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HORMONES AND SIGNALING
Departments of Gastroenterology, Hepatology, and Pathobiology, Cleveland Clinic, Lerner Research Institute and the Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio
Submitted 13 November 2006 ; accepted in final form 18 December 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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), and markers of satellite cell proliferation and differentiation were quantified. PCA rats did not gain body weight and had lower lean body mass, forelimb grip strength, and gastrocnemius muscle weight. The skeletal muscle expression of the mRNA of ubiquitin proteasome components was higher in PCA rats in the first 2 wk followed by a lower expression in the subsequent 2 wk (P < 0.01). The mRNA and protein of myostatin, activin 2B receptor, and CDKI p21 were higher, whereas IGF-I and its receptor as well as markers of satellite cell function (proliferating nuclear cell antigen, myoD, myf5, and myogenin) were lower at weeks 3 and 4 following PCA (P < 0.05). We conclude that PCA resulted in uninhibited proteolysis in the initial 2 wk. This was followed by an adaptive response in the later 2 wk consisting of an increased expression of myostatin that may have contributed to reduced muscle protein synthesis, impaired satellite cell function, and lower skeletal muscle mass.
lean body mass; satellite cell; myostatin
superfamily that is a negative regulator of muscle growth. Myostatin regulates myogenesis by inhibition of satellite cell activation (7, 23). Systemic administration of myostatin causes muscle wasting in mice, suggesting that it maybe directly involved in skeletal muscle loss (36). Skeletal muscle insulin-like growth factor (IGF)-I stimulates satellite cell function and protein synthesis, thereby countering the negative regulatory effects of myostatin (19, 20). Atrogin is a component of the ubiquitin proteasome pathway that mediates skeletal muscle proteolysis (13). In our previous study (7), we examined the expression of genes that regulate muscle mass only at 4 wk after PCA. Our data showed that the lower skeletal muscle mass was accompanied by an increase in the expression of myostatin, lower expression of IGF-I, lower expression of atrogin, and lower markers of satellite cell function (7). The time course of the changes in the expression of myostatin and the components of the ubiquitin proteasome pathway after PCA has not been examined. In the present study, we examined changes in the expression of regulators of skeletal muscle mass in PCA rats and compared them with those in sham-operated control rats at weekly intervals following surgery. Our data show that PCA results in higher expression of genes related to skeletal muscle proteolysis initially, followed by higher expression of myostatin and lower expression of markers of satellite cell function, resulting in lower skeletal mass. |
METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley rats (age: 8 wk, weight: 250–260 g) with end to side PCA or sham surgery (n = 24 in both groups) were obtained from Charles River Laboratories (Wilmington, MA). Following a recovery period for 3 days after the surgery, sham-operated control rats were pair fed similarly to the PCA rats. Animals were placed on standard rat chow (Harlan Teklad standard rat chow, no. 8604: 24.5% protein, 4.4% fat, and 3.93 kcal/g). Food and water intake were measured daily. Lean body mass and forelimb grip strength were measured at weekly intervals (14, 22). Food efficiency was calculated as the gain in total body weight per gram of food intake (9). Animals were killed at weekly intervals using intraperitoneal pentobarbitone. Blood was collected from the abdominal aorta in heparin- and EDTA-coated containers, and plasma was separated and stored at –80°C for biochemical, hormonal, and cytokine assays. Gastrocnemius and extensor digitorum longus muscles from both lower extremities and the liver were rapidly harvested, blotted free of blood, weighed, frozen in liquid nitrogen, and stored at –80°C for further assays. The kidneys, testes, and spleen were also harvested and weighed. All studies were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University (Cleveland, OH).
Measurement of Lean Body Mass Using Total Body Electrical Conductivity
Lean body mass was obtained using the total body electrical conductivity (TOBEC) body analyzer for small animals (SA-3000) fitted with the 114 x 318-mm measuring chamber (SA-3114, EC Systems, Springfield IL). After the warmup period, the phantom scan provided with the instrument was used to confirm reproducible data. Body weight and the length of the rat (tip of the nose to base of the tail) were measured prior to the test. Animals were placed in the carrier provided in a prone position. The consistency of the relative position of the head and limbs on the carrier between successive readings was ensured by marks placed on the carrier. Rectal temperatures were recorded using a thermocouple thermometer (Bio-Rad, Hercules, CA). This was done because alterations in body temperature affect TOBEC readings (30). The total energy absorption by the sample (E value) was obtained as a mean of nine replicate measurements for each animal. Animals were completely removed from the chamber between each reading. The lean body mass was obtained using the following manufacturer-recommended formula: (0.79174 x length0.81637) (E value0.53941) – 0.34685.
The length of the rat was measured from the tip of the nose to the base of the tail. The lean body mass was then validated using direct chemical carcass analysis.
Validation of TOBEC Measurements by Chemical Carcass Analysis
Male (n = 4) and female (n = 4) Sprague-Dawley rats aged 10–12 wk were included in a validation study. All animals were acclimatized for 7 days in the animal facility, and TOBEC measurements were performed after an overnight fast. Animals were then killed, and chemical carcass analysis was performed (5). In brief, animals were shaved, and the whole carcass was pulverized using a Vitamix industrial grade blender (Vitamix, North Olmsted, OH) and completely dried in a mechanical convection oven (Fisher Isotemp 500 series, Fisher Scientific Research, Pittsburgh, PA) at 60°C to reach a stable weight for 3 days. The water content in the carcass was calculated by the difference between the original wet weight and the final dry weight. A precisely weighed amount (0.5 g) of the powdered sample was mixed with 0.5 ml ethyl alcohol (100%) and 10 ml ethyl ether and shaken for 30 min in a vortex shaker followed by centrifugation at 1,800 rpm for 4 min, and the supernatant ether layer was removed. Lipids were extracted from the residue by an additional 10 ml ethyl ether. The residual pellet was air dried for 3 h and then placed in a convection oven at 60°C overnight. The dried sample was weighed three times at two hourly intervals until a constant weight was achieved. The fat content was calculated as the difference between the original weight and the dry weight of the sample after ether extraction. The fat mass and lean body mass were calculated from the whole body weight and the proportion of fat in the dried carcass. The TOBEC-derived lean body mass was significantly correlated (y = 0.835x + 9.189, r2 = 0.85, P < 0.01) with that measured by carcass analysis.
Grip Strength
Forelimb grip strength was measured using a computerized rat grip strength meter (model 1067CSX, Columbus Instruments, Columbus, OH). A series of pilot studies was performed in normal rats to ensure consistency and reproducibility of the method. In brief, an unsupported T bar was attached to the load cell with a sampling rate of 1,000 Hz. Animals were acclimatized to handling, after which time the grip strength was measured. The rat was held at a fixed angle by placing a metal plate in a horizontal position below the rat to ensure reproducible positions. The rat was pulled away from the bar with a constant force at medium speed by holding the base of the tail until the bar was released. The maximal force at which the animal released the bar was recorded. All readings were obtained for peak force generated and measured as pounds. Each animal was tested three times in succession, and the results were averaged. Measurements were repeated twice after a 30-min rest period. All measurements were performed by a single operator. Repeat measurements were done in random order.
Biochemical, Hormonal, and Cytokine Assays
Plasma amino acid concentrations were measured using an opthaldehyde derivative and precolumn derivatization with HPLC equipped with fluorescent detector (34). Plasma concentrations of bilirubin, ammonia, alanine aminotransferase, and aspartate aminotransferase were quantified by standard biochemical assays.
Plasma testosterone was measured in triplicate using a competitive enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI). The lowest detection limit was 3.9 pg/ml. The interassay coefficient of variation was 3.6%, and the intraassay coefficient of variation was 2.1% (all manufacturer's data). Free IGF-I in plasma was measured using a quantitative sandwich enzyme immunoassay kit (R&D Systems, St. Paul, MN). The intraassay coefficient of variation was 4.1%, and the interassay coefficient of variation was 4.3%. The minimum detectable limit was 3.5 pg/ml (all manufacturer's data). Plasma levels of insulin, leptin, TNF-, IL-1, IL-6, and the chemokine monocyte chemotactic protein (MCP) were quantified using the Bioplex suspension array system (Bio-Rad) using a rat cytokine immunoassay panel (Linco Research, St. Charles, MO).
Tissue Extraction and Processing
A part of the gastrocnemius muscle was homogenized, and RNA was extracted using TRI reagent per the manufacturer's protocol (Sigma Aldrich, St. Louis, MO). Briefly, using a Brinkmann tissue homogenizer (Brinkmann Instruments, Westbury, NY), 100 mg of gastrocnemius muscle were homogenized in 5 ml of TRI reagent (Sigma-Aldrich). Debris was removed by centrifugation, RNA was isolated and resuspended in diethyl pyrocarbonate-treated water, and the final concentration was determined by measuring absorption at 260 and 280 nm. Ten micrograms of total RNA from each sample were separated on a 1.2% formaldehyde agarose gel to verify the quality of the RNA. First-strand cDNA synthesis was performed using 1 µg of total RNA using a BD Clontech kit (Hercules, CA) per the manufacturer's protocol. The oligonucleotide primers used for the various genes were as published previously (7). Primers for proteasome C3, C5, and C9 and ubiquitin ligase E3 (atrogin and polyubiquitin) were used to quantify the expression of mRNA of the ubiquitin proteasome pathway genes (proteasome C3: upper 5'-CATTCAGCCCATCTGGTAAACTTGT-3' and lower 5'-CTGTACACCAAACCGATGTGCTT-3'; proteasome C5: upper 5'-CCTGTGCAGATGCGTTTCTCG-3' and lower 5'-AAAGCCACTGCAGCCAATTACTGTT-3'; proteasome C9: upper 5'-AGCTCAGTAAAGCGGCGCTGATCTG-3' and lower 5'-TCCCAAACAAGTGCCTGCGTGTC-3'; and polyubiquitin: upper 5'-TGGCTATTAATTCTTCAGTCTGC-3' and lower 5'-CATTTTTAACAGAGGTTCAGCTATT-3').
Quantitative Real-Time PCR
Real-time PCR for quantification of mRNA was performed on a Stratagene Mx 3000 P (Stratagene, La Jolla, CA) using a SYBR protocol on the fluorescence temperature cycler. Results were expressed as fold changes in expression of each gene in PCA animals compared with control sham animals using a relative quantification method (29). Relative expression of the gene of interest in PCA rats compared with the expression in sham-operated controls was calculated.
All real-time PCR products were then separated using 1.5% Tris-acetic acid agarose electrophoresis to confirm the product presence and size.
Western Blot Analysis
The expression of proteins of genes regulating skeletal muscle mass were quantified by Western blot assays as described previoulsy (7). In brief, muscle samples (100 mg each) were homogenized in 1 ml lysis buffer with protease inhibitor cocktail (Sigma-Aldrich). The homogenate was subsequently centrifuged at 10,000 g at 4°C for 30 min to remove tissue debris. The protein concentration of the supernatant was quantified using a Bio-Rad DC protein assay. The protein extract was boiled for 5 min with a 1:1 volume of Laemmeli loading buffer [125 mM Tris·HCl (pH 6.8), 20% glycerol, 4% SDS, 10% (wt/vol) -mercaptoethanol, and 0.05% bromophenol blue]. Twenty micrograms of protein from each sample were then separated using SDS-PAGE with a 4–20% gradient gel under reducing conditions. After an overnight electrotransfer to polyvinylidene difluoride membranes (Bio-Rad), membranes were stained with Ponceau S to confirm equal loading and uniformity of transfer, and they were then destained in Tris-buffered saline (TBS) with Tween [TBST; 0.05 M Tris (pH 7.4), 0.1 M NaCl, and 0.1% Tween 20 (Sigma-Aldrich)] and blocked in TBST containing 10% nonfat milk at room temperature for 4 h. Membranes were incubated in primary antibody in TBST overnight at 4°C followed by a wash in TBST. Membranes were then incubated in the appropriate secondary antibody conjugated to peroxidase for 1 h at room temperature. Membranes were washed in TBST, and horseradish peroxidase activity was detected using enhanced chemiluminescent reagent (Amersham Biosciences, Piscataway, NJ). Band intensities were quantified by densitometry (Bio-Rad GL710) using multianalyst software (Bio-Rad). Optical density data from Western blot analyses were compared between PCA and sham-operated animals after normalization for loading using cytoskeletal -actin protein. All primary and secondary antibodies were obtained from Santa Cruz Biotechnology (Sanata Cruz, CA) except for antimyostatin antibody, which was obtained from Bethyl Laboratories (Montgomery, TX).
Enzyme Assays
Alterations in the plasma kinetics of methionine and its metabolite have been reported with liver disease and PCA (3). The activity of hepatic enzymes involved in the demethylation, remethylation, and transsulfuration pathways of methionine metabolism were studied (3). In brief, liver samples were homogenized in enzyme buffer [10 mmol/l sodium phosphate (pH 7), 0.25 mol/l sucrose, 1 mmol/l sodium azide, 1 mmol/l EDTA, 0.1 mmol/l PMSF] and centrifuged at 14,000 g for 30 min. -Mercaptoethanol was added to an aliquot of the resulting supernatant to a final concentration of 10 mmol/l. The liver protein concentration was determined by the method of Lowry et al. using BSA as the reference standard.
Glycine-N-methyltransferase (GNMT) activity was measured by the addition of 250 µg protein to a reaction mixture containing 0.15 mmol/l Tris (pH 9), 5 mmol/l DTT, 1.25 mmol/l, glycine and 1 mmol/l S-adenosyl-L-[methyl-3H]methionine (SAM; 1 µCi/µmol) and incubated at 37°C for 30 min. Cold 10% trichloroacetic acid was added to the mixture to stop the reaction. Unreacted SAM was removed by the addition of charcoal (neutral) dextran and centrifuged at 10,000 g for 10 min. An aliquot of the supernatant was subjected to liquid scintillation counting.
Cystathionine-
-lyase activity was measured by the addition of 125 µg protein to a reaction mixture containing 100 mmol/l potassium phosphate buffer (pH 7.5), 4 mmol/l L-cystathionine, 0.125 mmol/l pyridoxal 5'-phosphate, 0.32 mmol/l NADH, and 1.5 units of lactate dehydrogenase in a 1.25-ml cuvette incubated at 37°C. The decrease in optical density at 340 nm due to the reduction of NADH to NAD+ was recorded with a spectrophotometer at 5-min intervals. Enzyme activity was calculated from the linear segment of the graphs.
Betaine-homocysteine methyltransferase (BHMT) activity was measured by the addition of 3 mg protein to a reaction mixture containing 35 µmol/l potassium phosphate buffer (pH 7.5), 6.5 µmol/l L-homocysteine, and 6.5 µmol/l [14CH3]betaine [
60,000 disintegrations/min (dpm) per 6.5 µmol/l] for a total volume of 1 ml and incubated at 37°C for 120 min. Ice-cold water was added to the mixture to stop the reaction. The reaction mixture was placed on a 3 x 0.9-cm column of Dowex 1-X4 (OH–) 100–200 mesh. The column was washed with 15 ml water to remove unreacted betaine, and products were eluted with 6 ml of 1 N HCl. The latter 3 ml of acid eluate was subjected to liquid scintillation counting.
The reaction mixture for cystathionine--synthase (CBS) activity assay contained 750 µg liver protein in 60 µmol/l Tris (pH 8.3), 1 µmol/l EDTA, 0.2 µmol/l pyridoxine-5-phosphate, 0.15 µmol/l S-adenosyl methionine, and 5 µmol/l serine (containing 75–90,000 dpm [14C]serine) and incubated at 37°C for 5 min. CBS activity assays were measured by the addition of 5 µmol/l homocysteine to the reaction mixture and incubated at 37°C for 120 min. The reaction was terminated by the addition of cold 10% trichloroacetic acid. Cystathionine (0.07 µmol/l) was added to the reaction mixture and centrifuged at 14,000 g for 10 min. The supernatant was placed on a 3 x 0.9-cm column of Dowex 50-X4 (H+) 200–400 mesh. The column was washed with 10 ml H2O, 20 ml 0.6N HCl, and 20 ml water to remove unreacted serine, and the product (cystathionine) was eluted with 8 ml of 3 N ammonium hydroxide. The acid eluate was subjected to liquid scintillation counting.
Statistical Analysis
Sample size was calculated based on our previous data showing that myostatin was increased at 4 wk in PCA rats (6). Since no data were available for gene expression at earlier time points, we used the data at 4 wk to calculate the number of animals needed. Therefore, six animals in each group were examined at the four time points. All data are expressed as means ± SE unless stated otherwise. Results were compared using a standard statistical package (SPSS 14, SPSS, Chicago, IL). One-way ANOVA was used for the comparison of data in PCA and sham-operated rats at each week. A paired Student's t-test was used to compare serial observations in the rats over time. A Mann-Whitney test was used to compare skewed data.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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All animals had similar weights prior to surgery. One week following PCA, whole body weight, lean body mass, and the liver, gastrocnemius muscle, extensor digitorum longus muscle of PCA rats were significantly less than those of pair-fed sham rats (Fig. 1 and Table 1). The whole body fat mass (Fig. 1), calculated as the difference between the whole body weight and lean body mass, was significantly lower in PCA rats (P < 0.01). During the 4 wk of observation, there were no significant increases in whole body weight, lean body mass, or liver or skeletal muscle weight in PCA rats, whereas a steady gain in these parameters was observed in pair-fed sham rats. There was a progressive loss of testes weight in PCA rats compared with sham-operated rats. Heart and kidney weights did not show significant changes over time in PCA or sham-operated rats (data not shown). The average food efficiency in PCA rats (–0.06 ± 0.02) was significantly less (P < 0.01) compared with that in sham-operated rats (0.2 ± 0.01). The grip strength of PCA rats was significantly less than that of sham-operated rats (P < 0.001). There were no changes in the grip strength of PCA rats through the 4 wk.
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Changes in plasma amino acid concentrations following PCA or sham surgery are shown in Table 2. Plasma concentrations of all amino acids were high at week 1 in sham-operated animals, suggesting an increase in protein breakdown. Subsequently, concentrations of all the amino acids gradually decreased and reached steady levels at weeks 3 and 4. In contrast, in PCA animals, branched-chain amino acids (BCAAs) were lower than those in sham rats in weeks 1 and 2 and were not different at weeks 3 and 4. The lack of an increase in BCAAs in PCA rats may suggest an increased metabolism of these amino acids. In contrast to BCAAs, plasma concentrations of phenylalanine were higher in PCA rats. Phenylalanine concentrations continued to remain higher in PCA rats compared with sham-operated rats in the subsequent weeks. Plasma threonine concentrations were lower in PCA rats at all 4 wk.
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-lyase and CBS) were not altered over the 4 wk (Table 3).
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Plasma ammonia levels were significantly higher (P < 0.05) in PCA rats compared with sham-operated rats throughout the 4 wk. Plasma bilirubin, alanine aminotransferase, aspartate aminotransferase, and albumin levels were not significantly different between the two groups (data not shown).
Hormones and Cytokines
Plasma testosterone levels (Table 4) were significantly lower in PCA rats compared with sham-operated rats (P < 0.05). In the sham-operated group, plasma testosterone levels were significantly lower (P < 0.05) at week 1 compared with weeks 2–4. Plasma insulin levels were significantly higher in sham-operated animals compared with PCA animals at weeks 1 and 2 after surgery (Table 4). Plasma IGF-I levels were similar in PCA and sham-operated rats and did not show significant alterations over time. Plasma leptin levels were lower in PCA rats compared with sham-operated rats at each week after PCA. Plasma TNF-, IL-1, and IL-6 did levels not show any specific patterns of alteration. Plasma levels of MCP-1 were significantly lower in PCA rats compared with sham-operated rats at weeks 1–3.
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Protein breakdown. Relative expressions of proteasome C3, C5, and C9, atrogin, and polyubiquitin mRNA in skeletal muscle are shown in Fig. 2. During week 1 and 2 after PCA, the expression of these components was significantly higher in the PCA group (P < 0.05). During weeks 3 and 4 after PCA, the expression of these components was lower in PCA rats (P < 0.05).
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) are shown in Fig. 5. Both IGF-I and IGF-I receptor- mRNA in muscles from PCA and sham-operated rats were similar at weeks 1 and 2. Subsequently, at weeks 3 and 4, they were lower in PCA compared with sham-operated rats (P < 0.05). Assays of the proteins by Western blot analysis were similar to the changes observed in the mRNA expression. IGF-I and IGF-I receptor proteins were lower at week 4 compared with earlier weeks in PCA rats, but no such pattern was observed in sham-operated rats (Fig. 6).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The metabolic and other alterations seen in the PCA rat are primarily the consequence of portacaval shunting and not directly related to hepatocellular injury. In fact, data in the literature (35) and those from our study show that the liver undergoes apoptotic changes as a result of PCA without evidence of inflammation or any significant alterations in liver-associated enzymes. In addition, we did not observe any changes in plasma levels of proinflammatory cytokines. As shown in the RESULTS, the alterations in plasma amino acid concentration were evident soon after the placement of the portacaval shunt. Thus, the observed changes in the skeletal muscle in PCA rats, which initially (weeks 1 and 2) may have been influenced by surgical trauma and altered nutrient intake, should be primarily attributed to portacaval shunting.
The failure to gain weight consisted of both lean body mass and fat mass. It could not be attributed to lower calorie intake of the PCA rats, since the control rats, which continued to gain weight, were pair fed and therefore received correspondingly similar amounts of nutrients. These alterations in growth and muscle mass cannot be attributed to gastrointestinal malabsorption (11, 28) but could be the result of overall changes in metabolism related to hyperammonemia, changes in energy expenditure as a consequence of changes in circulating cytokines and bile salts, or due to translocation of bacterial toxins (16, 18). Although not measured, a hypermetabolic state may be responsible for the lower food efficiency in PCA rats. Since previous data in rats have suggested that significant amounts of fatty acids are absorbed into the portal vein, PCA could potentially divert these fatty acids to the systemic circulation and impact skeletal muscle protein metabolism (24). However, the rat chow used in our study contained only 4.4% fat, or only 10% of the daily calorie intake, based on an average daily food intake of 25 g. Such a low fat intake is unlikely to impact skeletal muscle, even if diverted to the systemic circulation as a result of PCA.
The changes in plasma ammonia and amino acid concentration were similar to those reported previously (8, 16, 26). It is to be underscored that these changes appeared soon after PCA and remained unchanged during the 4 wk of observation. Therefore, the temporal changes in the expression of various genes in the skeletal muscle could not be related to alterations in blood ammonia or amino acid concentrations. Measurements of enzymes involved in sulfur-containing amino acids showed that GNMT and BHMT were higher after 2 wk of PCA. The biological significance of these changes is unclear and may be related to the hepatic atrophy and apoptosis after PCA.
Among the various hormones and cytokines measured, a significant change was observed only in plasma testosterone levels, which were significantly lower in PCA rats at all times compared with sham-operated rats. The reason for the lower plasma testosterone levels at week 3 in PCA rats compared with those at weeks 2 and 4 is unclear. However, examination of the data from weeks 1, 2, and 4 showed a pattern of progressive decline with time in PCA rats. The lower plasma testosterone levels in PCA have been shown to be the consequence of higher aromatase activity (9, 10). The specific organ system or the time of increase in aromatase activity in relation to PCA has not been identified.
The changes in the expression of the various genes in skeletal muscle protein breakdown and synthesis and in satellite cell function followed an interesting pattern. As shown in Fig. 3, the genes for ubiquitin proteasome system C3, C5, and C9 and atrogin were higher at weeks 1 and 2 and lower at weeks 3 and 4 in PCA rats compared with sham-operated rats. Since the ubiquitin proteins are transcriptionally regulated, the changes in mRNA can be related with functional activity (2, 25). These data suggest that PCA was followed by an increase in the rate of protein breakdown in skeletal muscle in weeks 1 and 2 after PCA. This increase could not be attributed to surgery or lower calorie intake alone in PCA animals, since their data were normalized to data of sham-operated pair-fed controls, suggesting that PCA resulted in an exaggerated proteolytic response to surgery in these animals. In weeks 3 and 4 after PCA, a lower or decreased rate of proteolysis can be inferred from the lower expression of ubiquitin genes (Fig. 3).
The expression of skeletal muscle IGF-I and its receptor (IGF-I receptor) followed a pattern of unaltered expression in weeks 1 and 2 after PCA and lower expression of IGF-I in weeks 3 and 4 after PCA. The mechanism of this pattern of IGF-I expression in the PCA rat may be related to the low plasma testosterone levels (21). It is possible that the regulatory role of testosterone on skeletal muscle IGF-I maybe related to a threshold effect with a critical plasma concentration below which IGF-I expression is reduced. Low plasma growth hormone levels in the PCA rat, as reported by a previous study (31), may also have been responsible for the lower mRNA levels of skeletal muscle IGF-I, but plasma growth hormone levels were not measured in the present study. Skeletal muscle expression of IGF-I regulates muscle mass by stimulating protein synthesis, inhibiting protein breakdown, and promoting satellite cell proliferation and differentiation. In weeks 1 and 2 after PCA, when the IGF-I expression was unaltered, the proteasome components were elevated. This suggests that the elevated proteolysis genes early in the course after PCA was not due to low muscle IGF-I levels. The low skeletal muscle IGF-I levels after 2 wk of PCA may contribute to the failure to gain skeletal muscle mass at this time. It is interesting to note that when the IGF-I expression was lower, proteolytic gene expression was also reduced. This suggests that IGF-I regulates skeletal muscle mass in the PCA rat by regulating protein synthesis and satellite cell function.
The present study showed that the expressions of markers of satellite cell proliferation (PCNA) and differentiation (myoD, myf5, and myogenin) in PCA rats were unaltered in weeks 1 and 2 after PCA but were lower in weeks 3 and 4 after PCA. Satellite cell proliferation and differentiation contribute to skeletal muscle growth and recovery following injury and denervation atrophy (27). Impaired satellite cell function contributes to sarcopenia of aging. The lack of any change in satellite cell function in weeks 1 and 2 after PCA suggests that the primary effect of PCA was not related to cell proliferation and differentiation. The present observations of lower satellite cell function at weeks 3 and 4 after PCA confirm our previous observation of impaired satellite cell function at 4 wk after PCA (7). Our observations suggest that impaired satellite cell function is involved in the failure of skeletal muscle growth after the initial 2 wk following PCA. The exact mechanism for the impaired satellite cell function after PCA is not known but may be related to the alteration in a number of factors that regulate satellite cell function after PCA (27). Plasma hormones that regulate satellite cell function include plasma testosterone and growth hormone, both of which are lower after PCA. Circulating IGF-I stimulates satellite cell function, but these levels were not significantly different during the time course after PCA. TNF- suppresses satellite cell differentiation, but in the present study we did not find any significant changes in plasma levels of TNF- following PCA (12).
We observed an increase in the expression of myostatin after the initial 2 wk following PCA. An increase in the expression of myostatin also accompanies the loss of skeletal muscle mass following immobilization, denervation, sepsis, and alcohol feeding (4). Myostatin inhibits satellite cell function and protein synthesis in skeletal muscle. Satellite cell function is inhibited by an increase in CDKI p21 after the binding of myostatin to its receptor, activin 2B receptor (7, 23). In the present study, alterations in the expression of myostatin were accompanied by similar changes in its intracellular mediator, CDKI p21. Our observations were similar to those reported previously showing that CDKI p21 mediates the effects of myostatin. Higher expression of myostatin after PCA may be related to the translocation of cytokines, endotoxins, or other "gut-derived" toxins that bypass the liver to enter the systemic circulation. However, our observations do not support this hypothesis because these changes occurred early and persisted after PCA, whereas myostatin levels increased only 2 wk after PCA. The mechanism of this delayed expression of myostatin 2 wk following PCA is unclear. It is possible that a critically low level of testosterone or lower expression of skeletal muscle IGF-I are responsible for the higher expression of myostatin after the initial 2 wk of PCA. An alternate mechanism alluded to earlier is related to a threshold effect of hormonal and amino acid changes. Another mechanism of these changes in skeletal muscle mass and gene expression in the muscle may be related to the hepatic atrophy in PCA rats. It is possible that once the liver mass is reduced to a critical volume, accompanied by a reduction in hepatic blood flow, hepatic function in terms of synthesis of regulatory proteins or clearance of gut-derived compounds may be reduced and consequently alters the expression of myostatin and IGF-I in skeletal muscle. The delayed rise in the expression of myostatin may also be the adaptive mechanism in the skeletal muscle compartment by which unregulated proteolysis is controlled.
We conclude that an accelerated protein breakdown in weeks 1 and 2 after PCA and lower protein synthesis and impaired satellite cell function likely are responsible for the failure of PCA animals to gain lean body mass and skeletal muscle weight after PCA in the subsequent weeks 3 and 4. We speculate that the changes after 2 wk of PCA may be the consequence of low skeletal muscle IGF-I expression. This results in lower protein synthesis and impaired satellite cell function and may also be responsible for the increase in myostatin. It is also possible that myostatin plays a temporizing role in this process by which the impaired satellite cell function and decrease in protein synthesis reduce the protein turnover and result in a lower steady state and continued failure of growth in this model. The clinical relevance of these observations are related to the failure of the cirrhotic patients to accrete lean body mass, and an understanding of these mechanisms may contribute to the development of targeted intervention strategies to overcome these changes in patients with cirrhosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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