Light aerobic physical exercise in combination with leucine and/or glutamine-rich diet can improve the body composition and muscle protein metabolism in young tumor-bearing rats Emilianne Miguel Salomão & Maria Cristina Cintra Gomes-Marcondes
Journal of Physiology and Biochemistry Official Journal of the University of Navarra, Spain ISSN 1138-7548 Volume 68 Number 4 J Physiol Biochem (2012) 68:493-501 DOI 10.1007/s13105-012-0164-0
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Author's personal copy J Physiol Biochem (2012) 68:493–501 DOI 10.1007/s13105-012-0164-0
ORIGINAL PAPER
Light aerobic physical exercise in combination with leucine and/or glutamine-rich diet can improve the body composition and muscle protein metabolism in young tumor-bearing rats Emilianne Miguel Salomão & Maria Cristina Cintra Gomes-Marcondes
Received: 25 October 2011 / Accepted: 13 March 2012 / Published online: 30 March 2012 # University of Navarra 2012
Abstract Nutritional supplementation with some amino acids may influence host’s responses and also certain mechanism involved in tumor progression. It is known that exercise influences body weight and muscle composition. Previous findings from our group have shown that leucine has beneficial effects on protein composition in cachectic rat model as the Walker 256 tumor. The main purpose of this study was to analyze the effects of light exercise and leucine and/or glutamine-rich diet in body composition and skeletal muscle protein synthesis and degradation in young tumor-bearing rats. Walker tumorbearing rats were subjected to light aerobic exercise (swimming 30 min/day) and fed a leucine-rich (3%) and/or glutamine-rich (4%) diet for 10 days and compared to healthy young rats. The carcasses were analyzed as total water and fat body content and lean body mass. The gastrocnemious muscles were isolated and used for determination of total protein synthesis and degradation. The chemical body composition changed with tumor growth, increasing body water and reducing body fat content and total body nitrogen. After tumor growth, the muscle
E. M. Salomão : M. C. C. Gomes-Marcondes (*) Department of Structural and Functional Biology, Biology Institute, State University of Campinas, UNICAMP, 13083-970 Campinas, São Paulo, Brazil e-mail:
[email protected]
protein metabolism was impaired, showing that the muscle protein synthesis was also reduced and the protein degradation process was increased in the gastrocnemius muscle of exercised rats. Although short-term exercise (10 days) alone did not produce beneficial effects that would reduce tumor damage, host protein metabolism was improved when exercise was combined with a leucine-rich diet. Only total carcass nitrogen and protein were recovered by a glutamine-rich diet. Exercise, in combination with an amino acid-rich diet, in particular, leucine, had effects beyond reducing tumoral weight such as improving protein turnover and carcass nitrogen content in the tumorbearing host. Keywords Leucine-rich diet . Glutamine-rich diet . Walker 256 tumour . Protein metabolism . Body composition
Introduction Cachexia is characterised by involuntary weight loss in patients that results in reduced survival [42]. The lean body mass loss occurs primarily in the skeletal muscle tissue and is directly proportional to the effects of tumor evolution. This results in a reduction in the patient’s physical performance and response to chemotherapy [43]. A decrease in protein synthesis and an increase in protein
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degradation, which alters total protein turnover balance, are major factors in tumor development [23, 43]. Such changes jeopardize vital functions by causing skeletal muscle loss, fatigue, weakness, and atrophy [30]. Aerobic exercise can improve physical conditioning and increase the quality of life of cancer patients [7]. The fatigue is one of the most severe symptoms in cancer patients, which compromises muscle resistance and health [9, 25]. In animals, moderate intensity physical exercise reduced the incidence of tumoral implants and tumor growth [2], although it remains uncertain that the modulatory effect of the exercise on the immune system could be the possible benefit found in tumor-bearing animals.. In addition, the branched chain amino acids (BCAAs) including valine, isoleucine and leucine are essential substrates and important regulators of total body protein synthesis [12]. They represent the largest sources of nitrogen for glutamine and alanine, which are also synthesized in diseases such as cancer [23, 43]. An increase in BCAA oxidation rate is common in a cancer-induced inflammatory response, which contributes to muscle loss [22]. In particular, leucine is used as an energy source by the skeletal muscle and can be transaminated and oxidized to produce acetyl-coenzyme A. Furthermore, the glutamine requirement, a nonessential amino acid, may be increased when catabolic processes are enhanced. This may not only occur in lymphocytes and small intestinal epithelial cells [22, 38, 52] but also in muscle tissue because glutamine may improve muscle protein synthesis in rats [4, 28, 33]. Wolfe [51] verified that the amino acids supplementation, in combination with exercise, increases protein synthesis and nitrogen balance. According to Segal et al. [37], well-supervised exercise can be an excellent coadjuvant therapy added to anti-cancer treatment and rehabilitation and can improve the physiological and psychological states of such patients. As exercise can benefit cancer patients, the main purpose of this work was to investigate whether high content leucine and/or glutamine dietary supplementation, in combination with light aerobic physical exercise, could counteract cachexia in our experimental model by improving lean body mass and muscle protein metabolism in tumor-bearing rats.
E.M. Salomão, M.C.C. Gomes-Marcondes
Methods Reagents All reagents were purchased from Sigma-Aldrich. Animals and diets Eighty Wistar male rats (35±2 days age), obtained from the animal facilities at the State University of Campinas, UNICAMP, Brazil, received diet and water ad libitum. Light and darkness cycles (12–12 h) and temperature (22±2°C) were controlled. The semipurified diets are in accordance with AIN93, the American Institute of Nutrition [35]. Normoprotein diet (C) comprised 18% protein (casein, as the sole source of protein, containing 0.46% alanine, 0.64% arginine, 1.22% aspartic acid, 0.37% cystine, 3.63% glutamic acid, 0.32% glycine, 0.46% histitine, 0.85% isoleucine, 1.54% L- leucine, 1.3% lysine, 0.46% methionine, 0.88% phenylalanine, 2.05% proline, 0.97% serine, 0.67% threonine, 0.93% tyrosine, and 3.46% L-glutamine). The leucine-rich diet (L) contained 18% protein and 3% L-leucine. The glutamine-rich diet (G) consisted of 18% protein and 4% L-glutamine. The leucine/glutamine-rich diet (LG) consisted of 18% protein, 3% leucine, and 4% glutamine. All diets contained nitrogen ranging between 13.2 and 15.6 mg N2/100 g diet. The C diet contained 20% casein, carbohydrate (39.7% cornstarch, 13.2% dextrin, and 10% sugar), fat (7% soy oil), fibers (5% cellulose micro fiber), salt mix (3.5%), and vitamin mix (1.0%) and was complemented with 0.3% cystine and 0.25% choline. All diets are equivalent in protein content and have the followed nitrogen content control 012.0 mg N2%, leucine 012.8 mg N2%, glutamine 013.6 mg N2%, and leucine/glutamine diet014.9 mgN2% [1]. The caloric adjustment of the amino acid-rich diets was made by reducing the equivalent amount of carbohydrates that corresponded to isocaloric diets. Therefore, the L, G, and LG diet contained similar amount of carbohydrate (L diet0 38.7% cornstarch, 12.2% dextrin, and 9% sugar; G diet037.7% cornstarch, 12.2% dextrin, and 9% sugar; LG diet036.9% cornstarch, 11% dextrin, and 8% sugar). The other ingredients had the same amount of fat, fibers, salt, and vitamin mix and cystine and
Author's personal copy Exercise, nutritional supplementation, and cancer
choline as normoprotein diet. The amino acids (L-leucine and L-glutamine) and cornstarch were kindly provided by Ajinomoto Interamericana and Corn Products Brazil Ingredients, respectively.
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carcasses (the whole animal without the gastrointestinal tract and tumor) were stored in -20°C for subsequent analysis of chemical body composition. Biochemical analyses
Tumor implantation Protein metabolism in vitro Approximately 0.25×106 Walker 256 carcinoma cells were implanted subcutaneously in the right flank of the rats [19]. The nontumor controls were injected with 0.5 ml 0.9% (w.v.) NaCl solution without anaesthesia. The general United Kingdom Coordinating Committee on Cancer Research (1998, UKCCCR [44] guidelines for animal welfare were followed, and the experimental protocols were approved by the institutional Committee for Ethics in Animal Research (CEEA.IB/UNICAMP, protocol # 465-4). Experimental protocols The rats were distributed into eight groups with ten animals/group. Animals, with or without the Walker tumor (W), were subjected to light physical exercise and fed diets with or without leucine (L) and/or glutamine (G) supplementation. Two groups were fed with semi-purified normoprotein diet (C, control rats; W, tumor-bearing rats). The other six groups were fed with a leucine-rich diet (L, nontumor-bearing rats and WL, tumor-bearing rats), a glutamine-rich diet (G, nontumor-bearing rats and WG, tumor-bearing rats) or a leucine/glutamine-rich diet (LG, nontumorbearing rats and WLG, tumor-bearing rats). The animals were housed in individual metabolic cages throughout experimentation. Body weight and food intake were recorded three times a week. Sedentary and pair fed groups were made to analyze the benefit effects produced by the exercise and also to avoid the food intake over the tumor effects, respectively, although these data were not shown. Three days after tumor injection, all rats were subjected to light exercise. The exercise protocol consisted of swimming exercise (30 min/day) for 10 days, without overload, under circulating water, in order to avoid the animals floating, at water temperature of 30±2°C, in a swimming pool (1 m3) [17]. The rats were sacrificed at 13–14 days after tumoral implant, and the gastrocnemius muscles were collected for protein synthesis and degradation analyses. The
Protein synthesis The gastrocnemius muscle was weighed and placed in Krebs–Henseleit buffer (KHB; 110 mM NaCl, 25 mM NaHCO 3 , 3.4 mM KCl, 1 mM CaCl2, 1 mM KH2PO4, 1 mM MgSO4, 5.5 mM glucose, 0.01% (w/v) albumin bovine, pH 7.4) and incubated at 37°C with continuous agitation and gassing with 95% O2 and 5% CO2 [13, 45]. After 30 min, the muscles were further incubated for 2 h in KHB supplemented with 5 μCi L[3H]-phenylalanine/ml (Amersham). The muscles were homogenized in 30% TCA and centrifuged at 10,000×g for 15 min at 4°C. The pellet was washed in 10% TCA, resuspended in 1 N NaOH and subjected to the protein content assay [3], and then measured radioactivity using liquid scintillation. The rate of protein synthesis was calculated and expressed in nmol of incorporate phenylalanine per microgram of precipitated protein per hour [45]. Protein degradation Contralateral gastrocnemius muscle was assayed for protein degradation, and data were expressed as arbitrary fluorescence unit of tyrosine released per micrograms of protein per hour. The muscle was previously incubated in RPMI 1640 at 37°C. After initial incubation, the medium was supplemented with cycloheximide (130 mg/ml; inhibitor of synthesis protein) for additional 2 h under 5% CO2 and 95% O2. Tyrosine release was measured in medium using a fluorimetric assay as described by Waalkes and Udenfriend [49]. Chemical body composition The carcass (without the gastrointestinal tract and tumor mass) was weighed and stored at -20°C for chemical analysis of body composition according to the method standardized by Gonçalves et al. [21]. The carcass water was expressed as the difference between the dry and wet weights. Total fat was extracted with petroleum ether using a Soxhlet apparatus. A homogenized aliquot of
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E.M. Salomão, M.C.C. Gomes-Marcondes
water and fat-extracted carcass was analyzed for total nitrogen content using a colorimetric micro-Kjeldahl method [1]. Another sample was subjected for alkaline digestion with sodium hydroxide and acidified tannic acid, and the collagen nitrogen was analyzsed using the Kjeldahl method [41]. Carcass total protein content was assessed by a colorimetric method [24]. Statistical analysis The results were expressed as the mean ± SEM. Data were analyzed statistically by two-way ANOVA, testing the effects of diet and tumor on body weight and chemical composition, food efficiency, and protein metabolism. Comparisons within control and tumor-bearing groups were performed using one-way ANOVA [15] followed by Bonferroni’s multiple comparison post hoc test (Graph Pad Prism software, v3.00 for Windows 98, USA). Data were considered statistically significant when the P value was less than 5%.
Results In the present work, we observed that the final body weight of W, WL, WG, and WLG rats was around
14%, 9%, 10%, and 11% smaller, respectively, when compared to the C group (Table 1). This reduction was smaller in the WL group as compared to the other tumor-bearing groups. A similar pattern was observed in body weight gain in the groups implanted with tumor (W, WL, WG, and WLG) as compared to the respective control groups. Food intake was reduced in all tumor-bearing animals, but the food efficiency ratio was only decreased in W and WLG groups (Table 1). Although the tumor weight was similar in all tumorbearing groups independent of the nutritional treatment, the tumor/body weight ratio was reduced in all treated tumor-bearing groups, being most apparent in WL rats (Table 1). These parameters were especially affected by tumor growth when analyzed by two-way ANOVA, which showed a P value equal to 0.0047. Chemical body composition was presented in Table 2. Carcass weight was significantly reduced in all tumorbearing groups. All tumor-bearing groups (W, WL, WG, and WLG) showed a significant increase in total body water as compared to the control C group. Body fat was greatly reduced in all tumor-bearing rats. This was most apparent in WL, WG and WLG groups (50%, 50% and 48% less fat mass than the control group). Tumor growth caused decreases in lean body mass, especially in W group compared to C, WL, and WLG groups.
Table 1 Effects of leucine-rich and/or glutamine-rich diets and exercise on body, carcass, tumor weights, and tumor/body weight rate of the studied groupsa Groups
Final weight (g)
Weight gain (%)b
Initial food efficiency ratioc
C
193.5±9.3
48.8±3.0
166.9±8.2
*
L
223.8±4.3
**
WL
176.5±6.3*
W
**
Final food efficiency ratio
0.27±0.02
0.22±0.01
14.0±1.5
*
0.32±0.02
-0.17±0.25*
55.6±3.0
**
0.27±0.01
0.15±0.01
0.20±0.01
0.30±0.07
13.5±4.0*
G
211.1±3.8
0.27±0.01
0.14±0.02
WG
174.7±5.8*
12.4±1.8*
0.20±0.01
0.18±0.09
LG
222.9±7.0**
57.9±3.3**
0.25±0.02
0.19±0.02
12.9±1.5*
0.22±0.02
-0.19±0.20
WLG
172.1±6.4
*
54.5±4.1
**
Tumor weight (g)
Tumor/body weight rate (%)d
26.0±2.5
15.8 1.3
23.1±2.0
10.3±1.0***
23.0±1.4
11.6±0.6***
22.2±1.6
11.6±0.8***
**
C Control exercised rats, W tumor-bearing exercised rats, L exercised rats fed with leucine-rich diet, WL tumor-bearing exercised rats fed with leucine-rich diet, G exercised rats fed with glutamine-rich diet, WG tumor-bearing exercised rats fed with glutamine-rich diet, LG exercised rats fed with leucine/glutamine-rich diet, WLG tumor-bearing exercised rats fed with leucine/glutamine-rich diet a
The data are the mean ± standard error of the mean, n010 rats/group
b
Body weight gain (%) was calculated as the final body weight divided by the body weight at the beginning of experiment
c
Food efficiency ratio was calculated as weight gain (g)/ food intake (g)
d
Rate of tumor weight/ carcass weight was calculated as the tumor weight percentage of the body weight
*
P
