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Departments of Experimental Pharmacology and Obstetrics and Gynecology (A.N., C.D.C., C.N.), University of Naples Federico II, 80131 Naples, Italy
Address all correspondence and requests for reprints to: Prof. Rosaria Meli, Department of Experimental Pharmacology, via D. Montesano 49, 80131 Naples, Italy. E-mail: meli{at}unina.it.
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Abstract |
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Introduction |
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Gender differences in body fat distribution implicate sex steroids in the regulation of adiposity. The role that estrogen plays in regulating body fat is confirmed by recent studies showing that mice lacking estrogen receptor-
(ER) or aromatase, the enzyme responsible for estrogen biosynthesis, have increased fat mass and hyperlipidemia (8, 9). Moreover, evidence indicates a direct genomic modulation of NPY neurosecretion by estrogens in hypothalamus (10); in particular, estrogen deficiency increases hypothalamic NPY and causes central leptin insensitivity (11).
The weight-reducing effect of leptin is mediated by leptin receptors (Ob-R), of which six alternatively spliced forms (Ob-Ra to Ob-Rf) have been identified, and mutations in Ob-R result in an obese phenotype, such as db/db mice, identical to that of ob mice (12). Obesity, from declining estrogen levels after menopause, increases the risk of heart disease, diabetes, and hypertension, and the severity and incidence of infectious illnesses (13). A large body of evidence suggests that estrogen or hormone replacement therapy may be a benefit in reducing the risk of heart disease in postmenopausal women, although adverse effects, such as an increased risk of endometrial and breast cancer, have been reported (14, 15). However, this benefit has not been confirmed in randomized clinical trials (i.e. Womans’s Health Initiative and Heart Estrogen/Progestin Replacement Study). Selective ER modulators are a family of compounds exerting estrogenic agonist or antagonist effects in a tissue-specific manner that have been introduced as therapy to target selective tissues. Raloxifene (RAL) is a nonsteroid benzothiophene that is an ER antagonist in the breast, like tamoxifene, but has a distinct profile on the skeleton, serum lipids, and uterine endometrium (16). Moreover, in postmenopausal women, RAL modulates peripheral insulin sensitivity and its hepatic clearance (17).
Estrogen insufficiency in humans can be modeled using ovariectomized (OVX) rats. This model is characterized by mild obesity and is useful to study how hypoestrogenism alters adiposity. The aim of this study was to examine how in mid- or long-term ovariectomy (OVX), 17ß-estradiol (E2) or RAL treatment alters leptinemia and Ob-R abundance within the hypothalamus and adipose tissue, thereby enhancing adaptation after the menopause.
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Materials and Methods |
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In the first set of experiments rats were divided into the three following groups: 1) sham-operated controls (SHAM); 2) OVX; and 3) OVX and E2 (OVX+E2). In the second set of experiments the three groups were: 1) SHAM; 2) OVX; and 3) OVX+RAL. At the onset of the study, OVX rats (average body weight of the cohort, 174 ± 2 g) were bilaterally OVX under anesthesia (ketamine, 100 mg/kg; xylazine, 5 mg/kg, ip). The SHAM animals were subjected to the same general surgical procedure as the OVX groups, except that the ovaries were not excised. In the first set of experiments treatment with E2 (25 µg/kg, sc, twice a week; Sigma-Aldrich Corp., St. Louis, MO) or vehicle (sesame oil) was initiated on d 2 of the study. In the second set of experiments rats were treated by gavage with RAL (3 mg/kg once daily; Lilly Research Laboratories, Indianapolis, IN) or vehicle administered on d 2 until the end of the experiments. In both sets of experiments the drug treatments were performed for 7 or 22 wk. A preliminary study was conducted for 4 wk.
Body weight, food intake, and body gain in fat
Throughout the treatment period, body weight and food intake were monitored once a week. At the end of both experimental periods, food intakes were cumulated. Bioelectrical impedance analysis was applied to body composition assessment at 4, 7, and 22 wk using a BIA 101 analyzer, modified for the rat (Akern, Florence, Italy). Fat-free mass was calculated using the bioelectrical impedance analysis (50 kHz) prediction equation of Ilagan et al. (18), and fat mass content was determined as the difference between body weight and fat-free mass.
Tissue collection and hematic parameters
SHAM rats at random stages of the estrous cycle, OVX, and E2- or RAL-treated rats were killed at 7 or 22 wk. Blood collected by cardiac puncture was centrifuged at 1500 x g at 4 C for 15 min, and sera were stored at –70 C for later biochemical and hormonal measurements.
Glucose, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, and cholesterol were quantified. The serum leptin concentration was measured using an RIA kit for rats according to the manufacturer’s instruction (Linco Research, Inc., St. Charles, MO).
Serum estradiol was determined with an ELISA kit (Abbott Laboratories, Abbott Park, IL). Whole hypothalamus [dissected according to the map of Glowinski and Iversen (19)] and sc white adipose tissue were excised and immediately frozen in liquid nitrogen.
Western blot analysis
The hypothalamus and sc adipose tissue (0.3 g) obtained from each animal were disrupted by homogenization on ice in lysis buffer [20 mM Tris-HCl (pH 7.5), 10 mM NaF, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonylfluoride, 1 mM Na3VO4, leupeptin, and 10 µg/ml trypsin inhibitor]. After 1 h, cell lysates were obtained by centrifugation at 100,000 x g for 15 min at 4 C. Protein concentrations were estimated by the Bio-Rad protein assay (Hercules, CA) using BSA as standard.
For Western blot analysis, 100 µg protein of all tissue lysate was dissolved in Laemmli sample buffer, boiled for 5 min, and subjected to SDS-PAGE (8% polyacrylamide). The blot was performed by transferring proteins from a slab gel to nitrocellulose membrane at 240 mA for 40 min at room temperature. The filter was then blocked with 1x PBS and 5% nonfat dried milk for 40 min at room temperature and probed with rabbit polyclonal Ob-R antibody (1:2000; Affinity Bioreagents, Golden, CO) or with a goat polyclonal anti-C-term Ob-Rb antibody (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in 1x PBS, 5% nonfat dried milk, and 0.1% Tween 20 at 4 C overnight. The secondary antibody (antirabbit or antigoat IgG-horseradish peroxidase conjugate; 1:2000 dilution) was incubated for 1 h at room temperature. Subsequently, the blot was extensively washed with PBS, developed using enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions, and exposed to Kodak X-OMAT film. To ascertain that blots were loaded with equal amounts of protein lysates, they were also incubated in the presence of the antibody against the ß-actin protein (Sigma-Aldrich Corp.). The protein bands on x-ray film were scanned and densitometrically analyzed with a model GS-700 imaging densitometer (Bio-Rad Laboratories, Milan, Italy).
Statistical analysis
All data are presented as the mean ± SEM. Statistical analysis was performed by ANOVA test for multiple comparisons, followed by Bonferroni’s test. Statistical significance was set at P < 0.05.
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Results |
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. OVX significantly reduced the estrogen level in OVX rats at both 7 and 22 wk (P < 0.05). As expected, in both experiments E2 supplementation increased the serum estrogen level (P < 0.05 vs. OVX), yielding an estrogen concentration at the physiological level (Fig. 1A). On the contrary, treatment with RAL did not restore the estrogen level (Fig. 1B).
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and 2, hypoestrogenism clearly leads to a significant increase in body weight at 4, 7, and 22 wk, and this change in body weight paralleled the increase in food intake. Moreover, the change in body weight was related to a variation in fat mass, which was significantly increased in OVX animals and was reversed by E2 (Table 1) and RAL (Table 2) at all treatment times.
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. OVX significantly increased serum cholesterol in short- and long-term studies (P < 0.01). E2 prevented this effect at 7 wk (P < 0.05), but not at 22 wk.
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Plasma triglycerides concentrations were not altered significantly by OVX with or without E2 replacement, but RAL treatment of OVX rats significantly increased plasma triglycerides values over those in intact or OVX untreated animals, but only in the short-term study. No significant change in serum glucose concentrations was observed with E2 or RAL compared with OVX controls.
Body weight and fat content changes caused by OVX were also accompanied by a variation in serum leptin concentration (Fig. 2). In the preliminary experiment, after 4 wk of treatment no significant differences among groups (n = 5/group) in serum levels of leptin were observed (SHAM, 1.76 ± 0.28; OVX, 1.78 ± 0.19; OVX+E2, 1.82 ± 0.08 ng/ml). As reported in Fig. 2, 7 wk after OVX, leptin serum levels were higher than SHAM values (P < 0.05). This effect was significantly reversed by E2 supplement. Similar findings were obtained from the long-term study, in which the increase in serum leptin level was more significant (P < 0.01) and was totally abolished by E2 replacement (Fig. 2A). RAL treatment of OVX rats had the same effect on leptinemia as E2 in both short- and long-term studies (Fig. 2B).
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Immunoblotting of all sc adipose tissue lysates was performed. It revealed all leptin receptor isoforms by a polyclonal antibody raised against an extracellular domain of rat Ob-R. In fact, several Ob-R-immunoreactive proteins with apparent relative molecular masses of approximately 75, 130, 150, and 210 kDa were detected (data not shown). Only the approximately 130-kDa bands, consistent with the predicted molecular mass of Ob-Rb based on amino acid composition (21), were modulated differently in eu- and hypoestrogenism conditions in sc adipose tissue in the short- and long-term study (Figs. 3 and 4). We confirmed the 130-kDa band as the long Ob-Rb isoform by immunoblot using the antibody that recognizes the C-terminal region of the receptor. Higher expression of Ob-Rb was seen in all OVX than in SHAM rats at 7 wk. Furthermore, when OVX rats were reconstituted with E2, these prominent bands were similar to those in SHAM animals (Fig. 3A).
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). The same effect was seen with RAL treatment. In fact, short-term RAL treatment down-regulated Ob-Rb expression, whereas long-term treatment up-regulated it (Fig. 4). In Figs. 3 and 4, representative immunoblots of tissue lysates from three rats for each group are shown.
Western blot analysis of the hypothalamus revealed no modulation of Ob-Rb expression among animal groups at 7 wk (data not shown), whereas in a long-term study the hypothalamic Ob-Rb expression strongly decreased in OVX rats and was significantly up-regulated after E2 or RAL treatment (Fig. 5, A and B, respectively), as evidenced by densitometric analysis of protein bands obtained from tissue lysates of all animals.
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Discussion |
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and ERß are expressed in adipose tissue, and evidence for the involvement of ER in white adipose tissue distribution and in the suppression of fat accumulation was provided by analyzing ER knockout mice (8). Altered fat distribution and weight gain are consequences of menopausal hypoestrogenism, but the mechanisms responsible are not completely known. The mild obesity induced by OVX in rats mimics estrogen insufficiency in humans, creating a useful model to study the mechanisms by which hypoestrogenism increases body fat and modifies leptin levels. A strong positive correlation between total fat mass and plasma leptin levels has been observed in humans and rodents (6, 23, 24), and a change in fat stores reflects changes in body weight. Our results are consistent with these previously published data.
In this study we provide evidence that time-dependent elevation of serum leptin levels by estrogen withdrawal is secondary to the increase in fat mass rather than the stimulatory role of estrogens on leptin secretion seen in vitro (25) and in vivo during the estrous cycle in the rat (26). Four-week OVX induces an increase in fat mass, not related to the leptin increase. Only at 7 and 22 wk after surgery is leptin modification observed. However, at 7 wk leptinemia is higher than in SHAM animals, and this effect is even more pronounced in the long-term study (22 wk). This late change in serum leptin concentrations strongly and positively correlates to time-dependent animal fat mass content, which is markedly increased between 7 and 22 wk, after chronic alteration of the hormonal status.
In our experiments treatment with E2 or RAL brings about a decline in serum leptin levels, reducing total fat mass and restoring body composition similar to that in euestrogenism. Other studies of serum leptin levels in OVX rats or postmenopausal women are consistent with our findings (26, 27, 28). Moreover, a recent study in woman showed increases in leptin and body mass index only 6 months after bilateral OVX, and treatment with E2 and RAL prevented both effects, suggesting that replacement therapy may correct changes in fat distribution and modification of leptin levels (29).
In addition, we demonstrate that the long form of Ob-R is modulated by mid- or long-term hypoestrogenism at both central and peripheral levels. Among the six alternatively spliced forms of Ob-R, Ob-Rb is responsible for the obese phenotype, as it is affected in the mutant db/db and is essential to the weight-reducing effect of leptin (30).
In the brain, Ob-Rb, ER, and ERß are found in the hypothalamus (31, 32). Moreover, colocalization of these receptors was evidenced in the neuronal population of the medial preoptic area, arcuate nucleus, ventromedial nucleus, and parvicellular paraventricular nucleus (33). These data predict that leptin and estradiol can interact in these neurons at a central level, as peripheral signals regulating the reproductive and metabolic homeostasis.
Moreover, it was found that estrogen deficiency caused overproduction of NPY in the hypothalamus and impaired central leptin sensitivity, even if at 12 wk after OVX no differences in hypothalamic Ob-Rb mRNA expression were found between OVX and SHAM animals, warranting leptin resistance (11).
In our experimental conditions after 22-wk OVX, we found a marked reduction of Ob-Rb in hypothalamus that, associated with the hyperleptinemia, supports central leptin insensitivity. The restoration of hypothalamic Ob-Rb expression by E2 or RAL treatment suggests the estrogenic agonistic effect of RAL in central nervous system.
In addition to leptin actions in the central nervous system, this hormone has direct effects on peripheral tissues. Among these, adipose tissue represents a target of leptin for regulation of energy store and fertility. Moreover, it is increasingly recognized that steroid hormone metabolism by adipose tissue is important in energy metabolism, including the regulation of fat mass and its distribution (34).
The observed increase in Ob-Rb expression in adipose tissue induced by short-term (7-wk) OVX may be due to the attempt to compensate for the developing obesity. However, in long-term OVX, this system is overwhelmed and desensitized, and the reduction of Ob-Rb, accompanied by a further increase in leptinemia, displays a breakdown of body weight homeostasis that is reversed by E2 replacement or RAL treatment.
This is the first report describing the effect of the selective estrogen receptor modulator RAL on serum leptin levels and Ob-Rb expression in hypothalamus and adipose tissue. The drug shows an estrogen agonistic effect on the fat mass homeostasis, modifying leptin levels and Ob-Rb expression similarly to E2 replacement. Therefore, leptin is only one factor that modulates food intake and fat mass, and an involvement of other hormones and neuropeptides cannot be excluded in RAL activity.
The modification of serum leptin levels and Ob-Rb expression in hypothalamus and adipose tissue by estrogen status identifies a close cross-talk between central and peripheral tissues in the regulation of body fat mass and weight gain.
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Acknowledgments |
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Footnotes |
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Abbreviations: E2, 17ß-Estradiol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NPY, neuropeptide Y; Ob-R, leptin receptor; OVX, ovariectomy, ovariectomized; RAL, raloxifene.
Received February 2, 2004.
Accepted for publication March 24, 2004.
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