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Effects of heat shock therapy on the maintenance of
the heat shock response of young adult and aged
female mice
Henrique Ribeiro Müller
Federal University of Rio Grande do Sul
Helena Trevisan Schroeder
Federal University of Rio Grande do Sul
Antônio Azambuja Miragem
Federal University of Rio Grande do Sul
Paulo Ivo Homem de Bittencourt Jr. ( pauloivo@ufrgs.br )
Federal University of Rio Grande do Sul
Research Article
Keywords: HSP70, estrogen, heat shock response, aging, menopause
Posted Date: June 3rd, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1714008/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Effects of heat shock therapy on the maintenance of the heat shock response
of young adult and aged female mice.
Authors: Henrique Ribeiro Müller1, Helena Trevisan Schroeder1, Antônio Azambuja Miragem2, Paulo
Ivo Homem de Bittencourt Jr.1*
ORCID IDs:
HRM: 0000-0002-9167-1875 (hribeiromuller@hotmail.com)
HTS:0000-0003-4000-3671 (helena.schroeder@hotmail.com)
AAM: 0000-0001-6253-8795 (antonio.miragem@iffarroupilha.edu.br)
PIHBJ: 0000-0003-1907-3341 (pauloivo@ufrgs.br)
Affiliations:
1 Department of Physiology, Federal University of Rio Grande do Sul, Porto Alegre, RS, 90050-170 Brazil
2 Federal Institute of Education, Science and Technology “Farroupilha”, Santa Rosa, RS, 98900-000 Brazil
Running title: Heat shock therapy and the heat shock response
*Correspondence address: Prof. P.I. Homem de Bittencourt Jr., Laboratory of Cellular Physiology, Department of
Physiology, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Rua Sarmento Leite 500, ICBS, 2nd
floor, suite 350, Porto Alegre, RS, 90050-170 Brazil. Phone: +55(51)33083151; fax: +55(51)33084555; e-mail:
pauloivo@ufrgs.br
Abstract
Menopause is an event resulting from the natural aging of the female organism, in which there is ovarian failure
that, consequently, leads to hypoestrogenism. With the gradual decrease of estrogen (E2) levels, women become more
susceptible to developing low-grade chronic inflammation. Low levels of E2 reduce the expression of genes that affect the
balance between energy expenditure and consumption, in addition to negatively affecting carbohydrate and lipid metabolism.
On the other hand, E2 has the ability to induce the expression of the HSP72 protein, one of the members of the 70 kDa family
of heat shock proteins, which are anti-inflammatory and cytoprotective, and whose expression is modulated by different types
of physiologically stressful situations, including heat stress. Therefore, during perimenopause, various homeostatic functions
based on E2-dependent expression of HSP70 may collapse. In this study, we investigated the effects of aging in female mice,
focusing on E2-mediated nitric oxide (NO)-induced heat shock response (HSR). We used female mice of the C57BL/6J
strain: young (4 months) and aged (16 months) adult animals, being submitted to the following treatments: HS (heat shock
treatment for 2 months once a week = 8 sessions with anesthesia) or SHAM (animals kept at room temperature and
anesthetized only). We observed that, with regard to the muscular HSP70 content in the gastrocnemius muscle of adult and
naturally aged females, there was a 59 % increase in the HS group as compared to the SHAM group in adult females; in the
group of aged females, there was a 77 % increase in the HS group compared to the SHAM group (p=0.0341). Still with regard
to HSP70 expression, we found a marked difference (p=0.0047) between the different ages of the animals that received the
HS, indicating a more accentuated increase in the muscular concentrations of this protein in the aged females. As an effect
of HS, we also observed an increase in 17β-Estradiol concentrations compared to the SHAM group (p=0.0467), being 83 %
in the HS group (4 months) and 80 % in the HS group (16 months). In total, the results suggest a potential use of HS therapy
as an alternative to hormone replacement therapy (HRT), especially its long-term safety has already been questioned. In
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addition, there are possible protective effects that are being re-established by the HSR pathway, until then suppressed due to
the effects of aging/natural menopause of female metabolism, which reduces the metabolic capacity, among other effects, to
maintain the hormonal status necessary for cytoprotective levels known.
KEYWORDS: HSP70; estrogen; heat shock response; aging; menopause.
INTRODUCTION
Aging, menopause, and hot flushes
Over the past few decades, the world population has been aging more and more (World Health Organization, 2020).
The decline in the fertility rate, linked to the increase in human life expectancy, from 48 years in the 1950s to a projection of
76 years in 2050 (Bloom et al., 2013), has been due to important advances within scientific fields, such as technological
development. Thus, there is an improvement in the means of production, increasing the availability of resources, food, and
medicines. This made longevity and population expansion a reality.
However, currently, the world scenario generates a warning signal. According to the projections of the World Health
Organization in one of its latest reports, published in 2020: the population is aging at an increasingly rapid pace, which will
make a large part of the population age very shortly. By 2019, the number of people aged 60 and over was 1 billion, a number
that will grow mainly in developing countries, thus reaching the mark of 1.4 billion in 2030 and 2.1 billion in 2050 (Newgard
& Sharpless, 2013; Kanasi, Ayilavarapu & Jones, 2016).
This significant change in the profile of the world population, alerts us to the need to carry out important adaptations
in structural sectors of our society, due to the great impact generated by such an increase, directly affecting health system s
and national economies, many of which have already are under severe pressure (Newgard & Sharpless, 2013). Thus, there is
a promotion of social well-being, even with these pronounced demographic changes, since the increase in life expectancy is
often unhealthy (Greco, Pietschmann, & Migliaccio, 2019).
Biology defines the concept of aging as a “time-dependent functional decline that affects most living organisms”
(Lopez et al., 2013). This natural process of living beings ends up triggering a series of imbalances in the body, inducing the
loss of physiological integrity, making the individual susceptible to developing pathologies, including cardiovascular
diseases, cancer, diabetes, and neurological disorders (Lopez et al., 2013; Kanasi., Ayilavarapu & Jones, 2016).
The biology of aging is a field that has always motivated several studies to investigate and understand the mechanics
of this process. As a result, certain theories were created, one of them being the senescence of aging. Biologically, the term
“cellular senescence” refers to the time when cell growth ceases (Hayflick & Moorehead, 1961). In an aging situation, the
cell replication process ends up being interrupted, causing stem cells, for example, to enter a permanent state of growth
interruption, thus, the accumulation of senescent cells. This can be due to a variety of problems, such as DNA damage, short
telomeres, oxidative stress, and/or changes in chromatin architecture. Thus, organs that require homeostatic cell replication
are compromised (Newgard & Sharpless, 2013).
In the case of an increase in oxidative stress, in senescent human endothelial cells for example, in addition to the
impairment of proteostasis, there is the accumulation of protein aggregates, a potential risk to cell function, thus becoming a
mechanism of deterioration of vascular function in function of aging, predisposing individuals to cardiovasc ular disease
(Hwang et al., 2019).
In addition to tissue homeostatic impairment, senescent cells can produce a series of potent and pleiotropic cytokines,
which make up the senescence-associated secretory phenotype (SASP), such as IL-6, IL-8, TNF-α, and VEGF- α, triggering
profound effects on local inflammation, angiogenesis, fibrosis, and wound healing (Newgard & Sharpless, 2013). Therefore,
aging, in addition to being associated with cellular senescence, is also associated with the production of pro-inflammatory
factors. Tissue dysfunction, together with an increase in inflammation, are related to SASP activity (Newgard & Sharpless,
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2013), promoting a series of aging-related phenotypes: such as a decline in immune system function (Rodriguez, Maoz &
Dorshkind, 2013), as well as decreased β cell regeneration in pancreatic islets (Kushner, 2013). These aging phenotypes are
called “inflammaging” (Kanasi, Ayilavarapu & Jones, 2016), and are a product of the autophagic capacity that negatively
affects the “cleaning activities” in cells, resulting in protein aggregation, mitochondrial dysfunction, oxidative stress, and
when associated with systemic inflammation, causes atherosclerosis, increased cortisol secretion (resulting in insulin
resistance in the muscles) and bone resorption (Salminen, Kaarnir anta & Kauppinen, 2012).
Under this aging context, when we turn our focus to women, a characteristic event that accompanies this process is
menopause. Characterized by follicular absence due to ovarian failure, it is a natural process of the body, and the age at which
it occurs is a powerful indicator of health outcomes in adult life (McCarthy & Raval, 2020). Thus, menopause leads to a
decline in ovarian estrogen and progesterone hormone production (Modi & Dhillo, 2018), triggering a series of changes in
hormonal status that are fundamental in maintaining the cytoprotection conferred by such endocrine pathways, as well as a
reduction in their metabolic capacity. These changes end up negatively influencing the woman's body, as it potentiates a pro-
inflammatory state, predisposing them to immunological disorders (Sharma et al., 2018; Benedusi et al., 2012; Kireev et al.,
2014). This is supported by studies that found a trend toward an increase in circulating pro-inflammatory cytokines (IL-6 and
TNF-alpha) in both natural and surgically induced menopause (Girasole et al., 1999; Pfeilschifter et al., 2002).
One of the biggest difficulties for women during the menopause period is frequently facing vasomotor symptoms
(VMS, from English: VasoMotor Symptoms) such as sweating and hot flashes. These, being highly uncomfortable over the
years (Prague et al., 2018). In post-menopause, about 70% of women have VMS, of which hot flashes are the most common;
strongly associated with disturbances at the hypothalamic level in thermoregulation (Rance et al., 2013).
Hot flushes, in humans, are characterized by brief episodes of a sudden increase in the sensation of body heat (Prague
& Dhillo, 2017), which can last from a few seconds to even more than 10 minutes in duration (Freedman, 1998). Concurrently,
mechanisms responsible for heat dissipation are activated, such as VMS and behavioral change, to reduce body temperature
(Rance et al., 2013). Because hot flashes directly affect women's quality of life, causing discomfort, the y end up being the
main reason why women seek medical intervention during climacteric (Hess et al., 2012; Padilla, 2018).
One of the phenomena that are increasingly becoming common due to population aging is the concomitant increase
in labor force participation among women aged 45 to 64 in OECD countries (Tilly, O'Leary & Russell, 2013; OECD, 2021).
This means that a large and growing number of women are going through the menopausal transition while exercising their
paid jobs (Riach & Jack, 2021). That is, the moment when this transition will be taking place is when the woman will be
experiencing key stages of her career, assuming leadership positions in their teams or areas (Nordling, 2022). This is
extremely important since the data obtained from a survey by the Japan Broadcasting Corporation (NHK) carried out in 2021,
is in line with this reasoning, showing us that: almost a fifth of respondents reported having considered giving up, or actually,
quitting their jobs due to the discomforts generated by menopause. This withdrawal process has a direct impact on sectors of
the economy. It is estimated that around 750,000 working women suffer from the negative effects of menopause on their
work environment, resulting in an estimated loss of ¥420 billion (US$3.2 billion) in the economy (Nordling, 2022). This
shows us that it is necessary to take a more attentive look on the part of employment organizations with government agencies
responsible for health, however, it is important to pay attention to the fact that: support for women at work can easily migrate
to a “management” approach to menopause. This results in consigning menopause to accolades in the HR sector, along with
“performance” awards in the workplace, which ultimately leads to the commodification of female bodies without actually
transforming cultures and practices in the long term (Riach & Jack, 2021).
It is extremely important to point out that aspects such as severity and duration of symptoms caused by menopause
are directly related to women's lifestyle factors. Obesity - particularly overweight - smoking and alcohol, play a key role in
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women's health in the short and long term. Therefore, the promotion of healthy habits, such as maintaining a balanced diet,
regular exercise, abstaining from smoking, and having a controlled alcohol intake, can be fundamental for later health and
well-being (Currie, Abernethy & Hamoda, 2020).
Among the available alternatives, to overcome the discomforts resulting from menopause, women have resorted to hormone
replacement therapy (HRT), which is very effective.
Variable in addition to side effects (Prague et al., 2018). Of the therapies that seek to stop the discomforts generated
by hot flashes, the most sought after by women is the replacement of E2. In addition to its eff ectiveness in controlling hot
flashes, HRT may also support bone health, reducing the risk of osteoporosis and fractures in adulthood (Currie, Abernethy
& Hamoda, 2020). However, therapies based on E2 replacement have had their long-term safety questioned recently
(Rossouw et al., 2013), as they have faced problems related to the risks associated with the treatment, including heart disease,
stroke, and blood clots and cancer of the liver. Furthermore, HRT cannot be initiated to prevent disease in postmenopausal
women (McCarthy & Raval, 2020). HRT can be offered or recommended, unless clinically inappropriate, its benefits, risks,
and side effects are variable for each woman. Therefore, whether or not to use HRT should be an individual choice (Currie,
Abernethy & Hamoda, 2020).
Among the problems associated with HRT treatment mentioned above, the one that has been most discussed and
widely documented is the slightly increased risk of breast cancer associated with HRT (Currie, Abernethy & Hamoda, 2020).
This increase is related to the duration of treatment and may decrease after discontinuation of HRT (NICE, 2015). In context,
breast cancer is the most common among women, with approximately 23 per 1,000 women in the general population aged
50 to 59 years who will suffer from breast cancer within 7.5 years (Currie, Abernethy & Hamoda, 2020). In addition, for
women using E2 and progesterone HRT, there are five extra cases of breast cancer in the same period (NICE, 2015). This
makes many women afraid to take HRT, due to the development of these possible cancer-related risks. As a possible solution
to this problem, the 2015 NICE (National Institute for Health and Care Excellence) guideline literature review on the
diagnosis and management of menopause concluded that optimizing the risk-benefit profile of HRT can potentially reduce
morbidity and mortality from breast cancer in women who need long-term HRT due to ongoing menopausal symptoms.
The main objective of HRT is to restore E2 concentrations previously suppressed due to menopause. This hormone
develops the natural and necessary protection against cerebrovascular diseases and its cytoprotective effects have already
been studied in different systems, such as the cardiovascular, bone, and brain (McCarthy & Raval, 2020). Its decline in
circulating levels due to menopause is directly related to an increased risk of developing cardiovascular disease, osteoporosis,
cancer, diabetes, stroke, sleep disorders, Alzheimer's disease, and cognitive decline (Stampfer & Colditz, 1991; Caldwell,
Yao & Brinton, 2015; Dalal & Agarwal, 2015).
Estrogen and the role of the Heat Shock Response in resolving inflammation
As we saw in the previous section, E2 has important cytoprotective properties for the organism. Coincidentally, this
hormone is also a powerful physiological inducer of the heat shock response (HSR), an anti-inflammatory biochemical
pathway that culminates in the activation of heat shock transcription factors (HSF), especially HSF-1, the only one of the
four HSFs that regulate the expression (Knowlton & Sun, 2001; van Why et al., 1999) of most heat shock proteins (HSP) of
the 70 kDa family (HSP70) (Stice et al., 2011). Adapted throughout evolution, heat shock proteins are the most abundant
proteins in the intracellular environment and, in stressfull situations, they can make up about 5 % of all cellular protein.
Recent studies in proximal tubular cells have shown that, after being administered to male rats, E2 modulates HSF-1 (Hosszu
et al., 2018), translocating it from the cytosol (inactive form) to the nucleus, activating the transcription of HSR genes
(Morano & Thiele, 1999).
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HSR is anti-inflammatory by its very nature, as it participates in a series of processes involving the constant
monitoring of polypeptide chains, such as transport of proteins through membranes, refolding due to aggregation and/or pro-
inflammatory protein misfolding, together with control of the activity of regulatory proteins (Bukao et al., 2006), since HSP70
blocks the activation of nuclear transcription factors of the κB family (NF-κB). And HSF1, by itself, restricts the transcription
of NF-κB-dependent inflammatory genes (Newsholme & Homem de Bittencourt, 2014). These processes are present from
synthesis on the ribosome, to the final formation of the protein's spatial structure. Therefore, it is often said that HSP70 plays
a chaperone role. According to the Oxford Dictionary, the term chaperone refers to an escort, usually an older woman,
responsible for the decorous behavior of a young single woman on social occasions (Malyshev, 2013).
The proteostasis promoted by these chaperones occurs through the sum of their specialties/functions, that is, not all
HSPs respond to specific stresses equally (Hwang et al., 2019). For example, HSP27 protects against heat and reactive oxygen
species (ROS) (Jolly & Morimoto, 2000); HSP72 is in charge of damage caused by ultraviolet radiation (Simon et al., 1995);
while HSP90 acts on signal transduction and regulation of HSF-1 activity (Jolly & Morimoto, 2000; Zou et al., 1998). Part
of the protective effect of HSR inducers lies in their ability to increase the production of the gaseous autacoid nitric oxide
(NO), which in turn is a powerful activator of HSF1 and HSR. On the other hand, chronic degenerative diseases of an
inflammatory nature (e.g. obesity, type 2 diabetes, cardiovascular diseases, neurodegenerative diseases) share in common the
progressive suppression of RRH (Rodrigues-Krause et al., 2012; Di Naso et al., 2015).
Although E2-deficient states are associated with chronic inflammatory diseases, very few studies have analyzed the
possible relationship of the effects of natural aging with HSR in menopausal women and their possible influence on chronic
inflammatory diseases, as well as possible unknown effects or subclinical metabolic changes that may be related to the decline
of E2, including hot flashes.
As proposed by Miragem and Homem de Bittencourt (2017), E2 can induce the expression of HSP72 (Stice et al., 2011), one
of the members of the 70 kDa family of heat shock proteins, which are anti-inflammatory and cytoprotective (e.g.,
cardioprotective, vasculoprotective, protein-accompanying, neuroprotective, antiatherosclerotic, antidiabetic) whose
expression is modulated by different types of physiologically stressful situations, including heat stress (Newsholme &
Homem de Bittencourt, 2014). It is therefore not surprising that during perimenopause (climacteric), various homeostatic
functions based on estrogen-dependent HSP70 expression begin to collapse.
Notably, only very few studies in the field of HSR have focused on menopause and other E2-restrictive situations,
as the reduced production of HSP70 together with its HSR is a favorable scenario for the development of chronic
inflammatory diseases. For example, suppression of HSR is associated with insulin resistance (Kurucz et al., 2002),
worsening of diabetes in obese patients (Rodrigues-Krause et al., 2012), with the severity of nonalcoholic fatty liver disease
( NAFLD) in humans (Di Naso et al., 2015) and with the spread of inflammation throughout the body (Newsholme & Homem
de Bittencourt, 2014; Leite et al., 2016; Bruxel et al., 2019; Bittencourt et al., 2019).
Furthermore, low HSP70 expression secondary to estrogen deficiency correlates with increased vulnerability age of
individuals to cardiovascular disease (CVD) (Tytell & Hooper, 2001), although aging itself can lead to reduced e xpression
of HSP70 (Stice et al., 2009; Leite et al., 2016).
Many (if not all) chronic degenerative diseases of a low-grade inflammatory nature, have at their bases a defective
ability to trigger an adequate HSR in the tissues. This leads to a state of ongoing unresolved inflammation that spreads
throughout all tissues, promoting a proliferative senescence status that positively feeds back inflammation through the
induction of pro-inflammatory cytokines and the potentiation of oxidative stress (Tchkonia et al., 2010; Kim et al., 2012;
Tchkonia et al., 2013; Newsholme & Homem de Bittencourt, 2014; Palmer et al., 2015; Schafer et al., 2017) . Interestingly,
the antidiabetic drug metformin inhibits senescence patterns in cellular models (Moiseeva et al., 2013) and alleviates ischemic
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retinopathy in vivo (Oubaha et al., 2016) by blocking NF-κB downstream pathways (Moiseeva et al., 2016). al., 2013;
Saengboonmee et al., 2017). Therefore, part of metformin's beneficial effects on diabetes can be explained by its ability to
block cellular senescence, thus freeing the HSR to start working again.
In addition to binding to nuclear receptors for E2 (ERΑ and ERΒ), 17-β-estradiol, the most potent form of estrogen
in humans (Hwang et al., 2019), also binds to non-classical estrogen receptors. Membrane E2 (mER), whereby E2 reverses
the inflammatory profile associated with senescence (Stout et al., 2017), while physiological inducers (e.g., exercise, heat
treatment), pharmacological inducers (e.g., cyclopentenone prostaglandins) and co -inducers (e.g., BGP-15, bimoclomol), as
well as transgenic induction of HSR, have all been shown to powerfully block chronic inflammatory states in vivo (Hargitai
et al., 2003; Ianaro et al., 2003; Homem de Bittencourt et al. ., 2007; Kokura et al., 2007; Chung et al., 2008; Choiet al., 2008;
Gupte et al., 2009; Bathaie et al., 2010; Gupte et al., 2011; Sapra et al., 2014; Karpe & Tikoo, 2014). Furthermore, using a
model of atherosclerosis in rats, subjected to heat shock (41.5 °C for 15 min) once a week for 8 weeks, a significant
interruption in the mortality rate of animals was observed, along with an impressive reduction in vascular disease and an
increase in blood flow and reversal of HSR suppression (Bruxel et al., 2019).
E2, on the other hand, enhances reendothelialization and endothelial function after vascular injury and delays
senescence in endothelial progenitor cells in spontaneously hypertensive rats (Imanishi et al., 2005). Thus, containing
vascular senescence, E2 is anti-inflammatory. However, the E2-dependent vasoprotective and anti-inflammatory effects on
the vessels of ovariectomized rats are effective only in young animals (Miller et al., 2007). Likewise, the efficacy of E2-
induced HSR augmentation in rat cardiomyocytes is perceived only in young, but not in elderly, ovariectomized animals
(Stice et al., 2011).
Furthermore, the neuroprotective effects of E2-based HRT against cognitive impairment are seen only in middle-aged
women, but not if HRT is started later in life (Whitmer et al., 2011). This may explain why, in cohort clinical trials, the
benefits or risks related to HRT are strictly dependent on the age of the women (Rossouw et al., 2002; Hodis et al., 2016).
Unfortunately, there is still no published study addressing the effects of aging on HSR in menopausal women and
its possible influence on chronic inflammatory diseases, as well as possible unknown or subclinical metabolic changes that
may be connected to various clinical manifestations, including hot flashes. This is possible because the hypothalamic areas
involved in thermoregulation (infundibular nucleus in humans and arcuate nucleus in other mammals) are the same ones
whose neurons are known to have their function altered after long-term E2 deficiency, particularly kisspeptin-neurokinin B
neurons. -dynorphin (KNDy) (Nakamura & Morrison, 2010; Nakamura, 2011; Rance et al., 2013). Interestingly, KNDy
(pronounced kændy) neurons also direct the neuroprotective expression of HSP70 (Chilumuri & Milton, 2013) and, in many
cases, via NO, even in the absence of estrogen, as discussed below.
In addition to acting on gonadotropin-releasing hormone (GnRH) neurons to control luteinizing hormone (LH)
secretion patterns (Rance et al., 2013; Skorupskaite et al., 2014), KNDy neurons transmit information and receive projections
of multiple nutrients/metabolic that signal specific hypothalamic nuclei to trigger appropriate autonomic responses. This may
explain many metabolic dysfunctions related to sex hormones, such as hypogonadotropic hypogonadism seen in obesity and
diabetes, in which KNDy neuron dysfunction is observed, as well as in polycystic ovary syndrome (PCOS), in which KNDy
neuron activity is exacerbated (Skorupskaite et al., 2014). The coincidence of obesity and the occurrence/severity of
vasomotor symptoms (Vasomotor Symptoms – VMS) closely resembles what is observed in patients with PCOS, who
develop obesity and insulin resistance (Dupont & Scaramuzzi, 2016), intrinsic characteristics of the syndrome and not just
its consequence (Stepto et al., 2013). Also according to the involvement of NO in these dysfunctions, insulin levels and
assessment by the homeostatic model (HOMA) of patients with PCOS are negatively correlated with NO production (Nácul
et al., 2007).
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Considering the above findings, we investigated the effects of aging in young and aged adult female mice, focusing
on E2-mediated NO production-induced HSR on tissue metabolism in the animals' liver, muscle, and adipose tissue.
Methods
Ethical Considerations
This study follows all the ethical rules established by the Arouca Law (Brazilian Federal Law nº 11794/2008) and
the 8th Edition of the Guide for the Care and Use of Experimental Animals published in the 2011 edition of the National
Research Council of the National Academies of the United States, according to ARRIVE guidelines (Kilkenny et al, 2010;
available at: https://www.nc3rs.org.uk/arriveguidelines). All studies were previously reviewed and approved by CEUA-
UFRGS (project n° 19874).
Statistics and Sample Size
The sample size was calculated to detect the smallest expected difference between groups (20 ± 10 ng/mL for the
main critical study variable, HSP70 expression) (Chung; Nguyen; Henstridge; Holmes et al., 2008). The statistical power of
80% was used for the calculation for a significance level of p<0.05 and the DIMAM 1.0 Sample Dimensioning software for
Windows from Editora Guanabara Koogan. The number of animals used for the execution of the project was the minimum
necessary to produce the conclusive result, saving the animal from suffering as much as possible, making a total of six animals
per group. The statistical program used for the analysis was GraphPad Prism 8. Data were tested for normality using the
Shapiro-Wilk test.
When parametric, two-way analysis of variance (ANOVA) with repeated measures analysis and Tukey's post hoc
test were performed. If considered non-parametric, the data were treated, performing transformation to logarithm in base ten,
to present a normal distribution and receive the same analyzes mentioned above.
Animals used
In this study, 34 female mice (Mus musculus) of the C57BL/6J strain aged 2 months were used, in addition to 16
aged females aged 14 months obtained from the Sectorial Animal Facility of the Department of Physiology, ICBS - UFRGS,
maintained by the Laboratory of Cell Physiology – FisCel. The animals remained in the same vivarium, in polypropylene
boxes (300 x 200 x 130 mm, totaling an internal area of 416 cm²). The vivarium had a light (artificial lighting)/dark cycle of
12 h, a temperature of 25 ± 2 °C, and relative humidity between 50 and 60%. The animals received water and food ad libitum
throughout the period. Under these conditions, the temperature inside their respective nests (ca. 28 °C) was kept close to
thermoneutrality for mice (Maloney; Fuller; Mitchell; Gordon et al., 2014).
Aging model
We chose to adopt the aging model considering that few studies in the literature mimic these conditions, usually
having the ovariectomy as the most used model.
In female mice of the C57BL6 strain, with advancing age, estrous cycles become prolonged, and irregular until they
cease completely (Collins, Laakkonen & Lowe, 2019). We call this reproductive senescence (climacteric), which differs from
menopause in that the deregulations resulting from this process are centrally mediated by changes in hypothalamic function
(Schimidt, 2018). But in terms of modeling, the natural age-induced decline in ovarian hormone production in the body is
more similar to menopause in women than ovariectomy surgery (Collins, Laakkonen & Lowe, 2019).
8
Experimental draw
Young female mice aged 2 months and aged 14 months started a chronic heat shock treatment, characterized by
bathing at 41 ºC for 15 min for 8 weeks, with one shock section per week. All animals were anesthetized with sodium
thiopental before the respective sessions, while those belonging to the SHAM group, instead of going to the bath, were kept
resting inside the boxes at a temperature of 37 ºC, or in a room with the air conditioning off and tungsten light to maintain
body temperature. These animals concluded the experiment at the following ages: 4 months for the young/adults and 16
months for the aged ones. After 48 h of the last shock session, the animals were submitted to the intraperitoneal glucose
tolerance test (ipGTT). Adding another 48 h after this test, the animals were sacrificed for the collection of tissue and blo od
samples. The young females, before being sacrificed, were submitted to the procedure of checking the estrous cycle, via
vaginal smear. The animals were allocated to the following groups according to treatment (n=17 for young/adult SHAM and
HS; n= 7 aged SHAM; n= 9 aged HS):
G1: SHAM (4 MONTHS);
G2: HS (4 MONTHS);
G3: SHAM (16 MONTHS);
G4: HS (16 MONTHS).
Figure 1 - Experimental design.
Heat Shock Protocol
For the heat shock procedure (hot tub), the animals were anesthetized with 2% sodium thiopental (m/v) i.p. (5 mg
for 100 g of the mouse; 2.5 µL/g of body weight, which represents about 60-80 µL per animal) ten minutes before the
procedure. The choice of anesthetic is based on the fact that it does not cause glycemic changes characteristic of most
anesthetics in use. The room temperature for the experiment was monitored and maintained at approximately 25 °C.
9
The temperature of the animals was monitored with a rectal thermometer (Minipa, São Paulo, SP; electrode
dimensions, 8 x 2 mm, in length and diameter, respectively), during the entire period in which the animals were kept in the
thermal bath with water at 40°C. After reaching a rectal temperature of 41 °C, the animals remained in the bath, with
continuous temperature monitoring (41-41.7 °C), for 15 min. During the heat shock, the animals had their hind legs and tail
immersed in the water, since especially the tail determines the regulation of temperature, losing heat in the exchange of
temperature with the environment. The procedure used is an adapted and standardized model in our laboratory (Bruxel;
Tavares; Zavarize Neto; De Souza Borges et al., 2019), similar to (Chung; Nguyen; Henstridge; Holmes et al., 2008).
The mice in the SHAM group, not subjected to heat shock, were also anesthetized (and taken as experimental
controls), remaining at room temperature with their body temperature-controlled, maintained between 36.5-37.5 °C, with the
aid of a rectal thermometer. . After heat shock and/or anesthesia, the animals were rehydrated with subcutaneous injection,
in the dorsal region, of sterile 0.9% saline buffer (2 mL for each 100 g of animal) and remained in the laboratory until recovery
from anesthesia.
Oral Glucose Tolerance Test (OGTT)
The oral glucose tolerance test (OGTT) was performed 48 h before the date of death of the animals in each group.
The animals were fasted during the day, 6 h before each test. Blood glucose measurements were performed by caudal
puncture, using 1 µL (one microliter) of blood for verification on the Accu-Chek glucometer (Roche).
To perform the OGTT test, a 50% (m/v) glucose solution was prepared in drinking water, and administered at a dose
of 1 g/kg of body weight, by gavage. To obtain the glycemic curve, the value of fasting blood glucose (6 h) was measured
after the animals remained in the laboratory for at least 30 min (time zero) and at times of 15, 30, 60, 90, and 120 min after
glucose administration. To calculate the incremental area under the curve (iAUC), used to assess the glycemic response to
glucose administration, enabling comparison between groups, the trapezium quadrature method was used (Wolever, 2006).
Thus, the area under the curve was calculated by the law of formation of the integral of a function in the Cartesian plane,
excluding from the calculation the areas below the fasting line, even when any point is less than the initial glycemia, using
the similarity of triangles.
Lee index
The Lee index, also known as the nutrient ratio, is a measure of obesity in rodents and takes into account the animal's
ability to convert dietary nutrients into fat mass. It is given by the quotient between the cube root of the weight (in grams)
and the nasoanal length (in centimeters) or by the quotient between the cube root of the animal's weight (in grams) and the
nasoanal length (in millimeters) multiplied by 10 (Lee, 1929).
Determination of the estrous cycle
On the last day of the experimental protocol, before the animals died, vaginal smears were collected to identify the
phase of the estrous cycle of young females participating in both treatments (SHAM and HS), according to the cytological
technique of vaginal lavage (MCLEAN et al., 2012). Washes were collected using a micropipette and 100 µL of sterile
double-distilled water. ¼ of the volume of this solution was expelled at the opening of the vaginal canal, and the procedure
was repeated 4-5 times to obtain a sufficient number of cells. The extracted wash was placed on a glass slide and the smear
was naturally dried at room temperature. After drying, the smears were stained with an aqueous solution of crystal violet dye
(gentian violet) at 0.1% (m/v). The slides containing the stained smears were immediately analyzed in an Olympus IX81
10
Inverted Microscope under 400X magnification and microphotographs were taken at the time of analysis to document the
cytology observed using a Samsung SM-N770F camera: F1.7; 1/250s; 4.32mm; ISO 50.
Death of Animals
The animals were killed after 12 h of fasting by decapitation in a rodent guillotine, without anesthesia, to obtain
whole blood and tissues for later analysis. The method chosen for euthanasia is based on the fact that it is an effective method
that produces minimal physiological changes in the tissues. In addition, and in particular, because we need to carry out cellular
and biochemical analyzes related to the expression of HSP70, no anesthetics or other substances were injected into the animals
at the time of decapitation, as the anesthetics commonly used in studies with experimental animals lead to intense
hyperglycemia in rodents (Brown et al., 2005; Saha et al., 2005), and glycemic changes interfere in the main parameter
measured, HSP70, whose plasma concentrations are dramatically reduced by the increase in glycemia (Chung et al., 2008).
The animals were decapitated in an environment exclusively intended for the death of the animals, with complete
cleaning of all the material between the death of one animal and another. After death, the animals were dissected for tissue
collection and disposal, they were placed in plastic bags identified with the biohazard symbol and frozen in a freezer (-20
°C), intended for this purpose, in the Physiology Laboratory. Cell Phone - FisCel from UFRGS. Following the institution's
weekly schedule, this biological material was sent to the collection service by a company bid by UFRGS, so that the waste
could be disposed of (autoclaving and sanitary landfill).
Morphometric Measurements
The following internal structures were weighed: heart, liver, gastrocnemius muscle, and visceral white adipose
tissue, and their data were expressed in mg/g of total animal mass.
Biochemical Measurements of Fasting Glucose
Biochemical analyzes of fasting glucose were performed on blood samples collected at the time of euthanasia of the
animals in heparinized tubes. Plasma was obtained after centrifuging whole blood for 15 min at 3,500 x g under refrigeration
(4 °C). Then, the samples were evaluated by colorimetric tests in an ELISA reader, according to the protocols and
recommendations of the manufacturers. Fasting plasma glucose measurements were performed using a LabTest colorimetric
test (catalog number: 133) and estrogen measurements using a specific Cayman brand kit (catalog number 501890).
Immunodetection of Protein Content
For determination of protein expression by Western Blotting, tissues were extracted and collected in a test tube
containing 0.1% (m/v) sodium dodecyl sulfate (SDS) solution and a cocktail of protease inhibitors [( 100 μM
phenylmethylsulfonyl fluoride (PMSF) in isopropanol, 20 μM N-tosyl-L-lysine chloromethyl ketone (TLCK), 2 μg/mL
aprotinin and 2 μg/mL leupeptin] and phosphatase (1 mM sodium orthovanadate, sodium molybdate 1 mM, 1 mM β-
glycerophosphate) to be homogenized at a rate of 5 mL/g of tissue in an Ultra 80 knife homogenizer. In the case of cell
precipitates from whole blood, the lysis buffer chosen was the RIPA Buffer (Sigma-Aldrich, R0278) and homogenization
took place in an ice bath in an ultrasonic processor model UIS250V (Hielscher Ultrasonics GmbH, Germany) equipped with
an LS24d3 sonotrode operating at 24 kHz and 75% of maximum power (= 9 W of final power in the tubes), for 30 s in pulses
of 0.5s each.
Then, the contents were centrifuged at 16,000 x g for 1 min at room temperature and the supernatant was collected.
The protein concentration was determined by the Bradford method (Bradford, 1976), and the samples were diluted in
11
electrophoresis buffer (50 mM Tris pH 6.8, 10% SDS (w/v), 10% glycerol (v/v), 2-mercaptoethanol 10% (v/v) and 2 mg/mL
bromophenol blue) and boiled for 5 minutes for complete denaturation. Equal amounts of protein (~40 μg per well) were
applied to a 10% or 15% polyacrylamide gel for separation for 2 hours using a constant electrical current at 100 V. The
vertical Slab Gel BIO-RAD Mini-Protean TetraCell (BIO-RAD Laboratories, Richmond, CA the USA) was filled with
running buffer containing 25 mM Tris and 1% SDS (w/v), pH 8.3. As a molecular weight marker , 5 µL of pre-stained
recombinant protein mixture (RPN800E, GE Health Care) per gel was used.
To perform the electrotransfer procedure, where the proteins are transferred to a nitrocellulose membrane (GE Health
Care-Amersham), the refrigerated BIO-RAD Blot Cell system was used at a constant 100 V for 2 h. Confirmation the success
of the procedures described above was confirmed by staining the nitrocellulose membranes with Ponceau Red S (Red
Ponceau S, 0.3% sodium salt, Sigma, in 3% trichloroacetic acid solution), which were then destained with TEN solutions
(Tris-EDTA-NaCl at respectively 50, 5 and 150 mM)-Tween 0.1% (v/v).
To determine the expression of HSP72 and HSP73, an anti-HSP70 monoclonal antibody (Sigma H5147) was used
that recognizes both the 72 kDa inducible form and the 73 kDa constitutive form; analysis of the cellular content of GAPDH
was used as a normalizer, obtained by incubation with peroxidase-containing conjugated anti-GAPDH antibody (Sigma
G9295) at 1:1000 dilutions.
Immunodetection was performed by chemiluminescence using Luminol, p-coumaric acid, and H2O2. The
chemiluminescence was photo-documented (60 photos, 1 photo/10 s) and the images were quantified using the ImageQuant
350 automatic system (GE Health Care).
Results
It is known that the duration of the estrous cycle of mice tends to be irregular. Instead of a 4-day cycle being
succeeded again by a 4-day one, there are chances of a 5-day cycle occurring (Schimidt, 2008). In addition, studies have
shown that females that are housed in all-female colonies tend to tend to cease the cycle, extending the diestrus state (Whitten,
1959; Van Der Lee & Boot, 1955; Van Der Lee & Boot., 1956).
To investigate the possible occurrence of these fluctuations, in addition to a possible impact on later analyses, such
as, for example, the verification of E2 levels, we chose to carry out the estrous cycle checking protocol, via vaginal smear in
adult female mice young, thus being able to establish the stage of the estrous cycle in the animals without altering their
reproductive status (McLean, et al., 2012).
By checking the slides, we detected that the animals of the SHAM group were mostly (82%) in the estrus (41%) or
metestrus (41%) phase, while in the group that received heat treatment, 85% of the animals were in estrus (62%) or metestrus
(23%). During the estrus phase, 17-β-E2 levels decrease and prolactin levels increase (Walmer et al., 1992; Parkening,
Collins, & Smith, 1982). The entry into the metestrus period coincides with a continuous increase in the levels of the hormone
progesterone (Walmer et al., 1992) and corresponds to the beginning of the human luteal phase (Mihm, Gangooly &
Muttukrishna, 2011). As progesterone levels begin to rise, there is a small increase in 17-β-estradiol levels in response to
corpus luteum activation (Walmer, et al., 1992; Appelgren, 1969; Sander, et al., 2009). These results give us an important
guide regarding the moment hormonal profile of these animals, helping to understand further analyses.
Below, in the figure, photographs of the histological slides, indicating the predominant cell types of each phase, as
well as the different phases of the estrous cycle in young adult mice, representatives of both groups: treated and untreated
with heat shock:
12
13
Figure 2 – Cytological evaluation of vaginal smears, having three main types of cells detected in the vagina: (A) nucleated
epithelial cells, (B) cornified squamous epithelial cells, and (C) leukocytes. Different stages of the estrous cycle of young
female mice, treated with shock (HS) or without (SHAM): (D) Proestrus – SHAM; (E) Estrus – SHAM; (F) Metaestrus –
SHAM; (G) Diestrus – SHAM; (H) Proestrus – HS; (I) Estrus – HS; (J) Metaestrus – HS; (K) Diestrus – HS. Images captured
by Samsung SM-N770F camera: F1.7; 1/250s; 4.32mm; ISO 50.
When performing the analysis of the samples by the SDS-PAGE electrophoresis technique and immunodetection of
HSP72 (HSPA1A) proteins, we could observe that concerning the muscular HSP70 content of the gastrocnemius of adult (4
months) and naturally aged (16 months) female mice ): we found an increase of 59 % in the HS group when compared to the
SHAM group in adult females; in the group of aged females, there is a 77% increase in the HS group compared to the SHAM
group, differing statistically (p=0.0341). Still concerning HSP, we found a significant difference (p=0.0047) between the
different ages of the animals that received heat shock, indicating a more accentuated increase in the muscular concentrations
of this protein in aged females.
Figure 3 – Protein content of HSP70 in the gastrocnemius muscle of young adult female mice (4 months) and aged (16
months) treated with shock (HS) or without (SHAM); Standardization performed by GAPDH. In detail the representative
image of the respective gels. Data expressed as mean ± DPM. 2-way ANOVA with post hoc Tukey repeated measures.
As an effect of the heat treatment, we also observed an increase in the concentrations of 17-β-Estradiol in the HS
groups compared to the SHAM group (p=0.0467), being 83% in the HS group (4 months) and 80% in the SH group. HS (16
months).
14
Figure 4 – Estradiol concentrations of young adult (4 months) and aged (16 months) female mice treated with shock (HS) or
without (SHAM); Data expressed as mean ± DPM. 2-way ANOVA with post hoc Tukey repeated measures.
With these results and taking into account that E2 is an inducer of HSP70 expression in females, we correlated these
two markers. From the point of view of Pearson's correlation, we did not find a difference, however, there is a behavior in
which, in SHAM animals, the protein expression drops, while in HS it rises, with this behavior being more visible in young
adults.
Figure 5 – Pearson's correlation between Estrogen and HSP70 concentrations. A – Correlation between the young adult
animals of the SHAM group. B - Correlation between the young adult animals of the HS group. C - Correlation between aged
adult animals of the SHAM group. D - Correlation between aged adult animals in the HS group.
15
When measuring the levels of iNOS in the gastrocnemius muscle, we verified that there was no statistical difference
in any of the comparisons, treatment (p=0.2886), and age (0.6826). As will be discussed below, one cannot rule out the
possibility, however, that the increases in NO-induced HSP70 expression are linked to NO production mediated by other
NOS, such as endothelial and neuronal, which are known to be present in muscle tissue.
Figure 6 – iNOS concentrations in the gastrocnemius muscle of young adult (4 months) and aged (16 months) female mice
treated with shock (HS) or without (SHAM); Standardization performed by GAPDH. In detail the representative image of
the respective gels. Data expressed as mean ± DPM. 2-way ANOVA with post hoc Tukey repeated measures.
Here the results of the weighing of the following tissues will be presented: gastrocnemius (figure 7), soleus (figure
8), visceral adipose tissue (figure 9), and liver (10) of the euthanized animals of each treatment and age group. Observing the
masses of the gastrocnemius and soleus muscles obtained, we found that aged females had higher weights when compared
to adults, indicating an expected difference (p=0.004 in the gastrocnemius and p=0.0013 in the soleus), due to the higher
body weights of these animals. compared to the younger ones.
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Figure 7 - Gastrocnemius muscle mass data from young (4 months) and aged (16 months) adult female mice treated with
shock (HS) or without (SHAM) – Raw data in milligrams; Data expressed as mean ± DPM. 2-way ANOVA with post hoc
Tukey repeated measures.
Figure 8 - Soleus muscle mass data from young (4 months) and aged (16 months) adult female mice treated with shock (HS)
or without (SHAM). – Raw data in milligrams; Data expressed as mean ± DPM. 2-way ANOVA with post hoc Tukey repeated
measures.
When analyzing the adipose tissue, concerning the treatment (SHAM vs HS), there was no difference (p=0.9999)
between the groups. When comparing the ages, we found a difference (p=0.0001) in both groups, both between young and
old in the SHAM group and the HS group, indicating a greater accumulation of adipose tissue as the animal ages. Still on the
17
adipose tissue, but now divided by the animal's weight, we found a statistical difference (p=0.0001) between the ages of the
animals, reinforcing what was discussed in the previous graph. Regarding treatments (SHAM vs HS), there was no difference
(p= 0.2693).
Figure 9 - Visceral adipose tissue mass data from young (4 months) and aged (16 months) adult female mice treated with
shock (HS) or without (SHAM). A – Raw data in milligrams; B – Normalization of the extracted tissue by the total mass of
the animals. Data expressed as mean ± DPM. 2-way ANOVA with post hoc Tukey repeated measures. * Differences are
shown in the graph.
When verifying the liver mass of these animals, we observed that the aged animals presented higher weights when
compared to the younger ones (p=0.0001).
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Figure 10 - Liver tissue mass data from young (4 months) and aged (16 months) adult female mice treated with
shock (HS) or without (SHAM) – Raw data in milligrams; Data expressed as mean ± DPM. 2-way ANOVA with post hoc
Tukey repeated measures. *Differences are shown in the graph.
There was no difference between the animals of the different groups when compared at the beginning of the
treatment (expected), nor at the end of the treatment. Differences were observed when animals from the same HS (p=000.3)
or SHAM (p=0.0001) group had their indices compared at the beginning and end of treatment. These results indicate that the
shock ended affecting these parameters. Since animals belonging to different groups showed the same response.
19
Figure 11 - Lee indices in young (4 months) and aged (16 months) adult female mice treated with heat shock (HS) or without
(SHAM) - Data expressed as mean ± DPM. 2-way ANOVA with post hoc Tukey repeated measures.
Analyzing the fasting glycemic values of the animals, we found no difference between groups (p=0.9221) and ages
(p=0.997).
Figure 12 - Fasting glucose (12 h) of young adult (4 months) and aged (16 months) female mice treated with shock (HS) or
without (SHAM). Data expressed as mean ± DPM. *p<0.05 between NC and HFD. 2-way ANOVA with post hoc Tukey
repeated measures.
Regarding the oral glucose tolerance test, no differences were observed in the areas under the curves between the
SHAM and HS animals, both in young and aged animals, as well as in the incremental area under the curve (p=0.4528)
(figure 13). Baseline blood glucose was relatively high in both controls and treated (~8-9 mM, when expected to be around
4.5-5.5 mM).
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Figure 13 - Oral glucose tolerance curve performed in young (4 months) and aged (16 months) female mice treated with
shock (HS) or without (SHAM) - Data expressed as mean ± DPM. * p<0.05 between NC and HFD. 2-way ANOVA with
post hoc Tukey repeated measures.
21
Figure 14 - Incremental area under the OGTT curve of young (4 months) and aged (16 months) female mice treated with
shock (HS) or without (SHAM) - Data expressed as mean ± SEM. 2-way ANOVA with post hoc Tukey repeated measures.
However, when we correlated the values corresponding to the incremental areas under the glycemic curves and the
estradiol concentrations, we found that the aged in the SHAM group, when compared to the young, tended to show a behavior
of decline in metabolic function linked to low estrogen. Surprisingly, when we turn our gaze to the elderly group that
performed the thermal shock, there is a standardization of the curve, equaling the other values of the young animals. This
suggests a possible factor of recovery of metabolic function as a function of heat treatment, reinforcing its proposal as a
therapy.
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Figure 15 – Pearson's correlation between the values of the incremental area under the curve and the concentrations of
Estradiol.
Discussion
Bilateral surgical removal of the ovaries, ovariectomy, is the most common method of inducing ovarian deficiency,
being referred to as a model of menopause (Koebele & Bimonte-Nelson, 2016). However, studies have pointed out that
ovariectomy has reduced protection in female rats since in females, HSP72 levels are higher than in males, but performing
ovariectomy, protein levels end up becoming similar between the sexes, highlighting the possible effect of E2 (Hosszu et al.,
2018). The same study, from the perspective of proximal tubular cells, also reports that an increase in the levels of HSF-1
protein, a modulator of HSP72 synthesis, was found in female rats, showing a considerable difference when compared to
ovariectomized males or females. This suggests that the absence of E2 impairs the HSF-1 response in both normal and
pathophysiological situations (Hosszu et al., 2018). In our study, to evaluate the effects of heat shock, we chose to use aged
female mice, simulating the condition of aging, climacteric and natural menopause of female physiology, in which the
metabolic capacity is reduced, among others to maintain the hormonal status required at known cytoprotective levels.
As previously discussed, menopause is one of the factors responsible for the decline in E2, so, as it induces the heat
shock pathway, HSP70 concentrations are expected to be suppressed. Previously, several studies have reported that in the
aging condition, there is a decrease in RRH along with proteostasis, being observed in several tissues and cells: such as the
liver, lymphocytes, heart, and rat arteries (Anckar & Sstonen, 2011; Faassen et al. al., 1989; Gutsmann-Conrad et al., 1998;
Söti & Csermely, 2002; Stice et al., 2011; Wang et al., 2006), however, being maintained in the brain an d skeletal muscle
(Carnemolla et al., 2014; Locke, 2000) even with aging. More recent studies in rodents have shown that: Loss of E2 in female
rats decreased baseline levels of HSP70, HSP27, and HSP90 in skeletal muscle (Wang et al., 2016). Under these conditions
of hypoestrogenism, we sought to verify a possible restoration of intramuscular HSP70 concentrations through heat treatment,
signaling a possible recovery of the HSR pathway.
In our results, we found an increase in HSP70 in the HS groups when compared to the CTRL, statistically differing
in aged animals (Figure 3). This aspect is reinforced as we also verified a significant difference when comparing the ages of
the animals that underwent the shock, indicating that the treatment increases HSP concentrations in the skeletal muscle, both
for young adults and foraged. However, the increase in muscle concentrations of this protein end up being more pronounced
in aged females, suggesting a recovery of the heat shock pathway, which until then had been suppressed due to
hypoestrogenism. This is surprising, considering that in previous studies, using the ovariectomy model, the induction of HSR
via E2 in rat cardiomyocytes was observed only in young animals and not in the elderly ovariectomized (Stice et al., 2011).
In addition, this increase in plasma concentrations of HSP70 was also recently observed, being independent of the
ovariectomized state, however, occurring in white adipose tissue and not in skeletal muscle. (Lissarassa et al., 2020).
In addition to the increase in HSP levels, the increase in 17-β-Estradiol concentrations, verified in our study (Figure
4), also stands out as an effect of heat shock. Which ends up being intriguing, since until then, studies that evaluated the
effects of hormonal treatment with E2 in rodents, both in males and in ovariectomized females, found that the hormone
attenuates the exercise-induced HSP70 and HSP72 response and has no effect on HSP27 in the muscles of the hind limbs
(Paroo, Dipchand & Noble, 2002; Paroo, Tiidus & Noble, 1999; Bombardier, et al., 2009). Furthermore, evidence has shown
that treatment with E2 at physiological concentrations did not improve cellular proteostasis status (Hwang et al., 2019). This
double increase in the concentrations of both that we found makes us think that E2 induces the expression of HSP70 in
females. Therefore, we correlated these two markers (Figure 5). From the point of view of Pearson's correlation, there was
no difference. However, it is notable the behavior in which SHAM animals have reduced protein expression, while in HS it
increases. This effect is most visible in young adults. Therefore, we can say that heat treatment, through the recovery of E2
23
concentrations, would increase the sensitivity of the heat shock pathway to estradiol, developing cytoprotection, and
preventing the development of a possible condition of pro-inflammatory. Preventing women from being more susceptible to
developing chronic low-grade inflammation diseases. Indicating heat treatment as a strong alternative to hormone
replacement therapy since its long-term safety has already been questioned (Rossouw et al., 2013).
As previously reported in this study, part of the protective effect of HSR inducers lies in their ability to increase the
production of the gaseous autacoid nitric oxide (NO), which in turn is a powerful activator of HSF1 and HSR. Therefore, we
evaluated the content of iNOS in the gastrocnemius, where no difference was found, both when comparing treatments and
ages. What, at first, ends up being curious, given the increases found in the concentrations of E2 and HSP72. However, recent
studies in spinal cord glial cells have shown that with the induction of inflammatory stimuli, there is an increase in iNOS
levels. However, this effect was reduced with heat shock (Clarke et al., 2019), suggesting that this is due to the induction of
HSP70, which limits the activation of NF-κB (Chen et al. 2006).
Furthermore, one cannot rule out the possibility that increases in NO-induced HSP70 expression are linked to NO
production mediated by other NOS, such as endothelial and neuronal (Yuste et al. 2015), which is known to be present in
tissue muscle. However, attention is also paid to the possibility of evaluating this same marker in another tissue, in the aorta,
since nitric oxide concentrations are directly related to vasoconstriction/dilation properties, in addition to being very possibly
correlated with thermogenesis and vasomotor symptoms. characteristic of menopause (sweating and hot flushes). In addition,
attention is paid to a possible formation of atherosclerotic plaque, since women may develop acceleration of the
atherosclerotic process, due to excessive loss of E2 (Knowlton et al., 2001), in addition to the fact that we have verified the
increase in fat deposits and liver mass in the animal (discussed below).
Previous evidence points to the modulating role of estrogens in maintaining muscle mass (Taaffe, et al., 2005;
Ronkainen, et al., 2009). This is because skeletal muscle has E2 beta-receptors in the cell membrane, cytoplasm, and nuclear
membrane (Jubrias et al., 1997). Muscle atrophy resulting from increased apoptosis is associated with biological aging.
Women who have developed a protective antioxidant capacity, in a hypoestrogenic condition, are subject to a decline in
protection after the menopausal transition (Knowlton & Korzick, 2014). This leads to muscle mass loss and muscle strength
generation, which is increasingly common at this age in women (Aloia et al., 1991; Meeuwsen, Samson & Verhaar, 2000;
Rolland et al., 2007; Bondarev, et al., 2018), which can be from 1 to 2% per year (Ferrucci et al., 1997). The name given to
this process of decreasing muscle mass is sarcopenia. A relatively recent condition, resulting from the accelerated drop in E2
concentrations, also ends up harming the health of bones, ligaments, collagen, cartilage, synovial membrane, and joint capsule
(Khadilkar, 2019). In addition to changes in hormonal status due to natural aging, possible causes that may lead to sarcopenia
include low levels of physical activity, reduced protein intake, and increased oxidative stress (Messier et al., 2011). Its
characterization is given by the atrophy of type II fibers, decrease in the number of motor units, and the accumulation of fat
inside the muscle, linked to insulin resistance and insulin-like growth factor I (Khadilkar, 2019).
What draws our attention to this condition is the accumulation of intramuscular fat, because in our results in tissue
variables, female mice aged 16 months had greater masses in the gastrocnemius (Figure 7) and soleus (Figure 8) muscles
when compared to 4-month-old adults. It is known that sarcopenia can progress to the state of sarcopenic obesity, in which
in addition to the decrease in lean mass, there is an increase in fat mass (Khadilkar, 2019). In humans, sarcopenia can end up
putting women at risk, as it is commonly exacerbated in overweight and obese individuals, resulting in morbidity due to the
increased incidence of lifestyle-related diseases, such as type 2 diabetes mellitus, hypertension, and metabolic rate (Zacker,
2006; Manini & Clark, 2012), as well as increased mortality (Khadilkar, 2019). This hypothesis gains strength while studies
have already found that, in postmenopausal women, when compared to younger women, a large amount of non-contractile
muscle tissue, such as intramuscular fat, is found (Jubrias et al., 1997). Thus, we believe that the increase found in the muscle
24
mass of aged females in our experiment may be due to sarcopenic obesity, however, future histological tests would be
necessary to prove it.
The ability of the skeletal muscle to generate force is considered an indication of injury recovery (Warren, Lowe &
Armstrong, 1999). Studies that have evaluated the recovery from a contraction-induced muscle injury in ovariectomized
young mice have shown incomplete strength recovery (Kosir et al., 2014), whereas, in treatments with estradiol, there was
an improvement in strength recovery after a traumatic injury by freezing (Le, et al., 2018). In addition to incomplete recovery
in skeletal muscle and loss of cytoprotection, under a state of hypoestrogenism, they are also associated with dyslipidemia
(Spangenburg & Jackson, 2013), oxidative stress (Sánchez-Rodríguez et al., 2012) and impairment of HSR (Stice et al., 2011;
Miragem & Homem de Bittencourt Jr, 2017). Heat shock proteins, together with mitochondria, protect skeletal muscle against
apoptosis, via E2 (Collins, Laakkonen & Lowe, 2019). Then, when they reduced expression, end up having their function
compromised and thus, make the cellular environment less susceptible to developing an adequate response for the repair of
the lesion in the skeletal muscle.
In addition to its effects on skeletal muscle, E2 contributes to the maintenance of energy balance and glucose
metabolism. With its decline due to menopause, lipid and carbohydrate metabolism ends up being negatively affected
(Kamada et al. 2011; Min et al. 2018; Lovejoy, 2003; Lizcano & Guzmán 2014), predisposing females, including humans,
to metabolic diseases. In ovariectomy models, it was observed that the resulting drop in this hormone led to a decline in the
expression of genes necessary for energy expenditure efficiency. These genes are related to fatty acid metabolism, as well as
in adipose tissue and skeletal muscle (Kamei et al. 2005). This leads to inflammatory disorders related to obesity, such as
increased oxidative profile (Lizcano & Guzmán 2014; Fernández-Sánchez et al. 2011), weight gain (Spangenburg & Jackson
2013; Sibonga et al. 2003) due to decreased basal metabolic rate and energy expenditure (Heine et al. 2000; Camporez et al.
2013), the loss of lean mass, added to a greater sedentary lifestyle and fat oxidation, together with more frequent treatments
with drugs such as steroids and insulin ( Lovejoy et al., 2008; Kalyani et al., 2009), and also by a suppressed HSR in metabolic
tissues (Newsholme & Homem de Bittencourt Jr 2014).
Relating these notes from the literature with our experimental model, we can infer that we are simulating the
condition of a sick animal because, in addition to the effects resulting from menopause/aging, there is also the influence of
the sedentary lifestyle of these animals due to confinement in a restricted space. , in addition to the free supply of food (even
if the diet is not hyperlipidemic) throughout their lives.
Hypoestrogenism due to menopause can lead to an increase in visceral adiposity (Messier et al., 2011). Evidence
shows that the risk of developing obesity (always associated with insulin resistance at some point), which is linked to other
cardiovascular diseases, increases by 86% in women after reaching 40 years of age, and 65% in women between 40 and 59
years of age. , and 74% in those over 60 years old (Schmidt, 2018). Therefore, the reduction of E2 could be an important
factor in the development of insulin resistance (Kennedy et al., 1997).
In a study that evaluated the effects of heat shock in ovariectomized rats, an increase in serum HDL levels was
observed, however, without modifying LDL levels (Lissarassa et al. 2020). In vitro studies showed that heat shock increased
the expression of genes related to fatty acid uptake and triacylglycerol synthesis in adipocytes (Qu et al. 2015), as well as the
demand for glycerol as an energy source (Qu & Ajuwon 2018). In our results, we verified that, concerning adipose tissue
(Figure 9), the main source of triacylglycerol storage, there was a significant increase in the weight of aged females, when
compared to adults. This is intriguing, as previous studies by our group in male LDL receptor knockout mice found that heat
shock reduced body weight gain, Lee index, and visceral obesity (Bruxel et al., 2018). . In our results, it was not verified
possible effects of heat shock on the response of the animals' body composition based on the parameter, Lee's Index. Since
the SHAM group showed the same trend presented by the HS group. In contrast, a study that investigated the effects of aging
25
in female mice also found a similar increase in visceral fat. Due to the redistribution of fat deposits (Schmidt, 2018), a process
that is characterized by hyperplasia and/or hypertrophy of adipocytes associated with changes in metabolic function (Graja
& Schulz, 2015), such as the imbalance between energy consumption and your expense.
Aging is linked to failures in the lipid storage process of adipose tissue, contributing to this redistribution of deposits
(Cartwright et al., 2007). These problems in lipid storage can also be seen at the infra hepatic level, influencing insulin
sensitivity in skeletal muscle (Schmidt, 2018), given that the woman may be developing a low-grade inflammation resulting
from the secretion of pro-inflammatory cytokines, these are not measured here.
In our results, when evaluating the hepatic mass of females (Figure 10), we verified a marked increase in aged
females, when compared to adults. This process of increasing liver weight was also verified in other studies with female mice
(Schmidt, 2018). The possible justification for this gain may be related to visceral fat, which in healthy individuals is a
determining factor for liver fat deposition (Filho et al., 2006).
From a metabolic perspective, even with significant changes in important tissue variables, indicating a possible
development of an inflammatory condition, when we evaluated the glycemia of the animals (Figure 12), we verified that
there was no difference. This ends up being intriguing, since this process does not correlate with the increase in fat deposits,
adipocyte hypertrophy, and greater liver weight in aged animals, due to the maintenance of glucose and triglyceride levels
within the pattern (Schimidt, 2018). Furthermore, when performing the glucose tolerance test (Figure 13), no differences
were observed in the areas under the curves between the SHAM and HS animals, both young and old (Figure 14). This may
have happened due to the females being cloistered, raising basal blood glucose levels in both controls and treated (~8-9 mm,
when expected would be around 4.5-5.5 mm). Previous studies by our group, when evaluating the effects of heat shock in
ovariectomy models, also did not verify alterations, both in glucose and insulin tolerance tests, leading to the reasoning that
while the suppression of RSH is allocated only in the tissue In adipose tissue and not in skeletal muscle, glycemic dysfunctions
will not be perceived, generating the hypothesis that hypoestrogenism alone is not enough to generate a state of insulin
resistance associated with the low level of intramuscular HSP70 (Lissarassa et al. 2020). New studies of glucose uptake in
the presence of insulin in incubations of muscle tissue obtained from these animals may shed new light on these findings.
However, one of the most intriguing effects of heat treatment that we found in our study was the correlation between
the values corresponding to the incremental areas under the glycemic curves and the estradiol concentrations (Figure 15).
The aged animals that did not do the treatment, showed a tendency to decrease the metabolic function linked to the low level
of E2. Surprisingly, there was a standardization of the curve carried out by the aged animals that underwent shock. This
indicated a recovery of the metabolic function of these animals, matching the patterns presented by the youngsters. Further
analyzes are still necessary to assist in the understanding of this process since we are still navigating a sea of uncertainties
and possibilities related to the mechanistic of these metabolic interactions. However, this suggests a possible factor in the
recovery of metabolic function due to heat treatment, which reverses the hypoestrogenism state resulting from menopause,
increasing the sensitivity of the HSR pathway to E2, reinforcing the proposal of heat treatment as a therapy.
Final considerations
It is known that reduced metabolic capacity is linked to aging and menopause. In our analyses, we verified that,
concerning tissue masses, more specifically in the muscles, differences were found only in comparisons related to the age of
the animals and not to the shock, indicating the possible development of sarcopenic obesity. When we turned our gaze to the
adipose tissue, we verified a significant increase in the tissue mass of aged females, due to the redistribution of fat deposits.
This may indicate possible metabolic dysfunctions related to lipid storage, as well as insulin resistance. In line with this result,
the increase in liver mass of aged females is also added. Directly correlated with visceral fat indices, a determining factor of
26
liver fat deposition. In other words, these changes resulting from aging and hypoestrogenism make women more susceptible
to developing low-grade inflammation.
However, in this study, we found that the performance of heat treatment in female mice promoted the reestablishment
of the HSR pathway since we verified an increase in the levels of intramuscular HSP72. This increase is due to the surprising
recovery of circulating E2 concentrations, which until then were suppressed due to hypoestrogenism resulting from
aging/natural menopause. E2 is an inducer of HSR, since, in the E2 x HSP72 ratio, there was a decrease in protein expression
due to the low hormone concentrations in the animals of the SHAM group, while in the treated animals, the observed behavior
was that of an increase. These results lead us to think that heat treatment may be increasing the sensitivity of the HSR pathway
to E2, resuming its cytoprotective effects. In addition, we also found that heat shock triggered a recovery of metabolic
function in treated animals. Reinforcing the proposal of thermal therapy as a strong alternative to hormone replacement
therapy whose long-term safety is questionable.
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