Estrogen receptor alpha is necessary in thymic development and estradiol-induced thymic alterations.

Estrogens affect the development, maturation, and function of multiple organ systems, including the immune system. One of the main targets of estrogens in the immune system is the thymus, which undergoes atrophy and phenotypic alterations when exposed to elevated levels of estrogen. To determine how estrogens influence the thymus and affect T cell development, estrogen receptor alpha (ERalpha) knockout (ERKO) mice were examined. ERKO mice have significantly smaller thymi than their wild-type (WT) littermates. Construction of ER radiation bone marrow chimeras indicated that the smaller thymi were due to a lack of ERalpha in radiation-resistant tissues rather than hemopoietic elements. ERKO mice were also susceptible to estradiol-induced thymic atrophy, but the extent of their atrophy was less than what was seen in WT mice. The estradiol-treated ERKO mice failed, however, to manifest alterations in their thymic CD4/CD8 phenotypes compared with WT mice. Therefore, ERalpha is essential in nonhemopoietic cells to obtain a full-sized thymus, and ERalpha also mediates some of the response of the thymus to elevated estrogen levels. Finally, these results suggest that in addition to ERalpha, another receptor pathway is involved in estradiol-induced thymic atrophy.     

Estrogen receptor knockout mice as a model for endocrine research

The biological effects of estrogen in mammalian target tissues are important for multiple organ systems including the male and female reproductive tract and the neuroendocrine, skeletal, and cardiovascular systems. Numerous physiological effects of estradiol are modulated by the estrogen receptor (ER), a Class I member of the nuclear receptor superfamily. However, more recent studies have also implicated nongenomic effects of estrogen, which may involve a membrane-binding site. The two forms of the ER are the classical estrogen receptor-alpha (ERalpha) and the more recently discovered estrogen receptor-beta (ERbeta). Gene-targeting techniques were used to generate mice lacking either functional ERalpha (alphaERKO), ERbeta (betaERKO), or both ERs (alphabetaERKO) to provide a model for evaluating estrogen receptor action. These knockout models provide a unique tool to study the effects of estrogen in the context of the whole animal and to discern the role of each ER in various tissues. The reproductive phenotypes as well as some of the nonreproductive phenotypes of the different ERKO models are summarized.     

Estrogen receptor alpha has a functional role in the mouse rete testis and efferent ductules

Previous studies of the estrogen receptor-alpha knockout (alpha ERKO) in the male mouse demonstrate that the rete testis and efferent ductules are targets of estrogen. Because the alpha ERKO mouse lacks a functional estrogen receptor alpha (ER alpha) throughout development, it was not known whether the morphological and physiological abnormalities observed in the alpha ERKO male were due to developmental defects or to dysfunctions concurrent with the lack of ER alpha in the tissue. This study was designed to determine if treatment of normal wild-type (WT) mice with the pure antiestrogen, ICI 182,780, (ICI) could reproduce the morphological characteristics seen in alpha ERKO mice. Thirty-day-old male mice were treated for 35 days with either castor oil or ICI. Age-equivalent alpha ERKO mice were used for comparison. Light microscopic examinations of the reproductive tracts revealed dramatic changes in the efferent ductules of treated mice: a 1.7-fold increase in luminal diameter, a 56% reduction in epithelial cell height, a 60% reduction in brush boarder height of nonciliated cells, and an apparent reduction of the number of observable lysosomes and endocytotic vesicles. Testes of ICI-treated mice showed swollen rete testes area (6.5 times larger than control) and a 65% reduction in rete testis epithelium height. However, there were no significant changes in body and testis weights. These results indicate that ER blockage with ICI in WT mice results in morphological changes of the efferent ductules resembling those seen in alpha ERKO siblings of the same age. Based on this study, we conclude that ER alpha has a functional role in the mouse reproductive tract and the aberrant morphology observed in the efferent ductules of the alpha ERKO mouse is likely the result of a concurrent response to the lack of functional ER alpha, and not solely due to the lack of ER alpha during early developmental times.     

The ratio of estrogen receptor alpha to estrogen receptor beta in adipose tissue is associated with leptin production and obesity

The loss of estrogen associated with menopause is suspected to play an important regulatory role in changes of fat metabolism and obesity. To evaluate the relationship between obesity and the ratio of estrogen receptor subtypes (ERalpha/ERbeta) in adipose tissues in pre- and postmenopausal women, we measured the anthropometric indices of 31 premenopausal women and 12 postmenopausal women. Serum samples, subcutaneous and omental adipose tissues were also obtained from study participants. Serum leptin, adiponectin, IL-6, and TNF-alpha levels were measured using ELISA methods. Real-time RT-PCR analysis was performed to detect and to compare mRNA levels of leptin and estrogen receptor subtypes (ERalpha and ERbeta) from adipose tissues. The ratio of abdominal subcutaneous to omental adipose tissue for the ER subtypes (Sc-Om ratio of the ER subtypes), i.e., subcutaneous ERalpha/ERbeta over omental ERalpha/ERbeta, showed significant correlations with anthropometric indices including BMI (r=0.801, p<0.05) and waist circumference (r=0.696, p<0.05) in both pre- and postmenopausal women. The Sc-Om ratio of the ER subtypes also had a significant correlation with the serum leptin level (r=0.735, p<0.05) as well as the mRNA level of leptin in omental adipose tissue (r=0.753, p<0.05). However, there were no significant differences between the pre- and postmenopausal groups with regard to the expressed level of ER subtypes. In conclusion, our study results showed that the ratio of ERalpha to ERbeta in adipose tissue was associated with obesity as well as the serum level and production of leptin in omental adipose tissue.     

Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract

Regulation of progesterone receptor (PR) by estradiol-17beta (E(2)) in mouse uterine and vaginal epithelia was studied. In ovariectomized mice, PR expression was low in both vaginal stroma and epithelium, but high in uterine epithelium. E(2) induced PR in vaginal epithelium and stroma, but down-regulated PR in uterine epithelium. Analysis of estrogen receptor alpha (ERalpha) knockout (ERKO) mice showed that ERalpha is essential for E(2)-induced PR expression in both vaginal epithelium and stroma, and for E(2)-induced down-regulation, but not constitutive expression of PR in uterine epithelium. Regulation of PR by E(2) was studied in vaginal and uterine tissue recombinants made with epithelium and stroma from wild-type and ERKO mice. In the vaginal tissue recombinants, PR was induced by E(2) only in wild-type epithelium and/or stroma. Hence, in vagina, E(2) induces PR directly via ERalpha within the tissue. Conversely, E(2) down-regulated epithelial PR only in uterine tissue recombinants constructed with wild-type stroma. Therefore, down-regulation of uterine epithelial PR by E(2) requires stromal, but not epithelial, ERalpha. In vitro, isolated uterine epithelial cells retained a high PR level with or without E(2), which is consistent with an indirect regulation of uterine epithelial PR in vivo. Thus, E(2) down-regulates PR in uterine epithelium through paracrine mechanisms mediated by stromal ERalpha.     

Gender-related differences in elite gymnasts: the female athlete triad.

High-intensity training can alter the normal pattern of pubertal development in elite gymnasts. We investigated sex hormones, the ob gene product leptin, body composition, nutrition, and eating habits in female and male elite gymnasts from national cadres to elucidate gender-related differences. Serum leptin levels were decreased, particularly in pubertal girls, and did not show the normal developmental pattern. After leptin levels were transformed into standard deviation scores, mainly pubertal female gymnasts had significantly lower values than normal controls of the same gender, pubertal stage, and body mass index. The percentage of body fat was reduced compared with a normal age-matched population in both genders but to a higher degree in female gymnasts. When leptin standard deviation scores were based on percent body fat instead of body mass index, mean values were still significantly decreased compared with those of normal controls: -1.05 in girls (P < 0.001) and -0.60 in boys (P = 0.025). In both genders, total energy consumption and nutritional intake were insufficient, although to a lesser extent in male gymnasts. Pubertal development is influenced to a different degree in female and male elite gymnasts. In contrast to their male counterparts, high-intensity training takes place during the sensitive phase of pubertal maturation in female gymnasts. Whereas the girls displayed low estrogen levels, hypoleptinemia, reduced body fat mass, insufficient caloric intake, and retarded menarche, the pubertal development of male gymnasts remained almost unaltered.     

Leptin levels and body composition of mice selectively bred for high voluntary locomotor activity

Selective breeding produced four replicate lines of high-runner (HR) mice that run on wheels for approximately 2.7 times more revolutions per day than four unselected control lines. Previous studies found that HR mice of both sexes have lower body fat (isotope dilution at 15 wk of age) and that males (females not studied) have smaller retroperitoneal fat pads (17 wk). HR mice also exhibit elevated plasma corticosterone and insulin-stimulated glucose uptake by some hindlimb muscles but apparently do not differ in circulating insulin or glucose levels (males at 18 wk). Given their lower body fat and higher activity levels, we hypothesized that HR mice would have lower circulating leptin levels than controls. Female mice were given wheel access for 6 d at 7 wk of age, as part of the routine wheel testing for the selective breeding protocol, and then were killed after one additional week without wheels to reduce possible acute effects of activity on leptin. As hypothesized, serum leptin levels were significantly lower in HR mice. ANCOVA indicated that leptin was strongly positively correlated with both total body fat (measured by ether extraction) and body mass change from weaning, but HR mice still had significantly lower adjusted leptin levels (ANCOVA). Within HR lines but not within control lines, individual variation in leptin levels was negatively correlated with amount or speed of wheel running measured a week before being killed. Growth from weaning to euthanasia and body dry mass were lower in HR mice than in controls, but absolute dry masses of the ventricles, liver, gut, and uterus plus ovaries did not significantly differ, nor did percentage of the total dry mass as fat. HR mice offer a novel model for studying the causes and consequences of physiologically relevant variations in serum leptin.     

A longitudinal study of leptin during development in the male rhesus monkey: the effect of body composition and season on circulating leptin levels

The objective of this study was to examine longitudinal changes in serum leptin concentrations during development and to correlate those changes with sexual development in male rhesus monkeys housed under natural environmental conditions. Blood samples were drawn from 8 control animals approximately every other month from 10 to 30 mo of age and thereafter monthly through 80 mo of age. Leptin levels declined through the juvenile period until the onset of puberty and were negatively correlated with body weight. Seven of the eight animals became sexually mature during the breeding season of their fourth year of life. Puberty was delayed in the other animal until the subsequent breeding season. There were no significant fluctuations in leptin levels prior to or in association with the pubertal rise in LH and testosterone (T) secretion. During the peripubertal period, levels of leptin varied between 2 and 3 ng/ml. The animal that exhibited delayed puberty had the lowest body weight and highest leptin levels during this period. With the achievement of sexual maturity, leptin levels varied seasonally, with peak levels in the late winter (Jan-Mar) and a nadir in the late summer (Aug-Sept). A late winter rise in leptin was also evident in most of the animals during Years 2 and 3, but not during Year 4. In the fall of Years 5 and 6, the seasonal rise in leptin concentrations lagged 3-4 mo behind the seasonal increase in LH and T. In the fall of Year 5, but not thereafter, leptin levels were positively related to percent body fat and negatively correlated with lean body mass. The data do not support the hypothesis that increasing leptin concentrations trigger the onset of puberty in the male rhesus monkey. During the juvenile period and after sexual maturation, but not during the peripubertal period, leptin secretion varied with season in the animals; but the environmental factors that cue or drive this rhythm remain to be determined.     

Leptin's Sexual Dimorphism Results from Genotype by Sex Interactions Mediated by Testosterone

Objective: Recent studies have reported the existence of marked sexual dimorphism in serum leptin levels in humans, with women having approximately three times the levels of men. As we have shown for other measures of adiposity, such sexual dimorphism can arise from a special case of genotype by environment interaction, that of genotype by sex interaction. Research Methods and Procedures: Using maximum likelihood-based variance decomposition techniques, we examined the genetic and environmental architecture of sexual dimorphism in serum leptin levels in 1147 Mexican Americans from the San Antonio Family Heart Study. Results: Both the genetic and environmental variances for this trait differed significantly between the sexes (p < 0.001 and p < 0.01, respectively), with women displaying larger values for both components. We found significant evidence that different genes influence variation in serum leptin levels between the two sexes (p = 0.05). Furthermore, this pattern of sexual dimorphism in serum leptin levels persisted even after accounting for the effects of either the percentage of body fat or total body fat. However, this pattern of sexual dimorphism was eliminated after accounting for the effects of testosterone. Discussion: These findings suggest that the sexual dimorphism seen in leptin levels is not simply explained as differences in total adiposity between the sexes. We conclude that the genes, which influence variation in serum leptin levels, are differentially expressed depending on sex, and that the sexes also show differences in response of the expression of this obesity-related trait to unmeasured residual effects.     

A role for the androgen receptor in follicular atresia of estrogen receptor beta knockout mouse ovary.

Estrogen receptor beta (ERbeta) is highly expressed, but ERalpha is not detectable in granulosa cells in the mouse ovary. In ERbeta knockout (BERKO) mice, there is abnormal follicular development and very reduced fertility. At 3 wk of age, no significant morphologic differences were discernable between wild type (WT) and BERKO mouse ovaries, but by 5 mo of age, atretic follicles were abundant in BERKO mice and there were very few healthy late antral follicles or corpora lutea. At 2 yr of age, unlike the ovaries of their WT littermates, BERKO mouse ovaries were devoid of healthy follicles but had numerous large, foamy lipid-filled stromal cells. The late antral and atretic follicles in BERKO mice were characterized by a high level of expression of the androgen receptor (AR) and IGF-1 receptor. These proteins were abundantly expressed in granulosa cells of preantral and early antral follicles in both genotypes, but their expression was extinguished in late antral follicles of WT mice. Healthy late antral follicles and corpora lutea were restored in BERKO ovaries after 15 days of treatment of mice with the antiandrogen flutamide. The results suggest that in the absence of ERbeta there was a loss of regulation of AR. Because androgens enhance recruitment of primordial follicles into the growth pool and cause atresia of late antral follicles, the inappropriately high level of AR probably is related to the follicular atresia and to the early exhaustion of follicles in BERKO mice.     

Effect of ovariectomy on adipose tissue of mice in the absence of estrogen receptor alpha (ERalpha): a potential role for estrogen receptor beta (ERbeta).

Adipose tissue deposition is highly responsive to estrogen; ovariectomy increases adipose deposition, and estrogen replacement reverses this. Estrogen receptor alpha (ERalpha) plays a major role in adipose tissue. ERalpha knockout (alphaERKO) mice show an increase in adipose tissue of over a 100 % compared to wild-type mice. However, alphaERKO mice undergo a 10-fold increase in 17beta-estradiol (E2), and persistent or even increased signaling through ERbeta could be a factor in obesity of alphaERKO mice. To test the hypothesis that ERbeta plays a role in adipose tissue, adult female alphaERKO mice were ovariectomized or sham-ovariectomized and fed a phytoestrogen-free diet. Ovariectomized mice were treated with vehicle or E2, and bodyweights and food consumption were measured. Mice were killed after 28 days and inguinal and parametrial fat pads collected. Sham-ovariectomized alphaERKO mice had increased body weight, ovariectomized alphaERKO mice showed a 6 % decrease, and E2 replacement restored body weight to sham levels. Fat pads of ovariectomized alphaERKO mice showed 45 % and 16 % decreases in weight and adipocyte circumference, respectively, compared to sham-ovariectomized or E2-replaced ovariectomized alphaERKO mice. Ovariectomized alphaERKO mice showed a trend towards decreased feed consumption that did not reach significance. Blood glucose levels were lower both before and after glucose injection in ovariectomized compared to sham alphaERKO mice, and E2 treatment reversed this. Insulin levels following glucose challenge were lower in ovariectomized compared to sham-ovariectomized alphaERKO mice, indicating that ovariectomy ameliorated the glucose intolerance and insulin resistance in alphaERKO mice. Immunohistochemical analysis revealed strong staining for ERbeta in adipose tissue. These observations indicate that removing E2/ERbeta signaling in alphaERKO mice by ovariectomy decreases body and fat-pad weights and adipocyte size, while improving insulin and glucose metabolism. ERbeta mediated effects on adipose tissue are opposite those of ERalpha, although E2 effects on adipose tissue are predominately through ERalpha.     

Increased adiposity in DNA binding-dependent androgen receptor knockout male mice associated with decreased voluntary activity and not insulin resistance

In men, as testosterone levels decrease, fat mass increases and muscle mass decreases. Increased fat mass in men, in particular central obesity, is a major risk factor for type 2 diabetes, cardiovascular disease, and all-cause mortality. Testosterone treatment has been shown to decrease fat mass and increase fat-free mass. We hypothesize that androgens act directly via the DNA binding-dependent actions of the androgen receptor (AR) to regulate genes controlling fat mass and metabolism. The aim of this study was to determine the effect of a global DNA binding-dependent (DBD) AR knockout (DBD-ARKO) on the metabolic phenotype in male mice by measuring body mass, fat mass, food intake, voluntary physical activity, resting energy expenditure, substrate oxidation rates, serum glucose, insulin, lipid, and hormone levels, and metabolic gene expression levels and second messenger protein levels. DBD-ARKO males have increased adiposity despite a decreased total body mass compared with wild-type (WT) males. DBD-ARKO males showed reduced voluntary activity, decreased food intake, increased serum leptin and adiponectin levels, an altered lipid metabolism gene profile, and increased phosphorylated CREB levels compared with WT males. This study demonstrates that androgens acting via the DNA binding-dependent actions of the AR regulate fat mass and metabolism in males and that the increased adiposity in DBD-ARKO male mice is associated with decreased voluntary activity, hyperleptinemia and hyperadiponectinemia and not with insulin resistance, increased food intake, or decreased resting energy expenditure.     

Middle-aged C57BL/6 mice have impaired responses to leptin that are not improved by calorie restriction

Midlife weight gain occurs in many species, suggesting that leptin signaling is impaired at middle age. To test this hypothesis, we measured changes in food intake and body composition in young (Y) and middle-aged (MA) C57BL/6 male mice infused subcutaneously with phosphate-buffered saline or leptin. Leptin-induced decreases in food intake and body fat were delayed in MA mice and associated with catabolism after longer treatment periods. Endogenous plasma leptin levels did not correlate with body fat in MA mice. Calorie restriction (CR) reduced body fat, plasma leptin, and insulin in MA mice to levels in Y mice but did not upregulate leptin sensitivity. CR mice did not respond to leptin doses that inhibited food intake in MA mice and reduced food intake and body fat in Y mice significantly below levels in CR mice. Plasma corticosterone was significantly higher in leptin-treated CR vs. MA mice. We conclude that MA C57BL/6 mice exhibit impaired leptin signaling and that CR, possibly by elevating glucocorticoids, impairs appetite control without improving the metabolic actions of leptin.     

Sexually dimorphic responses to fat loss after caloric restriction or surgical lipectomy

White adipose tissue is the principal site for lipid accumulation. Males and females maintain distinctive white adipose tissue distribution patterns. Specifically, males tend to accumulate relatively more visceral fat, whereas females accumulate relatively more subcutaneous fat. The phenomenon of maintaining typical sex-specific fat distributions suggests sex-specific mechanisms that regulate energy balance and adiposity. We used two distinct approaches to reduce fat mass, caloric restriction (CR), and surgical fat removal (termed lipectomy) and assessed parameters involved in the regulation of energy balance. We found that male and female mice responded differentially to CR- and to lipectomy-induced fat loss. Females decreased energy expenditure during CR or after lipectomy. In contrast, males responded by eating more food during food return after CR or after lipectomy. Female CR mice conserved subcutaneous fat, whereas male CR mice lost adiposity equally in the subcutaneous and visceral depots. In addition, female mice had a reduced capability to restore visceral fat after fat loss. After CR, plasma leptin levels decreased in male but not in female mice. The failure to increase food intake after returning to ad libitum intake in females could be due to the relatively stable levels of leptin. In summary, we have found sexual dimorphisms in the response to fat loss that point to important underlying differences in the strategies by which male and female mice regulate body weight.     

Effects of Exogenous Gonadal Steroids on Leptin Homeostasis in Rats

WU-PENG, SHARON, MICHAEL ROSENBAUM, MARGERY NICOLSON, STREAMSON C. CHUA, AND RUDOLPH L. LEIBEL. Effects of exogenous gonadal steroids on leptin homeostasis in rats. Obes Res. Background: In humans, circulating concentrations of the hormone leptin, normalized to body fat mass, are significantly higher in females compared to males. This experiment was designed to determine whether the administration of exogenous androgen or estrogen would significantly alter the relationship between plasma leptin and fat mass in rats. Methods: In the first experiment, plasma leptin and retro-peritoneal and parametrial (female)/epididymal (male) adipose tissue expression of leptin mRNA were measured in five male and five female 9. 5-week-old Sprague-Dawley rats. In a second experiment, gonadectomized 10. 5-week-old female Sprague-Dawley rats received 1 or 2 weeks of daily intraperitoneal injections (in oil) of 750 mg testosterone propionate, 2. 5 μg of estradiol benzoate or vehicle. At 0, 1, and 2 weeks, plasma concentrations of leptin, fat pad weight of parametrial and retroperitoneal fat pads, and leptin mRNA expression by Northern blot in retroperitoneal fat pads were determined. Daily weight and food intake of animals were monitored throughout the study. Results: Circulating leptin concentrations per unit of fat pad mass and leptin mRNA expression normalized to actin mRNA were higher in gonadally intact female compared to male rats. Compared to placebo, estrogen administration decreased food intake and body weight, but had no significant effect on leptin mRNA expression or on circulating leptin concentration. Testosterone administration increased body weight and decreased expression of leptin mRNA (only after 2 weeks), but did not change food intake or circulating leptin concentration. Conclusions: Administration of estrogen did not affect either leptin expression or the circulating concentration of leptin. Administration of androgen decreased expression of leptin mRNA. However, even after 2 weeks of testosterone administration to gonadectomized females, plasma leptin concentration, corrected for fat pad weight, was higher in gonadectomized females than in intact males, Thus, sex steroid-associated changes in plasma leptin concentration and leptin mRNA expression are not sufficient to explain the observed sexual dimorphism in plasma leptin concentrations in rats.     

Estrogen improved metabolic syndrome through down-regulation of VEGF and HIF-1α to inhibit hypoxia of periaortic and intra-abdominal fat in ovariectomized female rats

Metabolic syndrome (MBS), a cluster of metabolic abnormalities and visceral fat accumulation, increases cardiovascular risks in postmenopausal women. In addition to visceral fat, perivascular adipose tissue has been recently found to play an important role in vascular pathophysiology. Hence, the present study investigates the effects of estrogen on both intra-abdominal fat (visceral fat) and periaortic fat (perivascular fat) accumulation as well as hypoxia in ovariectomized female rats. Female rats were divided into sham operation, ovariectomy and ovariectomy with 17β-estradiol supplementation groups. Twelve weeks later, we found that estrogen improved MBS via reducing body weight gain, the weight of periaortic and intra-abdominal fat, hepatic triglyceride, and total serum cholesterol levels. Estrogen also increased insulin sensitivity through restoring glucose and serum leptin levels. For periaortic fat, western blot showed estrogen inhibited hypoxia by reducing the levels of VEGF and HIF-1α, which is consistent with the results from immunohistochemical staining. The correlation analysis indicated that perivascular fat had a positive correlation with body weight, intra-abdominal fat or serum total cholesterol, but a negative correlation with insulin sensitivity index. For intra-abdominal fat, real-time fluorescent RT-PCR showed estrogen improved fat dysfunction via reducing the levels of relative leptin, MCP-1 but increasing adiponectin mRNA. Estrogen reduced the levels of VEGF and HIF-1α to inhibit hypoxia but restored the levels of PPARγ and Srebp-1c, which are important for lipid capacity function of intra-abdominal fat. These results demonstrated estrogen improved MBS through down-regulating VEGF and HIF-1α to inhibit hypoxia of periaortic and intra-abdominal fat in ovariectomized female rats.     

A high fat diet-induced impaired glucose metabolism in mice with targeted deletion of calpain in osteoblasts

The ubiquitously expressed Calpains 1 and 2 belong to a family of calcium-dependent intracellular cysteine proteases. Both calpains are heterodimers consisting of a large subunit and a small regulatory subunit encoded by the gene Capns1. To investigate a role for the calpain small subunit in cells of the osteoblast lineage in vivo, we previously generated osteoblast-specific Capns1 knockout mice and characterized their bone phenotype. In this study, we further examined effects of low calcium and high fat diets on their bone, fat, and glucose homeostasis.Osteoblast-specific Capns1 knockout mice showed significantly reduced serum levels of total and uncarboxylated osteocalcin, and this was presumably due to their impaired bone formation and bone resorption. The reduced bone resorptive function of the mutant mice was also significant under a low calcium diet. Thus, these results suggest that reduced uncarboxylated osteocalcin levels of mutant mice were, at least in part, due to their osteoporotic bone with impaired bone resorptive function. Interestingly, unlike osteocalcin knockout mice, mutant mice on a normal chow diet were leaner than control littermates; this was likely due to their reduced food intake and overall lower energy homeostasis. To test this hypothesis, we next provided mutant mice with a high fat diet and further examined an effect of their reduced uncarboxylated osteocalcin levels on body composition and glucose metabolism. The average mean body weight of mutant mice became indistinguishable with that of controls after 2 weeks on a high fat diet, and continued to show an upward trend, at least, up to 6 weeks. Moreover, mutant mice on a high fat diet exhibited a significant increase in serum levels of leptin and resistin, adipocyte-specific adipokines, and developed impaired glucose tolerance. Collectively, mice with osteoporosis and reduced bone resorptive function showed reduced serum uncarboxylated osteocalcin levels and were susceptible to increase body adiposity and develop impaired glucose tolerance under a high fat diet.     

Long-term effects of ovariectomy on osteoporosis and obesity in estrogen-receptor-β-deleted mice

Untreated BERKO mice demonstrate few abnormalities in bone phenotype and recent ovariectomy has few effects on various bone characteristics in these mice. Long-term studies on the bone phenotype of intact and ovariectomized mice are unavailable. Using quantitative computed tomography (qCT), we determined various parameters of the metaphysis of the tibia in sham-ovariectomized (intact) and ovariectomized BERKO and wildtype mice. Body weight and estrogen-regulated fat were also measured. Mice underwent surgery (ovariectomy or sham) at 3 mo of age, and qCT analysis was performed every 2 to 4 mo until mice were 12 mo old. Ovariectomized wildtype mice gained body weight and their fat depot increased in size within 2 mo after ovariectomy. Obesity developed later in ovariectomized BERKO mice, which became significantly heavier than their wildtype counterparts. Ovariectomized wildtype mice lost trabecular density more rapidly than did ovariectomized BERKO mice, which did not show similar loss in trabecular density until at least 7 mo after ovariectomy. At the latest studied time point (9 mo after surgery), cortical area was significantly larger in ovariectomized BERKO mice than ovariectomized wildtype mice. The absence of ERβ in ovariectomized BERKO mice during the first 3 to 5 mo after ovariectomy had protective effects against obesity and trabecular rarification; this protective effect disappeared at later time points.     

Myocardial ischemia-reperfusion injury in estrogen receptor-alpha knockout and wild-type mice

We investigated the function of estrogen receptor-alpha in global myocardial ischemia and reperfusion injury in male estrogen receptor-alpha knockout (ERKO) and wild-type mice. Mouse hearts were subjected to 45 min of global ischemia followed by 180 min of reperfusion. The hearts were excised, cannulated, and maintained in a chilled (4 degrees C) cardioplegia solution until warm (37 degrees C) oxygenated Krebs-Henseleit bicarbonate buffer was perfused through the coronary arteries. ERKO hearts started beating later and had a higher incidence of ventricular fibrillation and/or tachycardia than control hearts. Coronary flow rate was significantly lower in ERKO hearts during the 90- and 120-min periods of reperfusion. Ca(2+) accumulation was significantly greater following 30, 90, 120, 150, and 180 min of reperfusion in ERKO hearts. Nitrite production was significantly less in ERKO hearts following 90, 120, and 150 min of reperfusion. Myocardial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was significantly lower in experimental ERKO hearts. Marked interstitial edema and contraction bands were seen in hematoxylin-eosin-stained sections of ischemia-reperfused ERKO hearts but not in control tissues. Hematoxylin-basic fuchsin-picric acid-stained sections from experimental ERKO hearts had fewer viable myocytes compared with controls. Transmission electron microscopy revealed swollen and fragmented mitochondria with amorphous and granular bodies, loss of matrix, and rupture of cristae in experimental ERKO hearts. This is the first demonstration that estrogen receptor-alpha plays a cardioprotective role in ischemia-reperfusion injury in males.