Fermented milk improves glucose metabolism in exercise-induced muscle damage in young healthy men
© Iwasa et al.; licensee BioMed Central Ltd. 2013
Received: 13 January 2013
Accepted: 12 June 2013
Published: 16 June 2013
This study investigated the effect of fermented milk supplementation on glucose metabolism associated with muscle damage after acute exercise in humans.
Eighteen healthy young men participated in each of the three trials of the study: rest, exercise with placebo, and exercise with fermented milk. In the exercise trials, subjects carried out resistance exercise consisting of five sets of leg and bench presses at 70–100% 12 repetition maximum. Examination beverage (fermented milk or placebo) was taken before and after exercise in double-blind method. On the following day, we conducted an analysis of respiratory metabolic performance, blood collection, and evaluation of muscle soreness.
Muscle soreness was significantly suppressed by the consumption of fermented milk compared with placebo (placebo, 14.2 ± 1.2 score vs. fermented milk, 12.6 ± 1.1 score, p < 0.05). Serum creatine phosphokinase was significantly increased by exercise, but this increase showed a tendency of suppression after the consumption of fermented milk. Exercise significantly decreased the respiratory quotient (rest, 0.88 ± 0.01 vs. placebo, 0.84 ± 0.02, p < 0.05), although this decrease was negated by the consumption of fermented milk (0.88 ± 0.01, p < 0.05). Furthermore, exercise significantly reduced the absorption capacity of serum oxygen radical (rest, 6.9 ± 0.4 μmol TE/g vs. placebo, 6.0 ± 0.3 μmol TE/g, p < 0.05), although this reduction was not observed with the consumption of fermented milk (6.2 ± 0.3 μmol TE/g).
These results suggest that fermented milk supplementation improves glucose metabolism and alleviates the effects of muscle soreness after high-intensity exercise, possibly associated with the regulation of antioxidant capacity.
KeywordsLactobacillus helveticus Delayed-onset muscle damage Inflammation Oxidative stress Antioxidant
Unaccustomed and strenuous exercise causes muscle damage that clinically presents as muscular pain and involves protein degradation and ultrastructural changes, a condition known as delayed-onset muscle damage. The exercise-induced muscle damage is caused by several factors, including mechanical stress, calcium accumulation, and oxidative stress [1–4]. It has been suggested that muscle functions, such as energy metabolism and power output, are difficult to maintain in damaged muscle. Previous studies have reported that glucose utilization as an energy substrate in whole body is decreased in muscle damage after exercise [5, 6], caused by an impairment of insulin-dependent glucose uptake in the damaged muscle .
It has been reported that oxidative stress and certain inflammatory cytokines impair glucose uptake via inactivation of insulin signaling pathways in muscle cells [7–9]. Infiltration of phagocytes into the damaged muscle is observed after strenuous exercise and an inflammatory response is implicated in the development of delayed-onset muscle damage [2, 10]. In addition, elevation of the levels of oxidative damage in cellular components is also observed in damaged muscle [1, 2]. Thus, inflammatory cytokines and oxidative stress can decrease insulin-dependent glucose uptake in exercise-induced damaged muscles . Therefore, we hypothesized that the decrease of glucose metabolism associated with muscle damage may be prevented by the suppression of inflammation and oxidative stress.
Fermented milk has several salutary effects, including prolonged lifespan, antihypertensive and antitumorigenic effects, and immune system regulation [12–15]. In addition, some types of fermented milk also possess anti-inflammatory and antioxidant properties [16–19]. Previously, we have shown that Lactobacillus helveticus–fermented milk prevents muscle damage induced by acute exercise via activation of antioxidative enzymes of skeletal muscle in an animal study , suggesting that fermented milk may prevent the impairment of glucose metabolism associated with muscle damage. Thus, the purpose of this study is to investigate the effect of fermented milk supplementation on glucose metabolism in damaged muscle after acute resistance exercise in humans.
Eighteen healthy young men who were not have the habituated to a regular exercise regimen were recruited to participate in this study. The characteristics of the subjects were follows: age, 21.6 ± 0.8 yr; height, 171.1 ± 1.5 cm; body weight, 59.9 ± 1.5 kg; body mass index, 20.5 ± 0.4 kg/m2; and body fat, 16.2 ± 0.8%. All subjects were free of signs, symptoms, and history of any overt chronic disease. None of the participants had a history of smoking and none were currently taking any medications or dietary supplements. This study was approved by the Ethics Committee of Kyoto Prefectural University, and all subjects signed a consent form after reading the design and protocol of the study.
The subjects participated in three trials of the study: rest with placebo intake (rest), exercise with placebo intake (placebo), and exercise with fermented milk intake (fermented milk) in a repeated-measures experimental design. These trials were performed in a random order by a counter-balanced design and were separated by at least six weeks in any individual subject to avoid the biasing of muscle damage. Subjects were also asked to refrain from caffeine and alcohol ingestion 24 h before each trial. Food intake was recorded on the day before the trial and the diet was repeated before each successive individual treatment.
Lactobacillus helveticus–fermented milk (Amiel S®, Calpis Co., Ltd., Tokyo, Japan) was used in the fermented milk trial. An equivalent dose of unfermented milk, with adjusted contents of protein (1.1%), fat (0%), carbohydrate (3.6%), and pH (3.75) to be equivalent with that of fermented milk, was used as a placebo beverage. Subjects consumed 200 mL of each beverage 3 times before and after exercise by the double-blinded method; therefore, they totally took energy: 102 kcal, protein: 6.6 g, fat: 0.0 g, and carbohydrate: 21.6 g / 600 mL.
After warm-up with a bicycle ergometer for 5 min and stretching, subjects undertook resistance exercise for 45 min. The resistance exercise was composed of leg and bench presses using a compound-type resistance training machine (Senoh Ltd., Chiba, Japan). Five sets of leg and bench presses were performed at a strength of 70–100% with a 12-repetition maximum (RM: maximum number of occurrence). This strength was determined using the methods of Drummond et al. . All subjects performed 10 repetitions at the load of 100% 12RM in 1–3sets and then the load of 70% 12RM in 4–5sets. The exercises were repeated at a pace of one repetition every 3 sec, with a 2-min interval between sets.
Indirect metabolic performance
Oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured using a breath-by-breath respiromonitor system (MetaMax 3B, Cortex, Leipzig, Germany). The respiratory quotient (RQ) and substrate utilization were calculated from the level of VO2 and VCO2, as described previously .
Blood and urine parameters
Blood lactate and glucose were measured using simple measuring instruments (Lactate Pro, GluTest; Arkray, Inc., Kyoto, Japan). The analysis of neutral fat, cholesterols (LDL-cholesterol, HDL-cholesterol, and total cholesterol), free fatty acid, high sensitivity C-reactive protein (hsCRP), and creatine phosphokinase (CPK) in serum was entrusted to FALCO Biosystems Corporation (Kyoto, Japan). Tumor necrosis factor α (TNF-α) and carbonyl protein levels in the serum were measured by using enzyme-linked immunosorbent assay (ELISA) kit (TNF-α: R&D Systems, MN, USA; carbonyl protein: BioCell, Auckland, NZ). Oxygen radical absorbance capacity (ORAC), a marker that reports antioxidant capacity, was measured by using the methods of Watanabe et al. . The concentration of 8-hydroxydeoxyguanosine (8-OHdG), a marker of DNA oxidative damage, was measured using an ELISA kit (Japan Institute for the Control of Ageing, Fukuroi City, Shizuoka, Japan) on the gathered urine, and the total amount of 8-OHdG was calculated by the volume of urine. Moreover, measurement of the creatinine was requested (FALCO Biosystems Ltd.) and used to correct the amount of 8-OHdG. These parameters cannot be analyzed for several subjects either because sample volume was insufficient or because subjects forgot to collect their urine samples.
All data were shown by mean value ± standard error. The significance level was assumed to be 5% (p < 0.05). The repeated-measures analysis of variance (ANOVA) was used to compare the date the 3 trials. In the index of 2–3 collection points per trial, such as blood glucose and lactate, a 2-way repeated-measures ANOVA was used. If ANOVA indicated a significance difference, a Tukey-Kramer test was used to determine the significance of the differences between mean values. Paired t-tests were used in the comparisons of muscle soreness and blood lactate between the two trials.
Muscle damage parameters and blood lactate
Comparison of muscle damage parameters
95.7 ± 7.2
192.8 ± 26.9**
152.3 ± 14.7*
Pectoralis major (score)
5.2 ± 0.5
4.3 ± 0.5#
3.3 ± 0.5
3.0 ± 0.5
Gluteus maximus (score)
5.6 ± 0.5
5.3 ± 0.5
Total score (score)
14.2 ± 1.2
12.6 ± 1.1#
Indirect metabolic performance
Blood glucose and serum lipids
Comparison of blood glucose level after oral glucose administration
89.1 ± 1.8
86.7 ± 2.4
90.3 ± 2.1
30 min (mg·dL-1)
147.9 ± 5.5
147.8 ± 5.9
156.7 ± 4.9
60 min (mg·dL-1)
132.4 ± 7.0
Comparison of serum lipids
LDL Cholesterol (mg·dL-1)
83.5 ± 5.1
88.4 ± 5.4
91.0 ± 5.5
HDL Cholesterol (mg·dL-1)
57.4 ± 3.4
58.8 ± 3.1
59.2 ± 3.1
Total Cholesterol (mg·dL-1)
157.9 ± 7.1
163.1 ± 5.3
163.6 ± 6.6
85.3 ± 11.9
79.5 ± 14.2
67.2 ± 6.5
Free fatty acids (mEq·L-1)
0.38 ± 0.04
0.47 ± 0.06
0.38 ± 0.05
Inflammation and oxidant stress parameters
Comparison of inflammatory factors and oxidant stress markers
Serum hsCRP (ng·mL-1)
88.9 ± 11.7
113.6 ± 22.1
79.3 ± 10.6
Serum TNF-α (pg·mL-1)
0.161 ± 0.002
0.166 ± 0.001
0.170 ± 0.004
Serum carbonyl protein (nmol·mg-1)
0.086 ± 0.003
0.089 ± 0.004
0.096 ± 0.003
Urine 8-OHdG (μg)
1.82 ± 0.16
1.70 ± 0.16
1.72 ± 0.19
The present study revealed the following main findings: 1) parameters of muscle damage were elevated on the day following acute resistance exercise; 2) the decrease of carbohydrate oxidation along with RQ was observed with exercise; and 3) the muscle soreness and metabolic changes were mitigated by the consumption of Lactobacillus helveticus–fermented milk in pre- and post-exercise. Previously, it had been unclear whether dietary intervention can improve metabolic impairment after muscle-damaging exercise. Our observations primarily demonstrate that dietary fermented milk improves the impairment of glucose metabolism associated with exercise-induced muscle damage in humans. Previously, consumption of milk (unfermented) partially attenuates the muscle damage ; therefore, the placebo trial, which used unfermented milk, may have also suppressed muscle damage to some extent. However, our results showed that fermented milk is more effective than milk.
Generally, it is well-known that a single bout of exercise elevates glucose uptake for a period of time post-exercise [25–27]. However, we have shown that insulin-mediated glucose uptake in muscle is decreased by muscle-damaging exercises, but not by non-muscle-damaging exercises . Therefore, the decreases of carbohydrate oxidation and respiratory quotient in the present study are suspected to be caused by insulin-dependent glucose uptake in damaged muscle. This decrease of glucose uptake is presumably due to a reduction of glucose transporter protein 4 (GLUT4) translocation via the insulin signaling pathway, which is the rate-limiting step in glucose metabolism. It has been reported that some inflammatory cytokines and chemokines, such as TNF-α and interleukin-1, attenuate the activity of insulin-mediated signaling in muscle cells [28, 29]. In addition, oxidative stress can also decrease glucose uptake by reducing the activity of insulin-mediated signaling [9, 30, 31]. In the damaged muscle on the day following the exercise, these cytokines and oxidative components are elevated [32–34], which could lead to the impairment of insulin-dependent glucose uptake. However, because we did not find any significant changes of inflammatory markers and oxidative products in serum and urine, the response is considered to be limited to muscle tissue, rather than the whole body, as suggested in a previous study .
Previously, we demonstrated that Lactobacillus helveticus–fermented milk attenuates delayed-onset muscle damage after acute exercise in rat . In this study, phagocyte infiltration and inflammatory cytokines expression, markers of inflammation in damaged muscle, were markedly reduced by the consumption of fermented milk. In addition, lipid peroxide levels were elevated after exercise, although the fermented milk uptake significantly reduced this elevation. Therefore, these observations suggest that fermented milk consumption mitigates inflammation and oxidative stress as well as muscle damage, which results in improvements in glucose metabolism by maintaining the insulin signaling pathway. In the present study, we found that ORAC, a marker of antioxidant capacity, was reduced in the placebo trial, but not in the fermented milk trial. Thus, the inhibitory effect of glucose metabolic impairment and muscle damage may be associated with elevated antioxidant levels caused by the consumption of fermented milk. Previously, we demonstrated that, in the skeletal muscle of rats, fermented milk upregulates expression of antioxidant enzymes, such as superoxide dismutase-2, catalase, and glutathione S-transferase α-1 . In addition, heat shock protein 70, a chaperone protein that can function as an antioxidant and an anti-inflammatory agent, was also elevated by the consumption of fermented milk . These observations suggest that fermented milk improves glucose metabolism and muscle damage at least, in part, by controlling endogenous antioxidant and anti-inflammation factors, a hypothesis that is supported by the ORAC results in the present study.
Although the detailed mechanisms of the effects of fermented milk on mitigating muscle damage remain unclear, small peptides present in fermented milk may be the causable agent, because fermented milk is more effective than unfermented milk. Fermented milk is manufactured by fermenting skim milk with a starter culture containing Lactobacillus helveticus. During this process, the proteins in skim milk are digested by Lactobacillus and converted into small peptides, which are more easily absorbed by the intestines compared to amino acids or large oligopeptides. Such peptides may also have additional physiological benefits aside from their use as a source of protein. Several studies have reported that peptides from fermented milk have various salutary effects, including an antihypertension effect, improvement of arterial stiffness, and immune regulation [12, 15, 35]. The present study suggests that small digested peptides in fermented milk may contribute to increasing the level of antioxidants in muscle. In future studies, we will attempt to detect the specific small peptides present after the consumption of fermented milk.
We found that fermented milk prevents glucose metabolic impairment and muscle soreness induced by acute resistance exercise in humans. The reduction of antioxidant capacity was suppressed by the consumption of fermented milk. These observations suggest that dietary fermented milk reduces impairment of glucose metabolism associated with exercise-induced muscle damage via an antioxidant effect. Dietary intake of fermented milk may be useful for persons who perform physical activity for health promotion. In future studies, further research is required to examine the detailed mechanisms of the effect of fermented milk in mitigating muscle damage along with the benefit to athletes.
Analysis of variance
Enzyme-linked immunosorbent assay
Glucose transporter protein 4
High sensitivity C-reactive protein
Oxygen radical absorbance capacity
Tumor necrosis factor alpha
Visual analog scale
Carbon dioxide production
This study was supported by Grants-in-Aid from the Japan Society of the Promotion of Science (23700776) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by research grants from Uehara Memorial Foundation. The authors thank Calpis Co., Ltd. for the gift of fermented milk.
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