Effects of dietary protein contents and habitual endurance exercise on supplemental leucine oxidation in mice

ABSTRACT

The effects of dietary protein contents and regular exercise on the oxidation of supplemented leucine were examined. In the short-term study, male BALB/cCrSlc mice were fed diets containing 0, 10, 20, 35, and 60% protein: energy ratios for 1 week. In the long-term study, exercised and sedentary mice were fed diets containing 20, 35, and 60% protein ratios for 9 weeks. After the feeding periods, the mice were a bolus administered oral supplements of l-[1-¹³C] leucine. Expired gas was analyzed, and oxidized leucine was expressed as a relative ¹³CO₂/¹²CO₂ ratio. In the short-term study, the peak ¹³CO₂/¹²CO₂ ratio significantly increased with diet protein concentrations. Moreover, the long-term study also showed that the peak ¹³CO₂/¹²CO₂ ratio was significantly increased by high protein diets in both exercised and sedentary mice. Our results indicate that supplemental leucine oxidation is associated with consumption of a high-protein diet, irrespective of exercise status.

Abbreviations: AUC: area under the curve; EX: exercise; RQ: respiratory quotient; SED: sedentary; VO₂/W: oxygen uptake per body weight

Graphical Abstract

The peak leucine oxidation increases with diet protein concentrations in short- and long-term study, irrespective of exercise status.

KEYWORDS:

• Leucine supplementation
• leucine oxidation
• high-protein diet
• endurance exercise
• mass spectrometric analysis

Leucine is a branched-chain amino acid, which is initially metabolized by muscle enzymes [1,2]. Leucine plays a regulatory role in muscle protein synthesis, which has been shown to activate the mammalian target of rapamycin (mTOR) pathway [3–5]. Although not enough evidence has been accumulated to conclude that leucine supplementation is effective in muscles of athletes, previous studies suggest that taking leucine partly promotes muscle recovery in injured athletes [6] and attenuates muscle membrane disruption in cyclists [7]. Moreover, two meta-analyses documented that leucine supplementation has beneficial effects on muscle mass in older individuals [8,9]. Therefore, leucine supplementation is a potential approach to attenuate skeletal muscle degradation.

Previous studies support a recommendation of leucine supplementation, especially after exercise [10], whereas how nutritional factors are associated with supplemental leucine metabolism has not been well-examined, either with or without exercise. Excess protein intake is reported to increase nitrogen excretion, suggesting that excess amino acids are metabolized as energy sources [11,12]. Although associations between dietary protein content and supplemental leucine oxidation have not been well examined, it was also reported that intravenous, whole body leucine oxidation was elevated by high-protein diets in neutered cats [13] and healthy adult men [14]. Therefore, it is likely that nutritional factors, especially protein intake, may influence supplemental leucine metabolism.

In addition to dietary conditions affecting leucine metabolism, the rate of leucine oxidation is increased during endurance exercise [15,16], and endurance athletes show high levels of leucine oxidation at rest [17]. Because these results suggest that endurance exercise induces whole leucine wastage, it is likely that habitual endurance exercise increases the need for leucine supplementation. Indeed, previous investigations have recommended high protein intake in endurance athletes [18,19]. Their results suggest the importance of leucine supplementation in habitual endurance exercise, even during rest; however, combined effects of dietary protein contents and endurance exercise on oral leucine oxidation have not been investigated previously.

We hypothesized that supplemental leucine oxidation is associated with dietary protein contents and endurance exercise. In the present study, we examined the effects of different protein contents and regular exercise on the oxidation of orally supplemented leucine in adult mice. The results provide evidence whether leucine supplementation is required, depending on dietary protein contents and habitual endurance exercise.

Materials and methods

Animals

Sixty-nine male wild-type BALB/cCrSlc mice (age, 8 weeks) were obtained from Shimizu Laboratory Animal Supply Co., Ltd (Kyoto, Japan). The mice were housed under controlled conditions of constant temperature (21°C–25°C), relative humidity (45%–65%), and light/dark cycle (12/12 h). The mice were exposed to light from 00:00 to 12:00 h, and exposed to dark from 12:00 to 00:00 h. The mice were supplied water ad libitum. This study was approved by the ethics committee of Ryukoku University.

Short-term study

After acclimatization for 1 week, the mice were divided into different protein diet groups. All pellet diets (D11080501, 10% protein: energy ratio; D12450B, 20% protein: energy ratio; D05060901, 35% protein: energy ratio; and D04080301, 60% protein: energy ratio), except the 0% protein pellet diet, were purchased from Research Diets Inc. (NJ, USA). The protein sources in the diets were casein and L-cystine (98.5 and 1.5%, respectively). Nutrient ingredient and composition of experimental diets were shown in Table 1, and the number of mice in each group was 7–15 (Table 2). Mice receiving nutrient-matched diet containing the 0% protein: energy ratio were fed AIN-93G-based diet (Oriental Yeast Co., Ltd., Chiba, Japan) containing corn starch instead of milk casein and cystine. The 0% protein diet was 10% for fat and 90% carbohydrates energy ratio, composed of 553 g; corn starch, 350 g; sucrose, 25 g; soybean oil, 20 g; lard, 50 g; cellulose, 35 g; AIN-93G mineral mix, 10 g; AIN-93 vitamin mix, 2 g; choline bitartrate. The mice were fed each diet ad libitum for 1 week prior to the metabolic study.

Table 1. Dietary composition.

Product number	D11080501		D12450B		D05060901		D04080301
	g%	kcal%	g%	kcal%	g%	kcal%	g%	kcal%
Protein	10.0	10	19.2	20	33.6	35	57.7	60
Carbohydrate	76.5	80	67.3	70	52.9	55	28.8	30
Fat	4.3	10	4.3	10	4.3	10	4.3	10
Total	90.8	100	90.8	100	90.8	100	90.8	100
kcal/g	3.85		3.85		3.85		3.85
Ingredient	g	kcal	g	kcal	g	kcal	g	kcal
Casein	100	400	200	800	350	1400	600	2400
L-Cystine	1.5	6	3.0	12	4.5	18	9.0	36
Corn Starch	516.5	2066	315	1260	244	976	112	448
Maltodextrin 10	35	140	35	140	29.5	118	35	140
Sucrose	250	1000	350	1400	275	1100	147	588
Cellulose, BW200	50	0	50	0	50	0	50	0
Soybean Oil	25	225	25	225	25	225	25	225
Lard	20	180	20	180	20	180	20	180
Mineral Mix S10026	10	0	10	0	10	0	10	0
DiCalcium Phosphate	13	0	13	0	13	0	13	0
Calcium Carbonate	5.5	0	5.5	0	5.5	0	5.5	0
Potassium Citrate, 1 H2O	16.5	0	16.5	0	16.5	0	16.5	0
Vitamin Mix V10001	10	40	10	40	10	40	10	40
Choline Bitartrate	2	0	2	0	2	0	2	0
Total	1055	4057	1055	4057	1055	4057	1055	4057

Table 2. Dietary composition, body weight and expired gas analysis in sedentary mice fed the diets for 1 week.

	0%	10%	20%	35%	60%
N	7	9	15	15	14
Dietary composition
Protein, %kcal	0	10	20	35	60
Fat, %kcal	10	10	10	10	10
Carbohydrate, %kcal	90	80	70	55	30
kcal/g	3.84	3.85	3.85	3.85	3.85
Body weight and dietary intake
Pre body weight, g	22.9 ± 0.7	24.2 ± 0.4	24.2 ± 0.2	24.2 ± 0.3	24.2 ± 0.2
Post body weight, g	19.2 ± 0.6*	24.4 ± 0.3	24.5 ± 0.2	24.2 ± 0.3	23.6 ± 0.3
Dietary intake, g/day	1.99 ± 0.11	2.37 ± 0.11	2.04 ± 0.12	2.18 ± 0.07	2.01 ± 0.01
^13CO2/^12CO2 ratio, ^13CO2/^12CO2 AUC, RQ and VO2 before and during 3 h metabolic trial
Baseline ^13CO2/^12CO2 ratio, %	1.00 ± 0.01	0.99 ± 0.01	0.97 ± 0.01	0.96 ± 0.01	0.95 ± 0.01
Relative ^13CO2/^12CO2 AUC ratio during trial, Fold change/180 min	42.0 ± 1.1	50.4 ± 3.1	53.1 ± 3.8**	55.5 ± 3.8**	60.6 ± 3.8**
Baseline RQ	0.73 ± 0.00	0.73 ± 0.00	0.73 ± 0.00	0.73 ± 0.00	0.73 ± 0.00
RQ during trial	0.72 ± 0.00	0.71 ± 0.00	0.71 ± 0.00	0.71 ± 0.00	0.71 ± 0.00
Baseline VO2/W, mL/min/kg	77.6 ± 1.7*	62.3 ± 1.4	60.9 ± 1.5	63.4 ± 1.6	62.8 ± 1.4
VO2/W during trial, mL/min/kg	66.4 ± 1.3*	54.8 ± 1.3	53.2 ± 0.9	57.5 ± 1.4	58.7 ± 1.4

Data are the mean ± SE. * indicates P < 0.05 compared with other groups. ** indicates P < 0.05 compared with 0% group. AUC; area under the curve, RQ; respiratory quotient, VO2/W; oxygen uptake per body weight.

Long-term study

Because the 140 g casein/kg diet (12% protein) was recommended in previous research [20], it is considered that mice fed 0 and 10% protein diets show dietary protein insufficiency, resulting in severe weight loss caused by protein catabolism throughout the long-term study. Therefore, mice fed 20, 35, and 60% protein diets were used for the long-term study which followed the short-term study. First, mice fed each diet were divided into exercise (EX) or sedentary (SED) groups (n = 7–11; Table 3). In the first week of the study, mice in the EX group were exercised on a treadmill (40% incline, 10 m/min, and 30 min/day; Mousebelt-2000; ARCO System, Chiba, Japan) two times. From the second week, the mice were exercised on a treadmill with high intensity for 7 weeks, and the exercise program comprised five exercise sessions per week. The mice exercised on the treadmill with a non-skid belt, set at a 40% incline; the treadmill speed was maintained at 5 m/min for the first 5 min and was increased to 10 m/min for the next 5 min. After 10 min, the speed was increased by 1 m/min every 1 min until the mice were exhausted [21].

Table 3. Body weight, organ weight, expired gas analysis in sedentary and trained mice fed the diets for 9 week.

							Exercise	Diet	Interaction
	SED 20%	SED 35%	SED 60%	EX 20%	EX 35%	EX 60%	P	P	P
N	7	7	7	11	11	10
Body and organ weights
Pre body weight, g	25.7 ± 0.2	25.8 ± 0.5	25.1 ± 0.3	25.3 ± 0.2	25.6 ± 0.3	24.8 ± 0.4	0.324	0.090	0.976
Post body weight, g	28.0 ± 0.4	28.2 ± 0.6	27.6 ± 0.5	30.1 ± 0.4	30.4 ± 0.3	29.8 ± 0.4	< 0.001	0.403	0.996
Liver, g	1.10 ± 0.03	1.09 ± 0.03	1.19 ± 0.03	1.28 ± 0.05	1.21 ± 0.03	1.31 ± 0.03	< 0.001	0.053	0.730
Kidney, g	0.49 ± 0.01	0.54 ± 0.02	0.60 ± 0.03	0.58 ± 0.03	0.65 ± 0.02	0.69 ± 0.03	< 0.001	0.002	0.936
Epididymal fat, g	0.23 ± 0.04	0.27 ± 0.03	0.17 ± 0.03	0.18 ± 0.01	0.17 ± 0.01	0.17 ± 0.02	0.006	0.035	0.074
Gastrocnemius muscle, mg	298 ± 2	285 ± 8	278 ± 10	315 ± 6	311 ± 5	302 ± 5	< 0.001	0.040	0.681
Plantaris muscle, mg	37.2 ± 1.1	36.2 ± 0.8	37.8 ± 0.8	41.9 ± 1.3	46.6 ± 2.1	43.7 ± 1.4	< 0.001	0.468	0.167
Soleus muscle, mg	16.4 ± 0.4	15.3 ± 1.1	16.0 ± 1.3	18.3 ± 0.5	18.6 ± 0.5	17.6 ± 0.8	< 0.001	0.769	0.505
Dietary intake, g/day	2.83 ± 0.10	3.05 ± 0.17	2.64 ± 0.22	2.97 ± 0.06	2.87 ± 0.05	2.89 ± 0.08	0.282	0.551	0.530
Running time, min/day				30.7 ± 0.4	30.4 ± 0.4	28.8 ± 0.7*
^13CO2/^12CO2 ratio, ^13CO2/^12CO2 AUC, RQ and VO2 before and during 3 h metabolic trial
Baseline ^13CO2/^12CO2 ratio, %	1.03 ± 0.00	1.03 ± 0.01	1.03 ± 0.00	1.04 ± 0.01	1.01 ± 0.01	1.01 ± 0.01	0.310	0.173	0.146
Relative ^13CO2/^12CO2 AUC ratio during trial, Fold change/180 min	50.8 ± 3.1	48.6 ± 4.9	49.3 ± 3.7	47.7 ± 1.9	50.3 ± 2.4	53.3 ± 2.8	0.733	0.769	0.523
Baseline RQ	0.75 ± 0.00	0.74 ± 0.00	0.74 ± 0.00	0.74 ± 0.00	0.75 ± 0.00	0.75 ± 0.00	0.228	0.758	0.006
RQ during trial	0.72 ± 0.00	0.72 ± 0.00	0.72 ± 0.00	0.72 ± 0.00	0.73 ± 0.00	0.73 ± 0.00	0.002	0.356	0.245
Baseline VO2/W, mL/min/kg	57.4 ± 2.6	61.7 ± 2.8	62.3 ± 1.9	58.1 ± 2.7	53.9 ± 1.2	54.7 ± 1.5	0.008	0.918	0.094
VO2/W during trial, mL/min/kg	48.9 ± 3.2	49.5 ± 1.5	56.9 ± 1.4	51.4 ± 2.7	48.8 ± 2.3	51.1 ± 2.8	0.538	0.165	0.307

Data are the mean ± SE. Asterisk indicates P < 0.05 among exercise group. AUC; area under the curve, EX; exercise group, RQ; respiratory quotient, SED; sedentary group, VO2/W; oxygen uptake per body weight.

Metabolic study

In both short- and long-term study, the mice were placed in metabolic cages at 9:00 h after fasting for 2 h. Their expired gas was analyzed using a CO₂ and O₂ mass-spectrometric analyzer (ARCO-2000; ARCO System). The gas analysis system has been previously described in detail [22–24]. In the long-term study, the metabolic measurements were performed at least 2 days after the previous exercise session to exclude the acute effects of exercise. After performing baseline measurements for 3 h, the mice were orally administered a single dose (0.8 mg/g body weight) of l-[1-¹³C] leucine (99 atom percent excess; Cambridge Isotope Laboratories, MA, USA) with water solution. Oxidation of the orally administered l-[1-¹³C] leucine was assessed based on the relative abundance of ¹³CO₂ (¹³CO₂/¹²CO₂ ratio). The administered leucine (0.8 mg/g body weight) was able to discriminate from the baseline ¹³CO₂/¹²CO₂ ratio. CO₂ and O₂ masses were analyzed for 3 h under fasting condition after l-[1-¹³C] leucine administration. After refeeding for 1 h, the CO₂ and O₂ mass were analyzed for 24 h with consumption of the indicated diets. Thus, the metabolic study was successively performed over 28 h. Respiratory quotient (RQ) was calculated from the expired volumes of CO₂ and O₂ (VCO₂ and VO₂, respectively) by using the formula VCO₂/VO₂, and oxygen uptake was expressed per body weight (VO₂/W).

Statistical analysis

All statistical analyses were performed using SPSS version 23.0 (SPSS Inc., IL, USA). Two-way repeated ANOVA (time × group) was used to examine differences in ¹³CO₂/¹²CO₂ ratio, VO₂/W, and RQ during metabolic measurements. Another two-way ANOVA (exercise × diet) was used to assess the main effects of variables on exercise and diet conditions in long-term study. A post hoc Tukey-Kramer’s test was used to identify significant differences among mean values when a significant main effect or interaction was identified. One-way ANOVA was used to assess differences in other variables. All measurements are presented as mean ± standard error. P < 0.05 was considered statistically significant.

Results

Effects of 1 week feeding different protein diets on supplemental leucine oxidation

Body weight between the groups was not different at the beginning of the study. After feeding for 1 week, body weights of mice fed the 0% protein diet were significantly lower than those of mice fed the other diets (P < 0.05; Table 2). However, no significant differences were observed in the body weights of mice fed the other protein diets for 1 week.

We observed that the 3-h baseline measurement values of the ¹³CO₂/¹²CO₂ ratio and RQ were not different among mice fed the different diets. Baseline oxygen uptake in mice fed the 0% protein diet was significantly higher than that of mice fed the other diets (P < 0.05; Table 2). The ¹³CO₂/¹²CO₂ ratio peaked at 30 min after supplementation of l-[1-¹³C] leucine, and returned to baseline at 90 min after the oral administration of l-[1-¹³C] leucine in mice in all the groups (Figure 1). Results of the two-way repeated ANOVA showed a significant higher ¹³CO₂/¹²CO₂ ratio for mice fed 60% protein diets than for mice fed 0% protein diets (P < 0.05). In addition, the peak ¹³CO₂/¹²CO₂ ratio was significantly higher for mice fed the 60% protein diet than for mice fed the 0, 10, and 20% protein diets (P < 0.05). The AUC of the ¹³CO₂/¹²CO₂ ratio was significantly higher for mice fed 35% or 60% protein diets than for mice fed the 0% protein diet (P < 0.05; Table 2).

Figure 1. Effects of the consumption of different protein diets for 1 week on oral leucine oxidation in mice; * indicates a significant difference (P < 0.05) compared with 0, 10, 20% protein diet groups.

Expired gas was monitored for 24 h without fasting (Figure 2). Results of the two-way repeated ANOVA with Tukey-Kramer’s test showed that the 24-h RQ was significantly lower in mice fed the 60% protein diet than in mice fed the other diets (P < 0.05). Moreover, the 24-h RQ was significantly lower in mice fed the 35% protein diet than in mice fed the 10 and 20% protein diets (P < 0.05). Mice fed the 0% protein diet showed significantly higher 24-h VO₂/W than mice fed the other diets (P < 0.05).

Figure 2. The 24-h RQ and VO₂/W in the short-term study; * indicates a significant difference (P < 0.05) compared with other groups, and ** indicates a significant difference (P < 0.05) compared with the 10 and 20% protein diet groups. RQ, respiratory quotient; VO₂/W, oxygen uptake per body weight.

Characteristics of exercised and sedentary mice fed different protein diets for 9 weeks

As shown in Table 3, average exercise time was significantly longer for EX mice fed the 20% protein diet than for EX mice fed the 60% protein diet (P < 0.05). After 9 weeks, the body weights of EX mice were significantly higher than those of SED mice (P < 0.05). The absolute weights of the soleus, plantaris, and gastrocnemius muscles were significantly higher in EX mice than in SED mice (P < 0.05). However, no significant differences in these skeletal muscles were observed in relative weight/100 g body weight between EX and SED mice. EX mice had lower absolute and relative epidydimal fat weights and higher absolute and relative kidney weights than SED mice (P < 0.05).

Effect of the 9-week intake of different protein diets on supplemental leucine oxidation in exercised and sedentary mice

Baseline measurements showed no significant differences in ¹³CO₂/¹²CO₂ ratios, and RQ between EX and SED mice (Table 3). Baseline VO₂/W was significantly higher in SED mice that than in EX mice (P < 0.05), but there was no significant difference in VO₂/W during 3 h after supplementation with l-[1-¹³C] leucine. RQ during the trial after leucine supplementation was slightly but significantly higher in the EX mice (P < 0.05).

Consistent with the results of the short-term study, the ¹³CO₂/¹²CO₂ ratio peaked at 30 min after oral l-[1-¹³C] leucine administration in both SED and EX mice (Figure 3). Results of two-way ANOVA for the peak ¹³CO₂/¹²CO₂ ratio showed a significant main effect of dietary protein concentration (P < 0.05), but not of exercise (P = 0.525). The post hoc test revealed that the peak ¹³CO₂/¹²CO₂ ratio was significantly higher in mice fed the 60% protein diet than in mice fed the 20% protein diet (P < 0.05).

Figure 3. Effects of the consumption of different protein diets and exercise for 9 weeks on oral leucine oxidation in mice; * indicate a significant different (P < 0.05) between the 20 and 60% protein diet groups. EX, exercise group; SED, sedentary group.

We next monitored 24-h expired gas, and lower 24-h RQ was obtained for both SED and EX mice fed the 60% protein diet compared with that for mice fed the other diets (P < 0.05; Figure 4). EX mice fed the 35% protein diet had lower 24-h RQ than both SED and EX mice fed the 20% protein diet (P < 0.05; Figure 4). The 24-h VO₂/W did not differ among the groups, and no significant differences in 24-h RQ and VO₂/W were observed between SED and EX mice fed the same protein diet.

Figure 4. The 24-h RQ and VO₂/W in the long-term study; * indicates a significant difference (P < 0.05) compared with mice fed the other protein diets, and ** indicates a significant difference (P < 0.05) compared with SED and EX mice fed the 20% protein diet. EX, exercise group; RQ, respiratory quotient; SED, sedentary group; VO₂/W, oxygen uptake per body weight.

Discussion

Our study examined the effects of different protein diets and endurance exercise on the metabolism of supplemental leucine in mice. Analysis of expired gas using a CO₂ and O₂ mass-spectrometric analyzer showed that peak leucine oxidation (peak ¹³CO₂/¹²CO₂ ratio) was associated with both short- and long-term dietary protein consumption. On the other hand, there was no significant difference in the peak leucine oxidation between EX and SED mice. These results suggest that supplemental leucine is rapidly metabolized as energy sources under enough protein intakes, and the peak oxidation was associated with dietary protein contents but not with endurance exercise.

Results of the short-term (1-week) study showed almost similar ¹³CO₂/¹²CO₂ ratio peaks in mice fed the 0 and 10% protein diets; however, the peak ¹³CO₂/¹²CO₂ ratio increased with an increase in other dietary protein concentration (Figure 1). Because the American Institute of Nutrition recommends that the diet of adult rodents should contain 12% protein concentration [20], the similar peak oxidation of leucine may be associated with insufficient protein intake in mice fed the 0 and 10% protein diets. Unlike the peak ¹³CO₂/¹²CO₂ ratio, the AUC of the ¹³CO₂/¹²CO₂ ratio was significantly lower in mice fed the 0% protein diet (Table 2). Therefore, the AUC, rather than the maximal oxidation capacity might be more correlated with a dietary protein deficit. In addition, because both the ¹³CO₂/¹²CO₂ peak and AUC increased with diet protein concentrations (Figure 1 and Table 2), the short-term study indicates that high protein diet induces leucine oxidation after a bolus of leucine intake, which may be related to supply above protein requirements.

In the short-term study, results of the 24-h metabolic measurements showed lower 24-h RQ for mice fed a low-carbohydrate and high-protein diet (Figure 2), which was consistent with that observed previously [25]. The results suggested that decreased glucose oxidation in these mice. Because no significant difference was observed in 24-h VO₂/W among mice fed 10–60% protein diets (Figure 2), the lower 24-h RQ was not be caused by increases in fat oxidation and energy expenditure. It is likely that the 24-h RQ is associated with protein oxidation as energy sources. In addition, the mice fed the 0% protein diet showed significantly higher 24-h VO₂/W than mice fed the other diets (Figure 2). This result was in concordance with the results of the baseline and trial VO₂/W in mice fed the 0% protein diet (Table 2). A study involving a rodent model of activity-based anorexia that received a limited amount of diet showed a significant increase in spontaneous exercise volume, which in turn decreased body weight [26]. Therefore, the increase in VO₂ consumption in mice fed the 0% protein diet might be because of poor nutritional status and/or weight reduction associated with higher physical activity in search of diet.

In the long-term study, no significant effects were observed in the baseline ¹³CO₂/¹²CO₂ ratio among all the groups, and thus the habitual dietary and exercise conditions did not influence the ¹³CO₂ levels. In addition, the long-term study showed that the peak ¹³CO₂/¹²CO₂ ratio after leucine supplementation increased with an increase in dietary protein concentration in both EX and SED mice (Figure 3), as seen in the short-term study. There was no significant difference in the peak ¹³CO₂/¹²CO₂ ratio between EX and SED mice. The EX mice showed higher weight gain, muscle mass, and fat loss than SED mice, suggesting that high-intensity treadmill exercise affects muscle anabolism in EX mice (Table 3). Although it has been reported that skeletal muscles metabolize branched chain amino acids, including leucine [2], the our results suggested that dietary protein contents had more influence on the peak leucine oxidation than changes in body composition.

In contrast, the long-term study showed that no significant association was observed between the AUC of the ¹³CO₂/¹²CO₂ ratio and dietary protein intake (Table 3). The results suggested that long-term intake of excess protein altered total leucine oxidation. Although the underlying mechanisms remain unclear, it was shown that a high protein diet induced gluconeogenesis in rodents and human [27–29]. Therefore, the lack of relationship between excess protein intake and supplemental leucine in the long-term study may be regulated by gluconeogenesis, resulting in the similar ¹³CO₂/¹²CO₂ AUCs among the groups. Although the relationship was not clear, slightly but significantly higher RQ after leucine supplementation was found in EX mice compared with SED mice (Table 3, average difference of 0.01, P < 0.05), suggesting that slightly more glucose might be metabolized during the 3 h after leucine supplementation in EX mice.

Twenty-four hours of metabolic chamber analysis revealed that the 24-h RQ and VO₂/W values obtained in the long-term study were similar to those obtained in the short-term study (Figure 4). The 24-h RQ was significantly lower in EX and SED mice fed high protein diets, and there was no significant difference in 24-h VO₂/W among the groups. In addition, the 24-h RQ and VO₂/W values were not significantly different between the EX and SED mice, suggesting that these values at rest were not affected by exercise status. It was reported that neither the 24-h RQ nor energy expenditure differed between male endurance athletes and untrained males [30]. These results suggested that, unlike after leucine supplementation, dietary protein contents have more influence than endurance exercise on 24-h RQ.

Our study has several limitations. First, because we sought to examine effects of endurance exercise and high protein diets on body composition and leucine supplementation, the EX mice performed high-intensity exercise, which induced muscle mass gain. Thus, we could not evaluate the relationship between low-intensity exercise and leucine supplementation. Second, this study used the experimental diets with varying ratio of protein and carbohydrate, whereas it reports that high-protein, low-carbohydrate diet increases plasma levels of glucoregulatory hormone such as cortisol [31]. Cortisol induces amino acid oxidation including leucine [32], and thus further research is needed to determine whether the effects of low-carbohydrate diet on gluconeogenic hormone alter the relationship between dietary protein intake and leucine oxidation. It was suggested that equal protein diets with varying ratios of fat and carbohydrate did not influence glucoregulatory hormones including cortisol, epinephrine, and norepinephrine [33]. Therefore, equal-protein, different carbohydrate diets may provide additional insight into relationship between dietary consumption and leucine oxidation. Third, the mice in the long-term study consumed sufficient dietary calories and protein. The results may be different from effects of leucine supplementation in humans who habitually consume lower energy and micronutrients intake, such as elderly individuals [34] and during weight loss, as well as those who require protein restriction, such as patients with kidney diseases [35]. Additional human studies are needed to examine the relationship between different protein intake and leucine metabolism.

In conclusion, peak leucine oxidation after leucine supplementation is associated with both short- and long-term protein intakes, and supplemental leucine oxidation does not correlate with exercise status and body composition. The results suggest that the peak leucine oxidation reflect differences in dietary protein contents irrespective of feeding duration and fitness level.

Author Contribution

HT, NA, and KI carried out animal experiments and measurements in the study. HT and KI participated in the design of the study. HT performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgments

This work was supported by a grant from the Kao Research Council for the Study of Healthcare Sciences (to HT). All authors declare that they do not have any conflicts of interest related to this study.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by research grant from Kao Research Council for the Study of Healthcare Sciences.