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DOI: 10.1530/JOE-15-0510
Journal of Endocrinology
http://joe.endocrinology-journals.org 2016 Society for Endocrinology
Printed in Great Britain
Published by Bioscientifica Ltd.
229:2
109–122
Abstract
Exercise plays a critical role in regulating glucose homeostasis and body weight.
However, the mechanism of exercise on metabolic functions associated with the CNS
has not been fully understood. C57BL6 male mice (n = 45) were divided into three
groups: normal chow diet, high-fat diet (HFD) treatment, and HFD along with voluntary
running wheel exercise training for 12 weeks. Metabolic function was examined
by the Comprehensive Lab Animal Monitoring System and magnetic resonance
imaging; phenotypic analysis included measurements of body weight, food intake,
glucose and insulin tolerance tests, as well as insulin and leptin sensitivity studies. By
immunohistochemistry, the amount changes in the phosphorylation of signal transducer
and activator of transcription 3, neuronal proliferative maker Ki67, apoptosis positive
cells as well as pro-opiomelanocortin (POMC)-expressing neurons in the arcuate area
of the hypothalamus was identied. We found that 12 weeks of voluntary exercise
training partially reduced body weight gain and adiposity induced by an HFD. Insulin
and leptin sensitivity were enhanced in the exercise training group verses the HFD
group. Furthermore, the HFD-impaired POMC-expressing neuron is remarkably restored
in the exercise training group. The restoration of POMC neuron number may be due
to neuroprotective effects of exercise on POMC neurons, as evidenced by altered
proliferation and apoptosis. In conclusion, our data suggest that voluntary exercise
training improves metabolic symptoms induced by HFD, in part through protected
POMC-expressing neuron from HFD and enhanced leptin signaling in the hypothalamus
that regulates whole-body energy homeostasis.
Exercise restores HFD-impaired
POMC-expression neuron
b t laing, k do and others
10.1530/JOE-15-0510
Voluntary exercise improves
hypothalamic and metabolic function
in obese mice
Brenton TLaing1,2, KhoaDo1,2,*, TomokoMatsubara1,2, David WWert2,
Michael JAvery1, Erin MLangdon1, DonghaiZheng1,2,3 and Hu Huang1,2,3,4
1Department of Kinesiology, East Carolina University, Greenville, North Carolina, USA
2East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina, USA
3 Human Performance Laboratory, Collage of Human Performance and Health, East Carolina University,
Greenville, North Carolina, USA
4Department of Physiology, East Carolina University, Greenville, North Carolina, USA
(*B T Laing and K Do contributed equally to this work)
Journal of Endocrinology
(2016) 229, 109–122
Research
Key Words
fhypothalamus
fexercise
fmetabolism
fmouse
fobesity
Correspondence
should be addressed to
H Huang
Email
huangh@ecu.edu
Introduction
Obesity is reaching epidemic proportions in North
America, affecting American society with increased
morbidity and mortality as well as economic cost
(Kopelman 2000). Obesity is usually associated with
defects of energy intake and energy expenditure, which
are tightly controlled by the CNS (Morton et al. 2006).
The CNS controls the important aspects of metabolism,
particularly in the hypothalamus, where neurons directly
229
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Research b t laing, k do and others Exercise restores HFD-impaired
POMC-expression neuron
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respond to physiological changes such as hunger and
satiety by secreted cytokines or hormones. Distinct nuclei
within the hypothalamus such as arcuate (ARC), the
paraventricular nucleus, the ventromedial hypothalamus
(VMH), the dorsomedial hypothalamus (DMH), and the
lateral hypothalamus share neuronal interconnections
to maintain body homeostasis (Mayers & Olson 2012).
Although many neurons in the hypothalamus regulate
metabolic functions, pro-opiomelanocortin (POMC)-
expressing neurons, located in the ARC area, is a key
regulator of energy metabolism. Genetic ablation of
Pomc neurons causes increased food intake and reduced
energy expenditure, leading to characteristics of the
obese phenotype such as increased body weight and
adiposity (Greenman et al. 2013, Zhan et al. 2013).
Conversely, activation of POMC neurons suppresses food
intake, increases energy expenditure, and induces the
characteristics of the lean phenotype, such as decreased
body weight and adiposity (Zhan etal. 2013). This indicates
that POMC neurons play critical roles in body weight
regulation. More specifically, because overnutrition and
high-fat diet (HFD) induce hypothalamic dysfunctions,
contributing to obesity and insulin resistance often
leading to type 2 diabetes (Zhang et al. 2008). HFD
specifically and preferentially affects POMC neurons in
the ARC. This suggests that POMC neurons are the target
and part of the mechanism of HFD-induced obesity and
diabetes (Li etal. 2012).
Exercise therapy is a proven and effective clinical
intervention for treating obesity and related diseases,
such as hyperlipidemia and type 2 diabetes mellitus
(Tremblay etal. 1985). Exercise stimulates glucose uptake
by skeletal muscle from the blood (Goldstein etal. 1953),
decreases fat content from the adipose tissue (Gollisch
et al. 2009), and prevents fat accumulation in the liver
(Jackson et al. 2011). Besides the effects of exercise on
peripheral tissues, voluntary exercise also improves brain
function. For instance, it enhances learning and memory
ability associated with the hippocampal area of the brain
to prevent cognitive dysfunction and Alzheimer’s disease
(Trejo etal. 2008, Fuss etal. 2010, Erickson etal. 2011).
Historical studies have demonstrated the effects of
exercise on the CNS that uses neurotransmitters and
trophic factors involved in energy homeostasis, such
as norepinephrine, γ-amino butyric acid, serotonin
(5-HT; Dishman 1997), and brain-derived neurotrophic
factor (BDNF; Neeper et al. 1996). Furthermore, 40-day
voluntary running wheel training significantly increases
neuropeptide Y gene expression in Sprague–Dawley male
rat ARC nucleus and DMH (Lewis etal. 1993). Recently,
it has been reported that hypothalamic melanocortin
receptor (MCR) expression has been associated with the
exercise activity and nonexercise activity thermogenesis
(Shukla et al. 2015). Although we have gained a better
understanding that hypothalamic MCR signaling is
highly regulated by the products of Pomc-expressing
neurons (Bagnol et al. 1999), little is known about how
exercise improves metabolic function via hypothalamic
POMC-expressing neurons. In light of this gap, we sought
to investigate the effects of voluntary exercise training
on whole-body metabolic parameters and hypothalamic
POMC neuron function in the diet-induced obese mice.
Materials and methods
Experimental animals
Eight-week-old C57BL6 male mice (n = 45) from Jackson
lab (The Jackson Laboratory, Bar Harbor, ME, USA)
were housed under controlled temperature and lighting
conditions of 20–22° and 12-h light:12-h darkness cycle.
Once the experimental protocol was initiated, all mice
were divided into three groups: chow group (control;
n = 15 with regular diet containing 26% protein, 14% fat,
and 60% carbohydrate), HFD group (n = 15, 16% protein,
58% fat, and 26% carbohydrate, Research Diets D12331;
Research Diets, Inc., New Brunswick, NJ, USA), and HFD
with exercise training (HFD + EX; n = 15, 16% protein,
58% fat, and 26% carbohydrate, Research Diets D12331)
and voluntary running wheel (TSE PhenoMaster System,
Bad Homburg, Germany) for 12 weeks. For the study of
voluntary wheel running, age-matched animals in the
HFD + EX group were placed in cages equipped with
running wheels for mice (TSE PhenoMaster), whereas
animals in the control group and HFD group were housed
in cages without running wheels for 12 weeks. Each cage
accommodated one mouse. All aspects of animal care
and experimentation were conducted in accordance with
the National Institutes of Health Guide for the Care and
Use of Laboratory Animals (National Institutes of Health
Publication no. 85-23, revised 1996) and approved by the
Institutional Animal Care and Use Committees of East
Carolina University (Greenville, NC, USA).
Energy intake, energy expenditure, and body
composition
Food intake was measured over a 5- to 7-day period,
and the data were combined, averaged, and analyzed.
Fresh pellets of food were provided every day to avoid
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Research b t laing, k do and others Exercise restores HFD-impaired
POMC-expression neuron
temperature-dependent spoilage to the HFD group, and
cages were changed every time that food weight was
measured. Any residual bits of food in the bedding were
included in measurements. Cumulative food intake data
were obtained by adding all intake measurements during
the study.
Fat and lean body mass were assessed using Echo
MRI (Echo Medical Systems, Houston, MA, USA).
Energy expenditure was measured by assessing oxygen
consumption and carbon dioxide production using an
indirect calorimetry with Comprehensive Lab Animal
Monitoring System (CLAMS; TSE PhenoMaster). Mice
were acclimated in the CLAMS chambers for 72 h before
data collection, and had free access to food and water for
the duration of the studies.
Glucose tolerance test and insulin tolerance test
Two weeks before the last day of the experiment,
an intraperitoneal glucose tolerance test (IPGTT)
and an intraperitoneal insulin tolerance test (IPITT)
were performed. After an overnight fast, IPGTT was
performed by intraperitoneal injection of a 20%
glucose solution (1 g/kg). Blood samples were collected
before and 15, 30, 60, 90, and 120 min after the
injection. For IPITT, after a 4-h fast, an intraperitoneal
injection of 1 IU/kg human rapid insulin (Eli Lilly) was
administered to the HFD-treated mice and 0.5 U/kg
human rapid insulin was administered to the chow
diet-treated mice. Blood samples were collected before
and 15, 30, 60, 90, and 120 min after the injection. For the
IPITT, the response of blood glucose levels was expressed
as a percentage of the values before insulin injections.
Morphological analysis of the liver and white
adipose tissue
Serial sections (5 µM thickness) were taken from the
post-fixed liver and epididymal fat, followed by
hematoxylin and eosin (H&E) staining as described
previously (Huang etal. 2013). The stained sections were
photographed digitally using an optical microscope (Leica
DM6000, Germany), and the images were transferred to
the computer medium.
Immunohistochemistry
For fluorescence detection of POMC, coronal brain
sections from 20-week-old mice in three groups were
generated, and immunohistochemistry was performed
as described previously (Huang etal. 2012). Briefly, brain
sections were incubated with antibody to POMC (Phoenix
Pharmaceuticals, Burlingame, CA, USA) and further
incubated with fluorescent-labeled secondary antibodies.
POMC-positive neurons throughout the mediobasal
hypothalamus were counted using ImageJ software (NIH,
Bethesda, MD, USA). Three serial sections were analyzed
in each mouse (n = 3).
Leptin-induced signal transducer and activator of
transcription 3 phosphorylation
Mice were injected with leptin (A.F. Parlow National
Hormone and Peptide Program, Torrance, CA, USA)
intraperitoneally (3 mg/kg) and killed 30 min later.
The brain sections were evaluated for phosphorylated-
STAT3 (pSTAT3) in hypothalamus neurons as described
previously (Huang et al. 2012). Briefly, brain sections
were incubated with an anti-pSTAT3 antibody (Cell
Signaling), followed by an anti-fluoresces-conjugated
rabbit antibody, pSTAT3 was then visualized under
an optical microscope (Leica DM6000). All pSTAT3-
immunoreactive ARC neurons were counted using ImageJ
software (NIH). Cells within the median eminence were
excluded from these analyses. Three serial sections were
analyzed in each mouse (n = 3).
Insulin signaling and immunoblotting analysis
After overnight fasting, mice were injected intraperi-
toneally with human insulin (10 unit/kg of body weight;
Humulin R, Eli Lilly) or saline and killed 10min later.
Gastrocnemius muscle was rapidly removed and snap
frozen in liquid nitrogen and stored at −80 °C until
analysis. Muscle lysis (60 µg protein) was separated
by SDS–PAGE and transferred onto nitrocellulose
membranes. The membranes were incubated with
polyclonal antibodies against phosphorylation of Serine
473 of AKT and total AKT (Cell Signaling). The bands
were visualized with enhanced chemiluminescence
and quantified by densitometry. The levels of
phosphorylation of AKT on Serine 473 were normalized
by total AKT protein levels.
Proliferative assay and tunnel assay
An endogenous proliferative marker Ki67 was used
to determine the neuronal proliferation. Briefly,
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Ki67 antibody (Abcam) was used for a single
immunolabeling study in brain sections among the
three groups, followed by an anti-fluoresces-conjugated
rabbit antibody. Ki67-positive cells throughout the
mediobasal hypothalamus were counted using ImageJ
software (NIH). Three serial sections were analyzed in
each mouse (n = 3).
A terminal deoxynucleotidyl transferase-mediated
dUTP nick end-labeling (TUNEL) assay was used to
identify double-stranded DNA fragmentation. Briefly,
coronal brain sections were washed in PBS, transferred to
blocking solution for 2 h, and then incubated in primary
POMC antibody (Phoenix Pharmaceuticals) overnight.
The next day, after washing, the sections were transferred
to secondary antibody for 2 h in light-deprived conditions.
After being washed in PBS, samples were incubated at 4°C
in permeability solution (PBS, 0.1% Triton-X, 0.1% sodium
citrate) for 2 min, and then incubated with TUNEL assay
solution (In Situ Cell Death Detection Kit, Fluorescein,
Sigma-Aldrich) for 1 h at 37°. After being washed in PBS,
all sections were mounted on slides with Vectashield
antifade reagent. Negative and positive controls for the
TUNEL assay were confirmed by staining the sections in
the same manner without primary antibody (negative
control) or pretreated with DNAse I (positive control).
Positive cells were counted in the ARC from slides (n = 3)
of each group.
Statistical analysis
Data are expressed as mean ±
s.e.m. Differences between
groups were compared for statistical significance by
ANOVA or Student’s t-test; P < 0.05 denoted significance.
Results
Long-term voluntary exercise training lowers body
weight gain and adiposity induced by HFD
To determine the effects of long-term voluntary running
wheel exercise training on body weight regulation and
adiposity, 45C57BL6 male mice were divided into three
groups (control, HFD, and HFD + EX) for 12 weeks. Figures
1A and B show the average daily locomotion activity and
running distance, respectively. Although HFD groups
show lowered locomotion activity compared with the
control group, the HFD + EX group shows significantly
increased daily running distance, indicating increased
total daily physical activity in the HFD + EX group. Next,
Figure 1
Long-term voluntary exercise training improves
HFD-induced body weight gain and adiposity.
(A) Average 24-h locomotion activity and
(B) running distance in the control, HFD, and
HFD + EX groups. (C) Body weight at the
beginning of study (aged 8 weeks) and (D) body
weight at the end of study (aged 20 weeks) in the
control, HFD, and HFD + EX groups. (E) Fat mass
and (F) lean mass in 18-week-old male mice.
(G) Epididymal, (H) perirental, and (I) mesenteric
fat contents at 20 weeks of age (n = 10); *P < 0.05
vs control group, #P < 0.05 vs HFD group. A full
colour version of this gure is available at
http://dx.doi.org/10.1530/JOE-15-0510.
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Research b t laing, k do and others Exercise restores HFD-impaired
POMC-expression neuron
we measured the body weight before and after the study,
as shown in Figs. 1C and D; there was no difference in
body weight among the three groups at the beginning
of the study. However, the HFD group significantly
increased body weight compared with the control group
(41.1 ± 0.4 vs 28.9 ± 1.1 g) after 12 weeks, and voluntary
exercise training significantly lowered HFD-induced
body weight gain (36.3 ± 1.7 vs 41.1 ± 0.4 g) at the end
of the study.
Echo MRI data revealed that 12 weeks of HFD
increased total fat mass significantly compared with
the control group (Fig. 1E; 8.1 ± 1.9 vs 2.5 ± 0.4 g). Data
also indicated that 12-week of voluntary running wheel
exercise training significantly reduced total fat mass
(6.1 ± 1.7 vs 8.1 ± 1.9 g) compared with the HFD group.
There was no significant difference in total lean mass
between the HFD groups (Fig. 1F).
By the end of the study, some regional fat pads were
harvested and fat contents were weighed. Fat contents
such as epididymal fat content (Fig. 1G; 0.61 ± 0.10 vs
0.75 ± 0.07 g), perineal fat content (Fig. 1H; 0.72 ± 0.06
vs 0.91 ± 0.03 g), as well as mesenteric fat (Fig. 1I;
0.47 ± 0.09 vs 0.67 ± 0.12 g) were significantly decreased
in the HFD + EX group compared with the HFD group.
Voluntary exercise training reduces body weight via
increased energy expenditure despite normal caloric
intake in HFDs.
Change in body weight is controlled by energy
intake and energy expenditure. To assess energy intake,
food was weighed daily. Daily and cumulative caloric
intake in all three groups was calculated at week 8
after the start date. At this point, although there was
a significant increase in calorie intake in the HFD
groups compared with the control group, we did not
observe any caloric intake difference between the
HFD and the HFD + EX groups (Fig. 2A and B). Energy
expenditure, as measured by oxygen consumption over
24 h, was significantly increased in the HFD + EX group
(5.32 ± 0.66 vs 4.72 ± 0.48 L/h/kg of lean mass) only
during the night (Fig. 2C and D). Similarly, the total
amount of carbon dioxide production over 24 h was also
significantly increased in the HFD + EX group compared
Figure 2
Energy intake and energy expenditure in control,
HFD, and HFD + EX groups. (A) Daily food intake
and (B) weekly food intake assessed on singly
housed male aged 12–13 weeks. *P < 0.05 vs
control group. (C) Hourly averages of oxygen
consumption and (D) corresponding 12-h average
during day and night phases of oxygen
consumption. (E) Hourly averages of carbon
dioxide production and (F) corresponding 12-h
average during day and night phases of carbon
dioxide production at the age of 18 weeks in HFD
and HFD + EX groups (n = 10), *P < 0.05 vs HFD
group. A full colour version of this gure is
available at http://dx.doi.org/10.1530/JOE-15-0510.
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with the HFD group (3.92 ± 0.62 vs 3.52 ± 0.39 L/h/kg
of lean mass) only during the night (Fig. 2E and F).
Long-term exercise training improves insulin
sensitivity in HFD
To determine whether long-term voluntary exercise
training can improve insulin sensitivity impaired by
HFD, we measured both fasted and fed status glucose
levels at 19 weeks of age. Although there was no
significant difference in fasting plasma glucose levels
between the HFD + EX and the HFD groups, fed plasma
glucose levels in the HFD + EX group were significantly
reduced compared with the HFD group (Fig. 3A and B).
A glucose tolerance test also revealed that glucose
tolerance was significantly improved in the HFD + EX
group versus the HFD group, especially after 30 and
60 min of glucose injection (Fig. 3C). The insulin
tolerance test also showed improvement associated
with exercise training, with peak differences after 15
and 30 min of the insulin injection. This indicates that
long-term exercise training improves systemic insulin
sensitivity (Fig. 3D).
In support of the notion that long-term exercise
training improves systemic insulin sensitivity in the
HFD + EX group, insulin signaling in skeletal muscle was
examined by immunoblotting for phosphorylation of
AKT (protein kinase B) in gastrocnemius. Figure 3E and
F shows that skeletal muscle phosphorylation of AKT
was significantly impaired in the HFD group compared
with the control group, and voluntary exercise training
remarkably reversed skeletal muscle phosphorylation
of AKT in the HFD + EX group, indicating that there
is significant improvement in skeletal muscle insulin
signaling (Fig. 3E and F).
Voluntary exercise training reduces HFD-induced lipid
accumulation in the liver and adipocytes size in white
adipose tissue
Histological analysis shows that 12 weeks of HFD
significantly increased lipid accumulation in the liver
revealed by H&E stain in liver sample sections, whereas
voluntary exercise training remarkably reduced lipid
accumulation in the liver (Fig. 4, left). In white adipose
tissue, the cell size in HFD group mice was significantly
Figure 3
Long-term voluntary exercise training improves
fed glucose levels and insulin sensitivity. (A) Fasted
and (B) fed glucose levels at the age of 19 weeks,
*P < 0.05 vs HFD group. (C) Glucose tolerance test
and (D) insulin tolerance test were accessed in the
control, HFD, and HFD + EX groups at the age of
18 and 19 weeks old, respectively (n = 15), *P < 0.05
vs HFD group. (E) The representative of western
blot and (F) normalized graphs show insulin
signaling in skeletal muscle by the end of the
study in the control, HFD, and HFD + EX groups
(n = 3); *P < 0.05 vs control group, #P < 0.05 vs HFD
group. A full colour version of this gure is
available at http://dx.doi.org/10.1530/JOE-15-0510.
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Research b t laing, k do and others Exercise restores HFD-impaired
POMC-expression neuron
increased compared with the control group; however,
voluntary exercise training reduced the adipocytes size
in the HFD+EX group (Fig. 4, right).
Effect of long-term voluntary exercise training on
HFD-impaired central leptin signaling
To determine whether the long-term voluntary running
wheel exercise training can improve the hypothalamic
function that controls energy metabolism, we measured
leptin-induced phosphorylation of STAT3 in the
hypothalamus. Leptin-induced phosphorylation of
STAT3 in the ARC and VMH was almost completely
blunted in the HFD group compared with the control
group (54 ± 3 vs 5 ± 1 counts per slice). Voluntary
exercise training partially restored leptin-induced
STAT3 phosphorylation in the HFD-treated mice (19 ± 2
vs 5 ± 1 counts per slice), suggesting that voluntary
exercise training improves central leptin signaling
(Fig. 5).
Effect of long-term HFD and voluntary exercise training
on POMC-expressing neurons
To determine the effect of HFD and exercise training on
POMC-expressing neurons, immunolabeling with an
anti-POMC antibody was assessed. It was found that 12
weeks of HFD significantly reduced the number of POMC
neurons in the hypothalamus (26 ± 3 counts per slice in
the control group vs 16 ± 2 counts per slice in the HFD
group); however, long-term voluntary exercise training
remarkably restored the number of POMC neurons in the
HFD + EX group (23 ± 2 counts per slice in the HFD+EX
group vs 16 ± 2 counts per slice in the HFD group; Fig. 6).
Long-term voluntary exercise training restores
HFD-damaged neuronal proliferation in the
hypothalamus
To elucidate the potential mechanism of HFD- and
exercise-induced POMC-expressing neuron alteration, an
endogenous proliferative marker Ki67 was used to determine
Figure 4
Liver and white adipose tissue morphology in
control, HFD, and HFD + EX groups. Liver sample
(left) and white adipose sample (right) (5 µM
thickness) were accessed by hematoxylin and
eosin staining by the end of the study (mice aged
20 weeks) in the control, HFD, and HFD + EX
groups (n = 3). A full colour version of this
gure is available at http://dx.doi.org/10.1530/
JOE-15-0510.
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neuronal proliferation. Under high-fat conditions, the Ki67-
positive cells showed significantly decreased proliferation
compared with the control group (8 ± 1 counts per slice in
the control group vs 2 ± 2 counts per slice in the HFD group).
It was found that 12 weeks of voluntary exercise training
significantly restored the loss of cell proliferation in the
hypothalamus (4 ± 1 counts per slice in the HFD + EX group
vs 2 ± 2 counts per slice in the HFD group) (Fig. 7). Long-term
voluntary exercise training reduces HFD-induced apoptosis in
POMC-expressing neurons in the hypothalamus. To further
investigate the potential mechanism of HFD- and exercise-
induced POMC-expressing neuron alteration, TUNEL assay
was performed to determine the neuronal apoptosis among
the three groups (Fig. 8). Although there was no apparent cell
apoptosis occurring in the ARC of the control group, 12-week
HFD significantly increased cell apoptosis, especially in the
ARC, and voluntary exercise training strongly protected
the HFD-induced apoptosis in this area. Furthermore, the
apoptosis that specifically occurred in the POMC neurons
was reduced by more than half in the exercise training group
with HFD (10 ± 3 counts per slice in the HFD group vs 4 ± 2
counts per slice in the HFD+EX group).
Discussion
Although over the past decade we have gained a better
understanding of CNS function in regulating food
Figure 5
Long-term voluntary exercise training improves
leptin signaling in arcuate of hypothalamus of
HFD group. (A) Phosphorylation of STAT3 of
hypothalamic sections from the control, HFD, and
HFD + EX groups at 20 weeks of age (n = 3).
(B) Quantication of leptin-induced pSTAT3 in
mediobasal hypothalamus among three groups,
*P < 0.05 vs control group, #P < 0.05 vs HFD group.
3 V, third ventricle; VMH, ventromedial
hypothalamus; scale bars represent 50 µM.
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Research b t laing, k do and others Exercise restores HFD-impaired
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intake and body weight homeostasis (Morton et al.
2006, Mayers & Olson 2012), a gap of knowledge
exists detailing how exercise training mechanistically
induces weight loss via neurological control of energy
balance and body weight in obese subjects. Given the
facts, certain areas within the hypothalamus, such as
ARC, VMH, DMH, and PVH, play important roles in
regulating systemic metabolic homeostasis. However,
to date, emerging evidence has shown that exercise
training-induced improvements are associated with
molecular changes that improve metabolic functions
in most peripheral tissues, such as increased glucose
uptake in skeletal muscle and adipose tissue as well as
decreased lipids accumulation in adipose tissue and
the liver, all of which contribute to enhanced insulin
sensitivity. Although there is an increasing amount of
studies demonstrating that exercise training enhances
the brain function, including the effects of exercise
on learning and memory in hippocampal neurons,
the role of exercise training in improving metabolic
function via CNS-mediated pathways has not yet been
fully understood. Thus, it is worthwhile to investigate
the CNS-associated mechanism(s) of exercise training
to improve metabolic function, particularly under
diet-induced obesity conditions. In this study, we
demonstrated that, first, HFD-induced body weight gain
and adiposity are reversed by voluntary exercise training
mainly through increased energy expenditure despite
normal energy intake, and secondly, these effects may
be associated with protection of POMC neurons and
Figure 6
Long-term voluntary exercise training increases
POMC-expressing neurons in the ARC of
the hypothalamus of HFD group.
(A) Immunouorescence of POMC-expressing
neurons of hypothalamic sections from the
control, HFD, and HFD + EX groups at 20 weeks of
age (n = 3). (B) Quantication of POMC-expressing
neurons in the mediobasal hypothalamus among
three groups, *P < 0.05 vs control group,
#P < 0.05 vs HFD group. 3 V, third ventricle; VMH,
ventromedial hypothalamus; scale bars represent
50 µM.
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enhanced hypothalamic function response to leptin by
voluntary exercise training under HFD conditions.
In the brain, POMC-expressing neurons are mainly
located in the ARC of the hypothalamus and in the
nucleus of solitary tract (NTS) in the brain stem.
Genetically, when a null mutation of Pomc gene is
generated by targeting a gene in embryonic stem cell,
hyperphagic and obesity phenotypes are displayed
(Yaswen et al. 1999). Human patients lacking POMC
also confirm this obesity phenotype (Kruse et al.
1998). Furthermore, a recent study has been published
by Zhan and coworkers using the designer receptor
exclusively activated by designer drugs system to
selectively remove POMC-expressing neurons in these
two areas. These researchers found that postnatal
ablation of POMC neurons in the ARC nucleus
(but not in the NTS) increased food intake, reduced
energy expenditure, and ultimately resulted in obesity
and metabolic and endocrine disorders (Zhan et al.
2013). Taken together, these findings indicate the
importance and necessity of ARC POMC-expressing
neurons in controlling whole-body homeostasis. An
HFD rapidly induces neuron injury and eventually
causes chronic inflammation in the hypothalamus, as
confirmed by obese human subjects’ MRI data (Thaler
et al. 2012). In the brain, POMC-expressing neurons
are specifically and preferentially affected by an HFD
in the ARC (Li et al. 2012). We hypothesize, thus, that
damage to a critical neuronal type (POMC) for body
weight control might play a role in obesity, and exercise
training may play a role to prevent the damage induced
by a HFD.
Figure 7
Long-term voluntary exercise training restores
HFD-damaged neuronal proliferation in
hypothalamus. (A) Immunouorescence of
Ki67-positive cells of hypothalamic sections from
the control, HFD, and HFD + EX groups at 20
weeks of age (n = 3). (B) Quantication of
Ki67-positive cells in the mediobasal
hypothalamus among three groups, *P < 0.05 vs
control group, #P < 0.05 vs HFD group. 3 V, third
ventricle; scale bars represent 50 µM.
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Research b t laing, k do and others Exercise restores HFD-impaired
POMC-expression neuron
POMC-expressing neurons control both energy intake
and energy expenditure. One interesting finding of this
study is that there is no food intake difference between
the HFD and HFD + EX groups observed, despite the fact
that exercise training restored POMC-expressing neurons
significantly. This might be due to counterpart effects,
because POMC neurons are not the only neurons affected
by an HFD. In fact, the orexigenic agouti-related peptide
(AgRP)-expressing neuron also resides in the ARC that
controls food intake, and ablation of AgRP neuron in adult
mice has been shown to result in significantly reduced
food intake (Luquet et al. 2005). It has also been reported
that an HFD induces apoptosis of AgRP neuron (Moraes
etal. 2009). These results suggest that long-term HFD and
exercise training may have broad effects on both orexigenic
and anorexigenic neurons in the ARC. Consistent with our
findings, it has been reported that 6-week voluntary exercise
training promotes leanness and prevents diet-induced
obesity by increasing energy expenditure but not energy
intake. These effects are associated with changes in the
CNS centers that control energy homeostasis, particularly
in the subset of neurons in the VMH, which is another
primary satiety center in the hypothalamus, further proved
the notion that exercise training may have broader effects
than just particular neurons in the hypothalamus. In the
same study, HFD-induced central leptin resistance, revealed
by measuring food intake after central administration of
leptin, was also significantly improved when followed by
voluntary exercise training (Krawczewski et al. 2011). This
suggests a potential mechanism of CNS-associated effects of
Figure 8
Long-term voluntary exercise training
reduces HFD-induced apoptosis in
POMC-expressing neurons in hypothalamus.
(A) Immunouorescence of apoptotic positive cells
and POMC-expressing neurons of hypothalamic
sections from the control, HFD, and HFD + EX
groups at 20 weeks of age (n = 3).
(B) Quantication of apoptotic positive cells in
POMC-expressing neurons in the mediobasal
hypothalamus among three groups. White arrows
indicate apoptotic positive cell in POMC
expressing neurons. *P < 0.05 vs control group,
#P < 0.05 vs HFD group. 3 V, third ventricle; scale
bars represent 50 µM.
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Research b t laing, k do and others Exercise restores HFD-impaired
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exercise training on metabolic function. Our study further
explored this notion by demonstrating central leptin
signaling by immunolabeling phosphorylation of STAT3,
a classic downstream pathway marker of leptin signaling
transduction in the hypothalamus. We found that 12-week
HFD treatment dramatically reduced pSTAT3 signals in
the ARC and VMH nuclei, and that this impairment was
significantly improved by voluntary exercise training. This
was also true with even shorter periods of exercise training
in diet-induced obesity rats. Patterson and coworkers
showed that 3 weeks of post-weaning exercise training
reduced body weight gain and adiposity in selectively bred
diet-induced obese rats, and that these effects are associated
with increased leptin-induced pSTAT3 expression in the
ARC area (Patterson et al. 2009). Leptin directly activates
hypothalamic POMC-expressing neurons (Cowley et al.
2001), and deficiency of leptin signaling pathway activation
in POMC-expressing neurons results in increased body
weight (Balthasar etal. 2004).
We investigated the effects of exercise training in POMC-
expressing neurons directly. A 12-week HFD significantly
decreased the number of POMC-expressing neurons, but
this decrease was not observed in the HFD + EX group. To
the best of our knowledge, this is the first study to show
that voluntary exercise training has a beneficial role on
POMC-expressing neuron turnover. Notably, turnover is
the balance between neurogenesis and neuronal death.
Most recently, neurogenesis has been described in the
hypothalamus and has been shown to participate in the
response of hypothalamic neuronal circuits to metabolic
status (Kokoeva etal. 2005). Emerging evidence suggests that,
in addition to the hippocampal area, active neurogenesis
takes place in other regions of the adult rodent brain,
including the hypothalamus, where a potential neurogenic
niche has been identified. In adults, neurogenesis occurs at
low rates in different areas of the brain. Moreover, the new
neurons produced through adult life seem to contribute to
physiological function of the entire body (Lee etal. 2012).
Neurogenesis in the ARC has shown to be essential for
reducing and sustaining reduced body weight, and an HFD
was shown to disrupt this neuronal proliferation process
in mice with diet-induced obesity (Li et al. 2012, McNay
etal. 2012). To investigate the potential role of exercise in
enhancing neuronal proliferation in the ARC that possibly
promotes POMC-expressing neurons, we measured the
endogenous proliferative marker Ki67’s expression in
the ARC area among the three groups. Consistent with
previous findings, we found that neurogenesis occurs in
adults at very low rates in different areas of the brain. We
could detect very few Ki67-positive cells in the ARC area
of mice in the control group; interestingly, there was a
significant reduction of Ki67-positive cells in the same area
of the HFD group. Voluntary exercise training significantly
restored the Ki67-positive cells in the hypothalamus.
However, one limitation of this study is that Ki67 can only
be detected in premature cells, thus making it impossible
to colabel along with mature cell markers to determine
their final destination. Although we observed that there
is a significant increase of Ki67-positive cells in the ARC
of the HFD + EX group compared with the HFD group, the
total net contribution to the increase of POMC-expressing
neurons associated with reduction of body weight and
adiposity remains mostly unclear. Future studies should
address the specificity of exercise training-induced
neuronal proliferation and weigh the contribution of these
proliferative cells that regulate whole-body metabolism.
Similar to our findings, Borg and coworkers have
recently reported that 7 days of exercise training
increased hypothalamic cell prolifera tion 3.5-fold
above the sedentary mice. However, blocking cell
proliferation via administration of the mitotic blocker
cytosine-1-β-d-arabinofuranoside (AraC) did not affect
food intake or body mass in obese mice, indicating
that the proliferation of new neurons is not required
for maintaining whole-body homeostasis by exercise
training (Borg et al. 2014). Therefore, to elucidate
the potential mechanism of nutrition and exercise
training in altering POMC-expressing neurons, we next
investigated neuronal death by using a TUNEL assay. It
has been reported that overnutrition, such as a long-term
HFD, could induce hypothalamic cell inflammation via
endoplasmic reticulum (ER) stress, and inflammatory
signal transduction can lead to the activation of apoptotic
signaling pathways (Zhang etal. 2008). In contrast to the
neuronal proliferative study, the TUNEL assay revealed
that although there is no obvious apoptosis occurring
in the control group, in the HFD group we found that
more cell apoptosis accumulated in the ARC area of the
hypothalamus, and most strikingly, we also observed that
voluntary exercise remarkably reduced neuronal apoptosis
compared with the HFD group, leading to a potential
protective mechanism in which exercise training rescues
HFD-induced neuronal loss. Along with our findings, Yi
and coworkers have reported that 26weeks of moderate
treadmill exercise training prevented Western-style diet-
induced hypothalamic inflammation by decreasing
microglia activation in the ARC, supporting the idea of
exercise training in repairing neuronal damage in the
hypothalamus (Yi et al. 2012). The possible molecular
mechanism of HFD-induced ER stress might be associated
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Research b t laing, k do and others Exercise restores HFD-impaired
POMC-expression neuron
with IKK-β/NF-κB pathway in the hypothalamus, which
an HFD could activate leading to a progression of ER stress
in the hypothalamus and therefore impairing insulin and
leptin signaling, thus resulting in energy imbalance (Zhang
etal. 2008). Thus, IKK-β/NF-κB in the hypothalamus is a
potential target pathway for exercise training-associated
benefits in the hypothalamus. Interestingly, interleukin
6 (IL6) is a cytokine that has both proinflammatory and
anti-inflammatory functions along with its metabolic
effects on food intake suppression, energy expenditure
induction, and body weight and adiposity reduction.
The actions of IL6 might mediate to suppression of
IKK-β/NF-κB activation in the hypothalamus and thus
help to maintain normal function (Ropelle et al. 2010).
Notably, the IL6 is a highly exercise inducible cytokine in
skeletal muscle (during contraction) as well as in neurons
located in the hypothalamus. Indeed, the increased
hypothalamic IL6 expression was observed in exercise-
trained rats (Ropelle et al. 2010), suggesting a possible
molecular mechanism that exercise improves metabolic
function at least partially via the IL6-IKK-β/NF-κB/ER
stress-mediated pathway, thus preventing HFD-induced
neuronal inflammation/apoptosis and eventual neuron
loss. BDNF is another element that influences neuronal
survival and differentiation (Binder & Scharfman 2004)
and has a strong metabolic function in regulating body
weight (Xu et al. 2003). It has been reported that HFD
reduces hippocampal levels of BDNF (Molteni et al.
2002). Interestingly, BDNF can be induced in the CNS by
exercise and exercise training (Molteni etal. 2004, Huang
etal. 2006), leading us to suspect that BDNF also plays an
important role in protecting neurons from HFD-induced
damages in the hypothalamus, which might be further
enhanced by exercise training. Taking these facts together,
it would be intriguing to investigate the relationship
between exercise-induced hypothalamic IL6 and BDNF
signaling in conjunction with hypothalamic function in
the context of energy homeostasis.
Overall, this study has demonstrated the effects of
voluntary exercise training on metabolic function that
may be associated with CNS-mediated pathways by
protecting POMC-expressing neurons and enhancing
leptin signaling in the ARC nucleus of the hypothalamus.
Although the cellular and molecular mechanisms
behind this phenomenon need to be explored further,
our findings regarding CNS-mediated pathways that
potentially mimic the effect of exercise training to
prevent hypothalamic neuron loss would make a highly
logical and desirable strategy for the prevention and
treatment of obesity in humans.
Declaration of interest
The authors declare that there is no duality of interest associated with this
manuscript.
Funding
This work was supported by start-up funds from East Carolina University
(Greenville, NC, USA) to H Huang.
Author contributions
B L, K D, T M, W D, M A, E L, and H H performed all the experiments and
analyzed data; D Z helped to perform the exercise training experiment.
H H designed and wrote the manuscript. All the coauthors reviewed and
approved the submission of the manuscript.
Acknowledgments
The authors thank Wendy Beachum and Ashley Busuda for their excellent
administrative help.
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Received in final form 11 February 2016
Accepted 1 March 2016
Accepted Preprint published online 1 March 2016
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