The effect of Katsura-uri (Japanese pickling melon, Cucumis melo var. conomon) and its derived ingredient methylthioacetic acid on energy metabolism during aerobic exercise

Human subjects

Eight healthy men (mean age, 21.4 ± 0.7 years; height, 172.3 ± 7.1 cm; body weight,
63.7 ± 4.9 kg; body mass index, 21.5 ± 2.1 kg/m
²
) were recruited as participants in a single-blind, crossover study, which was approved
by the ethics committee of Kyoto Prefectural University (No. 45). All the subjects
provided written informed consent. No subject had a current or prior chronic disease
or a history of smoking, and no subject was currently using any medication. Moreover,
no subject was habituated to a regular exercise regimen.

Test drink

Katsura-uri was cultivated and harvested after it was ripened in 2012–2013 in an open
field culture system at the Kyoto Prefectural Agricultural Research Institute (Kameoka,
Japan). After removal of the peels and seeds, the residual pieces were vacuum-packed
with a plastic bag and stored at ?25°C until ready for use in the experiment. Katsura-uri
drink was prepared by the following method: 500 g of frozen Katsura-uri fruit was
partially thawed, chopped, and mixed by using an electric automatic mixer with 10.6 g
of a sweetener (Pal Sweet; Ajinomoto, Tokyo, Japan). The placebo drink was made by
dissolving 6.0 g fructose, 5.5 g glucose, and 2.5 g sucrose in 484 g water, while
adjusting for the equivalent composition and amount of carbohydrates contained in
Katsura-uri (unpublished data), and adding 10.6 g Pal Sweet. Subjects were not informed
about the characteristics of Katsura-uri, which limited their ability to distinguish
between active and placebo drinks and removed bias. Thus, this study was performed
under a single-blind condition. The Katsura-uri drink and the placebo were prepared
on the day before each trial, and administered to subjects by pouring them into identical
cups; therefore, the outward appearance of the cup did not distinguish between Katsura-uri
and placebo drinks.

Human experiment design

The subjects were asked to fast, except for water consumption, from 22:00 the night
prior to the experiment. On the experiment day, all subjects consumed the same breakfast
(200 g of steamed rice and 170 ml of miso soup [Asage; Nagatanien, Tokyo, Japan])
at 8:30 to normalize the effects of a pre-exercise meal. Thirty minutes after drinking
the Katsura-uri drink or the placebo beverage at 10:00, all participants performed
a single session of steady-state cycling exercise for 30 min. The work load was gradually
increased by 10 W every 2 min beginning with 50 W until the heart rate reached the
predicted 40% of the maximum heart rate, which determined by using the Karvonen formula
(Karvonen 1957]), and this work load was maintained until the end of exercise. The measurement of
respiratory gas was initiated 15 min before exercise and continued until after exercise.
Blood glucose and lactate levels were measured at rest and after 15 and 30 min of
beginning exercise by using a simple finger stick blood test (Lactate Pro, Glu Test;
Arkray, Kyoto, Japan). There was a 1-week washout period prior to the test, and all
the subjects completed both testing conditions.

Indirect metabolic performance

Respiratory gas oxygen consumption (VO
₂
) and carbon dioxide production (VCO
₂
) were measured using a breath-by-breath respirometer system (Aeromonitor AE310S;
Minato, Osaka, Japan). To reduce breath-by-breath variation, these data were analyzed
using a mean value obtained every 60 s. The RQ and substrate utilization were calculated
from the level of VO
₂
and VCO
₂
, as described previously (Frayn 1983]).

Mice experimental design

This study complied with the guidelines of the Japanese Council on Animal Care, and
it was approved by the Committee for Animal Research of the University (M24-49). ICR
mice (7-week-old; Shimizu Laboratory Supplies, Kyoto, Japan) were acclimatized for
1 week in an air-conditioned (22 ± 2°C) room on a 12-h light/dark cycle (lights on
from 7:30 to 19:30). Mice were divided into the following 4 groups with 10 mice in
each group: sedentary, exercise with control, exercise with supplementation of 25 ppm
MTA, and exercise with supplementation of 250 ppm MTA.

MTA solutions were prepared with water to suitable concentration and given to mice
30 min before exercise (10 ?L/g body weight). Water was provided to control mice in
the same volume as MTA supplementation mice. After the oral administration, mice in
the exercise groups ran on a treadmill at 25 m/min for 30 min. Immediately after exercise,
intermuscular pH was measured under anesthesia, and blood concentrations of glucose
and lactate were obtained and measured (GluTest, LactatePro; Arkray) from the tail
vein. The gastrocnemius muscles and blood via cardiocentesis were collected. The sedentary
mice underwent the same blood and muscle collection procedures. Blood ammonia and
plasma NEFA levels were measured using assay kits, respectively (Wako, Osaka, Japan).

Glycogen content in skeletal muscle

Muscle tissues were homogenized in 0.3 M hypochlorous acid, incubated with amyloglucosidase
(25 mg protein/6 mL) and 20 mM sodium acetate, and quantified using a D-glucose measurement kit (F-kit, Roche Diagnostics, Basel, Switzerland).

Enzyme activity in skeletal muscle

Enzyme activity was measured using muscle homogenates. SDH and CPTI levels were measured
according to previously described protocols (Cooperstein et al. 1950]; Bieber et al. 1972]). PDH activity was measured using a commercial enzyme-linked sorbent assay kit (Abcam,
Cambridge, UK), and data was expressed as OD change at 450 nm for 15 min of enzymatic
reaction.

Acid-hydrolyzation from MTAE to MTA in artificial gastric juice

Five milligrams of MTAE (Alfa Aesar, Lancashire, UK) was dissolved with 5.0 mL of
artificial gastric juice (actual concentration of MTAE: 1 mg/mL or 7.45 mM). The tube
was incubated for 0.5–30 h at 37°C, a 200 ?L aliquot of the solution was withdrawn
at several time points, and residual MTAE and MTA (Matrix Scientific, Columbia, SC,
USA) formed in the artificial gastric juice were measured by using reverse-phase HPLC–UV.
The levels of MTAE and MTA was measured using an HPLC model LC-20AT with a SPD-20A
UV detector (Shimadzu, Kyoto, Japan). A YMC-pack Pro C18-RS (Ø4.6 × 150 mm) analytical
column was used for MTAE and MTA analyses based on reverse phase chromatography. A
10 ?L aliquot of sample was injected and isocratically eluted using 18% acetonitrile
in 0.1% trifluoroacetate mobile phase. The UV absorbance at 230 nm was used to detect
MTA at 3.5 min and MTAE at 23.5 min.

Statistical analysis

All data are presented as the mean ± SD. Differences between groups were evaluated
using a two-way analysis of variance. When significant interactions were detected,
post hoc multiple comparisons were conducted using the Bonferroni method. When data
were not normally distributed, differences between groups were tested using non-parametric
tests. To further assess time course comparisons, the mean differences in blood parameter
changes between conditions or time points as well as the 95% confidence intervals
were calculated. The correlation between substrate oxidation and lactate concentration
was evaluated by performing Spearman’s correlation analysis. P  0.05 was considered
statistically significant.