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Am J Physiol Cell Physiol 293: C1947-C1952, 2007. First published October 10, 2007; doi:10.1152/ajpcell.00269.2007
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

IFN-{gamma} does not mimic the catabolic effects of TNF-{alpha}

Melissa A. Smith, Jennifer S. Moylan, Jeffrey D. Smith, Wei Li, and Michael B. Reid

Department of Physiology, University of Kentucky, Lexington, Kentucky

Submitted 25 June 2007 ; accepted in final form 4 October 2007


   ABSTRACT
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 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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Cachexia is common in chronic inflammatory diseases and is attributed, in part, to an elevation of circulating proinflammatory cytokines. TNF-{alpha} is the prototype in this category. IFN-{gamma} is also thought to play a role, but the evidence supporting this model is primarily indirect. To determine the direct effects of IFN-{gamma} stimulation on muscle cells, we selected key components of the procatabolic signaling pathways by which TNF-{alpha} stimulates protein loss. We tested two hypotheses: 1) IFN-{gamma} mimics TNF-{alpha} signaling by increasing intracellular oxidant activity and activating MAPKs and NF-{kappa}B and 2) IFN-{gamma} increases the expression of the ubiquitin ligases atrogin1/MAFbx and muscle-specific ring finger protein 1 (MuRF1). Results showed that treatment with IFN-{gamma} at 60 ng/ml increased Stat1 phosphorylation after 15 min, indicating receptor activation. IFN-{gamma} had no effect on cytosolic oxidant activity, as measured by 2',7'-dichlorofluorescein oxidation. Nor did IFN-{gamma} activate JNK, ERK1/2, or p38 MAPK, as assessed by Western blot. Treatment for up to 60 min did not decrease I{kappa}B-{alpha} protein levels, as measured by Western blot analysis, or the DNA binding activity of NF-{kappa}B, as measured by EMSA. After 6 h, IFN-{gamma} decreased Akt phosphorylation and increased atrogin1/MAFbx and MuRF1 mRNA. Daily treatment for up to 72 h did not alter adult fast-type myosin heavy chain content or the total protein-to-DNA ratio. These data show that responses of myotubes to IFN-{gamma} and TNF-{alpha} differ markedly and provide little evidence for a direct catabolic effect of IFN-{gamma} on muscle.

skeletal muscle; cachexia; oxidative stress; C2C12; atrophy; tumor necrosis factor-{alpha}; interferon-{gamma}

CACHEXIA is a common feature of inflammatory diseases such as cancer, sepsis, chronic heart failure, rheumatoid arthritis, and chronic obstructive pulmonary disease. Cachexia contributes to the mortality of patients suffering from such diseases. Elevation of circulating proinflammatory cytokines is a hallmark of cachexia (11). Among these, TNF-{alpha} is typically considered the most relevant of the cytokines associated with inflammatory-mediated cachexia (34). TNF-{alpha} can directly stimulate protein loss mediated by an increase in ROS production via mitochondrial electron transport (23). The increased ROS production precedes the activation of NF-{kappa}B, which appears to increase the activity of the ubiquitin/proteasome pathway (35).

IFN-{gamma}, originally called macrophage-activating factor, is a proinflammatory cytokine that can have anti-inflammatory properties by modulating the production of other proinflammatory cytokines (30). As part of the adaptive immune response, IFN-{gamma} is produced by natural killer cells and lymphocytes (37). IFN-{gamma} has synergistic effects with TNF-{alpha} on the activation of NF-{kappa}B in cell culture and mouse models (31, 41, 42). In one report (28), mice inoculated with IFN-{gamma}-producing tumors developed cachexia. Upon administration of a monoclonal antibody against IFN-{gamma}, the mice stopped losing weight. In myotubes, TNF-{alpha} plus IFN-{gamma} reduces myosin protein and mRNA expression through a RNA-dependent mechanism (1). These studies support a procatabolic action for IFN-{gamma}.

However, little is known about the direct effects of IFN-{gamma} on skeletal muscle cells. The evidence supporting IFN-{gamma} as a catabolic cytokine is primarily indirect. In the present study, we evaluated whether IFN-{gamma} activates the procatabolic signaling pathways by which TNF-{alpha} acts directly on C2C12 myotubes to stimulate protein loss. We hypothesized that IFN-{gamma} treatment would mimic TNF-{alpha} signaling by increasing intracellular oxidant activity and activating both MAPK and NF-{kappa}B pathways.

Our second aim was to determine if direct IFN-{gamma} exposure induces muscle wasting in C2C12 myotubes. We hypothesized that IFN-{gamma} would increase the expression of atrogin1/MAFbx, a muscle-specific, ubiquitin ligase linked to protein loss, and would induce net catabolism as reflected by decreases in myosin heavy chain (MHC) content and the total protein-to-DNA ratio.


   EXPERIMENTAL PROCEDURES
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RESULTS
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Materials. Mouse recombinant IFN-{gamma} and TNF-{alpha} were purchased from R&D Systems (Minneapolis, MN). Antibodies to ERK, p38 MAPK, JNK, Stat1, phospho-Akt, I{kappa}B-{alpha}, and ubiquitin were purchased from Cell Signaling Technologies (Danvers, MA). Antibodies to atrogin and total Akt were purchased from ECM Biosciences (Versailles, KY). Muscle-specific ring finger protein 1 (MuRF1) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). MHC type II antibody was purchased from Chemicon (Temecula, CA). NF-{kappa}B consensus oligonucleotide and T4 polynucleotide kinase were purchased from Promega (Madison, WI). Primers for β-actin (forward: 5'-AGGCCCAGAGCAAGAGAGGTA-3' and reverse: 5'-CCATGTCGTCCCAGTTGGTAA-3'), atrogin1/MAFbx (forward: 5'-ATGCACACTGGTGCAGAGAG-3' and reverse: 5'-TGTAAGCACACAGGCAGGTC-3'), and MuRF1 (forward: 5'-ACGAGAAGAAGAGCGAGC-3' and reverse 5'-CTTGGCACTTGAGAGGAA-3') were purchased from Invitrogen (Carlsbad, CA).

Cell culture. C2C12 myoblasts were purchased from the American Type Culture Collection (Manassas, VA) and grown in DMEM supplemented with 10% FBS for 4 days. Upon 80% confluence, cells were serum restricted in DMEM with 2% heat-inactivated horse serum before and after treatment. Cells mature to myotubes on the fifth day after serum restriction. Mature myotubes were treated either with PBS + 0.1% BSA (control), 60 ng/ml IFN-{gamma}, or 6 ng/ml TNF-{alpha} and harvested for analyses. Serum circulating levels of IFN-{gamma} are in the picogram per milliliter range (38). In diseased patients, IFN-{gamma} remains in the picogram per milliliter range but is reported as elevated (19). For our experiments, we stimulated cells in the nanogram per milliliter range. Our logic is based on the experimental use of TNF-{alpha}, which has an EC50 of 0.05 ng/ml. The dosage used in cell culture was 100 times this EC50, based on clinical serum levels (26). Therefore, based on an EC50 of 0.3–0.9 ng/ml for recombinant IFN-{gamma}, we chose a dosage of 60 ng/ml.

Total protein and Western blot analysis. After treatment, myotubes were washed with PBS and scraped into 200 µl of 20 mM Tris·HCl (pH 7.5), 2 mM ATP, 5 mM MgCl2, and 1 mM DTT. Lysates were sonicated on ice and then heated at 98°C for 5 min. Equal amounts of protein were loaded in each lane of 4–15% Tris·HCl polyacrylamide gels and electrophoresed at 200 V for 50 min. Proteins were either dyed using Simply Blue (Invitrogen) and scanned for total protein or were transferred at 200 mA for 2 h to nitrocellulose membranes for Western blot analysis. Membranes were blocked in blocking buffer/PBS (Odyssey, Li-COR Biosciences, Lincoln, NE) for 1 h at room temperature and incubated with primary antibodies overnight, followed by four 5-min washes. Membranes were incubated with fluorescence-conjugated secondary antibodies in Odyssey blocking buffer plus Tris-buffered saline and 10% SDS for 45 min, followed by four 5-min washes. The membrane was then dried, and blots were scanned by a densitometer (Odyssey) to quantify differences. The intensity of each blot was adjusted to avoid saturation and demonstrate treatment-induced differences. Quantitative comparisons are best resolved by the data in the bar graphs, which were normalized for total protein and are expressed as a percentage of the respective control.

Cytosolic oxidant activity. As reported previously (32), the fluorochrome probe 2',7'-dichlorofluorescein (DCF) diacetate (DCFDA, Molecular Probes) was used to measure oxidant activity. Mature C2C12 myotubes were loaded with DCFDA (20 µM) for 15–45 min. Accumulation of the oxidized derivative DCF (480-nm excitation and 520-nm emission) was measured with the use of an epifluorescence microscope (Labophot-2, Nikon Instruments, Melville, NY) using commercial data acquisition and analysis software (Optimas 4.02, Bioscan, Edmonds, WA). Images were acquired in real time and stored in a desktop computer for later analysis of mean emission intensity.

EMSA. Cells were treated according to experimental conditions, and nuclear extracts were prepared following commercial nuclear and cytoplasmic extraction protocols (Pierce, Rockford, IL). NF-kB activity in nuclear extracts was determined using p32-labeled oligonucleotides (Ig{kappa} intronic {kappa}B site: 5'-CTCAACAGAGGGGACTTTCCGAGAGGCCAT-3'). Ten micrograms of nuclear protein were incubated with the NF-{kappa}B oligonucleotide followed by nondenaturing PAGE. Protein-DNA complexes were detected by autoradiography and quantified by densitometry.

RNA/DNA isolation and reverse transcription. Myotubes were collected and homogenized in 500 µl TRIzol reagent (Invitrogen), and RNA was extracted from myotubes following the TRIzol RNA extraction protocol. RNA concentrations were quantified at 260 nm by spectrophotometry. Samples were diluted to equal concentrations of RNA. Two microliters of RNA were used to synthesize cDNA at 37°C for 90 min with a thermocycler using 40 units of Moloney murine leukemia virus (M-MLV) reverse transcriptase, 4 µl of 5x M-MLV reaction buffer, 0.2 µl of 10 mM dNTP, 0.5 µg of random primer, and 12.4 µl of diethyl pyrocarbonate (DEPC)-treated H2O for a final volume of 20 µl (reagents from Promega). DNA was isolated following a genomic DNA isolation protocol using DNAzol reagent (Invitrogen). DNA concentrations were quantified at 260 nm by spectrophotometry.

Real-time PCR. mRNA was quantified using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). cDNA was obtained from the reverse transcription reaction of mRNA as described above. Real-time PCR analyses were performed in duplicate using 96-well plates with SYBR green master mix (Applied Biosystems). β-Actin was used as an endogenous control. Five microliters of cDNA were used as a template for real-time PCR; 1 µl forward primer and 1 µl reverse primer were added to 25 µl SYBR green master mix and 18 µl DEPC-treated water. Reactions were performed in a 50-µl reaction volume under the following conditions: initial step at 50°C for 2 min followed by 95°C for 10 min and 40 cycles of denaturation at 95°C for 15 s and hybridization and elongation at 60°C for 1 min. Primer sequences were as previously described in Materials. Threshold cycles for both the endogenous control (β-actin) and treatment (atrogin1/MAFbx and MuRF1) cDNA for each reaction were determined by Applied Biosystems Sequence Detection Software 1.3.

Statistical analysis. Data are expressed as means ± SE. Statistical analyses were performed using a paired Student's t-test or one-way ANOVA. Student's t-test was used for the comparison of two means; ANOVA was used for the comparison of multiple means. When ANOVA revealed significant differences, Tukey's post hoc test for multiple comparisons was performed. P values of <0.05 were considered significant.


   RESULTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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DISCUSSION
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C2C12 myotube responsiveness to IFN-{gamma}. As shown in Fig. 1, as a positive indicator for IFN-{gamma} signaling, we measured the phosphorylation of Stat1 since this early postreceptor signaling element appears to be critical for IFN-{gamma} responsiveness (18). Myotubes were treated with IFN-{gamma} for up to 60 min. Phosphorylated Stat1 was increased versus control after 5 min and remained elevated for the duration of the 60-min stimulation, whereas total Stat1 protein did not change over time.


Figure 1
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  		Fig. 1. IFN-{gamma} activates STAT1. C2C12 myotubes were exposed to IFN-{gamma} (60 ng/ml) for the indicated times. Top: cell lysates were analyzed by Western blot analysis using an antibody against the phosphorylated form of STAT1 and an antibody against total STAT1. Bottom: optical densities from 3 independent experiments were analyzed by one-way ANOVA followed by Tukey's post hoc test. *P < 0.05.

 
IFN-{gamma} and oxidant activity. Experiments were performed to determine whether IFN-{gamma} stimulation alters cytosolic oxidant activity, as measured by DCF oxidation (32). Myotube responsiveness was confirmed using TNF-{alpha} as a positive control. The data shown in Fig. 2 demonstrate that oxidant activity increased within 15 min of TNF-{alpha} exposure. In contrast, IFN-{gamma} had no effect on oxidant activity.


Figure 2
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  		Fig. 2. IFN-{gamma} does not increase oxidant activity in C2C12 myotubes. 2',7'-Dichlorofluorescein (DCF) emissions from DCF diacetate-loaded myotubes incubated for 15 min and exposed to IFN-{gamma} (60 ng/ml) or TNF-{alpha} [6 ng/ml, positive control (Con)] for 15 min are shown. In paired comparisons, DCF fluorescence was increased in response to TNF-{alpha} treatment, whereas no change occurred in response to IFN-{gamma} treatment (n = 8 comparisons/group). *P < 0.05.

 
MAPK activation. Figure 3 shows cytokine effects on MAPK phosphorylation including p38, JNK, and ERK. TNF-{alpha} treatment increased the phosphorylation of all three MAPK isoforms within 15 min. Within 60 min, phosphorylation returned toward basal levels; recovery was incomplete for p38 MAPK, whereas JNK phosphorylation overshot, dropping below the prestimulation value. Total protein levels of these enzymes were unaffected by TNF-{alpha} stimulation. IFN-{gamma} exposure did not alter the phosphorylation or total protein levels of p38, JNK, or ERK.


Figure 3
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  		Fig. 3. IFN-{gamma} does not increase MAPK activity in C2C12 myotubes. C2C12 myotubes were incubated for the indicated times with TNF-{alpha} (6 ng/ml, positive control), IFN-{gamma} (60 ng/ml), or vehicle (control). Top: cell lysates were analyzed with Western blot analysis using antibodies against the phosphorylated forms of p38 MAPK, ERK1/2, or JNK and antibodies against total p38, ERK1/2, or JNK. Bottom: optical density values normalized for total protein and expressed as percent control. Left: p38 MAPK; middle: ERK1/2; right: JNK. Optical density data from 3 independent experiments were analyzed by ANOVA for each group. *P < 0.05.

 
NF-{kappa}B signaling. The results shown in Fig. 4 demonstrate that TNF-{alpha} stimulation decreased I{kappa}B-{alpha} protein levels by 30% after 15 min, a change that reversed by 60 min. Concurrent with I{kappa}B-{alpha} changes, the DNA binding activity of NF-{kappa}B increased after 30 min. IFN-{gamma} had no effect on either I{kappa}B-{alpha} levels or NF-{kappa}B activity.


Figure 4
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  		Fig. 4. IFN-{gamma} does not activate NF-{kappa}B. C2C12 myotubes were exposed for the indicated times to TNF-{alpha} (6 ng/ml, positive control) or IFN-{gamma} (60 ng/ml). Nuclear extracts were incubated with an oligonucleotide containing an NF-{kappa}B consensus binding site. The NF-{kappa}B complex bound was then detected and quantified as described in EXPERIMENTAL PROCEDURES. Left: I{kappa}B-{alpha} protein; right: NF-{kappa}B DNA binding activity. Bars represent optical density values expressed as the percent control (untreated myotubes). Values are means for 3 independent experiments. *P < 0.05.

 
Akt signaling. The data shown in Fig. 5 demonstrate that IFN-{gamma} stimulation transiently increased Akt phosphorylation after 15 min and then decreased Akt phosphorylation after 6 h. Total Akt did not change. A decrease in Akt phosphorylation-dependent signaling is associated with increased atrogin1/MAFbx mRNA and muscle atrophy (36).


Figure 5
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  		Fig. 5. IFN-{gamma} alters Akt signaling. Top: IFN-{gamma} stimulation transiently increased Akt phosphorylation after 15 min and then decreased Akt phosphorylation after 6 h. Total Akt did not change. Bottom: optical density values were expressed as the percent control (untreated myotubes). Values are means for 3 independent experiments. *P < 0.05.

 
Atrogin1/MAFbx, MuRF1 mRNA, and protein balance. Atrogin1/MAFbx and MuRF1 are ubiquitin ligases that mediate muscle atrophy in a variety of inflammatory conditions and are well-recognized marker genes. As expected (12, 24), TNF-{alpha} increased atrogin1/MAFbx mRNA and MuRF1 mRNA (Fig. 6). Both were increased after 2 h of stimulation; MuRF1 remained elevated after 6 h. IFN-{gamma} also increased atrogin1/MAFbx and MuRF1 mRNA, producing a slower response that required 6 h to become evident. Neither atrogin1/MAFbx protein levels, MuRF1 protein levels, nor total protein ubiquitination were altered 24, 48, or 72 h after IFN-{gamma} treatment (data not shown). To assess the overall effects of IFN-{gamma} on protein balance, we examined cytokine effects on adult fast-type MHC content and the total protein-to-DNA ratio (Fig. 7). TNF-{alpha} induced a 30% loss of MHC and 8% loss of total protein to DNA. Despite the increase observed in atrogin1/MAFbx and MuRF1 mRNA expression, IFN-{gamma} actually increased MHC content by 25% and did not alter the total protein-to-DNA ratio.


Figure 6
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  		Fig. 6. IFN-{gamma} increases atrogin1/MAFbx (left) and muscle-specific ring finger protein 1 (MuRF1; right) mRNA expression. Myotubes were exposed to TNF-{alpha} (6 ng/ml, positive control) or IFN-{gamma} (60 ng/ml) for the indicated times. Total RNA was extracted, and cDNA was obtained from reverse transcription reaction. mRNA was quantified using real-time PCR. *P < 0.05.

 

Figure 7
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  		Fig. 7. IFN-{gamma} increases adult fast-type myosin heavy chain (MHC) and does not change the total protein-to-DNA ratio. Myotubes were exposed to TNF-{alpha} (6 ng/ml, positive control) or IFN-{gamma} (60 ng/ml) for 72 h. Top: cell lysates were analyzed with Western blot analysis using an antibody against total MHC. Bottom: optical density measurements were normalized to total protein and expressed as a percentage of vehicle control data. DNA was isolated and quantified by spectrophotometry. For total protein/total DNA, each treatment group was normalized to the control group at 1 day of treatment. Data represent means of 3 independent experiments. *P < 0.05.

 

   DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that skeletal muscle myotubes differ remarkably in the signaling events stimulated by IFN-{gamma} versus TNF-{alpha} treatment. The data suggest that IFN-{gamma} does not directly induce muscle protein catabolism in C2C12 myotubes. These findings advance our understanding of the physiological action of IFN-{gamma} and suggest that the catabolism attributed to IFN-{gamma} may require cofactors such as TNF-{alpha} or other inflammatory mediators.

In a variety of chronic wasting diseases, the development of immunodeficiency and cachexia are considered a lethal threat to the patients (5). IFN-{gamma} is often considered to be an instigator of immunodeficiency in these conditions, but its role in muscle wasting is less well understood. The enhanced production of IFN-{gamma} has been linked to weight loss, anemia, and depression (43). Early evidence that IFN-{gamma} promotes cachexia was obtained in a study (20) of T cell lymphoma patients treated with recombinant human IFN-{gamma}. Furthermore, IFN- {gamma} was linked to cachexia in mice that had been inoculated with IFN-{gamma}-producing tumors (28, 29). However, the simultaneous presence of both the tumor and IFN-{gamma} was necessary to induce cachexia in these animals. More recently, it has been demonstrated that IFN-{gamma} combined with TNF-{alpha} treatment leads to a decrease in MHC that was attributed to a corresponding decrease in MyoD mRNA in cultured myotubes (1). We measured a decrease in Akt phosphorylation after 6 h of IFN-{gamma} treatment, which could contribute to the potentiation of the catabolic effect of TNF-{alpha} by IFN-{gamma} when the two are used together. This differs from TNF-{alpha} stimulation alone, which promotes catabolic signaling in both animals (2, 6, 8, 13), isolated muscles (33), and cultured myotubes (21, 26). The evidence supporting a role for IFN-{gamma} as a catabolic stimulus was obtained from in vivo studies of patients and mice, complex conditions in which it is difficult to discriminate direct versus indirect effects. The direct action of IFN-{gamma} alone on catabolic signaling had not been tested in cultured myotubes. Importantly, our treatment range (ng/ml) in vitro was a magnitude higher than circulating levels of IFN-{gamma} (pg/ml) and yet failed to directly induce muscle catabolism. Our findings together with previous observations suggest that the cachexia linked to IFN-{gamma} is a result of complex systemic interactions and not a direct effect of IFN-{gamma} on muscle.

We hypothesized that IFN-{gamma} may influence catabolism by altering skeletal muscle oxidant activity. Increased ROS and oxidative stress are closely associated with loss of muscle mass in catabolic states (3, 9, 39). It has been demonstrated that TNF-{alpha} binds to surface receptors to increase ROS activity within skeletal muscle fibers (22) and activates protein kinases and redox-sensitive transcription factors, including p38 MAPK, ERK1/2, JNK, and NF-{kappa}B (14). The present study demonstrates that IFN-{gamma} stimulates early receptor signaling, as evidenced by the phosphorylation of Stat1, a transcriptional regulatory protein linked to IFN-{gamma} membrane receptors (18). However, IFN-{gamma} failed to elicit an increase in oxidant activity within C2C12 myotubes. Furthermore, IFN-{gamma} failed to activate p38 MAPK, ERK1/2, or JNK. These results are consistent with data from mouse macrophage cells (7) or microglial cells (17) in which direct IFN-{gamma} exposure did not activate MAPKs. Furthermore, in human macrophages, IFN-{gamma} exposure actually inhibited a lipopeptide-evoked MAPK response.

Activated NF-{kappa}B is required for cytokine-mediated MHC protein loss (14, 16, 26). In the canonical pathway, NF-{kappa}B is activated via release from the inhibitory protein I{kappa}B-{alpha}. Upon exposure to cytokines such as TNF-{alpha}, I{kappa}B-{alpha} protein is degraded, thereby permitting NF-{kappa}B to translocate to the nucleus and drive the transcription of catabolic genes. We showed that IFN-{gamma} treatment had no effect on I{kappa}B-{alpha} protein levels or NF-{kappa}B DNA binding activity. These results are consistent with observations in C2C12 myotubes showing that IFN-{gamma} alone does not activate NF-{kappa}B but can potentiate TNF-{alpha}-induced activation (40).

Important markers of catabolic gene expression in muscle include atrogin1/MAFbx and MuRF1 mRNA, key E3 ubiquitin ligases whose involvement in muscle atrophy are well established (4, 10, 15). Gomes et al. (15) and Bodine et al. (4) demonstrated that atrogin1/MAFbx and MuRF1 mRNA are upregulated in murine models of catabolism that included fasting, diabetes, cancer, renal failure, hindlimb suspension, immobilization, and denervation. Direct TNF-{alpha} exposure stimulates the expression of atrogin1/MAFbx by C2C12 myotubes (24). Exogenous infusion of TNF-{alpha} increases atrogin/MAFbx and MuRF1 mRNA in rat gastrocnemius muscle (12). Data in the present study are the first to show that TNF-{alpha} acts directly on muscle cells to stimulate the expression of MuRF1 and that IFN-{gamma} acts directly on muscle cells to stimulate the expression of atrogin1/MAFbx and MuRF1. These data appear to support IFN-{gamma} as a potential catabolic stimulus. However, according to Li et al. (24, 25), it is likely that atrogin1/MAFbx and NF-{kappa}B are both necessary to stimulate ubiquitin conjugation and protein degradation in muscle. Consistent with these observations, we found that IFN-{gamma} effects on atrogin1/MAFbx and MuRF1 mRNA did not alter atrogin1/MAFbx or MuRF1 protein levels, total protein ubiquitination, MHC protein levels, or the total protein-to-DNA ratio.

Our findings suggest that IFN-{gamma} alone is not a strong stimulus for muscle catabolism. IFN-{gamma} may simply exacerbate the effects of TNF-{alpha} (1), possibly through the regulation of Akt activity. Or, in some conditions, IFN-{gamma} may have anticatabolic actions. One study (27) has shown that the administration of recombinant IFN-{gamma} to patients with drug-resistant pulmonary tuberculosis increases the body mass index. In agreement with these findings, Tolosa et al. (40) demonstrated that IFN-{gamma} protects against TNF-{alpha}-induced cachexia through downregulation of TNF receptor 2. Clearly, systematic studies are needed to define the role of IFN-{gamma} as a possible synergistic factor in muscle catabolism.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-59878.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. Smith, Dept. of Physiology, Univ. of Kentucky, 800 Rose St., Rm. MS-508, Lexington, KY 40536-0298 (e-mail: masmit8{at}uky.edu)

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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