Keywords
testosterone - estradiol - adipose tissue - postmenopause
Palavras-chave
testosterona - estradiol - tecido adiposo - pós-menopausa
Introduction
Sexual hormones are involved in the balance of energy, and they play essential roles
in the control of food intake, energy metabolism and body weight.[1] Body changes observed during the transition to menopause and in the postmenopausal
period, with increasing body weight and a different pattern of fat distribution (transfer
of the main fat storage from the femoral-gluteal region to the abdominal region) are
examples of this involvement.[2]
[3]
The cell mechanisms implicated in this kind of change are not yet completely clear.[4]
[5] What has been revealed is that estrogen affects energy metabolism in a genomic manner
via the estrogen receptor (ER) or G protein-coupled estrogen receptors (GPERs).[6] Modulation of these receptors determines the action of anti-lipogens, the increase
in insulin sensitivity, glucose tolerance, and the decrease in body weight and visceral
mass.[1]
[6] Androgen receptors (ARs) are also present in the fat tissue, but there is less evidence
on their effect.[7]
[8] Some evidences associate androgens to lipogenesis stimulation and lipolysis inhibition
on white visceral fat.[1]
[8]
Estrogen replacement during the postmenopausal period is associated with the reduction
in visceral fat, the distribution of android fat mass and lower body mass index (BMI).[9]
[10] These effects determine a favorable metabolic profile with less risk of diabetes
and mortality.[11]
[12]
Different from estrogen, the impact resulting from androgen replacement on fat tissue
is less often applied. Few clinical trials,[8]
[13]
[14] most of them using heterogeneous methods, have evaluated the outcomes of this therapy
among women during the postmenopausal period.
Some concerns regarding the impacts of androgen replacement therapy in women's health
consider that supraphysiological doses may determine an inflammatory response on the
fat tissue. Such response, especially at a visceral site, is associated with diseases
such as resistance to insulin, dyslipidemia, diabetes, cardiovascular diseases and
stroke.[1]
[6]
The presents paper has the aim of studying the effects on fat tissue of doses of physiological
and supraphysiological testosterone associated or nor with estradiol. The authors
aim to show that high doses of testosterone define an unhealthy expansion of the visceral,
subcutaneous and intramedullary fat tissues.
Methods
Trial Design
In total 48 Wistar female rats (Rattus norvegicus albinus) were offered and cared for by the animal research facility of Faculdade de Medicina
do ABC, Brazil. The animals were fed using Nuvilab CR1 (NuVital Health, Long Beach,
NY, US) and water “ad libitum,” properly filtered in a feeding bottle. Artificial
lighting was controlled to obtain light/dark cycles of 12 hours each, and temperature
between 20 and 28°C, between 60% and 85% of air exchange/hour.
The animals underwent bilateral ovariectomy surgeries (OVXs). After the OVX, the animals
underwent vaginal colpocytology on a daily basis for two months. Colpocytology was
used to assess the cessation of the estrous cycle to evaluate the possibility of hypoestrogenism.
To confirm the possibility of hypoestrogenism, colpocytology was considered in the
diestrus phase for five days in a row.
Then, the animals were randomly divided into 6 groups consisting of 8 animals each.
Each group underwent the following hormone treatment: group P: placebo; group E2 + T:
estradiol (E2) 5 μg/female rat/day + testosterone 5 μg/female rat/day; group T: testosterone
5 μg/female rat/day; group E2 + TT: estradiol (E2) 5 μg/female rat/day + testosterone
30 μg/female rat/day; group TT: testosterone 30 μg/female rat/day; and group E2: estradiol
(E2) 5 μg/female rat/day.
The dose of testosterone dose was calculated to be 6 times higher than that of estradiol.
The rationality on this is that most commercial estradiol transdermal patches for
women release 50 mcg/day, and the only testosterone transdermal patch ever marketed
(Intrinsa, Warner Chilcott UK Ltda, Milbrook, Larne, UK) releases 300 mcg/day (6 times
lower).
During 5 weeks, each group was submitted to a corresponding hormone dose based on
the daily volume of 0,1 mL, which was applied by subcutaneous injections in the dorsal
region. The hormones were prepared and then diluted in sesame oil, Sesame oil was
also used by itself as a placebo.
The entire experiment was approved by the Animal Experimentation Ethics Committee
of Faculdade de Medicina do ABC (CEUA-FMAB, in Portuguese), under number 01/2016.
Material Collection
A few moments before the euthanasia, a blood sample was collected for glycaemia tests.
After the euthanasia, the animals had their right femurs collected, as well as their
visceral adipose and inguinal tissues. The femurs were kept in a 10% formaldehyde
solution for the histological and morphometric analyses. Adipose tissues were weighted
and stored in – 80°C for future analysis of peroxisome proliferator-activated receptors
(PPARs) gamma data using the real-time polymerase chain reaction (PCR) technique.
Histology
The femurs were fixed with 10% formaldehyde during 24 hours, and then they were decalcified
in a 7% ethylenediaminetetraacetic acid (EDTA) solution with 2% paraformaldehyde in
a 0.1-M phosphate buffer (pH 7.4) during 160 days at room temperature. The samples
were dehydrated in graded concentrations of ethanol, and then they were embedded in
paraffin. Serial 7-μm sections were made using a manual Leica RM-2245 (Leica Biosystems,
Nussloch, Germany) microtome, and they were stained with hematoxylin and eosin (H&E).
Morphometry
The morphometric analysis for the estimation of the volume density (Vv) of the intramedullary
adipocytes selected five photomicrographs of each group at a magnification of 100X
. For the evaluation of the adipocyte Vv, crosshairs of points superimposed on photomicrographs
were also needed, and the relation proposed by Weibel was used:[15]
-
Vv = P1/P, where
-
Vv = volume density of a given component;
-
P1 = number of incident points on the component studied;
-
P = total of incident points on the volume unit
PPAR Gamma Gene Expression RT-qPCR
The total RNA was extracted from ~ 1 cm2 of adipose tissue using QIAzol lysis reagent (Qiagen, Hilden, Germany). The amount
of RNA was determined using NanoDrope (Thermo Scientific, Waltham, MA, US) spectroscopy,
and diluted to a final concentration of 50 ηg/μl in 20 μl. In total, 1 μl of RNA was
used for the synthesis and amplification of complementary DNA (cDNA), which followed
the protocol of the high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City,
CA, US). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was
performed using the PPAR gamma gene (Mm00440940_m1), and the endogenous control GAPDH (Mm99999915_g1) followed the TaqMan Universal PCR Master Mix Kit (Applied Biosystems) using the
StepOne Real-Time PCR System (Life Technologies, Foster City, CA, US). Real-time PCR
reactions were conducted as follows: after a pre-denaturation and polymerase-activation
program (2 minutes at 50°C and 10 minutes at 95°C), 50 cycles, each one consisting
of 95°C for 15 seconds and of 60°C for 1 minute. The negative controls consisted of
wells in which the cDNA was absent. The relative expression of PPAR gamma/GAPDH was
calculated using the equation ΔCt, which expresses the difference between the number
of threshold cycles (Cts) of the target genes and the endogenous control.
Statistical Analysis
The results were calculated and analyzed by one-way analysis of variance (ANOVA) test
and the Tukey Test using the GraphPad Prism 5 (GraphPad Software, Inc., San Diego,
CA, US) software. Results ≤ 0.05 (p < 0.05) were considered relevant.
Results
By the end of the fifth week of treatment, we noticed that the average body weight
was different among the groups (p = 0.018), considering that group TT reached a higher final average weight of 307 ± 11.8 g,
and group E2 + TT presented the lowest average, 264 ± 6.9 g ([Table 1]).
Table 1
Comparison of the final weight and the visceral and inguinal fat of the study groups
|
Groups (mean ± standard error of the mean)
|
|
P
|
E2
|
T
|
E2 + T
|
TT
|
E2 +TT
|
p-value
|
|
Final weight (g)
|
291 ± 10.1
|
273 ± 5.3
|
271 ± 8.6
|
284 ± 8.5
|
307 ± 11.8
|
264.7 ± 6.9
|
0.018
|
|
Visceral fat (g)
|
6.5 ± 0.7
|
5.3 ± 0.5
|
3.8 ± 0.3
|
3.9 ± 0.7
|
10.3 ± 1.2*
|
4.5 ± 0.7
|
< 0.01
|
|
Inguinal fat (g)
|
2.6 ± 0.22***
|
2 ± 0.22
|
1.7 ± 0.13
|
1.4 ± 0.17
|
3.1 ± 0.2**
|
1.4 ± 0.13
|
< 0.01
|
Abbreviations: group E2, estradiol 5 μg/day; group E2 + T, estradiol 5 μg/day + testosterone
5 μg/day; group E2 + TT, estradiol 5 μg/day + testosterone 30 μg/day; group T, testosterone
5 μg/day; group TT, testosterone 30 μg/day; group P, placebo.
Notes: Tukey test: * = p < 0.05 (TT versus E2, T, E2 + T e E2 + TT); ** = p < 0.05 (TT versus T, E2 + T e E2 + TT); *** = p < 0.05 (P versus E2 + T e E2 + TT).
The average visceral fat weight was different among groups (p < 0.0001), and was observed to be much higher in group TT when compared with the
other groups. Nevertheless, regarding inguinal fat, the only group with a higher average
weight than that of group TT was group P. On the other hand, the groups that have
were submitted to both estrogen and testosterone presented an average weight in this
particular fat region that was considerably smaller than that of group P (p < 0.001), as seen in [Fig. 1A] and [B]. By using the micrometer, the number of adipocytes expressed in the intramedullary
region was shown to differ among groups (p = 0.012), presenting a significant difference between groups TT and E2 + TT (p < 0.05) ([Fig. 1C] and [Fig. 2]).
Fig. 1 (A) Weight of the visceral and (B) subcutaneous fat tissues of all treatment groups. (C) Distribution of the number of intramedullary adipocytes in all treatment groups.
* Represents the results with significance, that is, p < 0.05.
Fig. 2 Photomicrograph of the tibiae (b) with marrow adipose tissue (MAT) stained with hematoxylin
and eosin (H&E). (A) Group P; (B) group E2 + T; (C) group T; (D) group E2 + TT; (E) group TT; (F) group E2.
The PPAR gamma data did now show any statistical difference among the groups in any
of the adipose tissues analyzed. Despite that, a different behavior was noticed in
terms of data amongt tissues: regarding the subcutaneous tissues, the groups submitted
to isolated testosterone doses presented a higher average number than group; P regarding
the visceral tissue, all groups presented lower numbers than group P ([Fig. 3A] and [B]).
Fig. 3 Expression of the peroxisome proliferator-activated receptor (PPAR) gamma gene in
the subcutaneous and visceral adipose tissues in all treatment groups.
Discussion
The present study assessed the impact of the treatment on visceral, subcutaneous and
intramedullary fat tissues in 30-week-old female ovariectomized rats.
Two daily doses of testosterone were applied. One of the doses contained an equivalent
volume of estradiol (5 μg/day), and the other dose contained a volume of estradiol
6 times higher (30 μg/day) for over 30 days. The group treated with high doses of
testosterone showed a relevant visceral and subcutaneous fat growth in comparison
to the other groups, which is something that contributed to a heavier final weight
in this group. In a different manner, the group submitted to the lower dose showed
fat growth similar to the control groups (placebo and isolated estradiol).
The testosterone serum levels show a direct link with the growth in visceral fat tissue
in women going through postmenopause.[16]
[17]
[18] However, there are few studies assessing the effects of androgen replacement in
fat tissue during this period of a woman's life.[13]
[14]
[16]
[17]
[18]
[19]
[20] Despite applying a heterogeneous method, evidence confirms the idea that postmenopausal
women using androgen have increased lean mass and visceral fat.[1]
[3]
[14]
[19]
[20]
[21]
[22]
Androgen supplementation in animals has been observed to increase visceral fat.[23]
[24]
[25] There is a hypothesis that the estrogen serum level influence on this effect.[24] Iwasa et al[25] have evaluated such effect in female rats which have undergone ovariectomy regarding
the chronic doses of testosterone (associated or not to estradiol) and its link to
food intake, body weight and white fat weight.
Results found by Iwasa et al[25] are conflicting with our results. In comparison with groups treated with testosterone
or not and with no association with estrogen, Iwasa et al[25] observed a relevant reduction in the final weight, as well as in the weight of visceral
and subcutaneous fat in the group treated with testosterone. Their study concluded
that testosterone, once used apart from other elements, had an inhibitory effect on
weight gain and adiposity. This conclusion was not in line with the results of the
group treated with high doses of testosterone in our study, since the high dose determined
a considerable increase in adiposity. Moreover, the group submitted to low doses of
testosterone did now show any differences when compared with the control groups.
Iwasa et al[25] also observed that the association of testosterone to estrogen in ovariectomized
female rats determined an increase in body weight and in the weight of visceral and
subcutaneous fat. Their study concluded that testosterone lowered the inhibitory effect
of estrogen on body weight and adiposity. Despite that, our study, using a different
method, showed that the groups treated with estrogen had similar gains regarding weight
and adiposity, independent of testosterone. However, it is worth nothing that the
groups treated with both kinds of hormone have shown a relevant lower subcutaneous
fat weight in comparison to group P, which is something that did not occur with the
group treated with isolated estrogen.[25]
The difference in results of the study by Iwasa et al[25] and our study can be explained based on the period of treatment (35 days versus 16 days respectively) and the testosterone doses used. Considering the results from
both studies, it can be said that high doses of testosterone can stimulate excessive
adipogenesis, while low doses in association with estradiol can determine its inhibition.
The intramedullary fat tissue represents 70% of the total volume of bone marrow in
a healthy young adult,[26] being the third largest fat storage in the human body. The percentage of its contribution
to the total volume of body adipose tissue may vary from 1% to 30%.[26]
[27]
[28] Such tissue presents structural characteristics and lineage specific traits, which
suggests that marrow adipose tissue (MAT) adipocytes have a source different from
that of white and brown fat.[28] Therefore, it is interesting to observe any possible impacts resulting from the
testosterone treatment on this fat tissue.
When assessing the number of adipocytes in the bone marrow (which represents the amount
of fat in this region), an increase in the average number of adipocytes could be noticed
in the group treated with a high dose of testosterone, despite the difference not
being statistically relevant in relation to the control groups. Thereby, this fat
tissue site showed a behavior pattern similar to that of other fat sites when treated
with sex hormones.
It is worth saying, though, that the number of adipocytes was calculated based on
the entire femurs, and did not reveal any differences between the proximal and distal
regions. Such information is relevant when considering that there may be two forms
of MAT: variable (regulated - rMAT) and constant (constitutive - cMAT). The difference
between them is based on the bone marrow region and on the different response to external
stimuli. Besides, there are different kinds of development pattern, adipocyte size,
lipid saturation, and expression of transcription factors. The rMAT form is found
inside the red marrow (proximal region of the skeleton), and contains more saturated
fat and has a higher sensibility to external stimuli. The cMAT, on the other hand,
is located inside the yellow marrow (distal region of the skeleton), and presents
more resistance to external stimuli.[27]
The number of intramedullary adipocytes was calculated, and it was diffused in most
groups, reducing the possibility to determine the difference between them. Besides
that, there is evidence that ovariectomized rats have an increase in MAT,[27]
[28] something not showed in our study. The accuracy of the methodology for the histological
evaluation of the adipocytes was questioned,[29] and could be the reason for such an incoherent result. The coloration using osmium
tetroxide and further analysis using micro computed tomography (micro-CT) is considered
the standard procedure.[29] However, the risks of the toxicity related to osmium tetroxide and the lack of a
microtomograph kept the team from applying such methods.
The PPARs influence the gene network expression involved in adipogenesis, lipidic
metabolism, inflammation, and the maintenance of metabolic homeostasis. The PPAR gamma
specifically acts as a regulator on adipogenic media, being considered a fundamental
regulator for adipocyte differentiation.[30]
Estrogen has an impact on the transcriptional activity of PPRA gamma, and it inhibits
its effect on adipocyte differentiation.[31] On the other hand, the effect of androgen over the activity of PPAR gamma on the
fat tissue has not been determined. Compatible to what is mentioned in the technical
literature, the groups treated with estrogen in the present study revealed a reduced
PPAR gamma expression when compared with the control groups in both assessed fat tissues.[31]
The pattern of expression of PPRA gamma varied according to the location of the fat
in the groups treated with testosterone. Regarding subcutaneous fat, despite not being
statistically significant, the expression was higher than that of the control groups.
When considering visceral fat, it was lower in both groups.
Considering that PPRA gamma is involved in adipogenesis, it is expected that its expression
would be higher in those groups with higher adipogenesis.[30] This occurred in subcutaneous fat, and the TT group presented a higher fat growth
as well as the highest expression. Nevertheless, this did not occur in visceral fat,
in which the PPRA gamma expression was inhibited. Dysfunctional visceral adipose expansion
results in an inflammatory state and increases the release of inflammatory cytokines
and free fatty acids,[32] which worked as an inhibitor to the activity of PPRA gamma.[33]
[34]
The present study has some limitations. An individual assessment of each region of
the bone marrow could determine different results regarding intramedullary adipose
tissue, but this was not possible due to technical reasons. Moreover, the coloration
of the bone marrow using osmium tetroxide and further analysis using micro-CT is considered
to be the standard procedure for MAT analysis.[29] However, the risks of the toxicity related to osmium tetroxide and the lack of a
microtomograph kept the team from applying such methods in the present study.
Conclusion
High doses of testosterone replacement in OVX rats lead to an expansion of visceral,
subcutaneous and bone marrow fat. This phenomenon seems to be abrogated by estradiol
replacement. The increase in visceral fat is not linked to an increased PPAR gamma
expression.
