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Emerging role of estrogen in the control of cardiometabolic disease
Review
Emerging role of estrogen in the control of cardiometabolic disease Andrea Cignarella1, Mario Kratz2 and Chiara Bolego1 1 2
Department of Pharmacology and Anesthesiology, University of Padova, Largo Meneghetti 2, I-35131 Padova, Italy Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Mail stop M4-B402, Seattle, WA 98103, USA
Menopause is associated with an increased risk of cardiovascular and metabolic disease that is partly independent of aging. This increased risk is largely due to postmenopausal estrogen loss. Estrogen improves insulin sensitivity and ß-cell function. This is consistent with the increased risk of diabetes after menopause and the finding that menopausal hormone therapy (MHT) lowers the incidence of diabetes. Experimental data suggest that estrogen has anti-atherosclerotic and pro-thrombotic properties. This is consistent with observational and interventional studies suggesting that MHT reduces the risk of cardiovascular disease if initiated early in women with a low-risk profile, but might increase risk in older women and/or those with other risk factors (e.g. dyslipidemia). Future research focusing on improving prevention of cardiometabolic disease through MHT may help to identify agents with higher tissue- and estrogen receptor-isoform specificity than currently used hormones. Introduction Beyond controlling several processes in the reproductive system, estrogen is responsible for the normal function of the cardiovascular system [1]. Estrogen biosynthesis is initiated by the synthesis of the 19-carbon steroid hormone pregnenolone from cholesterol (Figure 1). This compound is converted to testosterone and then to the estrogens estrone and 17b-estradiol (the primary female sex steroid). Estrogen biosynthesis is catalyzed by aromatase, a microsomal member of the cytochrome P450 superfamily that introduces the characteristic phenolic ring. In premenopausal women, the ovaries are the principal source of 17bestradiol, which functions as a circulating hormone to act on distal target tissues. In men and postmenopausal women, when the ovaries cease to produce estrogens, 17b-estradiol is produced in several extragonadal sites (e.g. adrenal glands, adipose tissue) and acts locally as a paracrine factor [2]. It is unclear which adipose tissue cell type is the primary source of aromatase, and why estrogen is synthesized in adipose tissue. Estrogen exerts most of its biological actions via estrogen receptors (ERs) [3]. ERs are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily (Box 1). The menopausal transition is characterized by reduced serum concentrations of estrone and estradiol [9], and gradually developing testosterone predominance [9–11]. Corresponding author: Cignarella, A. ([email protected]).
Consistent with the widespread impact of estrogen on vascular function [12], the menopausal transition is associated with a gradual rise in the risk of cardiovascular disease (CVD) [13]. The increase with age is curvilinear so risk may be related not only to hormonal loss but more directly to aging [14]. Several metabolic changes are associated with the menopause independent of aging, such as increased abdominal fat mass and impaired glucose metabolism [15]. A lack of estrogen and progressive testosterone predominance might increase abdominal obesity. In postmenopausal women, there is a direct association between estrogen levels and body mass index [16]. Higher concentrations of estrogen in the serum of obese postmenopausal women may result from increased expression of aromatase in adipose tissue, which enhances conversion of androgens to estrogens [2]. Estrogens also display insulin-sensitizing properties and prevent failure of pancreatic ß-cells, so progressive loss of estrogen during menopause (possibly in concert with increased abdominal obesity) is associated with an increased risk of type-2 diabetes mellitus (T2DM) [15,17]. Menopausal hormone therapy (MHT) is a drug regimen used primarily to treat menopausal symptoms and prevent osteoporosis. In the combined regimen for women with an intact uterus, estrogen is prescribed with progestagen to reduce the development of endometrial cancer. Women without a uterus can take unopposed estrogen. In line with findings from preclinical studies, MHT has been shown to improve glycemic control [15] and endothelial function [18] in postmenopausal women. MHT has therefore been tested in postmenopausal women for the reduction of cardiometabolic risk. This notion encompasses the global risk of CVD and T2DM associated with traditional risk factors (lowdensity lipoprotein (LDL)-cholesterol, hypertension, age, male sex, smoking) taking into consideration the potential additional contribution of abdominal obesity and/or insulin resistance [19]. This article will tackle the role of estrogen in controlling metabolic risk factors for CVD and the potential for pharmacological targeting of ER to prevent the manifestation of cardiometabolic disease. The effects of estrogen metabolites (e.g. methoxy-estradiol) will not be covered. Effect of estrogen on CVD risk It has long been thought that estrogen exerts direct preventive effects on the risk of atherosclerosis and CVD in women. This hypothesis was based on the observation that premenopausal women have a significantly lower risk of CVD than men, until women undergo the menopausal
0165-6147/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2010.01.001 Available online 6 February 2010
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Figure 1. The metabolic pathway for estrogen. Estrogens and other steroid hormones are derived from cholesterol, with pregnenolone formed from cholesterol through the activity of cytochrome P-450 11A1. Other early steps in estrogen biosynthesis are the conversion of pregnenolone to dehydroepiandrosterone, and the conversion of progesterone to androstenedione. 3ß-hydroxysteroid dehydrogenases catalyze several reactions in the androgen pathway, leading to production of androstenedione and testosterone. The enzyme aromatase catalyses the final and key step (i.e. conversion of C19 steroids to estrogens). The ovarian granulosa cells express the highest levels of aromatase in premenopausal women, but the adipose tissue becomes the major aromatase-expressing site after menopause.
transition and experience estrogen loss [20]. Experimental studies suggest that estrogen regulates production of nitric oxide (a mediator involved in vascular function due to its vasodilating and anti-thrombotic properties) through genomic and nongenomic mechanisms mostly mediated by ERa Box 1. Signaling of estrogen receptors (ERs) Two known ER isoforms, ERa and ERb, are expressed in virtually every tissue in the body. They mediate distinct, overlapping or conflicting biological actions. The protein structure of ERa is highly homologous to that of ERb (particularly in the DNA- and ligandbinding domains). Upon ligand binding, ERs undergo a conformational change, allowing spontaneous dimerisation to form homo- or heterodimers. As a dimer, the ER binds to the estrogen response element (ERE) in the promoter region of target genes (Figure 2). Whereas the long-term regulation of gene expression takes hours-todays, estrogen mediates signaling events within seconds-to-minutes through activation of MAPK, PI3K and G-protein pathways [4]. Membrane versions of the classical ERs as well as the newly described G-protein-coupled estrogen receptor (GPER) [5,6] appear to account for such nongenomic effects. Natural hormones behave as full agonists of ERa and ERb in all tissues, so they induce beneficial and untoward effects if used for therapy. More selective pharmacological modulation of ERs is accomplished using plant-derived agents and synthetic compounds such as selective estrogen receptor modulators (SERMs). ER-selective agonists as exemplified by 4,40 ,400 -(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) [7] or 2,3bis(4-hydroxyphenyl)-propionitrile (DPN) [8] are under development.
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[12,21]. Estrogen therefore prevents endothelial dysfunction and vascular inflammation (reviewed in [12]). Certain estrogen effects might also be harmful, as shown by the increased risk of thromboembolism after oral estrogen therapy [22]. It is therefore not surprising that data on the relationship between MHT and CVD from observational studies and clinical trials are inconsistent. Basically, the direct vascular effects of endogenous estrogen protecting from CVD before menopause are not entirely reproduced by exogenous hormone administration. Complicating the relationship between estrogen and CVD is increasing evidence that the progressive testosterone predominance seen in menopause has unfavorable effects on CVD risk. The menopausal change in estrogen levels per se is weakly associated with a worsening of risk profile, but this association is stronger with testosterone predominance [10]. Effect of estrogen on T2DM risk Sensitivity of estrogen and insulin Individuals without functional aromatase activity or carriers of genetic variants that impair ERa signaling gradually develop insulin resistance and glucose intolerance [23,24]. Several single nucleotide polymorphisms in the ERa gene that predispose to insulin resistance, glucose intolerance, and increased risk of T2DM have been identified [25,26]. These findings indicate that physiological
Review Box 2. Obesity, inflammation and cardiometabolic risk The past decade has brought a wealth of new insights into the mechanistic links by which obesity causes cardiometabolic disease. Specifically, strong evidence suggests that adipose tissue inflammation constitutes the central link between excess body fat mass and metabolic complications such as insulin resistance and glucose intolerance [52]. In mice, alternatively activated, remodeling-type macrophages are present in the adipose tissue of lean, insulinsensitive animals [53]. These alternatively activated macrophages have important roles in tissue homeostasis because they remove dead cells [54] or are involved in angiogenesis [55]. They have also been described to have anti-inflammatory as well as insulinsensitizing properties [53–55]. By contrast, expansion of adipose tissue induces infiltration of classically activated macrophages that produce large amounts of proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) [53]. Infiltration of adipose tissue with these proinflammatory macrophages is associated with chronic low-grade inflammation, as well as insulin resistance and glucose intolerance [54]. Intriguingly, insulin sensitivity and glucose tolerance remain normal despite massive obesity in mice if the infiltration of adipose tissue with inflammatory macrophage subtypes is prevented [56]. Similarly, at least part of the increased risk of CVD seen in obesity might be due to inflammatory processes in adipose tissue. For example, inflammation in adipose tissue suppresses expression of the antiinflammatory, anti-atherogenic, and insulin-sensitizing hormone adiponectin in fat cells, and increases levels of cytokines and circulating C-reactive protein [57].
concentrations of estrogen, as well as intact ER signaling, are required for normal insulin sensitivity and glucose homeostasis. Accordingly, MHT in postmenopausal women improves insulin sensitivity and reduces diabetes risk [15,27,28]. It is unclear how these insulin-sensitizing effects of estrogen could be reconciled with the association of elevated concentrations of estrogen in the serum in states of insulin resistance such as pregnancy, polycystic ovary syndrome (PCOS), or obesity. We propose that elevated estrogen in these cases is not causally linked to insulin resistance. In pregnancy and PCOS, several estrogen-independent mechanisms underlying impaired sensitivity to insulin have been proposed [29,30]. In obesity, expansion of adipose tissue is associated with inflammation, leading to insulin resistance and increased synthesis of estrogen in adipose tissue (Box 2). Robust evidence that adipose tissuederived estrogen is involved in the etiology of insulin resistance is lacking. However, the effect of estrogen on insulin sensitivity is dose-dependent, and one could hypothesize that in these conditions a certain threshold is crossed beyond which estrogen contributes to insulin resistance. Accordingly, high concentrations of estradiol inhibit insulin-mediated glucose transport in cultured adipocytes, whereas low concentrations increase glucose uptake [31]. Physiological concentrations of estrogen therefore improve insulin sensitivity, and the menopausal fall in estrogen levels predisposes to insulin resistance. Estrogen and function of pancreatic b-cells Insulin resistance leads to glucose intolerance and T2DM if pancreatic ß-cells cannot meet the increased demand for insulin (‘ß-cell dysfunction’) [32]. Estrogen is increasingly recognized to play a part in this process [33]. A recent study showed that 17ß-estradiol protects ß-cells from oxidative injury in mice [34]. Similarly, treatment with 17ß-estradiol
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resulted in protection from proinflammatory cytokinemediated ß-cell death in vitro [34,35] through ERa activation [35]. Direct activation of ERa in vivo and in vitro by 17ß-estradiol at physiological concentrations regulates pancreatic insulin levels [36]. Accordingly, activation of ERa signaling by PPT improves glucose tolerance and insulin sensitivity in ob/ob mice [37]. Aromatase-deficient mice that cannot convert androgens to estrogens develop diabetes after exposure to streptozotocin (STZ), but physiological doses of estradiol prevent ß-cell apoptosis and diabetes [34]. The inflammatory process and ß-cell destruction resulting from STZ injection as used in the study described above might not be a good model for the spontaneous development of ß-cell dysfunction in mice and humans. ERa is not the sole estrogen-signaling pathway in ß-cells because 17ß-estradiol-mediated prevention of STZ-induced apoptosis in ERa-deficient mice is only partially abolished. Although 17b-estradiol acts on b-cells and interferes with insulin release mainly through ERa [36], the exact role of ERa and ERb in the regulation of islet function and insulin release has yet to be investigated. Whereas young ERß-deficient mice demonstrate normal glucose tolerance [38], decreased fasting glycemia is accompanied by signs of glucose intolerance in older mice [39]. The newly described extranuclear GPER, which is also expressed in ß-cells, mediates rapid 17ß-estradiol-induced insulin release [40] and confers protection from apoptosis [41]. The mechanisms by which estrogens affect insulin sensitivity through both ER isoforms and GPER also seem to involve regulation of the expression and activity of glucose transporters, in particular GLUT-4 [42] (Figure 2). Also relevant to understanding the protective effects of estrogen on b-cells are recent studies investigating the effects of 17ß-estradiol on innate immune systemmediated inflammation in multiple tissues [43]. 17ßestradiol exerts anti-inflammatory actions in different tissues and animal models [44,45] through inhibition of the synthesis of inducible nitric oxide synthase (iNOS), although this inhibitory activity is lost with concomitant overexpression of ERß [46]. The protective effects of ERa (but not ERß) on inflammation in the vascular wall have been demonstrated using ER-selective agonists in diabetic ER-knockout mice [47]. Thus, selective ERa targeting may reduce the inflammatory burden and improve ß-cell function. Impact of estrogen on adiposity, a risk factor for cardiometabolic disease An increased risk of cardiometabolic disease in women going through the menopausal transition could be due to an increase in abdominal adiposity. Changes in the metabolism of sex hormones lead to accumulation of excess fat in intra-abdominal adipose tissue depots. This could be partly mediated by the loss of estrogen because ERs are involved in the regulation of energy homeostasis and body composition [48,49]. As recently reported [50], the gain of weight and fat mass in ovariectomized mice on an obesogenic highfat diet is attenuated in mice treated with physiological doses of estradiol. Interestingly, deletion of the GPER gene leads to visceral obesity [51]. 185
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Figure 2. Outline of ER signaling pathways in target tissues relevant to cardiometabolic disease. Two estrogen receptors are known: ERa and ERb. Upon ligand binding, ERs undergo a conformational change, allowing spontaneous dimerisation to form homo- or heterodimers. As a dimer, the ER binds to the estrogen response element (ERE) in the promoter region of target genes in a broad spectrum of tissues and regulates their expression in the long-term. In addition, 17ß-estradiol induces rapid nongenomic effects by activating signaling pathways mediated, among others, by mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinases (PI3K) and G-proteins. Membrane versions of ERa and the newly described G-protein-coupled ER mainly account for these effects.
Aside from the possibility that the decline in estrogen levels per se is relevant for disease risk, there is evidence suggesting that progressive androgenicity of the hormonal milieu is a risk factor for the metabolic syndrome, a cluster of metabolic and CVD risk factors (e.g. dyslipidemia, impaired glucose homeostasis, deposition of abdominal adipose tissue) and high blood pressure [58,59]. In a cohort study of the natural history of the menopausal transition in nearly 1,000 women who never received hormone therapy nor had diabetes or metabolic syndrome at baseline, progressive testosterone predominance was significantly and independently linked to an increased risk of developing the metabolic syndrome during menopausal transition [10]. Whereas the onset of metabolic syndrome was not associated with levels of total estradiol or total testosterone, it was significantly associated with changes in bioavailable testosterone (defined as the ratio of total testosterone to the level of sex-hormone-binding globulin (SHBG)). In a recent study from the same research team, bioavailable testosterone was also associated with accumulation of visceral fat even after adjusting for insulin resistance [60]. The lack of ovary-derived estrogen after menopause might play a part in increased adiposity, which in turn might enhance estrogen synthesis in adipose tissue. Obese postmenopausal women have higher serum estrogen concentrations than lean postmenopausal women [61,62]. Inflammatory cytokines such as TNF-a induce aromatase expression in vitro [63], so the inflammatory process probably introduces into the adipose tissue a cell type that expresses aromatase or induces aromatase expression in a resident tissue cell (adipocyte, fibroblast). Estradiol has potent anti-inflammatory properties and suppresses the expression of proinflammatory cytokines such as IL-6 or TNF-a in macrophages and dendritic cells (summarized in [43]). Estrogen therefore may have a specific paracrine role in the inflammation of adipose tissue such as alleviating 186
potential tissue-damaging effects. Estradiol treatment of ovariectomized mice, while improving insulin sensitivity and glucose tolerance, is associated with increased inflammation of adipose tissue, suggesting that estrogens contribute to (rather than attenuate) inflammation [50]. In that study, the insulin-sensitizing effect of estradiol was sufficiently potent to overcome substantially increased inflammation in adipose tissue and reduced serum concentrations of adiponectin after estradiol treatment. The dual estrogenic modulation of the inflammatory process may be dependent upon the estrogen concentration, as well as other factors (e.g. type of tissue or nature of the antigen triggering inflammation) [43]. Even though it is implicated in postmenopausal breast cancer, adipose tissue-derived estrogen does not appear to meaningfully contribute to the obesity-associated postmenopausal increase in risk of cardiometabolic disease. MHT in the prevention of cardiometabolic disease: evidence from epidemiological and clinical studies The effect of long-term MHT on cardiac endpoints was explored in several observational studies in which women usually started MHT early in menopause and continued treatment for many years. MHT users of estrogen + progestin or estrogen alone appeared to have a lower risk of coronary heart disease than non-users in women with and without a history of heart disease [64]. In contrast, large trials such as the Heart and Estrogen/progestin Replacement Study (HERS) and the Women’s Health Initiative (WHI) failed to demonstrate the clinical benefit of MHT in postmenopausal women. The WHI trial assessed the effect of conjugated equine estrogen (CEE) alone or in combination with medroxyprogesterone acetate (MPA) in postmenopausal women on numerous health outcomes (CVD, osteoporosis, breast cancer). Overall results showed that neither therapy pro-
Review tects against coronary events (as had been suggested in earlier observational studies) and that combined therapy is associated with an increased risk of CVD events [65]. A favorable lipid status at baseline tended to predict better CVD outcomes if using CEE with or without MPA. Specifically, women with a baseline low-density lipoprotein/highdensity lipoprotein (LDL/HDL)-cholesterol ratio of >2.5 were at an increased risk of CVD from MHT, whereas there was no increased risk of CVD if the baseline LDL/ HDL-cholesterol ratio was <2.5 [66]. A potential explanation for these findings relies on the observation that the cholesterol metabolite 27-hydroxycholesterol competes with estrogen for ER binding in vascular cells [67]. This endogenous oxysterol interacts with and modulates the transcriptional activity of both ER isoforms, blocking estrogen action on the production of nitric oxide and endothelial function [68]. This would imply that postmenopausal women with a poor lipid profile have increased 27-hydroxycholesterol, whereas women with favorable lipid profiles might experience no harm (or even benefit) from MHT if their level of 27-hydroxycholesterol is low. Accordingly, the combination of MHT with a statin resulted in significant improvement of CVD compared with MHT alone in the HERS study [69]. Consistent with the divergent effects of MHT in women with high versus low serum lipids and the outcome of large trials in which MHT was initiated later than in most previous observational studies, MHT may be harmful in older women or in women with a worse CVD risk profile irrespective of age, who are therefore subject to the prothrombotic effects of MHT [13,70,71]. By contrast, a clinical benefit may arise if MHT is started in women with little atherosclerosis and preserved vascular function, although clinical trials do not provide substantive support. More recent analyses of the Nurses’ Health Study highlight an early increased risk associated with MHT use and a possible benefit according to years since menopause rather than age [72]. Differentiating between patients who might benefit and those whose CVD risk might be raised by MHT would be difficult [73,74]. Additional safety concerns (i.e. increased risk of breast cancer and lung cancer) are related to the supraphysiological dosages of estrogen applied in current MHT regimens. MHT appears to be an effective means of reducing the menopause-associated increase in the risk of diabetes mellitus. A recent population-based prospective cohort study suggests that MHT is associated with a reduced risk of developing diabetes [75]. During follow-up, 162 new cases were reported in the study population of nearly 9,000 women. Remarkably, the risk reduction was accentuated in women who used MHT for more than half of the follow-up time (5 years). Compared with individuals who never used MHT, diabetes risk was reduced by 62% by current use of MHT. These risk reductions are much greater than those observed in clinical trials, and it is likely that the observational data are confounded by factors such as compliance bias and survivor bias that would lead to an overestimate of potential benefit. Further observational data suggest a possible cardiovascular benefit for women with diabetes from MHT if they had not already had a recent myocardial infarction, where the risk seemed
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to increase with MHT exposure [76] in line with the above timing hypothesis. Most (but not all clinical trials) indicate that MHT improves glycemic control as assessed through measurements of fasting glucose or glycosylated hemoglobin levels in postmenopausal women with T2DM (reviewed in [15]). Accordingly, recent large-scale trials found a significantly lower incidence of diabetes in postmenopausal women on MHT [27,28]. Among the 2,029 women in HERS who had coronary disease but no diabetes at baseline, 6.2% of those receiving estrogen + MPA and 9.5% of those receiving placebo developed diabetes [27]. Similar results were obtained in a secondary analysis of the WHI trial [28], suggesting that MHT reduces the risk of developing diabetes mellitus. However, MHT has not been found to consistently improve glucose control in women with preexisting diabetes [15]. In fact, clinical trials have failed to show protective effects of MHT on CVD in diabetic women [77]. On these grounds, estrogens appear to be more useful as preventive rather than therapeutic agents. It is well documented that patients with diabetes (and women in particular) have an increased risk of CVD. For instance, in the 30-year follow-up of the Framingham Heart Study, the relative risks for all CVD or various types thereof were significantly greater for women with diabetes than for men with diabetes aged 35–64 years. The improved glucose tolerance and reduced diabetes risk with MHT are probably outweighed by the adverse cardiovascular consequences (e.g. thrombosis) [78] and potentially the interactions with serum lipoproteins as discussed above. Conclusions Despite our growing understanding of the mechanisms underlying its beneficial actions on metabolism, estrogenbased MHT induces deleterious effects such as an increased incidence of breast cancer and thromboembolic disease, thereby leading to an uncertain risk–benefit ratio. One of the most pressing questions is to understand which groups of patients might benefit most from currently used agents for MHT to reduce their cardiometabolic risk. Although controversial, evidence suggests that larger benefits might be elicited in younger postmenopausal women without established cardiometabolic disease. Pharmacological agents targeting ER-mediated pathways that can trigger the beneficial effects of physiological hormones while minimizing adverse effects are lacking. The two main ER isoforms appear to be differentially involved in the effects of estrogen on vascular function, insulin sensitivity and ß-cell health, thus selective targeting of ERa (possibly coupled to tissue-selective ER modulation) should be pursued [79]. Development of such new compounds appears to be a promising strategy for estrogen therapy. Acknowledgments A.C. has been recipient of a Fulbright Research Scholarship 2009-10.
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68 DuSell, C.D. and McDonnell, D.P. (2008) 27-Hydroxycholesterol: a potential endogenous regulator of estrogen receptor signaling. Trends Pharmacol. Sci. 29, 510–514 69 Herrington, D.M. et al. (2002) Statin therapy, cardiovascular events, and total mortality in the Heart and Estrogen/Progestin Replacement Study (HERS). Circulation 105, 2962–2967 70 Grodstein, F. et al. (2006) Hormone therapy and coronary heart disease: the role of time since menopause and age at hormone initiation. J. Womens Health (Larchmt) 15, 35–44 71 Rossouw, J.E. et al. (2007) Postmenopausal hormone therapy and risk of cardiovascular disease by age and years since menopause. JAMA 297, 1465–1477 72 Herna´n, M.A. et al. (2008) Observational studies analyzed like randomized experiments: an application to postmenopausal hormone therapy and coronary heart disease. Epidemiology 19, 766– 779 73 Alexandersen, P. et al. (2009) Long-term prevention with hormonereplacement therapy after the menopause: which women should be targeted? Womens Health (London Engl) 5, 637–647 74 Korljan, B. et al. (2010) The impact of hormone replacement therapy on metabolic syndrome components in perimenopausal women. Med. Hypotheses 74, 162–163 75 Pentti, K. et al. (2009) Hormone therapy protects from diabetes: the Kuopio osteoporosis risk factor and prevention study. Eur. J. Endocrinol. 160, 979–983 76 Ferrara, A. et al. (2003) Current use of unopposed estrogen and estrogen plus progestin and the risk of acute myocardial infarction among women with diabetes: the Northern California Kaiser Permanente Diabetes Registry, 1995–1998. Circulation 107, 43–48 77 Howard, B.V. et al. (2004) Postmenopausal hormone therapy is associated with atherosclerosis progression in women with abnormal glucose tolerance. Circulation 110, 201–206 78 Cushman, M. et al. (2004) Estrogen plus progestin and risk of venous thrombosis. JAMA 292, 1573–1580 79 Bolego, C. et al. (2006) Selective agonists of estrogen receptor isoforms: new perspectives for cardiovascular disease. Arterioscler. Thromb. Vasc. Biol. 26, 2192–2199
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Emerging role of estrogen in the control of cardiometabolic disease Andrea Cignarella1, Mario Kratz2 and Chiara Bolego1 1 2
Department of Pharmacology and Anesthesiology, University of Padova, Largo Meneghetti 2, I-35131 Padova, Italy Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Mail stop M4-B402, Seattle, WA 98103, USA
Menopause is associated with an increased risk of cardiovascular and metabolic disease that is partly independent of aging. This increased risk is largely due to postmenopausal estrogen loss. Estrogen improves insulin sensitivity and ß-cell function. This is consistent with the increased risk of diabetes after menopause and the finding that menopausal hormone therapy (MHT) lowers the incidence of diabetes. Experimental data suggest that estrogen has anti-atherosclerotic and pro-thrombotic properties. This is consistent with observational and interventional studies suggesting that MHT reduces the risk of cardiovascular disease if initiated early in women with a low-risk profile, but might increase risk in older women and/or those with other risk factors (e.g. dyslipidemia). Future research focusing on improving prevention of cardiometabolic disease through MHT may help to identify agents with higher tissue- and estrogen receptor-isoform specificity than currently used hormones. Introduction Beyond controlling several processes in the reproductive system, estrogen is responsible for the normal function of the cardiovascular system [1]. Estrogen biosynthesis is initiated by the synthesis of the 19-carbon steroid hormone pregnenolone from cholesterol (Figure 1). This compound is converted to testosterone and then to the estrogens estrone and 17b-estradiol (the primary female sex steroid). Estrogen biosynthesis is catalyzed by aromatase, a microsomal member of the cytochrome P450 superfamily that introduces the characteristic phenolic ring. In premenopausal women, the ovaries are the principal source of 17bestradiol, which functions as a circulating hormone to act on distal target tissues. In men and postmenopausal women, when the ovaries cease to produce estrogens, 17b-estradiol is produced in several extragonadal sites (e.g. adrenal glands, adipose tissue) and acts locally as a paracrine factor [2]. It is unclear which adipose tissue cell type is the primary source of aromatase, and why estrogen is synthesized in adipose tissue. Estrogen exerts most of its biological actions via estrogen receptors (ERs) [3]. ERs are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily (Box 1). The menopausal transition is characterized by reduced serum concentrations of estrone and estradiol [9], and gradually developing testosterone predominance [9–11]. Corresponding author: Cignarella, A. ([email protected]).
Consistent with the widespread impact of estrogen on vascular function [12], the menopausal transition is associated with a gradual rise in the risk of cardiovascular disease (CVD) [13]. The increase with age is curvilinear so risk may be related not only to hormonal loss but more directly to aging [14]. Several metabolic changes are associated with the menopause independent of aging, such as increased abdominal fat mass and impaired glucose metabolism [15]. A lack of estrogen and progressive testosterone predominance might increase abdominal obesity. In postmenopausal women, there is a direct association between estrogen levels and body mass index [16]. Higher concentrations of estrogen in the serum of obese postmenopausal women may result from increased expression of aromatase in adipose tissue, which enhances conversion of androgens to estrogens [2]. Estrogens also display insulin-sensitizing properties and prevent failure of pancreatic ß-cells, so progressive loss of estrogen during menopause (possibly in concert with increased abdominal obesity) is associated with an increased risk of type-2 diabetes mellitus (T2DM) [15,17]. Menopausal hormone therapy (MHT) is a drug regimen used primarily to treat menopausal symptoms and prevent osteoporosis. In the combined regimen for women with an intact uterus, estrogen is prescribed with progestagen to reduce the development of endometrial cancer. Women without a uterus can take unopposed estrogen. In line with findings from preclinical studies, MHT has been shown to improve glycemic control [15] and endothelial function [18] in postmenopausal women. MHT has therefore been tested in postmenopausal women for the reduction of cardiometabolic risk. This notion encompasses the global risk of CVD and T2DM associated with traditional risk factors (lowdensity lipoprotein (LDL)-cholesterol, hypertension, age, male sex, smoking) taking into consideration the potential additional contribution of abdominal obesity and/or insulin resistance [19]. This article will tackle the role of estrogen in controlling metabolic risk factors for CVD and the potential for pharmacological targeting of ER to prevent the manifestation of cardiometabolic disease. The effects of estrogen metabolites (e.g. methoxy-estradiol) will not be covered. Effect of estrogen on CVD risk It has long been thought that estrogen exerts direct preventive effects on the risk of atherosclerosis and CVD in women. This hypothesis was based on the observation that premenopausal women have a significantly lower risk of CVD than men, until women undergo the menopausal
0165-6147/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2010.01.001 Available online 6 February 2010
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Figure 1. The metabolic pathway for estrogen. Estrogens and other steroid hormones are derived from cholesterol, with pregnenolone formed from cholesterol through the activity of cytochrome P-450 11A1. Other early steps in estrogen biosynthesis are the conversion of pregnenolone to dehydroepiandrosterone, and the conversion of progesterone to androstenedione. 3ß-hydroxysteroid dehydrogenases catalyze several reactions in the androgen pathway, leading to production of androstenedione and testosterone. The enzyme aromatase catalyses the final and key step (i.e. conversion of C19 steroids to estrogens). The ovarian granulosa cells express the highest levels of aromatase in premenopausal women, but the adipose tissue becomes the major aromatase-expressing site after menopause.
transition and experience estrogen loss [20]. Experimental studies suggest that estrogen regulates production of nitric oxide (a mediator involved in vascular function due to its vasodilating and anti-thrombotic properties) through genomic and nongenomic mechanisms mostly mediated by ERa Box 1. Signaling of estrogen receptors (ERs) Two known ER isoforms, ERa and ERb, are expressed in virtually every tissue in the body. They mediate distinct, overlapping or conflicting biological actions. The protein structure of ERa is highly homologous to that of ERb (particularly in the DNA- and ligandbinding domains). Upon ligand binding, ERs undergo a conformational change, allowing spontaneous dimerisation to form homo- or heterodimers. As a dimer, the ER binds to the estrogen response element (ERE) in the promoter region of target genes (Figure 2). Whereas the long-term regulation of gene expression takes hours-todays, estrogen mediates signaling events within seconds-to-minutes through activation of MAPK, PI3K and G-protein pathways [4]. Membrane versions of the classical ERs as well as the newly described G-protein-coupled estrogen receptor (GPER) [5,6] appear to account for such nongenomic effects. Natural hormones behave as full agonists of ERa and ERb in all tissues, so they induce beneficial and untoward effects if used for therapy. More selective pharmacological modulation of ERs is accomplished using plant-derived agents and synthetic compounds such as selective estrogen receptor modulators (SERMs). ER-selective agonists as exemplified by 4,40 ,400 -(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) [7] or 2,3bis(4-hydroxyphenyl)-propionitrile (DPN) [8] are under development.
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[12,21]. Estrogen therefore prevents endothelial dysfunction and vascular inflammation (reviewed in [12]). Certain estrogen effects might also be harmful, as shown by the increased risk of thromboembolism after oral estrogen therapy [22]. It is therefore not surprising that data on the relationship between MHT and CVD from observational studies and clinical trials are inconsistent. Basically, the direct vascular effects of endogenous estrogen protecting from CVD before menopause are not entirely reproduced by exogenous hormone administration. Complicating the relationship between estrogen and CVD is increasing evidence that the progressive testosterone predominance seen in menopause has unfavorable effects on CVD risk. The menopausal change in estrogen levels per se is weakly associated with a worsening of risk profile, but this association is stronger with testosterone predominance [10]. Effect of estrogen on T2DM risk Sensitivity of estrogen and insulin Individuals without functional aromatase activity or carriers of genetic variants that impair ERa signaling gradually develop insulin resistance and glucose intolerance [23,24]. Several single nucleotide polymorphisms in the ERa gene that predispose to insulin resistance, glucose intolerance, and increased risk of T2DM have been identified [25,26]. These findings indicate that physiological
Review Box 2. Obesity, inflammation and cardiometabolic risk The past decade has brought a wealth of new insights into the mechanistic links by which obesity causes cardiometabolic disease. Specifically, strong evidence suggests that adipose tissue inflammation constitutes the central link between excess body fat mass and metabolic complications such as insulin resistance and glucose intolerance [52]. In mice, alternatively activated, remodeling-type macrophages are present in the adipose tissue of lean, insulinsensitive animals [53]. These alternatively activated macrophages have important roles in tissue homeostasis because they remove dead cells [54] or are involved in angiogenesis [55]. They have also been described to have anti-inflammatory as well as insulinsensitizing properties [53–55]. By contrast, expansion of adipose tissue induces infiltration of classically activated macrophages that produce large amounts of proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) [53]. Infiltration of adipose tissue with these proinflammatory macrophages is associated with chronic low-grade inflammation, as well as insulin resistance and glucose intolerance [54]. Intriguingly, insulin sensitivity and glucose tolerance remain normal despite massive obesity in mice if the infiltration of adipose tissue with inflammatory macrophage subtypes is prevented [56]. Similarly, at least part of the increased risk of CVD seen in obesity might be due to inflammatory processes in adipose tissue. For example, inflammation in adipose tissue suppresses expression of the antiinflammatory, anti-atherogenic, and insulin-sensitizing hormone adiponectin in fat cells, and increases levels of cytokines and circulating C-reactive protein [57].
concentrations of estrogen, as well as intact ER signaling, are required for normal insulin sensitivity and glucose homeostasis. Accordingly, MHT in postmenopausal women improves insulin sensitivity and reduces diabetes risk [15,27,28]. It is unclear how these insulin-sensitizing effects of estrogen could be reconciled with the association of elevated concentrations of estrogen in the serum in states of insulin resistance such as pregnancy, polycystic ovary syndrome (PCOS), or obesity. We propose that elevated estrogen in these cases is not causally linked to insulin resistance. In pregnancy and PCOS, several estrogen-independent mechanisms underlying impaired sensitivity to insulin have been proposed [29,30]. In obesity, expansion of adipose tissue is associated with inflammation, leading to insulin resistance and increased synthesis of estrogen in adipose tissue (Box 2). Robust evidence that adipose tissuederived estrogen is involved in the etiology of insulin resistance is lacking. However, the effect of estrogen on insulin sensitivity is dose-dependent, and one could hypothesize that in these conditions a certain threshold is crossed beyond which estrogen contributes to insulin resistance. Accordingly, high concentrations of estradiol inhibit insulin-mediated glucose transport in cultured adipocytes, whereas low concentrations increase glucose uptake [31]. Physiological concentrations of estrogen therefore improve insulin sensitivity, and the menopausal fall in estrogen levels predisposes to insulin resistance. Estrogen and function of pancreatic b-cells Insulin resistance leads to glucose intolerance and T2DM if pancreatic ß-cells cannot meet the increased demand for insulin (‘ß-cell dysfunction’) [32]. Estrogen is increasingly recognized to play a part in this process [33]. A recent study showed that 17ß-estradiol protects ß-cells from oxidative injury in mice [34]. Similarly, treatment with 17ß-estradiol
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resulted in protection from proinflammatory cytokinemediated ß-cell death in vitro [34,35] through ERa activation [35]. Direct activation of ERa in vivo and in vitro by 17ß-estradiol at physiological concentrations regulates pancreatic insulin levels [36]. Accordingly, activation of ERa signaling by PPT improves glucose tolerance and insulin sensitivity in ob/ob mice [37]. Aromatase-deficient mice that cannot convert androgens to estrogens develop diabetes after exposure to streptozotocin (STZ), but physiological doses of estradiol prevent ß-cell apoptosis and diabetes [34]. The inflammatory process and ß-cell destruction resulting from STZ injection as used in the study described above might not be a good model for the spontaneous development of ß-cell dysfunction in mice and humans. ERa is not the sole estrogen-signaling pathway in ß-cells because 17ß-estradiol-mediated prevention of STZ-induced apoptosis in ERa-deficient mice is only partially abolished. Although 17b-estradiol acts on b-cells and interferes with insulin release mainly through ERa [36], the exact role of ERa and ERb in the regulation of islet function and insulin release has yet to be investigated. Whereas young ERß-deficient mice demonstrate normal glucose tolerance [38], decreased fasting glycemia is accompanied by signs of glucose intolerance in older mice [39]. The newly described extranuclear GPER, which is also expressed in ß-cells, mediates rapid 17ß-estradiol-induced insulin release [40] and confers protection from apoptosis [41]. The mechanisms by which estrogens affect insulin sensitivity through both ER isoforms and GPER also seem to involve regulation of the expression and activity of glucose transporters, in particular GLUT-4 [42] (Figure 2). Also relevant to understanding the protective effects of estrogen on b-cells are recent studies investigating the effects of 17ß-estradiol on innate immune systemmediated inflammation in multiple tissues [43]. 17ßestradiol exerts anti-inflammatory actions in different tissues and animal models [44,45] through inhibition of the synthesis of inducible nitric oxide synthase (iNOS), although this inhibitory activity is lost with concomitant overexpression of ERß [46]. The protective effects of ERa (but not ERß) on inflammation in the vascular wall have been demonstrated using ER-selective agonists in diabetic ER-knockout mice [47]. Thus, selective ERa targeting may reduce the inflammatory burden and improve ß-cell function. Impact of estrogen on adiposity, a risk factor for cardiometabolic disease An increased risk of cardiometabolic disease in women going through the menopausal transition could be due to an increase in abdominal adiposity. Changes in the metabolism of sex hormones lead to accumulation of excess fat in intra-abdominal adipose tissue depots. This could be partly mediated by the loss of estrogen because ERs are involved in the regulation of energy homeostasis and body composition [48,49]. As recently reported [50], the gain of weight and fat mass in ovariectomized mice on an obesogenic highfat diet is attenuated in mice treated with physiological doses of estradiol. Interestingly, deletion of the GPER gene leads to visceral obesity [51]. 185
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Figure 2. Outline of ER signaling pathways in target tissues relevant to cardiometabolic disease. Two estrogen receptors are known: ERa and ERb. Upon ligand binding, ERs undergo a conformational change, allowing spontaneous dimerisation to form homo- or heterodimers. As a dimer, the ER binds to the estrogen response element (ERE) in the promoter region of target genes in a broad spectrum of tissues and regulates their expression in the long-term. In addition, 17ß-estradiol induces rapid nongenomic effects by activating signaling pathways mediated, among others, by mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinases (PI3K) and G-proteins. Membrane versions of ERa and the newly described G-protein-coupled ER mainly account for these effects.
Aside from the possibility that the decline in estrogen levels per se is relevant for disease risk, there is evidence suggesting that progressive androgenicity of the hormonal milieu is a risk factor for the metabolic syndrome, a cluster of metabolic and CVD risk factors (e.g. dyslipidemia, impaired glucose homeostasis, deposition of abdominal adipose tissue) and high blood pressure [58,59]. In a cohort study of the natural history of the menopausal transition in nearly 1,000 women who never received hormone therapy nor had diabetes or metabolic syndrome at baseline, progressive testosterone predominance was significantly and independently linked to an increased risk of developing the metabolic syndrome during menopausal transition [10]. Whereas the onset of metabolic syndrome was not associated with levels of total estradiol or total testosterone, it was significantly associated with changes in bioavailable testosterone (defined as the ratio of total testosterone to the level of sex-hormone-binding globulin (SHBG)). In a recent study from the same research team, bioavailable testosterone was also associated with accumulation of visceral fat even after adjusting for insulin resistance [60]. The lack of ovary-derived estrogen after menopause might play a part in increased adiposity, which in turn might enhance estrogen synthesis in adipose tissue. Obese postmenopausal women have higher serum estrogen concentrations than lean postmenopausal women [61,62]. Inflammatory cytokines such as TNF-a induce aromatase expression in vitro [63], so the inflammatory process probably introduces into the adipose tissue a cell type that expresses aromatase or induces aromatase expression in a resident tissue cell (adipocyte, fibroblast). Estradiol has potent anti-inflammatory properties and suppresses the expression of proinflammatory cytokines such as IL-6 or TNF-a in macrophages and dendritic cells (summarized in [43]). Estrogen therefore may have a specific paracrine role in the inflammation of adipose tissue such as alleviating 186
potential tissue-damaging effects. Estradiol treatment of ovariectomized mice, while improving insulin sensitivity and glucose tolerance, is associated with increased inflammation of adipose tissue, suggesting that estrogens contribute to (rather than attenuate) inflammation [50]. In that study, the insulin-sensitizing effect of estradiol was sufficiently potent to overcome substantially increased inflammation in adipose tissue and reduced serum concentrations of adiponectin after estradiol treatment. The dual estrogenic modulation of the inflammatory process may be dependent upon the estrogen concentration, as well as other factors (e.g. type of tissue or nature of the antigen triggering inflammation) [43]. Even though it is implicated in postmenopausal breast cancer, adipose tissue-derived estrogen does not appear to meaningfully contribute to the obesity-associated postmenopausal increase in risk of cardiometabolic disease. MHT in the prevention of cardiometabolic disease: evidence from epidemiological and clinical studies The effect of long-term MHT on cardiac endpoints was explored in several observational studies in which women usually started MHT early in menopause and continued treatment for many years. MHT users of estrogen + progestin or estrogen alone appeared to have a lower risk of coronary heart disease than non-users in women with and without a history of heart disease [64]. In contrast, large trials such as the Heart and Estrogen/progestin Replacement Study (HERS) and the Women’s Health Initiative (WHI) failed to demonstrate the clinical benefit of MHT in postmenopausal women. The WHI trial assessed the effect of conjugated equine estrogen (CEE) alone or in combination with medroxyprogesterone acetate (MPA) in postmenopausal women on numerous health outcomes (CVD, osteoporosis, breast cancer). Overall results showed that neither therapy pro-
Review tects against coronary events (as had been suggested in earlier observational studies) and that combined therapy is associated with an increased risk of CVD events [65]. A favorable lipid status at baseline tended to predict better CVD outcomes if using CEE with or without MPA. Specifically, women with a baseline low-density lipoprotein/highdensity lipoprotein (LDL/HDL)-cholesterol ratio of >2.5 were at an increased risk of CVD from MHT, whereas there was no increased risk of CVD if the baseline LDL/ HDL-cholesterol ratio was <2.5 [66]. A potential explanation for these findings relies on the observation that the cholesterol metabolite 27-hydroxycholesterol competes with estrogen for ER binding in vascular cells [67]. This endogenous oxysterol interacts with and modulates the transcriptional activity of both ER isoforms, blocking estrogen action on the production of nitric oxide and endothelial function [68]. This would imply that postmenopausal women with a poor lipid profile have increased 27-hydroxycholesterol, whereas women with favorable lipid profiles might experience no harm (or even benefit) from MHT if their level of 27-hydroxycholesterol is low. Accordingly, the combination of MHT with a statin resulted in significant improvement of CVD compared with MHT alone in the HERS study [69]. Consistent with the divergent effects of MHT in women with high versus low serum lipids and the outcome of large trials in which MHT was initiated later than in most previous observational studies, MHT may be harmful in older women or in women with a worse CVD risk profile irrespective of age, who are therefore subject to the prothrombotic effects of MHT [13,70,71]. By contrast, a clinical benefit may arise if MHT is started in women with little atherosclerosis and preserved vascular function, although clinical trials do not provide substantive support. More recent analyses of the Nurses’ Health Study highlight an early increased risk associated with MHT use and a possible benefit according to years since menopause rather than age [72]. Differentiating between patients who might benefit and those whose CVD risk might be raised by MHT would be difficult [73,74]. Additional safety concerns (i.e. increased risk of breast cancer and lung cancer) are related to the supraphysiological dosages of estrogen applied in current MHT regimens. MHT appears to be an effective means of reducing the menopause-associated increase in the risk of diabetes mellitus. A recent population-based prospective cohort study suggests that MHT is associated with a reduced risk of developing diabetes [75]. During follow-up, 162 new cases were reported in the study population of nearly 9,000 women. Remarkably, the risk reduction was accentuated in women who used MHT for more than half of the follow-up time (5 years). Compared with individuals who never used MHT, diabetes risk was reduced by 62% by current use of MHT. These risk reductions are much greater than those observed in clinical trials, and it is likely that the observational data are confounded by factors such as compliance bias and survivor bias that would lead to an overestimate of potential benefit. Further observational data suggest a possible cardiovascular benefit for women with diabetes from MHT if they had not already had a recent myocardial infarction, where the risk seemed
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to increase with MHT exposure [76] in line with the above timing hypothesis. Most (but not all clinical trials) indicate that MHT improves glycemic control as assessed through measurements of fasting glucose or glycosylated hemoglobin levels in postmenopausal women with T2DM (reviewed in [15]). Accordingly, recent large-scale trials found a significantly lower incidence of diabetes in postmenopausal women on MHT [27,28]. Among the 2,029 women in HERS who had coronary disease but no diabetes at baseline, 6.2% of those receiving estrogen + MPA and 9.5% of those receiving placebo developed diabetes [27]. Similar results were obtained in a secondary analysis of the WHI trial [28], suggesting that MHT reduces the risk of developing diabetes mellitus. However, MHT has not been found to consistently improve glucose control in women with preexisting diabetes [15]. In fact, clinical trials have failed to show protective effects of MHT on CVD in diabetic women [77]. On these grounds, estrogens appear to be more useful as preventive rather than therapeutic agents. It is well documented that patients with diabetes (and women in particular) have an increased risk of CVD. For instance, in the 30-year follow-up of the Framingham Heart Study, the relative risks for all CVD or various types thereof were significantly greater for women with diabetes than for men with diabetes aged 35–64 years. The improved glucose tolerance and reduced diabetes risk with MHT are probably outweighed by the adverse cardiovascular consequences (e.g. thrombosis) [78] and potentially the interactions with serum lipoproteins as discussed above. Conclusions Despite our growing understanding of the mechanisms underlying its beneficial actions on metabolism, estrogenbased MHT induces deleterious effects such as an increased incidence of breast cancer and thromboembolic disease, thereby leading to an uncertain risk–benefit ratio. One of the most pressing questions is to understand which groups of patients might benefit most from currently used agents for MHT to reduce their cardiometabolic risk. Although controversial, evidence suggests that larger benefits might be elicited in younger postmenopausal women without established cardiometabolic disease. Pharmacological agents targeting ER-mediated pathways that can trigger the beneficial effects of physiological hormones while minimizing adverse effects are lacking. The two main ER isoforms appear to be differentially involved in the effects of estrogen on vascular function, insulin sensitivity and ß-cell health, thus selective targeting of ERa (possibly coupled to tissue-selective ER modulation) should be pursued [79]. Development of such new compounds appears to be a promising strategy for estrogen therapy. Acknowledgments A.C. has been recipient of a Fulbright Research Scholarship 2009-10.
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