Many people from all age groups are currently involved in regular physical activity. Among them, children and adolescents deserve special attention (Ribeiro et al., 2010). Although moderate physical exercise has beneficial effect on growth and development, excessive exercise training could lead to opposite results (Caine et al., 2001; Georgepoulos et al., 2010).
Body growth processes (including bone, cartilage and muscle), demand a high amount of energy and nutrients. As such, nutritional deficits are expected to jeopardize growth as a whole. For instance, Yahya and Millward (1994) showed that short term protein deficits were capable of disbalancing the well-coordinated bone and muscle growth.
It is important to note that epiphysis cartilage development and growth consists of proteoglycan synthesis as well as cell multiplication (chondrogenesis). As such, proteoglycan synthesis is a measurement of cartilage and long bone growth. Indeed, Ribeiro et al. (2010) considered proteoglycan synthesis a biological marker of nutritional status.
Thinking about situations where the body faces energy/nutrient deficits, we can mention physical exercise. Exercise, single bout or regular training, characterizes a state of increased glucose uptake by skeletal muscle during (as an energy substrate) and after (for glycogen and protein synthesis) physical effort (Blomstrand and Saltin, 1999; Hargreaves, 1998; Tipton and Wolfe, 2001).
Considering the need for energy in protein synthesis, and consequently for body growth, there is a strict relationship between muscle protein synthesis and glycogen stores once glycogen is an available source of energy to the muscle (Newsholme and Leech, 1988). Muscle growth will occur adequately only if bone growth is guaranteed by an adequate nutrient supply (Yahya and Millward, 1994).
Branched-chain amino acids (BCAAs) are sources of nitrogen for the synthesis of non-essential amino acids such as glutamine and alanine (Tom and Nair, 2006). Leucine is a potent inhibitor of muscle protein degradation, enhances insulin secretion (Nicastro et al., 2011) and may also be an important precursor for gluconeogenesis in both liver and muscle (Araujo et al., 2006; Campos-Ferraz, data nonpublished).
To date, no studies have been conducted relating BCAAs supplementation (isoleucine, leucine and valine) on bone and cartilage development in growing trained animals. The few studies that used BCAA supplementation on cartilage development did not evaluate BCAA effects on training individuals. In one study with sedentary pigs, BCAA supplementation did not reduce severity of osteochondrosis lesions compared to the control group (Frantz et al., 2008).
With this in mind, we used an experimental model to reproduce the situation of young individuals submitted to a regular training in order to identify the effects of this practice in biomarkers of muscle and bone growth. Therefore, the aim of the present study was to evaluate if whether BCAA supplementation could feature any benefit regarding muscle and bone growth in growing rats submitted to aerobic exercise.
Male Wistar young rats (21 days old) were kept in individual cages on a 12 h dark/light cycle for 6 weeks. The animals were distributed into four experimental groups (n=8 each): supplemented trained (group Sup/Ex), control diet and trained (group Ctrl/Ex), supplemented diet and sedentary (group Sup/Sed) and control diet and sedentary (group Ctrl/Sed). The local Ethics Committee approved all experimental procedures, and the guidelines for animal care and use were followed (National Research Council, 1996). Both food intake and animal weight were measured three times a week. Protein ingestion was calculated based on total food intake (g) and protein content of the diet.
The control diet consisted of a standard ration designed for growing rodents by REEVES et al. (1993) and was prepared in our laboratory. It provided adequate levels of BCAA derived from casein, vitamins, minerals, fiber, carbohydrates and lipids. The protein content was assessed at 17% using the method provided by AOAC (1980). The supplemented diet was prepared using the same type of food as described above with the addition of BCAA (powdered, provided by Aji-No-Moto, Brazil) in order to reach 1.5 times the recommended level for each of the three amino acids (isoleucine, leucine and valine, 45 mg BCAA/kg body weight/day). The protein content of the supplemented diet was analyzed and resulted in 17.4% from the total energy content of the diet.
The swimming training protocol was that described by LANCHA Jr. et al. (1995), in which 10 individual PVC pools were connected to a boiler to allow the circulation of water and to keep the water temperature constant at 32DC. The first week of exercise consisted of an adaptation period during which the animals swam for 20 minutes. After this period, the animals started to swim longer (30 min-week 2, 40 min-week 3, 50 min-week 4 and 60 min-week 5, respectively) with a 5% body weight load attached to the tail, five times a week, always between 8 and 11 am. On the last week, if the rats were not able to stand the 5% body weight load, it would be reduced to 4%, to assure they completed 60 min of exercise.
Throughout the study, care was taken to avoid animals' unnecessary suffering. After the last training session, which was performed on the day before euthanasia, animals were fasted overnight. The animals were euthanized by decapitation. Blood was collected and handled in heparin-stabilized tubes, centrifuged at 2500g for 10 min at 4[degrees]C, and plasma was stored at -80[degrees]C. Soleus and gastrocnemius muscles were weighed, immersed in liquid nitrogen and stored at -80[degrees]C for further analysis.
A few minutes before euthanasia, body weight and body length were measured using a digital scale and a graded wooden device, respectively (Hughes and Tanner, 1970). After euthanasia, muscle, liver, tibia bone, tibia cartilage and adipose tissue were collected and weighed.
Glucose determination: Plasma glucose was determined according to Trinder (1969) using colorimetric-enzymatic reactions. Basically, glucose is oxidized to gluconic acid and [H.sub.2][O.sub.2] by glucose-oxidase. [H.sub.2][O.sub.2], in the presence of peroxidase, catalizes phenol oxidation with 4-amino-phenazon, giving a red color to the solution, whose peak absorbance is read at 505 nm.
Plasma aminogram: Plasma samples were deproteinized with 6% sulfosalicylic acid (1:5) and centrifuged at 9,000 g for 2 minutes, and the supernatant was diluted in NaS buffer, separated by ultra filtration and stored at 80[degrees]C. The concentrations of essential amino acids and ammonia were determined by reverse-phase HPLC (Busse and Carpenter, 1976).
Muscle protein and total RNA: Muscle protein content was determined according to Lowry et al. (1951). Protein was pretreated with copper (II) in a modified biuret reagent (alkaline copper solution stabilized with sodium potassium tartrate).The addition of Folin & Ciocalteu's phenol reagent forms chromogens that give increasing absorbance between 550 nm and 750 nm. Normally, absorbance at the peak (750 nm) or shoulder (660 nm) is used to quantify protein concentrations between 1-100 [micro]g/ml. In our case, bovine albumin was used to build the standard curve and read the samples at 660nm.
Total RNA concentration was determined in muscle samples as follows: protein in the homogenate was precipitated by the addition of an equal volume of 10% trichloroacetic acid (TCA) followed by two washings of the precipitate with 5% TCA to remove acid-soluble compounds. Lipids were removed by two washings with 95% ethanol: ether (3:1 v/v). The precipitate was suspended in 1.5 ml 0.5 N perchloric acid (PCA) and heated to 70C[degrees] for 20 minutes to hydrolyze DNA. The hydrolysis step was repeated with another 1.5 ml PCA. The samples...