Estrogen deficiency causes central leptin insensitivity and increased hypothalamic neuropeptide Y

Abstract

OBJECTIVE: Altered fat distribution is a consequence of menopause, but the mechanisms responsible are unknown. Estrogen insufficiency in humans can be modeled using ovariectomized rats. We have shown that increased adiposity in these rats is due to reduced physical activity and transient hyperphagia, and can be reversed with 17β-estradiol treatment. The aims of this study were to examine whether this altered energy balance is associated with circulating leptin insufficiency, central leptin insensitivity, decreased hypothalamic leptin receptor (Ob-Rb) expression, and/or increased hypothalamic neuropeptide Y (NPY).

METHODS: Plasma leptin levels, adipose tissue ob gene expression, energy balance responses to i.c.v. leptin, hypothalamic Ob-Rb expression and NPY concentration in five separate hypothalamic regions were measured in adult female rats after either ovariectomy or sham operations.

RESULTS: Obesity was not associated with hypoleptinemia or decreased ob gene expression in ovariectomized rats; however, it was associated with insensitivity to central leptin administration. Food intake was less suppressed and spontaneous physical activity was less stimulated by leptin. This was not due to decreased hypothalamic Ob-Rb expression. NPY concentration in the paraventricular nucleus of the hypothalamus was elevated in the ovariectomized rats, consistent with leptin insensitivity; however this effect was transient and disappeared as body fat and leptin levels increased further and hyperphagia normalized.

CONCLUSION: Impaired central leptin sensitivity and overproduction of NPY may contribute to excess fat accumulation caused by estrogen deficiency.

Introduction

Gender differences in body fat distribution implicate sex steroids in the regulation of adiposity. Premenopausal women tend to have a lower body or gynoid distribution of fat, while men and postmenopausal women tend to have an upper body or android distribution of fat.1,2 The latter is associated with a greater risk of cardiovascular disease and type 2 diabetes.3 Postmenopausal women who receive estradiol do not display the characteristic abdominal weight gain pattern usually associated with menopause,4 suggesting that estrogen insufficiency is largely responsible for the increase in adiposity during menopause. The role that estrogen plays in regulating body fat is also highlighted by recent studies showing that mice lacking estrogen receptor-α have increased fat mass,5 and the fact that mice6 or humans7 lacking aromatase, the enzyme responsible for estrogen biosynthesis, develop obesity and hyperlipidemia.

Post-menopausal women have 20–28% more body fat than pre-menopausal women.8,9 Similarly, removing the ovaries of female rats (ovariectomy) decreases plasma estrogen to a negligible level10 and results in a 22% increase in body weight after 10 weeks.11 The weight gain that occurs after ovariectomy in rats can be reversed by estrogen replacement,10,12 indicating that lack of estrogen causes the ovariectomy-induced obesity. Therefore, ovariectomy in rats creates a very useful model of mild obesity to study the mechanism by which estrogen insufficiency causes an increase in body fat in humans.

Lack of estrogen has the potential to affect body weight in several ways. Firstly, 17β-estradiol has been shown to increase both ob mRNA expression in 3T3 adipocytes,13 and leptin secretion in omental adipose tissue.14 The link between estrogen and leptin secretion is also evident from studies in women showing elevated leptin levels during the luteal phase of the menstrual cycle compared to the follicular phase when estrogen levels are lower.15,16 Therefore ovariectomy may alter body weight by reducing the amount of leptin secreted by adipose tissue, resulting in an inappropriately low satiety signal. Indeed, some studies have shown that ovariectomy decreases leptin mRNA expression in perirenal17 and perimetic adipose depots,18 although other studies have not been able to replicate these findings.19,20

Alternatively, lack of estrogen after ovariectomy may affect body weight regulation at a central level. Both estrogen receptor α and β are found in the hypothalamus of the brain, and mice deficient in estrogen receptor-α develop a marked increase in adipose tissue mass,5 consistent with a role for estrogen in the central regulation of body weight. It is possible that estradiol may regulate body weight by enhancing glucose uptake into the brain21 or by increasing insulin receptor number,22 processes that may influence recognition of the fed state.23,24 There is also some evidence that ovariectomy increases hypothalamic neuropeptide Y (NPY) expression25 and decreases hypothalamic corticotropin-releasing hormone (CRH) immunoreactivity,26 both of which would promote hyperphagia. These effects on NPY and CRH can be reversed by estradiol administration.27,28 A defect in leptin sensitivity could explain these abnormalities in NPY and CRH. Leptin insensitivity is a feature of many rodent models of obesity but the site of this defect varies. Both peripheral and central leptin insensitivity are present in the db/db mouse, the Zucker fa/fa rat and the agouti mouse, whereas peripheral leptin insensitivity predominates in NZO mouse.29,30,31 This suggests that the principal defect producing leptin insensitivity in the NZO mouse lies proximal to the hypothalamic leptin receptor. It is not known whether obesity caused by estrogen deficiency is associated with central leptin insensitivity and, if this is the case, whether this is due to defective leptin receptor function, as is the case in the db/db mouse and fa/fa rat.

The aim of the present study was to characterize changes in energy intake and energy expenditure over time after ovary removal in rats, and examine whether changes in leptin production, leptin sensitivity, leptin receptor expression, and hypothalamic NPY concentration explain these changes in energy balance.

Methods

Animals

Female hooded Wistar rats were housed at a constant temperature with a 12 h day/night cycle (06:00–18:00 h) They were given free access to a standard laboratory non-purified diet containing 77% of energy as carbohydrate, 20% protein and 3% of calories as fat (Barastock Products, Pakenham, Australia). At 12–14 weeks of age, rats were anesthetized with pentobarbital (60 mg/kg body weight, Nembutal, Boehringer Ingelheim, Artarmon, Australia) and given either an ovariectomy or sham operation. A subgroup of rats were subcutaneously implanted with a 17β-estradiol releasing pellet or placebo pellet (0.5 mg, Innovative Research of America, Ohio) when the ovariectomy or sham operations were performed (n=9–10 in each group). The success of the ovariectomy procedure was confirmed at the end of the study by measuring uterine weights and by determining plasma estradiol concentration using a double antibody radioimmunoassay (Diagnostic Products Corporation, CA, USA).

Energy balance measurements

Body weight and energy intake were measured daily for 12 weeks after the operation, and averaged every 4 days (over an estrus cycle). Ambulatory spontaneous physical activity levels and resting energy expenditure were measured 2, 7 and 12 weeks after the operation. These timepoints correspond to periods of rapid weight flux (2 weeks), short-term weight stability (7 weeks), and long-term weight stability (12 weeks). Physical activity levels were measured using a single cage animal activity monitor, which utilizes high-intensity modulated infrared light beams to non-invasively detect animal motion (Opto-Varimex Minor, Columbus Instruments, Columbus, Ohio, USA). Resting energy expenditure, fat oxidation and glucose oxidation rates were measured after an overnight fast using an indirect calorimeter (Columbus Instruments, Columbus, Ohio, USA).32

Plasma leptin and adipose tissue ob gene mRNA expression

To determine whether leptin deficiency preceded weight gain in the ovariectomized animals, plasma leptin concentrations were determined in fasted animals 5 and 10 days, and 2, 7 and 12 weeks after the operations using a rat leptin radioimmunoassay kit (Linco, MO, USA). Twelve weeks after the operations, samples of brown adipose tissue (BAT), subcutaneous and infrarenal white adipose tissue (WAT) were frozen in liquid nitrogen and stored at −80°C for ob mRNA expression measurement by Northern Blot analysis. This procedure was performed as previously described.33

Body composition

Dual-energy X-ray absorptiometry (DEXA, Hologic QDR 2000 W) was used to measure fat mass in abdominal and peripheral regions34 2 and 7 weeks after the operations. This was performed in a subgroup of animals lightly anesthetized with pentobarbital (55 mg/kg body weight). Terminal body composition was also measured in groups of rats sacrificed 2, 7 and 12 weeks after the operation. Single depot infrarenal and abdominal subcutaneous WAT and total interscapular BAT was weighed.

Hypothalamic NPY concentration

Groups of rats (n=9–14) were killed 2 and 7 weeks after the operation to measure hypothalamic NPY concentration. Brains were quickly removed and the hypothalamus was dissected on ice. Several subregions of the hypothalamus were dissected by dividing the hypothalamus into three coronal slices. The most caudal slice was divided into a ventral region containing the arcuate nucleus; the remainder of this slice was termed posterior hypothalamus. The intermediate slice was hemisected into a dorsal segment containing the paraventricular nucleus (PVN) and a ventral segment termed anterior hypothalamus. The most rostal slice was termed the preoptic region. Each subregion was weighed and stored at −80°C for later determination of NPY concentration. Sham and ovariectomized rat brains were always dissected at the same time and consistent tissue weights were achieved for each of the regions, with no difference in mass between treatment groups (data not shown).

NPY-like immunoreactivity from the various brain regions was extracted by boiling in 1 ml acetic acid (0.5 M), followed by homogenization and centrifugation (7500 rpm for 30 min at 4°C). The supernatant was decanted, weighed and a 50 µl sample was lyophilized then reconstituted with assay buffer (0.04 M sodium phosphate buffer containing 0.01 M EDTA, 0.1 M NaCl, 0.02% BSA, pH 7.3). NPY was measured with a specific radioimmunoassay developed by Morris et al35 using synthetic porcine NPY (10–1280 pg per tube, Auspep, Victoria, Australia) as standard. Incubation with NPY antibody was carried out overnight at 4°C. [125I]-NPY labeled with Bolton and Hunter reagent (2000 Ci/mmol, Amersham Australia) was added and the incubation continued overnight. Bound tracer was separated with sheep anti-rabbit second antibody and centrifugation. Samples were then counted for 10 min in the gamma counter (Crystal plus multidetector, Packard Australia). NPY was measured in triplicate and non-specific binding was determined for every other sample. The intra- and inter-assay coefficients of variation were 7 and 13%, respectively.

Energy balance response to central leptin administration

Twelve weeks after the ovariectomy or sham operation, a subgroup of rats (n=8 from each group) were anesthetized with intraperitoneal pentobarbital (60 mg/kg body weight) and subcutaneous buprenorphine hydrochloride (30 µg/kg body weight). An intracerebroventricular (i.c.v.) steel cannula was implanted in the lateral ventricle of the brain using a stereotaxic frame (0.7 mm posterior to bregma and 2 mm lateral to the midline). The position of this cannula was checked at the time of sacrifice using bromophenol blue dye. Once body weight had returned to pre-operative levels, rats were injected with 4 µl of i.c.v. saline or murine leptin (0.5 or 3 µg) in random order immediately before the dark period. For these experiments, the dark period began at 13:00 h and finished at 01:00 h each day. Each injection was followed by a 4 day wash-out period. Body weight and energy intake were measured at 1, 3, 20, 44, 68 and 96 h after the infusion. Spontaneous physical activity levels were also recorded at 1, 3, 20 and 44 h after the infusion.

Ob-Rb leptin receptor expression

Hypothalami were collected from fed rats 2 and 12 weeks after the operation (n=9–10) and homogenized in 600 µl Solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate pH 7.0, and 0.05% N-lauroylsarcoscine made up in 0.1% DEPC water). Total RNA was prepared using a modified protocol of Chomczynski and Sacchi.36 Briefly, to 600 µl of homogenates, 1 µl of 20 mg/ml glycogen, 60 µl of 2 M sodium acetate, 600 µl of DEPC-water (0.1%) saturated acid phenol and 180 µl of chloroform:isoamyl alcohol (49:1) were added, vortexed and left on ice for 10 min. Samples were then centrifuged at 13 000 rpm for 15 min at 4°C. The aqueous phase was added to 600 µl isopropanol and incubated overnight at −20°C. Samples were then centrifuged at 13 000 rpm for 15 min at room temperature and the pellet resuspended in 20 µl of DEPC water (0.1%).

Total RNA was reverse transcribed using the Promega Reverse Transcription Kit (Promega Corporation, Madison, WI, USA). Real-time polymerase chain reactions were used to semi-quantitatively determine the level of expression of Ob-Rb in each hypothalamic RNA sample. The ribosomal RNA primers and probe (Taqman® Ribosomal RNA kit, Perkin Elmer Applied Biosystems, Foster City, USA) included the forward primer 5 GCT GGA ATT ACC GCG GCT 3, the reverse primer 5 CGG CTA CCA CAT CCA AGG AA 3 and the VIC probe 5 VIC TGC TGG CAC CAG ACT TGC CCT C TAMRA 3. The Ob-Rb forward and reverse primers were 5 AAG CCT GAA ACA TTT GAG CAT CTT 3 and 5 TGC ATC GAC ACT GAT TTC TTC TG 3 respectively (All Geneworks Pty Ltd, Thebarton, SA, Australia), and the Ob-Rb probe was 5 FAM ATG TTC CAA ACC CCA AGA ATT GTT CCT GG TAMRA 3 (Perkin Elmer Applied Biosystems, Foster City, USA). A reaction mixture consisting of 12.5 µl of Taqman® universal PCR master mix (Perkin Elmer Applied Biosystems, Foster City, USA), forward and reverse primers, a probe and 2 µl of cDNA (diluted 1 in 8 in nuclease free water) or 2 µl of RNA (6.25 µg/mL) was made up to 25 µl with nuclease-free water. The primers and probe were used in a final reaction concentration of 50 nM for rRNA, while final reaction concentrations of 900 and 100 nM were used for primers and probe, respectively, for Ob-Rb.

Real-time polymerase chain reaction amplifications occurred using the following conditions: 50°C for 2 min then 95°C for 10 min, followed by 40 cycles of both 95°C for 15 s and 1 min at 60°C. Concentration of the target sequence was determined as the threshold cycle (the cycle at which emission intensity rises above baseline values), which is inversely proportional to the target sequence concentration.

Statistical analyses

Results are presented as mean±s.e.m. Most comparisons between ovariectomized and sham rats at different time points were made using two-way ANOVA with two factors—time and estrogen status—followed by post-hoc least-significant difference (LSD) tests to compare individual means (SPSS for Windows 8.0). Results from the leptin infusion studies were analyzed by repeated measures general linear model tests with two factors—estrogen status and leptin dose (SPSS for Windows 8.0). To detect individual differences, these were followed by either one-way ANOVA with post-hoc LSD tests (for comparisons between leptin doses), or Student t-tests (for single comparisons between ovariectomy and sham rats). The study protocol was approved by the Royal Melbourne Hospital's Animal Ethics Committee.

Results

Adiposity and energy balance

Figure 1 illustrates the increased rate of weight gain that occurred soon after the ovariectomy procedure. This rapid weight gain had stabilized by 7 weeks and thereafter weight remained 12–13% higher than sham animals (238±4 g vs 267±7 g at 12 weeks, n=24, P<0.001). DEXA measurements revealed that abdominal and peripheral fat were significantly increased in the ovariectomized animals at both 2 and 7 weeks after the operation. At 2 weeks, peripheral fat was significantly greater in ovariectomized (22.5±0.9 g) compared to sham animals (18.2±1.5 g, P<0.05, n=4 in all groups) and there was a trend for ovariectomized rats to have more abdominal fat (14.6±1.9 g) compared to sham rats (11.4±0.8 g, P=0.17). By 7 weeks, both peripheral and abdominal fat levels were significantly higher in ovariectomized compared to sham animals (32.6±2.7 vs 21.4±3.5 g peripheral fat, 25.6±3.9 vs 13.9±1.4 g abdominal fat, both P<0.05). In agreement with the DEXA analysis, subcutaneous fat depot weights were significantly heavier (64%, P<0.001) and there was a trend for infrarenal fat to be elevated in ovariectomized animals (P=0.09) at 2 weeks in the ovariectomized animals compared to sham animals (Table 1). In contrast, no differences in interscapular BAT weights between ovariectomized and sham animals were detected at 2 weeks. By 12 weeks, infrarenal WAT had increased by 152% (P<0.005), subcutaneous WAT had increased by 150% (P<0.0001), and interscapular BAT mass was 16% heavier in ovariectomized animals compared to sham animals (P<0.05, Table 1).

Figure 1
figure1

Body weight in ovariectomized rats (closed squares, n=24) and sham rats (open circles, n=24) expressed as a mean over 4 days±s.e.m. Significant differences in body weight were observed from day 10 onwards (*P<0.05).

Table 1 Mass of subcutaneous and infrarenal white adipose tissue (WAT) and interscapular brown adipose tissue (BAT) measured 2 or 12 weeks after ovariectomy (ovex) or sham operations

Table 2 illustrates the transient hyperphagia, persistent low levels of spontaneous physical activity, and unchanged resting energy expenditure in the ovariectomized rats compared to sham rats. Energy intake in estrogen-deficient rats was 16% higher only 4 days after the operation (P<0.005, n=24); however, this hyperphagia had disappeared by 7 weeks (Table 1), causing the rate of body weight gain to stabilize. Ambulatory physical activity levels were 22% lower in ovariectomized rats only 2 weeks after the operation (P<0.005, n=7–10) and remained at least 25% lower throughout the study (P<0.05, n=6–10). In contrast, resting energy expenditure was not lowered by ovariectomy when expressed either as kcal/day or kcal/day/kg body weight (Table 2). Rates of whole body fat oxidation and glucose oxidation were also not significantly different between ovariectomized and sham rats (Table 2).

Table 2 Energy intake, ambulatory spontaneous physical activity (SPA), resting energy expenditure (REE), fat oxidation and glucose oxidation, 2 weeks, 7 weeks and 12 weeks after ovariectomy (ovex) and sham operations

Subcutaneous implantation of a 17β-estradiol releasing pellet at the time of ovariectomy prevented all the abnormalities in energy balance seen in ovariectomised rats; estradiol-treated ovariectomised rats and sham rats had similar body weights, energy intakes and physical activity levels two and 12 weeks after the operations (data not shown). In contrast, treating sham-operated rats with estradiol had no effect on body weight, body fat or energy balance compared to sham rats treated with placebo pellets. Plasma estradiol levels in ovariectomized animals were negligible (0.06±0.01 pg/ml), while estrogen treatment of ovariectomized and sham rats increased circulating estradiol concentrations to a level within the range reported in the literature in intact rats37 (ovex estrogen-treated, 17±6 pg/ml, sham estrogen-treated, 20±5 pg/ml; sham, 2 pg/ml (diestrus) to 30 pg/ml (proestrus)).

Circulating leptin levels and ob gene expression

Ovariectomy did not lead to a decrease in circulating plasma leptin levels at any time point (Figure 2). Circulating leptin levels were not different between ovariectomized and sham animals despite significant weight gain in the ovariectomized animals only 10 days after ovariectomy (ovex, 209±3 g; sham, 196±3 g; P<0.01, n=24 in both groups). However, as body weight increased further, circulating leptin levels progressively increased so that by 7 and 12 weeks plasma leptin levels were 55 and 66% higher, respectively (both P<0.05), in the ovariectomized rats compared to the sham rats. At 12 weeks, ob mRNA expression was not different between ovariectomized and sham animals in infrarenal WAT, subcutaneous WAT or interscapular BAT (Table 3). 17β-estradiol treatment had no significant effect on ob mRNA expression in infrarenal WAT in either intact or ovariectomized animals; however estradiol treatment lowered ob gene expression in subcutaneous WAT in sham-treated (P<0.01), but not ovariectomized rats. In interscapular BAT adipose tissue, ob gene expression was lower in ovariectomized estradiol-treated rats compared to ovariectomized placebo rats (Table 3).

Figure 2
figure2

Circulating plasma leptin concentrations in ovariectomized rats (closed squares, n=10–16) and sham rats (open circles, n=10–14) expressed as mean±s.e.m. *P<0.05 ovariectomized vs sham rats.

Table 3 Ob mRNA expression (relative units) in infrarenal and subcutaneous white adipose tissue (WAT) and in interscapular brown adipose tissue (BAT) 12 weeks after ovariectomy (n=9–10) or sham operations (n=9)

Hypothalamic NPY concentration

Table 4 shows that hypothalamic NPY concentration in the paraventricular nucleus (PVN) was increased by 17% in ovariectomized rats compared to sham rats at 2 weeks (P<0.05) when the rats were clearly hyperphagic (Table 2). By 7 weeks after the operation, NPY concentration in the PVN of the ovariectomized animals had decreased (P<0.05) and was equivalent to sham levels (Table 4). This coincided with a normalization of energy intake (Table 2). No differences in hypothalamic NPY concentration were detected in any of the other hypothalamic regions at either time.

Table 4 Hypothalamic NPY concentration in the arcuate nucleus, paraventricular nucleus (PVN), anterior, posterior, pre-optic and total hypothalamus at 2 weeks (n=14 in both groups) and 7 weeks (n=9 in both groups) after ovariectomy (ovex) or sham operations

Central sensitivity to leptin

Figure 3 shows the effect of saline and leptin on body weight, energy intake and spontaneous physical activity over a 17 h period between 3 and 20 h after administration. This time period was chosen because the impact of leptin was greatest in sham rats between these time points. Saline infusion produced an unexpected decrease in body weight and energy intake in ovariectomized animals compared to sham animals (P<0.05, Figure 3A and Figure 3B). Interestingly, some other studies that have reported leptin insensitivity in obese rodents have shown a similar decrease in food intake in response to an i.c.v. saline infusion, which is absent in lean animals.38,39 The anorexia induced by saline in obese rodents is, however, much less dramatic than the anorexia that can be induced by leptin in lean rodents. In our study, energy intake in the ovariectomised rats after an i.c.v. saline infusion was 43 kJ or 28% less than energy intake in ovariectomized rats not given an i.c.v. saline infusion (109 vs 152 kJ/17 h), whereas 0.5 µg leptin is capable of causing a 74 kJ or 46% decrease in food intake in sham rats over the same time period (Figure 3B). Importantly, at both 0.5 and 3 µg of leptin, energy intake was suppressed to a greater extent in sham rats compared to ovariectomized rats (Figure 3B, P<0.05), suggesting that ovariectomy was associated with a decrease in leptin sensitivity. Further evidence of a blunted response to leptin in the ovariectomised rats can be seen in Figure 3C, which shows that infusion of both 0.5 and 3 µg of leptin significantly increased physical activity in sham animals compared to saline infusion (52 and 54%, respectively, both P<0.005), whereas leptin did not significantly increase spontaneous physical activity in the ovariectomized rats. At both leptin doses, ovariectomized animals were 27% less active than sham animals indicating a decrease in leptin sensitivity (Figure 3C).

Figure 3
figure3

Effect of saline, 0.5 and 3 μg i.c.v. leptin on body weight (A), energy intake (B) and ambulatory spontaneous physical activity (SPA) (C) over 17 h (between 3 and 20 h) after infusion. Data for sham animals is shown by open squares (n=8) while data for ovariectomized animals is shown by closed squares (n=8). *P<0.05 and **P<0.01 ovariectomized vs sham, §P<0.05 and §§P<0.005 sham given 0.5 and 3 µg leptin doses vs saline-infused sham rats.

Hypothalamic leptin receptor mRNA expression

Figure 4 shows that levels of mRNA expression of the Ob-Rb isoform of the leptin receptor in the hypothalamus were not significantly different between ovariectomized and sham animals at both 2 and 12 weeks.

Figure 4
figure4

Effect of ovariectomy on the expression of the Ob-Rb isoform of the leptin receptor in the hypothalamus 2 and 12 weeks after ovariectomy and sham operations. Data for sham animals is shown in white (n=7) while data for ovariectomized animals is shown in black (n=7–8). Ob-Rb expression is expressed in arbitrary units relative to rRNA as a mean±s.e.m.

Discussion

Many interesting observations about the effect of estrogen on body weight can be drawn from this study. In female rats, the rapid weight gain and resulting mild obesity induced by ovariectomy is due to increased food intake and decreased spontaneous physical activity, but not reduced resting energy expenditure. Normal resting energy expenditure in these rats might be expected based on the modest effect of ovariectomy on the mass of brown adipose tissue, a tissue that plays a critical role in energy homeostasis in the rat. Estradiol treatment completely normalized all the abnormalities in energy balance induced by ovariectomy making it likely that estrogen deficiency is responsible for the energy imbalance rather than other consequences of ovariectomy, such as reduced androgen or progesterone levels. Hyperphagia in the estrogen-deficient rats was transient, however, obesity was maintained in these rats by decreased spontaneous physical activity. The transient changes in energy intake coincided with transient changes in NPY concentration in the paraventricular nucleus of the hypothalamus. Increased body weight in estrogen-deficient rats was found to be associated with central leptin insensitivity.

Although data in the literature raises the possibility that hypoleptinemia contributes to the obesity induced by estrogen deficiency,13,14,15,16,17,18 we found no evidence of decreased leptin secretion in our ovariectomized rats. In fact, once body weight increased by more than 8%, plasma leptin levels began to rise, and by 7 weeks the rats were clearly hyperleptinemic with levels over 50% greater than sham rats. Our data confirms a recent study published by Pelleymounter et al20 showing that leptin levels are not independently altered by either estradiol administration or ovariectomy.

This hyperleptinemia associated with excess body fat suggests that estrogen-deficient rats may be leptin insensitive. Our study has confirmed this is the case by showing that the effect of central leptin administration on food intake and physical activity in ovariectomized rats is significantly less than sham rats. The presence of central leptin insensitivity in the ovariectomized rat indicates a defect at, or downstream of, the hypothalamic Ob-Rb leptin receptor. We found no differences in Ob-Rb mRNA expression between ovariectomized and sham animals, suggesting that a defect downstream of the leptin receptor itself may be responsible for inducing central leptin insensitivity. Our results contrast with Pelleymounter et al20 who showed that 14 days of peripheral leptin infusion (120 µg/day s.c.) produced similar weight loss in obese ovariectomized and lean sham mice. However, their studies on ovariectomized and sham mice, conducted before body weight diverged, showed that a much higher leptin dose of 5.6 µg/day was required for body fat reduction in ovariectomized mice compared to only 1.4 µg/day in sham mice (P<0.006) which can be interpreted as reduced leptin sensitivity in the ovariectomized mice. Differences in leptin sensitivity reported in our study and Pelleymounter's study may also be because estradiol levels fell to negligible values in our ovariectomized rats whereas they only fell by 59% in the ovariectomized mice.

We found that estrogen deficiency caused an increase in NPY concentration in the paraventricular nucleus of the hypothalamus where NPY nerve terminals originating in the arcuate nucleus are particularly dense.40 This complements other studies showing elevated NPY mRNA expression in the arcuate nucleus of the hypothalamus in ovariectomized animals.20 Our results are also consistent with those of Bonavera et al27 who showed that 17β-estradiol treatment decreases hypothalamic NPY levels selectively in the paraventricular nucleus in ovariectomized rats. Further suggestive evidence of a link between estrogen and NPY has been demonstrated by Sar et al41 who showed colocalization of 3 H-estradiol and NPY immunoreactivity in some neurons in the arcuate nucleus, which suggests a direct genomic modulation of NPY neurosecretion by estrogens in the hypothalamus. Alternatively, increased NPY may be a result of leptin insensitivity in our rats, since leptin has been shown to be a potent inhibitor of NPY synthesis, levels and release in the ARC-PVN projection.42,43,44 As ovariectomized rats progressively gained weight, we found that NPY concentration in the PVN returned to normal levels and this coincided with a normalization of energy intake in the rats. The observation that hyperphagia is transient in ovariectomized rats has been previously reported.45 The reason why food intake normalizes as the animals get fatter is not known, but may be because circulating leptin concentrations reach a sufficiently high level to inhibit NPY. Therefore NPY is one factor which modulates the food intake response to estrogen, however, our data does not rule out the possibility that other hormones (such as insulin and glucocorticoids) and neuropeptides (such as α-MSH, CART and MCH) also play a role.

Our finding that central leptin administration is capable of increasing physical activity in sham-operated rats is of interest because there is some discrepancy in the literature as to whether leptin is capable of altering physical activity in human subjects46,47 and an effect in animals has previously only been shown in the highly leptin sensitive ob/ob mouse.48

Physical inactivity rather than hyperphagia or reduced resting energy expenditure appeared to maintain body weight at an elevated level in estrogen-deficient rats. This data is consistent with longitudinal studies in women showing that subjects undergoing the transition to menopause have reduced physical activity compared to subjects who remained pre-menopausal.49 It is also consistent with studies showing reduced physical activity levels in obese aromatase-deficient mice that cannot synthesize endogenous estrogens.6 The reason why reduced estrogen is associated with inactivity is not known. It could be argued that reduced physical activity in the ovariectomized rats is simply a result of their increased body weight; however, we do not believe this is the case because body weight does not consistently correlate with activity level in our rats. For example, body weight in the ovariectomized rats at 2 weeks (212±3 g) was the same as body weight in the sham rats at 7 weeks (221±5 g, P=NS), yet the ovariectomized rats were still less active than the sham rats (ovex at 2 weeks: 24281±1090 vs sham at 7 weeks, 29933±2302 movements/day; P<0.05, Figure 1 and Table 2).

Unlike changes in food intake, the abnormality in physical activity caused by estrogen deficiency persisted over time. This may be because physical activity is less responsive to circulating leptin levels than food intake. In support of this, we found that the suppressive effect of 3 µg leptin on food intake was still evident after 44 h in sham rats, whereas the stimulatory effect on physical activity had disappeared, suggesting that leptin has a more robust effect on food intake than physical activity (data not shown).

Our study outlines some mechanisms that contribute to excess fat accumulation in states of estrogen insufficiency. One of these is impaired sensitivity to central leptin administration in the absence of altered Ob-Rb leptin receptor mRNA expression, suggesting defective leptin signaling lying downstream of the hypothalamic leptin receptor. We also determined that transient elevations in food intake caused by estrogen deficiency appear to be due, at least in part, to transient changes in hypothalamic NPY concentration. Finally, our study has identified two biological factors that influence spontaneous physical activity levels, estrogen deficiency and leptin treatment, which can decrease and increase spontaneous physical activity, respectively. This is of great interest given the paucity of information known about the biological regulation of spontaneous physical activity levels.

References

  1. 1

    Tremollieres FA, Pouilles JM, Ribot CA . Relative influence of age and menopause on total and regional body composition changes in postmenopausal women Am J Obstet Gynecol 1996 175: 1594–1600.

  2. 2

    Tchernof A, Poehlman ET, Despres JP . Body fat distribution, the menopause transition, and hormone replacement therapy Diabetes Metab 2000 26: 12–20.

  3. 3

    Lean ME, Han TS, Seidell JC . Impairment of health and quality of life in people with large waist circumference Lancet 1998 351: 853–856.

  4. 4

    Stevenson JC, Crook D, Godsland IF, Collins P, Whitehead MI . Hormone replacement therapy and the cardiovascular system. Nonlipid effects. Drugs 1994 47: 35–41.

  5. 5

    Heine PA, Taylor JA, Lubahn DB, Cooke PS . Increased adipose tissue in male and female estrogen receptor-a knockout mice Proc Natl Acad Sci USA 2000 97: 12729–12734.

  6. 6

    Jones MEE, Thorburn AW, Britt KL, Hewitt KN, Wreford NG, Proietto J, Oz O, Leury BJ, Robertson KM, Yao S, Simpson ER . Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity Proc Natl Acad Sci USA 2000 97: 12735–12740.

  7. 7

    Grumbach MM, Auchus RJ . Estrogen: consequences and implications of human mutations in synthesis and action J Clin Endocrinol Metab 1999 84: 4677–4694.

  8. 8

    Ley CJ, Lees B, Stevenson JC . Sex- and menopause-associated changes in body-fat distribution Am J Clin Nutr 1992 55: 950–954.

  9. 9

    Toth MJ, Tchernof A, Sites CK, Poehlman ET . Effect of menopausal status on body composition and abdominal fat distribution Int J Obes Relat Metab Disord 2000 24: 226–231.

  10. 10

    Nolan C, Proietto J . The effects of oophorectomy and female sex steroids on glucose kinetics in the rat Diabetes Res Clin Pract 1996 30: 181–188.

  11. 11

    Kakolewski JW, Cox VC, Valnstein ES . Sex differences in body-weight change following gonadectomy in rats Psychol Rep 1968 22: 547–554.

  12. 12

    Wade GN . Gonadal hormones and behavioral regulation of body weight Physiol Behav 1972 8: 523–534.

  13. 13

    Murakami T, Iida M, Shima K . Dexamethasone regulates obese expression in isolated rat adipocytes Biochem Biophys Res Commun 1995 214: 1260–1267.

  14. 14

    Casabiell X, Pineiro V, Peino R, Lage M, Camina J, Gallego R, Vallejo LG, Dieguez C, Casanueva FF . Gender differences in both spontaneous and stimulated leptin secretion by human omental adipose tissue in vitro: dexamethasone and estradiol stimulate leptin release in women, but not in men J Clin Endocrinol Metab 1998 83: 2149–2155.

  15. 15

    Riad-Gabriel MG, Jinagouda SD, Sharma A, Boyadjian R, Saad MF . Changes in plasma leptin during the menstrual cycle Eur J Endocrinol 1998 139: 528–531.

  16. 16

    Mannucci E, Ognibene A, Becorpi A, Cremasco F, Pellegrini S, Ottanelli S, Rizzello SM, Massi G, Messeri G, Rotella CM . Relationship between leptin and estrogens in healthy women Eur J Endocrinol 1998 139: 198–201.

  17. 17

    Machinal F, Dieudonne MN, Leneveu MC, Pecquery R, Giudicelli Y . In vivo and in vitro ob gene expression and leptin secretion in rat adipocytes: evidence for a regional specific regulation by sex steroid hormones Endocrinology 1999 140: 1567–1574.

  18. 18

    Yoneda N, Saito S, Kimura M, Yamada M, Iida M, Murakami T, Irahara M, Shima K, Aono T . The influence of ovariectomy on ob gene expression in rats Horm Metab Res 1998 30: 263–265.

  19. 19

    Watanobe H, Suda T . A detailed study on the role of sex steroid milieu in determining plasma leptin concentrations in adult male and female rats Biochem Biophys Res Commun 1999 259: 56–59.

  20. 20

    Pelleymounter MA, Baker MB, McCaleb M . Does estradiol mediate leptin's effects on adiposity and body weight? Am J Physiol 1999 276: E955–E963.

  21. 21

    Bishop J, Simpkins JW . Estradiol enhances brain glucose uptake in ovariectomized rats Brain Res Bull 1995 36: 315–320.

  22. 22

    Pedersen SB, Borglum JD, Moller-Pedersen T, Richelsen B . Effects of in vivo estrogen treatment on adipose tissue metabolism and nuclear estrogen receptor binding in isolated rat adipocytes Mol Cell Endocrinol 1992 85: 13–19.

  23. 23

    Livingstone C, Lyall H, Gould GW . Hypothalamic GLUT 4 expression: a glucose- and insulin-sensing mechanism? Mol Cell Endocrinol 1995 107: 67–70.

  24. 24

    Bruning JC, Gautam D, Burks DJ, Gillette J, Schubert M, Orban PC, Klein R, Krone W, Muller-Wieland D, Kahn CR . Role of brain insulin receptor in control of body weight and reproduction Science 2000 289: 2122–2125.

  25. 25

    Shimizu H, Ohtani K, Kato Y, Tanaka Y, Mori M . Withdrawal of estrogen increases hypothalamic neuropeptide Y (NPY) mRNA expression in ovariectomized obese rat Neurosci Lett 1996 204: 81–84.

  26. 26

    Haas DA, George SR . Gonadal regulation of corticotropin-releasing factor immunoreactivity in hypothalamus Brain Res Bull 1988 20: 361–367.

  27. 27

    Bonavera JJ, Dube MG, Kalra PS, Kalra SP . Anoretic effects of estrogen may be mediated by decreased neuropeptide-Y release in the hypothalamic paraventricular nucleus Endocrinology 1994 134: 2367–2370.

  28. 28

    Roy BN, Reid RL, Van Vugt DA . The effects of estrogen and progesterone on corticotropin-releasing hormone and arginine vasopressin messenger ribonucleic acid levels in the paraventricular nucleus and supraoptic nucleus of the rhesus monkey Endocrinology 1999 140: 2191–2198.

  29. 29

    Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P . Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks Science 1995 269: 546–549.

  30. 30

    Cusin I, Rohner-Jeanrenaud F, Stricker-Krongrad A, Jeanrenaud B . The weight-reducing effect of an intracerebroventricular bolus injection of leptin in genetically obese fa/fa rats. Reduced sensitivity compared with lean animals. Diabetes 1996 45: 1446–1450.

  31. 31

    Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, Friedman JM . Physiological response to long-term peripheral and central leptin infusion in lean and obese mice Proc Natl Acad Sci USA 1997 94: 8878–8883.

  32. 32

    Heffernan MA, Jiang WJ, Thorburn AW, Ng FM . Effects of oral administration of a synthetic fragment of human growth hormone on lipid metabolism Am J Physiol Endocrinol Metab 2000 279: E501–E507.

  33. 33

    Hope PJ, Turnbull H, Breed W, Morley JE, Horowitz M, Wittert GA . The effect of ovarian steroids and photoperiod on body fat stores and uncoupling protein 2 in the marsupial sminthopsis crassicaudata Physiol Behav 2000 69: 463–470.

  34. 34

    Thorburn AW, Baldwin ME, Rosella G, Zajac J, Fabris S, Song S, Proietto J . Features of Syndrome X develop in transgenic rats expressing a non-insulin responsive phosphoenolpyruvate carboxykinase gene Diabetologia 1999 42: 419–426.

  35. 35

    Morris MJ, Tortelli CF, Filippis A, Proietto J . Reduced BAT function as a mechanism for obesity in the hypophagic, neuropeptide Y deficient monosodium glutamate-treated rat Regulatory Peptides 1998 75–76: 441–447.

  36. 36

    Chomczynski P, Sacchi N . Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal Biochem 1987 162: 156–159.

  37. 37

    Brown-Grant K, Exley D, Naftolin F . Peripheral plasma oestradiol and luteinizing hormone concentrations during the oestrous cycle of the rat J Endocrinol 1970 48: 295–296.

  38. 38

    Seeley RJ, van Dijk G, Campfield LA, Smith FJ, Burn P, Nelligan JA, Bell SM, Baskin DG, Woods SC, Schwartz MW . Intraventricular leptin reduces food intake and body weight of lean rats but not obese Zucker rats Horm Metab Res 1996 28: 664–668.

  39. 39

    Widdowson PS, Upton R, Buckingham R, Arch J, Williams G . Inhibition of food response to intracerebroventricular injection of leptin is attenuated in rats with diet-induced obesity Diabetes 1997 46: 1782–1785.

  40. 40

    Akabayashi A, Watanabe Y, Wahlestedt C, McEwen BS, Paez X, Leibowitz SF . Hypothalamic neuropeptide Y, its gene expression and receptor activity: relation to circulating corticosterone in adrenalectomized rats Brain Res 1994 665: 201–212.

  41. 41

    Sar M, Sahu A, Crowley WR, Kalra SP . Localization of neuropeptide-Y immunoreactivity in estradiol-concentrating cells in the hypothalamus Endocrinology 1990 127: 2752–2756.

  42. 42

    Schwartz MW, Baskin DG, Bukowski TR, Kuijper JL, Foster D, Lasser G, Prunkard DE, Porte D Jr, Woods SC, Seeley RJ, Weigle DS . Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice Diabetes 1996 45: 531–535.

  43. 43

    Wang Q, Bing C, Al-Barazanji K, Mossakowaska DE, Wang XM, McBay DL, Neville WA, Taddayon M, Pickavance L, Dryden S, Thomas ME, McHale MT, Gloyer IS, Wilson S, Buckingham R, Arch JR, Trayhurn P, Williams G . Interactions between leptin and hypothalamic neuropeptide Y neurons in the control of food intake and energy homeostasis in the rat Diabetes 1997 46: 335–341.

  44. 44

    Lee J, Morris MJ . Modulation of neuropeptide Y overflow by leptin in the rat hypothalamus, cerebral cortex and medulla Neuroreport 1998 9: 1575–1580.

  45. 45

    Tarttelin MF, Gorski RA . Variations in food and water intake in the normal and acyclic female rat Physiol Behav 1971 7: 847–852.

  46. 46

    Salbe AD, Nicolson M, Ravussin E . Total energy expenditure and the level of physical activity correlate with plasma leptin concentrations in five-year-old children J Clin Invest 1997 99: 592–595.

  47. 47

    Farooqi IS, Jebb SA, Langmack G, Lawrence E, Cheetham CH, Prentice AM, Hughes IA, McCamish MA, O'Rahilly S . Effects of recombinant leptin therapy in a child with congenital leptin deficiency New Engl J Med 1999 341: 879–884.

  48. 48

    Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F . Effects of the obese gene product on body weight regulation in ob/ob mice Science 1995 269: 540–546.

  49. 49

    Poehlman ET, Tchernof A . Traversing the menopause: changes in energy expenditure and body composition Coronary Artery Dis 1998 9: 799–803.

Download references

Acknowledgements

We would like to thank Sue Fabris and Melanie Ball from the University of Melbourne for technical assistance, and Greg Collier from Deakin University for access to the real-time PCR equipment. This study was supported by a National Health and Medical Research Council of Australia Project Grant (970400) and a Novo Nordisk Regional Support Scheme Grant. Deborah Ainslie is supported by a Melbourne University Research Scholarship.

Author information

Correspondence to AW Thorburn.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ainslie, D., Morris, M., Wittert, G. et al. Estrogen deficiency causes central leptin insensitivity and increased hypothalamic neuropeptide Y. Int J Obes 25, 1680–1688 (2001). https://doi.org/10.1038/sj.ijo.0801806

Download citation

Keywords

  • estrogen
  • leptin receptor
  • neuropeptide Y
  • motor activity
  • energy intake
  • energy metabolism

Further reading