International Journal of Sport Nutrition and Exercise Metabolism, 2016, 26, 46 -54 http://dx.doi.org/10.1123/ijsnem.2015-0137 © 2016 Human Kinetics, Inc.
ORIGINAL RESEARCH
Amino Acid Mixture Enriched With Arginine, Alanine, and Phenylalanine Stimulates Fat Metabolism During Exercise Keisuke Ueda, Yutaka Nakamura, Makoto Yamaguchi, Takeshi Mori, Masayuki Uchida, and Satoshi Fujita Although there have been many investigations of the beneficial effects of both exercise and amino acids (AAs), little is known about their combined effects on the single-dose ingestion of AAs for lipid metabolism during exercise. We hypothesize that taking a specific combination of AAs implicated in glucagon secretion during exercise may increase fat metabolism. We recently developed a new mixture, d–AA mixture (D-mix), that contains arginine, alanine, and phenylalanine to investigate fat oxidation. In a double-blind, placebo-controlled crossover study, 10 healthy male volunteers were randomized to ingest either D-mix (3 g/dose) or placebo. Subjects in each condition subsequently performed a physical task that included workload trials on a cycle ergometer at 50% of maximal oxygen consumption for 1 hr. After oral intake of D-mix, maximum serum concentrations of glycerol (9.32 ± 6.29 mg/L and 5.22 ± 2.22 mg/L, respectively; p = .028), free fatty acid level (0.77 ± 0.26 mEq/L and 0.63 ± 0.28 mEq/L, respectively; p = .022), and acetoacetic acid levels (37.9 ± 17.7 µmol/L and 30.3 ± 13.9 µmol/L, respectively; p = .040) were significantly higher than in the placebo groups. The area under the curve for glucagon during recovery was numerically higher than placebo (6.61 ± 1.33 µg/L • min and 6.06 ± 1.23 µg/L • min, respectively; p = .099). These results suggest that preexercise ingestion of D-mix may stimulate fat metabolism. Combined with exercise, the administration of AA mixtures could prove to be a useful nutritional strategy to maximize fat metabolism. Keywords: preexercise nutrition, amino acid supplementation, metabolism In recent years, the incidence of obesity has sharply increased in developed countries, including Japan, as a result of overconsumption of calorie-rich diets and increasingly sedentary lifestyles (Watanabe et al., 2007). Obesity is known to be a strong risk factor for Type 2 diabetes mellitus, and insulin resistance is a key factor in defining Type 2 diabetes (Hartz et al., 1983; Larsson et al., 1981). Calorie reduction and regular physical activity are the most common strategies used for weight loss and weight control (National Institutes of Health Technology Assessment Conference Panel, 1993). Exercise is one important factor in the prevention of obesity (Jakicic, 2003). Exercise increases insulin sensitivity in skeletal muscle (Henriksen, 2002), so regular exercise is strongly encouraged to prevent obesity and Keisuke Ueda, Makoto Yamaguchi, Takeshi Mori, and Masayuki Uchida are with Food Science Research Laboratories, R&D Division, Meiji Co., Ltd., Odawara, Kanagawa, Japan. Yutaka Nakamura is with the Department of Physical Recreation, School of Physical Education, Tokai University, Hiratuka, Kanagawa, Japan. Satoshi Fujita is with the College of Sport and Health Science, Ritsumeikan University, Shiga, Japan. Address author correspondence to Keisuke Ueda at [email protected].
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is likely to be most effective for weight control when combined with improvements in eating habits. Dietary amino acid (AA) supplementation is another method used to reduce the risk of obesity. For example, arginine has been shown to decrease fat mass in obese rats (Fu et al., 2005), and leucine has been shown to stimulate protein synthesis (Lynch et al., 2002; Roh et al., 2003). Leucine (Cammisotto et al., 2005; Levy et al., 2000), alanine, aspartate, glycine, methionine, phenylalanine, and valine all stimulate the secretion of leptin in adipocytes (Cammisotto et al., 2005). Therefore, it may be possible to promote fat metabolism by ingesting certain combinations of AAs. It has been reported that ingestion or infusion of some AAs can lead to increased blood insulin or glucagon concentrations (Gannon et al., 2002; Gerich et al., 1976; Malaisse-Lagae et al., 1984; Müller et al., 1975; Nuttall et al., 2006). Bülow et al. (1998) reported that elevated glucagon concentrations lead to activation of lipoprotein lipase in muscle. Thus, we hypothesized that a specific combination of AAs may be implicated in glucagon secretion, and the attendant mechanism might promote more efficient fat metabolism depending on the combination of AAs administered. Although there have been many investigations of the beneficial effects of both exercise and AAs, little is known about their combined effects on
D-mix Stimulates Fat Metabolism During Exercise 47
the single-dose ingestion of AAs for lipid metabolism during exercise. We recently developed a new AA mixture called d–AA mixture (D-mix) that contains large amounts of arginine, alanine, and phenylalanine. Using a randomized, double-blind, placebo-controlled crossover trial, we aimed to assess two points related to D-mix: (a) Does oral administration of D-mix stimulate fat oxidation and, (b) if so, is glucagon involved in this potentiation?
Methods
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Subjects Ten healthy young men who exercised regularly were recruited as study volunteers. The average age (M ± SD) of the subjects was 20.6 ± 0.5 years; height was 176.7 ± 5.6 cm; body mass was 75.0 ± 7.8 kg; and body mass index was 24.0 ± 2.0 kg/m2. All study subjects provided written informed consent before participation in the study. Each subject continued his usual diet and refrained from smoking. Trials were conducted in Japan from March 2, 2013, to April 7, 2013. All participants underwent physical examinations and blood chemistry tests that included evaluations of platelet count, white blood cell count, red blood cell count, hemoglobin, hematocrit, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl transferase, total bilirubin, albumin, total protein, blood urea nitrogen, creatinine, uric acid, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, glucose, sodium, potassium, and chloride. The study was approved by the Ethics Committees of Tokai University and Meiji Institutional Review Board. The study was also performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
days. Dietary intake was self-recorded by the subjects from their first visit until their last visit. Subjects were instructed to refrain from binge eating, strenuous exercise, and drinking alcohol for 24 hr before each trial. They were also instructed to sleep more than 8 hours the evening before each visit. Subjects consumed meals with the same carbohydrate:fat:protein ratio of 71:16:13 until 2 hr of each trial. Individual trials were performed at a similar time of day for each subject (±3 hr) to avoid any circadian rhythm influences on the results. During the second and third visits, subjects participated in the main experimental trials. After measurement of blood pressure and heart rate, blood samples (21 ml) were drawn from the antecubital vein. Subjects were randomized to ingest either a cellulose capsule containing D-mix (3 g) as an active sample or an empty cellulose capsule as placebo with 50 ml of ordinary tap water (shown as 0 min). Treatments were switched later during the crossover portion of the study. The AA composition of D-mix (Kyowa Hakko Bio Co, Ltd., Tokyo, Japan) is shown in Table 1. After sitting for 30 min (rest period), subjects mounted a cycle ergometer and commenced cycling at a constant power output equivalent to 50% VO2max for 60 min (exercise period). After exercising, subjects rested for 60 min in a supine position (recovery period). Blood samples were collected every 15 min during rest and exercise and every 30 min during recovery. Heart rate was recorded and exhaled air samples were collected throughout the rest, exercise, and recovery phases. Blood pressure and heart rate were recorded again at the end of the recovery period. The Table 1 Proportion of the Amino Acid D-mix D-mix
Weight/volume %
L-arginine
40.7
L-alanine
18.9
L-phenylalanine
15.7
Experimental Procedure
Glycine
14.9
The study was conducted at Tokai University. Subjects made a total of three visits to the laboratory. During the first visit, a baseline blood sample (10 ml) was collected and evaluated. Maximal oxygen uptake VO2max (ml/kg/ min) and maximal heart rate (beats/min) were measured after incremental exercise on a cycle ergometer. Subjects continued cycling until exhaustion. Exhaustion was defined as meeting two or more of the following criteria: (a) Subjects felt they could no longer continue (more than 19 on the Borg scale), (b) the point at which oxygen consumption plateaued, (c) the ratio of respiratory exchange exceeded 1.1, (d) a heart rate of at least 200 beats/min (American College of Sports Medicine, 2006), (e) a self-imposed withdrawal from exercise, or all of these. The results of exhaustion testing were used to calculate the power output equivalent to 50% VO2max. The remaining two visits were separated by at least 6
L-prorin
2.24
L-lysine
1.7
L-tyrosine
0.92
L-threonine
0.92
L-leucine
0.87
L-valine
0.74
L-isoleicine
0.64
L-glutamic acid
0.51
L-tryptophan
0.48
L-histidine
0.43
L-serine
0.28
L-methionine
0.09
L-aspartic acid
0.03
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48 Ueda et al.
A
B
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Figure 1 — Study design: Study schedule (A) and schedule on experimental trial days (Visit 2 and Visit 3; B).
tests were conducted in a quiet, temperature-controlled (21 ± 2 °C), and humidity-controlled (45 ± 5%) room. The study design is summarized in Figure 1.
Exhaled Gas Analysis Exhaled oxygen and carbon dioxide concentrations were measured via the breath-by-breath method using a respiration metabolism monitor system (AE-310s, Minato Medical Science Co., Ltd., Osaka, Japan). All gas analyzers were calibrated before each trial. Values of more than 0–5 min were averaged and recorded as a value of 5 min. The same method was used, and values were recorded from 5 to 150 min.
Blood Sampling Whole blood from a sodium fluoride vacutainer was cooled to 4 °C for later analysis of glucose concentrations. Whole blood from the ethylenediaminetetraacetic acid (EDTA) vacutainer with added aprotinin was immediately centrifuged at 1,200 g for 10 min at 4 °C, and the plasma was then separated and immediately frozen at −80 °C for later analysis of glucagon concentrations. Whole blood from the EDTA vacutainer without added aprotinin was immediately centrifuged at 1,200 g for 10 min at 4 °C, and the plasma was then separated into two vials. One vial was cooled to 4 °C for later analysis of cortisol concentrations. The other vial was immediately frozen at −80 °C for later analysis of adrenaline and noradrenaline concentrations. Whole blood from the plain vacutainer was allowed to stand at room temperature for 20 min and then centrifuged at 1,200 g for 10 min at 4 °C, and the serum was separated into two vials. One vial was cooled to 4 °C for later analysis of free fatty acid (FFAs), growth hormone, and insulin concentrations. The other vial was frozen at −80 °C for later analysis of acetoacetic acid, 3-hydroxybutyrate, and glycerol concentrations. Whole blood from a regular syringe was transferred to a new vial to remove protein and then centrifuged at 1,200 g for 10
min at 4 °C; the deproteinized serum was frozen at −80 °C for later analysis of lactate concentrations. Glucagon concentrations were measured by double-antibody radioimmunoassay (Glucagon RIA SML, Euro-Diagnostica AB, Malmö, Sweden). Insulin was measured by chemiluminescent immunoassay (Architect Insulin, Abbott Japan, Japan). Blood glucose (Iatoro LQ GLU, Unitica, Japan), acetoacetic acid, 3-hydroxybutyrate (total ketone bodies Kainos, 3-HB Kainos, Kainos Co., Ltd., Japan), glycerol (Glycerol Colorimetric Assay Kit, Cayman Chemical, Ann Arbor, MI), and lactate (Detamina-LA, Kyowa Medex Co., Ltd., Japan) concentrations were measured by an enzymatic method. Blood FFAs (NEFA-SS Eiken, Eiken Chemical Co., Ltd., Japan) were measured by the enzyme ultraviolet method. Growth hormone (Access hGH, Beckman Coulter, Inc., Sharon Hill, PA) and cortisol (Access cortisol, Beckman Coulter, Inc.) concentrations were measured by chemiluminescent enzyme immunoassay. Adrenaline and noradrenaline were measured using high-performance liquid chromatography (HLC-725CAΠ, TOSOH Corp., Tokyo, Japan). Assays to measure glucagon, insulin, blood glucose, acetoacetic acid, 3-hydroxybutyrate, lactate, FFAs, growth hormone, adrenaline, and blood chemistry panels were performed at LSI Medience Corporation (Tokyo, Japan), and the glycerol assay was performed at Nikken Seil Co., Ltd. (Tokyo, Japan).
Statistical Analysis Data are presented as M ± SD and were analyzed using Microsoft Excel (Microsoft Corp., Redmond, WA). Areas under the curve for plasma concentrations compared with time were calculated with the use of the trapezium rule. Repeated-measures two-factor analysis of variance (ANOVA; Time × Condition) was used to examine differences among two trials. To specifically compare the peak responses of each blood and cardiorespiratory parameter between treatments, we also divided the experiment into three time phases (i.e., rest, exercise, and recovery period). Normal distribution of all variables was tested with the F test using StatView-J Version 5.0 software (Abacus Concepts, Berkeley, CA). The statistical significance of differences between the two trials were analyzed via paired-samples t test using Microsoft Excel if data were normally distributed and the Wilcoxon signed-rank test using StatView-J Version 5.0 software if not. Statistical significance was accepted at p < .05.
Results Cardiorespiratory Responses Cardiorespiratory responses after D-mix administration are summarized in Table 2. Two-factor ANOVA revealed significant main effects of time and no significant interaction. However, heart rate during exercise was numerically lower in the D-mix condition than in the placebo condition (p = .057). However, on examination respiratory
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D-mix Stimulates Fat Metabolism During Exercise 49
Table 2 Cardiorespiratory Responses of Participants During Rest, Exercise, and Recovery After consuming the Treatment Rest (0–30 min; M ± SD)
Exercise (>30–90 min; M ± SD)
Recovery (>90–150 min; M ± SD)
Response
D-mix
Placebo
D-mix
Placebo
D-mix
Placebo
p(EX)a
HR (bpm)
66 ± 8
69 ± 7
125 ± 11
128 ± 9
74 ± 8
76 ± 9
.057
RQ
0.90 ± 0.03
0.91 ± 0.04
0.96 ± 0.02
0.97 ± 0.03
0.87 ± 0.06
0.87 ± 0.05
.49
ΔRQ
-0.01 ± 0.03
0.00 ± 0.02
0.04 ± 0.03
0.06 ± 0.04
-0.04 ± 0.05
-0.01 ± 0.05
.11
Note. n = 10. bpm = beats per minute; HR = heart rate; RQ = respiratory quotient; ΔRQ = change in RQ. ap (EX) is the p value based on paired-sample t test for exercise data only.
quotient (RQ) and change in RQ (ΔRQ) did not differ between the two conditions.
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Biochemical Parameters Biochemical parameters after D-mix administration are shown in Figure 2, and maximum concentrations of each parameter measured during rest, exercise, and recovery are summarized in Table 3. Glycerol and FFAs are metabolites of fat, and acetoacetic acid and 3-hydroxybutyrate are ketone metabolites of FFAs. A two-factor ANOVA revealed significant main effects of time, and no significant interaction. However, the maximum concentrations of glycerol, FFAs, and ketone metabolites were significantly higher in the D-mix condition than in the placebo condition during exercise (p = .028 for glycerol; p = .022 for FFAs; p = .049 for 3-hydroxybutyrate; p = .040 for acetoacetic acid; Table 3). However, blood glucose and lactate measurements did not differ between the two conditions.
Circulating Hormones Circulating hormones after D-mix administration are shown in Figure 3, and the area under the curve (AUC) for each condition during rest, exercise, and recovery is summarized in Table 4. A two-factor ANOVA revealed significant main effects of time, and no significant interaction. However, the AUC for cortisol was significantly lower for the D-mix condition than for the placebo condition (p = .049), and the AUC for glucagon during recovery was numerically higher for the D-mix condition than for the placebo condition (p = .099). All other parameters for circulating hormones did not significantly differ between the two treatment conditions.
Discussion This randomized, double-blind, placebo-controlled crossover trial was conducted to assess the effects of D-mix during exercise. We conducted a two-factor ANOVA to compare the responses between the groups described in Figures 2 and 3. Overall significant differences were not observed between the groups.
We also divided the experiment into three time phases. Because we hypothesized that AAs may stimulate fat metabolism during exercise, it was necessary to carry out an independent analysis of the exercise phase. As a result, we found that during exercise ingestion of D-mix significantly elevated the maximum concentration of glycerol, FFAs, and ketone bodies compared with a placebo condition. These results suggested that fat metabolism increased after the ingestion of D-mix. Although there was a change in fat metabolites (glycerol, FFAs, ketone bodies), there was no effect on substrate metabolism as measured by RQ. However, RQ is just one indicator of progressing lipid oxidation in muscle. Thus, it is necessary to conduct further research on how D-mix affects lipid oxidation in muscle. It is well established that catecholamines such play an important role in the stimulation of lipolysis in human adipose tissue. Increased catecholamine concentrations in the blood stimulate cyclic adenosine monophosphate and hormone-sensitive lipase activity in skeletal muscle and in adipose tissue. Indeed, caffeine, which stimulates adrenalin secretion, is known to increase hormone-sensitive lipase activity and increase fat mobilization from skeletal muscles and adipose tissue, resulting in increased FFAs in the bloodstream (Ryu et al., 2001). Moreover, it has been shown that oral administration of an AA mixture derived from hornet larval saliva increases the concentrations of catecholamines in the blood, especially adrenaline (Uchida et al., 2008). In this study, concentrations of adrenaline and noradrenaline did not differ between the D-mix and placebo conditions. These results suggest that D-mix promoted lipid metabolism in an adrenaline-independent manner. There are also AAs that directly stimulate the sympathetic nerves. Yoshimatsu et al. (2002) reported that histidine accelerated lipolysis in white adipose tissue through activation of the sympathetic nerves. However, D-mix contains only 0.43% of histidine, so it is unlikely that lipid metabolism could be increased by stimulation of the sympathetic nervous system. Arginine accounts for 40% of the D-mix mixture. Arginine is known to stimulate the release of growth hormone (Collier et al., 2005), a molecule that has been suggested to increase fat oxidation. Hence, growth hormone may play a role in D-mix–associated fat metabolism. In this study, growth hormone concentrations did
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Figure 2 — Concentrations of biochemical assessed during the experimental trials: plasma glycerol (A), FFAs (B), 3-hydroxybutyrate (C), acetoacetic acid (D), glucose (E), and lactate (F). Values are M ± SD (n = 10). A = D-mix treatment; FFAs = free fatty acids; P = placebo treatment.
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D-mix Stimulates Fat Metabolism During Exercise 51
Table 3 Effects of Amino Acid Mixture on the Maximum Concentration of Biochemical Parameters Rest (0–30 min; M ± SD)
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Parameter
Exercise (>30–90 min; M ± SD) D-mix
Recovery (>90–150 min; M ± SD)
D-mix
Placebo
pa
Placebo
pa
D-mix
Placebo
pa
Glycerol (mg/L)
4.16 ± 5.78
3.44 ± 3.12
.70
9.32 ± 6.29
5.22 ± 2.22
.028
3.07 ± 1.94
2.69 ± 1.21
.63
FFAs (mEq/L)
0.42 ± 0.34
0.34 ± 0.16
.88
0.77 ± 0.26
0.63 ± 0.28
.022
0.64 ± 0.31
0.57 ± 0.32
.48
3-hydroxybutyrate (μmol/L)
20.5 ± 8.6
18.9 ± 6.2
.47
54.2 ± 47.2
34.8 ± 23.1
.049
163 ± 190
85.8 ± 95.1
.16
Acetoacetic acid (μmol/L)
27.9 ± 8.6
29.1 ± 11.0
.65
37.9 ± 17.7
30.3 ± 13.9
.040
83.9 ± 68.3
51.3 ± 30.8
.093
Glucose (mg/L)
1,040 ± 278
1,110 ± 181
.45
810 ± 89
835 ± 115
.41
870 ± 194
949 ± 216
.29
Lactate (mg/L)
118 ± 28.3
116 ± 17.3
.84
192 ± 54.1
198 ± 56.9
.56
97.4 ± 30.5
100.2 ± 24.2
.82
Note. n = 10. FFA—free fatty acid. ap value for the paired-sample t test if data were normally distributed and the Wilcoxon signed-rank test if not.
not differ between the D-mix and placebo conditions. This finding suggests that D-mix promoted fat metabolism via a growth hormone–independent mechanism. Ishidori et al. (1981) reported that oral ingestion of a mixture of arginine and lysine stimulated the secretion of insulin and growth hormone, but ingestion of either AA alone did not elicit the same effect. Hence, the stimulation of insulin and growth hormone by arginine may be affected by the composition of AAs to which it is added. The AUC for glucagon was numerically higher in the active group than the placebo group during recovery. These results suggest that ingestion of D-mix may have promoted the secretion of glucagon. It has been reported that ingestion or infusion of some AAs can lead to increased insulin or glucagon concentrations in the blood. For example, in a study by Nuttall et al. (2006), ingestion of phenylalanine increased both insulin and glucagon concentrations. Intravenously administered arginine and lysine have been shown to increase the circulating concentration of glucagon (Gerich et al., 1976). Alanine and glycine, but not valine, isoleucine, and leucine, have been observed to stimulate glucagon secretion (Gannon et al., 2002; Malaisse-Lagae et al., 1984; Müller et al., 1975). Therefore, a specific combination of AAs may stimulate the secretion of glucagon. D-mix is composed of arginine (41%), alanine (19%), phenylalanine (16%), and a lesser percentage of valine (0.74%), isoleucine (0.64%), and leucine (0.87%). Thus, we suggest that depending on the combination of AAs, such as those implicated in the secretion of glucagon, lipid metabolism may potentially be able to increase. However, there is a time lag between the increase of glycerol concentration and that of glucagon, which indicates that only the action of glucagon does not factor into lipolysis. Further study of the mechanism by which D-mix promotes lipid metabolism is necessary.
The concentration of glucose in the blood decreased and lactate concentrations increased from resting to the initiation of exercise. Although AAs cannot supply as much cellular energy as glucose and FFAs, the gluconeogenic process is an important pathway for utilization of AAs for energy. In the liver, the rate of gluconeogenesis from alanine is much higher than the rate from any other AA (Hood & Terjung, 1990; Wasserman et al., 1988). Alanine forms 19% of the D-mix concentration, and on this basis D-mix would be expected to cause a greater increase in circulating glucose than placebo. However, the concentration of glucose in the blood did not differ between the D-mix and placebo conditions. The administration of D-mix may have had an effect on gluconeogenesis in the liver, but this remains to be investigated. The AUC for cortisol during exercise was significantly lower in the D-mix condition than in the placebo condition. Cortisol is known to be elevated by acute stress, which can stimulate adrenocorticotropic hormone secretion (Levine, 1993; Oberbeck et al., 1998). In this study, although blood cortisol concentrations were suppressed, lipid metabolism increased after D-mix ingestion. However, it is unlikely that lipid metabolism increased via stimulation of cortisol secretion. Hainer et al. (1979) reported that intramuscularly administered glucagon decreased serum cortisol concentration, and this drop was not associated with the depressive effect of elevated growth hormone. The administration of D-mix may have had an effect on the decrease in cortisol concentration via promotion of glucagon secretion, but this remains to be confirmed. In conclusion, our results suggest that preexercise ingestion of D-mix stimulated fat metabolism. Combined with exercise, the administration of AA mixtures may
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Figure 3 — Concentrations of circulating hormones during the experimental trials: plasma glucagon (A), insulin (B), adrenaline (C), noradrenaline (D), growth hormone (E), and cortisol (F). Values are M ± SD (n = 10). A = D-mix treatment; P—placebo treatment
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D-mix Stimulates Fat Metabolism During Exercise 53
Table 4 Effects of Amino Acid Mixture on the Area Under the Curve for Circulating Hormones Rest (0–30 min; M ± SD) D-mix
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Glucagon (μg/L.min)
Placebo
2.90 ± 0.554 2.86 ± 0.570
Exercise (>30–90 min; M ± SD)
Recovery (>90–150 min; M ± SD)
pa
D-mix
Placebo
pa
D-mix
Placebo
pa
.88
6.60 ± 1.24
6.33 ± 1.21
.20
6.61 ± 1.33
6.06 ± 1.23
.099
Insulin (mU/L.min)
478 ± 348
590 ± 376
.46
300 ± 190
379 ± 181
.22
323 ± 322
481 ± 430
.33
Adrenaline (μg/L.min)
1.31 ± 0.79
1.41 ± 0.92
.55
6.68 ± 3.02
6.29 ± 2.59
.64
4.31 ± 2.02
3.66 ± 2.02
.36
Noradrenaline (μg/L-min)
8.16 ± 1.89
9.62 ± 3.37
.34
45.3 ± 8.94
44.9 ± 7.25
.87
27.9 ± 6.48
25.1 ± 6.81
.46
Growth hormone (μg/L.min)
55.5 ± 75.0
61.5 ± 113
.88
331 ± 302
359 ± 423
.86
188 ± 89.0
205 ± 217
.39
Cortisol (mg/L.min)
3.48 ± 0.85
3.71 ± 1.19
.33
5.87 ± 1.34
6.72 ± 2.12
.049
4.58 ± 0.85
5.04 ± 1.45
.20
Note. n = 10. ap value for the paired-sample t test if data were normally distributed and the Wilcoxon signed-rank test if not.
prove to be a useful nutritional strategy to maximize fat metabolism. On the basis of these preliminary findings, future studies of AA mixtures containing arginine, phenylalanine, and alanine appear to be warranted. A prominent limitation of this study is that we monitored only 10 subjects (all men). Therefore, future studies such as clinical trials in humans with no exercise habit are necessary. Acknowledgments All authors approved the final version of the article. We thank Ms. Shoko Takai for her technical analysis, Dr. Chiaki Sanbongi for his advice and valuable discussion during the development of this article, and Dr. Hiroyuki Itoh for providing encouragement throughout the study. The study was designed by KU, YN, MY, TM, MU, and SF; data were collected and analyzed by KU and YN; data interpretation and manuscript preparation were undertaken by KU, MY, and SF. KU, MY, TM, and MU are employees of Meiji Co.
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Collier, S.R., Casey, D.P., & Kanaley, J.A. (2005). Growth hormone responses to varying doses of oral arginine. Growth Hormone & IGF Research, 15, 136–139. PubMed doi:10.1016/j.ghir.2004.12.004 Fu, W.J., Haynes, T.E., Kohli, R., Hu, J., Shi, W., Spencer, T.E., . . . Wu, G. (2005). Dietary L-arginine supplementation reduces fat mass in Zucker diabetic fatty rats. Journal of Nutrition, 135, 714–721. PubMed Gannon, M.C., Nuttall, J.A., & Nuttall, F.Q. (2002). The metabolic response to ingested glycine. American Journal of Clinical Nutrition, 76, 1302–1307. PubMed Gerich, J.E., Charles, M.A., & Grodsky, G.M. (1976). Regulation of pancreatic insulin and glucagon secretion. Annual Review of Physiology, 38, 353–388. PubMed doi:10.1146/ annurev.ph.38.030176.002033 Hainer, V., Krejcik, L., Starka, L., & Urbanek, J. (1979). Glucagon-induced fall of serum cortisol levels: Effect of cyproheptadine pretreatment. Hormone and Metabolic Research, 11, 178–179. doi:10.1055/s-0028-1095767 Hartz, A.J., Rupley, D.C., Jr., Kalkhoff, R.D., & Rimm, A.A. (1983). Relationship of obesity level and body fat distribution. Preventive Medicine, 12, 351–357. PubMed doi:10.1016/0091-7435(83)90244-X Henriksen, E.J. (2002). Effects of acute exercise and exercise training on insulin resistance. Journal of Applied Physiology, 93, 788–796. PubMed Hood, D.A., & Terjung, R.L. (1990). Amino acid metabolism during exercise and following endurance training. Sports Medicine, 9, 23–35. PubMed doi:10.2165/00007256199009010-00003 Isidori, A., Monaco, A.L., & Cappa, M. (1981). A study of growth hormone release in man after oral administration of amino acids. Current Medical Research and Opinion, 7, 475–481. PubMed doi:10.1185/03007998109114287
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Jakicic, J.M. (2003). Exercise in the treatment of obesity. Endocrinology and Metabolism Clinics of North America, 32, 967–980. PubMed doi:10.1016/S0889-8529(03)00075-6 Larsson, B., Bjorntorp, P., & Tibblin, G. (1981). The health consequences of moderate obesity. International Journal of Obesity, 5, 97–116. PubMed Levine, S. (1993). The psychoendocrinology of stress. Annals of the New York Academy of Sciences, 697, 61–69. PubMed doi:10.1111/j.1749-6632.1993.tb49923.x Levy, J.R., Gyarmati, J., Lesko, J.M., Adler, R.A., & Stevens, W. (2000). Dual regulation of leptin secretion: Intra-cellular energy and calcium dependence of regulated pathway. American Journal of Physiology: Endocrinology and Metabolism, 278, E892–E901. PubMed Lynch, C.J., Patson, B.J., Anthony, J., Vaval, A., Jefferson, L.S., & Vary, T.C. (2002). Leucine is a direct-acting nutrient signal that regulates protein synthesis in adipose tissue. American Journal of Physiology: Endocrinology and Metabolism, 283, E503–E513. PubMed doi:10.1152/ ajpendo.00084.2002 Malaisse-Lagae, F., Welsh, M., Lebrun, P., Herchuelz, A., Sener, A., Hellerstrom, C., & Malaisse, W.J. (1984). The stimulus-secretion coupling of amino acid-induced insulin release: Secretory and oxidative response of pancreatic islets to L-asparagine. Diabetes, 33, 464–469. PubMed doi:10.2337/diab.33.5.464 Müller, W.A., Aoki, T.T., & Cahill, G.F.J. (1975). Effect of alanine and glycine on glucagon secretion in postabsorptive and fasting obese man. Journal of Clinical Endocrinology and Metabolism, 40, 418–425. PubMed doi:10.1210/ jcem-40-3-418 National Institutes of Health Technology Assessment Conference Panel. (1993). Methods for voluntary weight loss and control. Proceedings of NIH Technology Assessment Conference. Bethesda, Maryland, 30 March–1 April 1992. Annals of Internal Medicine, 119(7, Pt. 2), 641–770. PubMed Nuttall, F.Q., Schwein, K.J., & Gannon, M.C. (2006). Effect of orally administered phenylalanine with and without
glucose on insulin, glucagons and glucose concentrations. Hormone and Metabolic Research/Hormon- und Stoffwechselforschung/Hormones et Metabolisme, 38, 518–523. PubMed doi:10.1055/s-2006-949523 Oberbeck, R., Benschop, R.J., Jacobs, R., Hosch, W., Jetschmann, J.U., & Schurmeyer, T.H. (1998). Endocrine mechanisms of stress-induced DHEA-secretion. Journal of Endocrinological Investigation, 21, 148–153. PubMed doi:10.1007/BF03347293 Roh, C., Han, J., Tzatsos, A., & Kandror, K.V. (2003). Nutrient-sensing mTOR-mediated pathway regulates leptin production in isolated rat adipocytes. American Journal of Physiology: Endocrinology and Metabolism, 284, E322–E330. PubMed doi:10.1152/ajpendo.00230.2002 Ryu, S., Choi, S., Joung, S.S., Suh, H., Cha, Y.S., Lee, S., & Lim, K. (2001). Caffeine as a lipolytic food component increases endurance performance in rats and athletes. Journal of Nutritional Science and Vitaminology, 47, 139–146. PubMed doi:10.3177/jnsv.47.139 Uchida, M., Yoshida, K., & Wakabayashi, T. (2008). Effect of amino acid mixture derived from hornet larva on basal body temperature and blood catecholamine level in rats. Japanese Journal of Pharmacology, 36, 749–755. Wasserman, D.H., Williames, P.E., Lacy, D.B., Green, D.R., & Cherrington, A.D. (1988). Importance of intrahepatic mechanisms to gluconeogenesis from alanine during exercise and recovery. American Journal of Physiology, 254, E518–E525. PubMed Watanabe, S., Hojo, M., & Nagahara, A. (2007). Metabolic syndrome and gastrointestinal diseases. Journal of Gastroenterology, 42, 267–274. PubMed doi:10.1007/ s00535-007-2033-0 Yoshimatsu, H., Tsuda, K., Niijima, A., Tatsukawa, M., Chiba, S., & Sakata, T. (2002). Histidine induces lipolysis through sympathetic nerve in white adipose tissue. European Journal of Clinical Investigation, 32, 236–241. PubMed doi:10.1046/j.1365-2362.2002.00972.x
IJSNEM Vol. 26, No. 1, 2016
ORIGINAL RESEARCH
Amino Acid Mixture Enriched With Arginine, Alanine, and Phenylalanine Stimulates Fat Metabolism During Exercise Keisuke Ueda, Yutaka Nakamura, Makoto Yamaguchi, Takeshi Mori, Masayuki Uchida, and Satoshi Fujita Although there have been many investigations of the beneficial effects of both exercise and amino acids (AAs), little is known about their combined effects on the single-dose ingestion of AAs for lipid metabolism during exercise. We hypothesize that taking a specific combination of AAs implicated in glucagon secretion during exercise may increase fat metabolism. We recently developed a new mixture, d–AA mixture (D-mix), that contains arginine, alanine, and phenylalanine to investigate fat oxidation. In a double-blind, placebo-controlled crossover study, 10 healthy male volunteers were randomized to ingest either D-mix (3 g/dose) or placebo. Subjects in each condition subsequently performed a physical task that included workload trials on a cycle ergometer at 50% of maximal oxygen consumption for 1 hr. After oral intake of D-mix, maximum serum concentrations of glycerol (9.32 ± 6.29 mg/L and 5.22 ± 2.22 mg/L, respectively; p = .028), free fatty acid level (0.77 ± 0.26 mEq/L and 0.63 ± 0.28 mEq/L, respectively; p = .022), and acetoacetic acid levels (37.9 ± 17.7 µmol/L and 30.3 ± 13.9 µmol/L, respectively; p = .040) were significantly higher than in the placebo groups. The area under the curve for glucagon during recovery was numerically higher than placebo (6.61 ± 1.33 µg/L • min and 6.06 ± 1.23 µg/L • min, respectively; p = .099). These results suggest that preexercise ingestion of D-mix may stimulate fat metabolism. Combined with exercise, the administration of AA mixtures could prove to be a useful nutritional strategy to maximize fat metabolism. Keywords: preexercise nutrition, amino acid supplementation, metabolism In recent years, the incidence of obesity has sharply increased in developed countries, including Japan, as a result of overconsumption of calorie-rich diets and increasingly sedentary lifestyles (Watanabe et al., 2007). Obesity is known to be a strong risk factor for Type 2 diabetes mellitus, and insulin resistance is a key factor in defining Type 2 diabetes (Hartz et al., 1983; Larsson et al., 1981). Calorie reduction and regular physical activity are the most common strategies used for weight loss and weight control (National Institutes of Health Technology Assessment Conference Panel, 1993). Exercise is one important factor in the prevention of obesity (Jakicic, 2003). Exercise increases insulin sensitivity in skeletal muscle (Henriksen, 2002), so regular exercise is strongly encouraged to prevent obesity and Keisuke Ueda, Makoto Yamaguchi, Takeshi Mori, and Masayuki Uchida are with Food Science Research Laboratories, R&D Division, Meiji Co., Ltd., Odawara, Kanagawa, Japan. Yutaka Nakamura is with the Department of Physical Recreation, School of Physical Education, Tokai University, Hiratuka, Kanagawa, Japan. Satoshi Fujita is with the College of Sport and Health Science, Ritsumeikan University, Shiga, Japan. Address author correspondence to Keisuke Ueda at [email protected].
46
is likely to be most effective for weight control when combined with improvements in eating habits. Dietary amino acid (AA) supplementation is another method used to reduce the risk of obesity. For example, arginine has been shown to decrease fat mass in obese rats (Fu et al., 2005), and leucine has been shown to stimulate protein synthesis (Lynch et al., 2002; Roh et al., 2003). Leucine (Cammisotto et al., 2005; Levy et al., 2000), alanine, aspartate, glycine, methionine, phenylalanine, and valine all stimulate the secretion of leptin in adipocytes (Cammisotto et al., 2005). Therefore, it may be possible to promote fat metabolism by ingesting certain combinations of AAs. It has been reported that ingestion or infusion of some AAs can lead to increased blood insulin or glucagon concentrations (Gannon et al., 2002; Gerich et al., 1976; Malaisse-Lagae et al., 1984; Müller et al., 1975; Nuttall et al., 2006). Bülow et al. (1998) reported that elevated glucagon concentrations lead to activation of lipoprotein lipase in muscle. Thus, we hypothesized that a specific combination of AAs may be implicated in glucagon secretion, and the attendant mechanism might promote more efficient fat metabolism depending on the combination of AAs administered. Although there have been many investigations of the beneficial effects of both exercise and AAs, little is known about their combined effects on
D-mix Stimulates Fat Metabolism During Exercise 47
the single-dose ingestion of AAs for lipid metabolism during exercise. We recently developed a new AA mixture called d–AA mixture (D-mix) that contains large amounts of arginine, alanine, and phenylalanine. Using a randomized, double-blind, placebo-controlled crossover trial, we aimed to assess two points related to D-mix: (a) Does oral administration of D-mix stimulate fat oxidation and, (b) if so, is glucagon involved in this potentiation?
Methods
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Subjects Ten healthy young men who exercised regularly were recruited as study volunteers. The average age (M ± SD) of the subjects was 20.6 ± 0.5 years; height was 176.7 ± 5.6 cm; body mass was 75.0 ± 7.8 kg; and body mass index was 24.0 ± 2.0 kg/m2. All study subjects provided written informed consent before participation in the study. Each subject continued his usual diet and refrained from smoking. Trials were conducted in Japan from March 2, 2013, to April 7, 2013. All participants underwent physical examinations and blood chemistry tests that included evaluations of platelet count, white blood cell count, red blood cell count, hemoglobin, hematocrit, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl transferase, total bilirubin, albumin, total protein, blood urea nitrogen, creatinine, uric acid, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, glucose, sodium, potassium, and chloride. The study was approved by the Ethics Committees of Tokai University and Meiji Institutional Review Board. The study was also performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
days. Dietary intake was self-recorded by the subjects from their first visit until their last visit. Subjects were instructed to refrain from binge eating, strenuous exercise, and drinking alcohol for 24 hr before each trial. They were also instructed to sleep more than 8 hours the evening before each visit. Subjects consumed meals with the same carbohydrate:fat:protein ratio of 71:16:13 until 2 hr of each trial. Individual trials were performed at a similar time of day for each subject (±3 hr) to avoid any circadian rhythm influences on the results. During the second and third visits, subjects participated in the main experimental trials. After measurement of blood pressure and heart rate, blood samples (21 ml) were drawn from the antecubital vein. Subjects were randomized to ingest either a cellulose capsule containing D-mix (3 g) as an active sample or an empty cellulose capsule as placebo with 50 ml of ordinary tap water (shown as 0 min). Treatments were switched later during the crossover portion of the study. The AA composition of D-mix (Kyowa Hakko Bio Co, Ltd., Tokyo, Japan) is shown in Table 1. After sitting for 30 min (rest period), subjects mounted a cycle ergometer and commenced cycling at a constant power output equivalent to 50% VO2max for 60 min (exercise period). After exercising, subjects rested for 60 min in a supine position (recovery period). Blood samples were collected every 15 min during rest and exercise and every 30 min during recovery. Heart rate was recorded and exhaled air samples were collected throughout the rest, exercise, and recovery phases. Blood pressure and heart rate were recorded again at the end of the recovery period. The Table 1 Proportion of the Amino Acid D-mix D-mix
Weight/volume %
L-arginine
40.7
L-alanine
18.9
L-phenylalanine
15.7
Experimental Procedure
Glycine
14.9
The study was conducted at Tokai University. Subjects made a total of three visits to the laboratory. During the first visit, a baseline blood sample (10 ml) was collected and evaluated. Maximal oxygen uptake VO2max (ml/kg/ min) and maximal heart rate (beats/min) were measured after incremental exercise on a cycle ergometer. Subjects continued cycling until exhaustion. Exhaustion was defined as meeting two or more of the following criteria: (a) Subjects felt they could no longer continue (more than 19 on the Borg scale), (b) the point at which oxygen consumption plateaued, (c) the ratio of respiratory exchange exceeded 1.1, (d) a heart rate of at least 200 beats/min (American College of Sports Medicine, 2006), (e) a self-imposed withdrawal from exercise, or all of these. The results of exhaustion testing were used to calculate the power output equivalent to 50% VO2max. The remaining two visits were separated by at least 6
L-prorin
2.24
L-lysine
1.7
L-tyrosine
0.92
L-threonine
0.92
L-leucine
0.87
L-valine
0.74
L-isoleicine
0.64
L-glutamic acid
0.51
L-tryptophan
0.48
L-histidine
0.43
L-serine
0.28
L-methionine
0.09
L-aspartic acid
0.03
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48 Ueda et al.
A
B
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Figure 1 — Study design: Study schedule (A) and schedule on experimental trial days (Visit 2 and Visit 3; B).
tests were conducted in a quiet, temperature-controlled (21 ± 2 °C), and humidity-controlled (45 ± 5%) room. The study design is summarized in Figure 1.
Exhaled Gas Analysis Exhaled oxygen and carbon dioxide concentrations were measured via the breath-by-breath method using a respiration metabolism monitor system (AE-310s, Minato Medical Science Co., Ltd., Osaka, Japan). All gas analyzers were calibrated before each trial. Values of more than 0–5 min were averaged and recorded as a value of 5 min. The same method was used, and values were recorded from 5 to 150 min.
Blood Sampling Whole blood from a sodium fluoride vacutainer was cooled to 4 °C for later analysis of glucose concentrations. Whole blood from the ethylenediaminetetraacetic acid (EDTA) vacutainer with added aprotinin was immediately centrifuged at 1,200 g for 10 min at 4 °C, and the plasma was then separated and immediately frozen at −80 °C for later analysis of glucagon concentrations. Whole blood from the EDTA vacutainer without added aprotinin was immediately centrifuged at 1,200 g for 10 min at 4 °C, and the plasma was then separated into two vials. One vial was cooled to 4 °C for later analysis of cortisol concentrations. The other vial was immediately frozen at −80 °C for later analysis of adrenaline and noradrenaline concentrations. Whole blood from the plain vacutainer was allowed to stand at room temperature for 20 min and then centrifuged at 1,200 g for 10 min at 4 °C, and the serum was separated into two vials. One vial was cooled to 4 °C for later analysis of free fatty acid (FFAs), growth hormone, and insulin concentrations. The other vial was frozen at −80 °C for later analysis of acetoacetic acid, 3-hydroxybutyrate, and glycerol concentrations. Whole blood from a regular syringe was transferred to a new vial to remove protein and then centrifuged at 1,200 g for 10
min at 4 °C; the deproteinized serum was frozen at −80 °C for later analysis of lactate concentrations. Glucagon concentrations were measured by double-antibody radioimmunoassay (Glucagon RIA SML, Euro-Diagnostica AB, Malmö, Sweden). Insulin was measured by chemiluminescent immunoassay (Architect Insulin, Abbott Japan, Japan). Blood glucose (Iatoro LQ GLU, Unitica, Japan), acetoacetic acid, 3-hydroxybutyrate (total ketone bodies Kainos, 3-HB Kainos, Kainos Co., Ltd., Japan), glycerol (Glycerol Colorimetric Assay Kit, Cayman Chemical, Ann Arbor, MI), and lactate (Detamina-LA, Kyowa Medex Co., Ltd., Japan) concentrations were measured by an enzymatic method. Blood FFAs (NEFA-SS Eiken, Eiken Chemical Co., Ltd., Japan) were measured by the enzyme ultraviolet method. Growth hormone (Access hGH, Beckman Coulter, Inc., Sharon Hill, PA) and cortisol (Access cortisol, Beckman Coulter, Inc.) concentrations were measured by chemiluminescent enzyme immunoassay. Adrenaline and noradrenaline were measured using high-performance liquid chromatography (HLC-725CAΠ, TOSOH Corp., Tokyo, Japan). Assays to measure glucagon, insulin, blood glucose, acetoacetic acid, 3-hydroxybutyrate, lactate, FFAs, growth hormone, adrenaline, and blood chemistry panels were performed at LSI Medience Corporation (Tokyo, Japan), and the glycerol assay was performed at Nikken Seil Co., Ltd. (Tokyo, Japan).
Statistical Analysis Data are presented as M ± SD and were analyzed using Microsoft Excel (Microsoft Corp., Redmond, WA). Areas under the curve for plasma concentrations compared with time were calculated with the use of the trapezium rule. Repeated-measures two-factor analysis of variance (ANOVA; Time × Condition) was used to examine differences among two trials. To specifically compare the peak responses of each blood and cardiorespiratory parameter between treatments, we also divided the experiment into three time phases (i.e., rest, exercise, and recovery period). Normal distribution of all variables was tested with the F test using StatView-J Version 5.0 software (Abacus Concepts, Berkeley, CA). The statistical significance of differences between the two trials were analyzed via paired-samples t test using Microsoft Excel if data were normally distributed and the Wilcoxon signed-rank test using StatView-J Version 5.0 software if not. Statistical significance was accepted at p < .05.
Results Cardiorespiratory Responses Cardiorespiratory responses after D-mix administration are summarized in Table 2. Two-factor ANOVA revealed significant main effects of time and no significant interaction. However, heart rate during exercise was numerically lower in the D-mix condition than in the placebo condition (p = .057). However, on examination respiratory
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D-mix Stimulates Fat Metabolism During Exercise 49
Table 2 Cardiorespiratory Responses of Participants During Rest, Exercise, and Recovery After consuming the Treatment Rest (0–30 min; M ± SD)
Exercise (>30–90 min; M ± SD)
Recovery (>90–150 min; M ± SD)
Response
D-mix
Placebo
D-mix
Placebo
D-mix
Placebo
p(EX)a
HR (bpm)
66 ± 8
69 ± 7
125 ± 11
128 ± 9
74 ± 8
76 ± 9
.057
RQ
0.90 ± 0.03
0.91 ± 0.04
0.96 ± 0.02
0.97 ± 0.03
0.87 ± 0.06
0.87 ± 0.05
.49
ΔRQ
-0.01 ± 0.03
0.00 ± 0.02
0.04 ± 0.03
0.06 ± 0.04
-0.04 ± 0.05
-0.01 ± 0.05
.11
Note. n = 10. bpm = beats per minute; HR = heart rate; RQ = respiratory quotient; ΔRQ = change in RQ. ap (EX) is the p value based on paired-sample t test for exercise data only.
quotient (RQ) and change in RQ (ΔRQ) did not differ between the two conditions.
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Biochemical Parameters Biochemical parameters after D-mix administration are shown in Figure 2, and maximum concentrations of each parameter measured during rest, exercise, and recovery are summarized in Table 3. Glycerol and FFAs are metabolites of fat, and acetoacetic acid and 3-hydroxybutyrate are ketone metabolites of FFAs. A two-factor ANOVA revealed significant main effects of time, and no significant interaction. However, the maximum concentrations of glycerol, FFAs, and ketone metabolites were significantly higher in the D-mix condition than in the placebo condition during exercise (p = .028 for glycerol; p = .022 for FFAs; p = .049 for 3-hydroxybutyrate; p = .040 for acetoacetic acid; Table 3). However, blood glucose and lactate measurements did not differ between the two conditions.
Circulating Hormones Circulating hormones after D-mix administration are shown in Figure 3, and the area under the curve (AUC) for each condition during rest, exercise, and recovery is summarized in Table 4. A two-factor ANOVA revealed significant main effects of time, and no significant interaction. However, the AUC for cortisol was significantly lower for the D-mix condition than for the placebo condition (p = .049), and the AUC for glucagon during recovery was numerically higher for the D-mix condition than for the placebo condition (p = .099). All other parameters for circulating hormones did not significantly differ between the two treatment conditions.
Discussion This randomized, double-blind, placebo-controlled crossover trial was conducted to assess the effects of D-mix during exercise. We conducted a two-factor ANOVA to compare the responses between the groups described in Figures 2 and 3. Overall significant differences were not observed between the groups.
We also divided the experiment into three time phases. Because we hypothesized that AAs may stimulate fat metabolism during exercise, it was necessary to carry out an independent analysis of the exercise phase. As a result, we found that during exercise ingestion of D-mix significantly elevated the maximum concentration of glycerol, FFAs, and ketone bodies compared with a placebo condition. These results suggested that fat metabolism increased after the ingestion of D-mix. Although there was a change in fat metabolites (glycerol, FFAs, ketone bodies), there was no effect on substrate metabolism as measured by RQ. However, RQ is just one indicator of progressing lipid oxidation in muscle. Thus, it is necessary to conduct further research on how D-mix affects lipid oxidation in muscle. It is well established that catecholamines such play an important role in the stimulation of lipolysis in human adipose tissue. Increased catecholamine concentrations in the blood stimulate cyclic adenosine monophosphate and hormone-sensitive lipase activity in skeletal muscle and in adipose tissue. Indeed, caffeine, which stimulates adrenalin secretion, is known to increase hormone-sensitive lipase activity and increase fat mobilization from skeletal muscles and adipose tissue, resulting in increased FFAs in the bloodstream (Ryu et al., 2001). Moreover, it has been shown that oral administration of an AA mixture derived from hornet larval saliva increases the concentrations of catecholamines in the blood, especially adrenaline (Uchida et al., 2008). In this study, concentrations of adrenaline and noradrenaline did not differ between the D-mix and placebo conditions. These results suggest that D-mix promoted lipid metabolism in an adrenaline-independent manner. There are also AAs that directly stimulate the sympathetic nerves. Yoshimatsu et al. (2002) reported that histidine accelerated lipolysis in white adipose tissue through activation of the sympathetic nerves. However, D-mix contains only 0.43% of histidine, so it is unlikely that lipid metabolism could be increased by stimulation of the sympathetic nervous system. Arginine accounts for 40% of the D-mix mixture. Arginine is known to stimulate the release of growth hormone (Collier et al., 2005), a molecule that has been suggested to increase fat oxidation. Hence, growth hormone may play a role in D-mix–associated fat metabolism. In this study, growth hormone concentrations did
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Figure 2 — Concentrations of biochemical assessed during the experimental trials: plasma glycerol (A), FFAs (B), 3-hydroxybutyrate (C), acetoacetic acid (D), glucose (E), and lactate (F). Values are M ± SD (n = 10). A = D-mix treatment; FFAs = free fatty acids; P = placebo treatment.
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D-mix Stimulates Fat Metabolism During Exercise 51
Table 3 Effects of Amino Acid Mixture on the Maximum Concentration of Biochemical Parameters Rest (0–30 min; M ± SD)
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Parameter
Exercise (>30–90 min; M ± SD) D-mix
Recovery (>90–150 min; M ± SD)
D-mix
Placebo
pa
Placebo
pa
D-mix
Placebo
pa
Glycerol (mg/L)
4.16 ± 5.78
3.44 ± 3.12
.70
9.32 ± 6.29
5.22 ± 2.22
.028
3.07 ± 1.94
2.69 ± 1.21
.63
FFAs (mEq/L)
0.42 ± 0.34
0.34 ± 0.16
.88
0.77 ± 0.26
0.63 ± 0.28
.022
0.64 ± 0.31
0.57 ± 0.32
.48
3-hydroxybutyrate (μmol/L)
20.5 ± 8.6
18.9 ± 6.2
.47
54.2 ± 47.2
34.8 ± 23.1
.049
163 ± 190
85.8 ± 95.1
.16
Acetoacetic acid (μmol/L)
27.9 ± 8.6
29.1 ± 11.0
.65
37.9 ± 17.7
30.3 ± 13.9
.040
83.9 ± 68.3
51.3 ± 30.8
.093
Glucose (mg/L)
1,040 ± 278
1,110 ± 181
.45
810 ± 89
835 ± 115
.41
870 ± 194
949 ± 216
.29
Lactate (mg/L)
118 ± 28.3
116 ± 17.3
.84
192 ± 54.1
198 ± 56.9
.56
97.4 ± 30.5
100.2 ± 24.2
.82
Note. n = 10. FFA—free fatty acid. ap value for the paired-sample t test if data were normally distributed and the Wilcoxon signed-rank test if not.
not differ between the D-mix and placebo conditions. This finding suggests that D-mix promoted fat metabolism via a growth hormone–independent mechanism. Ishidori et al. (1981) reported that oral ingestion of a mixture of arginine and lysine stimulated the secretion of insulin and growth hormone, but ingestion of either AA alone did not elicit the same effect. Hence, the stimulation of insulin and growth hormone by arginine may be affected by the composition of AAs to which it is added. The AUC for glucagon was numerically higher in the active group than the placebo group during recovery. These results suggest that ingestion of D-mix may have promoted the secretion of glucagon. It has been reported that ingestion or infusion of some AAs can lead to increased insulin or glucagon concentrations in the blood. For example, in a study by Nuttall et al. (2006), ingestion of phenylalanine increased both insulin and glucagon concentrations. Intravenously administered arginine and lysine have been shown to increase the circulating concentration of glucagon (Gerich et al., 1976). Alanine and glycine, but not valine, isoleucine, and leucine, have been observed to stimulate glucagon secretion (Gannon et al., 2002; Malaisse-Lagae et al., 1984; Müller et al., 1975). Therefore, a specific combination of AAs may stimulate the secretion of glucagon. D-mix is composed of arginine (41%), alanine (19%), phenylalanine (16%), and a lesser percentage of valine (0.74%), isoleucine (0.64%), and leucine (0.87%). Thus, we suggest that depending on the combination of AAs, such as those implicated in the secretion of glucagon, lipid metabolism may potentially be able to increase. However, there is a time lag between the increase of glycerol concentration and that of glucagon, which indicates that only the action of glucagon does not factor into lipolysis. Further study of the mechanism by which D-mix promotes lipid metabolism is necessary.
The concentration of glucose in the blood decreased and lactate concentrations increased from resting to the initiation of exercise. Although AAs cannot supply as much cellular energy as glucose and FFAs, the gluconeogenic process is an important pathway for utilization of AAs for energy. In the liver, the rate of gluconeogenesis from alanine is much higher than the rate from any other AA (Hood & Terjung, 1990; Wasserman et al., 1988). Alanine forms 19% of the D-mix concentration, and on this basis D-mix would be expected to cause a greater increase in circulating glucose than placebo. However, the concentration of glucose in the blood did not differ between the D-mix and placebo conditions. The administration of D-mix may have had an effect on gluconeogenesis in the liver, but this remains to be investigated. The AUC for cortisol during exercise was significantly lower in the D-mix condition than in the placebo condition. Cortisol is known to be elevated by acute stress, which can stimulate adrenocorticotropic hormone secretion (Levine, 1993; Oberbeck et al., 1998). In this study, although blood cortisol concentrations were suppressed, lipid metabolism increased after D-mix ingestion. However, it is unlikely that lipid metabolism increased via stimulation of cortisol secretion. Hainer et al. (1979) reported that intramuscularly administered glucagon decreased serum cortisol concentration, and this drop was not associated with the depressive effect of elevated growth hormone. The administration of D-mix may have had an effect on the decrease in cortisol concentration via promotion of glucagon secretion, but this remains to be confirmed. In conclusion, our results suggest that preexercise ingestion of D-mix stimulated fat metabolism. Combined with exercise, the administration of AA mixtures may
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Figure 3 — Concentrations of circulating hormones during the experimental trials: plasma glucagon (A), insulin (B), adrenaline (C), noradrenaline (D), growth hormone (E), and cortisol (F). Values are M ± SD (n = 10). A = D-mix treatment; P—placebo treatment
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D-mix Stimulates Fat Metabolism During Exercise 53
Table 4 Effects of Amino Acid Mixture on the Area Under the Curve for Circulating Hormones Rest (0–30 min; M ± SD) D-mix
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Glucagon (μg/L.min)
Placebo
2.90 ± 0.554 2.86 ± 0.570
Exercise (>30–90 min; M ± SD)
Recovery (>90–150 min; M ± SD)
pa
D-mix
Placebo
pa
D-mix
Placebo
pa
.88
6.60 ± 1.24
6.33 ± 1.21
.20
6.61 ± 1.33
6.06 ± 1.23
.099
Insulin (mU/L.min)
478 ± 348
590 ± 376
.46
300 ± 190
379 ± 181
.22
323 ± 322
481 ± 430
.33
Adrenaline (μg/L.min)
1.31 ± 0.79
1.41 ± 0.92
.55
6.68 ± 3.02
6.29 ± 2.59
.64
4.31 ± 2.02
3.66 ± 2.02
.36
Noradrenaline (μg/L-min)
8.16 ± 1.89
9.62 ± 3.37
.34
45.3 ± 8.94
44.9 ± 7.25
.87
27.9 ± 6.48
25.1 ± 6.81
.46
Growth hormone (μg/L.min)
55.5 ± 75.0
61.5 ± 113
.88
331 ± 302
359 ± 423
.86
188 ± 89.0
205 ± 217
.39
Cortisol (mg/L.min)
3.48 ± 0.85
3.71 ± 1.19
.33
5.87 ± 1.34
6.72 ± 2.12
.049
4.58 ± 0.85
5.04 ± 1.45
.20
Note. n = 10. ap value for the paired-sample t test if data were normally distributed and the Wilcoxon signed-rank test if not.
prove to be a useful nutritional strategy to maximize fat metabolism. On the basis of these preliminary findings, future studies of AA mixtures containing arginine, phenylalanine, and alanine appear to be warranted. A prominent limitation of this study is that we monitored only 10 subjects (all men). Therefore, future studies such as clinical trials in humans with no exercise habit are necessary. Acknowledgments All authors approved the final version of the article. We thank Ms. Shoko Takai for her technical analysis, Dr. Chiaki Sanbongi for his advice and valuable discussion during the development of this article, and Dr. Hiroyuki Itoh for providing encouragement throughout the study. The study was designed by KU, YN, MY, TM, MU, and SF; data were collected and analyzed by KU and YN; data interpretation and manuscript preparation were undertaken by KU, MY, and SF. KU, MY, TM, and MU are employees of Meiji Co.
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