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Endocrinology, doi:10.1210/en.2007-0572
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Endocrinology Vol. 149, No. 3 1399-1406
Copyright © 2008 by The Endocrine Society

Impairment of the Natriuretic Peptide System in Follitropin Receptor Knockout Mice and Reversal by Estradiol: Implications for Obesity-Associated Hypertension in Menopause

Najara O. Belo, M. Ram Sairam and Adelina M. dos Reis

Department of Physiology and Biophysics (N.O.B., A.M.d.R.), Institute of Biological Sciences, Federal University of Minas Gerais, CEP 31270-901, Belo Horizonte, Minas Gerais, Brazil; Multidisciplinary Institute of Health (N.O.B.), Federal University of Bahia, 40170-290 Salvador, Bahia, Brazil; and Molecular Reproduction Research Laboratory (N.O.B., M.R.S.), Clinical Research Institute of Montreal, Quebec, Canada H3A 2B4

Address all correspondence and requests for reprints to: Dr. Adelina M. dos Reis, Departemento Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antonio Carlos 6627, CEP 31270–901, Belo Horizonte, Minas Gerais, Brazil. E-mail: adelina{at}icb.ufmg.br.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen is considered a major regulator of adipose tissue in females. Estrogen increases circulating levels of atrial natriuretic peptide (ANP), a hormone with renal and cardiovascular effects. The aim of this study was to determine the status of the natriuretic peptide system in female follitropin-receptor knockout (FORKO) mice that could be associated with obesity and hypertension observed in these mutants. Furthermore, estradiol treatment was used to reverse alterations observed. FORKO and wild-type (WT) mice received daily injections of estradiol for 4 d. On the fifth day, blood was collected for determination of plasma ANP levels, and selected tissues were collected for determination of ANP, natriuretic peptide receptor type-A (NPR-A) and type-C (NPR-C) gene expression by RT-PCR and binding of [125I]ANP by autoradiography. At 5 months of age, FORKO mice were heavier and had more adipose tissue than WT mice. FORKO mice had lower plasma ANP levels and atrial ANP gene expression and higher renal and adipocyte NPR-C gene expression than WT mice. Estradiol treatment reduced weight gain and increased atrial ANP synthesis as well as decreased ANP clearance NPR-C receptors, resulting in elevation of circulating ANP level. In conclusion, this study shows that FORKO females have an impaired natriuretic peptide system, which may contribute to the susceptibility of FORKO mice to developing age-related hypertension previously shown in these animals. This study establishes a relation between estrogen, adipose tissue, and ANP, which may have important implications in menopausal women.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN IS A MAJOR factor in regulating adipose development and deposition in females. Postmenopausal women typically experience increased fat mass (1), which correlates with the lower 17β-estradiol (E2) levels (2) and contributes to the development of several chronic diseases, including hypertension. Estrogen replacement therapy decreases visceral adipose tissue (3). Estrogen also induces a rise in the circulating levels of atrial natriuretic peptide (ANP) in postmenopausal women (4). Estradiol stimulates ANP secretion in isolated perfused rat atria (5) and increases gene expression and atrial concentration of ANP (6, 7). We have recently shown that ANP is involved in the reduction of blood pressure induced by E2 treatment in spontaneously hypertensive rats (8). The blood pressure reduction was accompanied by an increase of atrial ANP synthesis and content as well as release (8). ANP binds to specific natriuretic peptide receptors (NPR), either coupled (NPR-A) or uncoupled (NPR-C) with guanylyl cyclase activity. NPR-A mediates most of the biological effects of ANP, which exerts a wide array of effects on cardiovascular and renal function, such as natriuresis, diuresis, and antagonism of the renin-angiotensin and the sympathetic nervous systems (9). ANP is cleared from the circulation by NPR-C, which is abundantly expressed in kidneys and adipocytes (10, 11). Adipose tissue secretes ANP (12) and has natriuretic peptide binding sites (13). NPR-A and NPR-C have been identified in rat as well as in human adipose tissue (14, 15). The adipose tissue abundance of NPR-C (10) could explain the low circulating levels of ANP observed in obese individuals (16).

The FSH receptor knockout (FORKO) female mouse provides a useful model to examine the lack of estrogen’s effect in the development of common features observed in postmenopausal women, such as obesity and hypertension. Recent work has identified the FORKO mouse as a novel model of menopause-associated hypertension (17) that is not mediated through activation of the renin-angiotensin system. Further characterization of the factors involved in the development of obesity and hypertension in these mice may allow for a clearer understanding of the pathophysiology of the processes underlying the most frequent features found in menopause. FORKO female mice also are a very interesting model for investigating the effects of estrogen replacement therapy. Targeted disruption of the FSH receptor causes a gene dose-related endocrine and gametogenic abnormality in female mice. The resulting FORKO mutants have other characteristics similar to postmenopausal women such as infertility and skeletal abnormality (18). In FORKO mice lacking ovarian estrogen, the estrogen receptors remain fully functional. The administration of E2 induces uterine growth and reverses adipose tissue accumulation (19), showing that genetically altered FSH receptor mutants are an excellent experimental model to explore the effects of estrogen on different targets.

Obesity is a major risk factor for hypertension, and the mechanisms linking obesity to the development of hypertension have not been fully elucidated. The mechanisms underlying the increased blood pressure in FORKO mice do not appear to be angiotensin II dependent (17). Because the natriuretic peptide system plays a key role in the regulation of blood pressure, it has been speculated that obese individuals have an impaired natriuretic peptide system (20). Thus, we sought to determine alterations of the natriuretic peptide system, a family of hormones that usually have actions opposite to angiotensin II, to try to explain the development of hypertension in FORKO mice. In addition we studied the effect of estrogen replacement therapy in this model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The study was approved by the Animal Ethics Committee of the Clinical Research Institute of Montreal and carried out according to the recommendations of the Canadian Council for Animal Care. FORKO mice were obtained as previously described (21). Animals were housed under controlled temperature and constant cycle of light (12 h light, 12 h darkness), with food and water provided ad libitum. The female mice were genotyped by PCR according to methods described (18). Two- and 5-month-old mutants and wild-type (WT) mice were compared in each experiment.

Steroid hormone replacement therapy
The potential effect of estrogen replacement in FORKO female mice was studied by treating them with estradiol-17β (Sigma Chemical Co., St. Louis, MO) for a short period of time. FORKO or WT mice were given one daily sc injection of the hormone (1 µg) or olive oil (control) for 4 d and were killed 24 h after the last injection.

Collection of blood, atrial appendages, kidney, and adipose tissue
Mice were decapitated, and blood was collected in prechilled tubes containing 10–5 mol/liter EDTA, 10–5 mol/liter phenylmethylsulfonyl fluoride, and 0.5 x 10–5 mol/liter pepstatin A, reagents purchased from Sigma. Blood was centrifuged at 4 C, 1500 x g, for 15 min, and plasma was stored at –80 C until ANP and E2 determination by RIA. Right and left atrial appendages, kidneys, and mesenteric and parametrial adipose tissue were removed, frozen in liquid nitrogen, and stored at –80 C until used.

RT-PCR
RNA from right and left atrial appendages, adipose tissue, and kidney was extracted using Trizol, and 2 µg was reverse transcribed under standard conditions in a 20-µl reaction using Maloney murine leukemia virus reverse transcriptase. Forward and reverse primers for ANP, NPR-A, and NPR-C mRNA were based on sequences previously published. For amplifying ANP (425 bp, sequence 90–515) the oligonucleotides 5'-CAGCATGGGCTCCTTCTCCA-3' and 5'-TCCGCTCTGGGCTCCAATCCT-3' were used as forward and reverse primers, respectively (22). The forward and reverse primers used for amplifying NPR-A (632 bp, sequence 966-1598) were 5'-CTCAACATCACAGTAAATCACC-3' and 5'-CCTGAAGGCACCTGTCTCG-3' (23). And for NPR-C (492 bp, sequence 279–771), the primers were 5'-CTTCCAGGTGGCCTACGAA-3' and 5'-GGCACACATGATCACCACTC-3' (24). The PCR amplifications were performed with the following conditions: 95 C for 5 min followed by 29 cycles at 94 C for 30 sec, 60 C for 45 sec, and 72 C for 1 min for ANP and NPR-A and 31 cycles at 94 C for 30 sec, 58 C for 45 sec, and 72 C for 1 min for NPR-C. Final extension was performed for 10 min. All reaction products were separated on a 1.5% agarose gel and stained with ethidium bromide. β-Actin was amplified (514 bp) as an internal control in each PCR test using specific primers as described (25) to provide a semiquantitative assessment. The result is an average of three PCRs. The reaction conditions were identical to those described by Krishnamurthy et al. (26).

RIAs
To quantify plasma ANP levels, samples were extracted using Sep-Pak C-18 cartridges (Waters Corp., Milford, MA) as previously described (27). After drying in a Speed-Vac (Eppendorf 5301, Hamburg, Germany), the samples were dissolved in 500 µl phosphosaline buffer and the ANP levels were determined by RIA. E2 was determined using a Maia RIA kit (Adaltis Italia S.A., Rome, Italy). All samples were measured in the same assay where the intraassay coefficient of variation was 8%.

Autoradiography
ANP receptor autoradiography was described in detail previously (28). Essentially, mice were killed by decapitation, and the mesenteric adipose tissue was rapidly isolated and snap-frozen in isopentane at –18 C, mounted on cryostat chucks, and cut into 14-µm-thick sections at –30 C. Sections were thaw-mounted on precleaned gelatin-coated slides and then stored at –80 C until used. Frozen slide-mounted adipose tissue sections were preincubated for 15 min at 25 C in 50 mM Tris-HCl buffer (pH 7.4), containing 0.1% polyethylenimine to reduce binding of [125I]ANP to gelatin-coated slides. Sections were then incubated at 25 C with 50 mM Tris-HCl buffer (pH 7.4), containing 150 mM NaCl, 5 mM MgCl2, 40 pg/ml bacitracin, 0.5% BSA, and 50 pM [125I]ANP. The ability of 10–12 to 10–6 M des[Gln18, Ser19, Gly20, Leu21, Gly22]ANP-(4–23)-NH2 (C-ANF; Bachem, Torrance, CA) to displace specific radiolabeled NP binding was determined. C-ANF is a truncated ANP that binds only to NPR-C in mammals (29).

After 1 h incubation, slides were placed in racks and transferred sequentially through four rinses, 1 min each, of cold 50 mM Tris-HCl buffer (pH 7.4) and finally dipped in distilled water to wash out salts. Slides were rapidly dried and exposed to PhosphorImager (Fuji, BAS-1000), and the images were analyzed using the Image Gauge 3.12 software.

Statistical analysis
Data are expressed as mean ± SEM and analyzed using GraphPad software. Data were compared using one-way ANOVA followed by Newman-Keuls post hoc test. Correlations between E2 plasma levels and ANP plasma levels, E2 plasma levels and mesenteric adipose tissue weight, and ANP plasma levels and mesenteric adipose tissue weight were calculated using two-tailed Pearson’s correlation coefficients. Data were considered statistically significant at the P < 0.05 level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Table 1Go shows that body weight of female FORKO mice, at 2 months of age, are similar to WT mice. However, at 5 months of age, FORKO mice are significantly heavier than WT mice, showing a tendency to obesity. The weights of adipose tissues, retroperitoneal, mesenteric, or parametrial, in 2-month-old FORKO mice are similar to WT mice. However, at 5 months of age, female FORKO mice revealed an increased deposition of abdominal fat. Parametrial and mesenteric adipose tissues of FORKO mice were heavier than that of WT mice. No differences in retroperitoneal adipose tissue were observed between both genotypes.


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  		TABLE 1. Changes in body weight and adipose tissue weights of 2- and 5-month-old FORKO and WT female mice after E2 (1 µg/d) or vehicle (control) treatment for 4 d

 
Furthermore, the animals were submitted to short-term estrogen replacement therapy (E2). Plasma E2 level in FORKO mice, which was significantly lower than in WT mice, was significantly increased by E2 treatment and approached the same level as WT mice without treatment (Table 2Go). No differences in cardiac mass were observed between both genotypes (Table 2Go).


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  		TABLE 2. E2 plasma levels, uterine weight, and cardiac mass of female FORKO and WT mice after E2 (1 µg/d) or vehicle treatment for 4 d

 
E2 treatment of FORKO mice induced a 2.7-fold increase in uterine weight, indicating that FORKO mice were responsive to E2 (Table 2Go). In 5-month-old FORKO animals, E2 significantly decreased body weight and total fat mass as well as mesenteric adipose depot weight (Table 1Go). The reduction in adipose tissue weight of FORKO mice correlated with an increase of E2 plasma levels (Table 3Go). No alterations were observed in 2-month-old FORKO and WT mice and in 5-month-old WT mice (Table 1Go).


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  		TABLE 3. Pearson’s correlation coefficients (two-tailed) between plasma E2 levels and plasma ANP levels, plasma E2 levels and mesenteric adipose tissue weight, and plasma ANP levels and mesenteric adipose tissue weight in the overall population of FORKO mice treated with vehicle or E2 (1 µg/d) and among WT mice treated with vehicle or E2 (1 µg/d) for 4 d

 
Plasma ANP levels of FORKO mice were lower (~80% decrease) than that of WT mice. The E2 treatment induced a more than 100% increase in ANP plasma levels in FORKO compared with corresponding vehicle-treated mice (vehicle, 22.8 ± 6.1; E2, 58.2 ± 6.9 pg/ml; P < 0.05). A smaller albeit significant increase (of about 40%) in plasma ANP was observed in E2-treated WT animals (vehicle, 106.7 ± 16.8 pg/ml; E2, 143.6 ± 15.1; P < 0.05) (Fig. 1Go). The increase of plasma levels directly correlated with plasma E2 levels in FORKO and WT mice (Table 3Go).


Figure 1
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  		FIG. 1. Changes in circulating plasma ANP concentrations in FORKO (–/–) and WT (+/+) mice treated with vehicle (control) or E2 for 4 d. Values are the mean ± SEM of at least five animals. *, P < 0.05 vs. vehicle-treated WT or FORKO mice; **, P < 0.05 vs. vehicle-treated WT mice.

 
The plasma level of ANP depends on its rate of synthesis and/or degradation. FORKO mice presented lower ANP mRNA levels in both right and left atria than WT mice, indicating lower ANP synthesis. E2 treatment induced a large increase in ANP gene expression in right and left atria of FORKO (Fig. 2Go). E2 treatment increased ANP gene expression in both right and left atria of WT animals, but to a lower extent than in FORKO.


Figure 2
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  		FIG. 2. Expression of ANP gene in atria. The expression of this gene was examined at the mRNA level in right and left atria from 5-month-old FORKO and WT mice after E2 or (control) vehicle treatment for 4 d. RT-PCR analysis of ANP was done using primers specific for the mouse sequence as indicated in Materials and Methods. Top panel, Representative ethidium bromide visualization of RT-PCR products in right and left atria; bottom panel, ANP signal intensities were normalized to β-actin. n = 5 animals per group. *, P < 0.05 vs. vehicle-treated WT mice; **, P < 0.05 vs. vehicle-treated WT or FORKO mice.

 
The relative abundance and the changes of ANP receptor expression in kidney and adipose tissues were compared using RT-PCR. Kidneys of FORKO mice had significantly higher NPR-C mRNA than WT mice. A larger amount of clearance receptors together with lower ANP synthesis could account for the lower ANP plasma levels observed in FORKO mice. E2 treatment normalized NPR-C mRNA in FORKO kidneys. E2 treatment significantly increased the levels NPR-A mRNA in FORKO as well as in WT mice (Fig. 3Go). NPR-C gene expression in mesenteric adipose tissue of FORKO mice was much higher than that of WT mice, an effect normalized by E2 treatment (Fig. 4Go). No alteration in NPR-A and NPR-C gene expression in parametrial adipose tissue was observed after E2 treatment of FORKO or WT mice, but an increase in both transcripts was noted in retroperitoneal adipose tissue of FORKO. Mesenteric adipose tissue weight inversely correlated with ANP plasma levels in FORKO, but not in WT (Table 3Go).


Figure 3
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  		FIG. 3. Renal expression of NPR-A and NPR-C genes. The expression of these genes was examined at the mRNA level in kidneys from 5-month-old FORKO and WT mice after E2 or vehicle (control) treatment for 4 d. RT-PCR analysis of NPR-A and NPR-C was done using primers specific for the mouse sequence as indicated in Materials and Methods. Top panel, Representative ethidium bromide visualization of RT-PCR products; bottom panel, NPR-A and NPR-C signal intensities were normalized to β-actin. n = 5 animals per group. *, P < 0.05 vs. vehicle-treated WT mice; **, P < 0.05 vs. vehicle-treated WT or FORKO mice.

 

Figure 4
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  		FIG. 4. Adipose tissue expression of NPR-A and NPR-C genes. The expression of these genes was examined at the mRNA level in mesenteric adipose tissue from 5-month old FORKO and WT mice after E2 or vehicle (control) treatment for 4 d. RT-PCR analysis of NPR-A and NPR-C was done using primers specific for the mouse sequence as indicated in Materials and Methods. Top panel, Representative ethidium bromide visualization of RT-PCR products; bottom panel, NPR-A and NPR-C signal intensities were normalized to β-actin. n = 5 animals per group. *, P < 0.05 vs. vehicle-treated WT mice; **, P < 0.05 vs. vehicle-treated WT or FORKO mice.

 
To confirm that NPR-C gene expression in mesenteric adipose tissue was followed by NPR-C protein expression, the binding of NPR-C was investigated in mesenteric adipose tissue by in vitro autoradiography using C-ANF as specific ligand of this receptor. [125I]ANP bound reversibly but with high affinity to mesenteric adipose tissue from FORKO mice. Unlabeled C-ANF completely inhibited [125I]ANP binding to mesenteric adipose tissue of FORKO, but binding was only partially inhibited by 10–6 M C-ANF in WT mice, indicating up-regulation of NPR-C receptors in FORKO (Fig. 5Go).


Figure 5
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  		FIG. 5. Pseudocolor representation of [125I]ANP binding sites in FORKO and WT mice mesenteric adipose tissue. Total [125I]ANP binding and inhibition by 10–12 to 10–6 molar concentrations of unlabeled C-ANF in vehicle-treated WT mice (A), E2-treated WT mice (B), vehicle-treated FORKO mice (C), and E2-treated FORKO mice (D).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that in overweight estrogen-deficient FORKO mice, plasma ANP levels are lower than in WT and that these changes are influenced by both reduced ANP gene expression in atria and increased NPR-C mRNA in kidneys and mesenteric adipose tissue. These effects are reversed by E2 treatment, reinforcing the relationship between estrogen, fat deposit, and ANP, factors implicated in metabolic and cardiovascular alterations after menopause.

Five-month-old female FORKO mice were heavier than WT mice, showing a tendency to obesity, which is clearly indicated by the higher mass of parametrial and mesenteric adipose tissues. E2 treatment caused a significant decrease of body weight and also decreased total fat mass as well as mesenteric adipose depot weight. Estrogen is a major regulator of adipose tissue in females. Ovariectomy in rodents increases adiposity, and estrogen replacement decreases adipose tissue in rats and in postmenopausal women who have increased fat mass (30). Genetic models provide strong evidence that E2 modulates the amount of adipose tissue. Large increases in adipose mass are observed in both female estrogen receptor-{alpha} knockout mice and in female aromatase-deficient mice (31, 32). Estrogen has direct effects on adipocytes, acting by either decreasing lipogenesis through reduction of lipoprotein lipase activity (33) or indirectly by inducing the lipolytic enzyme hormone-sensitive lipase (HSL) (34). Estrogen also affects the natriuretic peptide system by stimulating ANP release (4, 8, 35) and by increasing gene expression and production of ANP in the heart (5, 6, 7). Although adipose tissue produces ANP (12) and expresses both receptors, NPR-A and NPR-C (11, 20), the effect of estrogen on NP receptors in adipose tissue is not known. However, it is well known that plasma ANP level is inversely correlated with fat mass and that obese and overweight individuals have lower plasma natriuretic peptide levels than individuals with normal body mass index (16, 36). Our results show high expression of NPR-A and NPR-C mRNA in all the depots of adipose tissue of both FORKO and WT mice. FORKO mice presented higher levels of mRNA for NPR-C in mesenteric adipose tissue compared with WT, and this expression was markedly reduced by E2 treatment. Similarly, high-affinity binding sites for [125I]ANP were found in mesenteric adipose tissue from FORKO mice, and this affinity was decreased by E2 treatment as well. Taken together, these studies demonstrate that NP receptors in adipose tissue are under estrogenic control and suggest that reduction of ANP clearance in this site results in elevated plasma ANP. It is interesting to note that fasting and subsequent weight reduction increase plasma ANP (37) and selectively inhibit adipose tissue NPR-C gene expression in rats (14). In this regard, high expression of NPR-C and high affinity of NPR-C binding sites in adipocytes could, in part, explain the low ANP levels present in FORKO mice (present study) as well as in obese individuals (16).

The low plasma ANP levels in FORKO mice may also be influenced by altered proteolysis by neutral endopeptidase (NEP). NEP is present on plasma membrane of human adipocytes and preadipocytes (38) and does not play a major role in the regulation of ANP-mediated lipolysis, but it is hypothesized that NEP might increase in obesity and contribute to the clearance of circulating natriuretic peptides (39). NEP activity in kidneys or adipose tissue has not been determined in the present studies, and we are unaware of any data on the effect of E2 on NEP in adipose tissue. The effect of E2 on NEP in other tissues is controversial. Estradiol increases NEP mRNA and activity in uterus but does not change it in kidney cortex and medulla of Sprague Dawley and heterozygous (mRen2) 27-transgenic hypertensive female rats (40). NEP mRNA and activity are increased during pregnancy in rat uterus (41, 42). Ovariectomy decreases NEP activity in the brain by 30%, and the decreased NEP activity can be ameliorated by estrogen-replacement therapy (43). These studies favor a positive effect of E2 on NEP in some but not all tissues. In contrast, Pinto et al. (44) have shown that E2 treatment decreases NEP mRNA in female Wistar rat uteri. At this point, we may only speculate that in FORKO mice, increased NEP activity would further reduce plasma ANP levels. More studies, however, are required to assess the influence of estrogen on NEP in multiple tissues, including adipose depots.

The cause and effect relation of the increased expression of NPR-C in adipose tissue of FORKO mice and obesity is not clear. Recent studies have raised the possibility that these relations may be bidirectional. Adipose tissue expresses NPR-A, which is the receptor responsible for the effects of ANP. Binding of ANP to NPR-A on adipocytes induces lipolysis in humans (45, 46). This new lipolytic pathway does not involve cAMP production but is associated with a cGMP-dependent mechanism that increases phosphorylation of the hormone-sensitive lipase (45, 46). Thus, low circulating ANP levels could lead to reduced lipolysis, contributing to development of obesity. At this stage, we cannot determine whether low plasma ANP levels followed or preceded obesity, but we may speculate that obesity appears first because the lipolytic action of ANP seems to be more important in primates (47). We have previously shown that ANP-dependent lipolysis in fat cells of male rats was site specific and that epididymal but not retroperitoneal adipose tissue was responsive to ANP (48), but the lipolytic action of ANP in rats was quite low when compared with humans (47). In the present work, we have observed no effect of ANP on lipolysis of adipocytes isolated from mesenteric, retroperitoneal, or parametrial adipose tissue (data not shown) of female mice. So, it seems unlikely that obesity in FORKO mice is a consequence of low plasma ANP levels.

The only fat depot of FORKO mice that showed weight reduction after a short E2 treatment was the mesenteric adipose tissue. Studies carried out in rodent models show that the effect of estrogens is location and hormonal status dependent (49). The differences in estrogen receptor expression reported by Rodriguez-Cuenca et al. (50), for example, suggest a higher estrogen sensitivity in visceral than in sc depots, which could explain the higher estrogen-stimulated lipolysis in visceral adipose cells, an effect that is absent in the sc depot (51). Another possibility to explain our observations is the short treatment duration. However, a longer treatment could also produce a reduction of parametrial depot weight (19). Besides, the mesenteric depot seems to be the most responsive adipose depot (52, 53).

Although the association between abdominal fat and hypertension is well known, the mechanisms linking obesity to the development of hypertension have not been fully established. Changes in sodium handling are considered to be a central feature of obesity-associated hypertension. Because ANP is involved in the regulation of sodium balance, it has been speculated that obese individuals have an impaired natriuretic peptide response. In fact, in rats made obese by a highly palatable diet, the same dose of ANP induced about a 3-fold lower increase in sodium excretion (54). The kidney is a major target organ for ANP, and the natriuresis and diuresis promoted by ANP binding to renal NPR-A result in lowering of blood pressure. The increased renal NPR-C in the present studies and subsequently lower plasma ANP levels imply attenuated renal action of ANP in FORKO mice. The reduction of renal action of ANP, associated with lower circulating levels of the hormone could contribute to blood pressure elevation. In fact, aging FORKO mice have higher arterial blood pressure than WT mice (17, 55). The natriuretic action of ANP was also attenuated in another model of obesity with increased blood pressure, the Zucker rats (56). It is also important to note that gene expression of NPR-C in human adipose tissue is greater in obese hypertensive than in obese normotensive subjects, thus implicating lower ANP levels in blood pressure elevation (20). In obese humans, during weight reduction induced by fasting, the reduction of blood pressure is accompanied by a considerable increase in diuresis and natriuresis and correlates with down-regulation of adipocyte NPR-C receptor (37), suggesting that subsequently elevated ANP can contribute to blood pressure reduction in obese subjects. Although it is beyond the scope of this study, it is tempting to speculate that low plasma ANP levels may contribute to the susceptibility of FORKO female mice to developing age-related hypertension (17, 55). Thus, it would be interesting to investigate in these mice the effects of long-term treatment with E2 alone or in combination with exogenous ANP on blood pressure.

In summary, the present studies show that FORKO female mice with a tendency to obesity have an impaired natriuretic peptide system and that E2 treatment improved this condition by increasing atrial ANP synthesis as well as by decreasing ANP clearance, most likely mediated by NPR-C receptors. These findings may have important implications for the treatment of postmenopausal cardiovascular and metabolic complications.


   Footnotes
 
This work was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Fundação de Amparo à Pesquisa do Estado de Minas Gerais, and Programa de Apoio a Núcleos de Excelência (Brazil) and Canadian Institutes for Health Research (Canada). N.O.B. was a recipient of a scholarship from CNPQ at the Programa de Pós-Graduação Ciências Biológicas: Fisiologia/Farmacologia, Universidade Federal de Minas Gerais.

Current address for N.O.B.: Multidisciplinary Institute of Health, Federal University of Bahia, Bahia, Brazil.

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online December 6, 2007

Abbreviations: ANP, Atrial natriuretic peptide; C-ANF, des[Gln18, Ser19, Gly20, Leu21, Gly22]ANP-(4–23)-NH2; E2, 17β-estradiol; FORKO, FSH receptor knockout; NEP, neutral endopeptidase; NPR, natriuretic peptide receptor; WT, wild-type.

Received May 1, 2007.

Accepted for publication November 29, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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