A series of polyphenols known as catechins are abundant in green tea, which is consumed mainly in Asian countries. The effects of catechin-rich green tea extract (GTE) on running endurance and energy metabolism during exercise in BALB/c mice were investigated. Mice were divided into four groups: nonexercise control, exercise control (Ex-cont), exercise + 0.2% GTE, and exercise + 0.5% GTE groups. Treadmill running time to exhaustion, plasma biochemical parameters, skeletal muscle glycogen content, β-oxidation activity, and malonyl-CoA content immediately after exercise were measured at 8–10 wk after the initiation of the experiment. Oxygen consumption and respiratory exchange ratio were measured using indirect calorimetry. Running times to exhaustion in mice fed 0.5% GTE were 30% higher than in Ex-cont mice and were accompanied by a lower respiratory exchange ratio, higher muscle β-oxidation activity, and lower malonyl-CoA content. In addition, muscle glycogen content was high in the GTE group compared with the Ex-cont group. Plasma lactate concentrations in mice fed GTE were significantly lower after exercise, concomitant with an increase in free fatty acid concentrations. Catechins, which are the main constituents of GTE, did not show significant effects on peroxisome proliferator-activated receptor-α or δ-dependent luciferase activities. These results suggest that the endurance-improving effects of GTE were mediated, at least partly, by increased metabolic capacity and utilization of fatty acid as a source of energy in skeletal muscle during exercise.
- malonyl-coenzyme A
- lipid metabolism
improvements of physical performance are important, both in sport and in the maintenance of quality of life in our increasingly aging society. Many attempts have been made to improve physical performance using nutritional approaches, as nutrients provide energy and regulate physiological processes associated with exercise. To date, the effects of food components, such as caffeine (3, 25), capsaicin (13), creatine (4), and amino acids (2), have been investigated; however, their efficacy in enhancing physical performance is limited and still controversial (12).
Fat is an important energy source for resting and contracting muscle (11, 23), and its effective utilization is thought to be important for the improvement of physical endurance (9). Wang et al. (27) reported that overexpression of peroxisome proliferator-activated receptor-δ (PPAR-δ) in skeletal muscle leads to an increase in running endurance, together with the upregulation of lipid metabolism-related molecules. Moreover, research on the effects of caffeine and capsaicin suggests that they improve endurance capacity by stimulating lipid metabolism (13, 25). Some studies reported that consumption of a high-fat diet for several days before exercise modified patterns of energy substrate utilization and had an impact on subsequent performance (9). Therefore, it is considered that the effective utilization of fat as an energy source by stimulating lipid catabolism leads to the saving of glycogen and results in an increase in endurance.
Green tea, one of the most popular beverages consumed in Asian countries, contains a class of polyphenols known as catechins, which consist mainly of epigallocatechin gallate, epicatechin gallate, gallocatechin, and epigallocatechin. Green tea and catechins have been reported to have a variety of nutritional and pharmacological properties, including anti-carcinogenic (30), anti-diabetic (17), and anti-atherogenic effects (18). Consequently, there is growing interest in the use of catechins for the treatment and prevention of diseases. Our laboratory has been studying the nutritional function of green tea extract (GTE) in humans and mice (20, 21) and recently showed that habitual intake of catechin-rich GTE prolongs the swimming times to exhaustion of mice in a current water pool experiment system (19).
The beneficial effects of GTE are assumed to be mediated, at least in part, by the stimulation of lipid catabolism in the liver and skeletal muscle. During the physiological process of exercise, the energy utilization balance between fat and carbohydrate is believed to be important in improving endurance levels. In this study, we hypothesized that GTE with lipid metabolism-modulating effects improves endurance capacity by stimulating fatty acid catabolism, particularly during exercise. To test our hypothesis and to further clarify the efficacy of GTE, we examined its effects on running endurance, whole body energy utilization during exercise, and the underlying mechanisms of lipid metabolism in BALB/c mice.
MATERIALS AND METHODS
GTE rich in tea catechins was prepared and analyzed, as described previously (19). In brief, green tea leaves (Camellia sinensis) were extracted with hot water, and the extract was reduced to a powder using the spray-dry method. This extract was dissolved in hot water and mixed with an equal volume of chloroform. The aqueous phase was recovered with three volumes of ethanol, and the extract was freeze-dried after the solvent was removed. The composition of catechins was measured by high-performance liquid chromatography (HPLC). Total catechin content was 81% (the sum of each catechin). The composition of the isolated tea catechins was epigallocatechin gallate (41%), epigallocatechin (23%), epicatechin gallate (12%), epicatechin (9%), gallocatechin (7%), gallocatechin gallate (4%), and others (4%). Caffeine content was 0.1%.
Animals and diets.
Six-week-old male BALB/c mice (Charles River, Kanagawa, Japan) were maintained at 23 ± 2°C under a 12:12-h light-dark regime (light from 0700 to 1900). They received a standard diet (CE-2, CLEA Japan, Tokyo, Japan) and had free access to drinking water. At 7 wk of age, they received running training, and their initial endurance levels were measured as described below. The mice were divided into four groups (n = 8), all of which were allowed unlimited access to water and given a synthetic diet containing 10% (wt/wt) fat, 20% casein, 55.5% potato starch, 8.1% cellulose, 2.2% vitamins, 0.2% methionine, and 4% minerals. The diet of the experimental animals was supplemented with 0.2 or 0.5% GTE. Animals were maintained on their respective diets for 10 wk. During the experiments, animals were cared for in accordance with the American Physiological Society Guiding Principles in the Care and Use of Animals. This study was approved by the Animal Care Committee of Kao Tochigi Institute, Japan.
Exercise and evaluation of endurance.
A 10-lane motorized rodent treadmill (Muromachi Kikai, Tokyo, Japan) was used to determine the running endurance capacity of the mice. At 7 wk of age, mice were run on a treadmill at an inclination of 7° and underwent a 5-day running program to enable them to run at 25 m/min as follows: 1st day: 5 m/min for 5 min, 10 m/min for 15 min, and 15 m/min for 10 min; 2nd day: 5 m/min for 5 min, 10 m/min for 10 min, and 15 m/min for 15 min; 3rd day: 10 m/min for 5 min, 15 m/min for 15 min, and 20 m/min for 10 min; 4th day: 15 m/min for 5 min, 20 m/min for 15 min, and 25 m/min for 10 min; and 5th day: 15 m/min for 5 min, 20 m/min for 5 min, and 25 m/min for 20 min.
At the age of 8–9 wk, running times to exhaustion were measured according to the following program, performed twice: 10 m/min for 6 min, 12 m/min for 2 min, 14 m/min for 2 min, 16 m/min for 2 min, 18 m/min for 2 min, 20 m/min for 2 min, 22 m/min for 2 min, 24 m/min for 2 min, 26 m/min for 2 min, and 28 m/min to the end.
To reduce the inherent variation in running capacity, those mice whose mean running times were 30% longer or shorter than the average were eliminated from the study. Also eliminated were mice whose running times varied greatly over two measurements. Using these criteria, 32 of 70 mice were selected and divided into experimental groups with similar running times and body weights.
At 9 wk of age, the mice were divided into four groups, and the feeding and exercise programs were begun. During the experimental period, mice were exercised on a treadmill three times a week at a speed of 15 m/min for 30 min, with the exception of the nonexercise (non-Ex) control mice. Eight weeks after the start of the experiment, the endurance capacity for running was measured as described above. A mouse was deemed to be fatigued when it was no longer able to continue to run. On the final day of the experiment, mice were exercised between 1300 and 1600, according to the following program: 10 m/min for 5 min, 15 m/min for 5 min, and 20 m/min for 30 min. Immediately after running, mice were killed and dissected.
Analysis of energy metabolism during exercise was carried out during the 8–9 wk of the experiment using an individual open-circuit indirect calorimeter (Oxymax; Columbus Instruments, Columbus, OH) equipped with a four-lane airtight rodent treadmill (Modular Treadmill System, Columbus Instruments), with one mouse per compartment.
Mice were deprived of food overnight, allowed access to the control synthetic diet for 1 h, and then accustomed to a treadmill chamber for 2 h. They were then made to run at a speed of 10 m/min for 5 min, 15 m/min for 5 min, and 20 m/min for 50 min. During this time, data for each chamber were collected every 5 min with a settling time of 45 s, a measurement time of 15 s, and the reference as room air. The respiratory exchange ratio (RER) was calculated from the measured values of oxygen consumption and carbon dioxide exhalation.
On the final day of the experiment, blood samples were collected from the postcaval vein of mice immediately after running. Plasma triglyceride, glucose, and nonesterified fatty acid (NEFA) concentrations were measured using the Triglyceride E-test, Glucose CII test, and NEFA-C test assay kits (WAKO, Osaka, Japan), respectively. Plasma lactate levels were determined using Lactate Pro (Arkley, Kyoto, Japan), according to the manufacturer’s instructions.
Muscle glycogen content was determined, as described by Hassid and Abraham (8). In brief, ∼100 mg of gastrocnemius muscle were digested in 300 μl of 30% KOH for 30 min at 100°C. Saturated sodium sulfate (50 μl) was added, and the glycogen was precipitated by adding 500 μl of 95% ethanol. The solution was heated to 100°C, stirred, then cooled, and centrifuged at 1,600 g. The supernatant was decanted, and the remaining alcohol was removed by heating. The pellet was dissolved in 200 μl of H2O and precipitated with 250 μl of 95% ethanol. After centrifugation at 1,600 g, the supernatant was decanted, and the remaining alcohol was removed by heating. Purified glycogen was hydrolyzed in 600 μl of 0.6 N HCl at 100°C for 2.5 h. The solution was cooled, and the glucose concentration was measured using the Glucose CII test kit. For calculating the amount of glycogen from the concentration of glucose in the hydrolyzed glycogen sample, a conversion factor of 0.93 was used.
Muscle malonyl-CoA content was measured as previously described using reversed-phase HPLC (5). Gastrocnemius muscle was homogenized on ice with four volumes of 5% sulfosalicyclic acid in 50 μM dithioerythritol and centrifuged at 600 g for 10 min. Aliquots of the supernatant (20 μl) were applied to a ODS Hypersil (C18) column (Shimazu, Kyoto, Japan) equipped with a ODS guard column.
Elution solvent A was 100 mM sodium phosphate and 75 mM sodium acetate, adjusted to pH 4.6. Solvent B was 70% solvent A in methanol. The elution was carried out at ambient temperature at a flow rate of 1.5 ml/min. The profile of the gradient was as follows: 0 min, 90% solvent A; 10 min, 60% solvent A; and 17.6 min, 10% solvent A. Standard malonyl-CoA (Sigma, St. Louis, MO) was prepared by dissolving in 5% sulfosalicyclic acid containing 50 μM dithioerythritol. HPLC chromatographic peaks were identified using a 254-nm UV detector (L-4250 UV-VIS detector: Hitachi, Japan).
Fatty acid β-oxidation activity measurement.
Fatty acid β-oxidation activity was measured as described previously (19) using [1-14C]palmitic acid as a substrate. Frozen gastrocnemius muscle was thawed and homogenized on ice using a Physcotron homogenizer (Microtech, Chiba, Japan) in 250 mM sucrose, and 1 mM EDTA in 10 mM HEPES (pH 7.2). Subcellular debris was removed by centrifugation at 600 g for 5 min, and the supernatant was used for assay. Values were expressed as percentages, taking levels in non-Ex mice as 100%.
Plasmids and transfection assay.
To generate the pBIND-GAL4-PPARαLBD and pBIND-GAL4-PPARδLBD chimeric receptor expression plasmids (14, 29), cDNAs encoding the ligand binding domains (LBDs) of the human PPAR-α (amino acids 167–468) and the rat PPAR-δ (amino acids 138–440) were amplified by polymerase chain reaction and subcloned into the pBIND-GAL4 expression plasmid (Promega, Madison, WI). The pG5luc reporter plasmid containing GAL4 binding site was obtained from Promega.
CV-1 cells were plated in 12-well plates in Dulbecco’s modified Eagle’s medium supplemented with 5% charcoal-treated fetal bovine serum (Thermo Trace, Melbourne, Australia). After 1 day, transfections were performed with SuperFect transfection reagent (QIAGEN), according to the manufacturer’s instruction. Briefly, transfection mixes for each well contained 6.25 μl SuperFect, 0.375 μg pBIND-GAL4-PPAR-LBD expression plasmid, and 0.375 μg pG5luc reporter plasmid. Cells were incubated in the transfection mixture for 3 h at 37°C in an atmosphere of 5% CO2. The cells were then incubated for 4 h in fresh Dulbecco’s modified Eagle’s medium (+5% charcoal-treated FBS). After treatment with or without each sample for 20 h, cells were lysed, then firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). We used Wy14643 (Sigma) as a positive control for PPAR-α and GW0742 (Sigma) for PPAR-δ. Catechins were purchased from Wako or Funakoshi (Tokyo, Japan).
All values are presented as means ± SE. Statistical analysis was conducted using unpaired t-tests or ANOVA and subsequently applying Tukey’s test (StatView: SAS Institute, Cary, NC). Two-way repeated-measures ANOVA was used to analyze the results of indirect calorimetric measurements. P < 0.05 was considered statistically significant.
Body weight and tissue weights.
The adipose tissue weights of the exercise control (Ex-cont) group of mice were significantly lower than those of mice that had not been exercised (Table 1), and body weight was slightly lower, although not significantly. Among the groups of mice that had been exercised, intake of the diet with GTE tended to decrease adipose tissue weight dose dependently, although not significantly. There were no differences in body weight among these groups.
Running endurance and energy metabolism during exercise.
Figure 1 shows the running times to exhaustion of the mice after 8 wk of the experiment. Compared with Ex-cont mice, the running times for mice fed 0.2 and 0.5% GTE were 21 and 30% longer, respectively.
To reveal the interaction between enhanced endurance and energy metabolism during exercise, we examined whole body energy metabolism of mice by indirect calorimetry. As the mice began to run, their V̇o2 gradually increased, although there was no difference between Ex-cont and GTE groups (Fig. 2, A and B). By contrast, the RER of GTE groups was significantly lower throughout the running period (P < 0.01, Fig. 2C). The average RER over the 60-min period following the start of exercise was 0.828 in the Ex-cont group and 0.813 in the 0.5% GTE group (Fig. 2D), indicating an increased utilization of lipids to generate running energy in GTE-fed mice.
Effects of GTE on blood components immediately after exercise.
We measured concentrations of blood components immediately after running to reveal the changes by exercise. Plasma lactate levels were significantly higher in Ex-cont mice than in the non-Ex group (Fig. 3A, P < 0.05). Supplementation of the GTE dose-dependently and significantly suppressed lactate production to levels approaching those of non-Ex mice (P < 0.05). In contrast, GTE dose-dependently increased plasma NEFA concentrations (Fig. 3B). Plasma glucose and triglyceride levels were low in mice that had been exercised compared with the non-Ex group (Fig. 3, C and D); however, the differences were not significant.
Effects of GTE on muscle glycogen content.
Muscle glycogen content was significantly lower in the Ex-cont group than in the non-Ex group, whereas supplementation with GTE suppressed the lowering of glycogen content in a dose-dependent manner (Fig. 4). In 0.5% of GTE-fed mice, the decrease in glycogen content was smaller by 84% than that in the Ex-cont group.
Effects of GTE on fatty acid β-oxidation in skeletal muscle.
Fatty acid β-oxidation activity in skeletal muscle was significantly increased in mice that had been exercised (Fig. 5) and further increased by the intake of GTE. Supplementation of the 0.5% GTE increased activity by 109 and 35% compared with the non-Ex and Ex-cont groups, respectively.
Effects of GTE on muscle malonyl-CoA content.
To reveal the mechanisms underlying the lipid oxidation-stimulating effect of GTE, we examined the effects of GTE on the malonyl-CoA content in skeletal muscle. Malonyl-CoA levels in skeletal muscle were significantly decreased after exercise (Fig. 6), consistent with the findings of a previous study (28). Furthermore, in mice whose diet was supplemented with 0.5% GTE, malonyl-CoA levels were significantly lower than those of non-Ex and Ex-cont mice. These results suggest that stimulation of β-oxidation activity by GTE is regulated by the amount of malonyl-CoA in skeletal muscle.
Effects of catechins on PPAR activation.
Transcription of many lipid metabolism-related enzymes is regulated by PPARs (15). We tested the possibility that eight kinds of catechins rich in GTE act as ligands for PPAR-α and -δ using the luciferase assay system. However, none of catechins showed a marked effect on luciferase activity, suggesting that catechins do not act as direct ligands, or are only weak ligands for PPAR-α and -δ (Fig. 7).
In this study, we examined the effects of GTE on running endurance in mice and demonstrated that dietary supplementation with GTE markedly improved endurance levels in association with an increase in lipid utilization during exercise. We suggest that the enhancement of lipid catabolism by GTE is mediated, at least in part, by a lowering of the malonyl-CoA content, thereby stimulating fatty acid oxidation in skeletal muscle. The results of this study support our laboratory’s previous finding that GTE improves swimming endurance in mice (19) and demonstrate that GTE also improves endurance in an alternative form of exercise.
Skeletal muscle mainly catabolizes fat and carbohydrate as sources of energy during exercise (11, 12). The significant reduction in RER and the high muscle glycogen content in mice that received a dietary supplement of GTE suggest enhanced utilization of fat as an energy source. In addition, plasma lactate concentrations decreased and muscle glycogen content was maintained higher in GTE-fed mice immediately after exercise (Figs. 3A and 4), suggesting that the intake of GTE suppresses the utilization of carbohydrate, thus sparing glycogen and resulting in an increase in running endurance.
Although the detailed molecular mechanism by which GTE enhances fat use during exercise remains to be elucidated, activation of lipid metabolism in skeletal muscle is believed to be a key factor. Fatty acid oxidation activity in skeletal muscle immediately after exercise was high in GTE-fed mice (Fig. 5), as in the resting condition in a previous report (19). These results indicate that the capacity for fatty acid oxidation in skeletal muscle was enhanced by the intake of GTE in combination with habitual exercise. We have confirmed that eight kinds of catechins that are the main constituents of GTE do not act as ligands for PPAR-α and -δ, which are transcription factors that regulate the expression of genes related to lipid metabolism (15), using the luciferase assay system (Fig. 7). Therefore, there is a possibility that the increase in muscle β-oxidation activity caused by GTE is posttranscriptional or secondary and not a result of direct induction of fatty acid oxidation-related enzymes at the PPAR-dependent transcriptional level.
It has been recognized that a decrease in the content of malonyl-CoA during exercise contributes to an increase in fat oxidation by relieving inhibition of carnitine-palmitoyltransferase I (24), secondary to activation of AMP-kinase and inhibition of acetyl-CoA carboxylase by phosphorylation. In the present study, we found that the malonyl-CoA content of skeletal muscle was significantly lower in GTE-fed mice than the Ex-cont group, suggesting that the marked decrease in the level of malonyl-CoA during exercise following the intake of GTE contributes to increased fatty acid oxidation. Moreover, we also showed that GTE causes a transient but repeated increase in the plasma fatty acid concentration during exercise (Fig. 3); therefore, repeated exposure to increased levels of plasma fatty acids during exercise might produce adaptive changes that stimulate fatty acid oxidation. In addition, this result suggests the involvement of adipose tissues in the GTE-dependent improvement of endurance levels.
Catecholamines released from sympathetic nerves during exercise promote lipid mobilization and thermogenesis via β3-adrenoreceptors (10, 22, 26). Because catechins abundant in GTE are reported to inhibit catechol-O-methyltransferase (the enzyme that degrades catecholamines) in vitro (1, 16), it is possible that GTE could prolong and augment the sympathetic stimulation of fat oxidation, especially in combination with exercise. In this context, GTE might influence lipid metabolism in adipose tissue and skeletal muscle during exercise, thereby affecting the energy balance and, hence, the endurance capacity. Some studies have shown that catechins act synergistically with caffeine in their enhancement of fat oxidation (6, 7). For this reason, we used GTE that did not contain caffeine in this study, to focus exclusively on the effects of tea catechins. Conversely, the simultaneous intake of GTE and caffeine has the possibility to effectively improve endurance by activating fat oxidation synergistically.
The clinical effect of GTE on endurance has not yet been confirmed in humans. However, our laboratory previously demonstrated that long-term intake of 0.2–0.5% GTE had an antiobesity effect in mice (20), and visceral fat mass was decreased following a 3-mo intake of 690 mg/day GTE in humans (21). Taking into consideration the findings of our antiobesity studies, one might expect a positive effect on endurance capacity upon drinking a proper amount of GTE, in combination with habitual exercise in humans.
In summary, we have shown that long-term intake of catechin-rich GTE, together with habitual exercise, is beneficial in improving endurance capacity, and that these effects might be attributed, at least in part, to the stimulation of whole body lipid metabolism. These results suggest that a sufficient intake of GTE, in combination with exercise, might improve endurance by modulating lipid metabolism and could potentially inhibit the development of obesity and associated lifestyle-related diseases.
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