Journal of Undergraduate Research
Volume 5, Issue 4 - January 2004

Dietary Arginine Supplementation Does Not Increase Body Weight or Leg Muscle Protein Concentration

Amy Fanning

ABSTRACT


Arginine, a conditionally essential amino acid, increases nitrogen retention in stressed animals. Arginine, when administered intravenously, stimulates growth hormone secretion. The aim of this study was to investigate the effects of oral arginine supplementation on leg muscle protein stores in lipopolysaccharide-treated vs. untreated mice. Forty mice were fed one of five diets: the AIN93G diet, the AIN93G diet with 2%, 4%, or 6% arginine, or the AIN93G diet isonitrogenous to 4% arginine. After fourteen days on the specified diet, the mice were sacrificed, and the leg muscle was harvested. Weight gain and protein concentration per gram of leg muscle were determined and analyzed. Average body weight gain was significantly lower (p < 0.05) in the mice fed a 6% arginine diet. There was no significant difference in protein concentration between lipopolysaccharide vs. untreated mice. Therefore, the two treatment groups were pooled to analyze protein concentration per gram tissue. Protein concentration per gram tissue was significantly lower (p <0.05) in mice fed 4% arginine diet than mice fed the AIN93G, the AIN93G- 2% arginine, or the AIN93G-isonitrogenous diet. These preliminary data suggest that dietary supplementation of arginine is not beneficial.

INTRODUCTION

Traditionally, arginine is classified as a non-essential amino acid because it can be synthesized in the body. However, animal studies suggest that during times of growth, stress, or injury, arginine is considered a conditionally essential amino acid. Arginine-free diets fed to laboratory animals during times of growth results in sub-optimal weight gain [1,2]. Supplemental arginine during times of growth, stress, or injury may be beneficial. One study by Sitren and Fisher showed that nitrogen retention in rats following femur fracture could be improved by supplementing a 20% casein diet with 2% arginine and 1% glycine [3]. Another study by Minuskin and Lavine showed that supplemental arginine does increase nitrogen retention in stressed laboratory rats [4].

In addition to the role of arginine in nitrogen retention, arginine also plays a role in the secretion of growth hormone. Growth hormone stimulates polyamine synthesis, and polyamines function in membrane transport, cell growth, cell proliferation, and cell differentiation [5]. Infusion of arginine monochloride into the bloodstream in normal subjects is followed by a rise in the plasma concentration of human growth hormone [6]. However, studies suggest that supplementation with oral arginine does not increase plasma growth hormone secretion [7,8].

The goal of this study is to investigate whether dietary arginine supplementation preserves muscle protein stores in lipopolysaccharide (LPS) treated mice. A secondary goal of this study is to determine an optimal level of oral arginine supplementation and to determine if this level increases muscle protein stores.

MATERIALS AND METHODS

Animals and Diets

Forty male one-month old CB6f1 mice were obtained from the Institute of Aging. The mice were randomized and caged in pairs in a 25… C light-controlled room (12 hour light: dark cycle). The mice were allowed to acclimate to their environment for fourteen days and were fed a standard stock diet during the acclimation period. After the two-week acclimation period, the mice were assigned to the AIN93G diet, the AIN93G diet with 2%, 4%, or 6% arginine, or the AIN93G diet isonitrogenous to 4%. (Table 1). The animals had access to food and tap water ad libitum, and food and water were replenished each day. Daily weights of each mouse were also obtained and recorded. The mice were fed the specified diet for a total of fourteen days. All procedures were approved though the University of Florida Institutional Animal Care and Use Committee.

Table 1
Diet Composition
Ingredient (g/kg)
AIN93G [9]
AIN93G with 2% Arginine
AIN93G witn 4% arginine
AIN93G with 6% Arginine
AIN93G
Isonitrogenous
to 4% Arginine
AIN93G(modified)*
900
900
900
900
900
Cornstarch
100
84
59
65
20
L-Arginine
0
16
41
35
0
L-Cysteine
0
0
0
0
1
Casein
0
0
0
0
79
*AIN93G (modified) = 1000g AIN93G-100g Cornstarch. All diets included equal amounts of AIN-93 vitamin and mineral mix, cellulose, chorline bitartate, maltodextrin, sucrose, soybean oil. and tert-butylhydroquinone.


LPS Challenge, Tissue Preparation, and Protein Assay

On the fourteenth day of the study, the mice were injected either a LPS (17.84 mg/kg) or 0.9% phosphate buffered saline (PBS). Four hours later, the animals were anesthetized with 30% halothane solution and sacrificed via cardiac puncture. The leg muscle was harvested from each animal, and the organs were frozen at -80… C until analyzed. The leg muscles (between the patella and the ankle) were removed from the bone and weighed. The leg muscle was homogenized in a 20% weight/volume PBS with 1 mM ethylene diamine tertaacetic acid and protease inhibitor cocktail (1:100) obtained from Sigma (St. Louis, MO). The BioRad Laboratories’ Detergent Compatible Protein Assay Kit (Richmond, CA) was used to determine the protein concentration of the leg muscle.


Statistical Analysis

Weight gain, protein concentration per gram of tissue, and total leg muscle weight were determined for each diet and treatment group and were analyzed using a two-way ANOVA with Duncan’s Multiple Range Test. Weight gain, protein concentration per gram of tissue, and total leg muscle weight were also analyzed using one-way ANOVA with Duncan’s Multiple Range Test. A p value < 0.05 indicates significance. Statistical analyses were done using the Statistical Analysis System (Version 8.2, SAS Institute, Cary, NC).

RESULTS

Performance Mean total leg muscle weights were not significantly different among the five diet groups (data not shown). However, the average body weight gain was significantly lower (p < 0.05) in the mice fed a 6% arginine diet. Average weight gain is shown in Figure 1. Protein concentration per gram of muscle tissue was significantly lower (p < 0.05) in mice fed a 4% arginine diet than mice fed the AIN93G (no additional arginine), the AIN93G with 2% arginine, or the AIN93G isonitrogenous diet (Figure 2).

Figure 1: Total body weight gain following 2 weeks on the respective diet before treatment. n=8 per diet group. Bars with different letters are significantly different at p<0.05
Figure 1. Total body weight gain following 2 weeks on the respective diet before treatment. n=8 per diet group. Bars with different letters are significantly different at p<0.05.


Figure 2: Leg muscle protein concentration per g tissue: PBS and LPS treated mice pooled, n=8 per diet group. Bars with different letters are significantly different at p<0.05

Figure 2. Leg muscle protein concentration per g tissue: PBS and LPS treated mice pooled, n=8 per diet group. Bars with different letters are significantly different at p<0.05.

DISCUSSION

The goal of this study was to investigate whether dietary arginine supplementation preserves muscle protein stores in LPS treated mice. However, the data do not support this hypothesis. One possible reason for this could be that the mice were euthanized before sepsis-induced muscle catabolism occurred. Each mouse treated with LPS was injected with a LD 50 dose (17.84 mg/kg) of the LPS toxin. LD 50, by definition, is a dose that will be lethal to 50% of the mice within 24 hours. Since the mice were subjected to the toxin for only four hours, a time point dictated by immune functions studies simultaneously being carried out on the mice, it may have not been a sufficient amount of time to observe muscle catabolism. If the time of the LPS exposure could have been lengthened, significant differences in the LPS vs. PBS treated mice would have been observed. Therefore, the data for the LPS and PBS treated animals were pooled and analyzed using a one-way ANOVA to determine the effect of oral arginine supplementation on muscle protein stores in unstressed mice.

A secondary goal of this study was to determine an optimal level of oral arginine supplementation and to determine if this level does increase muscle protein stores. The data suggest that supplementation at 6% arginine in the diet may not be accepted. This suggestion stems from significant differences in weight gain among the different diet groups. Weight gain in mice fed the AIN93G diet with 6% arginine was significantly lower than any other diet groups (Figure 1). One possible reason for this is the AIN93G with 6% arginine was unpalatable for the mice, so they did not consume sufficient amounts of calories, protein, carbohydrate, and fat that is required to gain weight. Another possible explanation for the difference in weight gain could be that the high level of arginine in the 6% diet could have had an adverse effect on the mice. One study by Saito showed that a 4% arginine diet supplemented to guinea pigs after burn actually increased mortality compared to 0%, 1%, and 2% arginine diets [10]. These data, along with the data from this study, suggest that dietary supplementation at 6% arginine may not be beneficial.

Protein concentration per mg tissue in the 6% arginine diet group was not significantly different from any other diet group. One possible reason for this is that the mice assigned to the 6% arginine diet may not have consumed enough arginine to make a significant difference in leg protein stores. Since the 6% ARG diet group gained the least amount of weight, it is probable that they did not consume the same amount of diet as the mice in other diet groups. Interestingly, protein concentration per gram muscle tissue was significantly lower (p<0.05) in mice fed a 4% arginine diet than mice fed the AIN93G (no additional arginine), the AIN93G with 2% arginine, or the AIN93G isonitrogenous diet. This result was also contradictory to the thought that supplemental arginine would enhance protein stores. One possible reason for this could be that the mice assigned to a higher percentage arginine diet could have been in a state of fluid retention. Since arginine is a precursor for creatine, the supplemented arginine could have increased muscle levels of creatine. If this did indeed occur, fluid retention in the muscle would be highly probable. In turn, this would cause the overall protein concentration per gram of tissue to decrease. Studies show that supplemental creatine does cause fluid retention. In one study by Zeigenfuss, total body water (TBW) in men increased 2% on the third day of creatine supplementation [11]. Additionally, arginine has been shown to stimulate insulin secretion [12], and insulin promotes the synthesis of glycogen. In this instance, the excess arginine in the diet could have induced glycogen synthesis in the muscle. Since glycogen is a hydrophilic substance, water would also be retained in the muscle cell, and would essentially decrease the overall protein concentration in the tissue.

Ultimately, this study did not show that arginine had a beneficial effect on protein stores. In fact, the data suggest that supplementing high levels of dietary arginine may have an adverse effect on weight gain and protein stores. Additional studies should investigate these results, and the biochemical mechanism that support these results. Since many athletes take individual amino acid supplements to increase lean body mass, it is essential to know if supplemental amino acids (specifically arginine) are harmful to humans.

REFERENCES

  1. Rose, W. C. The Nutritive Significance of the Amino Acids and Certain Related Compounds. Science, 1937. 86(2231): p. 298-300.

  2. Rose, W. C. & E. R Rice. The Significance of the Amino Acids in Canine Nutrition. Science, 1939. 90(2330): p. 186-187.

  3. Sitren, H. S. & H. Fisher. Nitrogen retention in rats fed on diets enriched with arginine and glycine. 1. Improved nitrogen retention after trauma. Br J Nutr, 1977. 37(2): p. 195-208.

  4. Minuskin, M. L., et al. Nitrogen retention, muscle creatine and orotic acid excretion in traumatized rats fed arginine and glycine enriched diets. J Nutr, 1981. 111(7): p. 1265-74.

  5. Nieves, C. & Langkamp, H. Arginine and immunity: a unique perspective. Biomed Pharmacother. 2002. 56: p. 471-482.

  6. Merimee, T. J., et al. Plasma growth hormone after arginine infusion. Clinical experiences. N Engl J Med, 1967. 276(8): p. 434-439.

  7. Marcell, T. J., et al. Oral Arginine does not stimulate basal or augment exercise- induced growth hormone secretion in either the young or older adults. The Journals of Gerontology. 1999. 54(8): p. 395-399.

  8. Chormiak, J. & J. Antonio. Use of amino acids as growth hormone releasing agents by athletes. Nutrition. 2002. 18: 657-661.

  9. Reeves, P. G., F. H. Neilsen, & G.C. Fahey, Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformation of the AIN-76A rodent diet. J Nutr, 1993. 123(11): p. 1939-51.

  10. Saito, H., et al. Metabolic and immune effects of dietary arginine supplementation after burn. Arch Surg, 1987. 122(7): p. 784-9.

  11. Ziegenfuss, T. N., L. M. Lowery, & P. W. R. Lemon. Acute fluid volume changes in men during three days of creatine supplementation. J Exerc Physiol, 1998. 1(3): p. 1-14.

  12. Floyd, J., et al. Stimulation of Insulin Secretion by Amino Acids. J Clinical Investigation, 1966. 45(9): p. 1487-1502.

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