J Appl Physiol 116: 674–682, 2014.
First published January 23, 2014; doi:10.1152/japplphysiol.00405.2013.
Effects of prepubertal-onset exercise on body weight changes up to middle
age in rats
Daisuke Shindo, Tomokazu Matsuura, and Masato Suzuki
Department of Laboratory Medicine, The Jikei University School of Medicine, Tokyo, Japan
Submitted 4 April 2013; accepted in final form 20 January 2014
body weight; body fat mass; lean body mass; energy metabolismrelated enzymes; UCP-3 mRNA
BETWEEN 1978 AND 2007, THE prevalence of obesity increased by
nearly threefold in young Japanese children (5– 6 yr) and
schoolchildren (7–17 yr) (44). Other developed countries have
experienced similar epidemics (24, 37), although they have
shown some signs of slowing (24, 44). The influence of
heredity on obesity has been demonstrated by family studies
(4, 39), especially Borjesson’s work on twins (4) and Stunkard
and colleagues’ research on adopted children (39). However,
reduced energy expenditure, particularly a lack of physical
activity, is also an important factor in the rapid weight gain
observed during the first year of life in infants born to overweight mothers (31). Research on an animal model genetically
predisposed to become obese found that prepubertal-onset
exercise treatment resulted in lowered body weight (BW) after
cessation of exercise, whereas a food-restriction treatment
Address for reprint requests and other correspondence: M. Suzuki, Dept. of
Laboratory Medicine, The Jikei Univ. School of Medicine, 3-25-8 Nishishimbashi, Minato-ku, Tokyo 105-8461, Japan (e-mail: [email protected]).
674
resulted in rapid BW gain after the restriction period (27).
Interestingly, sustained suppression of BW gain after exercise
cessation was not observed in adult rats placed on a similar
exercise regimen (22, 43).
Exercise-induced BW reductions are known to be regained
quickly after cessation of exercise regimens in both obese
humans and rodents (21, 42); such BW rebounds are at least
partly driven by reductions in resting metabolic rate (RMR)
and/or lean body mass (LBM) (21, 30). The latter is known as
the major determinant of RMR. One reason for this recidivism
might be a genetic predisposition to a particular BW in which
lost BW is regained to the predetermined BW. Indeed, individuals of many species are known to regulate BW from a
predetermined set point (11). However, studies have demonstrated that prepubertal-onset exercise may induce a sustained
decrease in the higher-predisposed BW set points in obese
individuals, even after the cessation of exercise regimens (15,
17, 18, 27).
To our knowledge, the mechanism of sustained suppression
of BW gain by prepubertal-onset exercise remains unsolved,
although this may be associated with increased “energy expenditure” in the skeletal muscles rather than decreased “energy
intake” (15, 17, 18, 27). Indeed, animal studies have demonstrated that prepubertal-onset exercise did not affect anabolic
or catabolic hypothalamic neuropeptide expression, both of
which are associated with energy intake behavior (20). Thus
we hypothesized that, regardless of an individual’s genetic
makeup, daily physical activity early in life maintains higher
levels of LBM and metabolic activity in the skeletal muscles,
as well as reduces the future likelihood of becoming obese. As
studies using an animal model of human obesity (13, 27) have
investigated the effects of prepubertal exercise on BW during
a relatively short term (⬃7 wk) in the developmental period, it
is also of interest to discern whether the effect would be
sustained in obese animals in later life.
In the present study, we examined whether maintenance of
prepubertal exercise-induced BW loss in genetically predisposed obese rats can be sustained until the period corresponding to middle age in humans. Furthermore, we investigated
whether inhibition of BW gain is facilitated by increased
energy metabolism. Our results further confirmed the benefits
of prepubertal exercise for inhibiting BW gain in obese individuals in later life.
METHODS
Animals. The Tokushima Research Institute (Otsuka Pharmaceutical, Tokushima, Japan) provided all 31 rats: 23 male Otsuka LongEvans Tokushima Fatty (OLETF) rats, which are characterized by
insulin resistance, accumulated intra-abdominal fat, dyslipidemia, and
type 2 diabetes (22, 23, 40), and 8 male Long-Evans Tokushima
Otsuka (LETO) rats, which were used as normal controls. As it has
been reported that running wheel activity efficiently prevented obesity
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Shindo D, Matsuura T, Suzuki M. Effects of prepubertal-onset
exercise on body weight changes up to middle age in rats. J Appl
Physiol 116: 674 – 682, 2014. First published January 23, 2014;
doi:10.1152/japplphysiol.00405.2013.—The present study was conducted to examine whether prepubertal-onset exercise might help
adults maintain long-term body weight (BW) reduction and increased
energy metabolism after the cessation of exercise. Furthermore, the
effects of the exercise regimen were compared with those of food
restriction. Twenty-three male obese-diabetic [Otsuka Long-Evans
Tokushima Fatty (OLETF)] rats were randomly assigned to prepubertal-onset exercise (Childhood-Ex), food restriction (ChildhoodDiet), and sedentary control (OLETF-Sed) groups. Childhood-Ex rats
exercised voluntarily every day using a rotating wheel, while the food
volume of the Childhood-Diet group was restricted to achieve a BW
similar to that recorded in the Childhood-Ex group. Both treatments
were conducted at 5–19 wk of age; after this period, the rats were kept
sedentary and allowed ad libitum food intake until 45 wk of age. BW
was significantly lower, and percent lean body mass was significantly
higher, in the Childhood-Ex group compared with those in the
Childhood-Diet and OLETF-Sed groups throughout maturation and
middle age after cessation of the interventions. The Childhood-Ex
group also demonstrated higher citrate synthase, succinate dehydrogenase, and phosphofructokinase activity levels, as well as uncoupling
protein-3 mRNA expression in skeletal muscle. This study revealed
that inhibited BW gain in an animal model of human obese diabetes
by prepubertal-onset exercise lasted for a long period after the completion of the exercise intervention. This effect may be facilitated by
increased energy metabolism. However, these benefits were not found
by prepubertal food restriction treatment. Importantly, to allow translation of our work, these novel insights need to be assessed in obese
human individuals.
Effects of Prepubertal Exercise in Rats
Shindo D et al.
675
(Aloka, Tokyo, Japan), was used to analyze contiguous 2-mm slice
images to quantify SFM, VFM, and LBM volumes (25). Values are
reported as %fat {[(SFM ⫹ VFM)/BW] ⫻ 100}, and %LBM [(LBM/
BW) ⫻ 100].
Analysis of biochemical components in the blood. When the rats
were 46 wk of age (ⱕ1 wk after completing the final blood sampling
and body composition analysis), they were placed on an overnight fast
and then anesthetized with pentobarbital (50 mg/kg ip) via a catheter
placed in the external jugular vein. We rapidly drew blood samples
(⬃10 ml), which were used to measure levels of serum free fatty acid,
TG, total cholesterol (Tcho), and HDL-cholesterol (HDL-C) using a
Hitachi 7600 –210 automatic analyzer (Hitachi, Tokyo, Japan). LDLcholesterol (LDL-C) levels were calculated using the Friedewald
equation (LDL-C, mg/dl ⫽ Tcho ⫺ HDL-C ⫺ TG/5).
Measurements of energy metabolism-related enzyme activity and
uncoupling protein-3 mRNA expression in the skeletal muscle. All rats
were euthanized by bloodletting, after which one quadriceps femoris
muscle per animal was removed and weighed. A portion of the muscle
was stored at ⫺80°C for later measurement of citrate synthase (CS),
succinate dehydrogenase (SDH), and phosphofructokinase (PFK).
Uncoupling protein-3 (UCP-3) mRNA expression was quantified by
real-time PCR using the ABI StepOnePlus system and software (8, 9,
14). CS, SDH, and PFK are the key regulatory enzymes in the
energy-generating metabolic pathway, while UCP-3 may serve as a
mitochondrial mediator of fatty acid metabolism in the muscle (5, 9).
Statistical analysis. All data are expressed as means ⫾ SE. Differences among the treatment groups were examined using one-way
ANOVA, followed by post hoc tests (Fisher’s protected least significant difference). Student’s paired t-tests were used to investigate
within-group changes in variables over the course of the study.
Pearson’s tests were used to examine correlations between parameters. A 95% confidence level was accepted as significant for all of the
statistical tests.
RESULTS
Changes in BW and food consumption. The mean BW of the
OLETF rats at 5 wk of age (135.3 ⫾ 6.5 g, n ⫽ 23) was
significantly higher than that of the LETO rats (118.3 ⫾ 1.8 g,
P ⬍ 0.05, n ⫽ 8); however, there were no differences in BW
among the three OLETF groups. As the rats aged (from 7 to 45
wk), gradual increases in BW were observed in the OLETFSed and LETO-Sed groups; however, rats in the OLETF-Sed
group had significantly higher BW compared with that of in
those in the LETO-Sed group (P ⬍ 0.001, Fig. 1A). The
Childhood-Ex group and Childhood-Diet group experienced
comparable changes in BW between 5 and 19 wk of age. From
15 wk of age, the mean BW in both the Childhood-Ex and
Childhood-Diet groups was significantly lower compared with
that in the OLETF-Sed group. At the end of the study period,
the mean BW in the Childhood-Ex, Childhood-Diet, and
OLETF-Sed groups were 460.8 ⫾ 15.3, 460.9 ⫾ 16.0, and
579.6 ⫾ 13.3 g, respectively. The mean BW was significantly
lower in the Childhood-Ex group than in the OLETF-Sed
group until the rats were 40 wk of age, after which this
difference in BW was only a trend. BW in the Childhood-Diet
group increased rapidly after completion of the food restriction
treatment and was significantly different from that in the
Childhood-Ex group when the rats were 24 wk of age. Surprisingly, however, it was not significantly different from that
in the OLETF-Sed group at 28 wk of age.
Absolute FI nearly plateaued in each group by the time the
rats were 8 wk of age (Fig. 1B). However, FI in the ChildhoodDiet group gradually increased during the latter half of the
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in OLETF rats, but not in Zucker fatty rats (32), we used this animal
model of human obesity in this study.
All rats were 4 wk old at the time of purchase. The rats were placed
in conventional plastic cages (36 ⫻ 26 ⫻ 20 cm) for 1 wk to become
acclimatized to the experimental environment. They were fed standard
solid rat chow (CE-II, CLEA, Tokyo, Japan) and tap water ad libitum
and were housed in dedicated facilities under specific pathogen-free
conditions. Temperature (20.5 ⫾ 1.2°C), humidity (43.0 ⫾ 7.9%),
lighting (0600 –1800), and air conditioning were controlled throughout the experimental period. This study was conducted in accordance
with the guiding principles of The Physiological Society of Japan. The
experimental protocol was approved by the Committee for the Care
and Use of Animals of the Jikei University School of Medicine.
Experimental design. It has been reported that a higher incidence of
obesity-related diabetes in humans appears from the maturation stage
to the middle-age period (35–50 yr of age). OLETF rats develop
diabetes around 20 wk of age, and their levels of serum glucose,
triglyceride (TG), and urine protein are known to dramatically increase around 30 wk of age (16, 35). Therefore, in the present study,
childhood (including adolescence) was defined as the period of 5–19
wk; adulthood (maturation stage) as the period of 20 –34 wk; and
middle age as the period after 34 wk. The OLETF rats were randomly
assigned to one of three groups: a childhood exercise group (Childhood-Ex, n ⫽ 7), in which the rats exercised at 5–19 wk of age; a
childhood food-restriction group (Childhood-Diet, n ⫽ 8), in which
food intake (FI) was limited while the rats were 5–19 wk of age; and
a sedentary control group (OLETF-Sed, n ⫽ 8), in which the rats were
kept sedentary and allowed food and tap water ad libitum throughout
the experiment. The Childhood-Ex group exercised voluntarily using
a rotating wheel made of wire mesh (20 ⫻ 20 ⫻ 25 cm, Shinano,
Tokyo, Japan) attached to the cage. To mimic a BW similar to that
recorded in the Childhood-Ex group, we restricted the FI of the
Childhood-Diet group to roughly 70 – 85% of the food volume consumed by the OLETF-Sed group (40). Rats in the Childhood-Ex and
Childhood-Diet groups were allowed to have resting periods and take
food freely when they were 20 – 45 wk of age. Eight male LETO rats
were assigned to the sedentary normal control group (LETO-Sed).
BW, FI, and running distance measurements. BW (g), FI (g),
volume of water consumed (ml), and running distance (m) were
measured weekly throughout the experiment. Running distance was
recorded by a cyclometer attached to the wheel’s axis, which recorded
the number of wheel revolutions per week.
Measurements of changes in fasting blood glucose, insulin, and
leptin levels. When the rats were 7, 12, 20, 27, 35, and 45 wk of age,
we placed them under diethyl ether anesthesia and collected blood
samples (⬃500 ␮l) from the retroorbital sinus; before sample collection, all rats were subjected to an overnight fast at 1800 – 0900 (24,
41). Serum specimens were stored at ⫺20°C until fasting serum
glucose (FSG; Glucose C II-test, Wako, Wako Pure Chemical Industries, Osaka, Japan), insulin (FSI; ELISA kit, Shibayagi, Gunma,
Japan), and leptin (FSL; Rat Leptin ELISA kit, Yanaihara Lab,
Shizuoka, Japan) levels could be examined. The homeostasis model
assessment of insulin resistance (HOMA-IR), which was developed to
assess hepatic insulin resistance in humans, was used to calculate
insulin resistance according to the following formula: HOMA-IR ⫽
FSI (␮U/ml) ⫻ FSG (mg/dl)/405. The resulting value was then used
to assess hepatic insulin resistance in rats according to the methods of
Anwer et al. (1).
Measurements of changes in whole body subcutaneous fat mass,
visceral fat mass, and LBM. Whole body subcutaneous fat mass
(SFM), visceral fat mass (VFM), and LBM were measured when the
rats were 4, 7, 12, 20, 27, 35, and 45 wk of age using a radiographic
computed tomography scan apparatus (25). The rats were anesthetized
via isoflurane inhalation 5– 6 h after the absorptive period. The
anesthetized rats were placed in the computed tomography scanner,
and the entire body of each was scanned along the body axis at 2-mm
intervals. Latheta software version 1.10 with a Latheta LCT-200
•
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Effects of Prepubertal Exercise in Rats
800
A Body weight
45
•
Shindo D et al.
B Food intake
40
700
g
35
600
30
OLETF-Sed
Childhood-Ex
Childhood-Diet
LETO-Sed
25
20
Post-treatment
Treatment
500
g
15
OLETF-Sed
Childhood-Ex
Childhood-Diet
LETO-Sed
280
C Food intake/BW
240
Treatment
Post-treatment
200
160
OLETF-Sed
Childhood-Ex
Childhood-Diet
LETO-Sed
120
200
80
Treatment
Post treatment
100
40
5
10
15
20
25
30
35
40
45
5
10
15
20
Week
25
30
35
40
45
Week
Fig. 1. Changes in body weight (BW; g; A), absolute quantity of food intake (g/day; B), and relative quantity of food intake per unit BW (g·kg⫺1·day⫺1; C)
throughout the experimental period (5– 45 wk of age). Values are means ⫾ SE. A: note that, in BW, significant differences (P ⬍ 0.05) were found between Otsuka
Long-Evans Tokushima Fatty (OLETF) obese-diabetic sedentary (OLETF-Sed) and prepubertal-onset exercise (Childhood-Ex) at 7– 40 wk of age, between
OLETF-Sed and Childhood-Diet at 7–28 wk of age, between Childhood-Ex and Childhood-Diet at 24 –26 wk of age, and between OLETF-Sed and Long-Evans
Tokushima Otsuka (LETO)-Sed at 5– 45 wk of age. B: in terms of food intake, significant differences (P ⬍ 0.05) were found between the OLETF-Sed and
Childhood-Ex groups at 34 –38 wk of age, between the OLETF-Sed and Childhood-Diet groups at 5–28 wk of age, between the Childhood-Ex and Childhood-Diet
groups at 5–20 and 24 –26 wk of age, and between the OLETF-Sed and LETO-Sed groups at 5– 45 wk of age. C: in relative quantity of food intake, significant
differences (P ⬍ 0.05) were found between the OLETF-Sed and Childhood-Ex groups at 7–33 wk of age, between the OLETF-Sed and Childhood-Diet groups
at 6 – 8, 11, and 12 wk of age, between the Childhood-Ex and Childhood-Diet groups at 5–33 wk of age, and between the Childhood-Ex and LETO-Sed groups
at 5–22 wk of age.
treatment until it was comparable to the FI in the OLETF-Sed
group. Directly after completion of the food-restrictive treatment, the FI in the Childhood-Diet group temporarily increased
but then remained constant. The Childhood-Ex group experienced a gradual reduction of FI from 27 wk of age onward; rats
in this group had significantly lower consumption from 35 wk
of age onward than individuals in the OLETF-Sed group. FI
relative to BW was significantly higher in the Childhood-Ex
group than in the other three groups from 7 to 33 wk of age
(Fig. 1C); however, this value did not vary significantly among
the OLETF-Sed, Childhood-Diet, and LETO-Sed groups. The
mean total running distance during the exercise intervention
period in the Childhood-Ex group was 350 ⫾ 20.0 km
(冱350 ⫾ 20.0 km). The amount of daily activity in the
OLETF-Sed and Childhood-Diet groups was not measured
throughout the experiment. Each animal in each experimental
group was allowed to move freely in a normal cage. However,
the Childhood-Ex group was given voluntary exercise opportunities using a rotator wheel attached to the normal cage. The
amount of activity in the normal cage was estimated to be
almost the same among the OLETF-Sed, Childhood-Diet, and
Childhood-Ex groups. The total amount of physical activity
during the exercise intervention period of the Childhood-Ex
group was the sum of the daily activity in the normal cage and
voluntary exercise (冱350 ⫾ 20.0 km). Therefore, it is evident
that the total amount of activity during the childhood intervention period was greater in the Childhood-Ex group than in the
OLETF-Sed and Childhood-Diet groups.
Changes in HOMA-IR and FSG, insulin, and leptin levels.
Changes in HOMA-IR as well as FSG, FSI, and FSL levels are
shown in Table 1. The mean HOMA-IR value in the Childhood-Ex group was consistent throughout the 15-wk treatment,
after which it was significantly lower than that in the OLTEFSed and Childhood-Diet groups. FSG levels were similar in all
four groups when the rats were 7–12 wk of age. At 20 wk of
age, however, FSG levels were significantly lower in the
Childhood-Ex and Childhood-Diet groups than in the OLETFSed group. Thereafter, the mean FSG level was significantly
lower in the Childhood-Ex group until the rats were 45 wk of
age. In contrast, the mean FSG level in the Childhood-Diet
group gradually increased over this period, approaching the
value recorded in the OLETF-Sed group. Throughout the
experiment, changes in FSI levels were similar to those of FSG
in the Childhood-Ex, Childhood-Diet, and OLETF-Sed groups.
J Appl Physiol • doi:10.1152/japplphysiol.00405.2013 • www.jappl.org
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300
g/kg
400
Effects of Prepubertal Exercise in Rats
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677
Shindo D et al.
Table 1. Changes in fasting serum glucose, insulin, and leptin concentrations, as well as HOMA-IR values
Treatment Period
7 wk
12 wk
20 wk
27 wk
35 wk
45 wk
8
7
8
8
89.1 ⫾ 4.3
88.8 ⫾ 2.8
82.9 ⫾ 1.5
82.9 ⫾ 1.2
88.5 ⫾ 5.1
84.1 ⫾ 5.9
85.7 ⫾ 4.4
83.6 ⫾ 2.1
123.1 ⫾ 10.2
93.3 ⫾ 7.2*
102.8 ⫾ 5.4†
84.1 ⫾ 2.8
141.8 ⫾ 7.0
117.0 ⫾ 3.9*
130.9 ⫾ 4.8
80.0 ⫾ 2.1
185.2 ⫾ 14.4
148.3 ⫾ 9.0*
167.3 ⫾ 8.1
112.7 ⫾ 3.6
229.4 ⫾ 22.1
182.2 ⫾ 16.8*
204.7 ⫾ 11.9
145.4 ⫾ 7.3
8
7
8
8
475.2 ⫾ 57.4
469.9 ⫾ 37.5
391.5 ⫾ 20.7
437.9 ⫾ 48.3
767.6 ⫾ 163.9
617.3 ⫾ 97.6
652.7 ⫾ 109.4
544.1 ⫾ 145.4
1,036.3 ⫾ 101.7
536.5 ⫾ 61.9*
844.1 ⫾ 154.3
586.7 ⫾ 99.0
1,327.1 ⫾ 88.2
745.6 ⫾ 169.8*
955.3 ⫾ 145.7*
491.8 ⫾ 88.2
1,327.2 ⫾ 45.5
868.3 ⫾ 182.0*
1,091.5 ⫾ 138.0
430.3 ⫾ 30.1
1,327.2 ⫾ 51.9
955.5 ⫾ 163.7*
1,234.5 ⫾ 133.8
560.4 ⫾ 101.9
8
7
8
8
452.9 ⫾ 7.6
447.0 ⫾ 19.1
465.3 ⫾ 19.3
426.7 ⫾ 19.8
745.5 ⫾ 31.8
427.6 ⫾ 17.7*
529.7 ⫾ 19.8*†
504.6 ⫾ 15.4
1,299.0 ⫾ 62.8
592.1 ⫾ 57.9*
622.8 ⫾ 23.6*
677.2 ⫾ 37.4
1,828.8 ⫾ 148.1
875.8 ⫾ 105.7*
1,601.4 ⫾ 119.8†
837.7 ⫾ 51.1
1,973.0 ⫾ 130.6
1,179.8 ⫾ 91.8*
1,788.5 ⫾ 80.7†
911.9 ⫾ 92.3
2,475.4 ⫾ 135.2
1,734.8 ⫾ 131.4*
2,465.3 ⫾ 77.1†
966.4 ⫾ 93.1
8
7
8
8
2.8 ⫾ 0.5
2.7 ⫾ 0.3
2.1 ⫾ 0.2
2.1 ⫾ 0.1
4.4 ⫾ 0.9
3.2 ⫾ 0.4
3.6 ⫾ 0.6
2.2 ⫾ 0.2
7.7 ⫾ 0.6
3.1 ⫾ 0.5*
5.6 ⫾ 1.1†
3.2 ⫾ 0.6
12.3 ⫾ 1.3
5.7 ⫾ 1.5*
8.0 ⫾ 1.3*
2.6 ⫾ 0.5
15.4 ⫾ 1.6
7.4 ⫾ 1.8*
11.6 ⫾ 1.7†
3.1 ⫾ 0.2
18.7 ⫾ 2.4
10.3 ⫾ 1.3*
15.8 ⫾ 2.4
5.2 ⫾ 0.9
Values are means ⫾ SE; n, no. of rats. HOMA-IR, homeostasis model assessment of insulin resistance; OLETF-Sed, Otsuka Long-Evans Tokushima
Fatty-sedentary; Childhood-Ex, childhood-exercise, Childhood-Diet, childhood-dietary; LETO-Sed, Long-Evans Tokushima Otsuka-sedentary. *P ⬍ 0.05 vs.
OLETF-Sed; †P ⬍ 0.05 vs. Childhood-Ex.
FSL levels gradually increased over time in the OLETFSed group, exhibiting a fivefold increase between 7 and 45
wk (Table 1). Indeed, FSL levels were much higher in the
OLETF-Sed group compared with those in the Childhood-Ex and Childhood-Diet groups throughout the 15-wk
treatment. However, levels increased markedly in the Childhood-Diet group after the treatment was completed; when
the rats were 27– 45 wk of age, FSL levels did not differ
between the Childhood-Diet and OLETF-Sed groups (Table
1). Levels in the Childhood-Ex group, on the other hand,
remained significantly lower throughout the remainder of
A
B
Serum lipids
500
the study, although they did increase slightly as the experiment progressed.
Serum lipid levels. Serum levels of Tcho, TG, HDL-C, and
LDL-C were significantly lower in the Childhood-Ex group
than in the OLETF-Sed and Childhood-Diet groups (Fig. 2).
Rats in the Childhood-Ex group had significantly higher
HDL-C-to-Tcho ratios compared with those for rats in the
OLETF-Sed group; however, LDL-C-to-Tcho ratios did not
differ significantly among the other three groups.
Changes in whole body SFM, VFM, and LBM. SFM, VFM,
%fat, and %LBM were comparable across groups (n ⫽ 23
Serum lipid ratio
0.8
400
OLETF-Sed
OLETF-Sed
Childhood-Ex
Childhood-Ex
Childhood-Diet
Childhood-Diet
0.6
LETO-Sed
LETO-Sed
Fig. 2. Effects of the Childhood-Ex and Childhood-Diet regimens on serum lipid levels (A)
and serum lipid ratios (B) during childhood.
Values are means ⫾ SE. Tcho, total cholesterol; TG, triglyceride; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; HDL-C/Tcho,
ratio of HDL-C to Tcho; LDL-C/Tcho, ratio of
LDL-C to Tcho. P ⬍ 0.05 vs. §Childhood-Ex,
Childhood-Diet, and LETO-Sed; *OLETFSed; †Childhood-Ex; &Childhood-Diet; and
#Childhood-Ex and Childhood-Diet.
%
mg/dl
300
0.4
200
0.2
100
0
Tcho
TG
HDL-C
LDL-C
0
HDL-C/Tcho
LDL-C/Tcho
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Serum glucose, mg/dl
OLETF-Sed
Childhood-Ex
Childhood-Diet
LETO-Sed
Serum insulin, pg/ml
OLETF-Sed
Childhood-Ex
Childhood-Diet
LETO-Sed
Serum leptin, pg/ml
OLETF-Sed
Childhood-Ex
Childhood-Diet
LETO-Sed
HOMA-IR
OLETF-Sed
Childhood-Ex
Childhood-Diet
LETO-Sed
n
Posttreatment Period
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Effects of Prepubertal Exercise in Rats
A
iment (OLETF groups: r ⫽ 0.830, P ⬍ 0.001; LETO group:
r ⫽ 0.660, P ⬍ 0.001; OLETF and LETO groups combined:
r ⫽ 0.830, P ⬍ 0.001). We also found a similar correlation
between the changes in SFM and FSG (OLETF groups: r ⫽
0.770, P ⬍ 0.001; LETO group: r ⫽ 0.690, P ⬍ 0.001; OLETF
and LETO groups combined: r ⫽ 0.80, P ⬍ 0.001). Changes
in FSL levels strongly correlated with the changes in %fat
(Fig. 5) and %LBM. The relationship between FSL level and
%fat in the OLETF group was described by the equation y ⫽
94.8x ⫺ 1,306 (r ⫽ 0.90, P ⬍ 0.001); for the LETO group, this
relationship was described as follows: y ⫽ 47.9x ⫺ 419.8 (r ⫽
0.83, P ⬍ 0.001).
FSL level was strongly correlated with serum TG level
measured at the completion of the experiment in both the
OLETF (r ⫽ 0.830, P ⬍ 0.001) and LETO (r ⫽ 0.925, P ⬍
0.001) groups. Rats in the OLETF group, however, displayed
a broader range of both FSL and TG levels relative to those
found in the LETO group. Serum TG levels were also closely
related to %fat (OLETF groups: r ⫽ 0.662, P ⬍ 0.001; LETO
group: r ⫽ 0.681, P ⬍ 0.001; OLETF and LETO groups: r ⫽
0.728, P ⬍ 0.001) and BW. Changes in HOMA-IR positively
correlated with BW (r ⫽ 0.742, P ⬍ 0.001) and VFM (r ⫽
0.817, P ⬍ 0.001) in the OLETF group. Although the same
C
Subcutaneous fat mass
Pre-treatment OLETF rats
Pre-treatment LETO rats
OLETF-Sed
Childhood-Ex
Childhood-Diet
LETO-Sed
Pre-treatment OLETF rats
Pre-treatment LETO rats
OLETF-Sed
Childhood-Ex
Childhood-Diet
LETO-Sed
40
%
60
g
% Fat
50
80
40
30
20
20
0
Pre
-treatment
B
Shindo D et al.
10
5
Pre
-treatment
Post-treatment period
Treatment period
D
Visceral fat mass
5
Post-treatment period
Treatment period
%LBM
100
200
160
90
g
%
120
80
80
70
40
0
4
Pre
-treatment
5
7
12
Treatment period
20
27
35
Post-treatment period
45 week
60
4 5
Pre
-treatment
7
12
Treatment period
20
27
35
45 week
Post-treatment period
Fig. 3. Changes in body composition throughout the experiment. A: subcutaneous fat mass (SFM). B: visceral fat mass (VFM). C: %fat (a quantity obtained by
dividing the sum of SFM and VFM by BW, then multiplying by 100). D: %lean body mass (LBM; a quantity obtained by dividing LBM by BW and then
multiplying by 100). Values are means ⫾ SE. P ⬍ 0.05 vs. §Childhood-Ex, Childhood-Diet, and LETO-Sed; *OLETF-Sed; †Childhood-Ex; &Childhood-Diet;
and #Childhood-Ex and Childhood-Diet.
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OLETF and n ⫽ 8 LETO rats) before the study (i.e., at 4 wk of
age). At 7 wk of age, we observed significantly lower SFM, VFM,
and %fat as well as significantly higher %LBM in the Childhood-Ex compared with values measured in both the OLETF-Sed
and Childhood-Diet groups (Fig. 3). These trends lasted until the
termination of the study, when the rats were 45 wk of age.
Energy metabolism-related enzyme activities and UCP-3
mRNA expression in skeletal muscle. At 46 wk of age, rats in
the Childhood-Ex group demonstrated higher levels of CS,
SDH, and PFK enzyme activity as well as greater UCP-3
mRNA expression compared with those in rats in the other
three groups (Fig. 4). No such differences were observed
among the OLETF-Sed, Childhood-Diet, and LETO-Sed
groups. UCP-3 mRNA expression levels were significantly
correlated with the activity of both PFK (r ⫽ 0.560, P ⬍
0.01) and SDH (r ⫽ 0.580, P ⬍ 0.01) in rats from the
OLETF-Sed, Childhood-Ex, and Childhood-Diet groups.
However, no significant correlations were observed between
enzyme activity and mRNA expression in the skeletal muscles of rats from the LETO group.
Correlations between body composition, serum leptin, and
metabolism-related parameters. Changes in VFM were significantly correlated with changes in FSG throughout the exper-
•
Effects of Prepubertal Exercise in Rats
•
679
Shindo D et al.
3.0
400
OLETF-Sed
Childhood-Ex
Childhood-Diet
LETO-Sed
OLETF-Sed
Childhood-Ex
Childhood-Diet
LETO-Sed
2.5
300
fold difference
μmol/g/min
2.0
200
Fig. 4. Activity of phosphofructokinase (PFK),
citrate synthase (CS), and succinate dehydrogenase (SDH), as well as uncoupling protein-3 (UCP-3) mRNA expression level in the
quadriceps femoris muscle at the completion
of the experiment. Values are means ⫾ SE.
§P ⬍ 0.05 vs. OLETF-Sed, Childhood-Diet,
and LETO-Sed.
1.5
1.0
100
0.5
0
PFK
CS
SDH
UCP-3 mRNA
general pattern was true for the LETO group, the relationships
between these variables were much weaker (BW: r ⫽ 0.389,
P ⬍ 0.01; VFM: r ⫽ 0.437, P ⬍ 0.01). Changes in serum free
fatty acid level were not significantly correlated with changes
in body composition in either the OLETF or the LETO group.
DISCUSSION
OLETF rats that engaged in childhood exercise maintained
significantly lower BWs and higher %LBM throughout the
maturation stage and middle age; this was not observed in rats
subjected to childhood food restriction. Rats placed on an
exercise regimen also displayed higher activity levels of energy
metabolism enzymes CS, SDH, and PFK, as well as greater
A
3,500
expression of UCP-3 mRNA in the skeletal muscle 25 wk after
the cessation of childhood exercise. Cumulatively, our results
suggest that the characteristics of energy metabolism obtained
through a childhood exercise regimen in an animal model of
human obese diabetes may be sustained more or less throughout maturation and middle age, which may contribute to the
sustained suppression of BW gain. Importantly, to allow translation of our work, these novel insights need to be assessed in
obese humans.
We found that absolute FI in the Childhood-Ex group
gradually decreased 9 wk after cessation of exercise. At present, we do not know the reason for this observation. However,
this is unlikely the main reason for the reduced BW and fat
B
3,500
: OLETF-Sed (r = 0.894, P < 0.001)
: Childhood-Ex (r = 0.876, P < 0.001)
FSL (pg/ml)
3,000
3,000
: Childhood-Diet (r = 0.906, P < 0.001)
2,500
2,500
2,000
2,000
1,500
1,500
1,000
1,000
500
500
Fig. 5. Relationship between fasting serum
leptin (FSL) and %fat in the OLETF (A) and
LETO (B) rat groups. The box indicates the
range of measurements observed in the
LETO rat group.
LETO-Sed (r = 0.830, P < 0.001)
0
0
15
20
25
30
% Fat
35
40
15
20
25
30
35
40
% Fat
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Effects of Prepubertal Exercise in Rats
Shindo D et al.
the termination of their exercise treatment, although confirmation of this hypothesis requires further investigation.
Our study also demonstrated that, even at 46 wk of age (e.g.,
25 wk after the cessation of childhood exercise), rats in the
Childhood-Ex group had 1.7- to 2.1-fold higher activity levels
of CS, SDH, and PFK in the quadriceps femoris muscle
relative to rats in the OLETF-Sed group; in contrast, the
activity of these enzymes did not differ among the OLETFSed, Childhood-Diet, and LETO-Sed groups. Extensive research on metabolic enzymes in the skeletal muscles of both
rodents and humans (9, 14, 36) identified CS as an important
regulatory enzyme in the energy-generating metabolic pathway; this has since been used as a metabolic marker for
assessing oxidative and respiratory capacity (36). With regard
to UCP-3 mRNA, the expression levels measured in the quadriceps femoris muscle upon the completion of the experiment
were 2.6- to 4.0-fold higher in the Childhood-Ex group than in
the other three groups. UCP-3, which is found only in muscles,
is thought to be involved in the regulation of energy expenditure (5). In both humans and rats, UCP-3 protein and/or UCP-3
mRNA expression in the skeletal muscle increases markedly
within 3 h after a single exercise session (12, 28). Furthermore,
among Pima Indians, UCP-3 mRNA expression in the skeletal
muscle is positively correlated with sleeping metabolic rate
(33). Taken together, these findings appear to suggest that
childhood exercise training produces a sustained adaptive
physiological response in skeletal muscle (5, 12, 28, 34), such
as increased energy metabolism and increased RMR. Further
studies are required to understand the mechanisms underlying
the sustained high levels of metabolism-related enzymes and
UCP-3 mRNA expression in skeletal muscle. Moreover,
changes in RMR need to be directly measured.
Rats in the OLETF groups displayed a broader range of FSL
values than those in the LETO group (Fig. 5). Circulating
leptin level closely relates to body fat stores (7), since increased adiposity induces greater leptin production. However,
the high leptin levels generally observed in obese individuals
do not induce loss of fat mass (7, 26). This apparent paradox is
described as “leptin resistance.”
Hypertriglyceridemia markedly impairs the rate of leptin
transport into the central nervous system (2). The broad range
of FSL levels observed in the OLETF group, as well as the
markedly higher correlation between FSL and TG levels (compared with the LETO group), may reflect reduced leptin activity in these rats; in other words, the OLETF rats may be more
leptin resistant than the LETO rats. Individuals in the Childhood-Ex group may have demonstrated long-term reductions in
leptin level because of their equally long-term reductions in
serum TG level, as well as their lower levels of body fat mass
induced by prepubertal-onset exercise. In the present study, the
strong relationship between FSL and TG levels may have been
driven by the over-accumulation of body fat, leading to hypertriglyceridemia and leptin resistance.
In summary, we speculate that prepubertal-onset exercise
affected the regulation of skeletal muscle mass and energy
metabolism in the skeletal muscle rather than the feeding
center in the hypothalamus. Although the exact mechanisms by
which prepubertal-onset exercise alters skeletal muscle properties remain to be determined, our findings further support the
concept that prepubertal-onset exercise induces a sustained
decrease in the higher-predisposed BW set points in obese
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mass parameters in the Childhood-Ex group. Indeed, BW and
all fat mass parameters after cessation of exercise in the
Childhood-Ex group demonstrated significantly lower values
than those in the Childhood-Diet and OLETF-Sed groups even
before showing significant reductions in FI in the Childhood-Ex group. Moreover, we found that absolute FI in the
Childhood-Diet group was lower than that in the Childhood-Ex
group up to ⬃10 wk after cessation of the interventions,
whereas BW in this group during this period demonstrated
higher levels than that in the Childhood-Ex group. These
findings indicate that absolute FI levels are not only the factor
for determining BW levels in OLETF rats.
In an earlier study in rats that are genetically predisposed to
become obese, Patterson et al. (27) found that early-onset
exercise in the postweaning period prevented the development
of obesity for up to 7 wk after exercise cessation, whereas rapid
increases in BW and intake were observed after the cessation
of a food-restriction treatment. However, exercise and food
restriction treatments have not been found (by these or any
other researchers, Ref. 20) to cause significantly different
changes in anabolic and catabolic hypothalamic neuropeptide
expression, suggesting that the hypothalamic roles in regulating energy intake might not be involved in the mechanisms
underlying the sustained suppression of BW gain after exercise
cessation.
In the case of exercise treatment in adult humans and obese
rats, BW gain is typically observed once exercise treatments
have been discontinued; no studies to date (21, 22, 42, 43) have
observed continuous suppression of BW after exercise cessation. This recidivism is explained by the set point theory for
BW (19), which states that individuals are predisposed to
return to a genetically determined BW. This process has been
associated with reductions of LBM and/or RMR in individuals
who have lost weight through exercise (21, 30). RMR is the
largest component (50 –70%) of daily energy expenditure (29),
and the major determinant of RMR is fat-free mass (LBM)
quantities. In the present study, we found that rats that engaged
in “childhood” exercise, but not food restriction, maintained
⬃10% lower BW and significantly higher %LBM compared
with individuals who performed no exercise (OLETF-Sed).
These results suggest that sustained higher %LBM may be an
important factor for contributing the long-term suppression of
BW gain in the Childhood-Ex group.
The question arises as to which mechanisms are involved in
the long-term maintenance of high levels of %LBM in the
Childhood-Ex group, even after the termination of their exercise treatment. This may be, in part, explained by the “muscle
memory” theory, which explains a phenomenon that individuals who have previously undergone athletic training quickly
acquire force when retraining (for example, Refs. 38, 41).
Bruusgaard et al. (6) previously reported that the major cause
of hypertrophy is the long-term (⬎3 mo after subsequent
denervation) maintenance of myonuclei that have been newly
recruited after overload. Thus “muscle memory” may be facilitated by a lasting change in the number of myonuclei in
muscle cells (6, 38, 41); furthermore, because the ability to
create myonuclei is impaired at older ages, individuals may
particularly benefit from strength training at an early age (10).
These mechanisms may be involved in the long-term maintenance of high levels of %LBM in the Childhood-Ex group after
•
Effects of Prepubertal Exercise in Rats
individuals even after cessation of exercise regimens. Additional future work might compare the effects of childhood
exercise and food-restriction treatments to those during adulthood (i.e., 20 –34 wk of age in OLETF rats). Such work would
be vital for confirming that the present results stem from
prepubertal onset of the exercise regimen.
ACKNOWLEDGMENTS
We thank Dr. Hidefumi Waki (Wakayama Medical University) for helpful
comments and editing of our manuscript. The OLETF and LETO rats were a
generous gift from the Tokushima Research Institute, Otsuka Pharmaceutical
(Tokushima, Japan).
GRANTS
This study was supported by Grants-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science and Technology of Japan (nos.
19500618, 21300256) to M. Suzuki.
DISCLOSURES
AUTHOR CONTRIBUTIONS
Author contributions: D.S. performed experiments; D.S. analyzed data;
D.S. prepared figures; T.M. drafted manuscript; T.M. edited and revised
manuscript; M.S. conception and design of research; M.S. interpreted results of
experiments; M.S. approved final version of manuscript.
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