PreprintPDF Available

Effects of heat shock therapy on the maintenance of the heat shock response of young adult and aged female mice

Preprints and early-stage research may not have been peer reviewed yet.

Abstract and Figures

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 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.
Content may be subject to copyright.
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. ( )
Federal University of Rio Grande do Sul
Research Article
Keywords: HSP70, estrogen, heat shock response, aging, menopause
Posted Date: June 3rd, 2022
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
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*
HRM: 0000-0002-9167-1875 (
HTS:0000-0003-4000-3671 (
AAM: 0000-0001-6253-8795 (
PIHBJ: 0000-0003-1907-3341 (
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:
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
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.
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,
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
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).
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
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).
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.
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: 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).
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):
G2: HS (4 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.
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
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
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).
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:
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
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
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.
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.
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
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
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).
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.
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
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).
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.
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
Figure 15 Pearson's correlation between the values of the incremental area under the curve and the concentrations of
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
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
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
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
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.
A.A. Knowlton, D.H. Korzick, Estrogen and the female heart, Mol. Cell. Endocrinol.
389 (1 2) (2014) 31 39.
A.M. Kosir, et al., Influence of ovarian hormones on strength loss in healthy and
dystrophic female mice, Med. Sci. Sports Exerc. 47 (2014) 1177 1187.
Abildgaard J, Tingstedt J, Zhao Y, Hartling HJ, Pedersen AT, Lindegaard B, et al. (2020) Increased systemic inflammation
and altered distribution of T-cell subsets in postmenopausal women. PLoS ONE 15(6): e0235174. https://doi.
org/10.1371 /journal.pone.0235174
Albakova, Z., Armeev, G. A., Kanevskiy, L. M., Kovalenko, E. I., &Sapozhnikov, A. M. (2020). HSP70 multi-functionality
in cancer. Cells, 9(3), 587.
Aloia JF, McGowan DM, Vaswani AN, Ross P, Cohn SH. Relationship of menopause to skeletal and muscle mass. Am J
Clin Nutr 1991;53(6):137883.
Anckar J, Sistonen L. Regulation of HSF1 function in the heat stress response: implications in aging and disease. Annu
Rev Biochem 80: 10891115, 2011. doi:10.1146/annurev-biochem-060809-095203. Carnemolla A, Labbadia JP, Lazell H,
Neueder A, Moussaoui S, Bates
Anderson, R. A., Skorupskaite, K., &Sassarini, J. (2019). The neurokinin B pathway in the treatment of menopausal hot
flushes. Climacteric, 22(1), 51-54.
Appelgren, L.E. Histochemical demonstration of drug interference with progesterone synthesis. Journal of reproduction
and fertility. 19, 185-186 (1969).
Bansal, R., & Aggarwal, N. (2019). Menopausal hot flashes: a concise review. Journal of Mid-life Health, 10(1), 6.
Bathaie SZ, Jafarnejad A, Hosseinkhani S, Nakhjavani M. The effect of hot-tub therapy on serum Hsp70 level and its
benefit on diabetic rats: a preliminary report. Int J Hyperthermia 2010;26:577-585.
Benedusi V, et al. A lack of ovarian function increases neuroinflammation in aged mice. Endocrinology.
Biglia, N., Cagnacci, A., Gambacciani, M., Lello, S., Maffei, S., & Nappi, R. E. (2017). Vasomotor symptoms in
menopause: a biomarker of cardiovascular disease risk and other chronic diseases?. Climacteric, 20(4), 306-312.
Bittencourt A, Schroeder HT, Rosa Porto R, Lemos Muller CH, Krause M, Homem de Bittencourt PI Jr. Heat shock response
to exercise in pancreatic islets of obese mice. Biochimie. 2019;167:
Bochaton, T., Paccalet, A., Jeantet, P., Crola Da Silva, C., Cartier, R., Prieur, C., ... &Ovize, M. (2020). Heat shock protein
70 as a biomarker of clinical outcomes after STEMI. Journal of the American College of Cardiology, 75(1), 122-124.
Bonaccorsi, G., Piva, I., Greco, P., &Cervellati, C. (2018). Oxidative stress as a possible pathogenic cofactor of post-
menopausal osteoporosis: Existing evidence in support of the axis oestrogen deficiency-redox imbalance-bone loss. The
Indian journal of medical research, 147(4), 341.
Bondarev, D., Laakkonen, E. K., Finni, T., Kokko, K., Kujala, U. M., Aukee, P., ... & Sipilä, S. (2018). Physical
performance in relation to menopause status and physical activity. Menopause, 25(12), 1432-1441.
Bruxel MA, Tavares AMV, Zavarize Neto LD, de Souza Borges V, Schroeder HT, Bock PM, Rodrigues MIL, Belló-Klein
A, Homem de Bittencourt PI Jr. Chronic whole-body heat treatment relieves atherosclerotic lesions, cardiovascular and
metabolic abnormalities, and enhances survival time restoring the anti-inflammatory and anti-senescent heat shock
response in mice. Biochimie. 2019 Jan;156:33-46.
Bukau B, Weissman J, Horwich A (2006) Molecular chaperones and protein quality control. Cell 125(3):443451
Burger HG, et al. Cycle and hormone changes during perimenopause: the key role of ovarian function. Menopause.
2008;15(4 Pt 1):603 12.
Caldwell CC, Yao J, Brinton RD. Targeting the prodromal stage of Alzheimer’ s disease: bioenergetic and
mitochondrial opportunities. Neurotherapeutics. 2015;12(1):66 80.
Chen HQ, Wu YF, Zhang YQ, Jin LN, Luo L, Xue B, Lu C, Zhang XR, Yin ZM (2006) Hsp70 inhibits lipopolysaccharide-
induced NF-kapp a B Activation Interacting with TRAF6 and inhibiting its ubiquitination. FEBS Lett 580:31453152
Chilumuri A, Milton NG. The Role of Neurotransmitters in Protection against Amyloid-β Toxicity by KiSS-1
Overexpression in SH-SY5Y Neurons. ISRN Neurosci 2013;2013:253210.
Choi M, Salanova B, Rolle S, Wellner M, Schneider W, Luft FC, Kettritz R. Short-term heat exposure inhibits
inflammation by abrogating recruitment of and nuclear factor B activation in neutrophils expos ed to chemotactic
cytokines. Am J Pathol 2008;172:367-777.
Chung, J., Nguyen, A. K., Henstridge, D. C., Holmes, A. G.,Chan, M. H., Mesa, J. L., Lancaster, G. I., Southgate, R. J.,
Bruce, C. R., Duffy, S. J. et al. (2008) HSP72 protects against obesity-induced insulin resistance. Proc. Natl. Acad. Sci.
U.S.A. 105, 17391744 CrossRef PubMed.
Clark, M. S., Suckling, C. C., Cavallo, A., Mackenzie, C. L., Thorne, M. A., Davies, A. J., & Peck, L. S. (2019). Molecular
mechanisms underpinning transgenerational plasticity in the green sea urchin Psammechinus miliaris. Scientific
reports, 9(1), 1-12.
Clarke, B. E., Gil, R. S., Yip, J., Kalmar, B., & Greensmith, L. (2019). Regional differences in the inflammatory and heat
shock response in glia: implications for ALS. Cell Stress and Chaperones, 24(5), 857-870.
Collaborative Group on Hormonal Factors in Breast Cancer. (2019). Type and timing of menopausal hormone therapy
and breast cancer risk: individual participant meta-analysis of the worldwide epidemiological evidence. The
Lancet, 394(10204), 1159-1168.
Collins, B. C., Laakkonen, E. K., & Lowe, D. A. (2019). Aging of the musculoskeletal system: How the loss of estrogen
impacts muscle strength. Bone, 123, 137-144.
D.R. Taaffe, et al., Estrogen replacement, muscle composition, and physical
function: the health ABC study, Med. Sci. Sports Exerc. 37 (10) (2005) 1741 1747.
Dalal PK, Agarwal M. Postmenopausal syndrome. Indian J Psychiatry. 2015; 57(Suppl 2): S222 32.
de Git, K. C., den Outer, J. A., WolterinkDonselaar, I. G., Luijendijk, M. C., Schéle, E., Dickson, S. L., & Adan, R. A.
(2019). Rats that are predisposed to excessive obesity show reduced (leptininduced) thermoregulation even in the
preobese state. Physiological reports, 7(14), e14102.
De Oliveira, A. A., Priviero, F., Webb, R. C., & Nunes, K. P. (2021). Impaired HSP70 expression in the aorta of female
rats: a novel insight into sex-specific differences in vascular function. Frontiers in Physiology, 12, 517.
Dhillo, M. M. W. S. (2018). Neurokinin 3 Receptor Antagonism: A Novel Treatment for Menopausal Hot Flushes.
Di Naso FC, Porto RR, Fillmann HS, Maggioni L, Padoin AV, Ramos RJ, Mottin CC, Bittencourt A, Marroni NA, Homem
de Bittencourt PI Jr. Obesity depresses the anti-inflammatory HSP70 pathway, contributing to NAFLD progression.
Obesity 2015;23:120-129.
Dupont J, Scaramuzzi RJ. Insulin signalling and glucose transport in the ovary and ovarian function during the ovarian
cycle. Biochem J 2016;473:1483-1501.
E. Bombardier, et al., Effects of ovarian sex hormones and downhill running on
fiber-type-specific HSP70 expression in rat soleus, J Appl Physiol (1985) 106 (6) (2009) 2009 2015.
El Khoudary, S. R., Aggarwal, B., Beckie, T. M., Hodis, H. N., Johnson, A. E., Langer, R. D., ... & American Heart
Association Prevention Science Committee of the Council on Epidemiology and Prevention; and Council on Cardiovascular
and Stroke Nursing. (2020). Menopause transition and cardiovascular disease risk: implications for timing of early
prevention: a scientific statement from the American Heart Association. Circulation, 142(25), e506-e532.
Eleazu, I. C., Jones-O’Connor, M., &Honigberg, M. C. (2020). The Impact of Premature Menopause on Future Risk of
Cardiovascular Disease. Current Treatment Options in Cardiovascular Medicine, 22(12), 1-11.
Elmallah, M. I., Cordonnier, M., Vautrot, V., Chanteloup, G., Garrido, C., & Gobbo, J. (2020). Membrane-anchored heat-
shock protein 70 (Hsp70) in cancer. Cancer Letters, 469, 134-141.
Faassen AE, O’Leary JJ, Rodysill KJ, Bergh N, Hallgren HM. Diminished heat-shock protein synthesis following mitogen
stimulation of lymphocytes from aged donors. Exp Cell Res 183: 326334, 1989. doi:10.1016/0014-4827(89)90393-5.
Fernandes, R. D., Hall, A., Ferguson, M., LorenzenSchmidt, I., Balasubramaniam, V., & Pyle, W. G. (2019). Cardiac
changes during the perimenopausal period in a VCDinduced murine model of ovarian failure. Acta
Physiologica, 227(1), e13290.
Ferrucci L, Guralnik JM, Buchner D, et al. Departures from linearity in the relationship between measures of muscular
strength and physical performance of the lower extremities: the Women’s Health and Aging Study. J Gerontol A Biol
Sci Med Sci. 1997;52:M27585.
G. Le, et al., A moderate oestradiol level enhances neutrophil number and activity in muscle after traumatic injury
but strength recovery is accelerated, J. Physiol. 596 (19) (2018) 4665 4680.
G.L. Warren, D.A. Lowe, R.B. Armstrong, Measurement tools used in the study of
eccentric contraction-induced injury, Sports Med. 27 (1) (1999) 43 59.
Girasole G, et al. Oestrogens prevent the increase of human serum soluble interleukin-6 receptor induced by
ovariectomy in vivo and decrease its release in human osteoblastic cells in vitro. Clin Endocrinol. 1999;51(6):801 7.
Gliemann, L., &Hellsten, Y. (2019). The exercise timing hypothesis: can exercise training compensate for the reduction
in blood vessel function after menopause if timed right? The Journal of Physiology, 597(19), 4915-4925.
Gowda, R., Jaffa, M., &Badjatia, N. (2018). Thermoregulation in brain injury. Handbook of Clinical Neurology, 157, 789-
GP. Contesting the dogma of an age-related heat shock response impairment: implications for cardiac -specific age-
related disorders. Hum Mol Genet 23: 36413656, 2014. doi:10.1093/hmg/ddu073.
Greco, E. A., Pietschmann, P., & Migliaccio, S. (2019). Osteoporosis and sarcopenia increase frailty syndrome in the
elderly. Frontiers in endocrinology, 10, 255.
Gupte AA, Bomhoff GL, Swerdlow RH, Geiger PC. Heat treatment improves glucose tolerance and prevents skeletal
muscle insulin resistance in rats fed a high-fat diet. Diabetes 2009;58:567-578.
Gupte AA, Bomhoff GL, Touchberry CD, Geiger PC. Acute heat treatment improves insulin-stimulated glucose uptake
in aged skeletal muscle. J Appl Physiol 2011;110:451-457.
Gutsmann-Conrad A, Heydari AR, You S, Richardson A. The expression of heat shock protein 70 decreases with cellular
senescence in vitro and in cells derived from young and old human subjects. Exp Cell Res 241: 404413, 1998.
doi:10.1006/excr.1998.4069. H421-H429.
Hargitai J, Lewis H, Boros I, Rácz T, Fiser A, Kurucz I, Benjamin I, Vígh L, Pénzes Z, Csermely P et al. Bimoclomol, a
heat shock protein co-inducer, acts by the prolonged activation of heat shock factor-1. Biochem Biophys Res Commun
Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res.1961;25:585621.
Hellsten, Y. (2019). Oestrogen, exercise and vascular function. The Journal of physiology, 597(19), 4871-4871.
Hess, R., Thurston, R.C., Hays, R.D., Chang, C.-C., Dillon, S.N., Ness, R.B., Bryce, C.L., Kapoor, W.N., and Matthews,
K.A. (2012). The impact of menopause on health-related quality of life: results from the STRIDE longitudinal study.
Qual. Life Res. 21, 535544.
Hodis HN, Mack WJ, Henderson VW, Shoupe D, Budoff MJ, Hwang-Levine J, Li Y, Feng M, Dustin L, Kono N et al.
Vascular Effects of Early versus Late Postmenopausal Treatment with Estradiol. N Engl J Med 2016;374:1221-1231.
Hosszu, A., Antal, Z., VeresSzekely, A., Lenart, L., Balogh, D. B. Szkibinszkij, E., … & Fekete, A. (2018). The role of
Sigma1 receptor in sexspecific heat shock response in an experimental rat model of renal ischaemia/reperfusion
injury. Transplant International, 31(11), 1268-1278.
Hwang, H. V., Lin, Y., Rebuffatti, M. N., Tran, D. T., Lee, L., Gomes, A. V., ... & Knowlton, A. A. (2019). Impaired
proteostasis in senescent vascular endothelial cells: a perspective on estrogen and oxidative stress in the aging
vasculature. American Journal of Physiology-Heart and Circulatory Physiology, 316(2),
I.B. Meeuwsen, M.M. Samson, H.J. Verhaar, Evaluation of the applicability of HRT
as a preservative of muscle strength in women, Maturitas 36 (1) (2000) 49 61.
Ianaro A, Maffia P, Cuzzocrea S, Mazzon E, Santoro MG, Di Rosa M, Ialenti A. 2-Cyclopenten-1-one and prostaglandin
J2 reduce restenosis after balloon angioplasty in rats: role of NF-kB. FEBS Lett 2003;553:21-27.
Imanishi T, Kobayashi K, Hano T, Nishio I. Effect of estrogen on differentiation and senescence in endothelial progenitor
cells derived from bone marrow in spontaneously hypertensive rats. Hypertens Res 2005;28:763-772.
Jolly C, Morimoto RI. Role of the heat shock response and molecular chaperones in oncogenesis and cell death. J Natl
Cancer Inst 92: 15641572, 2000. doi:10.1093/jnci/92.19.1564.
Jones, H., Bailey, T. G., Barr, D. A., France, M., Lucas, R. A., Crandall, C. G., & Low, D. A. (2019). Is core temperature
the trigger of a menopausal hot flush?. Menopause, 26(9), 1016-1023.
Jubrias SA, Odderson IR, Esselman PC, Conley KE. Decline in isokinetic force with age: muscle cross-sectional area and
specific force. Pflugers Arch 1997;434(3):24653.
Kalyani, R. R., Franco, M., Dobs, A. S., Ouyang, P., Vaidya, D., Bertoni, A., ... & Golden, S. H. (2009). The association of
endogenous sex hormones, adiposity, and insulin resistance with incident diabetes in postmenopausal women. The
Journal of Clinical Endocrinology & Metabolism, 94(11), 4127-4135.
Kanasi, E., Ayilavarapu, S., & Jones, J. (2016). The aging population: demographics and the biology of aging.
Periodontology 2000, 72(1), 13-18.
Karpe PA, Tikoo K. Heat shock prevents insulin resistance-induced vascular complications by augmenting angiotensin-
(1-7) signaling. Diabetes 2014;63:1124-1139.
Kaszubowska, L., Foerster, J., Kaczor, J. J., Schetz, D., Ślebioda, T. J., &Kmieć, Z. (2017). Expression of cellular protective
proteins SIRT1, HSP70 and SOD2 correlates with age and is significantly higher in NK cells of the oldest
seniors. Immunity & Ageing, 14(1), 1-13.
Kenny, G. P., Notley, S. R., & Gagnon, D. (2017). Direct calorimetry: a brief historical review of its use in the study of
human metabolism and thermoregulation. European journal of applied physiology, 117(9), 1765-1785.
Khadilkar, S. S. (2019). Musculoskeletal disorders and menopause. The Journal of Obstetrics and Gynecology of India,
69(2), 99-103.
Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG (2010) Improving Bioscience Research Reporting: The
ARRIVE Guidelines for Reporting Animal Research. PLoS Biol 8(6): e1000412. doi:10.1371/ journal.pbio.1000412
Kim G, Meriin AB, Gabai VL, Christians E, Benjamin I, Wilson A, Wolozin B, Sherman MY. The heat shock transcription
factor Hsf1 is downregulated in DNA damage-associated senescence, contributing to the maintenance of senescence
phenotype. Aging Cell 2012;11:617-627.
Kireev RA, et al. Melatonin and oestrogen treatments were able to improve neuroinflammation and apoptotic
processes in dentate gyrus of old ovariectomized female rats. Age (Dordr). 2014;36(5):9707.
Knowlton AA, Sun L. Heat-shock factor-1, steroid hormones, and regulation of heat-shock protein expression in the
heart. Am J Physiol Heart Circ Physiol 2001; 280: H455.
Kokura S, Adachi S, Manabe E, Mizushima K, Hattori T, Okuda T, Nakabe N, Handa O, Takagi T, Naito Y, Yoshida N,
Yoshikawa T. Whole body hyperthermia improves obesity-induced insulin resistance in diabetic mice. Int J
Hyperthermia 2007;23:259-265.
Kostrycki IM, Wildner G, Donato YH, Dos Santos AB, Beber LCC, Frizzo MN, Ludwig MS, Keane KN, Cruzat V, Rhoden
CR, Heck TG. Effects of High-Fat Diet on eHSP72 and Extra-to-Intracellular HSP70 Levels in Mice Submitted to
Exercise under Exposure to Fine Particulate Matter. J Diabetes Res. 2019 Jan 6;2019:4858740.
Krajewski-Hall, S. J., Blackmore, E. M., McMinn, J. R., & Rance, N. E. (2018). Estradiol alters body temperature
regulation in the female mouse. Temperature, 5(1), 56-69.
Krüger, K., Reichel, T., &Zeilinger, C. (2019). Role of heat shock proteins 70/90 in exercise physiology and exercise
immunology and their diagnostic potential in sports. Journal of Applied Physiology, 126(4), 916-927.
Kurucz I, Morva A, Vaag A, Eriksson KF, Huang X, Groop L, Koranyi L. Decreased expression of heat shock protein 72
in skeletal muscle of patients with type 2 diabetes correlates with insulin resistance. Diabetes 2002;51:1102-1109.
Kushner JA. The role of aging upon β cell turnover. J Clin Invest. 2013;123(3):990995.
Leite JSM, Cruzat VF, Krause MS, Homem de Bittencourt PI Jr. Physiological regulation of the heat shock response by
glutamine: implications for chronic low-grade inflammatory diseases in age-related conditions. Nutrire 2016;41:17.
Lissarassa, Y.P.S., Vincensi, C.F., Costa-Beber, L.C. et al. Chronic heat treatment positively impacts metabolic profile
of ovariectomized rats: association with heat shock response pathways. Cell Stress and Chaperones 25, 467479 (2020).
Locke M. Heat shock transcription factor activation and hsp72 accumulation in aged skeletal muscle. Cell Stress
Chaperones 5: 4551, 2000. doi:10.1379/1466-1268(2000)0050045:HSTFAA2.0.CO;2.
Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell 2013: 153: 11941217.
Lyon, M. S., & Milligan, C. (2019). Extracellular heat shock proteins in neurodegenerative diseases: new perspectives.
Neuroscience Letters, 711, 134462.
Magni, S., Succurro, A., Skupin, A., &Ebenhöh, O. (2018). Data-driven dynamical model indicates that the heat shock
response in Chlamydomonas reinhardtii is tailored to handle natural temperature variation. Journal of the Royal
Society Interface, 15(142), 20170965.
Malyshev, I. (2013). The functions of HSP70 in normal cells. In Immunity, tumors and aging: The role of HSP70 (pp.
15-29). Springer, Dordrecht.
Manini T, Clark B. Dynapenia and Aging: an Update. J Gerontol A Biol Sci Med Sci. 2012;67A(1):2840.
Marui, S., Masuda, Y., Kato, I., & Nagashima, K. (2020). Influence of exogenous and endogenous estrogen on
thermoregulatory responses to mild heat and the interaction with light and dark phases. The Journal of Physiological
Sciences, 70(1), 1-13.
McCarthy, M., & Raval, A. P. (2020). The peri-menopause in a woman’s life: a systemic inflammatory phase that
enables later neurodegenerative disease. Journal of neuroinflammation, 17(1), 1-14.
McLean, A. C., Valenzuela, N., Fai, S., & Bennett, S. A. (2012). Performing vaginal lavage, crystal violet staining, and
vaginal cytological evaluation for mouse estrous cycle staging identification. JoVE (Journal of Visualized Experiments),
(67), e4389.
Mehta, J. M., Chester, R. C., & Kling, J. M. (2019). The timing hypothesis: hormone therapy for treating symptomatic
women during menopause and its relationship to cardiovascular disease. Journal of Women's Health, 28(5), 705-711.
Messier, V., Rabasa-Lhoret, R., Barbat-Artigas, S., Elisha, B., Karelis, A. D., & Aubertin-Leheudre, M. (2011). Menopause
and sarcopenia: a potential role for sex hormones. Maturitas, 68(4), 331-336.
Mihm, M., Gangooly, S., &Muttukrishna, S. The normal menstrual cycle in women. Animal reproduction science. 124,
229-236 (2011).
Miller AP, Xing D, Feng W, Fintel M, Chen YF, Oparil S. Aged rats lose vasoprotective and anti-inflammatory actions
of estrogen in injured arteries. Menopause 2007;14:251-260.
Miragem AA, Homem de Bittencourt PI Jr. Nitric oxide-heat shock protein axis in menopausal hot flushes: neglected
metabolic issues of chronic inflammatory diseases associated with deranged heat shock response. Hum Reprod Update.
2017 Sep 1;23(5):600-628.
Modi, M., &Dhillo, W. S. (2019). Neurokinin 3 receptor antagonism: a novel treatment for menopausal hot
flushes. Neuroendocrinology, 109(3), 242-248.
Modi, M., &Dhillo, W. S. (2019, May). Neurokinin B and neurokinin-3 receptor signaling: promising developments in
the management of menopausal hot flushes. In Seminars in reproductive medicine (Vol. 37, No. 03, pp. 125-130). Thieme
Medical Publishers.
Moiseeva O, Deschênes-Simard X, St-Germain E, Igelmann S, Huot G, Cadar AE, Bourdeau V, Pollak MN, Ferbeyre G.
Metformin inhibits the senescence-associated secretory phenotype by interfering with IKK/NF-κB activation. Aging
Cell 2013;12:489-498.
Montecino-Rodriguez E, Berent-Maoz B, Dorshkind K. Causes, consequences, and reversal of immune system aging. J
Clin Invest. 2013; 123(3):958965.
Monteleone, P., Mascagni, G., Giannini, A., Genazzani, A. R., &Simoncini, T. (2018). Symptoms of menopauseglobal
prevalence, physiology and implications. Nature Reviews Endocrinology, 14(4), 199-215.
Moore AM, Prescott M, Campbell RE. Estradiol negative and positive feedback in a prenatal androgen-induced mouse
model of polycystic ovarian syndrome. Endocrinology 2013;154(2):796806.
Moore, A. M., Coolen, L. M., & Lehman, M. N. (2019). Kisspeptin/Neurokinin B/Dynorphin (KNDy) cells as integrators
of diverse internal and external cues: evidence from viral-based monosynaptic tract-tracing in mice. Scientific
reports, 9(1), 1-15.
Morano KA, Thiele DJ. Heat shock fator function and regulation in response to cellular stress, growth, and
differentiation signals. Gene Expr 1999; 7: 271
Morrison, S. F., & Nakamura, K. (2019). Central mechanisms for thermoregulation. Annual review of physiology, 81,
Mulyani, W. R. W., Sanjiwani, M. I. D., Sandra, I., Lestari, A. A. W., Wihandani, D. M., Suastika, K., ... &Manuaba, I. B.
A. P. (2020). Chaperone-based therapeutic target innovation: Heat shock protein 70 (HSP70) for Type 2 diabetes
mellitus. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 13, 559.
Nakamura K, Morrison SF. A thermosensory pathway mediating heat-defence responses. Proc Natl Acad Sci U S A
Nakamura K. Central circuitries for body temperature regulation and fever. Am J Physiol Regul Integr Comp Physiol
Newgard, C. B., & Sharpless, N. E. (2013). Coming of age: molecular drivers of aging and therapeutic opportunities.
The Journal of clinical investigation, 123(3), 946-950.
Newsholme P and Homem de Bittencourt PI Jr. Gut associated bacteria are critical to metabolism, inflammation and
health. Curr Opin Clin Nutr Metab Care 2016;19:245-249.
Newsholme, P. and de Bittencourt, Jr, P. I. (2014) The fat cell senescence hypothesis: a mechanism responsible for
abrogating the resolution of inflammation in chronic disease. Curr. Opin. Clin. Nutr. Metab. Care. 17, 295305 CrossRef
Newsholme, P. and Krause, M. (2014) Diet, obesity, and reactive oxygen species implications for diabetes and aging.
Systems Biol. Free Radicals Antioxid. 1, 13.
NICE guideline (NG23) 2015. Menopause: diagnosis and management. (
Nordling, L. (2022). Why menopause matters in the academic workplace. Nature, 605(7909), 381-384.
Novella, S., PérezCremades, D., Mompeón, A., & Hermenegildo, C. (2019). Mechanisms underlying the influence of
oestrogen on cardiovascular physiology in women. The Journal of physiology, 597(19), 4873-4886.
OECD. OECD Employment Outlook 2021: Navigating the COVID-19 Crisis and Recovery; OECD Publishing: Paris,
France, 2021. [CrossRef]
Oliveira, M. A., Lima, W. G., Schettini, D. A., Tilelli, C. Q., & Chaves, V. E. (2019). Is calcitonin gene-related peptide a
modulator of menopausal vasomotor symptoms? Endocrine, 63(2), 193-203.
P.H. Ronkainen, et al., Postmenopausal hormone replacement therapy modifies
skeletal muscle composition and function: a study with monozygotic twin pairs, J Appl Physiol (1985) 107 (1) (2009)
25 33.
Padilla, S. L., Johnson, C. W., Barker, F. D., Patterson, M. A., & Palmiter, R. D. (2018). A neural circuit underlying the
generation of hot flushes. Cell reports, 24(2), 271-277.
Palmer AK, Tchkonia T, LeBrasseur NK, Chini EN, Xu M, Kirkland JL. Cellular Senescence in Type 2 Diabetes: A
Therapeutic Opportunity. Diabetes 2015:64;2289-2298.
Parkening, T.A., Collins, T.J., & Smith, E.R. Plasma and pituitary concentrations of LH, FSH, and prolactin in aging
C57BL/6 mice at various times of the estrous cycle. Neurobiology of aging. 3, 31-35 (1982).
Pfeilschifter J, et al. Changes in proinflammatory cytokine activity after menopause. Endocr Rev. 2002;23(1):90119.
Prague, J. K., & Dhillo, W. S. (2017). Neurokinin 3 receptor antagonismthe magic bullet for hot
flushes?. Climacteric, 20(6), 505-509.
Prague, J. K., Roberts, R. E., Comninos, A. N., Clarke, S., Jayasena, C. N., Mohideen, P., ... & Dhillo, W. S. (2018).
Neurokinin 3 receptor antagonism rapidly improves vasomotor symptoms with sustained duration of
action. Menopause (New York, NY), 25(8), 862.
Prague, J. K., Roberts, R. E., Comninos, A. N., Clarke, S., Jayasena, C. N., Nash, Z., ... &Dhillo, W. S. (2017). Neurokinin
3 receptor antagonism as a novel treatment for menopausal hot flushes: a phase 2, randomised, double-blind, placebo-
controlled trial. The Lancet, 389(10081), 1809-1820.
Prague, J. K., Voliotis, M., Clarke, S., Comninos, A. N., Abbara, A., Jayasena, C. N., ... &Dhillo, W. S. (2019). Determining
the relationship between hot flushes and LH Pulses in menopausal women using mathematical modeling. The Journal
of Clinical Endocrinology & Metabolism, 104(9), 3628-3636.
Rance NE, Dacks PA, Mittelman-Smith MA, Romanovsky AA, Krajewski-Hall SJ. Modulation of body temperature and
LH secretion by hypothalamic KNDy kisspeptin, neurokinin B and dynorphin) neurons: a novel hypothesis on the
mechanism of hot flushes. Front Neuroendocrinol 2013:34;211-227.
Rannevik G, et al. A longitudinal study of the perimenopausal transition: altered profiles of steroid and pituitary
hormones, SHBG and bone mineral density. Maturitas. 1995;21(2):103 13.
Riach, K., & Jack, G. (2021). Women’s health in/and work: menopause as an intersectional experience. International
Journal of Environmental Research and Public Health, 18(20), 10793.
Rodrigues-Krause JC, Krause M, O'Hagan C, De Vito G, Boreham C, Murphy C, Newsholme P, Colleran G. Divergence of
intracellular and extracellular HSP72 in type 2 diabetes: does fat matter? Cell Stress Chaperones 2012:17;293-302.
Roland AV, Moenter SM. Prenatal androgenization of female mice programs an increase in firing activity of
gonadotropin-releasing hormone (GnRH) neurons that is reversed by metformin treatment in adulthood.
Endocrinology 2011;152(2):61828.
Roland AV, Nunemaker CS, Keller SR, Moenter SM. Prenatal androgen exposure programs metabolic dysfunction in
female mice. J Endocrinol 2010;207(2): 21323.
Rolland YM, Perry 3rd HM, Patrick P, Banks WA, Morley JE. Loss of appendicular muscle mass and loss of muscle
strength in young postmenopausal women. J Gerontol A: Biol Sci Med Sci 2007;62(3):3305.
Romanovsky, A. A. (2018). The thermoregulation system and how it works. Handbook of clinical neurology, 156, 3-43.
Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard
BV, Johnson KC, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal
results From the Women's Health Initiative randomized controlled trial. JAMA 2002:288;321-333.
S.V. Koebele, H.A. Bimonte-Nelson, Modeling menopause: the utility of rodents in translational behavioral
endocrinology research, Maturitas 87 (2016) 5 17.
Sabbah, N. A., Rezk, N. A., & Saad, M. S. (2019). Relationship between heat shock protein expression and obesity with
and without metabolic syndrome. Genetic Testing and Molecular Biomarkers, 23(10), 737-743.
Saengboonmee C, Seubwai W, Cha'on U, Sawanyawisuth K, Wongkham S, Wongkham C. Metformin Exerts
Antiproliferative and Anti-metastatic Effects Against Cholangiocarcinoma Cells by Targeting STAT3 and NF-ĸB.
Anticancer Res 2017:37;115-123.
Salminen A, Kaarniranta K, Kauppinen A. Inflammaging: disturbed interplay between autophagy and inflammasomes.
Aging (Albany NY) 2012: 4: 166175.
Sander, V.A., Facorro, G.B., Piehl, L., Rubin de Celis, E., & Motta, A.B. Effect of DHEA and metformin on corpus luteum
in mice. Reproduction. 138, 571-579 (2009).
Sapra G, Tham YK, Cemerlang N, Matsumoto A, Kiriazis H, Bernardo BC, Henstridge DC, Ooi JY, Pretorius L, Boey EJ et
al. The small-molecule BGP-15 protects against heart failure and atrial fibrillation in mice. Nat Commun 2014:5;5705.
Schafer MJ, White TA, Iijima K, Haak AJ, Ligresti G, Atkinson EJ, Oberg AL, Birch J, Salmonowicz H, Zhu Y, et al.
Cellular senescence mediates fibrotic pulmonary disease. Nat Commun 2017:8;14532.
Sharma N, et al. Association of periodontal inflammation, systemic inflammation, and duration of menopausal years
in postmenopausal women. Quintessence Int. 2018;49(2):12331.
Simon MM, Reikerstorfer A, Schwarz A, Krone C, Luger TA, Jäättelä M, Schwarz T. Heat shock protein 70 overexpression
affects the response to ultraviolet light in murine fibroblasts. Evidence for increased cell viability and suppression of
cytokine release. J Clin Invest 95: 926933, 1995. doi:10.1172/JCI117800.
Skorupskaite K, George JT, Anderson RA. The kisspeptin-GnRH pathway in human reproductive health and disease.
Hum Reprod Update 2014:20;485-500.
Skorupskaite, K., George, J. T., Veldhuis, J. D., Millar, R. P., & Anderson, R. A. (2018). Neurokinin 3 receptor antagonism
reveals roles for neurokinin B in the regulation of gonadotropin secretion and hot flashes in postmenopausal
women. Neuroendocrinology, 106(2), 148-157.
Somani, Y. B., Pawelczyk, J. A., De Souza, M. J., Kris-Etherton, P. M., & Proctor, D. N. (2019). Aging women and their
endothelium: probing the relative role of estrogen on vasodilator function. American Journal of Physiology-Heart and
Circulatory Physiology, 317(2), H395-H404.
Söti C, Csermely P. Chaperones come of age. Cell Stress Chaperones 7: 186190, 2002.
Stampfer MJ, Colditz GA. Estrogen replacement therapy and coronary heart disease: a quantitative assessment of the
epidemiologic evidence. Prev Med 20: 4763, 1991. doi:10.1016/0091-7435(91)90006-P.
Stefano, G. B., Esch, T., &Kream, R. M. (2019). Behaviorally-mediated entrainment of whole-body metabolic processes:
Conservation and evolutionary development of mitochondrial respiratory complexes. Medical science monitor:
international medical journal of experimental and clinical research, 25, 9306.
Stepto NK, Cassar S, Joham AE, Hutchison SK, Harrison CL, Goldstein RF, Teede HJ. Women with polycystic ovary
syndrome have intrinsic insulin resistance on euglycaemic-hyperinsulaemic clamp. Hum Reprod 2013:28;777-784.
Stice JP, Chen L, Kim SC, Jung JS, Tran AL, Liu TT, Knowlton AA. 17β-Estradiol, aging, inflammation, and the stress
response in the female heart. Endocrinology 2011:152;1589-1598.
Stice JP, Lee JS, Pechenino AS, Knowlton AA. Estrogen, aging and the cardiovascular system. Future Cardiol 2009:5;93-
Stout MB, Steyn FJ, Jurczak MJ, Camporez JG, Zhu Y, Hawse JR, Jurk D, Palmer AK, Xu M, Pirtskhalava T, et al.17α-
Estradiol Alleviates Age-related Metabolic and Inflammatory Dysfunction in Male Mice Without Inducing
Feminization. J Gerontol A Biol Sci Med Sci 2017:72;3-15.
Sturdee, D. W., Hunter, M. S., Maki, P. M., Gupta, P., Sassarini, J., Stevenson, J. C., & Lumsden, M. A. (2017). The
menopausal hot flush: a review. Climacteric, 20(4), 296-305.
Sullivan SD, Moenter SM. Prenatal androgens alter GABAergic drive to gonadotropin-releasing hormone neurons:
implications for a common fertility disorder. Proc Natl Acad Sci U S A 2004;101(18):712934.
Sun, F., Zuo, Y. Z., Ge, J., Xia, J., Li, X. N., Lin, J., ... & Li, J. L. (2018). Transport stress induces heart damage in newly
hatched chicks via blocking the cytoprotective heat shock response and augmenting nitric oxide production. Poultry
science, 97(8), 2638-2646.
Szeliga, A., Czyzyk, A., Podfigurna, A., Genazzani, A. R., Genazzani, A. D., &Meczekalski, B. (2018). The role of
kisspeptin/neurokinin B/dynorphin neurons in pathomechanism of vasomotor symptoms in postmenopausal women:
from physiology to potential therapeutic applications. Gynecological Endocrinology, 34(11), 913-919.
Takii, R., Fujimoto, M., Matsuura, Y., Wu, F., Oshibe, N., Takaki, E., ... &Nakai, A. (2017). HSF1 and HSF3 cooperatively
regulate the heat shock response in lizards. PLoS One, 12(7), e0180776.
Tan, C. L., & Knight, Z. A. (2018). Regulation of body temperature by the nervous system. Neuron, 98(1), 31-48.
Tchkonia T, Morbeck DE, Von Zglinicki T, Van Deursen J, Lustgarten J, Scrable H, Khosla S, Jensen MD, Kirkland JL. Fat
tissue, aging, and cellular senescence. Aging Cell 2010:9;667-684.
Tchkonia T, Zhu Y, van Deursen J, Campisi J, Kirkland JL. Cellular senescence and the senescent secretory phenotype:
therapeutic opportunities. J Clin Invest 2013:123;966-972.
Tilly, J.; O’Leary, J.; Russell, G. Older Women Matter: Harnessing the Talents of Australia’s Older Female Workforce;
Diversity Council Australia: Sydney, Australia, 2013.
Tytell M, Hooper PL. Heat shock proteins: new keys to the development of cytoprotective therapies. Expert Opin Ther
Targets 2001;5:267-287.
Van Der Lee, S. & Boot, L.M. Spontaneous pseudopregnancy in mice. Acta physiologica et pharmacologica Neerlandica.
4, 442-444 (1955)
Van Der Lee, S. & Boot, L.M. Spontaneous pseudopregnancy in mice. II. Acta physiologica et pharmacologica
Neerlandica. 5, 213-215 (1956).
van Why SK, Kim S, Geibel J, Seebach FA, Kashgarian M, Siegel NJ. Thresholds for cellular disruption and activation
of the stress response in renal epithelia. Am J Physiol 1999; 277: F227.
Wang J, He H, Yu L, Xia HH, Lin MCM, Gu Q, Li M, Zou B, An X, Jiang B, Kung H-F, Wong BCY. HSF1 down-regulates
XAF1 through transcriptional regulation. J Biol Chem 281: 24512459, 2006. doi:10. 1074/jbc.M505890200.
Wang, T. A., Teo, C. F., Åkerblom, M., Chen, C., Tynan-La Fontaine, M., Greiner, V. J., ... & Jan, L. Y. (2019).
Thermoregulation via temperature-dependent PGD2 production in mouse preoptic area. Neuron, 103(2), 309-322.
Whitehouse, L. M., McDougall, C. S., Stefanovic, D. I., Boreham, D. R., Somers, C. M., Wilson, J. Y., &Manzon, R. G.
(2017). Development of the embryonic heat shock response and the impact of repeated thermal stress in early stage
lake whitefish (Coregonusclupeaformis) embryos. Journal of Thermal Biology, 69, 294-301.
Whitmer RA, Quesenberry CP, Zhou J, Yaffe K. Timing of hormone therapy and dementia: the critical window theory
revisited. Ann Neurol 2011:69;163-169.
Whitten, W.K. Occurrence of anoestrus in mice caged in groups.The Journal of endocrinology. 18, 102-107 (1959).
Wilhelms DB, Dock H, Brito HO, Pettersson E, Stojakovic A, Zajdel J, Engblom D, Theodorsson E, Hammar ML and Spetz
Holm A-CE (2019) CGRP Is Critical for Hot Flushes in Ovariectomized Mice. Front. Pharmacol. 9:1452. doi:
World Health Organization. (2020). Decade of healthy ageing: baseline report. World Health Organization. Licença:
Yang, W. Z., Du, X., Zhang, W., Gao, C., Xie, H., Xiao, Y., ... & Shen, W. L. (2020). Parabrachial neuron types
categorically encode thermoregulation variables during heat defense. Science advances, 6(36), eabb9414.
Yuste JE, Tarragon E, Campuzano CM, Ros-Bernal F (2015) Implications of glial nitric oxide in neurodegenerative
diseases. Front Cell Neurosci 9:322
Z. Paroo, E.S. Dipchand, E.G. Noble, Estrogen attenuates postexercise HSP70 expression in skeletal muscle, Am J
Physiol Cell Physiol 282 (2) (2002) C245 C251.
Z. Paroo, P.M. Tiidus, E.G. Noble, Estrogen attenuates HSP 72 expression in
acutely exercised male rodents, Eur. J. Appl. Physiol. Occup. Physiol. 80 (3)
(1999) 180 184.
Zacker RJ. Health-related implications and management of sarcopenia. JAAPA. 2006;19(10):249.
Zhang, S., Osumi, H., Uchizawa, A., Hamada, H., Park, I., Suzuki, Y., ... & Tokuyama, K. (2020). Changes in sleeping
energy metabolism and thermoregulation during menstrual cycle. Physiological reports, 8(2), e14353.
Zilberman, J. M. (2018). Menopausia: Hipertension arterial y enfermedad vascular. Hipertensión y riesgo
vascular, 35(2), 77-83.
Zou J, Guo Y, Guettouche T, Smith DF, Voellmy R. Repression of heat shock transcription factor HSF1 activation by
HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell 94: 471480, 1998. doi:10.1016/S0092-
Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.
ResearchGate has not been able to resolve any citations for this publication.
Full-text available
This paper employs an intersectional lens to explore menopausal experiences of women working in the higher education and healthcare sectors in Australia. Open-text responses from surveys across three universities and three healthcare settings were subject to a multistage qualitative data analysis. The findings explore three aspects of menopause experience that required women to contend with a constellation of aged, gendered and ableist dynamics and normative parameters of labor market participation. Reflecting on the findings, the paper articulates the challenges of menopause as issues of workplace inequality that are rendered visible through an intersectional lens. The paper holds a range of implications for how to best support women going through menopause at work. It emphasizes the need for approaches to tackle embedded and more complex modes of inequality that impact working women’s menopause, and ensure that workforce policy both protects and supports menopausal women experiencing intersectional disadvantage.
Full-text available
Heat-shock protein 70 (HSP70) contributes to cellular calcium (Ca2+) handling mechanisms during receptor-mediated vascular contraction. Interestingly, previous studies have independently reported sex-related differences in HSP70 expression and Ca2+ dynamics. Still, it is unknown if sex, as a variable, plays a role in the impact that HSP70 has upon vascular contraction. To narrow this gap, we investigated if differences exist in the expression levels of HSP70 in the aorta, and if targeting this protein contributes to sex disparity in vascular responses. We report that, compared with male animals, female rats present a reduction in the basal levels of HSP70. More compelling, we found that the blockade of HSP70 has a greater impact on phenylephrine-induced phasic and tonic vascular contraction in female animals. In fact, it seems that the inhibition of HSP70 significantly affects vascular Ca2+ handling mechanisms in females, which could be associated with the fact that these animals have impaired HSP70 expression. Corroborating this idea, we uncovered that the higher sensitivity of female rats to HSP70 inhibition does not involve an increase in NO-dependent vasodilation nor a decrease in vascular oxidative stress. In summary, our findings reveal a novel mechanism associated with sex-specific differences in vascular responses to α-1 adrenergic stimulation, which might contribute to unraveling the network of intertwined pathways conferring female protection to (cardio)vascular diseases.
Full-text available
The present study aimed to determine the influence of estradiol (E2) and the interaction with circadian phases on thermoregulatory responses to mild heat in female rats. Heat loss and production during 3-h exposure to the environment at an ambient temperature of 28–34 °C were assessed by measuring abdominal temperature (Tabd), tail skin temperature, and oxygen consumption in ovariectomized rats with and without E2 replacement (OVX + E2 and OVX, respectively) and in control rats in the proestrus (P) and diestrus (D) phases. In the light phase, Tabd remained unchanged in all groups. Tabd increased in the dark phase, but was lower in the OVX + E2 and P groups than in the OVX and D groups. Oxygen consumption decreased at 34 °C, but to a lesser extent in the OVX + E2 group than in the OVX group. These results suggest that E2 activates thermoregulation in mild heat in the dark phase.
Full-text available
The peri-menopause or menopausal transition—the time period that surrounds the final years of a woman’s reproductive life—is associated with profound reproductive and hormonal changes in a woman’s body and exponentially increases a woman’s risk of cerebral ischemia and Alzheimer’s disease. Although our understanding of the exact timeline or definition of peri-menopause is limited, it is clear that there are two stages to the peri-menopause. These are the early menopausal transition, where menstrual cycles are mostly regular, with relatively few interruptions, and the late transition, where amenorrhea becomes more prolonged and lasts for at least 60 days, up to the final menstrual period. Emerging evidence is showing that peri-menopause is pro-inflammatory and disrupts estrogen-regulated neurological systems. Estrogen is a master regulator that functions through a network of estrogen receptors subtypes alpha (ER-α) and beta (ER-β). Estrogen receptor-beta has been shown to regulate a key component of the innate immune response known as the inflammasome, and it also is involved in regulation of neuronal mitochondrial function. This review will present an overview of the menopausal transition as an inflammatory event, with associated systemic and central nervous system inflammation, plus regulation of the innate immune response by ER-β-mediated mechanisms.
Full-text available
Heat defense is crucial for survival and fitness. Transmission of thermosensory signals into hypothalamic thermoregulation centers represents a key layer of regulation in heat defense. Yet, how these signals are transmitted into the hypothalamus remains poorly understood. Here, we reveal that lateral parabrachial nucleus (LPB) glutamatergic prodynorphin and cholecystokinin neuron populations are progressively recruited to defend elevated body temperature. These two nonoverlapping neuron types form circuits with downstream preoptic hypothalamic neurons to inhibit the thermogenesis of brown adipose tissues (BATs) and activate tail vasodilation, respectively. Both circuits are activated by warmth and can limit fever development. The prodynorphin circuit is further required for regulating energy expenditure and body weight homeostasis. Thus, these findings establish that the genetic and functional specificity of heat defense neurons occurs as early as in the LPB and uncover categorical neuron types for encoding two heat defense variables, inhibition of BAT thermogenesis and activation of vasodilation.
Full-text available
Low estrogen levels may predispose women to increased bodyweight and dyslipidemia. Previous studies from our laboratory suggest an involvement of depressed heat shock response (HSR) in this scenario because estrogen potently stimulates HSR. As heat treatment induces the expression of the anti-inflammatory heat shock proteins of the 70-kDa family (HSP70) and its accompanying HSR, we aimed to investigate whether chronic heat treatment promotes beneficial effects on biometric, lipid profile, oxidative stress, and HSR in ovariectomized rats. Wistar adult female rats (n = 32) were divided into four groups: control (C, n = 7), ovariectomized (OVX, n = 9), heat-treated (HT, n = 9), and heat-treated ovariectomized rats (OVX+HT, n = 7). HT and OVX+HT rats were anesthetized and submitted to heat treatment (once a week for 12 weeks) in a water bath (41 °C) to increase rats’ rectal temperature up to 41 °C for 15 min, while C and OVX animals were submitted to a 36 °C water bath. HT attenuated the weight gain induced by OVX and increased HDL cholesterol and triglyceride serum levels. Also, OVX rats showed increased total cholesterol and LDL cholesterol levels that were not influenced by HT. Interestingly, it was found that an overall trend for HT to decrease tissue catalase and superoxide dismutase antioxidant activities was paralleled by a decrease in malondialdehyde levels (indicative of lower lipoperoxidation), especially in the skeletal muscle. Surprisingly, OVX was not able to depress intracellular HSP70 expression in the skeletal muscle, as expected, and this remained unchanged with HT. However, chronic HT did enhance intracellular HSP70 contents in white adipose tissue of OVX animals. As both glucose and insulin tolerance tests were not affected by OVX, which was not modified by HT, we suppose that estrogen absence alone is not sufficient to determine a state of insulin resistance associated with low intramuscular HSP70 content.
Full-text available
Women with ovulatory menstrual cycles show an increase in body temperature in the luteal phase, compared with follicular phase, particularly during the night. Several, albeit not all, studies reported higher energy expenditure in the luteal phase compared with follicular phase. Q10 of biological reactions lies between 2.0 and 3.0, predicting a 7-12% increase in energy expenditure when body temperature rises by 1°C. In this study, temperature dependence of energy expenditure was assessed by comparing changes in sleeping energy expenditure and thermoregulation with menstrual cycle in 9 young females. Energy expenditure was measured using a metabolic chamber, in which sleep was recorded polysomnographically, and core body temperature and skin temperature were continuously monitored. Distal-to-proximal skin temperature gradient was assessed as an index of heat dissipation. In the luteal phase, a significant increase in average core body temperature (+0.27°C) and energy expenditure (+6.9%) were observed. Heat dissipation was suppressed during the first 2 hr of sleep in the luteal phase, compared with follicular phase. Rise in basal body temperature in the luteal phase was accompanied by increased energy expenditure and suppressed heat dissipation. The 6.9% increase in metabolic rate would require a Q10 of 12.4 to be attributable solely to temperature (+0.27°C), suggesting that energy expenditure in the luteal phase is enhanced through the mechanism, dependent and independent of luteal-phase rise in body temperature presumably reflects other effects of the sex hormones.
Full-text available
Neurons in the hypothalamic arcuate nucleus (ARC) that co-express kisspeptin, neurokinin B and dynorphin (KNDy cells) are essential for mammalian reproduction as key regulators of gonadotropin-releasing hormone (GnRH) secretion. Although multiple endogenous and exogenous signals act indirectly via KNDy neurons to regulate GnRH, the identity of upstream neurons that provide synaptic input to this subpopulation is unclear. We used rabies-mediated tract-tracing in transgenic Kiss1-Cre mice combined with whole-brain optical clearing and multiple-label immunofluorescence to create a comprehensive and quantitative brain-wide map of neurons providing monosynaptic input to KNDy cells, as well as identify the estrogen receptor content and peptidergic phenotype of afferents. Over 90% of monosynaptic input to KNDy neurons originated from hypothalamic nuclei in both male and female mice. The greatest input arose from non-KNDy ARC neurons, including proopiomelanocortin-expressing cells. Significant female-dominant sex differences in afferent input were detected from estrogen-sensitive hypothalamic nuclei critical for reproductive endocrine function and sexual behavior in mice, indicating KNDy cells may provide a unique site for the coordination of sex-specific behavior and gonadotropin release. These data provide key insight into the structural framework underlying the ability of KNDy neurons to integrate endogenous and environmental signals important for the regulation of reproductive function.
Employers in some countries are stepping up efforts to support staff through this life stage. Others need to follow suit. Employers in some countries are stepping up efforts to support staff through this life stage. Others need to follow suit.