Nutritional Effect of Vespa Amino Acid Mixture on Endurance exercise in Swimming Mice

Introduction
Much is known about the influence of foods on exercise activity. In a recent study of endurance exercise by MacLean et al. 12~, a high carbohydrate (CHO) diet was found to produce prolonged exercise with high concentrations of glucose and lactate in the blood.

In many cases of endurance exercise 6,14,17, the concentration of blood lactate increases rapidly just before exhaustion, while blood glucose levels increase temporarily at the start of exercise, then gradually decrease to exhaustion. Excess production and accumulation of lactate in either muscle or blood brings about acidosis’ 6, while a decrease in blood glucose levels directly suppresses the functioning of the central nervous system. Either situation lead to the inability to continue exercise and thus can be said to promote fatigue conditions. It is therefore important to preserve glucose homeostasis and lactate degradation in order to increase exercise activity.

Supplementation with amino acids, especially branched chain amino acids (BCAA), improves exercise activity. apparently by preventing the catabolism of muscular proteins during exercise (4). However, it is not known whether supplementation with specific components of other amino acids will improve physiological condition as well as exercise performance.

There are some living creatures that consume amino acid mixtures in nature. One example is the family of hornets. Adult hornets are not able to consume solid foods because of their constricted trunks. The meat ball of insects to be preyed on by adult hornets is brought back to the nest where it is fed to the larvae. A nutritional exchange between the meat ball and larval saliva, that is trophallaxis, is performed between adults and larvae.

The giant hornet, V. mandarinia, covers an area with a radius of 2 Km in hunting a prey. Adult hornets continue their hunting at flying velocities over 30 Km/hr all day long, resulting in daily flight distances of about 100 Km; Their wings must support a weight of over 3 g if they carry a meat boal, since their body weights, heavy among flying insects, are commonly over 2 g. The question arises then as to how they produce the flight energy for their hunting, and how they reduce the fatigue brought on by heavy endurance exercise. The answer may exist in the ecological habits of their social life, for example, trophallaxis. Larval saliva may contain the secret that sustains hornet flight.

Our studies show clearly that larval saliva consists mainly of amino acids, the composition of which is similar among the five hornet species found in Japan (1). In this study, we prepared an amino acid mixture identical to that in the larval saliva of V. mandarinia, and analyzed the nutritional effect of these amino acid nutrients on endurance exercise in swimming mice.

Materials and Methods
Materials
Tryptophan and perchioric acid (FCA) were purchased from Wako Chemical Co. (Tokyo, Japan). Diagnostic kits and reagents for measuring blood lactate and glucose were purchased from Sigma Chemical Co. (St. Louis, MO.. USA) and Boehringer Mannheim (Mannheim. Germany), respectively. Glucose and -Haemo-sol were from Iwaki -Pharm. Co. (Tokyo, Japan) and Raemo-Sol Co. (Baltimore, MD, USA), respectively. All amino acids except tryptophan were from Kyowa Hakko Kogyo Co. (Tokyo, Japan). Hornet larval saliva was collected from V. mandarinia larvae by the method previously reported (6), and frozen at -80’C until used.

Methods
Preparation of Nutrients
An amino acid mixture with the composition of hornet larval saliva from V. mandarinia was prepared as 1.8% VAAM (Vespa amino acid mixture) -as shown in Table 1. As a positive control, 1.8% CAAM (casein amino acid mixture), with the same composition as the major casein component (C 1 a) from cow milk was prepared as shown in Table 1.

Effects of VAAM administration on the changes of blood lactate and glucose by exercise.
The compositions of amino acid mixtures derived from VAAM, including EAAM (essential amino acid mixture), -EAAM1, EAAM2, EAAM 3, and IEAAM 4 are also listed in Table 1.

Animals
Untrained mice (male; ddy), aged 4 to 10 weeks, were fasted for 16 hrs at room temperature (24C) and then administered various nutrients.

Optimum dose of nutrients by oral administration
Solution containing 1.8% VAAM at 0, 12.5, 25.0,37.5, and 50.0 per gram body weight were each administered to five 5 week-old mice previously fasted for 16 hrs. The mice were then allowed to rest for 60 mm at room temperature (24’G). Several seconds before the start of a swim, the mice were rinsed and washed with I % Haemo-Sol solution to de-aerate the skin hair. Mice administered different doses of 1.8% VAAM were started to placed at 5 mm intervals in a river pool containing 0.01% Haemo-Sol at 40 with a constant water flow of 8m/min (Fig. 1). A maximum of five mice were in the pool at any time. A swimming exercise was stopped when the mouse sank to the bottom of the pool with air bobbling from its nose. The optimum doses of 1.8% VAAM were found to be 25.0 ul and 37.5 ul/g body weight as shown in Fig. 2. In the following experiments, 37.5 ul/g body weight was chosen for administration.

Optimum mouse age
Either 37.5 ul/g body weight of 1.8% VAAM or distilled water (DW) was administered to 4 (is 18g), 5(17-21g). 8(26-30g) and 10(32-35 g) week-old fasted mice (5 mice per age group). The mice were allowed to rest for 60 mm at room temperature and the swimming exercise was performed as -described above. For mice administered VAAM, the mean swimming-times were 101 mm in 4 week-old mice, 171 mm in 5 week-old mice, 50 mm in 8 week-old mice and 93 mm in 10 week-old mice. The mean times in mice administered DW were for 53 mm in 4 week-old mice, 91 mm in 5 week-old mice, 58 mm in 8 week-old mice and 69 mm in 10 week-old mice. Thus, it was shown that 5 week-old mice were able to swim for the longest time.

Optimum resting time after administration of nutrients
Five week old fasted mice were administered 37.s-pl/g body weight of 1.8% VAAM, 1.8% CAAM or DW The mice were then allowed to rest for 0, 15, 30, 60. 120 or 180 mm at room temperature -prior to being placed in the pool. Swimming times w-ere -then measured as described above. The optimum resting time was found to be 30min for mice receiving 1.8% VAAM or DW -as shown in Fig. 3. The resting time was therefore fixed at 30 min. in subsequent experiments.

Optimum temperature for swim
Thirty minutes before swimming, 5 week-old fasted mice were administered 37.5 ul/g body weight of 1.8% VAAM or DW (n = 5) and placed in the river pool at 25, 30,35, 40 or 45. The optimum swimming temperature was found to be 35C as shown in Fig. 4. At 45C, the mice stopped swimming within a couple minutes. Based -on these results, the swimming conditions in our experiments were set as -follows : five week-old mice were administered nutrients at 37.5 ul/g body weight allowed to the rest for 30 min. after administration, and placed a water temperature at 35C.

Assay for blood lactate
In order to assay of blood lactate and glucose levels, mice were administered nutrients, then a weight (0.3 g) was attached onto the tail. The mice were then placed in the river pool for 30 mm under the conditions described above. Under these conditions, mice administered DW were exhausted in about 60 mm. After the swimming session, the mice were quickly anesthetized with ether, and blood was obtained from the abdominal vein within 1 mm. Fifty micro liters of the blood -was mixed with 100 u1 of 6% perchloric acid (PCA) – mixed well and centrifuged at 2.000 rpm for l0 min. One hundred microliters of the supernatant was reacted with 900 uI of lactate dehydrogenase solution containing nicotinamide adenine d inucleotide for 30 mm at 37 using Sigma diagnostic kit. Absorbance at 340 nm was measured by a Shimadzu UV-150-02 spectrometer.

Assay for blood glucose
Twenty microliters of blood was mixed well with 40 p1 of 6% PCA. and the mixture was centrifuged at 2,000rpm for l0min. Thirty microliters of the supernatant was reacted with 900 p1 of an enzyme solution containing hexokinase, glucose 6 phosphate dehydrogenase. and nicotinamide adenine dinucleotide phosphate using a diagnostic reagent kit (Boehringer Mannheim). The reaction mixture was incubated for 30 mm at 37’C. and the absorbance at 340 nm was measured by a Shimadzu UV-150-02 spectrometer.

Assay for muscular lactate
Mice exercised as described above for the assay of blood lactate were quickly exsanguinated and the leg muscles were immediately frozen in liquid nitrogen. The frozen muscles were crushed in a mortar and pestle, then homogenized with a Polytron homogenizer for 2 mm. The homogenate was centrifuged at 15,OOOXg for 30min at 4. The supernatant was denatured with 6 % PCA and centrifuged again at 2,OOOrpm. The supernatant was assayed for lactate described for the blood lactate analysis.

Statistics
All data are means f SEM. unless otherwise noted. The Student paired t test was used for testing the significance of differences between related samples of the same subject, and for testing the significance of differences between samples of the same subject obtained at different times during the exercise bouts.

Results
Effects of VAAM, CAAM. glucose. DW and amino acid nutrients containing VAAM -components on maximum swimming times In mice The effects of several orally administrated nutrients on the maximum swimming times obtained in mice undergoing endurance exercise were measured. The swimming times in mice receiving 0.9% VAAM corresponding to the concentration in hornet larval saliva and 1.8% VAAM (Nut. no. 2) were significantly longer than in mice receiving DW (Nut. no.26), 1.8% CAAM (Nut. no.3), or 10% glucose (Nut. no.25) (p <-0.05) as shown in Table 2. The total intake of nutrients by a 20 g mouse was about 75 mg in the case of 10% glucose, but only about 14 mg in -the case of the 1.8% amino acid nutrients. In spite of the smaller amount of VAAM intake, the swimming times were prolonged. In comparison to CAAM, which has the desirable nutritional balance for mammalian growth. VAAM contains large amounts of threonine, proline. glycine and tryptophan, but little aspartic acid, serine, or glutamic acid, and no cystine or methionine. This suggests that there is a fundamental difference in the amino acid requirements between exercise and growth. It is thus considered that the peculiar amino acid composition of VAAM might be markedly related to the prolongation of swimming times. Swimming times were measured following the administration of several amino acid nutrients in which the compositions were changed from that of VAAM keeping the molar ratios fixed (Table 1).

However, no nutrients prolonged the swimming times better than VAAM (Table 2). The swimming times in mice receiving proline+glycine. EAAM (Nut. no.9). EAAM+proline (Nut. no.10) and EAAM+glycine (Nut. no.11) were close to that of mice receiving VAAM. These results suggest that the prolongation of swimming time is a reinforcement by several amino acids. Furthermore. the molar ratio of the amino acids in VAAM must play an important role in the effect. This inference is supported by the fact that the administration of insoluble VAAM at high concentration did not prolong swimming times (data are not shown).

Effects of VAAM, CAMM, glucose, and DW on blood concentrations of lactate and glucose in exercising mice

The concentration of blood lactate at the start of the swim was influenced by the administered nutrients and showed slightly little differences as follows:
2.69 +/- 0.l2mMol (n = 35) for DW. 2.84 +/-0.l3mMol (n=20) for .8% CAAM and 2.39 +/-0.l3mMol (n=20) for 1.8% VAAM. After swimming for 3Omin with an 0.3g tail weight. the blood lactate concentration in mice administered 1.8% VAAM was slightly increased (Fig. 5), but was still lower than the starting concentrations in mice receiving other nutrients. However, the post-swim concentrations in both DW and 1.8% CAAM administered mice increased markedly (p <0.05).

At the same time, blood glucose concentrations were also measured. Pre-exercise blood glucose levels were about 4.5 mMol for DW. L8% CAAM and 1.8% VAAM. After exercise, the value decreased slightly for 1.8% VAAM. but largely for DW and 1.8% CAAM (Fig. 6) than those of pre-exercise. In case of 10% glucose and 1.8% VAAM+10% glucose, pre-exercise blood glucose levels were very high, hut the concentrations decreased sharply after swimming, although they were still higher than for nutrients without glucose (Fig. 6). The suppressive effect on the decrease of blood glucose levels by VAAM was also present despite the presence or absence of administered glucose. Post-swim blood glucose levels decreased to 85.8% of starting levels in mice administered DW. a comparatively small decrease. However, if it is considered that DW causes simultaneous decreases in swimming times and increases in lactate concentration,-the result may be shown less active glucose metabolism than with other nutrients. Following exercise, blood glucose levels in mice receiving 10% glucose and 1.8% VAAM+10% glucose decreased -to 61.2% and 61.8%, respectively, of pre-swim levels. As with the increases in blood lactate, the decrease -in blood glucose for these nutrients was very similar. However, for 1.8% VAA-M blood glucose levels decreased only to 89.4% of-pre-swim levels, very small in comparison with the decrease-to 66.3% for 1.8% CAAM.

Considering the compositional differences between VAAM and CAAM. acidic and sulfur containing amino acids, such as glutamic acid, aspartic acid, cyctine and methionine, present in large amounts in CAAM. but rare in VAAM. may act to suppress maximum exercise times and changes in blood -composition during exercise. Glucose homeostasis during exercise brought about by VAAM, as found in these experiments, may prevent hypoglycemia due to exercise. These effects of VAAM would lead to the prolongation of exercise ability of 10% glucose or 1.8% VAAM + 10% glucose resulted in an extremely elevated starting blood lactate concentration. Compared with 10% glucose, however, 1.8% VAAM+10% glucose clearly decreased the post-swim blood lactate concentration despite the presence of glucose (Fig. 5). The ratios of the increases in blood lactate concentrations after exercise in mice by administered different nutrients were 106.1% for 1.8% VAAM. 117.3% for 10% glucose. 117.8% for 1.8% VAAM+/-% glucose. 123.2% for DW, and 129.4% for 1.8% CAAM. Lactate production in mice receiving VAAM was definitely lower than in mice receiving other nutrients.

Muscular lactate concentration in exercising mice administered VAAM, CAAM, glucose or DW
Concentrations of muscular -lactate in the legs of mice undergoing the same swimming exercise were analyzed. Administration of 1.8% VAAM brought about lower muscular lactate concentrations than other -nutrients (Table 3). Muscular lactate concentrations correlated with blood lactate concentrations in mice receiving each nutrient.

Differences in blood concentrations of glucose and lactate in exercising mice administered amino acid nutrients containing VAAM components, and relationship between these concentrations.
To analyze which amino acids cause the effect of VAAM in exercise. blood concentrations of lactate and glucose were measured after the administration of several amino acid -nutrients. Administrations of glycine (Nut. no.3), EAAM (Nut. no. 9), VAAM-Pro (Nut. no.16), and VAAM-(Met. Asp, Ser) (Nut. no.20) produced low concentrations of blood lactate; however, they also produced low concentrations of blood glucose.