A proinflammatory tumor that activates protein degradation sensitizes rats to catabolic effects of endotoxin

Michelle L. Mackenzie,1 Nathalie Bedard,3 Simon S. Wing,3 and Vickie E. Baracos1,2

1Department of Agricultural, Food, and Nutritional Science; 2Department of Oncology, University of Alberta, Edmonton, Alberta, Canada; and 3Polypeptide Laboratory, Department of Medicine, McGill University, Montreal, Quebec, Canada

Submitted 4 February 2005 ; accepted in final form 27 May 2005


    ABSTRACT
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 ABSTRACT
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Chronic or acute inflammation may participate in the etiology of cancer cachexia. To investigate the interaction between tumor and a secondary inflammatory stimulus on muscle wasting, rats with and without tumors (Yoshida ascites hepatoma) received low doses of endotoxin (LPS, 400 µg/kg sc) or saline. Nitrogen balance was measured 24 h before and after LPS/saline. Epitrochlearis muscle was used to measure in vitro protein metabolism, and gastrocnemius muscle was used for quantification of the mRNA for components of the ubiquitin proteolytic pathway. The YAH reduced muscle mass (P = 0.002), increased muscle protein degradation (P = 0.042), and elevated mRNA expression of components of the ubiquitin proteolytic pathway (P < 0.01) including ubiquitin, ubiquitin-conjugating enzyme E214k, and ubiquitin ligases muscle RING Finger 1 and atrogin-1. Although the selected low dose of LPS had no impact on protein metabolism in control rats, LPS in rats bearing YAH caused weight loss (P = 0.0007), lowered nitrogen balance (P = <0.0001), and increased muscle protein degradation (P = 0.0336). In conclusion, the presence of a tumor can potentiate whole body and muscle-specific catabolic losses of protein in response to a stimulus that is not catabolic in healthy animals. This effect might be dependent on the inflammatory nature of the tumor.

muscle wasting; inflammation; ubiquitin proteolytic pathway


MUSCLE WASTING IN ADVANCED CANCER is associated with impaired functioning, intolerance to chemotherapy, and mortality (30, 37). Numerous factors may have a role in the development of muscle wasting; however, the end result is a shift in the catabolic-anabolic balance that promotes the breakdown of body protein (1).

The Yoshida ascites hepatoma (YAH) 130 in rats is a tumor with rapid growth and progressive development of cachexia. YAH induces a loss of skeletal muscle mass through an enhanced rate of muscle protein degradation involving the activation of the intracellular ATP-ubiquitin-dependent proteolytic pathway (3). This pathway involves three enzymes that ubiquinate proteins for degradation by the 26S proteasome (36). Ubiquitin-activating enzyme (E1) activates and transfers ubiquitin to an ubiquitin-conjugating enzyme (E2), which cooperates with an ubiquitin protein ligase (E3) to conjugate ubiquitin to the substrate. E3 is responsible for recognizing and targeting proteins for degradation. Several types of E3s have been identified, and at least three have been found to have increased expression in states of muscle atrophy, including YAH (22). Of these ligases, muscle-specific RING Finger 1 (MuRF-1) and atrogin-1/MAFbx have higher upregulation in muscle atrophy, suggesting a role in disease-associated muscle wasting (21).

In tumor models, including YAH, proinflammatory cytokines are clearly established to be mediators of protein catabolism. Levels of tumor necrosis factor-{alpha} (TNF-{alpha}) are increased in physiological fluids (35), and a causal role in elevated catabolism is demonstrated through the use of highly specific interventions, such as anti-TNF-{alpha} antibodies, soluble TNF-{alpha} receptors, and drugs that block TNF-{alpha} production, such as pentoxifylline (8, 9, 23). There is some evidence that the TNF-{alpha} response to endotoxin is exaggerated in tumor-bearing rats compared with non-tumor-bearing rats (7). Related to this finding, studies using endotoxin have demonstrated an increase in its lethality in rats bearing the Ward colon tumor (17) and mice bearing the Lewis lung carcinoma (26).

The presence of activated protein degradation and an enhanced sensitivity of tumor-bearing animals to proinflammatory signals would be expected to result in an enhanced catabolic response in tumor-bearing animals. Here, we hypothesized that in the YAH, bacterial endotoxin would elicit a disproportionately large activation of muscle protein catabolism compared with non-tumor-bearing animals. To test this hypothesis, we used a low-dose endotoxin to determine the impact of a mild catabolic stimulus on muscle wasting, nitrogen balance, and skeletal muscle protein degradation in rats bearing the YAH and non-tumor-bearing controls. Low doses of endotoxin (400 µg/kg body wt) produce a mild response that includes fever, metabolic response, and cytokine receptor gene expression in muscle (16, 38).


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Study Design

All studies were conducted in accordance with the Canadian Council on Animal Care Guidelines and were approved by the institutional Animal Policy and Welfare Committee. Male Sprague-Dawley rats (n = 28; Charles River, St. Constant, QC, Canada) were used as hosts for the YAH. Rats were housed in individual wire metabolic cages throughout the study period. The room was temperature and humidity controlled with a 12:12-h light-dark cycle. All rats consumed a diet of ground laboratory chow (LabDiet 5001; PMI Nutrition International, St. Louis, MO) that met all rodent nutrient requirements (28). Weight and feed intake were measured daily.

Rats were randomly allocated to one of four treatment groups after a 1-wk adaptation to environment and diet. Treatment variables were tumor and low-dose endotoxin (LPS). Tumor cells, maintained in liquid nitrogen, were initially implanted into nonstudy rats (n = 2). Ascites fluid was harvested after 5 days of tumor growth and injected immediately into study rats (n = 14). YAH (50 µl ip) was implanted on study day 0. After 4 days of tumor growth, treatment with saline or LPS (400 µg of Escherichia coli O55:B5/kg sc) was given. Twenty-four hours later, rats were killed by CO2 asphyxiation. Urine and feces were collected on study days 3 and 4 for determination of nitrogen (N) balance. Muscles (gastrocnemius and soleus) were dissected, weighed, and frozen in liquid N. Gastrocnemius muscle was used for expression of components of the ATP-ubiquitin proteolytic pathway. Epitrochlearis muscle was dissected and incubated for determination of protein synthetic and degradation rates.

Analysis

N balance. Urine and feces were collected for two separate 24-h periods before and after LPS/saline treatment. Feces were dried at 60°C for 48 h and ground. Total urine and feces collected in each period were weighed. N content of urine, feces, and diet were determined using the macro-Kjeldahl method (5). N balance was calculated by subtracting the N output (total urinary N plus total fecal N) from dietary N intake (feed intake x N content of feed). Absorbed N was calculated for each rat by subtracting fecal N content from N intake. The difference between absorbed N and urinary N was used to determine the amount of absorbed N retained and was expressed as a percentage.

Muscle incubations. Muscle incubations and analysis were similar to methods described previously (3, 34). The epitrochlearis is a small, thin muscle, making it ideal for muscle incubations. After dissection of both epitrochlearis muscles, each was placed in a tube containing 3 ml of medium. All chemicals listed below are from Sigma (St. Louis, MO) unless otherwise noted. The medium was composed of Krebs-Ringer bicarbonate buffer (119 mM NaCl, 4.8 mM KCl, 1.25 mM MgSO4, 25 mM NaHCO3, 1.2 4mM NaHPO4, 1.0 mM CaCl2, 2 mM HEPES, pH 7.4), glucose (8 mM), insulin (0.01 U/ml), bovine serum albumin (0.1% wt/vol), leucine (0.17 mM), isoleucine (0.1 mM), and valine (0.2 mM). Muscles were initially incubated for 30 min at 37°C with 95% O2-5% CO2. Muscles were then transferred to another tube containing 3 ml of medium, with the addition of either [3H]phenylalanine (0.5 uCi/ml) and phenylalanine (1 mM) or cycloheximide (0.5 mM) followed by incubation for 2 h under the same conditions as described above. At the end of the incubation, muscles were transferred to tubes containing 2% perchloric acid (PCA), and 0.5 ml of 16% PCA was added to the medium to make a 2% PCA solution. Samples were frozen at –80°C.

In vitro muscle protein synthesis and degradation. Muscle protein synthesis was determined using [3H]phenylalanine incorporation into muscle protein. Muscles incubated in [3H]phenylalanine and phenylalanine were homogenized in 3 ml of 5% trichloroacetic acid (TCA) and centrifuged for 20 min. The supernatant containing the intracellular soluble fraction was removed, and after the pellet was washed three times in 5% TCA the protein was dissolved overnight at room temperature in 1.5 ml of Soluene-350 (40–60% toluene, 30–40% dimethyl diakyl quaternary ammonium hydroxide, and 5–10% methanol; Packard Instruments, Meriden, CT). Disintegrations per min (dpm) in the intracellular and protein fractions were measured using a {beta}-counter (Beckman LS 5801; Beckman Instruments, Irvine, CA). Intracellular phenylalanine concentration was measured using high-performance liquid chromatography (HPLC) with precolumn o-phthaldialdehyde (OPA) derivatization (32). Intracellular specific activity (dpm·3 ml–1·nmol Phe–1·3 ml–1) was used as the precursor pool for muscle protein synthesis. Muscle protein concentration (mg/mg muscle) was measured using LECO FP-428 Nitrogen/Protein determinator (Leco, St. Joseph, MI), and protein mass (protein concentration x muscle mass) was used to calculate the specific activity of muscle protein (dpm/mg protein). Protein synthesis rate was calculated as nanomole phenylalanine incorporated into protein/milligram protein over the 2 h.

Muscle protein degradation was measured using the amount of phenylalanine released from muscle when protein synthesis was blocked by cycloheximide. The phenylalanine concentration of the medium was determined using HPLC and OPA derivatization. Degradation rate was expressed as nanomole phenylalanine released/milligram protein over the 2 h.

ATP-ubiquitin-dependent proteolytic system mRNA and protein expression. Levels of mRNA for components of the ATP-ubiquitin-dependent proteolytic system (ubiquitin, E214k, MuRF-1, and atrogin-1/MAFbx) were measured using Northern blot analysis on RNA extracted from frozen gastrocnemius muscles using the guanidinium isothiocynate-CsCl method. Electrophoresis of muscle RNA on 1% agarose-containing formaldehyde was followed by transfer onto nylon membranes and cross-linked to the membranes by ultraviolet light. Membranes were hybridized with cDNA probes encoding for ubiquitin, E214k, MuRF-1, atrogin-1/MAFbx, or 18s rRNA. Quantification was by densitometric scanning of autoradiographs or by phosphorimager analysis, and values for ubiquitin system transcripts were normalized to the 18s rRNA to correct for differences in sample loading and transfer to membrane.

Protein levels of atrogin-1/MAFbx were measured by Western blotting. Gastrocnemius muscles were homogenized by using a Polytron in PBS containing 1% NP-40 and protease inhibitors. After centrifugation at 10,000 g, proteins from the soluble fraction were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with the primary atrogin-1/MAFbx antibody (gift of Regeneron Pharmaceuticals, Tarrytown, NY), followed by a secondary antibody for 1 h. Phosphorimager analysis was used for quantification, and values were normalized to GAPDH to correct for differences in sample loading and transfer to membrane.

Statistical Analysis

Statistical Analysis System (SAS, version 8.2; SAS Institute, Cary, NC) was used for statistical analysis. Data are expressed as means ± SE. Data were log-transformed if not normally distributed, and multiple variances were used if group variances were not homogenous. The effects of tumor before the saline/LPS administration were determined using one-way ANOVA in the SAS mixed procedure (weight, intake, and N balance, day 3). For the remaining analysis, treatment (tumor, LPS) and interaction effects were determined using two-way ANOVA in the SAS mixed procedure. Differences among treatment groups were identified using t-test. Statistical significance was considered at P < 0.05.


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Effects of the Yoshida Hepatoma

Body weight (Table 1) was not different between control and tumor-bearing animals at the start of the study, but the growth of the ascites tumor resulted in a higher body weight at the start of study days 4 (main effect P = 0.049) and 5 (P = 0.005 vs. control). However, muscle mass (Table 2) was reduced (gastrocnemius P = 0.002) in the tumor-bearing group, suggesting nontumor weight loss. Body weight increased during the final 24 h (P = 0.007). Tumor-bearing rats had a reduction in feed intake (Fig. 1) on study days 3 (main effect P = 0.01) and 4 (P = 0.002).


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Table 1. Initial body weight and changes in body weight before (day 4) and after (day 5) LPS or saline administration in tumor-bearing and non-tumor-bearing controls

 

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Table 2. Muscle weights 24 h after LPS or saline administration in tumor-bearing rats and non-tumor-bearing controls

 


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Fig. 1. Daily feed intake in tumor-bearing [Yoshida ascites hepatoma (YAH)] and nontumor-bearing (Control) rats over 5 days of treatment. Low-dose endotoxin (LPS) or saline was given at the start of study day 4 (n = 7 rats per group). *Significant YAH effect (ANOVA, P < 0.05). Groups with same letter are not significantly different (P < 0.05).

 
N intake, loss, and balance data are shown in Table 3 and Fig. 2. On study day 3, YAH-bearing rats compensated for a lower total N absorbed (P = 0.005) through a reduction in urinary N loss (P = 0.0008) and thus maintained N balance at levels similar to that of controls. Despite a similar pattern on study day 4 (P = 0.0009 for N absorbed and P < 0.0001 for urinary N), the tumor-bearing group had a lower N balance compared with day 3 (P = 0.038).


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Table 3. Nitrogen balance before and after LPS or saline administration in tumor-bearing and non-tumor-bearing controls

 


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Fig. 2. Nitrogen absorbed (A) and %absorbed nitrogen retained (B) in tumor-bearing (YAH) and nontumor-bearing (Control) rats during 24 h before (day 3) and after (day 4) low-dose endotoxin (LPS) or saline administration (n = 7 rats per group). A: significant main effect (ANOVA, P < 0.05) of tumor days 3 and 4. B: significant main effect of LPS and interaction effect (tumor x LPS) day 4. For each day, groups with same letter are not significantly different (P < 0.05). *Significant difference between study day 4 and day 3 for that group (paired t-test, P < 0.05).

 
YAH increased muscle protein degradation (P = 0.0028) by 37% (Table 4). Expression of ubiquitin and E214k mRNA (Fig. 3) was elevated in the YAH group (main effect P = 0.003 and 0.0113, respectively). The two ubiquitin ligases (Figs. 3 and 4) measured also had higher expression in the tumor-bearing group: the atrogin-1/MAFbx ligase mRNA and protein were increased almost 5-fold (main effect P < 0.0001) and 1.7-fold (main effect P = 0.0001), respectively, and MuRF-1 mRNA was 1.5-fold higher (main effect P = 0.0116).


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Table 4. In vitro skeletal muscle protein degradation 24 h after LPS (+LPS) or saline (–LPS) administration in YAH-bearing rats (+tumor) and non-tumor-bearing controls (–tumor)

 


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Fig. 3. A: mRNA expression of components of ATP-ubiquitin proteolytic system in gastrocnemius muscle from tumor-bearing (YAH, n = 13) and nontumor-bearing (Control, n = 14) rats 24 h after low-dose endotoxin (LPS) or saline administration. LPS had no effect in either Control or YAH rats; therefore, data were pooled. YAH was in the 5th day of tumor growth. Results are expressed as %control. E2, ubiquitin conjugating enzyme; MuRF-1, muscle-specific RING Finger 1; *Significant main effect of tumor (ANOVA, P < 0.05). B: representative Northern blots for the components of ATP-ubiquitin proteolytic system and 18S rRNA for Control and YAH rats given LPS or saline, with analyzed bands indicated. mRNA levels were standardized using 18S rRNA.

 


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Fig. 4. A: atrogin-1/MAFbx protein in gastrocnemius muscle from tumor-bearing (YAH) and nontumor-bearing (Control) rats 24 h after low-dose endotoxin (LPS) or saline. YAH was in the 5th day of growth. Results are expressed as %control. There was a main effect of tumor (ANOVA, P = 0.0001). Group size: Control, n = 4; Con + LPS, n = 7; YAH, n = 3; YAH + LPS, n = 7. Groups with same letter are not significantly different. B: representative Western blots for atrogin-1/MAFbx (40.9 kDa) with molecular mass markers on the left. Indicated band migrates at ~38 kDa, consistent with it being atrogin-1/MAFbx, given the error of molecular mass estimation based on electropheretic migration of prestained standard markers. In support of this assignment, this band, like the mRNA for atrogin-1/MAFbx (see Ref. 4), is present only in skeletal muscle and heart, but not in brain, kidney, lung, liver, testis, and spleen (data not shown). Protein levels were standardized using GAPDH.

 
Effects of LPS Injection in Non-Tumor-Bearing Animals

Mean body and muscle weight of rats that received LPS did not differ from those that received saline in the control groups (Table 1). However, unlike the control group, body weight did not increase during the 24 h after LPS administration. Despite a decrease in N intake (P = 0.039) and loss (P = 0.0106 for urinary N and P = 0.0214 for fecal N) compared with controls, LPS alone did not significantly affect the total N absorbed, the percentage retained, or N balance (Table 3). As well, muscle protein content (Table 2), degradation (Table 4), and expression of ATP-ubiquitin-dependent system components were not affected by LPS in control rats (Ub mRNA, P = 0.7; E2 mRNA, P = 0.5; MuRF-1 mRNA, P = 0.4; atrogin-1/MAFbx mRNA, P = 0.7; and atrogin-1/MAFbx protein, P = 0.6), suggesting that in the healthy state the dose of LPS used does not promote a catabolic response in muscle protein.

Synergy Between YAH and LPS

There was a significant interaction effect (P = 0.0228) between tumor and LPS treatment on weight (P = 0.0028) and weight gain (P = 0.0068). Tumor-bearing rats receiving LPS had a lower weight (P = 0.013) and weight gain (P = 0.0007) compared with the tumor-bearing rats that received saline despite similar feed intake (Table 1 and Fig. 1).

LPS stimulated N loss in the tumor-bearing rats, as indicated by a reduction in N balance (P = 0.0015) in the 24 h after LPS administration (Table 3). This lowering of N retention was attributed to both a decrease in N intake (P = 0.0013) and total N absorbed (P = 0.0014) without a change in N loss. N absorbed was similar to that in rats bearing YAH alone (Fig. 2); however, there was a drastic reduction in the percentage of absorbed N retained in the tumor-bearing rats given LPS (P = 0.0051 vs. YAH alone). Tumor-bearing rats that received LPS were the only group with N balance significantly lower than controls (P = < 0.0001 vs. control and P = 0.0034 vs. LPS).

There was a main effect of YAH on the weight (P = 0.0263) and protein content (P = 0.0125) of the epitrochlearis muscle (Table 2); however, only the tumor-bearing rats that received LPS had lower values compared with controls (P = 0.0265 for weight and P = 0.041 for protein content). Protein synthesis rates were not affected by either tumor implantation or LPS treatment (CON: 0.970 ± 0.070; CON + LPS: 1.084 ± 0.051; YAH: 1.111 ± 0.094; YAH + LPS: 1.008 ± 0.097 nmol Phe·mg protein–1·2 h–1). The major effect of the study treatments on protein turnover was on the process of protein degradation, which was strongly elevated by the tumor (+38%) and further elevated 18% by LPS treatment in the tumor-bearing group (P = 0.0336 vs. YAH alone) (Table 4). However, this activation of catabolism above that induced by the tumor alone was not associated with any further increase in the mRNA expression of the elements of the ATP-ubiquitin proteolytic system studied (ubiquitin, P = 0.4; E2, P = 0.7; MuRF-1, P = 0.8; atrogin-1/MAFx, P = 0.9) or protein levels of atrogin-1/MAFbx (Fig. 4; P = 0.3). Because LPS did not have an effect on the mRNA expression of ubiquitin, E214k, MuRF-1, or atrogin-1/MAFbx, nor protein level of atrogin-1/MAFbx, the data were pooled into two groups: control or YAH bearing (Fig. 3).


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The primary catabolic effects of tumors on skeletal muscle protein have been well established in animal models (2), and here we hypothesized a possible sensitization of protein catabolism to secondary factors in the tumor-bearing state. Our study shows that a superimposed inflammatory stimulus in the tumor-bearing state results in a larger catabolic response than would be expected on the basis of the independent responses to tumor and endotoxin alone. Low-dose endotoxin reduced nitrogen retention and elevated skeletal muscle proteolysis when a tumor known to be associated with catabolism induced by TNF-{alpha} and eicosanoids was present. The tumor model used produces an inflammatory response. Thus activation of proteolytic systems by a primary inflammatory response might predispose skeletal muscle to catabolism by a secondary stimulus such as endotoxin.

Endotoxin produces a classic response mediated by proinflammatory cytokines (15). A variety of studies have established evidence for a heightened sensitivity to endotoxin in various pathological states. Studies using a "two-hit" model in rats have shown that activation of inflammatory genes by hemorrhagic shock primes macrophages for an exaggerated cytokine (TNF{alpha}) response to low-dose endotoxin (11, 29). This theme is seen in the literature on tumor-bearing animals. Rats bearing the Ward colon tumor had a mortality rate of 83% compared with 8% in control rats in the 24 h after the administration of endotoxin (5 mg/kg) (17). A dose-response study in mice bearing the Lewis lung carcinoma is also associated with an increased lethality of endotoxin (26). The LD50 of endotoxin decreased from 700 µg in healthy mice to 60 and 1 µg in mice with 2.7-cm3 and 6.0-cm3 tumors, respectively (26). Rats bearing a Yoshida sarcoma given 1 mg/kg endotoxin exhibited a plasma TNF-{alpha} response 45-fold higher than in nontumor-bearing controls (7), and it is easy to imagine reaching a threshold for endotoxic shock under these conditions. Collectively, these studies suggest a sharp left shift in the endotoxin dose-toxicity relationship in the tumor-bearing state, which may be related to an amplified production of TNF-{alpha}. TNF-{alpha} mediates endotoxin-induced shock as well as a catabolic response in skeletal muscle (24, 25). The high levels of TNF-{alpha} production in tumor-bearing animals may account not only for increased mortality but also a shift in the catabolic response to endotoxin.

Animals with YAH experienced a large increase in muscle protein catabolism associated with an induction of proteolytic gene expression. Although changes in muscle protein synthetic rates were not observed in the current study, the catabolic state of in vitro muscle incubations might have masked any alterations by tumor or endotoxin. In agreement with previous studies that have used this model (3, 21), gene expression for key enzymes in the ubiquitin-proteasome pathway were elevated. At least four E3s have been found to have increased expression in states with muscle atrophy (20, 22). These include E3{alpha}/Ubr1, a second Ubr1 homolog E3{alpha}II, MuRF-1, and atrogin-1/MAFbx (also known as SCF atrogin-1). Studies in gene knockout mice and differential gene analysis in rat models of atrophy indicate that atrogin-1/MAFbx and MuRF-1 are involved in the enhanced protein degradation associated with muscle wasting (4, 21). Mice with atrogin-1/MAFbx gene deletion demonstrate a 56% reduction in denervation-induced muscle wasting, whereas the reduction in MuRF-1 gene-deficient mice was 36% (4). Atrogin-1/MAFbx mRNA expression displayed the greatest degree of YAH-induced elevation, along with an increase in protein levels, in our study. Despite the higher rates of skeletal muscle protein breakdown, endotoxin in YAH-bearing rats did not increase the expression of genes involved in ubiquitin conjugation, suggesting that other mechanisms of increasing substrate flux through the pathway are involved. The relative contribution of different ligases to muscle wasting associated with tumors or endotoxin is not known. Two muscle specific ligases, MuRF-1 and atrogin-1/MAFbx, were examined in this study, and the protein level was determined only for atrogin-1/MAFbx. Therefore, it is possible that other ligases were more responsive to endotoxin in the tumor-bearing rats. E3{alpha}II mRNA expression is elevated in response to YAH as well as TNF (20), and thus may have a role in sensitizing skeletal muscle of tumor-bearing rats to endotoxin. It is also possible that the increased expression of genes in the conjugation pathway is a part of an increase in the infrastructure within proteolytic pathways that allows for more catabolism to take place upon the arrival of a second stimulus, in this case endotoxin. The second stimulus may, for example, cause enhanced gene translation, posttranslational activation of enzymatic activity, or proteasome regulation, which are sites downstream of gene transcription that may affect proteolytic rates. As well, the availability of substrates for ubiquitin conjugation may be elevated by endotoxin.

There is some evidence of a synergistic effect of catabolic factors on skeletal muscle protein breakdown. A low dose of cortisol, which had a minimal effect on muscle protein catabolism in normally active healthy young men, elevated skeletal muscle protein breakdown rates threefold in the same subjects after bed rest for 2 wk (14). Although both inactivity and cortisol stimulate muscle protein breakdown, coadministration results in muscle protein breakdown greater than expected from the sum of both. Endotoxin at relatively high doses (10 mg/kg) in rats has been shown to increase skeletal muscle breakdown through activation of the ATP-ubiquitin proteolytic pathway (6, 7). In the design of the present study, we selected a much lower dose of endotoxin, which would be expected to cause little or no perturbation in protein metabolism. Accordingly, the effects on N metabolism elicited by low-dose endotoxin in healthy control rats were minimal; however, like the study of muscle sensitivity to cortisol, muscles of tumor-bearing rats had an enhanced degradative response to endotoxin. Our related work with the same low dose of endotoxin that demonstrated an induction of TNF and IL-6 receptors in skeletal muscle (38) provides a possible basis for this sensitization. This pattern of enhanced sensitivity of muscle protein degradation to catabolic factors may be an important component of complex wasting disorders where tumor, inflammation or infection, inactivity, elevated levels of glucocorticoids, and other factors may frequently be simultaneously present. The present study highlights the complexity involved in defining the mechanism responsible for and treatment of muscle wasting in disease states. The etiology of wasting may not be attributable to a single factor but rather to a series of factors that disastrously potentiate each other's catabolic effects, and the magnitude of the overall effect cannot be predicted on the basis of the separate effects of each.

The unfortunate outcomes of tumor and endotoxin/inflammation may be a feature of tumors that are themselves associated with some degree of inflammatory mediator production or host inflammatory response. The YAH used here and the other tumor models where enhanced sensitivity to endotoxin was demonstrated (7, 26) are associated with an inflammatory response. Many human tumors that are associated with wasting of skeletal muscle are associated with indices of inflammation (12, 13, 33), and these in turn are known to have prognostic significance and to be related to shortened survival (10, 31). The present study relates clinically to the metabolic response to infections in cancer patients. Infections are very common in patients with cancer (27) and are often the cause of death in advanced-cancer patients (18, 19). Our results generate the speculation that episodes of infection or inflammation may be associated with exaggerated catabolic responses in the tumor-bearing state.


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This work was supported by a Natural Sciences and Engineering Research Council of Canada Grant and Canadian Institutes of Health Research Grant MT-12121. AntiMAFbx antibody was a gift from Regeneron Pharmaceuticals.


    FOOTNOTES
 

Address for reprint requests and other correspondence: V. Baracos, Dept. of Oncology, Univ. of Alberta, Cross Cancer Institute, 11560 University Ave., Edmonton, AB, Canada T6G 1Z2 (email: vickieb{at}cancerboard.ab.ca)

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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  1. Baracos VE. Hypercatabolism and hypermetabolism in wasting states. Curr Opin Clin Nutr Metab Care 5: 237–239, 2002.[CrossRef][ISI][Medline]
  2. Baracos VE. Regulation of skeletal-muscle-protein turnover in cancer-associated cachexia. Nutrition 16: 1015–1018, 2000.[CrossRef][ISI][Medline]
  3. Baracos VE, DeVivo C, Hoyle DH, and Goldberg AL. Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma. Am J Physiol Endocrinol Metab 268: E996–E1006, 1995.[Abstract/Free Full Text]
  4. Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, Poueymirou WT, Panaro FJ, Na E, Dharmarajan K, Pan ZQ, Valenzuela DM, DeChiara TM, Stitt TN, Yancopoulos GD, and Glass DJ. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294: 1704–1708, 2001.[Abstract/Free Full Text]
  5. Bradstreet RB (Editor). The Kjeldahl Method for Organic Nitrogen. New York: Academic, 1965, p. 1–8.
  6. Chai J, Wu Y, and Sheng ZZ. Role of ubiquitin-proteasome pathway in skeletal muscle wasting in rats with endotoxemia. Crit Care Med 31: 1802–1807, 2003.[CrossRef][ISI][Medline]
  7. Combaret L, Tilignac T, Claustre A, Voisin L, Taillandier D, Obled C, Tanaka K, and Attaix D. Torbafylline (HWA 448) inhibits enhanced skeletal muscle ubiquitin-proteasome-dependent proteolysis in cancer and septic rats. Biochem J 361: 185–192, 2002.[CrossRef][ISI][Medline]
  8. Combaret L, Ralliere C, Taillandier D, Tanaka K, and Attaix D. Manipulation of the ubiquitin-proteasome pathway in cachexia: pentoxifylline suppresses the activation of 20S and 26S proteasomes in muscle from tumor-bearing rats. Mol Biol Rep 26: 95–101, 1999.[CrossRef][ISI][Medline]
  9. Costelli P, Carbo N, Tessitore L, Bagby GJ, Lopez-Soriano FJ, Argiles JM, and Baccino FM. Tumor necrosis factor-alpha mediates changes in tissue protein turnover in a rat cancer cachexia model. J Clin Invest 92: 2783–2789, 1993.[ISI][Medline]
  10. Falconer JS, Fearon KC, Ross JA, Elton R, Wigmore SJ, Garden OJ, and Carter DC. Acute-phase protein response and survival duration of patients with pancreatic cancer. Cancer 75: 2077–2082, 1995.[ISI][Medline]
  11. Fan J, Marshall JC, Jimenez M, Shek PN, Zagorski J, and Rotstein OD. Hemorrhagic shock primes for increased expression of cytokine-induced neutrophil chemoattractant in the lung: role in pulmonary inflammation following lipopolysaccharide. J Immunol 161: 440–447, 1998.[Abstract/Free Full Text]
  12. Fearon KC, McMillan DC, Preston T, Winstanley FP, Cruickshank AM, and Shenkin A. Elevated circulating interleukin-6 is associated with an acute-phase response but reduced fixed hepatic protein synthesis in patients with cancer. Ann Surg 213: 26–31, 1991.[ISI][Medline]
  13. Fearon KC, Hansell DT, Preston T, Plumb JA, Davies J, Shapiro D, Shenkin A, Calman KC, and Burns HJ. Influence of whole body protein turnover rate on resting energy expenditure in patients with cancer. Cancer Res 48: 2590–2595, 1988.[Abstract]
  14. Ferrando AA, Stuart CA, Sheffield-Moore M, and Wolfe RR. Inactivity amplifies the catabolic response of skeletal muscle to cortisol. J Clin Endocrinol Metab 84: 3515–3521, 1999.[Abstract/Free Full Text]
  15. Fink MP and Heard SO. Laboratory models of sepsis and septic shock. J Surg Res 49: 186–196, 1990.[CrossRef][ISI][Medline]
  16. Goodman MN. Tumor necrosis factor induces skeletal muscle protein breakdown in rats. Am J Physiol Endocrinol Metab 260: E727–E730, 1991.[Abstract/Free Full Text]
  17. Grossie VB and Mailman D. Influence of the Ward colon tumor on the host response to endotoxin. J Cancer Res Clin Oncol 123: 189–194, 1997.[ISI][Medline]
  18. Homsi J, Walsh D, Panta R, Lagman R, Nelson KA, and Longworth DL. Infectious complications of advanced cancer. Support Care Cancer 8: 487–492, 2000.[ISI][Medline]
  19. Inagaki J, Rodriguez V, and Bodey GP. Causes of death in cancer patients. Cancer 33: 568–573, 1974.[ISI][Medline]
  20. Kwak KS, Zhou X, Solomon V, Baracos VE, Davis J, Bannon AW, Boyle WJ, Lacey DL, and Han HQ. Regulation of protein catabolism by muscle-specific and cytokine-inducible ubiquitin ligase E3{alpha}-II during cancer cachexia. Cancer Res 64: 8193–8198, 2004.[Abstract/Free Full Text]
  21. Lecker SH, Jagoe RT, Gilbert A, Gomes M, Baracos V, Bailey J, Price SR, Mitch WE, and Goldberg AL. Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. FASEB J 18: 39–51, 2004.[Abstract/Free Full Text]
  22. Lecker SH. Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways? Curr Opin Clin Nutr Metab Care 6: 271–275, 2003.[CrossRef][ISI][Medline]
  23. Llovera M, Garcia-Martinez C, Lopez-Soriano J, Agell N, Lopez-Soriano FJ, Garcia I, and Argiles JM. Protein turnover in skeletal muscle of tumour-bearing transgenic mice overexpressing the soluble TNF receptor-1. Cancer Lett 130: 19–27, 1998.[CrossRef][ISI][Medline]
  24. Llovera M, Garcia-Martinez C, Agell N, Lopez-Soriano FJ, and Argiles JM. TNF can directly induce the expression of ubiquitin-dependent proteolytic system in rat soleus muscles. Biochem Biophys Res Commun 230: 238–241, 1997.[CrossRef][ISI][Medline]
  25. Mathison JC, Wolfson E, and Ulevitch RJ. Participation of tumor necrosis factor in the mediation of gram negative lipopolysaccharide-induced injury in rabbits. J Clin Invest 81: 1925–1937, 1988.[ISI][Medline]
  26. Matthys P, Heremans H, Opdenakker G, and Billiau A. Anti-interferon-{gamma} antibody treatment, growth of lewis lung tumours in mice and tumour-associated cachexia. Eur J Cancer 27: 182–187, 1991.[ISI][Medline]
  27. Nagy-Argen S and Haley HB. Management of infections in palliative care patients with advanced cancer. J Pain Symptom Manage 24: 64–70, 2002.[CrossRef][ISI][Medline]
  28. National Research Council. Nutrient Requirements of Laboratory Animals (4th Ed.). Washington, DC. National Academy of Science, 1995.
  29. Powers KA, Woo J, Khadaroo RG, Papia G, Kapus A, and Rotstein OD. Hypertonic resuscitation of hemorrhagic shock upregulates the anti-inflammatory response by alveolar macrophages. Surgery 134: 312–318, 2003.[CrossRef][ISI][Medline]
  30. Ross PJ, Ashley S, Norton A, Priest K, Waters JS, Eisen T, Smith IE, and O'Brien MER. Do patients with weight loss have a worse outcome when undergoing chemotherapy for lung cancers? Br J Cancer 90: 1905–1911, 2004.[CrossRef][ISI][Medline]
  31. Scott HR, McMillan DC, Forrest LM, Brown DJ, McArdle CS, and Milroy R. The systemic inflammatory response, weight loss, performance status and survival in patients with inoperable non-small lung cancer. Br J Cancer 87: 264–267, 2002.[CrossRef][ISI][Medline]
  32. Sedgwick GW, Fenton TF, and Thompson JR. Effect of protein precipitating agents on the recovery of plasma free amino acids. Can J Anim Sci 71: 953–957, 1991.[ISI]
  33. Staal-van den Brekel AJ, Schols AM, Dentener MA, ten Velde GP, Buurman WA, and Wouters EF. The effects of treatment with chemotherapy on energy metabolism and inflammatory mediators in small-cell lung carcinoma. Br J Cancer 76: 1630–1635, 1997.[ISI][Medline]
  34. Strelkov AB, Fields AL, and Baracos VE. Effects of systemic inhibition of prostaglandin production on protein metabolism in tumor-bearing rats. Am J Physiol Cell Physiol 257: C261–C269, 1989.[Abstract/Free Full Text]
  35. Tessitore L, Costelli P, and Baccino FM. Humoral mediation for cachexia in tumour-bearing rats. Br J Cancer 67: 15–23, 1993.[ISI][Medline]
  36. Tisdale MJ. Cachexia in cancer patients. Nat Rev Cancer 2: 862–871, 2002.[CrossRef][ISI][Medline]
  37. Vigano A, Dorgan M, Buckingham J, Bruera E, and Suarez-Almazor M. Survival prediction in terminal cancer patients: a systematic review of the medical literature. Palliat Med 14: 363–374, 2000.[CrossRef][ISI][Medline]
  38. Zhang Y, Pilon G, Marette A, and Baracos VE. Cytokines and endotoxin induce cytokine receptors in skeletal muscle. Am J Physiol Endocrinol Metab 279: E196–E205, 2000.[Abstract/Free Full Text]




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