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Increase in insulin secretion and decrease in muscle degradation by fat-free milk intake are attenuated by physical exercise
Clinica Chimica Acta 484 (2018) 21–25
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Increase in insulin secretion and decrease in muscle degradation by fat-free milk intake are attenuated by physical exercise
T
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Tetsuo Yamadaa, , Masami Matsuzakia, Akira Tanakab a b
Department of Nutrition and Dietetics, Kanto Gakuin University, Yokohama, Japan Laboratory of Clinical Nutrition and Medicine, Kagawa Nutrition University, Sakado, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Fat-free milk Exercise C-peptide immunoreactivity Insulin 3-Methyl-histidine Catecholamine
Background: Protein intake, particularly branched chain amino acids (BCAAs), and exercise have opposing actions on insulin secretion, but the same action on protein anabolism. We examined the effects of BCAA-rich fatfree milk intake and/or exercise on levels of insulin secretion and indices related to muscle protein metabolism in order to assess the potency of dietary and exercise therapies against metabolic and locomotive disorders. Methods: Eight adult female volunteers participated in all four 24 h experiments; control diet intake with or without exercise, and fat-free milk-containing diet intake with or without exercise. Fat-free milk was replaced with one-sixth of all foods in the control diet. Exercise was set at an equal-energy level as fat-free milk. Urine and fasting blood samples were collected for each experiment. Results: Urinary C-peptide immunoreactivity excretion and serum insulin levels were significantly higher, but urinary 3-methyl-histidine excretion levels were significantly lower with low urinary adrenaline and dopamine excretion in the fat-free milk-containing diet than in the control diet. These findings were reduced by exercise with high urinary adrenaline and noradrenaline excretion. Conclusions: BCAA-rich fat-free milk intake enhanced insulin secretion and suppressed muscle protein degradation, but these effects are attenuated by exercise accompanied with increase in catecholamine secretion.
1. Introduction Metabolic syndrome has always been identified as a risk factor for coronary heart disease and stroke, which are closely linked to obesity and unhealthy lifestyle [1–3]. In contrast, the term “sarcopenia” was coined by Rosenberg in 1989 to denote the age-related decline in muscle mass and function [4,5], and recently, the concept of locomotive syndrome has been suggested in relation to bone disorder and sarcopenia [6,7]. Furthermore, sarcopenic obesity has been defined as a condition that encompasses sarcopenia and obesity [8,9]. Both nutrition and exercise are key factors involved in metabolic and locomotive syndrome [2,3,10,11]. Regarding metabolic syndrome, obesity, impaired glucose tolerance, hyperlipidemia and hypertension are frequently improved by energy-restricted diet and/or aerobic exercise, which directly promotes glucose and free fatty acid (FFA) consumption. During acute aerobic exercise, glucose transporter 4 increases glucose intake into muscle cells [12], serum FFA level increases [13], triglyceride (TG) level decreases [14] and lipoprotein lipase (LPL) activity is promoted [15]. The cumulative training effects of aerobic exercise are also understood [16,17]. Regarding locomotive syndrome, sufficient
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energy and adequate nutrients such as protein, n-3 polyunsaturated fatty acids, calcium and vitamin D, are required [9–11]. In addition, mechanically loaded and/or resistance exercise is typically used to increase bone mineral density and muscle mass [7,9–11], indirectly improving dysfunctional glucose and lipid metabolism. It is known that insulin lowers blood glucose and stimulates protein anabolism. While it is necessary to avoid excessive secretion of insulin from the viewpoint of metabolic syndrome, insulin is also considered to contribute to increases in muscle mass. Exercise reduces insulin secretion [18] and promotes body protein anabolism [19,20]. On the other hand, proteins, particularly branched chain amino acids (BCAAs), are utilized for energy [21], body protein maintenance [22] and anabolism [23], but simultaneously produce insulinotropic effects [24–26]. Thus, BCAA intake and exercise have opposing actions on insulin secretion, but the same action on protein metabolism. Therefore, it is significant to investigate each and combined effect of BCAA intake and exercise. In the present study, we examined the effects of BCAAs-rich fat-free milk intake or exercise, and the effects of exercise under fat-free milk intake on serum insulin, urinary C-peptide immunoreactivity (CPR) excretion and indices related to muscle protein metabolism in order to
Corresponding author at: 1-50-1 Mutsuura-higashi Kanazawa-ku, Yokohama 236-8503, Japan. E-mail address: [email protected] (T. Yamada).
https://doi.org/10.1016/j.cca.2018.05.017 Received 31 December 2017; Received in revised form 25 April 2018; Accepted 8 May 2018 Available online 09 May 2018 0009-8981/ © 2018 Elsevier B.V. All rights reserved.
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with exercise.
assess the potency of dietary and exercise therapies against metabolic and locomotive disorders.
2.2.3. Procedure of exercise Exercise tolerance was tested before starting the study by gradually increasing the work rate on a bicycle ergometer. Heart rate was recorded by telemetry (DS-3400; Fukuda Denshi Co., Ltd., Tokyo, Japan). Expired gas before and at steady state during exercise was gathered using a Douglas bag, and then gas volume was measured using a dry gas meter (DC-5A; Shinagawa Corporation, Tokyo, Japan). Expired oxygen and carbon dioxide concentrations were measured using an expired gas monitor (Portable Gas Monitor AR-1; Arco System Inc., Kashiwa, Japan). Relationships among additional energy expenditure calculated based on oxygen intake and the respiratory exchange ratio, work rate (kilopond of bicycle ergometer) and heart rate were determined for each individual. In the exercise experiment, participants expended 352 kcal by pedaling a bicycle ergometer at a target intensity of 40–50% of maximal oxygen intake for 101 ± 4 min split between the morning and afternoon. In the non-exercise experiment, the participants performed only normal daily activities.
2. Materials and methods 2.1. Subjects Eight healthy adult female volunteers [age, 22 ± 0 years; height, 159 ± 1 cm; weight, 52.8 ± 2.4 kg; mean ± standard error of the mean (SEM)] provided written, informed consent to participate in all procedures associated with the study, which proceeded according to the Declaration of Helsinki (1964; amended 2013). 2.2. Experimental procedure The Research Ethics Committee of Kanto Gakuin University approved the study. 2.2.1. Experimental protocol The study comprised four experiments. Eight subjects participated in all four experiments in random order; control diet intake, control diet intake with exercise, fat-free milk-containing diet intake and fat-free milk-containing diet intake with exercise. Each experiment was conducted at interval of one week or more. Participants were fed experimental diets in all four experiments, and exercised in two experiments under the control diet or fat-free milk-containing diet. In every experiment, the participants ate the prescribed supper on the previous evening and stayed two nights at the metabolic unit to complete each experiment.
2.2.4. Environmental conditions Room temperature during the experimental period was maintained between 25 and 26 °C on the dry-bulb thermometer and between 22 and 23 °C on the wet-bulb thermometer. 2.2.5. Sample collection and measurement Nitrogen contents of breakfast, lunch, supper and fat-free milk were determined by the Kjeldarl method. Urine samples were collected at 6:00–8:00 (baseline before each experiment), 8:00–14:00, 14:00–20:00 and 20:00–8:00 the following morning to measure creatinine [29], urea nitrogen (UN) [30], CPR (Chemiluminescent enzyme immunoassay, Lumipulse Presto C-peptide; Fujirebio Inc., Tokyo, Japan), 3-methylhistidine (3-MH) [31], adrenaline, noradrenaline and dopamine [32] levels. Fasting blood samples were collected early in the morning at the end of each experiment to measure blood glucose (BG) [33], serum immunoreactive insulin (IRI) [34], FFA [35], TG [36], remnant-like particle-cholesterol (RLP-C) [37], UN [30], insulin-like growth factor-1 (IGF-1: Immunoradiometric assay, IGF-1 (Somatomedin C) IRMA DAIICHI; Fujirebio Inc., Tokyo, Japan) and cortisol (Electro chemiluminedcence immunoassay, ECLusys reagent Cortisol II; Roche Diagnostics K. K., Tokyo, Japan) levels. Homeostasis model assessment for insulin resistance (HOMA-IR) was calculated by multiplying fasting BG (mg/dl) by IRI (μU/ml) by 1/405.
2.2.2. Experimental diet For control diet intake, the diet comprised the following foods; soft rolls, sausages (Vienna), ketchup, potato salad, vegetable juice, spaghetti, salted cod sauce, sweet corn (boiled), salted butter, pepper (mixed, ground), vitamin mixture beverage, custard pudding, European plums (dried), well-milled rice, soy protein processed food, miso (lightyellow type), kanze-fu, spinach (leaves, boiled) and soy sauce. The composition of the experimental diet was based on the recommended dietary allowance or adequate intake published in the Dietary Reference Intakes for Japanese [27]. The calculated values per day on the Standard Tables of Food Composition in Japan [28] were as follows: energy, 2100 kcal; protein, 63.8 g (BCAAs, 9.9 g); lipid, 67.8 g; carbohydrate, 302.2 g and the PFC ratio was 12.2:29.1:58.7. For fat-free milk-containing diet intake, the experimental diet was reduced to approximately five-sixths of control diet by reducing all foods in proportion to the energy intake, and fat-free milk was replaced with approximately one-sixth of control diet. Fat-free milk was given with every three meals and its volume was decided as the likely level in daily life, i.e., equivalent energy amount in 175 ml of ordinary milk per meal. The participants consumed experimental diets (energy, 2100 kcal; protein, 88.9 g (BCAAs, 15.3 g); lipid, 57.0 g; carbohydrate, 302.3 g and the PFC ratio was 17.4:24.5:58.2) that contained fat-free milk at 352 kcal. In control diet intake with exercise, no extra energy corresponding to addition energy expended during exercise was added. In fat-free milk-containing diet with exercise, the participants consumed control diet and 352 kcal of fat-free milk corresponding to extra energy (energy, 2452 kcal; protein, 99.6 g (BCAAs, 17.0 g); lipid, 68.4 g; carbohydrate, 352.9 g and the PFC ratio was 16.6:25.1:58.2). The calculated values of other amino acids (lysine, sulfur amino acids (methionine, cystine: SAAs), aromatic amino acids (phenylalanine, tyrosine: AAAs)) relatively relevant to protein intake in each experiment were as follows: lysine, 2.8 g; SAAs, 2.2 g; AAAs, 4.8 g in control diet intake and control diet intake with exercise, lysine, 4.9 g; SAAs, 2.9 g; AAAs, 7.2 g in fat-free milk-containing diet intake, lysine, 5.3 g; SAAs, 3.3 g; AAAs, 8.0 g in fat-free milk-containing diet intake
2.3. Statistical analysis Results are expressed as means ± SEM. All statistical analyses were performed using SPSS14.0 for Windows. We applied the Wilcoxon signed-ranks test to examine differences between control diet intake and control diet intake with exercise, fat-free milk-containing diet intake, fat-free milk-containing diet intake with exercise, and between fat-free milk-containing diet intake with or without exercise. Significance was established at p < 0.05 in all analyses. 3. Results There was no significant difference in the timing of the menstrual cycle in each experiment (when the whole menstrual cycle was taken as 100%, at 45 ± 10 percentile point, control diet intake; at 67 ± 11 percentile point, control diet intake with exercise; at 69 ± 10 percentile point, fat-free milk-containing diet intake; at 57 ± 9 percentile point, fat-free milk-containing diet intake with exercise). 22
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Table 1 Comparison of excretion levels of the urinary metabolic parameters in each experiment of control diet intake (Control), control diet intake with exercise (Control + Ex), fat-free milk-containing diet intake (Fat-free milk-containing) and fat-free milk-containing diet intake with exercise (Fat-free milk-containing + Ex). Variable Creatinine (mg) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Ex UN (mg) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Ex CPR (μg) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Ex 3-MH (μmol) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Ex Adrenaline (μg) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Ex Noradrenaline (μg) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Dopamine (μg) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Ex
6:00–8:00
8:00–14:00
14:00–20:00
20:00–8:00
8:00–20:00
8:00–8:00
90 91 92 91
316 314 287 292
265 262 268 267
528 530 504 515
581 576 555 559
1108 1106 1059 1074
± ± ± ±
63 49 40 52
± ± ± ±
5 4 4 3
± ± ± ±
18 21 12 12
± ± ± ±
23 22 20 27
± ± ± ±
28 19 20 21
± ± ± ±
38 35 26 36
535 625 627 660
± ± ± ±
51 30⁎ 58 31⁎
1695 1662 1839 2043
± ± ± ±
113 137 124 141⁎
1608 1643 2202 2247
± ± ± ±
150 182 148⁎ 232⁎
2787 3023 3504 3853
± ± ± ±
217 140 252⁎ 228⁎
3303 3305 4041 4290
± ± ± ±
236 263 175⁎ 262⁎
6090 6328 7545 8143
± ± ± ±
430 357 386⁎ 427⁎
1.64 2.11 2.22 2.00
± ± ± ±
0.23 0.32⁎ 0.23⁎ 0.24
27.05 21.54 32.85 28.70
± ± ± ±
4.27 2.30 1.98 3.64
28.93 21.15 37.03 28.61
± ± ± ±
2.64 2.31⁎ 3.64⁎ 4.17
26.03 23.60 29.80 28.30
± ± ± ±
2.13 2.16 2.77 3.08
55.98 42.69 69.88 57.31
± ± ± ±
6.03 3.15 4.16 6.92
82.00 66.28 99.68 85.61
± ± ± ±
7.74 4.86 6.66⁎ 7.81#
13.2 13.6 13.6 14.1
± ± ± ±
0.8 0.6 0.8 0.7
52.9 50.3 47.3 49.9
± ± ± ±
3.8 2.9 2.1 2.7
39.6 38.1 38.5 40.3
± ± ± ±
3.2 3.0 2.8 4.4
77.5 78.0 66.4 73.1
± ± ± ±
4.0 2.6 3.5⁎ 3.6
92.4 88.4 85.8 90.2
± ± ± ±
6.2 4.4 3.5 6.2
170.0 166.4 152.2 163.3
± ± ± ±
9.8 6.0 6.1⁎ 9.0
0.58 0.48 0.40 0.42
± ± ± ±
0.17 0.13 0.08 0.11
2.77 3.32 2.23 2.60
± ± ± ±
0.60 0.86 0.42 0.55
2.51 2.77 2.10 3.02
± ± ± ±
0.56 0.49 0.39 0.68#
2.79 3.21 2.10 2.58
± ± ± ±
0.54 0.58 0.48 0.52
5.28 6.09 4.34 5.62
± ± ± ±
1.14 1.30 0.79 1.22#
8.07 9.30 6.44 8.20
± ± ± ±
1.62 1.85 1.25⁎ 1.73#
6.3 6.4 5.3 5.7
± ± ± ±
0.9 0.6 0.6 0.6
27.5 32.3 22.5 26.2
± ± ± ±
3.5 5.5 2.8 2.6
23.5 29.6 20.9 28.1
± ± ± ±
3.3 4.8 2.3 4.5#
35.4 40.9 30.1 35.6
± ± ± ±
4.6 4.5 4.3 4.3#
51.0 61.9 43.4 54.4
± ± ± ±
6.4 9.8 4.6 7.1
86.4 ± 10.8 102.8 ± 14.2 73.5 ± 8.5 90.0 ± 11.2#
41 40 34 36
± ± ± ±
5 3 2 3
197 182 150 167
± ± ± ±
18 11 9⁎ 9
211 202 177 202
± ± ± ±
24 15 9 21
403 374 317 364
± ± ± ±
29 11 9⁎ 26
408 384 328 369
± ± ± ±
39 22 13⁎ 28
811 758 644 733
± ± ± ±
66 27 17⁎ 49
UN, urea nitrogen; CPR, C-peptide immunoreactivity; 3-MH, 3-methyl-histidine. ⁎ p < 0.05 compared with Control. # p < 0.05 compared with Fat-free milk-containing. Table 2 Comparison of the blood metabolic parameters in each experiment. Variable
Control
Control + Ex
Fat-free milk-containing
Fat-free milk-containing + Ex
BG (mg/dl) IRI (μU/ml) FFA (mEq/l) TG (mg/dl) RLP-C (mg/dl) UN (mg/dl) IGF-1 (ng/ml) Cortisol (μg/dl) HOMA-IR
85 ± 2 5.0 ± 0.8 0.41 ± 0.06 89 ± 15 3.8 ± 0.7 9.4 ± 0.5 234 ± 15 19.7 ± 2.9 1.07 ± 0.17
84 ± 2 5.3 ± 0.6 0.52 ± 0.04 75 ± 15 3.2 ± 0.4 11.1 ± 0.8⁎ 229 ± 16 17.6 ± 1.3 1.09 ± 0.12
85 ± 2 6.8 ± 0.9⁎ 0.47 ± 0.04 76 ± 9 3.4 ± 0.5 11.5 ± 0.7⁎ 252 ± 18 17.6 ± 2.0 1.43 ± 0.20⁎
85 ± 2 6.6 ± 1.3 0.46 ± 0.04 64 ± 6⁎# 3.1 ± 0.4 13.4 ± 0.8⁎# 240 ± 17 17.9 ± 2.3 1.39 ± 0.29
BG, blood glucose; IRI, serum immunoreactive insulin; FFA, free fatty acid; TG, triglyceride; RLP-C, remnant-like particle-cholesterol; UN, urea nitrogen; IGF-1, insulin-like growth factor-1; HOMA-IR, Homeostasis model assessment for insulin resistance. ⁎ p < 0.05 compared with Control. # p < 0.05 compared with Fat-free milk-containing.
6:00–8:00, no differences were observed at 8:00–14:00. When compared with control, urinary CPR excretion levels were significantly smaller or tended to be smaller in control + Ex (14:00–20:00, p < 0.05; 8:00–20:00, p = 0.078) but were larger in milk intake (14:00–20:00 and for 24 h, p < 0.05; 8:00–20:00, p = 0.055), and were significantly smaller in milk intake with exercise (milk intake + Ex) than in milk intake alone for 24 h. Urinary 3-MH excretion levels were significantly smaller in milk intake than in control at 20:00–8:00 and for 24 h. Urinary adrenaline and dopamine excretion levels were significantly smaller or tended to
3.1. Urinary metabolic parameters Table 1 shows a comparison of urinary excretion levels of creatinine, UN, CPR, 3-MH and catecholamine 3 fractionations. Urinary creatinine excretion levels were almost the same in all experiments. In contrast, UN excretion levels were significantly larger after 14:00 in fatfree milk-containing diet intake (milk intake) than in control diet intake (control). Although urinary CPR excretion levels were significantly smaller in control than in control with exercise (control + Ex) or in milk intake at 23
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serum UN levels appeared to be due to the increase in amino acid catabolism by increasing the turnover rate of body protein metabolism [19,38]. The insulin-sparing action of exercise with control diet was also observed, and decreases in CPR levels of plasma and urinary excretion with exercise have been reported [39,40]. For exercise under fat-free milk intake, further elevation in serum UN was observed, suggesting a combined effect from increased protein intake and exercise. The insulin-sparing action of exercise based on the results of urinary CPR excretion was also observed for fat-free milk intake. In addition, serum TG levels were simultaneously decreased. We previously reported a strong positive correlation between decreased levels in serum TG and IRI [41]. For muscle protein metabolism, levels of serum IGF-1 (anabolism marker) [42,43] and cortisol (catabolism marker) [44] were not affected by fat-free milk intake and/or exercise in this acute experiment. More intense, amount or longer duration exercise may be needed to detect changes in these indices. 3-MH indicates the rate of muscle protein breakdown, and is released into blood, before being excreted in urine [45]. In this study, urinary 3-MH excretion levels were decreased by fat-free milk intake accompanied by decreases in urinary adrenaline and dopamine excretion. Nagasawa et al. [46] reported the suppression of myofibrillar protein degradation, i.e., the decline in serum 3-MH, was regulated by dietary proteins, and was not synchronized with changes in the serum concentration of insulin and corticosterone in the rat study. Based on the results of urinary catecholamine excretion, sympathetic action may have been suppressed by fat-free milk intake. The absence of these 3-MH-decreasing effects during exercise under fatfree milk intake in this study may have been caused by the increase in the muscle protein turnover rate [47,48]. In the present study, which was carried out to observe the acute effects of fat-free milk intake and/or exercise for 24 h, fat-free milk intake brought about an increase in UN levels of serum and urinary excretion and insulin secretion, and a decrease in the levels of urinary 3-MH and catecholamine excretion. Although urinary nitrogen excretion is not increased immediately after increase in protein intake level, the lower ratio of urinary UN excretion levels to total nitrogen intake levels indicates at least temporary retention of nitrogen in the body. These results suggest that fat-free milk intake can accelerate protein anabolism and suppress catabolism. Exercise under fat-free milk intake attenuated the changes in insulin secretion, urinary 3-MH and catecholamine excretion. Therefore, exercise may be able to increase muscle mass and strength without the action of insulin, and this insulinsparing action itself has beneficial effects against metabolic syndrome. As mentioned above, dietary protein promotes insulin secretion. For that reason, it has been disputed whether intake of protein, particularly BCAAs, is associated with an increased risk of type 2 diabetes mellitus based on the findings concerning the BCAA degradation pathway and the mammalian target of rapamycin (mTOR) [49–53]. A moderately high protein diet has a positive impact [54,55], whereas excessive dietary protein intake has adverse effects [56]. It is the case that exercise attenuates the insulinotropic effects of protein, particularly with BCAA intake. For that reason, protein intake in cooperation with exercise is recommended to prevent or improve both metabolic and locomotive syndrome. At the same time in this study, it is also necessary to consider effects of amino acids other than BCAAs in fat-free milk. Although they are calculated values on the Standard Tables of Food Composition, fat-free milk-containing diet contained not only 55% more BCAAs but also 75% more lysine compared to control diet. It is reported that lysine has suppressing effects of muscle protein degradation [57], and other amino acids may be involved. Moreover, the present study was conducted in young healthy and lean women. Therefore, obtained results cannot be easily extrapolated against metabolic and locomotive disorders. Further investigation, including the dynamic state of circulating amino acids and longer study periods in addition to selection of subjects will be necessary to determine the proper intake levels of amino acids
be smaller in milk intake than in control, i.e., adrenaline was lower at 8:00–14:00 (p = 0.078), 14:00–20:00 (p = 0.055), 20:00–8:00 (p = 0.055) and for 24 h (p < 0.05), and dopamine was significantly lower after 8:00 except for 14:00–20:00 (p = 0.078). On the other hand, in milk intake + Ex, as compared to milk intake alone, both urinary adrenaline and noradrenaline excretion levels were significantly larger at 14:00–20:00 and for 24 h. 3.2. Blood metabolic parameters Table 2 shows a comparison of levels of BG, serum IRI, FFA, TG, RLP-C, UN, IGF-1, cortisol and HOMA-IR. BG levels were almost the same after all experiments. Serum IRI levels were significantly higher after milk intake than after control. HOMA-IR levels showed similar results as serum IRI levels. Serum FFA and RLP-C levels did not show significant differences. Serum TG levels tended to be lower (p = 0.086) after control + Ex than after control alone, and were significantly lower after milk intake + Ex than after milk intake alone. Serum UN levels were significantly higher after control + Ex and milk intake than after control, and were also significantly higher after milk intake + Ex than after milk intake alone. On the other hand, neither serum IGF-1 nor cortisol levels showed significant differences. 3.3. Urinary UN excretion and total nitrogen intake levels The ratio of urinary UN excretion levels to total nitrogen intake levels was significantly lower in milk intake than in control, and no differences were observed between milk intake with or without Ex (Table 3). 4. Discussion While hyperinsulinemia promotes various complicating disorders related to metabolic syndrome, insulin also contributes to muscular hypertrophy. Exercise exerts an insulin-sparing action [18] and promotes body protein anabolism [19,20]. On the other hand, BCAAs, particularly leucine, are utilized for energy [21], body protein maintenance [22] and accretion [23], but simultaneously increase insulin secretion [24–26]. In the present study, we examined the effects of BCAA-rich fat-free milk intake or exercise, and the effects of exercise under fat-free milk intake on the levels of serum insulin, urinary CPR excretion and indices related to muscle protein metabolism. Because energy-restricted diet and/or exercise are frequently used against metabolic syndrome, no extra energy corresponding to addition energy expended during exercise was added in control diet intake with exercise. Regarding locomotive syndrome, sufficient energy and adequate nutrients and/or exercise is recommended. For that reason, extra energy was added in fat-free milk-containing diet with exercise. Based on the results of urinary creatinine excretion, urine samples were precisely gathered in all experiments. With intake of the fat-free milk-containing diet, urinary UN excretion levels increased after 14:00 and serum UN levels were elevated the next morning. At the same time, urinary CPR excretion levels increased at 14:00–20:00, for 24 h and serum insulin levels were also elevated. These results were consistent with the findings regarding protein intake and insulin secretion [24–26]. In the exercise experiment with control diet, the elevation in Table 3 Comparison of the ratio of urinary urea nitrogen excretion levels to total nitrogen intake levels (%). Control
Control + Ex
Fat-free milkcontaining
Fat-free milkcontaining + Ex
66.0 ± 4.7
68.6 ± 3.9
57.0 ± 2.9⁎
55.1 ± 2.9⁎
⁎
p < 0.05 compared with Control. 24
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and appropriate exercise.
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Shimomura, Clofibrate-induced reduction of plasma branched-chain amino acid concentrations impairs glucose tolerance in rats, JPEN 36 (2012) 337–343. [53] C.J. Lynch, S.H. Adams, Branched-chain amino acids in metabolic signalling and insulin resistance, Nat. Rev. Endocrinol. 10 (2014) 723–736. [54] P.M. Piatti, F. Monti, I. Fermo, L. Baruffaldi, R. Nasser, G. Santambrogio, M.C. Librenti, M. Galli-Kienle, A.E. Pontiroli, G. Pozza, Hypocaloric high-protein diet improves glucose oxidation and spares lean body mass: comparison to hypocaloric high-carbohydrate diet, Metabolism 43 (1994) 1481–1487. [55] D.K. Layman, J.I. Baum, Dietary protein impact on glycemic control during weight loss, J. Nutr. 134 (2004) 968S–973S. [56] C.C. Metges, C.A. Barth, Metabolic consequences of a high dietary-protein intake in adulthood: assessment of the available evidence, J. Nutr. 130 (2000) 886–889. [57] T. Sato, Y. Ito, T. 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Declarations of interest None. Acknowledgements This work was supported by JSPS KAKENHI Grant Number JP15K00852. We are also grateful to Fuji Oil Co., Ltd. for providing the foodstuffs used in the study. References [1] J. Mayer, D.W. Thomas, Regulation of food intake and obesity, Science 156 (1967) 328–337. [2] J. de Toro-Martín, B.J. Arsenault, J.P. Després, M.C. Vohl, Precision nutrition: a review of personalized nutritional approaches for the prevention and management of metabolic syndrome, Nutrients 9 (2017) E913. [3] C. Ostman, N.A. Smart, D. Morcos, A. Duller, W. Ridley, D. Jewiss, The effect of exercise training on clinical outcomes in patients with the metabolic syndrome: a systematic review and meta-analysis, Cardiovasc. Diabetol. 16 (2017) 110. [4] R. Roubenoff, V.A. Hughes, Sarcopenia: current concepts, J. Gerontol. A Biol. Sci. Med. Sci. 55 (2000) M716–M724. [5] I.H. Rosenberg, Sarcopenia: origins and clinical relevance, J. Nutr. 127 (1997) 990S–991S. [6] K. Nakamura, A “super-aged” society and the “locomotive syndrome”, J. Orthop. Sci. 13 (2008) 1–2. [7] K. Nakamura, T. Ogata, Locomotive syndrome: definition and management, Clin. Rev. Bone Miner. Metab. 14 (2016) 56–67. [8] V. Santilli, A. Bernetti, M. Mangone, M. Paoloni, Clinical definition of sarcopenia, Clin. Cases Miner. Bone Metab. 11 (2014) 177–180. [9] P. JafariNasabian, J.E. Inglis, O.J. Kelly, J.Z. Ilich, Osteosarcopenic obesity in women: impact, prevalence, and management challenges, Int. J. Womens Health 9 (2017) 33–42. [10] W. Evans, Functional and metabolic consequences of sarcopenia, J. Nutr. 127 (1997) 998S–1003S. [11] O.C. Witard, C. McGlory, D.L. Hamilton, S.M. Phillips, Growing older with health and vitality: a nexus of physical activity, exercise and nutrition, Biogerontology 17 (2016) 529–546. [12] J.O. Holloszy, A, forty-year memoir of research on the regulation of glucose transport into muscle, Am. J. Physiol. Endocrinol. Metab. 284 (2003) E453–E467. [13] K. Rodahl, H.I. Miller, B.Jr. Issekutz, Plasma free fatty acids in exercise, J. Appl. Physiol. 19 (1964) 489–492. [14] L.A. Carlson, F. Mossfeldt, Acute effects of prolonged, heavy exercise on the concentration of plasma lipids and lipoproteins in man, Acta Physiol. Scand. 62 (1964) 51–59. [15] H. Lithell, J. Orlander, R. Schele, B. Sjodin, J. Karlsson, Changes in lipoproteinlipase activity and lipid stores in human skeletal muscle with prolonged heavy exercise, Acta Physiol. Scand. 107 (1979) 257–261. [16] J.R. Zierath, Invited review: exercise training-induced changes in insulin signaling in skeletal muscle, J. Appl. Physiol. 93 (2002) 773–781. [17] S.F. Crouse, B.C. O'Brien, P.W. Grandjean, R.C. Lowe, J.J. Rohack, J.S. Green, Effects of training and a single session of exercise on lipids and apolipoproteins in hypercholesterolemic men, J. Appl. Physiol. 83 (1997) 2019–2028. [18] S.R. Bird, J.A. Hawley, Update on the effects of physical activity on insulin sensitivity in humans, BMJ Open Sport Exerc. Med. 2 (2017) e000143. [19] M.J. Rennie, R.H. Edwards, S. Krywawych, C.T. Davies, D. Halliday, J.C. Waterlow, D.J. Millward, Effect of exercise on protein turnover in man, Clin. Sci. 61 (1981) 627–639. [20] M.J. Rennie, R.H. Edwards, C.T. Davies, S. Krywawych, D. Halliday, J.C. Waterlow, D.J. Millward, Protein and amino acid turnover during and after exercise, Biochem. Soc. Trans. 8 (1980) 499–501. [21] D.J. Millward, J.L. Bowtell, P. Pacy, M.J. Rennie, Physical activity, protein metabolism and protein requirements, Proc. Nutr. Soc. 53 (1994) 223–240. [22] D.A. MacLean, T.E. Graham, B. Saltin, Branched-chain amino acids augment ammonia metabolism while attenuating protein breakdown during exercise, Am. J. Phys. 267 (1994) E1010–E1022. [23] S.R. Kimball, The role of nutrition in stimulating muscle protein accretion at the molecular level, Biochem. Soc. Trans. 35 (2007) 1298–1301. [24] S. Berger, N. Vongaraya, Insulin response to ingested protein in diabetes, Diabetes 15 (1966) 303–306. [25] S.H. Holt, J.C. Miller, P. Petocz, An insulin index of foods: the insulin demand generated by 1000-kJ portions of common foods, Am. J. Clin. Nutr. 66 (1997) 1264–1276. [26] F.Q. Nuttall, M.C. Gannon, Metabolic response of people with type 2 diabetes to a high protein diet, Nutr. Metab. (Lond.) 1 (2004) 6. [27] Ministry of Health and Welfare, Japan, Dietary Reference Intakes for Japanese, DAIICHI Shuppan publishing, Tokyo, 2015, p. 2014.
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Increase in insulin secretion and decrease in muscle degradation by fat-free milk intake are attenuated by physical exercise
T
⁎
Tetsuo Yamadaa, , Masami Matsuzakia, Akira Tanakab a b
Department of Nutrition and Dietetics, Kanto Gakuin University, Yokohama, Japan Laboratory of Clinical Nutrition and Medicine, Kagawa Nutrition University, Sakado, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Fat-free milk Exercise C-peptide immunoreactivity Insulin 3-Methyl-histidine Catecholamine
Background: Protein intake, particularly branched chain amino acids (BCAAs), and exercise have opposing actions on insulin secretion, but the same action on protein anabolism. We examined the effects of BCAA-rich fatfree milk intake and/or exercise on levels of insulin secretion and indices related to muscle protein metabolism in order to assess the potency of dietary and exercise therapies against metabolic and locomotive disorders. Methods: Eight adult female volunteers participated in all four 24 h experiments; control diet intake with or without exercise, and fat-free milk-containing diet intake with or without exercise. Fat-free milk was replaced with one-sixth of all foods in the control diet. Exercise was set at an equal-energy level as fat-free milk. Urine and fasting blood samples were collected for each experiment. Results: Urinary C-peptide immunoreactivity excretion and serum insulin levels were significantly higher, but urinary 3-methyl-histidine excretion levels were significantly lower with low urinary adrenaline and dopamine excretion in the fat-free milk-containing diet than in the control diet. These findings were reduced by exercise with high urinary adrenaline and noradrenaline excretion. Conclusions: BCAA-rich fat-free milk intake enhanced insulin secretion and suppressed muscle protein degradation, but these effects are attenuated by exercise accompanied with increase in catecholamine secretion.
1. Introduction Metabolic syndrome has always been identified as a risk factor for coronary heart disease and stroke, which are closely linked to obesity and unhealthy lifestyle [1–3]. In contrast, the term “sarcopenia” was coined by Rosenberg in 1989 to denote the age-related decline in muscle mass and function [4,5], and recently, the concept of locomotive syndrome has been suggested in relation to bone disorder and sarcopenia [6,7]. Furthermore, sarcopenic obesity has been defined as a condition that encompasses sarcopenia and obesity [8,9]. Both nutrition and exercise are key factors involved in metabolic and locomotive syndrome [2,3,10,11]. Regarding metabolic syndrome, obesity, impaired glucose tolerance, hyperlipidemia and hypertension are frequently improved by energy-restricted diet and/or aerobic exercise, which directly promotes glucose and free fatty acid (FFA) consumption. During acute aerobic exercise, glucose transporter 4 increases glucose intake into muscle cells [12], serum FFA level increases [13], triglyceride (TG) level decreases [14] and lipoprotein lipase (LPL) activity is promoted [15]. The cumulative training effects of aerobic exercise are also understood [16,17]. Regarding locomotive syndrome, sufficient
⁎
energy and adequate nutrients such as protein, n-3 polyunsaturated fatty acids, calcium and vitamin D, are required [9–11]. In addition, mechanically loaded and/or resistance exercise is typically used to increase bone mineral density and muscle mass [7,9–11], indirectly improving dysfunctional glucose and lipid metabolism. It is known that insulin lowers blood glucose and stimulates protein anabolism. While it is necessary to avoid excessive secretion of insulin from the viewpoint of metabolic syndrome, insulin is also considered to contribute to increases in muscle mass. Exercise reduces insulin secretion [18] and promotes body protein anabolism [19,20]. On the other hand, proteins, particularly branched chain amino acids (BCAAs), are utilized for energy [21], body protein maintenance [22] and anabolism [23], but simultaneously produce insulinotropic effects [24–26]. Thus, BCAA intake and exercise have opposing actions on insulin secretion, but the same action on protein metabolism. Therefore, it is significant to investigate each and combined effect of BCAA intake and exercise. In the present study, we examined the effects of BCAAs-rich fat-free milk intake or exercise, and the effects of exercise under fat-free milk intake on serum insulin, urinary C-peptide immunoreactivity (CPR) excretion and indices related to muscle protein metabolism in order to
Corresponding author at: 1-50-1 Mutsuura-higashi Kanazawa-ku, Yokohama 236-8503, Japan. E-mail address: [email protected] (T. Yamada).
https://doi.org/10.1016/j.cca.2018.05.017 Received 31 December 2017; Received in revised form 25 April 2018; Accepted 8 May 2018 Available online 09 May 2018 0009-8981/ © 2018 Elsevier B.V. All rights reserved.
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with exercise.
assess the potency of dietary and exercise therapies against metabolic and locomotive disorders.
2.2.3. Procedure of exercise Exercise tolerance was tested before starting the study by gradually increasing the work rate on a bicycle ergometer. Heart rate was recorded by telemetry (DS-3400; Fukuda Denshi Co., Ltd., Tokyo, Japan). Expired gas before and at steady state during exercise was gathered using a Douglas bag, and then gas volume was measured using a dry gas meter (DC-5A; Shinagawa Corporation, Tokyo, Japan). Expired oxygen and carbon dioxide concentrations were measured using an expired gas monitor (Portable Gas Monitor AR-1; Arco System Inc., Kashiwa, Japan). Relationships among additional energy expenditure calculated based on oxygen intake and the respiratory exchange ratio, work rate (kilopond of bicycle ergometer) and heart rate were determined for each individual. In the exercise experiment, participants expended 352 kcal by pedaling a bicycle ergometer at a target intensity of 40–50% of maximal oxygen intake for 101 ± 4 min split between the morning and afternoon. In the non-exercise experiment, the participants performed only normal daily activities.
2. Materials and methods 2.1. Subjects Eight healthy adult female volunteers [age, 22 ± 0 years; height, 159 ± 1 cm; weight, 52.8 ± 2.4 kg; mean ± standard error of the mean (SEM)] provided written, informed consent to participate in all procedures associated with the study, which proceeded according to the Declaration of Helsinki (1964; amended 2013). 2.2. Experimental procedure The Research Ethics Committee of Kanto Gakuin University approved the study. 2.2.1. Experimental protocol The study comprised four experiments. Eight subjects participated in all four experiments in random order; control diet intake, control diet intake with exercise, fat-free milk-containing diet intake and fat-free milk-containing diet intake with exercise. Each experiment was conducted at interval of one week or more. Participants were fed experimental diets in all four experiments, and exercised in two experiments under the control diet or fat-free milk-containing diet. In every experiment, the participants ate the prescribed supper on the previous evening and stayed two nights at the metabolic unit to complete each experiment.
2.2.4. Environmental conditions Room temperature during the experimental period was maintained between 25 and 26 °C on the dry-bulb thermometer and between 22 and 23 °C on the wet-bulb thermometer. 2.2.5. Sample collection and measurement Nitrogen contents of breakfast, lunch, supper and fat-free milk were determined by the Kjeldarl method. Urine samples were collected at 6:00–8:00 (baseline before each experiment), 8:00–14:00, 14:00–20:00 and 20:00–8:00 the following morning to measure creatinine [29], urea nitrogen (UN) [30], CPR (Chemiluminescent enzyme immunoassay, Lumipulse Presto C-peptide; Fujirebio Inc., Tokyo, Japan), 3-methylhistidine (3-MH) [31], adrenaline, noradrenaline and dopamine [32] levels. Fasting blood samples were collected early in the morning at the end of each experiment to measure blood glucose (BG) [33], serum immunoreactive insulin (IRI) [34], FFA [35], TG [36], remnant-like particle-cholesterol (RLP-C) [37], UN [30], insulin-like growth factor-1 (IGF-1: Immunoradiometric assay, IGF-1 (Somatomedin C) IRMA DAIICHI; Fujirebio Inc., Tokyo, Japan) and cortisol (Electro chemiluminedcence immunoassay, ECLusys reagent Cortisol II; Roche Diagnostics K. K., Tokyo, Japan) levels. Homeostasis model assessment for insulin resistance (HOMA-IR) was calculated by multiplying fasting BG (mg/dl) by IRI (μU/ml) by 1/405.
2.2.2. Experimental diet For control diet intake, the diet comprised the following foods; soft rolls, sausages (Vienna), ketchup, potato salad, vegetable juice, spaghetti, salted cod sauce, sweet corn (boiled), salted butter, pepper (mixed, ground), vitamin mixture beverage, custard pudding, European plums (dried), well-milled rice, soy protein processed food, miso (lightyellow type), kanze-fu, spinach (leaves, boiled) and soy sauce. The composition of the experimental diet was based on the recommended dietary allowance or adequate intake published in the Dietary Reference Intakes for Japanese [27]. The calculated values per day on the Standard Tables of Food Composition in Japan [28] were as follows: energy, 2100 kcal; protein, 63.8 g (BCAAs, 9.9 g); lipid, 67.8 g; carbohydrate, 302.2 g and the PFC ratio was 12.2:29.1:58.7. For fat-free milk-containing diet intake, the experimental diet was reduced to approximately five-sixths of control diet by reducing all foods in proportion to the energy intake, and fat-free milk was replaced with approximately one-sixth of control diet. Fat-free milk was given with every three meals and its volume was decided as the likely level in daily life, i.e., equivalent energy amount in 175 ml of ordinary milk per meal. The participants consumed experimental diets (energy, 2100 kcal; protein, 88.9 g (BCAAs, 15.3 g); lipid, 57.0 g; carbohydrate, 302.3 g and the PFC ratio was 17.4:24.5:58.2) that contained fat-free milk at 352 kcal. In control diet intake with exercise, no extra energy corresponding to addition energy expended during exercise was added. In fat-free milk-containing diet with exercise, the participants consumed control diet and 352 kcal of fat-free milk corresponding to extra energy (energy, 2452 kcal; protein, 99.6 g (BCAAs, 17.0 g); lipid, 68.4 g; carbohydrate, 352.9 g and the PFC ratio was 16.6:25.1:58.2). The calculated values of other amino acids (lysine, sulfur amino acids (methionine, cystine: SAAs), aromatic amino acids (phenylalanine, tyrosine: AAAs)) relatively relevant to protein intake in each experiment were as follows: lysine, 2.8 g; SAAs, 2.2 g; AAAs, 4.8 g in control diet intake and control diet intake with exercise, lysine, 4.9 g; SAAs, 2.9 g; AAAs, 7.2 g in fat-free milk-containing diet intake, lysine, 5.3 g; SAAs, 3.3 g; AAAs, 8.0 g in fat-free milk-containing diet intake
2.3. Statistical analysis Results are expressed as means ± SEM. All statistical analyses were performed using SPSS14.0 for Windows. We applied the Wilcoxon signed-ranks test to examine differences between control diet intake and control diet intake with exercise, fat-free milk-containing diet intake, fat-free milk-containing diet intake with exercise, and between fat-free milk-containing diet intake with or without exercise. Significance was established at p < 0.05 in all analyses. 3. Results There was no significant difference in the timing of the menstrual cycle in each experiment (when the whole menstrual cycle was taken as 100%, at 45 ± 10 percentile point, control diet intake; at 67 ± 11 percentile point, control diet intake with exercise; at 69 ± 10 percentile point, fat-free milk-containing diet intake; at 57 ± 9 percentile point, fat-free milk-containing diet intake with exercise). 22
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Table 1 Comparison of excretion levels of the urinary metabolic parameters in each experiment of control diet intake (Control), control diet intake with exercise (Control + Ex), fat-free milk-containing diet intake (Fat-free milk-containing) and fat-free milk-containing diet intake with exercise (Fat-free milk-containing + Ex). Variable Creatinine (mg) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Ex UN (mg) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Ex CPR (μg) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Ex 3-MH (μmol) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Ex Adrenaline (μg) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Ex Noradrenaline (μg) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Dopamine (μg) Control Control + Ex Fat-free milk-containing Fat-free milk-containing + Ex
6:00–8:00
8:00–14:00
14:00–20:00
20:00–8:00
8:00–20:00
8:00–8:00
90 91 92 91
316 314 287 292
265 262 268 267
528 530 504 515
581 576 555 559
1108 1106 1059 1074
± ± ± ±
63 49 40 52
± ± ± ±
5 4 4 3
± ± ± ±
18 21 12 12
± ± ± ±
23 22 20 27
± ± ± ±
28 19 20 21
± ± ± ±
38 35 26 36
535 625 627 660
± ± ± ±
51 30⁎ 58 31⁎
1695 1662 1839 2043
± ± ± ±
113 137 124 141⁎
1608 1643 2202 2247
± ± ± ±
150 182 148⁎ 232⁎
2787 3023 3504 3853
± ± ± ±
217 140 252⁎ 228⁎
3303 3305 4041 4290
± ± ± ±
236 263 175⁎ 262⁎
6090 6328 7545 8143
± ± ± ±
430 357 386⁎ 427⁎
1.64 2.11 2.22 2.00
± ± ± ±
0.23 0.32⁎ 0.23⁎ 0.24
27.05 21.54 32.85 28.70
± ± ± ±
4.27 2.30 1.98 3.64
28.93 21.15 37.03 28.61
± ± ± ±
2.64 2.31⁎ 3.64⁎ 4.17
26.03 23.60 29.80 28.30
± ± ± ±
2.13 2.16 2.77 3.08
55.98 42.69 69.88 57.31
± ± ± ±
6.03 3.15 4.16 6.92
82.00 66.28 99.68 85.61
± ± ± ±
7.74 4.86 6.66⁎ 7.81#
13.2 13.6 13.6 14.1
± ± ± ±
0.8 0.6 0.8 0.7
52.9 50.3 47.3 49.9
± ± ± ±
3.8 2.9 2.1 2.7
39.6 38.1 38.5 40.3
± ± ± ±
3.2 3.0 2.8 4.4
77.5 78.0 66.4 73.1
± ± ± ±
4.0 2.6 3.5⁎ 3.6
92.4 88.4 85.8 90.2
± ± ± ±
6.2 4.4 3.5 6.2
170.0 166.4 152.2 163.3
± ± ± ±
9.8 6.0 6.1⁎ 9.0
0.58 0.48 0.40 0.42
± ± ± ±
0.17 0.13 0.08 0.11
2.77 3.32 2.23 2.60
± ± ± ±
0.60 0.86 0.42 0.55
2.51 2.77 2.10 3.02
± ± ± ±
0.56 0.49 0.39 0.68#
2.79 3.21 2.10 2.58
± ± ± ±
0.54 0.58 0.48 0.52
5.28 6.09 4.34 5.62
± ± ± ±
1.14 1.30 0.79 1.22#
8.07 9.30 6.44 8.20
± ± ± ±
1.62 1.85 1.25⁎ 1.73#
6.3 6.4 5.3 5.7
± ± ± ±
0.9 0.6 0.6 0.6
27.5 32.3 22.5 26.2
± ± ± ±
3.5 5.5 2.8 2.6
23.5 29.6 20.9 28.1
± ± ± ±
3.3 4.8 2.3 4.5#
35.4 40.9 30.1 35.6
± ± ± ±
4.6 4.5 4.3 4.3#
51.0 61.9 43.4 54.4
± ± ± ±
6.4 9.8 4.6 7.1
86.4 ± 10.8 102.8 ± 14.2 73.5 ± 8.5 90.0 ± 11.2#
41 40 34 36
± ± ± ±
5 3 2 3
197 182 150 167
± ± ± ±
18 11 9⁎ 9
211 202 177 202
± ± ± ±
24 15 9 21
403 374 317 364
± ± ± ±
29 11 9⁎ 26
408 384 328 369
± ± ± ±
39 22 13⁎ 28
811 758 644 733
± ± ± ±
66 27 17⁎ 49
UN, urea nitrogen; CPR, C-peptide immunoreactivity; 3-MH, 3-methyl-histidine. ⁎ p < 0.05 compared with Control. # p < 0.05 compared with Fat-free milk-containing. Table 2 Comparison of the blood metabolic parameters in each experiment. Variable
Control
Control + Ex
Fat-free milk-containing
Fat-free milk-containing + Ex
BG (mg/dl) IRI (μU/ml) FFA (mEq/l) TG (mg/dl) RLP-C (mg/dl) UN (mg/dl) IGF-1 (ng/ml) Cortisol (μg/dl) HOMA-IR
85 ± 2 5.0 ± 0.8 0.41 ± 0.06 89 ± 15 3.8 ± 0.7 9.4 ± 0.5 234 ± 15 19.7 ± 2.9 1.07 ± 0.17
84 ± 2 5.3 ± 0.6 0.52 ± 0.04 75 ± 15 3.2 ± 0.4 11.1 ± 0.8⁎ 229 ± 16 17.6 ± 1.3 1.09 ± 0.12
85 ± 2 6.8 ± 0.9⁎ 0.47 ± 0.04 76 ± 9 3.4 ± 0.5 11.5 ± 0.7⁎ 252 ± 18 17.6 ± 2.0 1.43 ± 0.20⁎
85 ± 2 6.6 ± 1.3 0.46 ± 0.04 64 ± 6⁎# 3.1 ± 0.4 13.4 ± 0.8⁎# 240 ± 17 17.9 ± 2.3 1.39 ± 0.29
BG, blood glucose; IRI, serum immunoreactive insulin; FFA, free fatty acid; TG, triglyceride; RLP-C, remnant-like particle-cholesterol; UN, urea nitrogen; IGF-1, insulin-like growth factor-1; HOMA-IR, Homeostasis model assessment for insulin resistance. ⁎ p < 0.05 compared with Control. # p < 0.05 compared with Fat-free milk-containing.
6:00–8:00, no differences were observed at 8:00–14:00. When compared with control, urinary CPR excretion levels were significantly smaller or tended to be smaller in control + Ex (14:00–20:00, p < 0.05; 8:00–20:00, p = 0.078) but were larger in milk intake (14:00–20:00 and for 24 h, p < 0.05; 8:00–20:00, p = 0.055), and were significantly smaller in milk intake with exercise (milk intake + Ex) than in milk intake alone for 24 h. Urinary 3-MH excretion levels were significantly smaller in milk intake than in control at 20:00–8:00 and for 24 h. Urinary adrenaline and dopamine excretion levels were significantly smaller or tended to
3.1. Urinary metabolic parameters Table 1 shows a comparison of urinary excretion levels of creatinine, UN, CPR, 3-MH and catecholamine 3 fractionations. Urinary creatinine excretion levels were almost the same in all experiments. In contrast, UN excretion levels were significantly larger after 14:00 in fatfree milk-containing diet intake (milk intake) than in control diet intake (control). Although urinary CPR excretion levels were significantly smaller in control than in control with exercise (control + Ex) or in milk intake at 23
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serum UN levels appeared to be due to the increase in amino acid catabolism by increasing the turnover rate of body protein metabolism [19,38]. The insulin-sparing action of exercise with control diet was also observed, and decreases in CPR levels of plasma and urinary excretion with exercise have been reported [39,40]. For exercise under fat-free milk intake, further elevation in serum UN was observed, suggesting a combined effect from increased protein intake and exercise. The insulin-sparing action of exercise based on the results of urinary CPR excretion was also observed for fat-free milk intake. In addition, serum TG levels were simultaneously decreased. We previously reported a strong positive correlation between decreased levels in serum TG and IRI [41]. For muscle protein metabolism, levels of serum IGF-1 (anabolism marker) [42,43] and cortisol (catabolism marker) [44] were not affected by fat-free milk intake and/or exercise in this acute experiment. More intense, amount or longer duration exercise may be needed to detect changes in these indices. 3-MH indicates the rate of muscle protein breakdown, and is released into blood, before being excreted in urine [45]. In this study, urinary 3-MH excretion levels were decreased by fat-free milk intake accompanied by decreases in urinary adrenaline and dopamine excretion. Nagasawa et al. [46] reported the suppression of myofibrillar protein degradation, i.e., the decline in serum 3-MH, was regulated by dietary proteins, and was not synchronized with changes in the serum concentration of insulin and corticosterone in the rat study. Based on the results of urinary catecholamine excretion, sympathetic action may have been suppressed by fat-free milk intake. The absence of these 3-MH-decreasing effects during exercise under fatfree milk intake in this study may have been caused by the increase in the muscle protein turnover rate [47,48]. In the present study, which was carried out to observe the acute effects of fat-free milk intake and/or exercise for 24 h, fat-free milk intake brought about an increase in UN levels of serum and urinary excretion and insulin secretion, and a decrease in the levels of urinary 3-MH and catecholamine excretion. Although urinary nitrogen excretion is not increased immediately after increase in protein intake level, the lower ratio of urinary UN excretion levels to total nitrogen intake levels indicates at least temporary retention of nitrogen in the body. These results suggest that fat-free milk intake can accelerate protein anabolism and suppress catabolism. Exercise under fat-free milk intake attenuated the changes in insulin secretion, urinary 3-MH and catecholamine excretion. Therefore, exercise may be able to increase muscle mass and strength without the action of insulin, and this insulinsparing action itself has beneficial effects against metabolic syndrome. As mentioned above, dietary protein promotes insulin secretion. For that reason, it has been disputed whether intake of protein, particularly BCAAs, is associated with an increased risk of type 2 diabetes mellitus based on the findings concerning the BCAA degradation pathway and the mammalian target of rapamycin (mTOR) [49–53]. A moderately high protein diet has a positive impact [54,55], whereas excessive dietary protein intake has adverse effects [56]. It is the case that exercise attenuates the insulinotropic effects of protein, particularly with BCAA intake. For that reason, protein intake in cooperation with exercise is recommended to prevent or improve both metabolic and locomotive syndrome. At the same time in this study, it is also necessary to consider effects of amino acids other than BCAAs in fat-free milk. Although they are calculated values on the Standard Tables of Food Composition, fat-free milk-containing diet contained not only 55% more BCAAs but also 75% more lysine compared to control diet. It is reported that lysine has suppressing effects of muscle protein degradation [57], and other amino acids may be involved. Moreover, the present study was conducted in young healthy and lean women. Therefore, obtained results cannot be easily extrapolated against metabolic and locomotive disorders. Further investigation, including the dynamic state of circulating amino acids and longer study periods in addition to selection of subjects will be necessary to determine the proper intake levels of amino acids
be smaller in milk intake than in control, i.e., adrenaline was lower at 8:00–14:00 (p = 0.078), 14:00–20:00 (p = 0.055), 20:00–8:00 (p = 0.055) and for 24 h (p < 0.05), and dopamine was significantly lower after 8:00 except for 14:00–20:00 (p = 0.078). On the other hand, in milk intake + Ex, as compared to milk intake alone, both urinary adrenaline and noradrenaline excretion levels were significantly larger at 14:00–20:00 and for 24 h. 3.2. Blood metabolic parameters Table 2 shows a comparison of levels of BG, serum IRI, FFA, TG, RLP-C, UN, IGF-1, cortisol and HOMA-IR. BG levels were almost the same after all experiments. Serum IRI levels were significantly higher after milk intake than after control. HOMA-IR levels showed similar results as serum IRI levels. Serum FFA and RLP-C levels did not show significant differences. Serum TG levels tended to be lower (p = 0.086) after control + Ex than after control alone, and were significantly lower after milk intake + Ex than after milk intake alone. Serum UN levels were significantly higher after control + Ex and milk intake than after control, and were also significantly higher after milk intake + Ex than after milk intake alone. On the other hand, neither serum IGF-1 nor cortisol levels showed significant differences. 3.3. Urinary UN excretion and total nitrogen intake levels The ratio of urinary UN excretion levels to total nitrogen intake levels was significantly lower in milk intake than in control, and no differences were observed between milk intake with or without Ex (Table 3). 4. Discussion While hyperinsulinemia promotes various complicating disorders related to metabolic syndrome, insulin also contributes to muscular hypertrophy. Exercise exerts an insulin-sparing action [18] and promotes body protein anabolism [19,20]. On the other hand, BCAAs, particularly leucine, are utilized for energy [21], body protein maintenance [22] and accretion [23], but simultaneously increase insulin secretion [24–26]. In the present study, we examined the effects of BCAA-rich fat-free milk intake or exercise, and the effects of exercise under fat-free milk intake on the levels of serum insulin, urinary CPR excretion and indices related to muscle protein metabolism. Because energy-restricted diet and/or exercise are frequently used against metabolic syndrome, no extra energy corresponding to addition energy expended during exercise was added in control diet intake with exercise. Regarding locomotive syndrome, sufficient energy and adequate nutrients and/or exercise is recommended. For that reason, extra energy was added in fat-free milk-containing diet with exercise. Based on the results of urinary creatinine excretion, urine samples were precisely gathered in all experiments. With intake of the fat-free milk-containing diet, urinary UN excretion levels increased after 14:00 and serum UN levels were elevated the next morning. At the same time, urinary CPR excretion levels increased at 14:00–20:00, for 24 h and serum insulin levels were also elevated. These results were consistent with the findings regarding protein intake and insulin secretion [24–26]. In the exercise experiment with control diet, the elevation in Table 3 Comparison of the ratio of urinary urea nitrogen excretion levels to total nitrogen intake levels (%). Control
Control + Ex
Fat-free milkcontaining
Fat-free milkcontaining + Ex
66.0 ± 4.7
68.6 ± 3.9
57.0 ± 2.9⁎
55.1 ± 2.9⁎
⁎
p < 0.05 compared with Control. 24
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and appropriate exercise.
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