Effect of alanyl-glutamine supplementation on plasma and tissue glutamine concentrations in rats submitted to exhaustive exercise

Nutrition 22 (2006) 564 –571 www.elsevier.com/locate/nut

Basic nutritional investigation

Effect of alanyl-glutamine supplementation on plasma and tissue glutamine concentrations in rats submitted to exhaustive exercise Marcelo Macedo Rogero, M.Sc., Julio Tirapegui, Ph.D.*, Rogerio Graça Pedrosa, M.Sc., Inar Alves de Castro, Ph.D., and Ivanir Santana de Oliveira Pires, B.Chem. Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil Manuscript received September 9, 2005; accepted November 11, 2005.

Abstract

Objective: We investigated the effect of supplementation with L-glutamine and L-alanyl-Lglutamine (DIP) on the plasma and tissue glutamine concentrations of exercise-trained rats immediately and 3 hours after a single exercise session until exhaustion. Methods: Thirty-six male rats were divided into six groups, and then subdivided into groups submitted only to the exhaustion test: control (CON-EXA, n ⫽ 6), glutamine (GLN-EXA, n ⫽ 6) and DIP-EXA (n ⫽ 6), or to the exhaustion test followed by a recovery period lasting 3 hours: control (CON-REC, n ⫽ 6), glutamine (GLN-REC, n ⫽ 6) and DIP-REC (n ⫽ 6). The training protocol consisted of bouts of swimming exercise (60 min·day⫺1) for 6 weeks. During the last 21 days, before sacrifice, the glutamine and DIP groups received a daily dose of 1 g·kg⫺1 of glutamine and 1.5 g·kg⫺1 of DIP, respectively. The GLN-REC and DIP-REC groups were also supplemented immediately after the exhaustion test. Concentrations of glutamine, glutamate, glucose and ammonia in plasma and of glutamine, protein and glycogen in liver and muscle were evaluated. Results: The time to exhaustion did not differ between groups. A higher concentration of glutamine in the gastrocnemius and soleus muscles was observed for the DIP-EXA group compared to the CON-EXA and GLN-EXA groups (P ⬍ 0.05). The DIP-REC group presented a higher plasma and liver glutamine concentration than the CON-REC group (P ⬍ 0.05). Muscle glutamine and protein concentration was higher in both the GLN-REC and DIP-REC groups compared to the CON-REC group (P ⬍ 0.05). Conclusions: Chronic supplementation with DIP promoted a higher muscle glutamine concentration than chronic supplementation with glutamine immediately after exercise. However, no significant difference in plasma or tissue glutamine concentrations was observed between acute supplementation with glutamine and DIP during the post-exhaustive exercise recovery period. © 2006 Elsevier Inc. All rights reserved.

Keywords:

Alanyl-glutamine; Supplementation; Dipeptides; Exhaustive exercise; Rat

Introduction Glutamine is the most abundant free amino acid in human muscle and plasma and is also found at relatively

Marcel Macedo Rogero, M.Sc., was supported by fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil. * Corresponding author. Tel.: ⫹55-11-3091-3309; fax: ⫹55-11-38154410. E-mail address: [email protected] (J. Tirapegui). 0899-9007/06/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2005.11.002

high concentrations in many human tissues [1]. Quantitatively, the most relevant tissue in the synthesis, storage, and release of glutamine is skeletal muscle, which plays a fundamental role in the maintenance of plasma glutamine concentration [2]. Glutamine is an amino acid essential for many important homeostatic functions and for the optimal functioning of a number of tissues in the body, in particular the immune system and the gut [3– 6]. However, during various catabolic states including infection, surgery, trauma, acidosis, and exhaustive exercise, glutamine homeostasis is placed under stress, and glu-

M. M. Rogero et al. / Nutrition 22 (2006) 564 –571

tamine reserves, in particular those of skeletal muscle, are depleted [7]. The decrease in plasma glutamine concentration observed after exhaustive exercise may result from increased glutamine extraction by the liver (for gluconeogenesis and urea formation) or from an increased rate of glutamine utilization by the kidneys and immune system cells [5] or a decreased rate of glutamine release from skeletal muscle [5]. Several studies involving supplementation of glutamine in its free form have been carried out in individuals who underwent exhaustive exercise [8 –13] based on the hypothesis that a decreased plasma glutamine concentration after intense and prolonged exercise may contribute to an increased susceptibility to infection [2,14,15]. While some studies have supported this hypothesis [8,9,10], other investigators have not observed an effect of glutamine supplementation on the immune function of athletes [11,12,13]. In addition, glutamine supplementation has been suggested to stimulate an increase in muscle protein synthesis [16,17] and glycogen resynthesis [18,19] and to improve performance [20]. The constraints of administering free glutamine are its limited solubility and its instability in aqueous solution [21]. This problem can be overcome by using glutamine derivatives, such as alanyl-glutamine (DIP) and alanylglutaminyl-glutamine, which are more stable than glutamine at low pH and at the high temperatures normally used to sterilize these enteral and parenteral solutions. In addition, these di- and tripeptide derivatives provide a larger number of glutamine molecules at the physiologic osmolality needed for oral solutions [22]. A recent study [23] in sedentary rats using DIP found that acute oral supplementation with DIP promoted a greater increase in plasma glutamine concentration and that chronic supplementation increased muscle and liver glutamine stores compared with supplementation with free glutamine. However, the effects of acute and chronic oral DIP supplementation on the plasma and tissue concentrations of glutamine after exhaustive exercise have not been clearly established. Thus, this study investigated whether supplementation with DIP is more effective than supplementation with free glutamine with respect to concentrations of glutamine in plasma, liver, and the soleus and gastrocnemius muscles in exercisetrained rats immediately and 3 h after a single exercise session until exhaustion.

Materials and methods Animals and diets Adult male albino Wistar rats weighing 250 to 300 g from the Animal House of the Faculty of Pharmaceutical Sciences, University of São Paulo, were used. The animals were housed in individual cages and fed a ration prepared according to the 1993 recommendations of the American

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Institute of Nutrition for adult rats [24]. The rats were maintained in a room at an ambient temperature of 22 ⫾ 2°C and a relative humidity of 55 ⫾ 10% under a 12-h light/12-h dark cycle (lights on at 7:00 PM), with water and rations available ad libitum throughout the experiment. Food intake was monitored three times a week in all groups. All animals were allowed to adapt to the experimental conditions for 1 wk before the beginning of the experimental protocol. After the adaptation period, the animals were weighed and randomized into six groups, which were then subdivided into groups that were subjected to the exhaustion test (experiment 1), control (CON-EXA, n ⫽ 6), glutamine (GLN-EXA, n ⫽ 6), and DIP-EXA (n ⫽ 6), and groups that were subjected to the exhaustion test followed by a 3-h recovery period (experiment 2), control (CON-REC, n ⫽ 6), glutamine (GLN-REC, n ⫽ 6), and DIP-REC (n ⫽ 6). The experimental protocol was approved by the ethics committee for animal research, Faculty of Pharmaceutical Sciences, University of São Paulo. Supplementation protocol The animals were supplemented with glutamine (Ajinomoto Interamericana Indústria e Comércio Ltda, São Paulo, Brazil) or DIP (Fórmula Medicinal, Suporte Nutricional e Manipulação Ltda, São Paulo, Brazil) through a gastric tube (gavage) during the final 21 d of the training protocol, immediately after the end of the daily swimming session. The GLN-REC and DIP-REC groups were also supplemented immediately after the end of the exhaustion test. The amount of DIP administered (1.5 g/kg of body weight) was calculated so that the total amount of glutamine was the same as that of glutamine administered in its free form (1 g of glutamine/kg of body weight). Control animals received water by gavage. Animals in the present study ingested on average 250 mg/d of dietary glutamine. In addition, animals in the supplemented groups (GLN-EXA, DIP-EXA, GLN-REC, and DIP-REC) received on average an additional 350 mg/d of glutamine, for a total intake of 600 mg/d of glutamine. Training protocol The training period lasted 6 wk and consisted of 60-min daily sessions five times per week. Swimming was always performed in water at a temperature of 32°C between 9:00 and 11:00 AM. During the first week of training, all animals underwent a swimming adaptation period without weights. On the first day of the second week of training, a test protocol was used to determine the lactate threshold (LT) and the initial tail weight to be used for subsequent training. A second LT test was carried out on the first day of the fifth week for correcting training weight size. The LT test was carried out according to the protocol described by Marquezi et al. [25] and consisted of swimming exercises with progressive overload through weights attached to the animal’s

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tail, corresponding to 4%, 5%, 6%, 7%, and 8% of the body weight of each animal for 3-min periods, separated by 1-min resting periods. During the resting periods, 25-␮L blood samples were collected from the tail vein into heparinized capillary tubes for determination of lactate concentration. The LT for each animal was calculated based on the point of inflection of the graph when plotting lactate concentration against the corresponding exercise workload. Exercise to exhaustion On the last day of the experiment, the animals underwent the exhaustion test. Exhaustion was defined to have been reached whenever the animal stayed submerged for a period longer than 10 s. The CON-EXA, GLN-EXA, and DIPEXA groups were killed immediately after the end of the exhaustion test, whereas the CON-REC, GLN-REC, and DIP-REC groups were supplemented immediately after the end of the exhaustion test with water, glutamine, and DIP by gavage, respectively, and killed 3 h later. Sample processing and measurements All animals were killed by decapitation. Blood was collected and centrifuged for plasma and serum separation, which were stored at ⫺80°C in a freezer for subsequent determination of corticosterone, glutamine, glutamate, glucose, and ammonia concentrations. Immediately after death, the liver and gastrocnemius and soleus muscles were removed and frozen in liquid nitrogen for later determination of protein, glutamine, and glycogen concentrations. Plasma glutamine and glutamate concentrations were determined according to the method described by Lund [26] and plasma ammonia concentration was measured with a commercial kit (Sigma Diagnostics, St. Louis, MO, USA). Corticosterone was measured by radioimmunoassay using a commercial kit (Rat Corticosterone [125I] assay system, Amersham, Little Chalfont, Buckinghamshire, UK). Plasma glucose was measured by a colorimetric method using a commercial kit obtained from CELM (São Paulo, Brazil). Tissue glutamine was extracted as described by Sahlin et al. [27]. Frozen muscle (gastrocnemius and soleus) and liver samples were pulverized on aluminum plates under the constant addition of liquid nitrogen, weighed, and immediately homogenized in 0.5 mol/L of ice-cold perchloric acid. Samples were then centrifuged at 3000 rpm at 4°C for 15 min. The supernatant was neutralized with KHCO3 and glutamine concentration was determined as described by Lund [26]. Mean values are reported as micromoles of glutamine per gram of fresh tissue and as micromoles of glutamine per milligram of protein. Muscle and liver protein concentrations were determined in homogenized tissue according to the method described by Lowry et al. [28], and values were compared with a standard albumin curve (Sigma Chemical Company, St. Louis, MO, USA). Liver and muscle glycogen was determined as described by

Hassid and Abraham [29]. The concentration of blood lactate used for LT determination was obtained by an electrochemical technique (Lactate Analyzer Yellow Springs Instruments 1500 Sport, Yellow Springs, OH, USA) after stabilization in sodium fluoride (4.7 mM). Statistical analysis The normality and variance homogeneity of the variables were tested by dispersion graphs and the Hartley, Cochran, and Bartlett tests, respectively, with an ␣ value of 0.05. Results are expressed as mean ⫾ standard deviation. The three treatments of experiments 1 and 2 were subjected to one-way analysis of variance followed by Tukey’s Honestly Significant Difference test. Effects before (experiment 1) and after (experiment 2) acute supplementation were evaluated by t test for dependent variables. All calculations and graphs were obtained by using Statistica 6.0 (StatSoft Inc., Tulsa, OK, USA). Results Food intake (CON 21.4 ⫾ 1.3 g/d, GLN 21.4 ⫾ 2.1 g/d, DIP 20.6 ⫾ 1.0 g/d), total daily energy intake (CON 322.1 ⫾ 19.6 kJ/d, GLN 322.1 ⫾ 31.7 kJ/d, DIP 310.1 ⫾ 15.1 kJ/d), and the final weight of animals (CON 345.8 ⫾ 26.0 g, GLN 343.9 ⫾ 27.6 g, DIP 344.3 ⫾ 25.8 g) did not differ across groups, indicating energy balance in the animals. There was also no difference in time to exhaustion across groups (CON 140 ⫾ 35 min, GLN 135 ⫾ 30 min, DIP 144 ⫾ 40 min). Further, the mean exercise workload (⬃6%) in the exhaustion test was similar across groups. Experiment 1 Plasma concentrations of ammonia and glucose and serum concentration of corticosterone did not differ across exhaustion groups (Table 1). However, plasma glutamate concentration was higher (46%) in the DIP-EXA group than in the GLN-EXA group immediately after the exhaustion test (Table 1). In addition, plasma glutamine concentration was found to be higher (63%) in the DIP-EXA group than in the CON-EXA group (Table 1). The DIP-EXA group presented a higher glutamine concentration in gastrocnemius and soleus muscles than did the CON-EXA and GLNEXA groups (Table 2). Muscle protein concentration was found to be significantly higher in the DIP-EXA than in the CON-EXA group (Table 2). Supplementation with glutamine and DIP was associated with a higher concentration of liver glycogen when compared to the CON-EXA group (Table 2). Experiment 2 The GLN-REC group presented a lower plasma glutamate concentration than did the CON-REC and DIP-REC

Table 1 Effects of chronic (experiment 1) and acute (experiment 2) supplementation with GLN and DIP on plasma and serum parameters of exercise-trained rats subjected to a single EXA followed by a 3-h REC* Parameters

Experiment 2 †

CON-EXA (n ⫽ 6)

GLN-EXA (n ⫽ 6)

DIP-EXA (n ⫽ 6)

P

CON-REC (n ⫽ 6)

GLN-REC (n ⫽ 6)

DIP-REC (n ⫽ 6)

P†

12.45 ⫾ 2.07

13.07 ⫾ 3.48

13.84 ⫾ 1.75

0.648‡

2.28 ⫾ 0.22

2.20 ⫾ 0.40

2.69 ⫾ 0.44

0.069‡

5.07 ⫾ 1.55

6.66 ⫾ 1.36

5.23 ⫾ 1.40

0.144‡

⫺81.69 (P ⫽ 0.001) 8.03 ⫾ 1.04

⫺83.19 (P ⫽ 0.008) 7.54 ⫾ 0.92

⫺80.53 (P ⫽ 0.001) 7.88 ⫾ 1.45

0.758‡

0.31 ⫾ 0.09ab

0.26 ⫾ 0.04a

0.38 ⫾ 0.07b

0.036

58.29 (P ⫽ 0.016) 0.20 ⫾ 0.03a

13.22 (P ⫽ 0.291) 0.17 ⫾ 0.02b

50.65 (P ⫽ 0.020) 0.23 ⫾ 0.01a

0.001

0.84 ⫾ 0.22a

1.17 ⫾ 0.13ab

1.37 ⫾ 0.41b

0.038

⫺33.96 (P ⫽ 0.019) 0.74 ⫾ 0.23a

⫺35.65 (P ⫽ 0.002) 0.91 ⫾ 0.25ab

⫺39.75 (P ⫽ 0.001) 1.09 ⫾ 0.17b

0.047

428 ⫾ 73

387 ⫾ 72

444 ⫾ 57

0.347‡

⫺11.62 (P ⫽ 0.401) 194 ⫾ 37

⫺22.31 (P ⫽ 0.075) 216 ⫾ 61

⫺20.72 (P ⫽ 0.210) 177 ⫾ 64

0.491‡

⫺54.59 (P ⫽ 0.005)

⫺44.27 (P ⫽ 0.001)

⫺60.22 (P ⫽ 0.001)

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Plasma ammonia (mmol/L) %⌬ Plasma glucose (mmol/L) %⌬ Plasma glutamate (mmol/L) %⌬ Plasma glutamine (mmol/L) %⌬ Serum corticosterone (nmol/L) %⌬

Experiment 1

%⌬, percentage difference between experiment 1 (EXA) and experiment 2 (REC) (significantly different when P ⬍ 0.05); CON, control; DIP, L-alanyl-L-glutamine; EXA, exercise until exhaustion; GLN, L-glutamine; REC, recovery period * Results are means ⫾ standard deviations. Values in the same row followed by the same letter are significantly different (P ⬍ 0.05). † Probability value for groups. ‡ Not significant (P ⬍ 0.05).

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568

Table 2 Effect of chronic (experiment 1) and acute (experiment 2) supplementation with GLN and DIP on tissue parameters of exercise-trained rats subjected to a single EXA followed by a 3-h REC* Parameters

Experiment 1 CON-EXA (n ⫽ 6)

GLN-EXA (n ⫽ 6)

DIP-EXA (n ⫽ 6)

P

CON-REC (n ⫽ 6)

GLN-REC (n ⫽ 6) 4.78 ⫾ 0.83b

DIP-REC (n ⫽ 6) 5.65 ⫾ 1.29b

P†

2.20 ⫾ 0.49a

1.80 ⫾ 0.58a

3.07 ⫾ 0.41b

0.002

2.82 ⫾ 0.73a

7.33 ⫾ 0.59a

7.07 ⫾ 1.69a

9.95 ⫾ 1.26b

0.013

28.11 (P ⫽ 0.202) 8.10 ⫾ 0.73

165.66 (P ⫽ 0.001) 9.48 ⫾ 1.44

83.93 (P ⫽ 0.006) 9.40 ⫾ 1.36

2.73 ⫾ 0.41

2.84 ⫾ 0.33

3.11 ⫾ 0.86

0.531‡

24.8 ⫾ 7.4

17.6 ⫾ 6.0

26.3 ⫾ 3.7

0.050‡

10.51 (P ⫽ 0.126) 3.79 ⫾ 1.11a 38.69 (P ⫽ 0.116) 32.84 ⫾ 9.79

34.13 (P ⫽ 0.080) 5.32 ⫾ 0.84ab 87.37 (P ⫽ 0.002) 43.97 ⫾ 10.41

⫺5.52 (P ⫽ 0.343) 6.62 ⫾ 1.88b 112.77 (P ⫽ 0.018) 45.89 ⫾ 11.79

32.7 ⫾ 7.3

25.3 ⫾ 4.8

30.3 ⫾ 10.8

0.290‡

32.42 (P ⫽ 0.166) 46.80 ⫾ 16.52

149.83 (P ⫽ 0.001) 41.84 ⫾ 7.34

74.49 (P ⫽ 0.009) 60.85 ⫾ 19.55

0.118‡

43.12 (P ⫽ 0.184) 8.73 ⫾ 1.49a

65.38 (P ⫽ 0.011) 11.07 ⫾ 1.17b

100.83 (P ⫽ 0.039) 12.43 ⫾ 1.55b

0.001

0.001

0.122‡

0.009 0.107‡

9.15 ⫾ 1.57a

10.29 ⫾ 0.61ab

11.67 ⫾ 0.63b

0.003

8.60 ⫾ 1.93

11.36 ⫾ 0.92

10.42 ⫾ 0.67

0.074‡

⫺4.62 (P ⫽ 0.230) 8.55 ⫾ 2.20a

7.60 (P ⫽ 0.146) 12.76 ⫾ 0.43b

6.54 (P ⫽ 0.391) 10.99 ⫾ 0.52ab

0.001

0.11 ⫾ 0.03

0.09 ⫾ 0.02

0.09 ⫾ 0.03

0.168‡

⫺0.63 (P ⫽ 0.973) 0.17 ⫾ 0.05

12.28 (P ⫽ 0.013) 0.25 ⫾ 0.07

5.48 (P ⫽ 0.224) 0.18 ⫾ 0.05

0.042‡

0.09 ⫾ 0.03a

0.34 ⫾ 0.10b

0.24 ⫾ 0.07b

0.001

50.35 (P ⫽ 0.128) 0.12 ⫾ 0.03a 33.37 (P ⫽ 0.058)

176.03 (P ⫽ 0.004) 0.63 ⫾ 0.16b 86.56 (P ⫽ 0.012)

94.93 (P ⫽ 0.008) 0.32 ⫾ 0.12b 32.42 (P ⫽ 0.073)

0.001

%⌬, percentage difference between experiment 1 (EXA) and experiment 2 (REC) (significantly different when P ⬍ 0.05); CON, control; DIP, L-alanyl-L-glutamine; EXA, exercise until exhaustion; GLN, L-glutamine; REC, recovery period * Results are means ⫾ standard deviations. Values in the same row followed by the same letter are significantly different (P ⬍ 0.05). † Probability value for groups. ‡ Not significant (P ⬍ 0.05).

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Gastrocnemius muscle GLN (␮mol GLN/ g fresh tissue) %⌬ Soleus muscle GLN (␮mol GLN/g fresh tissue) %⌬ Liver GLN (␮mol GLN/g fresh tissue) %⌬ Gastrocnemius GLN/protein ratio (nmol GLN/mg protein) %⌬ Liver GLN/protein ratio (nmol GLN/ mg protein) %⌬ Gastrocnemius protein (mg protein/100 mg fresh tissue) %⌬ Liver protein (mg protein/100 mg fresh tissue) %⌬ Gastrocnemius muscle glycogen (mg/100 mg tissue) %⌬ Liver glycogen (mg/100 mg tissue) %⌬

Experiment 2 †

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groups (Table 1), whereas plasma and liver glutamine concentrations were higher in the DIP-REC group than in the CON-REC group (Tables 1 and 2). The GLN-REC and DIP-REC groups showed higher glutamine and protein concentrations in the gastrocnemius muscle and higher liver glycogen concentration than did the CON-REC group (Table 2). In addition, liver protein concentration was higher in the GLN-REC group than in the CON-REC group (Table 2). Comparison of results between experiments 1 and 2 showed that plasma ammonia and glutamate concentrations and serum corticosterone were significantly lower in the REC groups than in the respective EXA groups (Table 1). Plasma glucose concentration was higher in the CON-REC and DIP-REC groups than in the CON-EXA and DIP-EXA groups (Table 1). It should be noted that there was no significant difference in any tissue parameter (liver and muscle) between the CON-REC and CON-EXA groups (Table 2). Glutamine concentration and glutamine/protein ratio in the liver and gastrocnemius muscle were higher in the GLN-REC and DIP-REC groups than in the GLN-EXA and DIP-EXA groups, respectively (Table 2). The GLNREC and DIP-REC groups presented a higher muscle glycogen concentration than did the GLN-EXA and DIP-EXA groups (Table 2). Liver protein and glycogen concentration were higher in the GLN-REC group than in the GLN-EXA group (Table 2).

Discussion Long-term supplementation with glutamine or DIP did not influence food intake in the groups studied, a finding that allowed us to evaluate the exclusive effect of supplementation on glutamine metabolism in trained rats that underwent exhaustive exercise. With respect to experiment 1, we observed that long-term supplementation with DIP promoted a higher plasma glutamine concentration compared with the CON-EXA group. This result can be explained by the higher glutamine concentration in the gastrocnemius and soleus muscles observed in animals in the DIP-EXA group immediately after the exhaustion test compared with the CON-EXA group. This is because skeletal muscle is the tissue most involved in the synthesis, storage, and release of glutamine and, hence, plays a fundamental role in the maintenance of plasma glutamine concentration [2]. However, plasma glutamine concentration may not reflect the concentration of glutamine in muscle because in this study plasma glutamine concentration did not differ between the GLN-EXA and DIP-EXA groups, whereas the concentration of glutamine in the soleus and gastrocnemius muscles was found to be significantly higher in the DIPEXA group than in the GLN-EXA group. This difference between the GLN-EXA and DIP-EXA groups might be due to the fact that long-term supplementation with DIP promoted a greater supply of glutamine to the bloodstream and,

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hence, to muscle tissue than did supplementation with glutamine. This fact was reported in a recent study on sedentary rats supplemented with DIP [23], which showed a greater kinetic response of plasma glutamine concentration and a higher muscle glutamine concentration compared with animals supplemented with free glutamine, indicating that ingestion of DIP is effective in providing glutamine to the peripheral circulation despite the extensive utilization of this amino acid by the splanchnic bed. We should point out that the dipeptide DIP is absorbed in its intact form by the luminal membrane due to the presence of a different intestinal transport mechanism for dipeptides compared with that of free amino acids [30]. According to Adibi [31], dipeptides are transported across the intestinal epithelium by the oligopeptide transporter PepT-1, which shows a higher absorption rate for dipeptides than for free amino acids. Thus, supplementation with DIP may result in a higher intestinal uptake rate compared with glutamine supplementation. Associated with this fact, the high intracytoplasmic concentration of dipeptidase in the enterocyte might favor an increased stoichiometric release of glutamine and alanine and, hence, promote a greater kinetic response of plasma glutamine concentration [31]. Moreover, the higher plasma glutamate concentration in the DIP-EXA group compared with the GLN-EXA group may be related to the higher concentration of glutamine in the gastrocnemius and soleus muscles observed in this group. In muscle, there is continuous glutamate uptake and glutamine release, with glutamate uptake accounting for about half of the glutamine release, characterizing part of the glutamine-glutamate cycle [32,33]. Glutamine is produced from glutamate and ammonia by the enzyme glutamine synthetase in muscle tissue [34]. Further, during the initial period of moderate to intense dynamic exercise, there is a one- to three-fold increase in the uptake of glutamate from the bloodstream by the muscle cell compared with the resting period and a two- to nine-fold increase in the release of glutamine and alanine [35]. Thus, we suggest that the higher plasma concentration of glutamate observed in the DIP-EXA group is the result of a lower need for uptake of this amino acid by muscle tissue, in view of the fact that the DIP-EXA group presented a higher glutamine concentration in muscle tissue. Experiment 2 was carried out to determine the effect of acute supplementation with glutamine or DIP administered immediately after the exhaustion test to chronically supplemented animals. Except for plasma glutamate concentration, no significant difference in the other blood or tissue parameters was observed between the GLN-REC and DIPREC groups. This finding might be related to the time between acute supplementation and death of REC animals, which was 3 h after the exhaustion test in the present experiment. Studies have demonstrated that acute oral supplementation with glutamine or DIP stimulates an increase in plasma glutamine concentration 10 to 90 min after supplementation; however, in both interventions, plasma glu-

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tamine concentration returned to basal values within 90 to 120 min after supplementation [23,36,37]. Similarly, we observed no significant difference in plasma glutamine concentration 3 h after acute supplementation with glutamine or DIP compared with values obtained immediately after the exhaustion test. Therefore, an increase in plasma glutamine concentration is not observed exactly 3 h after supplementation with glutamine or DIP, whether or not it is carried out after exercise. However, the higher glutamine concentration in the gastrocnemius muscle observed in the GLN-REC and DIP-REC groups compared with the GLN-EXA and DIPEXA groups suggests that both supplementations are effective nutritional interventions in terms of providing glutamine to the peripheral circulation despite extensive utilization of this amino acid by the splanchnic bed during the recovery period after exhaustive exercise. Considered as a whole, the higher muscle glutamine concentration induced by long-term supplementation with DIP might indicate potential beneficial physiologic effects in individuals engaged in intensive and prolonged training. Muscle glutamine concentration possibly acts on the volume [38] and synthesis of glutathione [39] and protein by the muscle cell [16,17], factors related to muscle injury and exercise-induced local inflammatory processes. It also favors the maintenance of plasma glutamine concentration, a fact that is related to the functionality of high-turnover tissues such as the intestine and immune system [2– 6].

Conclusion Our results suggested that long-term supplementation with DIP promotes a higher glutamine concentration in the gastrocnemius and soleus muscles immediately after the exhaustion test compared with supplementation with free glutamine. However, no difference in plasma or tissue glutamine concentration was observed between acute supplementation with glutamine and DIP during the recovery period after exhaustive exercise.

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