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The role of estrogen and estrogen receptor-α in male adipose tissue
Molecular and Cellular Endocrinology 178 (2001) 147– 154 www.elsevier.com/locate/mce
The role of estrogen and estrogen receptor-a in male adipose tissue Paul S. Cooke a,*, Patricia A. Heine a, Julia A. Taylor b, Dennis B. Lubahn b a
Department of Veterinary Biosciences, Uni6ersity of Illinois, 2001 S. Lincoln A6enue, Urbana, IL, 61802 USA b Department of Biochemistry and Child Health, Uni6ersity of Missouri, Columbia, MO, 65211 USA
Abstract Males and females both express estrogen receptor (ER) in white adipose tissue (WAT), and estrogens appear to play an important role in regulating WAT in females. However, the role of ER in male WAT was unclear. In this review, we describe our work, which used wild type (WT) and ERa-knockout (aERKO) male and female mice to determine the role of ERa in regulating WAT and brown adipose tissue (BAT). There were progressive increases in WAT with advancing age in aERKO compared with WT males; weights of various WAT depots in aERKO males were increased by more than 100% compared with WT controls during adulthood. Conversely, BAT weight was similar in aERKO and WT males at all ages. Adipocyte areas and numbers were also increased in WAT from aERKO compared with WT males. Compared with WT controls, aERKO females also had increases in WAT. The aERKO mice also had insulin resistance and impaired glucose tolerance, similar to humans lacking ERa or aromatase. The obesity in aERKO males appeared to involve decreased energy expenditure rather than hyperphagia. In summary, ERa absence causes adipocyte hyperplasia and hypertrophy in WAT, but not BAT, and is accompanied by insulin resistance and glucose intolerance in both males and females. These results are the first evidence that the estrogen/ERa signaling system is critical in female and male WAT deposition, and may have clinical implications. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Adipocyte; Estrogen receptor beta; Insulin resistance; Glucose intolerance; Obesity
1. Introduction Androgens are the primary regulator of male reproductive tract development, growth and function. Sex steroids such as testosterone also have effects on muscle and adipose tissue development. For example, lean body mass is increased by testosterone, and testosterone is lipolytic, as shown by increases in lean body mass and decreased fat in hypogonadal men treated with a testosterone replacement gel (Wang et al., 2000). Similarly, testosterone treatment in female-to-male transsexuals resulted in decreased subcutaneous fat and adipocyte size (Elbers et al., 1999). In contrast, the role of estrogen in both reproductive and non-reproductive organs of the male has been unclear, despite suggestive evidence over the years that it may be important. The recent development of knockout mice lacking aromatase, estrogen receptor-a (ERa) or ERb (Lubahn * Corresponding author. Tel.: + 1-217-3336825; fax: +1-2172441652. E-mail address: [email protected] (P.S. Cooke).
et al., 1993; Fisher et al., 1998; Krege et al., 1998; Couse et al., 1999) and the identification of humans lacking aromatase or ERa (Smith et al., 1994; Grumbach and Auchus, 1999) have allowed novel insights into the role of estrogen, and ERa and b. Some phenotypic changes were predictable, such as uterine and vaginal hypoplasia in aERKO mice (Lubahn et al., 1993). Other phenotypic changes in humans or mice lacking ER were unexpected and indicated previously unknown estrogen effects. For example, lack of epiphysial plate closure in men without ERa or aromatase (Smith et al., 1994; Morishima et al., 1995; Carani et al., 1997) revealed that estrogen played an obligatory role in this process in males. The partial sex reversal in ovaries of female mice lacking both ERa and ERb (Couse et al., 1999) is perhaps the clearest example of unanticipated effects resulting from absence of ER, and how analysis of animals lacking ER can advance understanding of ERa and ERb function in different tissues. We have recently observed that ERa knockout (aERKO) males have pronounced increases in white
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adipose tissue (WAT). This increase in WAT in aERKO males (Heine et al., 2000), which is the focus of the present review, was unexpected based on prevailing concepts of the role of ER in the male. Like the effects described above, this work may lead to elucidation of a critical role of ERa in the male not known earlier. In this review, we examine earlier work suggesting a role for ER in adipose tissue development, then discuss our data showing that absence of ERa leads to increased WAT in male mice. Initial results directed toward establishing the mechanism of this effect are also presented.
2. Clinical importance of obesity Obesity is a major human health problem, the incidence of which is increasing and reaching epidemic proportions in some Western countries. For example, obesity in Americans has risen from 12.8% in 1962 to 22.5% in 1998, and 55% of the population is considered overweight (reviewed in Taubes, 1998). Of special concern is the increase of obesity in children in developed countries (Flegal, 1999), since childhood obesity predisposes a person to adult obesity. Obesity is associated with increased Type II diabetes, hypertension, sleep apnea, heart disease and certain cancers (Must et al., 1999). People with a body-mass index (BMI in weight in kilograms divided by the square of the height in meters) of 30 or more have up to a 100% increase in mortality compared with those with a body-mass index below 25 (Calle et al., 1999), and obesity is responsible for 300 000 deaths per year in the US (Allison et al., 1999). Therefore, factors regulating development and function of WAT are a major public health concern.
3. Regulation of adipose tissue in food animals Regulation of WAT in food animals is also important because of concerns over high fat consumption in human diets, which has adverse health effects. Over 90% of US heifers and steers receive growth-promoting implants containing estrogenic and androgenic compounds. Animals treated with estrogens such as estradiol 17-b (E2) or the synthetic estrogen zeranol have decreased fat and increased muscle (Moran et al., 1991). Due to concerns about effects on humans resulting from hormonal residues in meat, the European Union presently bans importation of US beef from animals treated with hormonal implants. The mechanism by which estrogen implants decrease fat is unclear and these treatments were developed empirically. Clearly, it is desirable to raise leaner food animals due to beneficial effects lower fat levels have for consumers
of the meat, but there is also a pressing need to better understand how these hormonal implants work.
4. Adipose tissue differentiation and development There are two types of adipose tissue: brown and white. Brown adipose tissue (BAT) is primarily responsible for heat regulation; it occurs in limited body sites and is more extensive in newborns and hibernating animals. WAT is found throughout the body and functions as a storage site for energy reserves. WAT consists of adipocytes, stromal cells and blood vessels. Each adipocyte contains one lipid inclusion body, which is variable in size and composed of fatty acids. Pre-adipocytes originate from mesenchymal tissue and undergo initial proliferation; they do not possess morphological features or the unique proteins typical of mature adipocytes, nor store significant fat (Cornelius et al., 1994). As they differentiate into adipocytes, their mitosis stops and they begin to show typical adipocyte morphology and fat storage. Once cells differentiate into mature adipocytes, increases in WAT usually result from hypertrophy of existing adipocytes, rather than adipocyte hyperplasia. Differentiation of adipocytes typically occurs by puberty, though some precursor cells are maintained throughout life (Van and Roncari, 1987; Hauner et al., 1989).
5. The effects of estrogens on adipose tissue in females Evidence from both humans and laboratory animals suggests that estrogens are important regulators of WAT in females. Ovariectomy of rodents increases WAT, and estrogen treatment reverses this (reviewed in Wade et al., 1985). Similar data in humans indicate that post-menopausal women have increased WAT compared with pre-menopausal women, and estrogen replacement results in decreased WAT (Tchernof et al., 1998). Female WAT expresses both ERa and the recently discovered second estrogen receptor, ERb (Wade and Gray, 1978; Pederson et al., 1991, 1992; Crandall et al., 1998). The relative role of each of these receptors is unknown. Likewise, the mechanism by which estrogen regulates WAT is unclear, although both human and animal studies suggest estrogen may affect glucose tolerance and insulin resistance in females by direct actions on adipocytes to increase insulin receptor expression (Bailey and Matty, 1972; Bailey and AhmedSorour, 1980; Pederson et al., 1992; Lindheim et al., 1994; Tchernof et al., 1998). Estrogen also appears to act on WAT through alteration of leptin production (Machinal et al., 1999) and hypothalamic leptin receptor expression (Bennett et al., 1998).
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6. Does estrogen have effects on male adipose tissue? ER in reproductive and non-reproductive organs of developing and adult males has been extensively described (Stumpf and Sar, 1976). The recent discovery of ERb (Kuiper et al., 1996) and subsequent work showing its wide distribution in males indicated that ERb may play a role in males. However, definitive data establishing a role for ER in normal male development has been elusive, despite extensive reports showing deleterious effects of fetal and neonatal estrogen on development of reproductive and non-reproductive organs. Production and circulating levels of estrogen in men and males of other species is typically lower than in females (Kelch et al., 1972). Was this low circulating level of estrogen normally sufficient to activate ER and produce effects in adipose and other male tissues? Or were circulating levels so low that E2/ERa signaling was not activated and played no significant role in males despite the extensive distribution of ER and known deleterious effects of exogenous estrogens during early development? More specifically for this review, does ER have a role in male WAT? As in females, male adipose tissue expresses ER (Pederson et al., 1991), and ER is also expressed in other male organs associated with satiety and feeding, such as hypothalamus and pituitary. Estrogen treatment decreased adipocyte size in male rats (Pederson et al., 1991) and estrogen and anti-androgen treatment of male-to-female transsexuals altered adipocyte size and body fat deposition (Elbers et al., 1999). But in both cases it was unclear if estrogen effects were direct or also reflected changes in testosterone levels or action. In addition, estrogen had effects on proliferation and differentiation of preadipocytes from female rats, but did not have a similar effect in male preadipocytes. Dieudonne et al., 2000). Therefore, it was unclear if estrogen binding to ERa and/or ERb normally plays any role in regulating the amount of WAT in males, and if absence of one or both ERs would result in phenotypic effects in male WAT.
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creases in parametrial, perirenal and inguinal WAT of 103 54 and 96%, respectively, compared with WT females of the same age. In contrast, BAT weights were not different in aERKO and WT females. These results indicated that ERa is critical for regulating deposition of WAT in females, confirming earlier literature, suggesting estrogen regulates WAT in females. Unexpectedly, WAT in aERKO males also showed dramatic increases compared with WT controls (Fig. 1). Increased WAT was seen in aERKO males as early as 30 days of age, when clear increases in epididymal fat pad weight were detected. The increased WAT deposition became more pronounced with age, and in aERKO males at 9– 12 months of age weights of epididymal, perirenal and inguinal WAT depots were all more than 100% greater than WT controls (Heine et al., 2000). As in females, BAT weights were similar in aERKO and WT males. The increased WAT in aERKO males did not reflect a general increase in body weight and organ size, since body and kidney weight increases in aERKO males showed only small increases that were far less than those in WAT. The increased WAT reflected both increased adipocyte hypertrophy and hyperplasia (Heine et al., 2000). Areas of epididymal and perirenal adipocytes from 180 day old aERKO males were increased approximately 20% compared with WT (Figs. 1 and 2). Inguinal adipocytes also showed a trend toward an increase, but this did not reach statistical significance. In addition, adipocyte number was 80–170% greater in various fat pads of 180 day old aERKO males compared with WT (Heine et al., 2000). Despite large increases in adipose tissue, male aERKO mice do not show increased food consumption
7. Adipose tissue is increased in aERKO male mice The development of aERKO mice provided a powerful tool to examine the role of E2/ERa signaling in development and function of various organs and tissues (Lubahn et al., 1993). We have recently used male and female aERKO mice to determine if absence of ERa expression produces phenotypic changes in WAT and BAT in either sex (Heine et al., 2000). Our results indicate that ERa absence results in marked increases in WAT in 90 day old aERKO females compared with wild-type (WT) controls (Heine et al., 2000); the aERKO females had significant in-
Fig. 1. Relative weight of the epididymal fat pad and numbers and sizes of epididymal adipocytes in 180 day old aERKO males and WT mice. Data are expressed as percent of WT controls. Epididymal WAT weight, adipocyte number and size were all significantly (PB 0.05) greater than the age-matched control values, and n ] 5 for all groups.
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Fig. 2. Histological sections of epididymal WAT from 180 day old WT and ERa knockout (KO) males, showing the increased adipocyte size seen in aERKO males.
(Heine et al., 2000). However, indirect calorimetry measurements indicated that energy expenditure was significantly reduced in aERKO males (Heine et al., 2000). This reduced energy expenditure in the face of continued normal food consumption may be a critical aspect of the mechanism by which obesity develops in these animals. It has been unclear if estrogen plays any role in normal male WAT development, as discussed above. Our data indicating that absence of ERa in males results in marked increases in WAT during postnatal life indicates that E2/ERa signaling is also important for normal development and function of WAT in males. These results are the first to indicate that overall WAT amounts, as well as adipocyte number and size are, dramatically altered in the absence of ERa. This appears to be a critical effect based on the magnitude of the increases in WAT in aERKO males. The observed WAT effects in aERKO mice may not result solely from lack of ERa. Changes in ERa/ERb ratio could be important, or absence of heterodimer formation by ERa and ERb (Cowley et al., 1997) or other putative estrogen receptors (Das et al., 1997) in aERKO mice could play a role in development of the obese phenotype. Similarly, the several-fold increase in circulating E2 levels in aERKO females (Couse and Korach, 1999) could produce increased signaling through ERb or other estrogen receptors (Das et al., 1997) and potential effects on WAT, though this would not appear to be a factor in the increased WAT in aERKO males, since E2 levels are similar in WT and aERKO males (Couse and Korach, 1999). Testosterone levels are increased in aERKO mice (Couse and Korach, 1999), and testosterone levels are also elevated in other situations associated with estrogen deficiency and increased fat deposition, such as in women who are post-menopausal or suffering from polycystic ovary syndrome (Tchernof et al., 1998;
Legro, 2000). There is an association between hyperandrogenism and hyperinsulinism (Diamond et al., 1988), and increases in insulin levels could contribute to obesity. However, the overall effects of testosterone are lipolytic, as shown by testosterone treatment of hypogonadal men and female-to-male transsexuals (Elbers et al., 1999; Wang et al., 2000), suggesting that the increase in testosterone in aERKO males is not a major factor in the obese phenotype observed in these animals. Similarly, corticosterone levels are similar in juvenile and adult aERKO males (Yellayi et al., 2000), suggesting that this hormone is not involved in the obese phenotype of aERKO males. However, changes in insulin levels, as discussed below, or other unknown secondary hormonal effects could be a factor in the development of the obese phenotype in aERKO mice. The recent development of mice lacking either ERb or both ERa and ERb (Krege et al., 1998; Couse et al., 1999) should provide useful tools to definitively establish the role of ERb and ERa/ERb interactions in regulating WAT. Female aERKO mice show moderate insulin resistance and impaired glucose tolerance, a phenomenon seen to a lesser degree in aERKO males also (Heine et al., 2000). These findings are consistent with earlier reports that ovariectomy results in impaired glucose tolerance in mice, and that estradiol replacement reversed these effects (Ahmed-Sorour and Bailey, 1980; Bailey and Ahmed-Sorour, 1980) and the reported beneficial effects of estrogen replacement on glucose tolerance in post-menopausal women (Lindheim et al., 1994; Tchernof et al., 1998); the altered glucose tolerance and insulin resistance in the aERKO mice could be a major factor in the etiology of the increased WAT in both sexes. The insulin resistance and impaired glucose tolerance, as well as the increase in male WAT, in mice lacking ERa was similar to those reported for the aromatase knockout (ArKO) mouse (Jones et al.,
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2000), and the insulin resistance and glucose tolerance findings in aERKO and ArKO mice parallel observations in humans lacking ERa or aromatase (Smith et al., 1994; Morishima et al., 1995; Grumbach and Auchus, 1999), emphasizing the utility of the mouse models for these human conditions.
8. Potential clinical applications Obesity is a chronic disease whose etiology is poorly understood. Obvious treatment modalities such as diet and exercise frequently produce modest and transient benefits, especially in cases of severe obesity. Our emerging understanding of the role of ERa in WAT may offer new approaches to treatment of this disease, which could be combined with present and future treatment options. There has been intense recent interest in the potential health benefits of phytoestrogens, estrogenic compounds found in soybeans and other plants. These compounds may also be beneficial in regulating WAT in humans. People in Far Eastern countries have lower incidences of breast and prostate cancer, heart disease, osteoporosis and menopausal symptoms than residents of Western countries (Adlercreutz et al., 1995; Adlercreutz and Mazur, 1997). The incidence of obesity in the Far East is also markedly less than in Western countries; for example, adult obesity in Thailand is less than 4% (Deurenberg et al., 1998; Taubes, 1998). The cause of these differences in obesity has not been definitively established, but may reflect differences in diet (Far Eastern diets typically contain less animal protein and less fat than Western diets), genetics and other factors. Phytoestrogens may play a major role in the health benefits of soy (Patisaul and Whitten, 1999). The primary phytoestrogen in soy is the isoflavone genistein (reviewed in Patisaul and Whitten, 1999), and other isoflavones such as daidzein are also found in high concentrations in soy (Franke et al., 1995). Genistein and some other isoflavones act as weak estrogens (Clarke et al., 1996; Kuiper et al., 1997). Genistein binds ERa, although genistein has typically been reported to have 1/1000 or less of the potency of E2 (Kuiper et al., 1998). Despite the low estrogenic potency of genistein, it is potentially capable of exerting physiological effects due to its high concentrations in some human diets. Asian diets that are high in soy may result in daily consumption of 1 mg of isoflavones per kg body weight, and plasma concentrations of 1 mM have been reported in Japanese subjects consuming a high soy diet (Adlercreutz et al., 1995; Adlercreutz and Mazur, 1997). Earlier studies in menopausal women have established that estrogen replacement decreases WAT (Tch-
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ernof et al., 1998), and our present data indicates this effect may involve ERa. Our data in aERKO males suggests that a similar effect could occur in men (Smith et al., 1994). An intriguing question is whether genistein and other phytoestrogens could modulate the amount of WAT in humans. Could the ability of genistein and other phytoestrogens to signal through ERa be one contributor to the lower obesity levels seen in populations that consume high amounts of these compounds? Nogowski et al. (1998) have shown that genistein can act on adipocytes to strongly depress both their basal and insulin-induced lipid synthesis and that genistein can have effects on triglyceride and free fatty acid concentrations in the serum. Similarly, we have recently shown that administration of genistein to mice inhibits the normal post-ovariectomy increase in adipose tissue (Naaz and Cooke; unpublished), suggesting that genistein in food could be a negative regulator of the amount of adipose tissue. These findings clearly do not establish that genistein or related compounds could decrease WAT levels in humans, but do suggest that this needs to be addressed experimentally.
9. A new paradigm for the role of estrogen in the male Estrogens are critical for female reproduction, but their role in male reproductive and other organs was unclear despite wide distribution of ER in males and measurable levels of circulating estrogen in males. Recent insights from both humans and mice lacking ERa or aromatase have necessitated a reevaluation of this area and have revealed important actions for estrogen in the male. Estrogen is critical for fluid reabsorption in efferent ductules of male mice (Hess et al., 1997), and plays a role in male germ cell development in ArKO mice (Robertson et al., 1999). Epiphysial plates in men lacking ERa or aromatase do not close, revealing a role for estrogens in this important process in males (Smith et al., 1994; Morishima et al., 1995; Carani et al., 1997; Grumbach and Auchus, 1999). Low bone mineral density and osteoporosis have also been reported in men lacking either ERa or aromatase, suggesting that wellknown estrogen effects on bones in females also occur in males (Smith et al., 1994; Morishima et al., 1995; Carani et al., 1997). These men also have insulin resistance and altered lipid profiles, further indicating a normal role for estrogen in males (reviewed in Grumbach and Auchus, 1999). In addition, ERa deficiency in a human male has been reported to be accompanied by early onset of cardiovascular disease (Sudhir et al., 1997a) and dysfunction in vasodilation (Sudhir et al., 1997b), and male aERKO mice are more adversely affected by cardiac ischemia/reperfusion injury than males expressing ERa (Zhai et al., 2000). Aromatase deficiency and the consequent loss of endogenous estro-
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gen production does not appear to alter gender identity or sexual orientation, though it may have a role in regulating the amount of sexual activity (Carani et al., 1999). ERa is also critical for maintaining the size of the male thymus in mice during postnatal life (Staples et al., 1999; Yellayi et al., 2000). Thus, the emerging consensus is that ERa plays critical roles in the development and adult function of many male organs and tissues, and it is likely that additional roles for ERa will be elucidated in the near future.
10. Summary and conclusions In summary, our results are the first to show that lack of ERa produces large increases in WAT in male mice. The unexpected large stimulatory effect on WAT deposition in aERKO males emphasizes that ERa plays a previously unappreciated role in male WAT development, and similar recent results in other organs indicate that estrogen is a key regulator of both reproductive and non-reproductive organs in the male. The aERKO mouse may be useful for study of factors regulating obesity and the role of estrogen and ERa in this process, and this work may have relevance both for human obesity and regulation of fat in food animals.
Acknowledgements This work was supported by NIH grants AG 15500 (to P.S.C.) and ES 08272 (to D.B.L.), and Animal Health and Disease Research Funds from the University of Illinois (to P.S.C. and P.A.H.). The authors thank Megan Borgstrom and Sri Yellayi for technical and genotyping assistance, respectively, David Schaeffer for statistical analysis and Martha Hufford for providing animals.
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The role of estrogen and estrogen receptor-a in male adipose tissue Paul S. Cooke a,*, Patricia A. Heine a, Julia A. Taylor b, Dennis B. Lubahn b a
Department of Veterinary Biosciences, Uni6ersity of Illinois, 2001 S. Lincoln A6enue, Urbana, IL, 61802 USA b Department of Biochemistry and Child Health, Uni6ersity of Missouri, Columbia, MO, 65211 USA
Abstract Males and females both express estrogen receptor (ER) in white adipose tissue (WAT), and estrogens appear to play an important role in regulating WAT in females. However, the role of ER in male WAT was unclear. In this review, we describe our work, which used wild type (WT) and ERa-knockout (aERKO) male and female mice to determine the role of ERa in regulating WAT and brown adipose tissue (BAT). There were progressive increases in WAT with advancing age in aERKO compared with WT males; weights of various WAT depots in aERKO males were increased by more than 100% compared with WT controls during adulthood. Conversely, BAT weight was similar in aERKO and WT males at all ages. Adipocyte areas and numbers were also increased in WAT from aERKO compared with WT males. Compared with WT controls, aERKO females also had increases in WAT. The aERKO mice also had insulin resistance and impaired glucose tolerance, similar to humans lacking ERa or aromatase. The obesity in aERKO males appeared to involve decreased energy expenditure rather than hyperphagia. In summary, ERa absence causes adipocyte hyperplasia and hypertrophy in WAT, but not BAT, and is accompanied by insulin resistance and glucose intolerance in both males and females. These results are the first evidence that the estrogen/ERa signaling system is critical in female and male WAT deposition, and may have clinical implications. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Adipocyte; Estrogen receptor beta; Insulin resistance; Glucose intolerance; Obesity
1. Introduction Androgens are the primary regulator of male reproductive tract development, growth and function. Sex steroids such as testosterone also have effects on muscle and adipose tissue development. For example, lean body mass is increased by testosterone, and testosterone is lipolytic, as shown by increases in lean body mass and decreased fat in hypogonadal men treated with a testosterone replacement gel (Wang et al., 2000). Similarly, testosterone treatment in female-to-male transsexuals resulted in decreased subcutaneous fat and adipocyte size (Elbers et al., 1999). In contrast, the role of estrogen in both reproductive and non-reproductive organs of the male has been unclear, despite suggestive evidence over the years that it may be important. The recent development of knockout mice lacking aromatase, estrogen receptor-a (ERa) or ERb (Lubahn * Corresponding author. Tel.: + 1-217-3336825; fax: +1-2172441652. E-mail address: [email protected] (P.S. Cooke).
et al., 1993; Fisher et al., 1998; Krege et al., 1998; Couse et al., 1999) and the identification of humans lacking aromatase or ERa (Smith et al., 1994; Grumbach and Auchus, 1999) have allowed novel insights into the role of estrogen, and ERa and b. Some phenotypic changes were predictable, such as uterine and vaginal hypoplasia in aERKO mice (Lubahn et al., 1993). Other phenotypic changes in humans or mice lacking ER were unexpected and indicated previously unknown estrogen effects. For example, lack of epiphysial plate closure in men without ERa or aromatase (Smith et al., 1994; Morishima et al., 1995; Carani et al., 1997) revealed that estrogen played an obligatory role in this process in males. The partial sex reversal in ovaries of female mice lacking both ERa and ERb (Couse et al., 1999) is perhaps the clearest example of unanticipated effects resulting from absence of ER, and how analysis of animals lacking ER can advance understanding of ERa and ERb function in different tissues. We have recently observed that ERa knockout (aERKO) males have pronounced increases in white
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adipose tissue (WAT). This increase in WAT in aERKO males (Heine et al., 2000), which is the focus of the present review, was unexpected based on prevailing concepts of the role of ER in the male. Like the effects described above, this work may lead to elucidation of a critical role of ERa in the male not known earlier. In this review, we examine earlier work suggesting a role for ER in adipose tissue development, then discuss our data showing that absence of ERa leads to increased WAT in male mice. Initial results directed toward establishing the mechanism of this effect are also presented.
2. Clinical importance of obesity Obesity is a major human health problem, the incidence of which is increasing and reaching epidemic proportions in some Western countries. For example, obesity in Americans has risen from 12.8% in 1962 to 22.5% in 1998, and 55% of the population is considered overweight (reviewed in Taubes, 1998). Of special concern is the increase of obesity in children in developed countries (Flegal, 1999), since childhood obesity predisposes a person to adult obesity. Obesity is associated with increased Type II diabetes, hypertension, sleep apnea, heart disease and certain cancers (Must et al., 1999). People with a body-mass index (BMI in weight in kilograms divided by the square of the height in meters) of 30 or more have up to a 100% increase in mortality compared with those with a body-mass index below 25 (Calle et al., 1999), and obesity is responsible for 300 000 deaths per year in the US (Allison et al., 1999). Therefore, factors regulating development and function of WAT are a major public health concern.
3. Regulation of adipose tissue in food animals Regulation of WAT in food animals is also important because of concerns over high fat consumption in human diets, which has adverse health effects. Over 90% of US heifers and steers receive growth-promoting implants containing estrogenic and androgenic compounds. Animals treated with estrogens such as estradiol 17-b (E2) or the synthetic estrogen zeranol have decreased fat and increased muscle (Moran et al., 1991). Due to concerns about effects on humans resulting from hormonal residues in meat, the European Union presently bans importation of US beef from animals treated with hormonal implants. The mechanism by which estrogen implants decrease fat is unclear and these treatments were developed empirically. Clearly, it is desirable to raise leaner food animals due to beneficial effects lower fat levels have for consumers
of the meat, but there is also a pressing need to better understand how these hormonal implants work.
4. Adipose tissue differentiation and development There are two types of adipose tissue: brown and white. Brown adipose tissue (BAT) is primarily responsible for heat regulation; it occurs in limited body sites and is more extensive in newborns and hibernating animals. WAT is found throughout the body and functions as a storage site for energy reserves. WAT consists of adipocytes, stromal cells and blood vessels. Each adipocyte contains one lipid inclusion body, which is variable in size and composed of fatty acids. Pre-adipocytes originate from mesenchymal tissue and undergo initial proliferation; they do not possess morphological features or the unique proteins typical of mature adipocytes, nor store significant fat (Cornelius et al., 1994). As they differentiate into adipocytes, their mitosis stops and they begin to show typical adipocyte morphology and fat storage. Once cells differentiate into mature adipocytes, increases in WAT usually result from hypertrophy of existing adipocytes, rather than adipocyte hyperplasia. Differentiation of adipocytes typically occurs by puberty, though some precursor cells are maintained throughout life (Van and Roncari, 1987; Hauner et al., 1989).
5. The effects of estrogens on adipose tissue in females Evidence from both humans and laboratory animals suggests that estrogens are important regulators of WAT in females. Ovariectomy of rodents increases WAT, and estrogen treatment reverses this (reviewed in Wade et al., 1985). Similar data in humans indicate that post-menopausal women have increased WAT compared with pre-menopausal women, and estrogen replacement results in decreased WAT (Tchernof et al., 1998). Female WAT expresses both ERa and the recently discovered second estrogen receptor, ERb (Wade and Gray, 1978; Pederson et al., 1991, 1992; Crandall et al., 1998). The relative role of each of these receptors is unknown. Likewise, the mechanism by which estrogen regulates WAT is unclear, although both human and animal studies suggest estrogen may affect glucose tolerance and insulin resistance in females by direct actions on adipocytes to increase insulin receptor expression (Bailey and Matty, 1972; Bailey and AhmedSorour, 1980; Pederson et al., 1992; Lindheim et al., 1994; Tchernof et al., 1998). Estrogen also appears to act on WAT through alteration of leptin production (Machinal et al., 1999) and hypothalamic leptin receptor expression (Bennett et al., 1998).
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6. Does estrogen have effects on male adipose tissue? ER in reproductive and non-reproductive organs of developing and adult males has been extensively described (Stumpf and Sar, 1976). The recent discovery of ERb (Kuiper et al., 1996) and subsequent work showing its wide distribution in males indicated that ERb may play a role in males. However, definitive data establishing a role for ER in normal male development has been elusive, despite extensive reports showing deleterious effects of fetal and neonatal estrogen on development of reproductive and non-reproductive organs. Production and circulating levels of estrogen in men and males of other species is typically lower than in females (Kelch et al., 1972). Was this low circulating level of estrogen normally sufficient to activate ER and produce effects in adipose and other male tissues? Or were circulating levels so low that E2/ERa signaling was not activated and played no significant role in males despite the extensive distribution of ER and known deleterious effects of exogenous estrogens during early development? More specifically for this review, does ER have a role in male WAT? As in females, male adipose tissue expresses ER (Pederson et al., 1991), and ER is also expressed in other male organs associated with satiety and feeding, such as hypothalamus and pituitary. Estrogen treatment decreased adipocyte size in male rats (Pederson et al., 1991) and estrogen and anti-androgen treatment of male-to-female transsexuals altered adipocyte size and body fat deposition (Elbers et al., 1999). But in both cases it was unclear if estrogen effects were direct or also reflected changes in testosterone levels or action. In addition, estrogen had effects on proliferation and differentiation of preadipocytes from female rats, but did not have a similar effect in male preadipocytes. Dieudonne et al., 2000). Therefore, it was unclear if estrogen binding to ERa and/or ERb normally plays any role in regulating the amount of WAT in males, and if absence of one or both ERs would result in phenotypic effects in male WAT.
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creases in parametrial, perirenal and inguinal WAT of 103 54 and 96%, respectively, compared with WT females of the same age. In contrast, BAT weights were not different in aERKO and WT females. These results indicated that ERa is critical for regulating deposition of WAT in females, confirming earlier literature, suggesting estrogen regulates WAT in females. Unexpectedly, WAT in aERKO males also showed dramatic increases compared with WT controls (Fig. 1). Increased WAT was seen in aERKO males as early as 30 days of age, when clear increases in epididymal fat pad weight were detected. The increased WAT deposition became more pronounced with age, and in aERKO males at 9– 12 months of age weights of epididymal, perirenal and inguinal WAT depots were all more than 100% greater than WT controls (Heine et al., 2000). As in females, BAT weights were similar in aERKO and WT males. The increased WAT in aERKO males did not reflect a general increase in body weight and organ size, since body and kidney weight increases in aERKO males showed only small increases that were far less than those in WAT. The increased WAT reflected both increased adipocyte hypertrophy and hyperplasia (Heine et al., 2000). Areas of epididymal and perirenal adipocytes from 180 day old aERKO males were increased approximately 20% compared with WT (Figs. 1 and 2). Inguinal adipocytes also showed a trend toward an increase, but this did not reach statistical significance. In addition, adipocyte number was 80–170% greater in various fat pads of 180 day old aERKO males compared with WT (Heine et al., 2000). Despite large increases in adipose tissue, male aERKO mice do not show increased food consumption
7. Adipose tissue is increased in aERKO male mice The development of aERKO mice provided a powerful tool to examine the role of E2/ERa signaling in development and function of various organs and tissues (Lubahn et al., 1993). We have recently used male and female aERKO mice to determine if absence of ERa expression produces phenotypic changes in WAT and BAT in either sex (Heine et al., 2000). Our results indicate that ERa absence results in marked increases in WAT in 90 day old aERKO females compared with wild-type (WT) controls (Heine et al., 2000); the aERKO females had significant in-
Fig. 1. Relative weight of the epididymal fat pad and numbers and sizes of epididymal adipocytes in 180 day old aERKO males and WT mice. Data are expressed as percent of WT controls. Epididymal WAT weight, adipocyte number and size were all significantly (PB 0.05) greater than the age-matched control values, and n ] 5 for all groups.
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Fig. 2. Histological sections of epididymal WAT from 180 day old WT and ERa knockout (KO) males, showing the increased adipocyte size seen in aERKO males.
(Heine et al., 2000). However, indirect calorimetry measurements indicated that energy expenditure was significantly reduced in aERKO males (Heine et al., 2000). This reduced energy expenditure in the face of continued normal food consumption may be a critical aspect of the mechanism by which obesity develops in these animals. It has been unclear if estrogen plays any role in normal male WAT development, as discussed above. Our data indicating that absence of ERa in males results in marked increases in WAT during postnatal life indicates that E2/ERa signaling is also important for normal development and function of WAT in males. These results are the first to indicate that overall WAT amounts, as well as adipocyte number and size are, dramatically altered in the absence of ERa. This appears to be a critical effect based on the magnitude of the increases in WAT in aERKO males. The observed WAT effects in aERKO mice may not result solely from lack of ERa. Changes in ERa/ERb ratio could be important, or absence of heterodimer formation by ERa and ERb (Cowley et al., 1997) or other putative estrogen receptors (Das et al., 1997) in aERKO mice could play a role in development of the obese phenotype. Similarly, the several-fold increase in circulating E2 levels in aERKO females (Couse and Korach, 1999) could produce increased signaling through ERb or other estrogen receptors (Das et al., 1997) and potential effects on WAT, though this would not appear to be a factor in the increased WAT in aERKO males, since E2 levels are similar in WT and aERKO males (Couse and Korach, 1999). Testosterone levels are increased in aERKO mice (Couse and Korach, 1999), and testosterone levels are also elevated in other situations associated with estrogen deficiency and increased fat deposition, such as in women who are post-menopausal or suffering from polycystic ovary syndrome (Tchernof et al., 1998;
Legro, 2000). There is an association between hyperandrogenism and hyperinsulinism (Diamond et al., 1988), and increases in insulin levels could contribute to obesity. However, the overall effects of testosterone are lipolytic, as shown by testosterone treatment of hypogonadal men and female-to-male transsexuals (Elbers et al., 1999; Wang et al., 2000), suggesting that the increase in testosterone in aERKO males is not a major factor in the obese phenotype observed in these animals. Similarly, corticosterone levels are similar in juvenile and adult aERKO males (Yellayi et al., 2000), suggesting that this hormone is not involved in the obese phenotype of aERKO males. However, changes in insulin levels, as discussed below, or other unknown secondary hormonal effects could be a factor in the development of the obese phenotype in aERKO mice. The recent development of mice lacking either ERb or both ERa and ERb (Krege et al., 1998; Couse et al., 1999) should provide useful tools to definitively establish the role of ERb and ERa/ERb interactions in regulating WAT. Female aERKO mice show moderate insulin resistance and impaired glucose tolerance, a phenomenon seen to a lesser degree in aERKO males also (Heine et al., 2000). These findings are consistent with earlier reports that ovariectomy results in impaired glucose tolerance in mice, and that estradiol replacement reversed these effects (Ahmed-Sorour and Bailey, 1980; Bailey and Ahmed-Sorour, 1980) and the reported beneficial effects of estrogen replacement on glucose tolerance in post-menopausal women (Lindheim et al., 1994; Tchernof et al., 1998); the altered glucose tolerance and insulin resistance in the aERKO mice could be a major factor in the etiology of the increased WAT in both sexes. The insulin resistance and impaired glucose tolerance, as well as the increase in male WAT, in mice lacking ERa was similar to those reported for the aromatase knockout (ArKO) mouse (Jones et al.,
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2000), and the insulin resistance and glucose tolerance findings in aERKO and ArKO mice parallel observations in humans lacking ERa or aromatase (Smith et al., 1994; Morishima et al., 1995; Grumbach and Auchus, 1999), emphasizing the utility of the mouse models for these human conditions.
8. Potential clinical applications Obesity is a chronic disease whose etiology is poorly understood. Obvious treatment modalities such as diet and exercise frequently produce modest and transient benefits, especially in cases of severe obesity. Our emerging understanding of the role of ERa in WAT may offer new approaches to treatment of this disease, which could be combined with present and future treatment options. There has been intense recent interest in the potential health benefits of phytoestrogens, estrogenic compounds found in soybeans and other plants. These compounds may also be beneficial in regulating WAT in humans. People in Far Eastern countries have lower incidences of breast and prostate cancer, heart disease, osteoporosis and menopausal symptoms than residents of Western countries (Adlercreutz et al., 1995; Adlercreutz and Mazur, 1997). The incidence of obesity in the Far East is also markedly less than in Western countries; for example, adult obesity in Thailand is less than 4% (Deurenberg et al., 1998; Taubes, 1998). The cause of these differences in obesity has not been definitively established, but may reflect differences in diet (Far Eastern diets typically contain less animal protein and less fat than Western diets), genetics and other factors. Phytoestrogens may play a major role in the health benefits of soy (Patisaul and Whitten, 1999). The primary phytoestrogen in soy is the isoflavone genistein (reviewed in Patisaul and Whitten, 1999), and other isoflavones such as daidzein are also found in high concentrations in soy (Franke et al., 1995). Genistein and some other isoflavones act as weak estrogens (Clarke et al., 1996; Kuiper et al., 1997). Genistein binds ERa, although genistein has typically been reported to have 1/1000 or less of the potency of E2 (Kuiper et al., 1998). Despite the low estrogenic potency of genistein, it is potentially capable of exerting physiological effects due to its high concentrations in some human diets. Asian diets that are high in soy may result in daily consumption of 1 mg of isoflavones per kg body weight, and plasma concentrations of 1 mM have been reported in Japanese subjects consuming a high soy diet (Adlercreutz et al., 1995; Adlercreutz and Mazur, 1997). Earlier studies in menopausal women have established that estrogen replacement decreases WAT (Tch-
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ernof et al., 1998), and our present data indicates this effect may involve ERa. Our data in aERKO males suggests that a similar effect could occur in men (Smith et al., 1994). An intriguing question is whether genistein and other phytoestrogens could modulate the amount of WAT in humans. Could the ability of genistein and other phytoestrogens to signal through ERa be one contributor to the lower obesity levels seen in populations that consume high amounts of these compounds? Nogowski et al. (1998) have shown that genistein can act on adipocytes to strongly depress both their basal and insulin-induced lipid synthesis and that genistein can have effects on triglyceride and free fatty acid concentrations in the serum. Similarly, we have recently shown that administration of genistein to mice inhibits the normal post-ovariectomy increase in adipose tissue (Naaz and Cooke; unpublished), suggesting that genistein in food could be a negative regulator of the amount of adipose tissue. These findings clearly do not establish that genistein or related compounds could decrease WAT levels in humans, but do suggest that this needs to be addressed experimentally.
9. A new paradigm for the role of estrogen in the male Estrogens are critical for female reproduction, but their role in male reproductive and other organs was unclear despite wide distribution of ER in males and measurable levels of circulating estrogen in males. Recent insights from both humans and mice lacking ERa or aromatase have necessitated a reevaluation of this area and have revealed important actions for estrogen in the male. Estrogen is critical for fluid reabsorption in efferent ductules of male mice (Hess et al., 1997), and plays a role in male germ cell development in ArKO mice (Robertson et al., 1999). Epiphysial plates in men lacking ERa or aromatase do not close, revealing a role for estrogens in this important process in males (Smith et al., 1994; Morishima et al., 1995; Carani et al., 1997; Grumbach and Auchus, 1999). Low bone mineral density and osteoporosis have also been reported in men lacking either ERa or aromatase, suggesting that wellknown estrogen effects on bones in females also occur in males (Smith et al., 1994; Morishima et al., 1995; Carani et al., 1997). These men also have insulin resistance and altered lipid profiles, further indicating a normal role for estrogen in males (reviewed in Grumbach and Auchus, 1999). In addition, ERa deficiency in a human male has been reported to be accompanied by early onset of cardiovascular disease (Sudhir et al., 1997a) and dysfunction in vasodilation (Sudhir et al., 1997b), and male aERKO mice are more adversely affected by cardiac ischemia/reperfusion injury than males expressing ERa (Zhai et al., 2000). Aromatase deficiency and the consequent loss of endogenous estro-
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gen production does not appear to alter gender identity or sexual orientation, though it may have a role in regulating the amount of sexual activity (Carani et al., 1999). ERa is also critical for maintaining the size of the male thymus in mice during postnatal life (Staples et al., 1999; Yellayi et al., 2000). Thus, the emerging consensus is that ERa plays critical roles in the development and adult function of many male organs and tissues, and it is likely that additional roles for ERa will be elucidated in the near future.
10. Summary and conclusions In summary, our results are the first to show that lack of ERa produces large increases in WAT in male mice. The unexpected large stimulatory effect on WAT deposition in aERKO males emphasizes that ERa plays a previously unappreciated role in male WAT development, and similar recent results in other organs indicate that estrogen is a key regulator of both reproductive and non-reproductive organs in the male. The aERKO mouse may be useful for study of factors regulating obesity and the role of estrogen and ERa in this process, and this work may have relevance both for human obesity and regulation of fat in food animals.
Acknowledgements This work was supported by NIH grants AG 15500 (to P.S.C.) and ES 08272 (to D.B.L.), and Animal Health and Disease Research Funds from the University of Illinois (to P.S.C. and P.A.H.). The authors thank Megan Borgstrom and Sri Yellayi for technical and genotyping assistance, respectively, David Schaeffer for statistical analysis and Martha Hufford for providing animals.
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