PLOS ONERESEARCH ARTICLE

Effects of long-term childhood exercise anddetraining on lipid accumulation in metabolicrelated organs
Son Tien Nguyen ID1, Naoto Fujita ID1*, Takaya Oshima1, Misuzu Nishihira1,
Haruya Ohno ID2, Masayasu Yoneda3, Susumu Urakawa1
1 Department of Musculoskeletal Functional Research and Regeneration, Graduate School of Biomedicine
and Health Sciences, Hiroshima University, Kasumi, Minami-ku, Hiroshima, Japan, 2 Department of
Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University,
Kasumi, Minami-ku, Hiroshima, Japan, 3 Department of Preventive Medicine for Diabetes and LifestyleRelated Diseases, Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi,
Minami-ku, Hiroshima, Japana1111111111
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OPEN ACCESS
Citation: Nguyen ST, Fujita N, Oshima T, Nishihira
M, Ohno H, Yoneda M, et al. (2022) Effects of longterm childhood exercise and detraining on lipid
accumulation in metabolic-related organs. PLoS
ONE 17(6): e0270330. https://doi.org/10.1371/
journal.pone.0270330
Editor: Clemens Fürnsinn, Medical University of
Vienna, AUSTRIA
Received: February 28, 2022
Accepted: June 9, 2022
Published: June 24, 2022
Copyright: © 2022 Nguyen et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information
files.
Funding: The authors received no specific funding
for this work.

* fujitan@hiroshima-u.ac.jp

Abstract
The preventive effects of regular exercise on obesity-related health problems are carriedover to the non-exercise detraining period, even when physical activity decreases withaging. However, it remains unknown whether regular childhood exercises can be carriedover to adulthood. Therefore, this study aimed to investigate the effects of long-term childhood exercise and detraining on lipid accumulation in organs to prevent obesity in adulthood. Four-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats were used
as obese animals. OLETF rats were allocated into sedentary and exercise groups: exercise
from 4- to 12-week-old and detraining from 12- to 20-week-old. At 12-week-old immediately
after the exercise period, regular exercise completely inhibited hyperphagia, obesity,
enlarged pancreatic islets, lipid accumulation and lobular inflammation in the liver, hypertrophied adipocytes in the white adipose tissue (WAT), and brown adipose tissue (BAT) whitening in OLETF rats. Additionally, exercise attenuated the decrease in the ratio of muscle
wet weight to body weight associated with obesity. Decreased food consumption was maintained during the detraining period, which inhibited obesity and diabetes at 20-week-old
after the detraining period. Histologically, childhood exercise inhibited the enlargement ofpancreatic islets after the detraining period. In addition, inhibition of lipid accumulation wascompletely maintained in the WAT and BAT after the detraining period. However, the effectiveness was only partially successful in lipid accumulation and inflammation in the liver.
The ratio of muscle wet weight to body weight was maintained after detraining. In conclusion, early long-term regular exercise effectively prevents obesity and diabetes in childhood,and its effectiveness can be tracked later in life. The present study suggests the importanceof exercise during childhood and adolescence to inhibit hyperphagia-induced lipid accumulation in metabolic-related organs in adulthood despite exercise cessation.

Competing interests: The authors have declaredthat no competing interests exist.

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Introduction
Childhood obesity is a non-communicable disease that has drastically increased in prevalence
worldwide, with an increase from 30 million children under aged 5 years affected in 2000 tonearly 39 million in 2020 [1]. Furthermore, childhood obesity is usually carried over to adulthood, with a 1.3 fold increase in the risk of developing adulthood obesity compared with nonobese children [2]. In addition, children with obesity have a higher risk of developing metabolic disorders and disabilities, such as diabetes, in later life [3]. Therefore, the treatment andprevention of childhood obesity are of particular importance.
Clinically, treatment for childhood obesity aims to alleviate or reverse lipid accumulation in
the whole body, with lifestyle modification being the cornerstone [4]. Exercise is effective inpreventing and treating childhood obesity [5]. However, children who are overweight andobese tend to have trouble adhering to a continual exercise program, with a high dropout rateof approximately 50% [6]. In addition, strict adherence to exercise regimens is difficult for children due to injuries, determination, desire, and design of exercise programs [7], or recently,
the coronavirus disease 2019 (COVID-19) lock-down. Therefore, discontinuation of exercise
could induce a detraining effect, which may negatively impact children’s metabolic health.
In adults with obesity, detraining has been shown to partly or totally attenuate the metabolic
benefits of exercise, with a significant increase in lipid accumulation, insulin resistance, andinsulin levels [8, 9]. A study by Weinsier et al. showed that in postmenopausal obese women,
although exercise could reduce their body weight to the level matched to lean counterparts,
detraining increased their body weight significantly compared to lean counterparts [8]. Fatouros
et al. also found that 6-month detraining following 6-month exercise in obese men reversed
body mass index, leptin, adiponectin, glucose, and insulin levels to the baseline measurements[9]. In contrast, in children with obesity, long-term exercise intervention could prolong its effectiveness in preventing obesity and obesity-related metabolic complications. A study by GarciaHermoso et al. involving children with obesity aged 8 to 11 years outlined that 3-year exercise
training could sustain low %fat mass and body mass index after 6-months of detraining [10].
Another study by Reinehr et al. involving children with obesity aged 6 to 14 years outlined that
1-year exercise training could sustain low body mass index, insulin levels, and insulin resistanceindex at 1-year follow-up [11]. However, information regarding lipid accumulation in children
with obesity undergoing detraining is limited. Lipid accumulation during the detraining period
occurs in the main lipid supply organs, such as the intra-abdominal white adipose tissue (WAT),
brown adipose tissue (BAT), and liver. Moreover, lipid accumulation in organs increases insulin
resistance, which is similar to a vicious cycle. Therefore, the question arises whether there are
unique changes in lipid accumulation in organs under the effects of exercise in childhood. However, studies on lipid accumulation during childhood detraining have mostly focused on individual organs. Hence, there is a need to investigate lipid accumulation in the organs.
Compared to human studies, animal studies have directly examined lipid accumulation in
many organs. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an obesity model characterized by hyperphagia due to the loss of cholecystokinin receptor-1 in the dorsomedial
hypothalamus, leading to decreased satiety [12]. The natural progression of obesity and obesity-related metabolic complications in OLETF rats reflects human childhood obesity, leading
to metabolic health problems in adulthood. OLETF rats exhibit an early development of mild
obesity after weaning, with overeating and sedentariness, which could be prevented by lifestylemodification with diet and exercise. Obesity in OLETF rats gradually leads to glucose intolerance, and insulin resistance at around 12-week-old and then diabetes and chronic obesity
complications occur no earlier than 20-week-old (or adulthood), similar to those of humanobesity [13]. Therefore, OLETF rats are a suitable model for investigating the influence of

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exercise on childhood obesity and obesity-related complications later in life. Given the unclear
effects of long-term childhood exercise and detraining on lipid accumulation, we conducted astudy on young OLETF rats subjected to long-term wheel running. The purpose of the present
study was to investigate the influence of long-term childhood exercise and detraining on the
progression to obesity, focusing on lipid accumulation in the peripheral organs as a whole andwhether the effectiveness of childhood exercise was carried over to adulthood after a period ofdetraining. These results further confirmed the merits of long-term childhood exercise in preventing obesity and metabolic disorders.

Materials and methods
Experimental design
This study was approved by the Institutional Animal Care and Use Committee of Hiroshima University (A19-163) and conducted under the Hiroshima University Regulations for Animal Experimentation. All experiments were performed in accordance with the National Institute of Health
Guidelines for the Care and Use of Laboratory Animals. The health of the rats was monitored
twice a day. If the rats expressed the specific signs such as severe weight loss, tachypnea, and lethargy, we judged the signs as terminal. The rats that showed the signs (i.e., rats nearing death) were
humanely euthanized by an overdose of sodium pentobarbital to minimize the distress.
Four-week-old male OLETF rats and age-matched male Long-Evans Tokushima Otsuka(LETO) rats were used as the obese and non-obese control groups, respectively. OLETF rats
were randomly assigned to the sedentary (OLETF Sed, n = 12) and exercise (OLETF Ex,
n = 12) groups. All rats were housed in three and provided standard chow and water ad libitum. The housing conditions were maintained as follows: ambient temperature of 22 ± 2˚Cand under a 12–12 light-dark cycle, with the light on from 08:00 and off from 20:00.

Exercise protocol
OLETF rats in the OLETF Ex group ran freely on a running wheel for 12 h/day from 20:00 to
08:00 and returned to their home cages from 08:00 to 20:00. The rats exercise for 8 weeks from
4- to 12-week-old and non-exercise for 8 weeks from 12- to 20-week-old. The rats freely
accessed food and water during running time, and ambient temperature and humidity conditions were maintained the same as in their home cages. Running duration, running distance,
and average and maximum running velocities were automatically recorded with the magnetic
sensor attached to the running cage and stored in a corresponding digital meter during theexercise period (S1 Fig).

Oral glucose tolerance test
An oral glucose tolerance test (OGTT) was performed 1 week prior to sacrificing at 20-weekold. The rats were fasted for 12 h prior to testing. For each rat, a dose of 2 g glucose/kg bodyweight was inserted via an esophageal tube. Blood glucose from the lateral caudal vein wasmeasured at four time points during the test using an ACCU-CHECK ST meter (Roche,
Tokyo, Japan), including the fasting point, at 30, 60, and 120 min after glucose administration.
Blood was aspirated at the fasting point into heparinized microhematocrit tubes and centrifuged at 3000 rpm for 10 min, and the plasma was stored at –80˚ C until further analyses.

Measurements of plasma triacylglycerol, free fatty acid, and insulin
Triacylglycerol (TAG), free fatty acid (FFA), and insulin levels during fasting were quantified
from the OGTT plasma samples. TAG and FFA levels were measured using

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spectrophotometric assay kits (290–63701 and 294–63601, respectively; Wako, Osaka, Japan).
Insulin levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit(M1101; Morigana, Yokohama, Japan).

Tissue sampling
All rats in the OLETF Ex group fasted for 5 h before sampling and after the last exercise andwere euthanized by an overdose of sodium pentobarbital. The pancreas, gastrocnemius muscle, liver, epididymal white adipose tissue (eWAT), and interscapular BAT were harvested andfrozen in nitrogen or fixed in 4% paraformaldehyde in 0.1 M phosphate buffer.

Histological analysis
The pancreatic head embedded in paraffin was cut into 5 μm and stained with hematoxylin
and eosin (HE). Histological images were used to measure the cross-sectional area of the pancreatic islets, and the diameter was calculated from these values. Images of 20–30 pancreatic
islets for each rat were counted for measurement.
Serial transverse sections of the muscle belly of the gastrocnemius lateralis were stained foradenosine triphosphatase (ATPase, pH 4.1) and succinate dehydrogenase (SDH) activities tocategorize the muscle fibers as type I, IIA, or IIB. The muscle fibers stained darkly with ATPaseand SDH were categorized as type I, lightly with ATPase, darkly with SDH were categorized as
type IIA, and lightly with both ATPase and SDH were categorized as type IIB. Two random
images with >100 muscle fibers were chosen in each superficial and deep layer to measure thecross-sectional area of the muscle fibers.
Paraffin sections of the left lateral lobe of the liver were stained with HE, and the images
were used to measure the percentage of liver steatosis and degree of lobular inflammation. The
area in which lipid droplets occurred inside the hepatocytes with microvesicular or macrovesicular structures was defined as the liver steatosis area, and four to five images, including portal
triads, were used to measure the percentage of liver steatosis. A × 200 image was divided into
100 μm2 squares, and the percentage of liver steatosis was calculated as all squares that contained liver steatosis areas per total squares in the section. Lobular inflammation was calculated as the total number of inflammatory foci showing focal mononuclear cells around thevein or artery per section. In addition to histological analysis, the hepatic TAG content wasmeasured using spectrophotometric assay kits (290–63701, Wako). Total lipids from liver samples were extracted according to Folch’s method [14].
Paraffin sections of the eWAT and BAT were stained with HE. Regarding the eWAT histology, images stained with HE were used to measure the adipocytes’ diameter. Three to five random images with approximately 300 adipocytes were selected to measure the adipocytes’ area,
and the diameter was calculated from these values. Adipocytes surrounded by mononuclear
cells were defined as crown-like structures (CLS), and the number of CLS was calculated as thetotal number of CLS per section. Histological parameters such as the cross-sectional area,
diameter, and the number in the pancreas, skeletal muscle, liver, and eWAT were quantified
using ImageJ software (NIH, Bethesda, MD, USA).

Statistics
The data were expressed as the mean ± standard error (SEM). Two-way analysis of variance(ANOVA) with Bonferroni post hoc test or one-way ANOVA with Tukey’s post hoc test wasused to determine significant differences among the groups. Statistical significance was set at P
<0.05. The data were processed using SPSS software (IBM SPSS Statistics version 19.0, IBM
Japan, Tokyo, Japan).

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Results
Changes in body weight and food consumption
From 4- to 5-week-old, the body weight was almost the same between the OLETF Sed groupand LETO rats (Fig 1A). Body weight increased weekly, with a steeper growth curve in the
OLETF Sed group than in LETO rats. After 6-week-old, the value was significantly higher in
the OLETF Sed group than in LETO rats. Food consumption was significantly higher in the
OLETF Sed group than in LETO rats throughout the experimental period (Fig 1B).
During the exercise period from 4- to 12-week-old, food consumption was significantly
lower in the OLETF Ex group than in the OLETF Sed group. As a result, body weight increasedin the OLETF Ex group during the exercise period but significantly lower in the OLETF Ex
group than in the OLETF Sed group after 7-week-old.
Food consumption in the OLETF Ex group at 13-week-old (i.e., a week after the last exercise) was significantly increased compared with that at 12-week-old (i.e., immediately after thelast exercise). However, food consumption in the OLETF Ex group was still significantly lower
than that in the OLETF Sed group at all the subsequent time points. In addition, at 13-weekold, the body weight in the OLETF Ex group was significantly higher than that in the 12-weekold but remained lower than that in the OLETF Sed group. The body weight gradually
increased in the OLETF Ex group after 14-week-old. However, at the end of the experimental
period, the body weight in the OLETF Ex group was still significantly lower than that in the
OLETF Sed group.

Biomedical markers changes
Plasma TAG (Fig 2A) and FFA (Fig 2B) levels were significantly higher in the OLETF Sed
group than in LETO rats at 12- and 20-week-old. At 12-week-old (i.e., immediately after the
exercise period from 4- to 12-week-old), plasma TAG levels were significantly lower in the
OLETF Ex group than in the OLETF Sed group, whereas plasma FFA levels were almost thesame between the OLETF Ex and OLETF Sed groups. However, at 20-week-old (i.e., after the
detraining period from 12- to 20-week-old), the values were significantly lower in the OLETF
Ex group than in the OLETF Sed group.

Fig 1. Changes in body weight. Body weight (A) and daily food consumption (B). Data are illustrated as mean ± SEM. � and † show significantly
different between the OLETF Sed group vs. LETO rats and the OLETF Sed vs. OLETF Ex groups, respectively, P < 0.05, two-way ANOVA. n = 5–6 pergroup.
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Fig 2. Changes in blood biomarkers. Fasting plasma triacylglycerol (A) and free fatty acid (B), glucose homeostasis during
the oral glucose tolerance test at 20-week-old (C), and fasting plasma insulin concentrations at 20-week-old (D). wo, weekold. Data are illustrated as mean ± SEM. � and † show significantly different from age-matched LETO rats and age-matched
the OLETF Sed group, respectively, P < 0.05, one way ANOVA. n = 5–6 per group.
https://doi.org/10.1371/journal.pone.0270330.g002

Regarding glucose homeostasis at 20-week-old, hyperglycemia was observed in the OLETF
Sed group, with significantly higher blood glucose levels at fasting and after glucose administration than in LETO rats (Fig 2C). Although blood glucose levels at fasting and 120 min after
glucose administration were significantly higher in the OLETF Sed group than in LETO rats,
the values in the OLETF Ex group were significantly lower than those in the OLETF Sed
group, almost the same in LETO rats. The values in the OLETF Ex group at 30 and 60 min
after glucose administration were also significantly lower than those in the OLETF Sed groupbut not in LETO rats. Additionally, fasting plasma insulin levels at 20-week-old were significantly higher in the OLETF Sed group than in LETO rats (Fig 2D). These values were significantly lower in the OLETF Ex group than in the OLETF Sed group.

Histological changes in the pancreas
At 12-week-old, the islets were oval, and there was no lipid accumulation in the islets of all thegroups (Fig 3A). However, large islets with diameters from 350 to 400 μm were observed onlyin the OLETF Sed group but not in the OLETF Ex group (Fig 3B).
At 20-week-old, distorted large islets with a multilobular shape were observed in the
OLETF Sed group, whereas the shape of the large islets was still oval in the OLETF Ex group.
In addition, no lipid accumulation was observed in the islets of all groups (Fig 3A). Huge islets
with diameters ranging from 350 to 400 μm were still observed only in the OLETF Sed group,and no islets in the OLETF Ex group exceeded 300 μm in diameter. (Fig 3B).

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Fig 3. Histological changes in the pancreas. Representative pancreas images stained with hematoxylin and eosin (A) and distribution of
islets diameter (B). wo, week-old. Data are illustrated as mean ± SEM.
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Histological changes in the skeletal muscle
At 12-week-old, although there were no significant differences in the muscle wet weight
between the OLETF Sed and LETO rats (Fig 4A), the ratio of muscle wet weight to body weight
was significantly lower in the OLETF Sed group than in LETO rats (Fig 4B). In addition, themuscle wet weight was significantly lower in the OLETF Ex group than in the OLETF Sed
group. Conversely, the ratio of muscle wet weight to body weight was significantly higher in
the OLETF Ex group than in the OLETF Sed group. At 20-week-old, the muscle wet weight
and the ratio of muscle wet weight to body weight were significantly lower in the OLETF Sed
group than in LETO rats. However, the ratio of muscle wet weight to body weight was still significantly higher in the OLETF Ex group than in the OLETF Sed group.
The gastrocnemius lateralis was composed of type I, IIA, and IIB fibers in all the groups(Fig 4C). There were no significant differences in the cross-sectional areas of type I, IIA, andIIB fibers among all the groups at 12- and 20-week-old (Fig 4D).

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Fig 4. Histological changes in the skeletal muscle. Gastrocnemius muscle wet weight (A), the ratio of muscle wet weight to body weight(B), representative gastrocnemius lateralis images (C), and cross-sectional area of each muscle fiber type (D). wo, week-old; BW, bodyweight; SDH, succinate dehydrogenase. I, IIA, and IIB denote type I, IIA, and IIB fibers, respectively. Data are illustrated as mean ± SEM. �and † show significantly different from LETO rats and the OLETF Sed group, respectively, P < 0.05, one way ANOVA. n = 5–6 per group.
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Histological changes and triacylglycerol contents in the liver
The liver wet weight was significantly higher in the OLETF Sed group than in LETO rats at 12and 20-week-old (Fig 5A). However, at 12-week-old, the value was significantly lower in the
OLETF Ex group than in the OLETF Sed group. In addition, these values were still significantly lower in the OLETF Ex group than in the OLETF Sed group at 20-week-old.
Histologically, at 12-week-old, microvesicular steatosis in the liver was observed only in the
OLETF Sed group (Fig 5B). Liver steatosis (Fig 5C) and lobular inflammation (Fig 5D) weredetected in the OELTF Sed group but not in the OLETF Ex group. At 20-week-old, liver histology showed microvesicular and macrovesicular steatoses scattered in the hepatic lobules in the
OLETF Sed group (Fig 5B). Microvesicular steatosis with a few macrovesicular steatosis andinflammatory foci near the center of hepatic lobules were detected in the OLETF Ex group.

Fig 5. Histological changes and TAG contents in the liver. Liver wet weight (A), representative liver images stained withhematoxylin and eosin (B), percentage of liver steatosis (C), number of lobular inflammatory foci (D), and hepatic TAG contents(E). TAG, triacylglycerol; wo, week-old. A black arrow and a black arrowhead indicate macrovesicular steatosis andmicrovesicular steatosis, respectively. A yellow arrow indicates an inflammatory focus. Data are illustrated as mean ± SEM. � and† show significantly different from age-matched LETO rats and age-matched the OLETF Sed group, respectively, P < 0.05, one
way ANOVA. n = 5–6 per group.
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The percentage of liver steatosis in the OLETF Ex group was significantly lower than that in
the OLETF Sed group but not in LETO rats (Fig 5C). The number of inflammatory foci in the
OLETF Ex group was significantly lower than that in the OLETF Sed group but significantly
higher in LETO rats (Fig 5D).
Hepatic TAG content was significantly higher in the OLETF Sed group than in LETO rats
at 12- and 20-week-old (Fig 5E). However, at 12-week-old, these values were significantly
lower in the OLETF Ex group than in the OLETF Sed group. In addition, at 20-week-old, thehepatic TAG content in the OLETF Ex group was still significantly lower than that in the
OLETF Sed group, which was significantly higher than that in LETO rats.

Histological changes in the white adipose tissue
The eWAT wet weight was significantly higher in the OLETF Sed group than in LETO rats at
12- and 20-week-old (Fig 6A). At 12-week-old, the value was significantly lower in the OLETF
Ex group than in the OLETF Sed group. In addition, at 20-week-old, the value was still significantly lower in the OLETF Ex group than in the OLETF Sed group.
Histologically, at 12-week-old, larger adipocytes were always observed in the OLETF Sed
group but rarely in the OLETF Ex group (Fig 6B). The distribution of adipocyte diameter
shifted to the right in the OLETF Sed group, with diameters ranging from 95 to 155 μm in the
OLETF Sed group but not in the OLETF Ex group (Fig 6C). No CLS was observed in anygroup (Fig 6D). From 12- to 20-week-old, an increased adipocyte size was histologicallyobserved in all groups (Fig 6B). At 20-week-old, adipocytes with diameters ranging from 125
to 240 μm were observed in the OLETF Sed group but not in the OLETF Ex group (Fig 6C).
Additionally, CLS was less abundant in the OLETF Ex group than in the OLETF Sed group,and the number of CLS was significantly lower in the OLETF Ex group than in the OLETF Sed
group (Fig 6D).

Histological changes in the brown adipose tissue
The BAT wet weight was significantly higher in the OLETF Sed group than in LETO rats at
12- and 20-week-old (Fig 7A). However, at 12-week-old, the value was significantly lower in
the OLETF Ex group than in the OLETF Sed group. In addition, at 20-week-old, the value wasstill significantly lower in the OLETF Ex group than in the OLETF Sed group.
Histologically, BAT whitening was observed in all groups at 12-week-old, BAT whitening
was observed especially in the periphery of the adipose lobule (Fig 7B). Although whitened
unilocular adipocytes were more abundant and larger in the OLETF Sed group, smaller whitened and multilocular adipocytes were observed in the OLETF Ex group. At 20-week-old, thewhitened unilocular adipocytes in the OLETF Sed group were evenly scattered throughout the
adipose lobule, whereas the localization in the OLETF Ex group appeared to be more restricted
to almost the periphery of the lobule.

Discussion
The present study revealed that childhood exercise completely inhibited hyperphagia and subsequent obesity, including histological lipid accumulation in the liver, WAT, and BAT, during
the exercise period, regardless of non-weight loss. Additionally, childhood exercise attenuatedthe loss of muscle mass in obesity and prevented the enlargement of pancreatic islets in diabetes. A study by Crissey et al. involving adult OLETF rats exercised from 12- to 20-week-old
also exhibited a significant decrease in lipid accumulation in the liver, WAT, and BAT [15].
However, body weight marginally decreased compared to baseline in their study. The differences in weight loss between childhood and adulthood could be due to the developmental

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Fig 6. Histological changes in the epididymal white adipose tissue. eWAT wet weight (A), representative eWAT images stained withhematoxylin and eosin (B), distribution of adipocyte diameter (C), and number of crown-like structures (D). eWAT, epididymal white
adipose tissue; wo, week-old. Black arrows indicate crown-liked structure. Data are illustrated as mean ± SEM. � and † show significantly
different from age-matched LETO rats and age-matched the OLETF Sed group, respectively, P < 0.05, one way ANOVA. n = 5–6 per group.
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Fig 7. Histological changes in the brown adipose tissue. BAT wet weight (A) and representative BAT images stainedwith hematoxylin and eosin (B). BAT, brown adipose tissue; wo, week-old. Data are illustrated as mean ± SEM. � and †show significantly different from age-matched LETO rats and age-matched the OLETF Sed group, respectively,
P < 0.05, one way ANOVA. n = 5–6 per group.
https://doi.org/10.1371/journal.pone.0270330.g007

stages, with a steeper growth velocity in childhood. Childhood exercise inhibits the development of obesity without negatively affecting children’s growth. Although both food restrictionand exercise in childhood could prevent the development of obesity, the effectiveness of foodrestriction could not be maintained after cessation. Shindo et al. found that in childhood
OLETF rats both early food restriction and exercise interventions reduced body weight to thesame level, however, after stopping interventions, only formerly exercised OLETF rats maintained lower both body weight and fat mass parameters compared with OLETF sedentary rats

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Effects of childhood exercise and detraining on lipid accumulation in metabolic-related organs[16]. The differences between the two kinds of intervention were partly attained to the superior
suppression of de novo lipogenesis and an enhancement of oxidizing redundant metabolic
fuels of early exercise intervention [17]. These findings indicated that besides the reduction inbody weight, childhood exercise could uniquely prolong its effectiveness in preventing obesity
due to the inhibition of lipid accumulations in metabolic-related organs.
The present study revealed that childhood exercise maintained lower food intake during
the detraining period and prevented obesity and diabetes, even though a slight glucose intolerance was found after glucose administration. In addition to maintaining higher muscle massand smaller pancreatic islets, childhood exercise histologically inhibited lipid accumulation in
the WAT and BAT after the detraining period. However, the effectiveness was only partiallysuccessful in fat accumulation and inflammation in the liver. A study by Yasari et al. involving
adult rats with obesity, exercised for 8 weeks and then detrained for 6 weeks, showed thatdetraining completely reversed the effectiveness of exercise with rapid increases in intraabdominal fat deposition, the diameter of adipocytes in the WAT, and activity of lipoprotein
lipase to a level not different from that of age-matched sedentary rats [18]. Moreover, in
10-week-old mice fed a high-fat diet, although 8-week exercise reduced significantly fat masscompared, no significant difference was observed between formerly exercised mice and sedentary mice after 8-week subsequent detraining [19]. The results suggest that detraining in adulthood reverses the inhibition of lipid accumulation in the WAT induced by regular exercise,
which disagrees with the results of the present study using young rats. The number of adipocytes increases during childhood and adolescence, which remains constant in adulthood [20].
However, in childhood obesity, if adipocytes in the WAT are enlarged with excessive food consumption and hypertrophy exceeds the critical limitation, overwhelming hypertrophy inhibits
an increase in adipocyte number and simultaneously secretes pro-inflammatory cytokines in
the presence of CLS, contributing to systemic inflammation and peripheral insulin resistance[21, 22]. In the present study, childhood exercise inhibited hyperphagia during the exercise
period, resulting in a normal distribution of adipocyte diameters immediately after the exercise
period. Therefore, OLETF rats in the Ex group could show a normal increase in adipocyte
number during the exercise period from 4- to 12-week-old. The number of adipocytes could
compensate for hypertrophy during the detraining period and inhibit inflammation, which
contributes to maintaining normal insulin sensitivity.
Whole metabolic functions during the detraining period may also contribute to the inhibition of obesity and diabetes from childhood to adolescence. Physiologically, studies found thatdaily energy expenditure increase in the vicinity of puberty and remains stable during adulthood [23, 24]. Thermogenesis in the BAT contributes to an increase in resting metabolic rateearly in life [25–27]. However, its activity declines with aging, which is histologically known as
BAT whitening. In addition to aging, obesity accelerates BAT whitening. A study in a model ofrats with obesity in the prediabetes stage showed lipid accumulation in the BAT with a positiveenergy balance [28]. The whitening process decreases mitochondrial functions with the downregulation of uncoupling protein-1 and vascularity in the BAT, thereby impairing thermogenesis and resting metabolic rate [29]. The present study showed that childhood exercise
inhibited BAT whitening after the detraining period, suggesting increased lipid metabolism
during childhood and adolescence. The resting metabolic rate, especially in adults, depends onheat production in the BAT and skeletal muscle mass, almost equivalent to lean body mass.
Compared with their lean counterparts, in humans and rats with obesity, children with obesity
show normal or increased lean body mass, but adults with obesity show decreased lean bodymass [16, 30, 31]. In the present study, the ratio of muscle wet weight to body weight was
higher in the OLETF Ex group than in the OLETF Sed group at 20-week-old, which suggested
that childhood exercise maintained higher metabolic-related organs after the detraining

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period. Therefore, lipid metabolic function after the detraining period could be increased byinhibiting BAT whitening and maintaining skeletal muscle mass during childhood exercise.
Whitening process leads to a reduction in uncoupling protein expression and activity of BAT[29], which negatively affects lipolysis and FFA oxidation [32]. Additionally, skeletal muscle
was found positively correlated with the two important enzymes in circulating lipid metabolism (i.e., lipoprotein lipase and glycosylphosphatidylinositol anchored high-density lipoprotein binding protein 1) [33]. In obesity, a low resting metabolic rate contributes to a positiveenergy balance [34]. Therefore, lower food intake could inhibit BAT whitening and increaseenergy consumption while maintaining skeletal muscle mass.
After the detraining period, childhood exercise diminished food intake and lipid accumulation in adipose tissue, even though it was only partially suppressed by inflammation and lipid
accumulation in the liver. It is known that childhood exercise attenuates lipid accumulation in
the liver, whereas the effectiveness of adult exercise on lipid accumulation in the liver waned
after the detraining period. Specifically, in a study by Yasari et al. involving adult rats with obesity, hepatic TAG content in formerly exercised rats increased to a level significantly higher
than that in sedentary rats after 6 weeks of detraining [18]. Lipid accumulation in the liverstems from two sources: 75% from non-hepatic FFAs (i.e., from adipose tissues and diet) and
25% from de novo lipogenesis [35]. The partial success of childhood exercise in inhibiting lipid
accumulation in the liver after detraining is likely due to de novo lipogenesis. However, thepresent study showed that childhood exercise completely inhibited lipid accumulation in adipose tissues, suggesting a reduction in circulating FFAs in the liver. In contrast to childhood
exercise, the effect of adulthood exercise is often incomplete on lipid accumulation in adipose
tissues [36, 37]. Incomplete effectiveness of adulthood exercise on lipid accumulation in adipose tissues could contribute to lipid reaccumulation in the liver after the detraining period. In
contrast, childhood exercise completely inhibits lipid accumulation in adipose tissues, resulting in partial preservation of lipid accumulation in the liver after the detraining period.
The present study revealed that long-term childhood exercise attenuated body weight gain,
glucose intolerance, and lipid accumulation in the organs carried over to adulthood. Nevertheless, this study had some limitations. First, the study focused on the imaging and variability ofsome indirect reflections of metabolism. Future studies on the molecular mechanisms of lipid
metabolism in each organ after childhood detraining are needed to clarify this. Second, thestudy participants were completely obese because of genetic defects. Additionally, a study on
5-week-old non-obese Wistar rats found that 10-week detraining following 10-week exercise
led to no significant changes in body weight compared with non-exercise rats [38]. Therefore,
the interpretation of the results for other models requires specific studies for each specificobese model, and future studies are needed to clarify whether there are differences in detraining in both obese and non-obese childhood models. Third, the follow-up time was only 8
weeks post-exercise in OLETF rats; thus, it was impossible to confirm an effect on weight andmetabolism beyond this time. However, obesity and metabolic disorders in individuals withgenetic obesity require regular dietary and exercise interventions throughout childhood to prevent them in adulthood. In addition to childhood obesity, gestational diabetes mellitus could
pose risk in the development of metabolic disorders and obesity in offspring. The present
study showed the merits of childhood exercise in preventing obesity and its related consequences. It is interesting to repeat the experiment in both maternal individuals and their offspring to clarify the advantages of exercise in full prevention of obesity. In conclusion, theresults suggested that long-term childhood exercise was effective in the long-term preventionof obesity and diabetes, possibly due to the inhibition of both BAT whitening and reduction ofskeletal muscle mass, and adequate inhibition of hyperphagia. Additionally, childhood exercise
inhibited lipid accumulation in the WAT and BAT and attenuated lipid accumulation in the

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liver after the detraining period, suggesting the importance of exercise during childhood andadolescence in inhibiting hyperphagia-induced lipid accumulation in metabolic-related organs
in adulthood.

Supporting information
S1 Fig. Wheel running parameters. Total running duration per day (A), total running distance per day (B), average running velocity in a day (C), and maximum running velocity in aday (D). Data are illustrated as mean ± SEM. n = 12.
(TIF)
S1 Dataset.
(XLSX)

Acknowledgments
This study was conducted at the Analysis Center of Life Science, Natural Science for Basic
Research and Development, Hiroshima University. We would like to acknowledge the technical support and help provided by the institution’s staff members.

Author Contributions
Conceptualization: Naoto Fujita.
Data curation: Naoto Fujita, Haruya Ohno, Masayasu Yoneda.
Formal analysis: Son Tien Nguyen, Takaya Oshima, Misuzu Nishihira, Haruya Ohno,
Masayasu Yoneda.
Investigation: Son Tien Nguyen, Takaya Oshima, Misuzu Nishihira.
Methodology: Naoto Fujita.
Project administration: Naoto Fujita.
Resources: Naoto Fujita, Susumu Urakawa.
Supervision: Naoto Fujita, Haruya Ohno, Masayasu Yoneda.
Writing – original draft: Son Tien Nguyen, Naoto Fujita.
Writing – review & editing: Son Tien Nguyen, Naoto Fujita, Haruya Ohno, Masayasu
Yoneda.

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