Introduction

The sex-bias in basic and clinical research and the vulnerability of menopausal women

Sex is the main determinant of our physical attributes, the structure of our brains, our behavior, the susceptibility, progression, and response to diseases, and our own conception of self [1]. Despite this sexual identity, biomedical scientists have ignored the clear and important differences between males and females [2], i.e., analysis of the scientific literature in numerous disciplines shows that there is an indisputable sex-bias: “males are studied much more than females, in both animal and human research”. In fact, drug development is based on research on males, even for diseases that are more frequent in females, and despite evidence that drug metabolism and efficacy differ in the two sexes [1]. For this reason, the National Institutes of Health in the United States recently ordered the inclusion of both sexes in clinical trials and basic research [3], with the aim of promoting gender-specific therapies that can contribute to developing individualized therapies in the future [2].

Sexual identity is highly influenced by hormones. Hormones are the most amazing molecules in biochemistry, i.e., they are the only ones that can achieve the formation, growing, differentiation, functioning, behavior, and reparation of an entire organism, acting at minimal concentrations [4,5,6,7]. Moreover, sex hormones are responsible for sexual differentiation (phenotype and behavior), and participate importantly in the maintenance of metabolic homeostasis [8, 9]. Among sex hormones, estrogens distinguish themselves for their protector role on cardio-metabolism, playing a very important function in the regulation of body fat distribution, and maintenance of metabolic health through several mechanisms, like (1) reduction in food intake maintaining an anorexigenic tone and improving satiety signals [10, 11], (2) regulating body fat distribution, favoring the subcutaneous and gluteal-femoral fat depot and preventing the visceral storage [10, 12], (3) promoting browning of adipose tissue, increasing the catabolism of fatty acids and glucose [10, 13, 14], while improving insulin and leptin sensitivity [15, 16], (4) acting as anti-inflammatory and antioxidant agents that prevent endothelial dysfunction, vascular inflammation and atherosclerosis [17,18,19,20,21], and (5) stimulating the release of calcitonin gene-related peptide (CGPR) from perivascular nerves, and nitric oxide production in blood vessels promoting anti-hypertensive actions [22,23,24] (Fig. 1). Considering all these positive effects of estrogens on cardio-metabolism, it seems reasonable to suppose that a hypoestrogenic state could promote the development of metabolic syndrome (MS) through several mechanisms, like visceral obesity development, and the resulting inflammatory and oxidative states, which together with atherogenic dyslipidemia contributes and/or aggravates a simultaneous development of insulin resistance. All these conditions can lead to endothelial dysfunction and hypertension, which in turn promotes complications such as myocardial infarction and stroke, or the development of other chronic diseases like type two diabetes (T2D), which increase the risk of mortality in menopausal women [11, 15] (Fig. 2). In this way, estrogens have been assigned as main responsible for prolonged life span and cardiometabolic health in premenopausal women, and represent the main biological advantage in nature compared to males.

Fig. 1
figure 1

Estrogen pathways for metabolic regulation and body weight maintenance. Acting mainly by estrogen receptors alpha (ERα), estrogens reduce food intake increasing leptin sensitivity, and the potency of anorectic signals like cholecystokinin (CCK) and pro-opiomelanocortin (POMC), at the same time estrogens reduce the signaling of orexigenic molecules like neuropeptide Y (NPY) and ghrelin, also modulate brown adipose tissue (BAT) thermogenesis through AMPK → β3 → UCP1 pathway, and promote mitochondrial biogenesis. In subcutaneous adipose tissue (AT), estrogens increase the expression of the antilipolytic α2 adrenoceptors, and augment the activity of lipoprotein lipase (LPL) promoting the uptake and storage of fatty acids, meanwhile in visceral adipose tissue increase lipolytic β1-2 adrenoceptors and reduce LPL activity, increasing lipolysis and preventing the visceral storage of fat in the abdominal cavity. Estrogens, also promote hyperplasic adipose tissue expansion (related with an increase in insulin sensivity) over hypertrofic procces (related to oxidative stress, fibrosis and immflamation). Regarding energetic metabolism, estrogens increase insulin sensitivity improving glucose uptake through specific transporters, and aughmenting glucose catabolism by activation of key enzymes, also increase lipid catabolism by lipolysis and β-oxidation, and reduce total colesterol and LDL fraction, preventing the development of atherogenic dyslipidemia. Estrogens can act as scavengers of free radicals by it self, but their antioxidants effects also are mediated by ERα activation and upregulation of enzymatic and no enzymatic antioxidant defenses, reduction of reactive species oxygen (ROS) production by renin-angiotensin system (RAS)/NADPH oxidase inhibition, and indirectly, promoting a decrease in the expression of iNOS, and levels of inflammatory markers (TNFα, IL-1β, IL-6, MCP-1). Finally, estrogens promote vascular relaxation through PI3K/Akt/eNOS pathway activation and calcitonin gene-related peptide (CGPR) release

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Fig. 2
figure 2

The reduction of estrogens signaling promotes the development of metabolic syndrome and increases mortality. Hypoestrogenism promotes the development of metabolic syndrome (MS) through visceral obesity development, and the resulting inflammatory and oxidative states, which together with atherogenic dyslipidemia, contributes and/or aggravates a simultaneous development of insulin resistance. All these conditions can lead to endothelial dysfunction, hypertension, cardiovascular disease (CVD), stroke, or type 2 diabetes (T2D), increasing the risk of mortality in menopausal women seriously

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Among estrogens, 17β-estradiol (E2) is the most abundant and potent in humans [24], its production in females before the hormonal decline mainly occurs in the ovaries after the aromatization of androgenic precursors by aromatase. After estrogenic deficit and in males, estradiol is synthesized in extragonadal sites, such as the adipose tissue, where it acts locally as a paracrine or intracrine factor [12]. The protector effects of estrogens result from their interaction with classical estrogen receptors (ERs), ERα and ERβ, as well as the more recently identified G-protein coupled receptor 30 (GPER30)/G-protein estrogen receptor 1 (GPER1), and exert their actions via both genomic and non-genomic mechanisms [17]. However, the majority of cardiometabolic protector effects for estrogens are mediated by ERα, which is in higher proportion than ERβ in key tissues for metabolic regulation; indeed, estrogen receptor alpha knockout (ERαKO) mice show severe alterations in their metabolism suggesting that the beneficial effects of E2 are mainly mediated by this receptor and not by ERβ since ERβKO mice do not show important alterations in these parameters [11, 15]. Like the other sex hormones, E2 is transported from circulation to target tissues by sex-hormone binding globulin (SHBG), whose diminished concentrations during estrogenic deficiency (menopause, Poly Cystic Ovary Syndrome) are highly related with insulin resistance and MS development [25]. Actually, it has been amply reported that the risk for developing chronic diseases such as central obesity, T2D, hypertension, hepatic steatosis, and cardiovascular disease or MS is higher in males than in females just before estrogenic deficiency, and it is well known that this prevalence shifts during the menopausal transition and after that. Clinical consequences of cardiovascular disease usually manifest 7–10 years later in women than in men when the protector effects of estrogens are lost. In this condition, the risk of metabolic disturbances and cardiovascular events, such as myocardial infarction and stroke, increase importantly and tend to have a more severe prognosis and a higher mortality in women [26,27,28].

Unfortunately, despite menopause and its metabolic and cardiovascular consequences affect all women, some of them for decades, there is not an urgent and priority concern in the agenda of governments’ health services or in the research field to face this growing problem [11, 26]. Hence in this review, we propose the use of an animal model that adequately represents the contribution of estrogenic deficiency in the pathophysiology of MS, this model will allow the research and development of new and safe therapeutic strategies for metabolic syndrome during menopause, an increasing public health problem that affects a large segment of the female population.

Metabolic syndrome during menopause

Menopause is an inevitable component of aging in women; it is defined as the permanent cessation of the menstrual cycle owing to the loss of ovarian reproductive function, either occurring spontaneously by follicular atresia or secondary to other conditions such as the removal of the ovaries or chemotherapy [26]. The transition to menopause is characterized by metabolic changes that promote the development of MS [29]. The MS is related to a set of cardiovascular, renal and hepatic risk factors that favor the development of T2D. In general, there is a consensus to obtain a clinically useful definition that includes the presence of at least three of the following factors: abdominal obesity, fasting hyperglycemia, hypertension, dyslipidemia, and insulin resistance [30,31,32,33]. Currently, oxidative stress, chronic low-grade inflammation, and a prothrombotic state have also been considered [34,35,36], as well as a decrease in SHBG [37, 38]. Observational studies have shown that the prevalence of MS is lower in women at reproductive age in comparison with men of the same age. However, this advantage disappears along the transition stage to the establishment of menopause [38, 39], where the incidence of MS increases 2–3fold [40, 41]; even several reports show that cardiovascular risk and mortality for coronary heart disease increase up to 4–5 times in postmenopausal women [42, 43], indicating that the loss of estrogens is fundamental in the development of the main risk factors associated with MS. Accordingly, evidence suggests that estrogenic deficiency in menopause is a predictor of MS independent of aging [43]. These issues are especially important because women will spend at least a third of their lives in a postmenopausal state.

The changes that promote the development of MS during and after the menopausal transition are a consequence of hypoestrogenic status which produces an increase in android body fat, a decrease in muscle mass, and an important predisposition to develop insulin resistance [44,45,46]; thereby, the increase in visceral adipose tissue contributes to the rise in the production of cytokines and the establishment of a pro-inflammatory state (Fig. 2) [47]. In this regard, when all MS factors are adjusted for age in women, only abdominal obesity and insulin resistance were associated significantly with a postmenopausal status, and it is independent of general adiposity before postmenopausal state [41, 48]. In addition, if women are overweight at the time of entering menopause, there is a higher probability of developing MS over the years [37].

In menopausal women the prevalence of MS is higher in those subjected to ovariectomy, compared with who had natural menopause [49], probably the removal of ovaries in women under 45 years promotes an acute reduction of estrogen that causes the accelerated appearance of menopause, along with the development of obesity, an increase in the production of atherogenic lipoproteins, and oxidation of LDL [50, 51]; there is also an elevation in arterial vascular tone as a consequence of the reduction of vasodilator peptides, and a rise in vasoconstrictor peptides [52]. Likewise, these women have a higher risk of developing T2D and cardiovascular disease compared with their counterparts in the general population [51]. Therefore, the sudden change in the hormonal status resultant from surgery promotes the development of cardiovascular risk factors such as obesity, hypertension, dyslipidemia, and insulin resistance [44, 53, 54]. On the other hand, the decrease in estrogen production during menopause has been associated with increased androgen production (testosterone), which promotes the elevation of blood pressure, triglycerides, and increased risk of developing insulin resistance favoring the manifestation of MS [37, 38, 55]. Notwithstanding, visceral obesity, and hypertriglyceridemia, in addition to the metabolic implications that have in menopause, are also associated with the appearance of other factors, for example, dry skin, depression, and lack of sexual desire [56]. Thus, in summary, menopause is associated with the onset and progression of the main components of MS and other factors that significantly affect the quality of life of women in a hypoestrogenic state.

Inflammatory markers and their involvement in Metabolic Syndrome (MS) development

Inflammation is one of the main risk factors recently considered as a big contributor in the physiopathology of metabolic syndrome; indeed, its role as the link between obesity, insulin resistance, atherogenic dyslipidemia, and cardiovascular disease begins to emerge as a key in the promotion and development of several diseases [57,58,59,60]. Obesity promotes a low-grade activation of the innate immune system and the development of inflammation that contributes significantly to the onset of alterations present in MS [57, 58]. The main source of proinflammatory cytokines in MS are adipocytes and macrophages, where the latter may have a change in their phenotype that makes them more proinflammatory, and together with leading to an overproduction of proinflammatory cytokines that initially promote localized inflammation and subsequently propagates to systemic level [57,58,59]. In this way, adipocytes and macrophages can produce proinflammatory cytokines such as interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and the tumor necrosis factor-alpha (TNF-α) that have been closely associated with the pathophysiology of MS. These proinflammatory cytokines can act paracrine or autocrine manner, promoting their own release through positive feedback that allows them to initiate and maintain the inflammatory state for long periods of time [59]. The main molecular mechanisms through which IL-1β, IL-6 and TNF-α exert their effects have been mainly associated with the activity of transcription factors such as NF-κB, JNK, and STAT, related with the proinflammatory effects of obesity, development of insulin resistance, and endothelial dysfunction which also have played a central role in the development of metabolic disorders such as T2D [57,58,59,60].

Use of estrogenic dysfunction models in the study of factors associated with the metabolic syndrome

Currently, there is a wide need to understand the pathophysiology of various metabolic disorders present in large part of the population worldwide, mainly those associated with MS and T2D. For this reason, it is important to develop animal models that mimic these pathologies and provide information on their development, progression, and prevention. Thereby, experimental models of metabolic alterations induced by the consumption of hypercaloric diets [61], transgenic models, and drugs, especially in rodents, have been widely used to understand their relationship with several alterations associated with MS and its subsequent complications [62, 63]. In this sense, these models allowed to scrutinize a wide variety of diseases related to MS, such as hypertension, cardiomyopathy, atherosclerosis, kidney damage, hepatic steatosis, Alzheimer and insulin resistance [64,65,66,67,68,69]. Notably, due to the importance of estrogens in metabolism, the use of animal models for loss, deficiency or resistance to estrogens, like ovariectomized rats and mice, aromatase knockout (ARKO) mice, and ERαKO mice has been used to evaluate many of the factors associated with MS (Table 1) [14].

Table 1 Studies of metabolic syndrome development into hypoestrogenic state without other treatment. Summary of models used, metabolic syndrome factors present and main pathologies or alterations associated
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Ovariectomized rats and mice develop the majority of metabolic syndrome risk factors

After ovariectomy, there is an increase in visceral adipose tissue storage, which promotes serious metabolic disturbances that include insulin resistance, dyslipidemia, hyperleptinemia, and lower adiponectin concentrations; these metabolic derangements also occurring during menopause and may trigger cardiovascular disease in women [70]. Supporting this evidence, there are several ovariectomy-induced hypoestrogenic animal models, which reported that ovariectomy per se promotes the development of obesity, glucose intolerance, insulin resistance, atherogenic-dyslipidemia, oxidative stress and inflammation, mimicking the features of the MS during menopause [71, 72]. On the other hand, the estrogenic dysfunction animal models such as the obese ARKO mice [73,74,75], or the ERαKO mice only develop some of the MS factors, and frequently they must combine with the administration of a hypercaloric diet to promote or aggravate the development of all metabolic syndrome risk factors [14, 76, 77] (Table 1). By this way, is clear that ovariectomy model develop most of the MS risk factors (Fig. 3 and Table 1), which signal it as one of the most representative models of metabolic syndrome during a hypoestrogenic state like menopause.

Fig. 3
figure 3

Ovariectomized rodents as a menopausal metabolic syndrome model. Estrogenic deficit promotes the development of main risk factors of metabolic syndrome

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Ovariectomy as a menopausal metabolic syndrome model

Among those models of hypoestrogenism or menopause are: (1) Natural reproductive senescence, (2) Ovariectomy, and (3) Ovotoxins to accelerate ovarian failure; all are used depending on the stage along with the establishment of menopause that it wants to study [78, 79].

Similar to menopause in women, rodent natural reproductive senescence (estropause) shows a dysregulation in hypothalamic-pituitary–gonadal (HPG) axis, morphofunctional ovarian changes, gonadal hormone fluctuations and irregular fertility [79]. However, only 25% of estropause rodents show a similar hormonal profile with menopausal women, the rest of animals maintain a constant estrus state characterized for high and sustained levels of estradiol and progesterone, contrary to the low levels maintained during menopause [78]. Another important difference between aging female rodents and women is the presence of mature ovarian follicles in estropause rodents, while in menopausal women the ovarian failure is complete [79]. In fact, many researchers finally conduct an ovariectomy in aged rodents to avoid these discrepancies [78]. Additionally, for the high rates of mortality, and because the model needs a long time for implementation, it requires rat strains with high longevity such as the Fischer-344, Sprague Dawley or Long Evans [79].

Other model more recently reported, and implemented is the 4-vinylcyclohexene diepoxide, or VCD, considered as a model of transitional menopause because it selectively depletes the non-growing ovarian follicle pool via atresia, resulting in follicular depletion, and eventual ovarian failure. This model shows an ovarian, and hormone profile more similar to the majority of women undergoing menopause and retain their reproductive organs in the post-reproductive life stage, the animals also present some neurologic and cardiometabolic alterations associated with hypoestrogenic state during menopause. However, its implementation has important disadvantages, in first place, VCD is toxic at high doses, and the regimen of VCD administration requires around fifteen series of intraperitoneal injections ranging from 80 to 160 mg/kg, which also result stressing for the animals, even it was reported a reduction in body weight during injections, another consideration is the elevated costs of implementation since VCD is used in high and repetitive doses [78, 79].

Finally, the model considered as the “gold standard” in the study of hypoestrogenism or menopause is that induced by ovariectomy [79], since it has several advantages over others, among them the easy implementation, cost reduction, rapid manifestation of hypoestrogenism, and the most important, ovariectomy itself is the most effective model of hypoestrogenism in promoting the development of the main risk factors of MS, compared to the natural senescence or chemical models that present the development of few MS factors. i.e.; ovariectomy promotes the development of visceral obesity, insulin resistance, oxidative stress, inflammation, atherogenic dyslipidemia, hepatic steatosis and hypertension [71, 80,81,82,83] (Table 1). Ovariectomized rats also develop other complications present in menopausal women like atherosclerotic lesion [84,85,86,87], osteoporosis, and cognitive decline [78, 79]. On the contrary, it has the drawback that it does not contemplate the menopause transition period because the hormonal change is very sudden, and the loss of the ovaries reduces the production of hormones such as testosterone. Nevertheless, as mentioned before, hypoestrogenism induced by ovariectomy shares the main characteristics of human menopause, such as a similar hormonal profile, HPG axis dysregulation, as well as cognitive and cardio-metabolic alterations that are present in menopausal women that maintain their reproductive system intact [78, 79].

Among the different models of metabolic syndrome [61, 63, 68], and in humans [88], high-caloric diets, sedentarism, and obesity are independently associated with MS, in contrast to hypoestrogenism (ovariectomy model) that per se generates metabolic syndrome (this proposal). However, due to the wide use of ovariectomy in combination with hypercaloric diets to develop metabolic disorders, most authors have dismissed the results offered by ovariectomy itself; given more importance to the combination with hypercaloric diets for MS development [89,90,91,92,93,94]. However, hypoestrogenism-induced MS could yield interesting and necessary data in understanding metabolic complications originated by menopause, because estrogens play a crucial role in regulating metabolic-energetics, while their loss leave the body “unprotected” favoring the development of MS independently of the diet used. Accordingly, the success of hypoestrogenism induced by ovariectomy as a MS model could depend on several factors that need a deeper evaluation, like (1) rodent species: Wistar and Sprague–Dawley rats present the greatest development of MS factors, (2) age of the animals at the time of surgery: pre-puber, young, adult or old; an adult age is the best to perform the surgery and achieve the successful establishment of MS, (3) time in which the animals are in hypoestrogenic state: after 12 weeks the main factors associated with MS do appear (Table 1). Therefore, a combination of all these variables could allow the effective achievement of a MS model.

Conclusion

As mentioned, MS-induced by hypercaloric diets can promote the display of multiple pathologies [62, 63]; in this sense, it should be noticed that ovariectomy-induced MS favors the manifestation of pathologies similar to those with hypercaloric diets or a combination of both; for example, cardiomyopathy, hepatic steatosis, kidney damage, inflammatory state and hypertension [65, 70, 71, 81, 83, 95,96,97,98], which also have been described in menopause, and that could be a consequence from a predominant hypoestrogenic state [29, 99,100,101]. Taken together, we suggest that ovariectomy per se can be used as a "menopausal metabolic syndrome model", mainly because the physiopathology of MS that develops in menopause could be different to the one with radical changes in diet; even the main features of menopausal metabolic syndrome could be compared with other MS models. In this way, a more accurate approximation should be made to the development of MS in postmenopausal women, which might imply the search for more specific treatments for this population.