Scientific Studies

Scientific Study 1:

Effects of Vespa Amino Acid Mixture (VAAM) Isolated from Hornet Larval Salivaand Modified VMM Nutrients on Endurance Exercise in Swimming Mice - Improvement in Performance and Changes of Blood Lactate and Glucose

TAKASHI ABE*, YOSHIMI TAKIGUCHI**, MASAHIRO TAMURA*, JUNKO SHIMURA* and KEN-ICHI YAMAZAKI**
* Institute of Physical and Chemical Research. Hirosawa 2-1, Wako-shi. Saitama, Japan
351.
**R & D Laboratories L Nippon Steel Co. 1618 Ida, Nakahara-Ku, Kawasaki, Japan 211
Japanese Journal of physical Fitness and Sports Medicine
(Vol.44 No.2 APR. 1995)

Abstract


For endurance exercise in swimming mice. 1.8% VAAM (Vespa Amino Acid Mixture) which has the same amino acid-components as hornet (Vespa mandarinia) larval saliva, 1.8% casein amino acid mixture (CAAM). 10% glucose, or amino acid mixtures in which the amino acids were varied while -maintaining the same molar ratio as VAAM were administered orally to mice. Mice receiving 1.8% VAAM showed significantly longer maximum swimming times than mice receiving other nutrients. Among these nutrients - mixtures of proline, glycine, and essential amino acid mixture (EAAM) from the VAAM component. showed maximum times near those with VAAM. In swimming exercise in mice earring of 0.3g tail weight, mice administered 1.8% VAAM showed lower blood lactate concentrations and higher blood glucose concentrations than mice receiving other nutrients. Mice receiving 1.8% VAAM also had lower lactate concentrations in muscle as well as blood. This suggests that VAAM suppresses lactate production and glucose catabolism during exercise. The effects of hornet larval saliva were stronger than those of VAAM. VAAM therefore showed the major effect of the saliva. The results suggest that VAAM improves physiological condition during endurance exercise. A positive correlation was observed between the blood concentrations of lactate and glucose in exercising mice administered various nutrients (r=0.779). This suggests metabolic equilibration between glucose and lactate during exercise. A positive correlation (r=0.507) was also found between the maximum swimming time and blood glucose concentration. Maximum swimming times were high. est at low (Ca. 2.5 mMol) and high (Ca. 4.0 mMol) blood lactate concentrations in high blood glucose concentrations. These facts support that glucose-homeostasis is important in prolonged exercise. (Jpn. J. Phys. Fitness Sports Med. 1995.44.: 225-238)

Key Words : Endurance exercise. Blood lactate. Blood glucose. Hornet larval saliva, Amino acid nutrient.

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 aminoacid 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. mandannia' 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 deaerate 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 microliters 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 Iactate
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.

Scientific Study 2:

The Activation of Fatty Acid Metabolism by Vespa Amino Acid Mixture (VAAM) and Related Nutrients during Endurance Exercise in Mice

Takashi ABE(1), Mihoko INAMORI(1), Kouji IIDA(2), Masahiro TAMURA(1), Yoshimi TAKIGUCHI (3), and Kaneaki YASUDA(3)

(1) The Institute of Physical and Chemical Research, Horosawa 2-1, Wako-Shi, Saitama, Japan 351
(2) Nutritional Laboratory, Central Research Institute, Meiji Milk Products Co., 1-21-3, Sakae-cho, Higashimurayama-Shi, Tokyo, Japan 189
(3) R&D Lab, I, Nippon Steal Co., 1618 Ida, Nakahara-ku, Kawasaki, Japan 211

Abstract
ABE, T. INAMORI, M. IIDA, K. TAMURA, M. TAKIGUCHI, Y. and YASUDA K. Tha Activation of Fatty Acid Metabolism by Vespa Amino Acid Mixture (VAAM) and Related Nutrients during Endurance Exercise in Mice. Adv. Exerc. Sports Physiol., Vol. 3 No. 1 pp 35-44, 1997. The action of Vespa amino acid mixture (VAAM) on fatty acid metabolism was analyzed as changes in blood biochemical indices during endurance exercise in swimming mice. In response to the oral ingestion of VAAM, but not other nutrients, the concentrations of serum NEFA, blood ketone bodies, and plasma noradrenaline (NA) increased significantly during endurance exercise. The same mice showed the suppression of increase in blood lactate and decrease in blood glucose. Under similar exercise conditions, a relatively low plasma insulin concentration and an increase in the pyruvate/lactate low plasma ratio were observed simultaneously compared to other nutrients. A strong correlation (r=0.794) was found betweem the blood glucose and lactate concentrations in mice ingesting various nutrients other than VAAM. Compositional anyalyses suggest that the excretion of plasma NA and adrenaline (A) are stimulated by different amino acid compositions, but a constant ratio of both catecholamines was secreted following feeding with either VAAM or VAAM 8. We also showed a high correlation (r=0.746) between the inductions of serum NEFA and the secretion of plasma NA by various nutrients. These results suggest that VAAM suppresses glucose oxidation, increases fatty acid oxidation, and also enhances the aerobic metabolism through the hormonal activation of NA during endurance exercise.

Key words: Catecholamines, NEFA, Acetoacetate, Glucose, Lactate, Endurance Exercise, VAAM.

Introduction
Minimizing fatigue, which significantly limits exercise performance, is one of the most important subjects in exercise sciences. Fatigue during exercise has been mainly attributed to a rise in blood lactate levels, a reduction in blood glucose levels, and the depletion of muscle glycogen.

It is well known that fatigue and exercise performance are markedly influenced by food intake. Many studies of foods that contribute to energy yield during exercise have been conducted. Many such studies have dealt with carbohydrtes, including fructose (17, 26), glucose (17, 25, 26, 32), glucose polymer (25), maltodextrins (10, 32) and corn starch (17). Others have studied fatty acids (10, 32), proteins (6, 20) and amino acids, especially branched chain amino acids (5, 7, 13, 24, 34). A carbohydrate-rich diet results in high levels of both plasma glucose and lactate, but lower plasma NEFA levels during endurance exercise. A fat and protein-rich diet, however, produces low levels of plasma NEFA levels (23). Protein supplementation prevents the decrease in plasma levels of branched chain amino acids (BCAA), which contributes to energy production during endurance exercise (6, 20). BCAA ingestion also protects muscle protein from catabolism (7).

On the other hand, very active muscles, such as flight muscles, exist in nature. Hornets, for example, have very strenuous muscles that can be trembled at over 1,000 cycles per minute and can lift a weight of over 3 g. The muscle works continuously all day long and hornets fly distances of over 70 km at 30 km per hour (1). We do not, however, understan the metabolic mechanisms that prevent the occurence of fatigue during such hard flying exercise. The answer might lie in the special food intake of hornets. Adult hornets, which are among the most developed of social insects, ingest only liquid food comprising an amino acid mixture obtained from larvae during trophallaxis (1). This probably represents a kind of food evolution in which the substances for ingestion change depending on the development stage of the animal, progressing from hard solids to soft gels and liquids. Among relatively differentiated animals, such as insects some species ingest mainly liquid diets. in previous study, we found a major antifatigue component, the amino acid nutrient Vespa Amino Acid Mixture (VAAM), from the saliva of Vespa mandarinia larvae (1). It has been shown that VAAM suppresses the decrease in blood glucose and the increase in blood lactate concentrations during endurance exercise and elongates swimming time in mice (2). The question arises as to what fuels are used for exercise energy. Blood glucose and lactate changes brought about by exercise after the ingestion of VAAM suggest that glucose is not a major source of energy for exercise (2). As another energy source, plasma NEFA is mainly used during endurance exercise. An increase in plasma NEFA, as well as ketone bodies, is accepted to indicate that the exercise is associated with an increased capacity to oxidize fas, probably caused in part by the increase in the activities of skeletal muscle oxidative enzymes (16). This is in agreement with the hypothesis that the exercise-induced increase in the oxidative capacity of skeletal muscles leads to an increase in the utilization of fatty acids (14). Further, the ability to carry out liolysis during exercise leads frequently to an improvement in performance; therefore, the induction of blood NEFA during exercise is one of the most important issues of endurance athletes. However, it is not well understood what nutrients induce lipolysis during exercise. From these points of view, the major effect of VAAM of serum NEFA levels has been analyzed with respect to energy metabolism, including hormonal regulation and the amino acid composition nest for the induction of serum NEFA.

Material and methods
Animals
Male ddY strain mice, 6 weeks of age (17-22g body weight, 408 mice) (Saitama Animals Supply Co., LTD), were used without any pretraining exercises as in a previous study (2). Treatment of the animals was in accordance with the guidelines of the Institute of Physical and Chemical Research Committee Following NIH (USA) Guidelines. Swimming was performed at 35°C at a pool flow rate of 5.m/min as in previous experiments (2). The mice had 0.3g weights attached to their tails during swimming. THe 16hr fasting schedule and oral administration of nutrients at 37.5 u l/g body weight were performed in the same manner as previously descrivbed (2). Mice were administered each nutrient 30 min before exercise. Endurance swimming was carried out for 30 or 60 minutes in the river pool. After swimming, blood was taken quickly from an abdominal vein or artery.

Preparation of nutrients
VAAM, casein amino acid mixture (CAAM), and essential amino acid mixture (EAAM), and other modified VAAM nutrients used in these experiments are listed in Table 1.

Blood Assays

Blood concentration of lactate and glucose after swimming for 60min were analyzed by the lactate dehydrogenase and hexokinase methods, respectively, as in the previous study (2). Blood pyruvate levels just after swimming for 30 min were measured by an enzymatic spectrophotometric method using lactate dehydrogenase and a Sigma diagnostics kit, Pyruvate (Sigma Chemical Co., St. Louis, MO, USA). Serum prior to exercise (0 min) and after 30 and 60 min of swimming by a modification of a colorimetric procedure as follows. Forty microliters of mouse serum was mixed with 400 u l of 50mMol Na-phosphate reaction buffer, pH 7.0 containing 5mMol MgCl2, 1.5mMol 4-aminoantipyrine, 0.73mMol CoA, 4.5mMol ATP, 0.27U acyl CoA synthetase, and 2.7U ascorbate oxidase, and the mixtures were incubated for 10min at 37°C. To the enzyme reaction mixture was added 800 u l of dye-enzyme solution containing 1.2mMol 3-methyl-N-ethyl-N-(2-hydroxyethyl)-anoline and 2.92mMol N-ethylmaleimide, 6.8U peroxidase, and 5.5U acyl CoA oxidase, and the mixtures were incubated for 10min at 37°C. Enzyme activity was measured at OD 550nm. The amounts of NEFA in mEq were calculated using oleic acid as a standard. Blood ketone bodies in sedentary mice and those swimming for 30min were measured enzymatically with acetoacetate by a modification of the spectrophotometric method followed of mellanby and Williamson (27). Li-acetoacetate was used as a standard. Serum insulin antibody complex method using the Glazyme Insulin-EIA Test (Wako Chemical Co., Osaka, Japan). Catecholamines, including adrenaline (a) and noradrenaline (NA), after 60 minutes of swimming were determined bu high performance liquid chromatography (HPLC) with flourescence detection. Before HPLC analysis, 1 ml of blood from the carotid artery was mixed with 0.1,,ole EDTA-Na, and the plasma was deproteinated with 1N HC1)4. Plasma catecholamines were adsorbed onto 50mg of activated alumina packed in aSepacol mini column (Seikagaku Kogyo Chemical Co., Tokyo, Japan) under basic conditions, and then extracted with 0.4N acetic acid after the clumn was washed well with distilled water (yield 80%). The extract was lyophilized and redissolved in 30 u l of 4N acetic acid. Twenty microliters of the sample was applied to ODS-HPLC (4.5x250mm). The separated A and NA were oxidized with potassium ferricynate with strong base at 50°C and detected as hydroxyindole flourescence (Ex. 380nm, Em. 480nm). The minimal detectable levels were 0.1 pmol/ml for both A and NA.

Chemicals
Adrenaline (A), non adrenaline (NA), Li-ace-toacetate, ATP, peroxidase and D-(-)-3-hydroxybutyrate dehodrogenase were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The reduced form of nicotinamide denine dinucleotide (NADH) and coenzyme A (CoA) or ascorbate oxidase were provided by Oriental Yeast Chemical Co. (Tokyo, Japan). Tryptophane, HClO4, 4-aminoantipyrine, 3methyl-N-ethyl-N-(2-hydroxyethyl)-aniline, EDTA-Na and oleic acid were purchased from Wako Chemical Co. (Osaka, Japan). All amino acids except tryptophan were from Jyowa Hakko Kogyo Co. (Tokyo, Japan). Acetyl CoA synthetase and acetyl CoA oxidase were from Toyobo (Osaka, Japan). N-Ethylmaleimide was from Eastman Kodal Co., (New Have, CT. USA). Aluminum oxide (Woelm Nutral W-200) was prepared by M. Woelm Pharma (Eshwege, Germany).

Statistics
All data are presented as mean+- SE. The effects of nutrients on swimming time to exercise were assessed by a 1x2 ANOVA. The paried student's t test was used to test the significance if differences between related samples from the same mouse. Repeated measures ANOVA with a subsequent Bonferoni test was used to test the significance of differences in the mean values of blood biochemical indices. The significance level for all analyses was set at p<0.05.

Results
Effects of VAAM, CAAM and Glucose on NEFA induction during swimming exercise

Serum NEFA concentrations, which were 0.85 +- 0.03 mEq/L in resting mice (n=8) after the fasting for 16hrs, were slightly increased to 0.90+-0.03mEq/L by the ingestion of 1.8% VAM, but not changes by distilled water (DW) (0.87 +- 0.03mEq/L) or 1.8% CAAM (0.83 +- 0.04mEq/L), and decrease slightly to 0.65 +- 0.01mEq/L by 20% glucose administered 30min before exercise (Fig 1). After 30min of continuous swimming, serum NEFA concentrations were significantly increased by VAAM or DW ingestion, while it was increased gradually in CAAM or glucose ingestion. During further swimming up to 60min, serum NEFA concentration in mice that received VAAM or DW remained constant at about 1.60mEq/L, but the concentrations in mice receiving CAAM or glucose increased continuously to low levels of 1.15 +- 0.06mEq/L or 0.74 +- 0.06mEq/L, respectively (Fig. !). Blood concentrations of lactate and glucose were also analyzed in the same swimming mice (Table 2). Blood lactate concentrations were elevated in mice receiving CAAM, glucose or DW, but decreased in mice receiving VAAM. Blood glucose concetrations decreased in mice receiving VAAM. Both blood lactate and glucose concentrations showed responses similar to those described in our previous study (2). On the other hand, blood levels of ketone bodies formed by the oxidation of fatty acids were analyzed under the same exercise conditions. Following the ingestion of 10% glucose, the resting blood concentration of ketone bodies was very low at 73.59+-9.30u Mol; after exercising for 30min, the level was still low at 119.12+-26.5u Mol. Despite the 62.7% increase over the resting level (see Fig. 2). In the case of 1.8% CAAM or DW ingestion, the blood ketone body concetration at rest was 230.95 +- 33.83u Mol or 247.69 +- 33.95 u Mol, respectively. In both cases, there was a very slight increase during exercise of 3.2% and 7.7%, respectively. In mice ingesting 1.8% VAAM, the blood ketone body concentration was 248.11 +- 13.35 u Mol at rest, and a significant 60.3% increase was observed with exercise. During 60min exercise, as shown in Table 2, plasma insulin concentrations were lower I mice that ingested VAAM than in those receiving CAAM or glucose. On the other hand, the ration of pyruvate to lactate was about 1.7 times higher in mice ingesting VAAM than in those receiving CAAM.

Serum NEFA induction by modified VAAM nutrients during swimming exercise

To analyze the amino acid compositions most effective in increasing NEFA concentrations during exercise, modified VAAMs maintaining the original VAAM compositions were fed to mice. Upon endurance swimming for 60min, the major amino acid components, proline, glycine and threonine, alone induced lower concentrations of serum NEFA than those induced by 1.8% VAAM (Table 3). Prolinewas especially effective in increasing the NEFA concentration in comparision with EAAM + proline or EAAM. The exclusion of more than one amino acid (VAAM 1,2,5) resulted in a decrease in NEFA concentration. It is suggesting that the composition and ratios of VAAm components would influence the increase of serum NEFA concentration during exercise. Further investigation with VAAM 6 and VAAM 7 again showed clearly that proline is the neccesary component for maintaining high concentrations of serum NEFA. Finally, in comparative experiments with VAAM 8 and 9, it was found that the most effective amino acids in VAAM for the induction of serum NEFA were proline, alanine, caline, leucine, and lysine. Under the same exercise conditions, the correlation between blood glucose and lactate concentrations was better (r=0.794) than the concentration between serum NEFA and blood glucose and r=0.526 for lactate, respectively).

Correlation between plasma catecholamine excretion and serum NEFA induction by VAAm and other amino acid nutrients during swimming exercise

The hormonal effect on serum NEFA induction was analyzed with respect to catecholamines. The concentrations of both plasma A and NA were increased under the same swimming conditions as in the NEFA induction experiment (Table 4). The molar ration in plasma was always lower for A (32-43%) than NA (57-68%) in exercising mice who ingested amino acid nutrients and DW. The increase in the concentration of plasma A and the proportion of A in the total catecholamine content were higher following 1.8% VAAM or DW ingestion (42-43%) than NA (57-68%) in exercising mice who ingested amino acid nutrients and DW. The increase in the concentration of plasma A and the proportion of A in the total catecholamine content were higher following 1.8% VAAM or DW ingestion (42-43%), but lower following 1.8% VAAM8 or 1.8% CAAM ingestion (32-36%) as show in Table 4. The ratio of NA to A was lower in the former case (about 1.3) than the latter (about 2). This difference is caused by the lower level of A, which then produces the low total catecholamine concentration following VAAM 8 or CAAM ingestion. The total catecholamine content showed no correlation to the induction of serum NEFA or the elongation of swimming time (2). The best correlation with NEFA induction was found for NA (r=0.746) but was negative for A (r=-0.039) (Table 4). The extent of NEFA inductionby each nutrient was analyzed for the effect on the ration of NA to A (Fig.3). The enhancement of NZ induction corresponded to an increase in the NA/A ration as represented by the slope constant. And the parallel induction of both catecholamines was observed quantitatively. At the same time, a larger increase in NA or a greater induction of serum NEFA was related to an increase in the correlation coefficient between A and NA (r=0.848 for VAAM8, r=0.145 for DW). As for catecholamine induction, although the levels of plasma NA and A were similar following either VAAM or DW ingestion, exercise performance was not improved by DW ingestion. This supports conjecture that VAAM has different effects than DW, such as the improvement of fat oxidation, an antifatigue effect in brain, etc.

Discussion

It is well known that the oxidation of fatty acids is passively activated by an increase in serum NEFA. In comparison with the ingestion of VAAM, glucose, or DW, the fact that VAAM induced high serum NEFA and blood ketone body levels, in contrast to the suppression of blood glucose decrease and blood lactate increase (Figs. 1 and 2, Table 2)(2), might be expected to encourage lipolysis and the subsequent activation of fatty acid oxidation during endurance exercise. It is to be expected that glucose oxidation does not progress at low plasma insulin levels, so that higher insulin levels cause more glucose oxidation, resulting in low levels of blood glucose. This strongly suggests that the energy supply in endurance exercised mice receiving VAAM depends on fatty acid oxidation but not on the activation of glucose as in the case of CAAM, because the decrease in glucose consumption corresponds to a lower level of insulin, and higher blood glucose and lower blood lactate levels show the suppression of glycosis. Further, the ratio of pyruvate to lactate in blood reflects an enhancement in aerobic metabolism, which also increases during exercise in mice receiving VAAM (Table 2). These metabolic changes induced by VAAM are analogous to the progressive induction of fatty acid oxidation with training adaptation (5, 19, 33).

The serum NEFA concentrations induced by these selected amino acids (especially VAAM 6, 8 and 9) are over the risky concentration of 2 mEq/L (9, 29). The serum NEFA inducing effect of these nutrients is therefore very close to the physiological maximum of metabolic adaptation of the exercising subject. However, it might be avoid the risk of membrane perturbation because almost all the induced serum NEFA is bound to lipoproteins in blood. Thus, this high concentration of serum NEFA would enhance fatty acid oxidation.

The high serum NEFA levels caused by the ingestion of DW during exercise probably reflects fasting conditions. The concentrations of blood glucose, lactate and pyruvate, and serum insulin were also lower in mice ingesting DW than in mice ingesting other nutrients (Table 2). However, the concentrations of serum NEFA and plasma catecholamines were similar to those in mice ingesting VAAM, but the concentration of blood ketone bodies was a little lower. Thus, the physiological conditions in the case of DW ingestion represent a state of extreme hunger and excitement following continuous exercise after fasting for 16 hours. Under starvation conditions, the high blood levels of ketone bodies are remarkably reduced by the injection of glucose. This suggests that glucose supplementation suppresses fatty acid oxidation and/or liposis as was observed in previous experiments (2); in other words, glucose takes priority as the energy source for oxidative metabolism over all other nutrients. Despite the depletion of energy stores by strenuous exercise, the ingestion of VAAM brought about higher levels of blood glucose, fatty acid oxidation and aerobic metabolism, thus producing better performance than other nutrients. Additionally, the same effect of VAAM, that is the suppression of the increase in blood lactate levels and the decrease in blood glucose levels during exercise, was found with glucose supplementation desptie the high blood glucose levels, as shown in the previous study (2). These results suggest that the effect of VAAM is not altered by starvation.

In experiments using various amino acids, the relationship between the concentration of serum NEFA and blood glucose or lactate was not strong, although similar for both (r=0.536 for blood glucose, r=0.526 for blood lactate) Table 3) However, there was a good correlation between the concentrations of blood glucose and lactate in this study (r=0.749) (Table 3) and also the previous study (r=0.779)(2). The correlation was generally found regardless of the state of rest or exercise and nutrient ingestion. However, the effect of VAAM goes against its trend, that is, produces high blood glucose and low blood lactate levels during exercise as shown in our previous study (2). With the administration of various nutrients, blood glucose concentrations showed a slight correlation with an improvement in performance (r=0.0507). This improvement in performance was found at low (2.5 mMol) and middle high (4.0mMol) concentrations of blood lactate. This suggests that high blood glucose concentrations, but do not always lead to an improvement in performance.

All the compositional studies suggest that glycien contributes to the suppression of the deccrease in blood glucose, but has little effect on the decrease in blood lactate. It might be expected that glycine is metabolized to two ways; one is threonine which is metabolized to propionyl CoA, thus finally might reduce the production of lactate and suppresses the decrease in glucose. Another way is serine which produces pyruvate, then also lactate. It was also found that serum NEFA induction by leucine (VAAM 8) and isoleucine (VAAM 9) is similar, so that these amino acids may be interchangeable. Detailed compositional analyses show that the induction of serum NEFA is not equivalent to the effect of VAAM, but rather represents only a partial effect. Synthetic nutrients are not better than either of VAM 6, 8 or 9. However, compositional changes in VAAM show that at a minimum, the components of VAAM are required for serum NEFA induction and higher levels of plasma NA and A (Tables 3 and 4). The results show the importance of both the compositional ration and components of VAAM for its effects. Effective components, such as VAAM 6 to 9, contain large amounts of branched amino acids, which are utilized by muscle cells. This suggests that the peculiar nutritional effect of VAAM, high levels of serum NEFA and blood glucose and low levels of blood lactate during exercise, is produced by a special balance of amino acid composition brough about exclusively by nature. The dietary function of the complicated amino acid composition of VAAM is not known completely at present. The complicated composition of VAAM may affect the transfer of some information to cells or organs as an amino acid language. Future composition in certain specific food functions.

A comparative compositions study of catecholeamine induction suggests that the induction of plasma A requires another special amino acid composition,a s shown for plasma NZ by VAAM 8. Further, plasma A probably has another effect besides the induction of serun MEFA (Table 4). These phenomena show tha apparent response of both hormones to special amino acid nutrients, and suggest that these amino acid nutrients play an important role in serum NEFA induction through plasma NA activation. Considering that high concentrations of serum NEFA lead to more fatty acid oxidation, it is possible that lipolysis during exercise is brought about by an increase in fat consumption. However, the inductions of serum NEFA and plasma NA do not always show a close relationship to one another or to improvements in blood glucose as shown by, for example, the suppressive effect on the increase in blood biochemical indices during exercise as shown by, for example, the suppressive effect on the increase in blood lactate or decrease in blood glucose as shown in Tables 3 and 4, and in the previous study (2). The fact that both catecholeamines are hardly distinguishable from eahc other strongly suggest that the ration of plasma NA to A must be newarly equal (NA/A=1.1) and the correlation coefficient between them high (r=0.862), in other words, their excretion in a nearly equal ration in each individual is a minimum requirement for exercise improvements (Fig 3). Thissuggests that plasma A plays an important role in the improvement of performance. The synchronous stimulation of organs controlled by both a – and b receptors is required for optimal exercise performance. In fact, plasma NZ activates fatty acid hydrolysis in fat bodies (5, 35) and glycogen degration in liver (18). Plasma A, in the meantime, induces the hydrolysis of muscle TG (3). Thus, VAAM is a multifunctional complex that probably controls complicated physiological functions of exercise.

The effect of VAAM as a metabolic controller during endurance exercise can be thought of as follows: VAAM absorbed from the intestines stimulates the adrenergic system, maybe the adrenal, leading to increases in MA and A. As shown in Table 4 and Fg 3, the ratios and correlation coefficients between NZ and A are obviously higher with VAAM than with CAAM ingestion. A large increase in NA has been found to interfere with the development of hypoglycaemia directly by stimulating the production of glucose through hepatic glycogenolysis, but A does not appear to be critical for the prevention of hypoglycemia during exercse. The adrenergic system and the cyclic AMP cascade play crucial roles in the activation of hormone-sensitive TG lipase and the subsequent TG hydrolysis in adipose tissues (4,5, 34). The intercellular lipoprotein lipase activity in each type of rat muscle is increased by A (28). The pattern of plasma A is similarly and significantly correlated with that of serum NEFA and with glycerol concentration (29). There is other eveidence that the adrenergic system also plays an important role in activating the lipolysis of muscle TG (12). Further studies of agonists and antagonists of b – adrenergic receptors strongly suggest that the process is probably controlled exclusively by the adrenergic system (3). The higher levels of serum NEFA and blood ketone bodies induced by plasma NZ and A (Figs 1 and 2, Tables 2 and 3) would produce excess amounts of acetyl CoA by b – oxidation. An activation of both lipolysis and oxidation is found in training adaptation of skeletal muscle (33). Endurance training in particular increases the capacity of muscle to oxidize fats derived from muscle Ts (19). For the control of energy in the fatty acid metabolism, this adaptation increases the uptake and oxidation of serum NEFA, and concomitantly bring s about a decrease in glucose uptake. The activation of fat hydrolysis and fatty acid oxidation during endurance exercise after VAAM ingestion is likely, therefore to be a kind of metabolic adaptation from the untrained tot he trained condition. This might be important for the improvement of exercise performance. Glucose uptake and oxidation decrease because of the higher glucose level and lower plasma insulin level (Table 2) (2). In fact, this response is also obeserved in trained subjects, that is, glucose uptake by skeletal muscle is decreased late in the exercise period despite higher blood glucose concentrations (8, 33). The reason for this lower glucose uptake in trained subjects during exercise is not readily apparent. One possibility is that increased fat oxidation in trained subjects leads to a citrate-mediated inhibition of phosphofructokinase (21). At the same time, glycerol, as a counterpart to fat hydrolysis, is probably metabolized mainly in the liver, which has a high activity of glycerol kinase (22). The rate of utilization by the liver is directly proportional to its concentration (31). This metabolic regulation, which is responsible for the activation of lipid hydrolysis following VAAM ingestion, finally brings about both the decrease in lactate production and the maintenance of glucose levels (Table 2). Certainly, the higher pyruvate/lactate ratios demonstrate aerobic metabolism (see Table 2). The suppressive production of lactate during exercise following VAAM ingestion could lead to the increas in serum NEFA, because lactate, which lowers the pH (15), increases the re-esterification of serum NEFA in adipose tissue (11). The lactate concentration increases in contracting muscles, and muscle pH is further reduced as lactate accumulates. The effect would be then further potentiated by the concomitant reduction in muscle pH. Lowering the pH also reduces the lipolysis stimulated bu NA, ACTH, and glucagon (30). Lactate as the end product of anaerobic glycolysis would be expected to inhibit NEFA supplementation into muscle cells. Under such circumstances, the lactate could be involved in the metabolism of fats and carbohydrates. These findings suggest that VAAM causes a shift from carbohydrate to fat combustion. These metabolic responses to VAAM ingestion during endurance exercise would prevent the occurrence of fatigue. It is thus considered that the complex effects of VAAM, its anti-fatigue effects, finally result in an improvement in exercise performance such as elongation of swimming time (2).

Footnote
The data reported in this study were presented at the Annual Meetings of the Japanese Biochemical Society between 1990 and 1993.