Keywords adiposity - dyslipidemia - experimental - menopause - treadmill
Introduction
Menopause is characterized by a decrease in estrogen production, a hormone that stimulates
lipolysis and influences the production of lipoproteins leading to an increase in
visceral adipose tissue (VAT).[1 ]
[2 ]
[3 ] Thus, weight gain associated with climacteric leads to obesity and related diseases
such as dyslipidemia, diabetes, and cardiovascular diseases.[1 ]
[3 ]
Lipid storage is possible due to the increase in number (hyperplasia) and size (hypertrophy)
of VAT adipocytes.[4 ] In obese individuals, the VAT storage capacity is exceeded, and the lipids are deposited
in other organs (e.g., liver, heart, pancreas, and kidneys), which can be damaged
in a process named lipotoxicity.[5 ]
[6 ] The expansion of VAT compresses the blood vessels leading to a deficit in the supply
of oxygen, nutrients, and hormones. The hypoxia caused by VAT stimulates inflammatory
markers and oxidative stress, as well as, the accumulation of immune system cells,
mainly macrophages.[7 ]
[8 ]
[9 ]
Regular physical exercise promotes positive physiological adaptations and minimizes
the effects of aging and menopause by reducing the levels of triglycerides and low-density
lipoprotein cholesterol (LDL-cholesterol), aiding in the loss of visceral fat. Aerobic
exercise is recommended in a non-pharmacological fashion for the prevention and treatment
of diseases associated with weight gain (e.g., dyslipidemia).[1 ]
[10 ]
[11 ]
[12 ] However, a few studies evaluated the direct effects of aerobic exercise in adipose
tissue when associated with estrogen deprivation and dyslipidemia.
Therefore, this study aims to analyze the effects of aerobic exercise on the visceral
adipose tissue, through morphometric, stereological, and immunohistochemical techniques,
of female LDL-receptor knockout ovariectomized mice.
Materials and Methods
Division of Animals
This study was approved by the Ethics Committee in Research of the São Judas Tadeu
University under protocol number 058/2007. We used 24 genetically modified female
mice, knockout of the LDL receptor (LDL-knockout group) and 24 wild female mice (C57BL/6J)
(control group) purchased from the Laboratory Animal Center of São Judas Tadeu University,
São Paulo, Brazil. Animals were kept in cages in a room with controlled temperature
(22–24°C) and a light/dark cycle of 12/12 hours. All mice were fed with standard chow
and water ‘ad libitum .’ The animals were randomly divided into six groups (n = 8/per group): sedentary control (SC), sedentary control ovariectomized (SCO), trained
control ovariectomized (TCO), LDL-knockout sedentary (KS), LDL-knockout sedentary
ovariectomized (KOS), and LDL-knockout trained ovariectomized (KOT).
Experimental Procedures
Ovariectomy was performed at 9 months as previously reported.[13 ] Aerobic physical training began 7 days after ovariectomy for 4 weeks on a treadmill
with progressive load (1 hour per day, 5× per week, and 50–70% of maximal running
speed) as previous reported.[12 ]
[13 ] At the end of the training protocol, animals were euthanized. Blood was collected
in tubes without anticoagulant and centrifuged at 3000 rpm at room temperature for
10 minutes to obtain the serum of each animal to determine the following biochemical
parameters using enzymatic colorimetric assay with spectrophotometric analysis: (A)
glucose, (B) triglycerides, and (C) total cholesterol. Each biochemical parameter
was performed in duplicate as previous described.[12 ]
A laparotomy was performed in which the VAT was removed, weighted, and sectioned.
The samples were fixed in 10% buffered formalin for 24 hours. Afterward, the tissue
was transferred to a 70% ethyl alcohol solution, dehydrated in increasing ethanol
series, diaphanized in xylol, and embedded in paraffin. Four non-serial 6 μm thick
sections were obtained per animal and stained with hematoxylin and eosin for light
microscopy analysis (Zeiss, ×400 magnifications). Additionally, immunohistochemical
stain techniques were used for metalloproteinases 2 (MMP-2, Santa Cruz Biotechnology-sc-13595)
and 9 (MMP-9, Santa Cruz Biotechnology-sc-21733), macrophages (F4/80, Abcam-ab6640),
cyclo-oxygenase-2 (COX-2, Abcam-ab15191), caspase-3 (Caspase-3, GeneTex-GTX22302),
8-hydroxy-2'-deoxyguanosine (8-OHdG, Santa Cruz Biotechnology-sc-66036), and vascular
endothelial growth factor (VEGF, MyBioSource-MBS859873). Thus, sections were washed
with a PBS-Tween solution and incubated with biotinylated secondary antibody, avidin-chain
enzyme, stained with DAB, and lastly with hematoxylin. Ten images per section were
evaluated in each animal (i.e., 40 images per animal). The slides were analyzed using
a light microscopy to identify the presence of a dark brown precipitate and photographed
for quantitative analysis with the aid of the ImageJ software. We analyzed the numerical
density of adipocytes and adipocyte area (µm2 ) through morphometric techniques and with the aid of the Axio Vision 4.8 software
(Zeiss). Using the area, we classified the adipocytes size as: small (<1485.47 µm2 ), medium (between 1485.48 µm2 and 3921.81 µm2 ), and large (>3921.82 µm2 ). Stereology was used for the volume densities of adipocytes, interstitium, blood
vessels with the aid of the ImageJ software (version 1.47-National Institutes of Health)
in a test system of 252 points.[14 ]
Statistical Analysis
Data are expressed as mean ± standard error (SEM). The variables had their normality
and homogeneity verified by the Kolmogorov–Smirnov and Levene tests, respectively.
We used one-way analysis of variance (ANOVA) with Tukey's posttest. The generalized
linear model (GLzM) was used to establish the influence of independent variables (dyslipidemia,
ovariectomy, and physical training) on the differences found between the groups. All
statistical analyses were performed with the aid of the SPSS software (version 21).
P < 0.05 was considered significant.
Results
[Table 1 ] shows that there was no significant difference between initial body mass (IM), final
body mass (FM), and their difference (FM-IM). In contrast, we observed that ovariectomy
promotes a significant increase in visceral adipose tissue (VAT %) in sedentary groups
(SCO and KOS) when compared to non-ovariectomized groups (SC and KS) and physical
training led to a significant reduction in ovariectomized animals adiposity (TCO and
KOT groups).
Table 1
Initial body mass (IM), final body mass (FM), difference between the masses (FM-IM)
and percentage of visceral adipose tissue in relation to final mass (%VAT)
SC
SCO
TCO
KS
KOS
KOT
IM (g)
22.50 ± 0.3
22.90 ± 0.2
22.40 ± 0.1
22.90 ± 0.4
22.70 ± 0.4
23.40 ± 0.2
FM (g)
24.14 ± 0.2
23.80 ± 0.2
23.40 ± 0.4
23.20 ± 0.4
24.90 ± 0.9
25.50 ± 0.4
FM-IM (g)
1.40 ± 0.1
1.40 ± 0.3
0.90 ± 0.4
0.30 ± 0.2
1.90 ± 0.5
1.20 ± 0.1
VAT %
2.30 ± 0.04
3.20 ± 0.02a
1.90 ± 0.04ab
2.60 ± 0.1ac
3.11 ± 0.02abcd
1.70 ± 0.04abde
Values represent mean ± SEM. a
p < 0.05 vs. SC; b
p < 0.05 vs. SCO; c
p < 0.05 vs. TCO; d
p < 0.05 vs. KS; e
p < 0.05 vs. KOS.
We observed that ovariectomy led to an increase in glucose, triglyceride, and total
cholesterol levels; however, physical exercise contributed to the reduction of these
parameters in both control and dyslipidemic animals ([Table 2 ]).
Table 2
Biochemical parameters: glucose (mg/dL), triglycerides (mg/dL), and total cholesterol
(mg/dL) in the studied groups
Groups
Glucose (mg/dL)
Triglycerides (mg/dL)
Total cholesterol (mg/dL)
SC
82.4 ± 18.0
30.0 ± 7.8
69.0 ± 14.8
SCO
98 ± 12
73.9 ± 17.0a
180.0 ± 69.0a
TCO
88.8 ± 13.0
27.0 ± 16.0b
83.0 ± 17.9b
KS
86 ± 20.0
110.0 ± 20.8abc
221.0 ± 52.8ac
KOS
128 ± 6.0
177.0 ± 7.5abcd
242.9 ± 5.0ac
KOT
103.0 ± 44.8
88 ± 20.0ace
158.8 ± 2.0ace
Values represent mean ± SEM. a
p < 0.05 vs. SC, b
p < 0.05 vs. SCO; c
p < 0.05 vs. TCO; d
p < 0.05 vs KS, e
p < 0.05 vs. KOS.
In the morphometric analysis, both dyslipidemia, ovariectomy, and exercise significantly
influenced the numerical density and area of the adipocytes ([Table 3 ]). Regarding size distribution, dyslipidemic groups had a higher percentage of small
adipocytes and a lower percentage of large adipocytes when compared with control groups.
Additionally, ovariectomy increased the percentage of small adipocytes and decreased
medium and large adipocytes, while training reduced small adipocytes and increased
medium and large adipocytes ([Fig. 1 ]). Stereology showed that training was the main factor that significantly influenced
the differences between the groups regarding the density of adipocyte volume ([Table 3 ]). However, both dyslipidemia and moderate training were key factors for the volume
density of interstitium. In contrast, dyslipidemia significantly decreased the blood
vessels density when compared to the non-ovariectomized animals. Representative images
are shown in [Fig. 2 ].
Fig. 1 Frequency distribution of small (<1485.47 µm2 ), medium (between 1485.48 µm2 and 3921.81 µm2 ) and large (>3921.82 µm2 ) adipocytes.
Fig. 2 Representative images stained with hematoxylin and eosin of the visceral adipose
tissue (VAT) of the groups. Scale bar = 20 µm.
Table 3
Morphometric and stereological parameters showing the adipocytes numerical density
(No.), area (µm2 ), and volume density (%), as well as insterstitium (%) and blood vessels (%)
SC
SCO
TCO
KS
KOS
KOT
Morphometry
Numerical density (No.)
23.20 ± 1.19
69.76 ± 7.86a
37.57 ± 2.55a
38.76 ± 2.12a
54.97 ± 4.78ac
35.36 ± 1.30a
Area (µm2 )
2879.12 ± 67.46
1969.36 ± 60.16a
2306.58 ± 41.77ab
2214.72 ± 45.60ab
1686.60 ± 41.62acd
2082.92 ± 44.30abce
Stereology
Adipocytes (%)
86.99 ± 0.44
86.31 ± 1.18a
92.78 ± 0.27ab
91.86 ± 0.43ab
89.15 ± 0.98ac
89.60 ± 0.57acd
Interstitium (%)
11.08 ± 0.44
9.06 ± 0.48a
6.01 ± 0.25ab
6.32 ± 0.27ab
7.48 ± 0.35a
8.86 ± 0.28cde
Blood vessels (%)
1.87 ± 0.18
1.00 ± 0.15a
1.09 ± 0.14
0.62 ± 0.18abc
0.39 ± 0.12abc
0.61 ± 0.11ac
No.: number. Values represent mean ± SEM. a
p < 0.05 vs. SC, b
p < 0.05 vs. SCO; c
p < 0.05 vs. TCO; d
p < 0.05 vs KS, e
p < 0.05 vs. KOS.
We observed that ovariectomy and sedentarism increased immunohistochemical parameters,
as well as, 8-OHdG, caspase-3, COX-2, MMP-9, and VEGF when compared to non-ovariectomized
sedentary animals ([Table 4 ] and [Fig. 3 ]). Nevertheless, ovariectomized animals submitted to a routine of exercise, significantly
modulating these parameters.
Fig. 3 Representative images of immunohistochemical staining for caspase-3, F4/80, COX-2,
MMP-2, MMP-9, VEGF and 8-OHdG between the groups. Scale bar = 20 µm.
Table 4
Immunohistochemical (%) analysis between the groups
SC
SCO
TCO
KS
KOS
KOT
8-OHdG (%)
0.70 ± 0.80
2.30 ± 0.15a
0.74 ± 0.88b
1.28 ± 0.19b
3.20 ± 0.23acd
1.94 ± 0.26ace
Caspase-3 (%)
0.72 ± 0.23
1.38 ± 0.02a
0.43 ± 0.01ab
0.83 ± 0.14bc
2.68 ± 0.14acd
0.88 ± 0.06ace
COX-2 (%)
0.77 ± 0.86
3.25 ± 0.28a
1.28 ± 0.15b
1.67 ± 0.12abc
3.72 ± 0.22acd
2.69 ± 0.19ac
F4/80 (%)
0.57 ± 0.05
2.14 ± 0.16a
0.53 ± 0.46b
1.00 ± 0.11bc
1.86 ± 0.13acd
0.84 ± 0.66be
MMP-2 (%)
0.80 ± 0.72
2.49 ± 0.22a
1.09 ± 0.72b
1.04 ± 0.79b
1.23 ± 0.12b
1.11 ± 0.13b
MMP-9 (%)
0.80 ± 0.54
1.88 ± 0.15a
1.84 ± 0.85a
2.39 ± 0.20a
3.07 ± 0.20abc
2.05 ± 0.11a
VEGF (%)
2.18 ± 0.15
1.15 ± 0.83a
2.32 ± 0.16b
2.32 ± 0.20b
0.96 ± 0.72acd
4.35 ± 0.21abcde
Note: Values represent mean ± SEM. a
p < 0.05 vs. SC, b
p < 0.05 vs. SCO; c
p < 0.05 vs. TCO; d
p < 0.05 vs KS, e
p < 0.05 vs. KOS.
Discussion
In our study, ovariectomized animals had higher VAT, glucose, triglycerides, and total
cholesterol when compared to non-ovariectomized animals. These results corroborate
with the literature, which demonstrates the role of estrogen in the regulation of
energy homeostasis.[15 ] Estrogen reduction is considered a risk factor for obesity and other comorbidities
such as insulin resistance, type 2 diabetes, dyslipidemia, and cardiovascular diseases.[16 ]
[17 ] Our data also demonstrated that aerobic exercise is an important intervention for
significantly decreasing the adiposity of ovariectomized animals and for the reduction
of serum levels of glucose, triglycerides, and total cholesterol because exercise
promotes increased the lipolysis of adipose tissue and improvements in metabolic homeostasis,
regardless of the loss of changes in body weight.[18 ]
[19 ]
The sedentary ovariectomized groups (SCO and KOS) showed an increase in the number
of adipocytes and a great variation in the area of these cells when compared to the
SC and KS groups, respectively. However, exercise training decreased adipocytes numerical
density, and increased adipocytes area. Low levels of estrogen increase the area of
adipocytes as previously demonstrated in studies with mice and humans.[4 ]
[9 ] However, physical exercise allows a decrease in the levels of fat stored in the
VAT, thus the adipocytes decrease in size.[1 ]
[20 ]
[21 ] Rodrigues et al[22 ] working with Sprague–Dawley females showed that ovariectomy increased adipocyte
hypertrophy and that training decreased the area of fat cells.
It is known that adipose tissue has high plasticity and maintains its ability to expand
throughout the life of animals through hypertrophy and hyperplasia.[23 ]
[24 ] The balance between these two mechanisms is an important factor for lipid storage
homeostasis and for adipocyte metabolism because inadequate VAT expansion is related
to dyslipidemia and other disorders such as insulin resistance and changes in adipocytokine
secretion.[25 ]
[26 ]
Hyperplasia was seen as a process that would occur only during early stages of development
and that hypertrophy would have a greater influence on the growth of adipose tissue.[23 ] However, recent research in rodents has proven that during prolonged caloric excess,
new adipocytes may emerge contributing to the expansion of the VAT.[24 ] Smaller adipocytes caused by hyperplasia, in contrast, are associated with increased
angiogenesis, which reduces hypoxia and, consequently, inflammation in adipose tissue.[24 ] In addition, an increase in adipocyte size has been linked to diseases such as insulin
resistance, dyslipidemia and impaired adipocytokine secretion that represents a marker
for adipose tissue dysfunction.[25 ]
[26 ]
[27 ]
The simultaneous presence of large and small cells is associated with biochemical
differences where large and hypertrophic adipocytes are associated with a greater
secretion of inflammatory markers (e.g., COX-2) and less secretion of anti-inflammatory
factors when compared to smaller adipocytes.[24 ] However, future studies analyzing the inflammatory markers are needed. According
to Welte,[28 ] small adipocytes are associated with an increased rate of proliferation and large
adipocytes, in contrast, are related to the accumulation of fat in mature adipocytes.
In addition, small adipocytes are considered especially important to avoid the metabolic
decline associated with obesity because adipogenesis not only distributes excess calories
among newly formed adipocytes but also reduces the number of hypertrophic adipocytes
and, therefore, the secretion of pro-inflammatory factors.[24 ] Thus, the results of our study showed that ovariectomy, by promoting an increase
in the number of adipocytes and inflammatory markers in the SCO and KOS groups, is
directly related to the hyperplasia process, whereas physical exercise, by decreasing
the number of fat cells, is associated with lipolysis, anti-inflammatory factors,
and the maturation of the remaining adipocytes.
Stereological analysis showed that physical exercise decreased the volume density
of adipocytes and increased the volume density of interstices in a statistically significant
way in the dyslipidemic groups. Ovariectomy did not significantly influence these
parameters, but it did contribute to a decrease in vascularization and an increase
in the infiltration of macrophages in VAT. The increase in the volume density of vessels,
VEGF and the decrease in the volume density of macrophages demonstrate that physical
exercise is an effective factor in improving VAT vascularization. However, future
studies should analyze the effect of different exercise intensities on VAT.
As the adipocytes expand, they impair vascularization and, consequently, the diffusion
of oxygen and nutrients to the adipose tissue via matrix remodeling (e.g., metalloproteinases),[7 ]
[9 ] in addition to triggering immune responses and contributing to chronic low-grade
inflammation and oxidative stress.[24 ]
[29 ] Under normal conditions, adipose tissue is minimally infiltrated by immune cells
responsible for maintaining its integrity.[8 ]
[30 ] However, during obesity, overexpression of monocyte chemotactic protein 1 (MCP-1)
causes infiltration of macrophages in the VAT, increase oxidative stress, metalloproteinases
activity, and greater differentiation of adipocytes.[8 ]
[30 ] In addition, the compression of blood vessels due to the expansion of VAT and the
consequent oxygen deficit increases the amount of macrophages (chemotaxis), increasing
inflammatory mediators.[8 ]
[31 ] Physical exercise, in addition to reducing VAT by increasing the oxidation of fatty
acids through lipolysis, increases vascularity, blood flow, and the expression of
anti-inflammatory cytokines and maintain oxidative balance. Such anti-inflammatory
effect contributes to the decrease of macrophage infiltration.[1 ]
[12 ]
[32 ] Thus, further studies should focus in analyze different exercise intensities to
show these parameters.
Conclusion
We can conclude that in animal models exercise is considered a treatment and prevention
of dyslipidemia, a condition related to hypoestrogenism due to menopause because the
decrease in the area of adipocytes provides an increase in vascularization, which
reduces hypoxia and, consequently, chronic inflammation, oxidative stress, and matrix
remodeling. However, more biochemical and molecular studies analyzing the inflammatory
pathways are needed to corroborate our findings mainly in humans using different intensities
of exercise.
