Effect of eicosapentaenoic acid, protein and amino acids on protein synthesis and degradation in skeletal muscle of cachectic mice

Atrophy of skeletal muscle reduces both the quality and quantity of life of patients with cancer cachexia. Loss of muscle mass is thought to arise from a reduction in protein synthesis combined with an enhanced rate of protein degradation, and few treatments are available to counteract this process. Eicosapentaenoic acid (EPA) has been shown to attenuate the enhanced protein degradation, but to have no effect on protein synthesis. This study examines the effect of EPA combined with a protein and amino-acid supplementation on protein synthesis and degradation in gastrocnemius muscle of mice bearing the cachexia-inducing MAC16 tumour. Muscles from cachectic mice showed an 80% reduction in protein synthesis and about a 50-fold increase in protein degradation compared with muscles from nontumour-bearing mice of the same age and weight. Treatment with EPA (1 g kg−1) daily reduced protein degradation by 88%, but had no effect on protein synthesis. Combination of EPA with casein (5.35 g kg−1) also had no effect on protein synthesis, but when combined with the amino acids leucine, arginine and methionine there was almost a doubling of protein synthesis. The addition of carbohydrate (10.7 g kg−1) to stimulate insulin release had no additional effect. The combination involving the amino acids produced almost a doubling of the ratio of protein synthesis to protein degradation in gastrocnemius muscle over that of EPA alone. No treatment had a significant effect on tumour growth rate, but the inclusion of amino acids had a more significant effect on weight loss induced by the MAC16 tumour than that of EPA alone. The results suggest that combination therapy of cancer cachexia involving both inhibition of the enhanced protein degradation and stimulation of the reduced protein synthesis may be more effective than either treatment alone.

Atrophy of skeletal muscle in cancer cachexia results in weakness (asthenia) and death. Muscle mass is controlled by the rate of protein synthesis and the rate of protein degradation, which in the adult are normally in balance, so that muscle mass remains constant. In cachexia whole body protein turnover is increased (Carmichael et al, 1980), while in skeletal muscle protein synthesis is decreased (Lundholm et al, 1976), while protein degradation is elevated (Lundholm et al, 1982). Attempts to increase protein synthesis, for example, by elevation of substrate flux, in the absence of inhibitors of protein degradation have not increased lean body mass (Evans et al, 1985). Protein degradation in cancer cachexia has been attributed predominantly to an increased expression of the ubiquitin -proteasome proteolytic pathway (Lecker et al, 1999). One of the few agents capable of attenuating protein degradation in cachexia is the polyunsaturated fatty acid, eicosapentaenoic acid (EPA), which downregulates the increased gene expression of key regulatory components of the ubiquitin -proteasome pathway in skeletal muscle (Whitehouse et al, 2001). Although EPA attenuates the increased protein degradation in cancer cachexia, it has no effect on the depression of protein synthesis (Beck et al, 1991). Thus, EPA attenuates the development of weight loss in cachectic patients with pancreatic cancer, but weight gain is minimal (Wigmore et al, 2000). However, when patients were administered EPA together with a high-protein high-energy supplement weight gain was seen and this arose solely from an increase in lean body mass (Barber et al, 1999).
This suggests a mechanism to counter muscle atrophy in cancer cachexia by the inhibition of protein degradation, combined with a stimulus for protein synthesis. Muscle protein synthesis has been shown to be initiated by essential amino acids, particularly the branched chain amino-acid (BCAA) leucine (Anthony et al, 2000). Stimulation of protein synthesis requires an optimal plasma profile of amino acids and occurs through a mechanism that involves the stimulation of the eukaryotic initiation factor (eIF)-binding protein 1. Arginine is a conditionally essential amino acid, which has been shown to be deficient in tumour-bearing mice (Vissers et al, 2002). Arginine may improve the delivery of amino acids to muscle (Robertson et al, 1994), and thus may increase protein synthesis. Methionine is another essential amino acid, which may be rate limiting in protein synthesis. It has recently been shown (Tesseraud et al, 2003) that simultaneous addition of two amino acids, leucine and methionine, are critical in controlling protein translation. Methionine is not only a building block for proteins but is also the primary amino acid needed to initiate protein synthesis (Bradshaw et al, 1998). In addition, synthesis of acute phase proteins, which has been correlated with the loss of body mass in patients with lung and gastrointestinal cancers (McMillan et al, 1998), alters requirements for amino acids, since these proteins contain relatively high levels of sulphur-containing amino acids (Reeds et al, 1997). Lower plasma levels of leucine and methionine have been reported in the serum of weight-losing mice bearing the MAC16 tumour (Beck and Tisdale, 1989).
The purpose of the present study was to investigate the effect of protein (casein) and amino acids (leucine, arginine and methionine) together with EPA on protein synthesis and degradation in gastrocnemius muscle of mice bearing the MAC16 colon adenocarcinoma (Bibby et al, 1987). An additional group received carbohydrate, to stimulate insulin release, which should facilitate the uptake of amino acids into muscle and stimulate protein synthesis.

Animals
Pure strain male NMRI mice were bred in our own colony and fed economy rodent breeder diet (Special Diet Services, Essex, UK) and water ad libitum. The diet contained 19.2% crude protein and 4.3% fat. Animals were implanted subcutaneously (s.c.) in the flank with fragments of the MAC16 tumour by means of a trochar, selecting from donor animals with established weight loss (Bibby et al, 1987). All animal experiments followed a strict protocol, approved by the British Home Office, and the ethical guidelines that were followed meet the standards required by the UKCCR guidelines (Workman et al, 1998). Weight loss was evident 10 -12 days after transplantation and the animals were randomised into groups of 12 when the weight loss was about 5%. Six animals were used for protein synthesis and six for protein degradation. A nontumour-bearing group formed an additional control. Cachectic mice received olive oil as a control, EPA (1.0 g kg À1 ), EPA (1.0 g kg À1 and casein protein 5.35 g kg À1 ), EPA (1.0 g kg À1 , casein 5.35 g kg À1 , leucine 25.5 mg, arginine 12 mg, methionine 5 mg) and EPA (1.0 g kg À1 , casein 5.35 g kg À1 , amino acids as above and 10.7 g kg À1 carbohydrate). The supplements were administered twice daily in 50 ml doses. All treatments were administered daily peritoneally (p.o.) by gavage for a 4-day period and protein synthesis and degradation were determined on the 4th day. The concentrations of protein, amino acids and carbohydrate were equivalent to those consumed by patients taking the recommended dosage of Resource s Support (Novartis Medical Nutrition). Tumour volumes were measured daily by means of calipers, and were recorded as a percentage of the starting tumour volume. Body weight was measured daily and recorded as the change in body weight from the start of the experiment. At the end of the experiment, the animals were humanely killed and the gastrocnemius muscles were rapidly removed for further analysis.

Protein synthesis and degradation in gastrocnemius muscle
The method for the determination of protein synthesis and degradation in gastrocnemius muscle has been described previously (Beck et al, 1991). Briefly, protein synthesis was measured by the incorporation of L-[4 À3 H]phenylalanine during a 2 h period in which isolated gastrocnemius muscles were incubated at 371C in RPMI 1640 without phenol red and saturated with O 2 : CO 2 (19 : 1). After incubation, muscles were rinsed in nonradioactive medium, blotted and homogenised in 4 ml 2% perchloric acid. The rate of protein synthesis was calculated by dividing the amount of protein-bound radioactivity by the amount of acid-soluble radioactivity.
For protein degradation assays animals from the same group as used to measure protein synthesis were administered i.p. with 0.4 mM L-[4 À3 H]phenylalanine in phosphate-buffered saline (100 ml) 24 h prior to the assay. Isolated gastrocnemius muscles were extensively washed with 0.9% NaCl and RPMI 1640 medium before measuring the release of radioactivity into RPMI 1640 over a 2 h period. The protein-bound radioactivity was determined by homogenising in 2% perchloric acid as above and the rate of protein degradation was calculated by dividing the amount of [ 3 H]phenylalanine radioactivity released into the incubation medium during the 2 h incubation period by the specific activity of protein-bound [ 3 H]phenylalanine.

Statistical analysis
Results are expressed as mean7s.e.m. Differences were determined by one-way analysis of variance (ANOVA) followed by Tukey -Kramer multiple comparison test. P-values less than 0.05 were considered significant.

RESULTS
The effects of the various treatments on protein synthesis and protein degradation in gastrocnemius muscle of cachectic mice bearing the MAC16 tumour is shown in Figure 1. The concentration of EPA used was suboptimal in order to discern synergistic interactions. Control animals showed high levels of protein synthesis and little protein degradation, which was expected, since they were growing steadily. There was a significant depression in protein synthesis and a large increase in protein degradation in muscles of cachectic mice bearing the MAC16 tumour, as reported previously (Beck et al, 1991). EPA treatment significantly reduced protein degradation, but had no effect on protein synthesis. There was no significant effect of any of the other treatments on protein degradation compared with that of EPA alone. There was a small, but nonsignificant elevation in protein synthesis in animals receiving casein in addition to EPA. However, when casein was administered together with leucine, arginine and methionine, there was a significant increase in protein synthesis above that seen in cachectic mice or cachectic mice administered EPA. Interestingly, this effect was not seen in the presence of carbohydrate. The ratio of protein synthesis to degradation was improved by all supplement treatments over that of EPA alone (Figure 2), although it never reached the values observed in nontumour-bearing mice.
The effect of the various treatments on body weight loss is shown in Figure 3. Both EPA and EPA þ casein reduced total body weight loss, but this did not reach significance except at the 4-day time point. However, the addition of both amino acids and amino acids þ carbohydrate to the EPA þ casein regime caused a significant reduction in weight loss compared with nontreated controls. None of the treatments had a significant effect on tumour volume (Figure 4).  Figure 2 Ratio of protein synthesis to protein degradation in gastrocnemius muscle of cachectic mice bearing the MAC16 tumour after being subjected to the nutritional regimes detailed in Figure 1 for a 4-day period. Effect of nutritional supplementation on tumour growth in mice bearing the MAC16 tumour. Tumour size has been normalised to 100% at the start of the experiment. Animals (n ¼ 12) were randomised to receive daily p.o. treatments of olive oil (X), EPA ('), EPA þ casein (J), EPA þ casein þ amino acids (K) or EPA þ casein þ amino acids þ carbohydrate (&) at the concentrations indicated in the Materials and methods section. There was no significant difference between the groups.

DISCUSSION
The mechanism for the increased protein degradation in skeletal muscle in cancer cachexia has been suggested as an increased expression of the ubiquitin -proteasome proteolytic pathway (Williams et al, 1999). Various cytokines, such as tumour necrosis factor-a or interferon gamma (Llovera et al, 1998), have been shown to increase transcripts of ubiquitin in skeletal muscle, but the importance of these cytokines as mediators of the weight loss in human cancer cachexia is controversial (Maltoni et al, 1997). In cancer cachexia, a sulphated glycoprotein, proteolysis-inducing factor (PIF) produced by cachexia inducing tumours (Todorov et al, 1996) may be more important in skeletal muscle atrophy. Proteolysis-inducing factor stimulates loss of the myofibrillar protein myosin directly by increasing the expression of proteasome subunits and the ubiquitin-conjugating enzyme (E2 14k ) (Gomes-Marcondes et al, 2002). The process is initiated by the activation of PIF of an intracellular signalling cascade in muscle involving the release of arachidonic acid from membrane phospholipids and metabolism to 15-hydroxyeicosatetraenoic acid (15-HETE) (Smith et al, 1999), and involving the activation of the transcription factor nuclear factor-kB (NF-kB) (Whitehouse and Tisdale, 2003). Eicosapentaenoic acid attenuates this process, both by inhibiting the formation of 15-HETE (Smith et al, 1999) and by stabilising the IkB/NF-kB cytosolic complex and nuclear accumulation of NF-kB (Whitehouse and Tisdale, 2003), preventing the increase in proteasome expression and degradation of myofibrillar proteins. Proteasome-mediated proteolysis is independent of the amount of protein consumed, so that simple nutritional supplementation would not be expected to prevent muscle catabolism as observed in mice bearing the MAC16 tumour. However, leucine has been shown to inhibit the expression of genes of the proteasome pathway in muscle of cachectic rats, but not in those of rats after starvation (Busquets et al, 2002). Branched chain amino acids also directly inhibit the proteasome 'chymotrypsinlike' enzyme activity, the predominant proteolytic activity of the bsubunits of the proteasome in skeletal muscle, but not in the liver (Hamel et al, 2003). Despite this in the present study, there was no evidence for attenuation of protein degradation by leucine/aminoacid mixture below that induced by EPA alone in gastrocnemius muscle of cachectic mice bearing the MAC16 tumour.
In addition to the effect on protein degradation PIF also inhibits protein synthesis in muscle, but the mechanism by which this occurs is not known, although it is attenuated by insulin at physiological concentrations and below, but not by EPA (Smith et al, 1999). The action of insulin suggests that PIF may inhibit translation, for example, by dephosphorylation of translation factors or the S6 ribosomal protein. Branched chain amino-acid, and leucine in particular, enhance muscle protein synthesis through the activation of the mRNA-binding step in translation initiation in skeletal muscle, but not in the liver (Yoshizawa, 2004). The action of leucine is mediated by the activation of the ribosomal protein S6 kinase and by phosphorylation of initiation factors, suggesting that it may, like insulin, overcome the PIFinduced inhibition of protein synthesis. This study shows that while casein alone did not significantly stimulate protein synthesis in gastrocnemius muscle of cachectic mice, there was a significant increase in protein synthesis with the addition of the leucine/ arginine/methionine mix. This was not further enhanced by the carbohydrate mix, although leucine and arginine have been previously shown to stimulate insulin secretion (Malaisse, 1984), and insulin plays a permissive role in leucine-induced protein synthesis (Yoshizawa, 2004). Thus treatment of cancer patients with a combination of EPA, protein and amino acids, particularly leucine, would be expected to attenuate protein degradation and stimulate protein synthesis in skeletal muscle, which should lead to improvements both in the quality of life and survival, and this combination is currently under clinical evaluation.