) abolished the acid-induced increase in PD (Figure 6). A more physiological IGF-I supplement (1 nmol/l) transiently prevented acid-induced PD during the first 7 h (Figure 7) but not in longer incubations (Figure 6). The re-emergence of acid-induced PD after 7 h with 1 nmol/l IGF-I was almost prevented (B in Figure 8) if fresh medium with 1 nmol/l IGF-I was added at intervals to replenish free IGF-I and remove putative inhibitors such as IGFBPs. As predicted, this suppression by IGF-I was reversed when 5 nmol/l DEX was added with the IGF-I (C in Figure 8).

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Fig. 7. Suppression of acid-induced PD by IGF-I in short incubations (n = 4). PD rate was determined over 7 h commencing on day 8. Open bars denote incubation at pH 7.1, hatched bars pH 7.4. All media contained 2% DFBS. *P < 0.05 vs corresponding value at pH 7.4. #P < 0.05 vs value without IGF-I at pH 7.4.
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Fig. 8. Effect of replenishing culture medium supplemented with 1 nmol/l IGF-I on the acid-induced rise in the PD rate. Rates pooled from three experiments with three to four replicate wells in each, determined over 31 h. (A) Continuous incubation in medium with an initial IGF-I supplement of 1 nmol/l for 31 h as in Figure 6. (B) Parallel cultures in which fresh medium was added at t = 7 and 21 h. (C) Parallel cultures treated as in (B) but with addition of 5 nmol/l DEX. Each data point is the difference between an adjacent pair of pH 7.1 and 7.4 culture wells. *P < 0.05 vs A. #P < 0.05 vs B.
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Discussion
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The requirement for glucocorticoid: direct or indirect?
The previously reported complete abolition by adrenalectomy of acid-induced PD in muscle in vivo and its restoration by glucocorticoid [3,4] implies a key role for glucocorticoid in the catabolic action of acid. In contrast, the failure of adrenalectomy in the original studies to prevent acid-induced weight loss in rats (table 1 in May et al. [4]), the failure of a high dose of RU38486 to block the wasting effects of acidosis in vivo (Table 1 and Figure 1), the relatively small increase in glucocorticoid secretion observed during moderate metabolic acidosis (Table 1), and the weak response of acid-induced PD to glucocorticoid alone in L6 cells (Figures 4 and 5) all suggest that glucocorticoid is not the sole determinant of protein wasting during metabolic acidosis. The observation of direct effects of extracellular pH on PD in L6 cells in vitro [6] (Figures 48) suggests that low pH itself is important, possibly by inhibiting the pH-sensitive System A amino acid transporters [10]. The strong suppression of this response to pH in vitro by IGF-I (Figures 68) and its reversal by glucocorticoid (Figure 8), suggest that glucocorticoids effect could be indirect, occurring through an endogenous anabolic factor such as IGF-I.
If this is true then the observed differences in weight gain across the four groups of rats in Table 1 (in spite of comparable food intake) arise through variation in the severity of acidaemia [through differences in acid loading and through RU38486 effects on acid excretion (see below)]. These effects of acidaemia must be distal to food intake, possibly acting on the supply of ingested nutrients to the cells through acid-inhibitable transporters such as System A as discussed elsewhere [10].
Why are glucocorticoid effects in L6 smaller than in adrenalectomized rats?
Even though enhancement of acid-induced PD by DEX was clearly demonstrated in L6 cells (Figures 5B and 8), the response was not as dramatic as in adrenalectomized rats in vivo [3,4]. The anti-IGF-I actions of glucocorticoid in vivo are well documented and involve at least four mechanisms, some of which are absent in L6 cultures. First, circulating IGF-I is decreased, in part because of decreased synthesis in liver [11,12] and other tissues through blockade of IGF-I induction by growth hormone [13]. Secondly, endogenous IGF-I expression in muscle may also be decreased [12]. Thirdly, the free concentration of IGF-I may decrease because of increased output of the glucocorticoid-inducible IGF-binding protein IGFBP1 from liver [14]. Fourthly, IGF-I action on skeletal muscle cells is blunted by impairment of intracellular signalling through p70 S6 kinase [15]. Of these four mechanisms, only the last has been demonstrated in L6 cells [15] and the first and third are not applicable in isolated muscle cell cultures in vitro.
Is the glucocorticoid receptor a suitable target for therapy?
In spite of blockade of 80% of the glucocorticoid receptors in gastrocnemius, RU38486 failed to prevent the wasting disorder in metabolic acidosis (Table 1 and Figure 1A). This failure is unlikely to arise from incomplete receptor blockade, because the same dose of RU38486 did prevent the muscle wasting induced in non-acidotic rats by a high dose of exogenous glucocorticoid [5]. This dose of RU38486 also apparently blunted acid-induced renal hypertrophy in the present study (Figure 1B). It is possible that an anabolic effect of RU38486 in muscle might have been observed if the rats in the present study had received a larger acid load (as in Figure 2A) sufficient to allow direct measurement of increased muscle PD. However, this also seems an inadequate explanation because the severity of acidosis achieved was close to that in chronic uraemia [2] and led to clear weight loss (Table 1), muscle wasting (Figure 1A) and increased ubiquitin mRNA (Table 1) which all failed to improve with RU38486.
Unexpectedly RU38486 seemed to worsen the wasting disorder in two ways (Table 1). First, in non-acidotic rats, muscle Tyr output increased (Table 1). The reason is unknown but may relate to the reproducible longer term suppression of PD observed with low-dose glucocorticoid in vitro (Figure 4) and to the myopathy [16], myalgia, rhabdomyolysis and loss of the muscle differentiation marker creatine phosphokinase (EC 2.7.3.2) [17] reported on prolonged corticosteroid deprivation in vivo, effects which are at least partly attributable to glucocorticoid [17]. This increase in muscle Tyr output was accompanied by a smaller (statistically insignificant) increase in ubiquitin mRNA (Table 1). The reason for this quantitative discrepancy between the two catabolic markers is unknown, but it should be noted that the ubiquitin mRNA and Tyr output were measured in different muscles, and probe early and late events, respectively, in the process of protein catabolism. The large increase in Tyr output observed in EDL of non-acidotic rats receiving RU38486 probably does not reflect a corresponding increase in PD in all muscles, as the anticipated marked decline in body weight was not observed in these rats (Table 1).
Secondly, the already impaired 15 day weight gain in acidotic rats was worsened by the drug (Table 1). The same effect of glucocorticoid withdrawal is observed in adrenalectomized rats (table 1 in May et al. [4]), i.e. in contrast to the effect of adrenalectomy on acid-induced PD, adrenalectomy seems to worsen the effect of acid-loading on body weight. As food intake was not significantly affected in the present study (Table 1), this did not arise from greater acid intake in the RU38486 animals (Table 1). It appears however that RU38486 did reach functionally significant concentrations in the rats kidneys, with an apparent blunting of acid-induced renal hypertrophy (Figure 1B). There is abundant evidence that in kidney glucocorticoid stimulates ammoniagenesis and renal acid excretion and that RU38486 blunts urinary NH4+ excretion in acid-loaded rats [18]. Such a renal action of the drug may explain the statistically significant fall in arterial pH in the acid-loaded RU38486 group (Table 1). A related problem is encountered in adrenalectomy experiments [19] and necessitates giving sodium supplements [3,4,19] to prevent acidosis [19]. Clearly in renal patients, in whom acid excretion is already seriously impaired, such acid retention would not be a desirable feature of glucocorticoid antagonism.
A final reason for caution over the idea that glucocorticoid is the key determinant of acid-induced protein wasting is that, to date, the enhancing effect of glucocorticoid has only been shown at an arterial pH of
7.15 in vivo [3,4] and an extracellular pH of 7.1 in vitro [20] (Figures 5 and 8). The effects of glucocorticoid in adrenalectomy experiments in vivo were also observed in the context of complete corticosteroid deprivation which may lead to myopathies [16,17] and necessitates sodium supplementation [3,4,19] as discussed above. It may therefore be unwise to extrapolate from these results to the more moderate acidosis observed in chronically uraemic subjects with intact adrenal glands in whom plasma [HCO3] is rarely <17 mM, and respiratory compensation leads to an arterial pH no lower than 7.3 [2]. The failure of RU38486 to block wasting in moderate acidosis in Table 1 and Figure 1A conceivably arises therefore through some difference (possibly related to IGFs or IGFBPs) between the glucocorticoid requirement in moderate acidosis, and severe acidosis in the context of adrenalectomy.
In conclusion, glucocorticoid receptor blockade in moderate acidosis in vivo fails to blunt acid-induced wasting, and glucocorticoid alone (even in severe acidaemia in vitro) exerts only a weak effect on acid-induced PD. In contrast, IGF-I is a potent determinant of acid-induced PD in vitro and is antagonized by glucocorticoid. Glucocorticoid action in metabolic acidosis may therefore be indirect, acting by antagonizing the anabolic effects of IGF-I (and also possibly in vivo other insulin like-factors such as insulin and IGF-II). In the absence of complete adrenalectomy, glucocorticoid effects may therefore be too indirect and weak to be a useful therapeutic target.
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Acknowledgments
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The cDNA probe for ubiquitin was a kind gift from Dr S. R. Price, Renal Division, Emory University School of Medicine, Atlanta, GA, USA. The cDNA probe for cyclophilin was a kind gift from SmithKlein Beecham UK. The authors gratefully acknowledge support for this work through project grants ref. R29/1/97 from the National Kidney Research Fund, ref. 059828/Z/99/Z from the Wellcome Trust, and ref. 452001 from the Renal Care and Research Association. S.G. 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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References
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Received for publication: 30.10.02
Accepted in revised form: 21. 2.03