Laura Trapani

Francesca Violo

Valentina Pallottini

Hypercholesterolemia and 3-hydroxy 3-methylglutaryl Coenzyme A reductase regulation in aged female rats

Keywords: Ageing, AMPK, estropause, estradiol, hypercholesterolemia, HMGR

Coronary heart disease is less prevalent in pre-menopausal women than in men, but increases at the onset of menopause. This delay is due to estrogen protective effects. The rise of cholesterolemia is one of the main risk factors for coronary disease. Since 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is the rate-limiting enzyme of the cholesterol biosynthetic pathway, it plays a pivotal role in cholesterol homeostasis maintenance. Aim of this study is to investigate whether HMGR is involved in the cholesterolemia increase that occurs during aging, and to consider its potential role as a target for estrogen protective effects. "In vivo" studies have been performed using the livers of 12-month-old female rats (whose estrogen level decrease is comparable to the one detected at the occurrence of human estropause), 12-month-old female rats treated with 17--estradiol, and 3-month-old untreated male and female rats. The results indicated hypercholesterolemic status and a significant increase of HMGR activity according to a reduced activation of AMPK detected in treated rats compared to controls. Furthermore, 17-estradiol treatment reduced HMGR activity restoring AMPK activation. These findings highlight the correlation between estrogen and HMGR short-term regulation, and suggest the presence of another mechanism underlying the protective role of estrogen in age-related diseases.

Introduction

Coronary heart disease and other vascular diseases account for approximately half of all deaths in women. Although the underlying pathophysiological processes (atherosclerosis and thrombosis) are similar, deaths and other clinical end-points are significantly delayed in women compared to men. Under age 55, the incidence of CVD (Cardiovascular Diseases) in women is one third of that in men; however, at age 75, the incidence is essentially the same in both genders [START_REF] Skafar | Female sex hormones and cardiovascular disease in women[END_REF]. The loss of female sex hormones after menopause contributes to the striking increase in the incidence of cardiovascular morbidity and mortality after menopause. Estrogen replacement therapy improves lipoprotein profiles in postmenopausal women, but this accounts for less than half of the cardioprotective effects of estrogen replacement therapy. The cardioprotective benefits of estrogens include a positive impact on plasma lipids (increased high density lipoprotein (HDL), decreased total cholesterol, triglycerides, low density lipoprotein (LDL), and LDL oxidation) [START_REF] Skafar | Female sex hormones and cardiovascular disease in women[END_REF][START_REF] Moorthy | Estradiol and progesterone treatments change the lipid profile in naturally menopausal rats from different age groups[END_REF], anti-platelet and antioxidant effects and preservation of endothelium-mediated vasodilatation [START_REF] Maxwell | Women and heart disease[END_REF][START_REF] Moorthy | Administration of estradiol and progesterone modulate the activities of antioxidant enzyme and aminotransferases in naturally menopausal rats[END_REF]. These effects may be mediated via genomic and/or nongenomic mechanisms [START_REF] Skafar | Female sex hormones and cardiovascular disease in women[END_REF]. Moreover, studies performed on animal models of estrogen deficiency have indicated a disruption of lipid homeostasis [START_REF] Heine | Increased adipose tissue in male and female estrogen receptor -a knockout mice[END_REF].

Cholesterol is both synthesized by cells and taken in via food. It plays an essential role in the synthesis of new membranes, the turnover of lipids in existing membranes, and the biosynthesis of products such as steroid hormones and bile acids (Martini and Pallottini, 2007). The liver is the principal site for cholesterol homeostasis maintenance [START_REF] Dietschy | Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans[END_REF], which involves the regulation of many processes, such as biosynthesis, via 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, E.C. 1.1.1.34) activity, uptake through low-density lipoprotein receptors (LDLr), lipoprotein release in the blood, storage by esterification, degradation and conversion into bile acids [START_REF] Weber | Maintaining cholesterol homeostasis: sterol regulatory element-binding proteins[END_REF].

As the central enzyme of the cholesterol biosynthetic pathway, HMGR can catalyze the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate (a four-electron oxidoreduction) [START_REF] Friesen | The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases[END_REF], and is tightly regulated [START_REF] Goldstein | Regulation of the mevalonate pathway[END_REF].

The enzyme is subject both to short-and long-term regulations. Short-term regulation of HMGR is achieved phosphorylation and dephosphorylation reactions, both of which can affect enzyme activity. The phosphorylation of residue S872 of HMGR decreases its catalytic activity. Removal of the phosphate by HMGR phosphorylase reactivates the enzyme [START_REF] Beg | Phosphorylation of hepatic 3-hydroxy-3methylglutaryl coenzyme A reductase and modulation of its enzymic activity by calciumactivated and phospholipid-dependent protein kinase[END_REF][START_REF] Omkumar | Modulation of Syrian hamster 3-hydroxy-3-methylglutaryl-CoA reductase activity by phosphorylation Role of serine 871[END_REF]. HMGR activation (dephosphorylation) is mediated by protein phosphatase 2A (PP2A), which regulates a significant network of cellular events [START_REF] Janssens | Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling[END_REF]. Aside from short-term regulation, HMGR is subject to transcriptional, translational, and post-translational control [START_REF] Xu | Dual roles for cholesterol in mammalian cells[END_REF]. This control can result in changes of over 200-fold in enzyme levels as a function of intracellular sterol levels and cholesterol uptake by LDLr [START_REF] Goldstein | Protein sensors for membrane sterols[END_REF]. Both LDLr and HMGR are produced in response to the activation of the Sterol Regulatory Element Binding Proteins (SREBPs) in the liver, particularly SREBP-2 [START_REF] Brown | The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor[END_REF][START_REF] Horton | SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver[END_REF].

The long-term regulation system consists of a pair of membrane-bound proteins, Scap (SREBP cleavage activating protein) and Insig 1 and 2 (Insulin-induced gene), which directly bind sterols and thereby sense their concentrations in the membranes of the endoplasmic reticulum (ER) [START_REF] Goldstein | Protein sensors for membrane sterols[END_REF].

Several hormones act to alter the expression of hepatic HMGR in animals. These include insulin, glucagon, glucocorticoids, thyroid hormone and estrogens [START_REF] Ness | Feedback and hormonal regulation of hepatic 3-hydroxyl-3methylglutaryl Coenzyme A reductase: the concept of cholesterol buffering capacity[END_REF].

However, available data regarding to estrogen effects on HMGR regulation remain controversial.

Initially, a role for physiologic levels of estrogen as a positive effector of HMGR activity was reported by Carlson and coworkers (Carlson et al. 1980). Other studies show that pharmacological 17-β-estradiol treatment in male rats "in vivo" induces a decline in hepatic HMGR activity after six hours and rapid LDLr induction [START_REF] Marino | Activation of IP3-protein kinase C-α signal transduction pathway precedes the changes of plasma cholesterol, hepatic lipid metabolism and induction of low-density lipoprotein receptor expression in 17-β-oestradiol-treated rats[END_REF]. Furthermore, other studies provide evidence that LDLr and HMGR are respectively up-and down-regulated by 17-β-estradiol, which induces growth inhibition or apoptosis in the DLD-1 colon cancer cell line [START_REF] Messa | Estrogenic regulation of cholesterol biosynthesis and cell growth in DLD-1 human colon cancer cells[END_REF]. Research also reveals that estrogens are able to modulate LDLr transcription through a mechanism mediated by tyrosine kinase signaling in HepG2 cells [START_REF] Distefano | Role of tyrosine kinase signalling in estrogen-induced LDL receptor gene expression in HepG2 cells[END_REF].

Hypercholesterolemia is one of the primary risk factors for CVD, whose incidence is highly related to aging. Almost 25% of men and 42% of women older than 65 years have a serum total cholesterol level greater than 240 mg/dL [START_REF] Grundy | Cholesterol lowering in the elderly population. Coordinating Committee of the National Cholesterol Education Program[END_REF]. The mechanisms behind this agerelated increase in plasma cholesterol are still incompletely understood. Of particular interest are findings indicating a gradual decline in the fractional clearance of LDL from the circulation with age [START_REF] Finch | Genetics of aging[END_REF][START_REF] Holliday | Ageing research in the next century[END_REF][START_REF] Carnes | Biological evidence for limits to the duration of life[END_REF] and evidence of the reduced expression of hepatic LDLr with increasing age in some species [START_REF] Korpelainen | Variation in the heritability and evolvability of human lifespan[END_REF][START_REF] Gudmundsson | Inheritance of human longevity in Iceland[END_REF]. The age-related disruption of cholesterol maintenance could be due to perturbations in homeostasis, which, with progressive declines in physiological capacity and reduced ability to respond adaptively to environmental stresses, are a primary characteristic of elderly patients. In 24month-old ad libitum-fed male rats, the hepatic HMGR is fully activated and cholesterol synthesis is increased; cholesterol content is unchanged in the liver, but increased in the blood [START_REF] Marino | Age-related changes of cholesterol and dolichol biosynthesis in rat liver[END_REF]. In fact, HMGR tends to be completely dephosphorylated and activated [START_REF] Marino | Age-related changes of cholesterol and dolichol biosynthesis in rat liver[END_REF] by the rise of reactive oxygen species (ROS) that occurs during aging [START_REF] Pallottini | Age-related HMG-CoA reductase deregulation depends on ROS-induced p38 activation[END_REF].

There is little published research on putative physiological sex-differences in cholesterol homeostasis-related proteins [START_REF] Abul-Hajj | Stimulation by estradiol benzoate of hepatic beta-hydroxy-betamethylglutaryl coenzyme a reductase in normal and ovariectomized rats[END_REF][START_REF] Carlson | Evidence that physiologic levels of circulating estrogens and neonatal sex-imprinting modify postpubertal hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase activity[END_REF][START_REF] Choi | Age-related changes in lipid metabolism in rats: the consequence of moderate food restriction[END_REF][START_REF] Hewitt | The aromatase knockout mouse presents with a sexually dimorphic disruption to cholesterol homeostasis[END_REF][START_REF] Kojima | Gender-related difference in altered gene expression of a sterol regulatory element binding protein, SREBP-2, by lead nitrate in rats: correlation with development of hypercholesterolemia[END_REF]. Recent data suggest that sex differences in HMGR expression and regulation depend upon variation in regulatory proteins and are related to the presence of estrogen. Some research has shown that HMGR levels and activity are lower in adult female rats than in male rats. This feature seems to depend on estrogen-dependent Insig 2 levels, which can affect enzyme transcription and degradation [START_REF] De Marinis | Sex differences in hepatic regulation of cholesterol homeostasis[END_REF]. We know of no report to date that analyzes the lack of estradiol during female ageing and putative changes in the protein able to regulate cholesterol homeostasis maintenance, as most existing research has examined males. This paper evaluates HMGR regulation in estropausal rats to determine if estrogen protective effects on the lipid profile are also mediated via HMGR (estrogen-dependent) regulation.

As an experimental model, 12-month-old female Wistar rats were treated with 17estradiol.

At the beginning of estropause (12 months of age), female rats --like women --cycle irregularly, but after cycling ceases, 17-β estradiol levels in rats --unlike those in women and mice --fall not to zero, but to steady moderate levels (for two months) and then increase again [START_REF] Berkley | Endometriosis-induced vaginal hyperalgesia in the rat: effect of estropause, ovariectomy, and estradiol replacement[END_REF]. So we can consider 12-month-old female rats to be in a similar condition to that occurring in women at the onset of menopause. Thus, in our experimental model, the putative connection between estrogen decrease, hypercholesterolemia and HMGR activity was investigated.

To this end, the different expression patterns of hepatic proteins involved in HMGR long-and short-term regulation, HMGR activity and ROS production were investigated. The estrogen effects were evaluated treating 12-month-old female rats with pharmacological doses of 17-estradiol to assess the involvement of HMGR in the protective role played by estrogen.

Materials and Methods

Materials

All materials used were obtained from commercial sources, and were of the highest quality available. All materials with no specified source were obtained from Sigma-Aldrich.

Animals

Three-months-old male and female and 12-month-old female Rattus norvegicus Wistar (Morini, Italy) rats were housed under controlled temperature (20±1 °C), humidity (55±10%), and light (from 07.00 to 19.00 h). The animals had free access to food and water. Twelve-month-old female rats were injected intraperitoneally with 1 mg/kg of 17-estradiol 3-benzoate (Sigma Chemical Co.) in vehicle (1 ml/kg dimethyl sulfoxide (DMSO) in saline). Control animals received an equal volume of vehicle. Sampling was then performed at 24 h after the estradiol injection. All female rats were anaesthetized with ether in a fume cupboard. Plasma was obtained from blood collected in EDTA (1 mg/ml blood). Livers were removed and rapidly frozen in liquid nitrogen.

Four animals per group were used, and livers were independently analyzed in duplicate. The

HMGR activity

The assay was carried out via the radioisotopic method, following the production of [14C]mevalonate from 3-[14C]-HMG-CoA (3-[ 14 C]-HMG-CoA; specific activity 57.0 mCi/mmol; GE Healthcare, Little Chalfont, UK). Microsomes were prepared in the presence or absence of NaF according to the method of [START_REF] Bruscalupi | True uncoupling between cholesterol synthesis and 3-hydroxy-3-metylglutaryl-coenzyme A reductase in a early stage of liver regeneration[END_REF]. NaF was used to block lysosomal phosphatase, thus permitting the analysis of expressed HMGR, while microsomes without NaF were useful in evaluating total HMGR activity. Microsomes (100 µg) were incubated in the presence of cofactors (20 mM glucose-6-phosphate, 20 mM NADP sodium salt, 1 IU glucose-6-phosphate dehydrogenase, and 5 mM dithiothreitol). The assay, in a final volume of 200 µl, was started by the addition of 10 µl (0.088 µCi/11.7 nmol) of 3-[ 14 C]-HMG-CoA. The radioactivity of the produced [ 14 C]-mevalonate, isolated by chromatography on AG1-X8 ion exchange resin (Bio-Rad Laboratories), was counted. An internal standard (3-[ 3 H]-mevalonate, specific activity 24.0 mCi/mmol; GE Healthcare) was added to calculate the recovery.

Protein levels analysis

Protein levels were analyzed via Western blotting. An analysis of Insig 1, Insig 2, HMGR and Scap was performed on microsomes prepared as described previously. Total lysates and total membranes were prepared according to Martini et al. (2007). Western blot analysis of LDLr was performed on total membranes while the analysis of SREBP 2 (N-terminus), AMPK, P-AMPK and PP2A was performed on total lysates. Table 1 describes the sources and the antibodies used for the Western blotting assays. Twenty micrograms of protein from solubilized membranes were resolved by 12% (for Insig 1, Insig 2, AMPK, P-AMPK) and 7% (for SREBP 2, LDLr, HMGR and Scap) SDS-PAGE at 100 V for 60 min. The proteins were subsequently transferred electrophoretically onto nitrocellulose for 80 min at 100 V. The nitrocellulose membrane was treated with 3% BSA in 138 mMNaCl, 27 mMKCl, 25 mM Tris-HCl, 0.05% Tween-20 (pH 6.8), and probed at 4°C overnight with primary antibodies, then for 1 h with secondary antibodies. The nitrocellulose membrane was then stripped with Restore Western Blot Stripping Buffer (Pierce Chemical, Rockford, IL, USA) for 10 min at room temperature and reprobed with anti-tubulin antibody (MP Biomedicals, Solon, OH, USA). Bound antibodies were visualized using enhanced chemoluminescence detection (GE Healthcare). All images derived from Western blotting were analyzed with ImageJ (NIH, Bethesda, MD, USA) software for Windows. Each reported value was derived from the ratio between arbitrary units obtained by the protein band and the respective tubulin band (chosen as a housekeeping protein).

Plasma cholesterol analysis

The plasma cholesterol content was assessed through the colorimetric CHOD-POD kit according to manufacturer's instructions (Assel, Rome, Italy). In brief, by the catalysis of cholesterol esterase and cholesterol oxidase, cholesterol ester is catalyzed to yield H 2 O 2 , which oxidates 4-Aminoantipyrine with phenol to form a colored dye of quinoneimine. The absorbency increase is directly proportional to the concentration of cholesterol

Measurement of Reactive Oxygen Species

ROS were detected in liver homogenates by incubating 2 mg protein with 10 M dichlorodihydrofluorescein diacetate (DCF; Molecular Probes, Eugene, OR, USA) for 30 min at 37°C in the dark. Samples were then allowed to equilibrate for an additional 60 min. We generally followed the procedures previously published for homogenates and perfused tissues to obtain a overall sense of an increase in oxidative stress [START_REF] Kehrer | The use of fluorescent probes to assess oxidative processes in isolated-perfused rat heart tissue[END_REF][START_REF] Al-Mehdi | Fluorescence microtopography of oxidative stress in lung ischemia-reperfusion[END_REF]. Fluorescence was measured under continuous gentle magnetic stirring at 37°C in a Perkin Elmer LS-50B spectrofluorimeter. Excitation wavelengths were set at 498 nm and emission was set at 530 nm using 5 and 10 nm slits, respectively. A Fenton-type reaction was induced by the addition of 10 M FeSO 4 and revealed the capability to continue producing unstable ROS species.

Plasma estrogen analysis

Plasma estrogen content was assessed using the ELISA method in Laboconsult (Rome, Italy).

Statistical analysis

Each mean was derived from four different experiments performed in duplicate. The statistical analysis consisted of ANOVAs followed by the Tukey-Kramer post test. P values under 0.05 were considered statistically significant. All the statistical analyses were performed using GraphPad Instat3 (Graphpad Software Inc., La Jolla, CA, USA) software for Windows.

Results

To analyze the role of HMGR and its regulatory network during estropause, the validity of the experimental model first had to be confirmed, moreover data on 3 month-old male rats to confirm previous and already published [START_REF] De Marinis | Sex differences in hepatic regulation of cholesterol homeostasis[END_REF] results obtained on sex-related differences in hepatic regulation of cholesterol homeostasis have been added. Thus, 17-estradiol levels were analyzed. As shown in Figure 1 (Panel a), the amount of 17-estradiol decreases significantly in 12-month-old female rats compared to 3-month-old rats. In the attempt to evaluate some of the metabolic parameters of estropausal rats, cholesterol and glucose plasma contents were assessed. As illustrated in Figure 1, a significant increase in cholesterolemia occurred in 12-month-old female rats with respect to 3-month-old rats (Panel b), whereas no differences in glycemia were observed (Panel c).

Because cholesterolemia is dependent upon the presence and functionality of LDLr on hepatocyte membranes, LDLr membrane exposure was evaluated. Figure 2 (Panel a) shows a slight but statistically significant decreased exposure of LDLr on hepatocyte membranes in estropausal rats with respect to 3-month-old female rats. The decreased LDLr exposition, by itself, cannot explain the hypercholesterolemia occurring in 12-month-old female rats, though. So HMGR levels were evaluated to determine whether some variations occurred and combined to induce hypercholesterolemia. Figure 2 (Panel b) illustrates that HMGR protein levels are understood to be higher in 3-month-old males than in female rats, no significant differences were observed between estropausal and 3-month-old female rats, even though a slight increment can be detected.

Although no significant differences in HMGR levels have been found between estropausal and 3-month-old female rats, the increase could suggest the involvement of proteins affecting longterm regulation of HMGR. Thus, the protein levels of Scap (the escort protein for SREBPs), SREBP 2 (the transcription factor able to induce hmgr expression), and Insig were evaluated, as illustrated in Figure 3.

The obtained data show no differences in Scap protein levels in 12-month-old rats with respect to the younger controls (Panel a); higher (even though not significant) n-SREBP 2 protein levels in 3-month-old male rats with respect to female counterparts and estropausal rats (Panel b); and no changes in Insig 1 protein levels (Panel c), although lower Insig 2 protein levels were observed in 3-month-old male rats with respect to their female counterparts and estropausal rats (Panel d).

Taking into account the absence of substantial differences in HMGR protein levels and HMGR regulatory network, it can be gathered that the hypercholesterolemia detected in estropausal rats cannot be attributed to variations in the long-term regulation of HMGR. Thus, our interest has been focused on the short-term regulation of HMGR to determine if hypercholesterolemia in estropausal rats could be due to variations in HMGR activation state. As described above, HMGR activity is regulated by phosphorylation/dephosphorylation: the unphosphorylated enzyme represents the active form of HMGR and the phosphorylated enzyme the inactive one. The enzyme activity was measured in microsomes prepared in the presence or absence of NaF to monitor the total and expressed activity, respectively. The obtained data indicate a higher activation state of HMGR in 12-month-old female rats with respect to younger controls shown both in the table and in Fig. 4, Panel a. Since previous works [START_REF] Pallottini | Age-related HMG-CoA reductase deregulation depends on ROS-induced p38 activation[END_REF] have demonstrated that the increased HMGR activity is due to increased ROS content, the reactive oxygen species levels were measured in our experimental rats (data not shown). Although slight variations were observed, none were statistically significant; thus, the increased activation state of HMGR cannot be attributed to ROS content modification.

To determine whether the increased HMGR activity was due to variations in the proteins involved in the rapid phosphorylation/dephosphorylation mechanisms, the AMPK activation state and the PP2A catalytic sub-unit protein levels were measured. AMPK was observed to be lower when phosphorylated and activated in estropausal rats compared with controls (Fig. 4, Panel b), whereas no differences in PP2A were observed (Fig. 4,Panel c).

To determine whether the previously observed variations were ascribable to the estrogen levels falling, 12-month-old female rats were treated with a single pharmacological dose of 17estradiol (1 mg/kg). At first, estrogen plasma content was checked in 12-month-old female rats treated with 17-estradiol. As shown in Figure 5, estradiol treatment restored the hormone concentrations to the levels observed in 3-month-old female rats.

As such, in the older 17-β estradiol-treated female rats, plasma cholesterol content (Figure 6 Subsequently, the effects of 17-estradiol treatment on HMGR expressed, total activity and activation state was evaluated (Figure 7). In estradiol-treated estropausal rats, the HMGR activation state results were similar those detected in controls. The 17-estradiol effects were later evaluated for the proteins involved in short-term regulation of HMGR. As illustrated in Figure 8 (Panel a), treated animals demonstrated a higher phosphorylation and activation state for AMPK compared to 12-month-old female rats, even though AMPK activation was not completely restored (compared to 3-month-old female rats). In addition, 17-estradiol treatment did not have any observable effect on PP2A protein levels (Figure 8, Panel b).

Discussion

The aim of this work was to evaluate the putative correlation between the variations in the HMGR regulatory network and estrogen declines during the ageing process, at the beginning of female rat estropause, and to determine whether this was related to the occurrence of hypercholesterolemia and the eventual involvement of HMGR in the protective role of estrogen against age-related increases in plasma cholesterol.

We chose, as an experimental model, 12 month-old female rats because only at 12 months is estrogen behavior similar to that occurring in human females at the onset of menopause [START_REF] Berkley | Endometriosis-induced vaginal hyperalgesia in the rat: effect of estropause, ovariectomy, and estradiol replacement[END_REF].

Data obtained from estropausal rats (in which estrogen levels decrease) indicate a higher plasma cholesterol amount and HMGR activation state and a lower level of LDLr membrane exposure with respect to controls. This event contributes to decreases in cholesterol uptake and a rise in cholesterol synthesis, which supports the correlation between hypercholesterolemia, ageing and estropause. The higher HMGR activation state does not depend on a rise in ROS content, as in aged-match male rats [START_REF] Straniero | Stimulation of autophagy by antilipolytic drugs may rescue rodents from ageassociated hypercholesterolemia[END_REF], but seems to depend on the lower AMPK activation state observed in 17-β estradiol deficiency (a condition that occurs at the beginning of estropause), thus triggering the HMGR phosphorylation decrease and enzyme activation.

The 17-estradiol treatment of older female rats restores both proper cholesterolemia and the HMGR activation state, along with LDLr exposure on the plasma membrane. Moreover, AMPK activation results were higher than those in control animals, but activation was not completely restored. However, the increase of the AMPK activation state induced by 17-estradiol treatment seems to be sufficient to phosphorylate HMGR and to restore its proper activity. The enhancement of estradiol-induced AMPK activation is supported also by other data in the literature [START_REF] Schulz | Estradiol-mediated endothelial nitric oxide synthase association with heat shock protein 90 requires adenosine monophosphatedependent protein kinase[END_REF].

Our results demonstrate that the estradiol decrease occurring at the onset of estropause does not affect the long-term regulation of HMGR, but rather its short-term modulation via AMPK activation-state control. Thus, the existence of a relationship between estrogen changes and cholesterolemia modulation (the latter connected to the HMGR activity) seems to occur in older female rats.

Our work suggests how the protective role played by estrogens on the lipid profile is carried out not only through the increase of HDL and the decrease of LDL plasma content and oxidation -as previously assessed by numerous papers in the literature [START_REF] Skafar | Female sex hormones and cardiovascular disease in women[END_REF][START_REF] Maxwell | Women and heart disease[END_REF][START_REF] Moorthy | Estradiol and progesterone treatments change the lipid profile in naturally menopausal rats from different age groups[END_REF])--but also through the modulation of the AMPK activation state, which, in turn, controls both the normal HMGR activation state and cholesterol synthesis.

Restoration of normal HMGR activation should restore physiological production of cholesterol in cells and, thus, the correct LDLr membrane exposure, depending on cellular sterol content, as described by the classical model generally accepted [START_REF] Goldstein | Protein sensors for membrane sterols[END_REF]Espenshade ANOVA followed by the Tukey-Kramer test versus 3-month-old female rats. ### =p<0.0001 from ANOVA followed by the Tukey-Kramer test versus 12-month-old female rats.

Table 1

List of antibodies used for western blotting assay.

Primary Antibody

Sources Secondary Antibody Sources

  experiments were performed according to ethical guidelines for the conduct of animal research (Ministero della Salute, Official Italian Regulation N°. 116/92, Communication to Ministero della Salute n° 391/121).

,

  Panel a) and LDLr exposure on liver tissue membranes (Figure 6 Panel b) were analyzed. The data indicate that both cholesterolemia and LDLr exposure in treated animals were comparable to the young rats.

Figure legend Figure 1 .

 legend1 Figure legend

Figure 2 .

 2 Figure 2. LDLr and HMGR levels in the livers of 3-month-old male and in 3-and 12month-old female rats. The figure illustrates a typical Western blot and densitometric analysis of LDLr (a) and

Figure 3 .

 3 Figure 3. Scap, SREBP 2, Insig 1, and Insig 2 levels in 3-month-old male and 3-and 12month-old female rat livers. The figure shows a typical Western blot and densitometric analysis of Scap (a), SREBP 2 (b),

Figure 4

 4 Figure 4 HMGR, AMPK activation state and PP2A catalytic sub-unit levels in 3-monthold male and 3-and 12-month-old female rat livers.In Panel a, the HMGR activity, table on the top, and activation state are represented . In Panel b, the AMPK activation state is expressed as the ratio between the densitometric analysis of P-AMPK and AMPK is shown. Panel c illustrates a typical Western blot and densitometric analysis of PP2A protein levels. All experiments were conducted in the livers of 3-month-old male and 3-and

Figure 5 .

 5 Figure 5. Plasma 17-estradiol levels in 3-month-old, 12 month-old and 17-estradioltreated 12-month-old female rats.The figure illustrates plasma 17-estradiol levels assayed in 3-and 12-month-old females and

Figure 6 .

 6 Figure 6. Cholesterolemia and LDLr levels in 3-month-old, 12-month-old and in 17estradiol-treated 12-month-old female rats. Panel a shows plasma cholesterol levels. Panel b illustrates a typical Western blotting and the densitometric analysis of hepatic LDLr protein levels. The experiments were conducted in 3-month-

Figure 7 .

 7 Figure 7. HMGR activity and activation state in the liver of 3-, 12-month-old and 12month-old 17-estradiol-treated female rats.

Figure

  Figure shows the HMGR activity, table on the top, and activation state (ratio between +NaF

Figure 8

 8 Figure 8 AMPK activation state and PP2A catalytic sub-unit levels in the liver of 3-, 12month-old and 12-month-old 17-estradiol-treated female rats. Panel a represents a typical Western blot and densitometric analysis of the AMPK activation state expressed by the ratio between P-AMPK and AMPK. Panel b represents a typical Western blot and densitometric analysis of PP2A catalytic sub-unit levels. Protein levels were analyzed in 3-and

and Huges, 2007). In the light of these observations, we can conclude that during the female rat aging process, the diminished AMPK activation state, triggered by estrogen declines at the onset of estropause, can induce hypercholesterolemia. Thus, due to its strict dependence on the AMPK activation state, HMGR can be considered as another mediator of the protective effects of estrogen.

Acknowledgments

The Authors wish to thank past and present members of their laboratories who contributed data and discussions to the ideas presented here. In particular, the invaluable and dedicated work of our mentor Prof. Anna Trentalance is warmly acknowledged.


Paul Vigne

Dr Christian Frelin
email: cfrelin@unice.fr

Food presentation modifies longevity and the beneficial action of dietary restriction in Drosophila

Keywords: Drosophila, Nutrition, Ageing, Dietary restriction, Longevity

Recent studies have indicated that flies respond to dilute food solutions by compensatory feeding. The existence of compensation mechanisms calls for a reconsideration of the relationships between diet, feeding behaviour and longevity. This study shows that flies fed on liquid diets, sense sucrose and yeast nutrients and adapt to changes in the quantity and presentation of the two nutrients. They have a marked preference for sucrose and regulate their sucrose intake more tightly than their yeast intake.

ACCEPTED MANUSCRIPT

These preferences are not modified as fly age from 1 day to 30 days. Compensatory feeding suppresses the beneficial action of dietary restriction on longevity when flies are fed on liquid diets supplemented with yeast extracts. Flies which are given the choice to feed on separate yeast and sucrose food sources were longer lived than flies fed on nutrient mixtures. We conclude that flies sense and respond to specific nutrients and that food presentation is a major factor which determines the sensitivity of flies to dietary restriction.

Introduction.

The relationships between diet and longevity are of increasing interest. The observation that flies fed on dilute sources of food are long lived has suggested that dietary restriction promotes longevity [START_REF] Partridge | Dietary restriction in Drosophila[END_REF]Partridge, 2007). This conclusion assumes that flies ingest nutrients in proportion of their concentrations in their food. Recent papers have challenged this view. First flies respond to specific macronutrients such as protein and carbohydrates and not to the calorie content of the food [START_REF] Mair | Calories do not explain extension of life span by dietary restriction in Drosophila[END_REF][START_REF] Min | Counting calories in Drosophila diet restriction[END_REF], Vigne et al., 2007a, b, Skorupa et al., 2008). Second flies can change their feeding behaviour in response to changes in their diet [START_REF] Carvalho | Compensatory ingestion upon dietary restriction in Drosophila melanogaster[END_REF][START_REF] Ja | Prandiology of Drosophila and the CAFÉ assay[END_REF]. Feeding behaviour by small insect has long been difficult to analyze. It can now be assessed using the capillary feeding assay [START_REF] Ja | Prandiology of Drosophila and the CAFÉ assay[END_REF]. This assay has recently been used to show that caloric restriction per se does not promote longevity of flies [START_REF] Lee | Ageing and reproduction in Drosophila: new insights from nutritional geometry[END_REF].

We previously reported that aged, 30 day old, flies do not respond to dietary restriction by an increased longevity (Vigne and Frelin, 2007a). Whether their unresponsiveness is due to a modified feeding behaviour or to a modified sensitivity to nutrients was not defined. This study uses a capillary feeding assay to analyse the feeding behaviour of flies and its possible alterations with ageing. The results indicate that the

ACCEPTED MANUSCRIPT

feeding behaviour of flies is more complex than previously thought. They suggest that feeding behaviour must be carefully defined when considering the relationship between nutrition and longevity.

Materials and Methods.

2.1 Fly stocks.

w 1118 flies (Bloomington stock Center) were reared in 300 ml bottles filled with 30 ml of standard food medium (8.2% cornmeal, 6.2% sucrose, 1.7% heat inactivated baker's yeast and 1% agar supplemented with 3.75 g/l methyl 4-hydroxybenzoate) in humidified, temperature controlled chambers at 25°C and 60 % relative humidity and under a 12:12 light: dark cycle. Experiments were performed using male flies. Young flies were 1 day old. Aged flies were 10, 20 or 30 day old. During ageing, flies were maintained on standard food.

Dietary conditions

Diets were composed of sucrose and yeast extract (Fluka 70161). Diets were labelled using the following convention. A "10S10Y" diet means that flies were fed with a mixture of 10% sucrose and 10% yeast and had "no choice" to select their nutrients. A "10S-10Y" diet means that flies were exposed to separate sources of 10% sucrose and 10% yeast and could select which nutrient to consume. 5S and 10S diets corresponded to 5% and 10% sucrose solutions. A 10Y diet corresponded to a 10% yeast solution.

Capillary feeding assay.

30 ml plastic vials were used. A piece of paper towel wetted with 2 ml of distilled water was introduced down to the bottom of the tubes. It had two functions. It provided a source of water that was independent of feeding and moist conditions to prevent evaporation of feeding solutions. Flies in groups of 6 were added to the tubes and tubes were sealed with rubber stoppers (SubA seal, Sigma) in which three holes had been drilled. Food was provided by one (or two) 75 µl micro capillary tube (Hirschmann). The third hole was used to insert a syringe needle for pressure compensation and gas exchanges. Capillaries were filled with 40 µl of liquid diet and inserted through the stopper via truncated 200 µl pipette tips. A mineral oil overlay (about 5 µl) was used to prevent evaporation from the top of the capillaries. In the "choice" experiments two capillaries were used. They were filled either with sucrose or yeast solutions and the flies were allowed to select which source of food to use.

They will be called "self selecting flies". Only one capillary was used in the "no choice" experiments. It was filled with pure yeast, pure sucrose or mixed yeast/sucrose solutions.

Flies were allowed to recover from anaesthesia for 6 hours and maintained at 21°C. The height of the liquid column in the capillaries was measured twice a day using callipers and during 2-3 consecutive days.

Figure 1A plots liquid intakes (using a 5S solution) as a function of time and of the number of flies used (between 3 and 9). Intakes were linearly related to time. The slopes of the representations were measured and used to calculate daily volumes intakes. Figure 1B shows that daily intakes were linearly related to the number of flies used. The representation extrapolated to a value of 2 µl / day in the absence of flies. This value represented the rate of evaporation from the bottom of the capillaries and was used to correct all intake data. This evaporation cannot be neglected. It limits the precision of ingestion rates and imposes important constraints to the design of the experiments. The conditions used (6 flies per tube fed with a single capillary) provided the best precision. Precision was lowest when the amount of ingested liquid was the lowest, for instance when rich, 10S or 10Y solutions were used. No evidence for a dependence of food consumption on fly number could be obtained, as reported by [START_REF] Ja | Prandiology of Drosophila and the CAFÉ assay[END_REF]. Different results have been reported for flies fed on agar based food [START_REF] Wong | Quantification of food intake in Drosophila[END_REF].

Survivorship of flies fed on liquid solutions.

Flies were fed with liquid solutions as described above for the capillary feeding assay with two differences; (i) groups of 10 flies were used and (ii) two capillaries per vials were used. Vials were maintained at 21°C and dead flies were scored every day. Liquid solutions in the capillaries were changed twice a week.

Survivorship of flies fed on semi solid media.

Three types of semi solid food mixtures were prepared: (i) 10% sucrose in 2% agar, (ii) 10% yeast in 2% agar and (iii) 10% yeast and 10% sucrose in 2% agar. Lyophilized yeast was used in these experiments. All mixtures were supplemented with 3.75 g/l of methyl 4hydroxybenzoate. Mixtures were layered in Petri dishes and 1 cm 2 squares of jellified food patches (height ~ 0.3 mm) were cut with a razor blade. Two patches were glued onto the side of the plastic tubes with a 2% agar solution and at a distance of 1.5 cm from the bottom of the tubes. Male, 1 day old, flies were added in group of 10 to the tubes and tubes were left in an upright position at 25°C. Dead flies were counted everyday. In the "choice" conditions flies were given the choice to forage on a patch of sucrose, a patch of yeast and agar (which provides an independent source of water). In the "no choice" condition, flies could only forage on patches of mixed sucrose/yeast or on agar. Flies were transferred to new patches of food twice a week.

Statistical analyses

Means were compared using unpaired t tests or two way ANOVA with Benferroni correction. Survivorship curves were compared using the log rank test. The GraphPad Prism 4 software was used. P values < 0.01 were considered as statistically significant.

Results

Influence of ageing on feeding behaviour

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The feeding behaviour of aging male flies was analysed using a capillary feeding assay. Four age classes were selected: 1, 10, 20 and 30 days. Flies were exposed to a pure sucrose solution, to a mixed sucrose/yeast solution or to independent sources of sucrose and yeast. Figure 2 compares sucrose intakes by flies exposed to rich diets (10S, 10S10Y or 10S-10Y) or to poorer, two fold diluted diets (5S, 5S5Y or 5S-5Y). Under all conditions, we observed that flies on the diluted diet consumed larger volumes of the nutritive solutions, which indicated compensatory feeding. As a consequence, net daily ingestions of sucrose were similar (Figure 2). Compensatory feeding was observed at all ages from 1 to 30 days.

Different results were obtained with pure yeast diets. The daily intake of yeast by 1 day old flies fed on a 10Y solution was 39 ± 2 µg (n = 8). It was 2 times larger (77 ± 6 µg, n = 12) when flies were fed with a diluted 5Y solution (p < 0.01). Thus, young flies overcompensated for the dilution of a yeast solution. Aged, 30 day old flies were different. The daily intakes were 85 ± 12 µg (n = 6) and 20.5 ± 3.0 µg (n = 7) when flies were fed on a 10Y diet and a 5Y diet respectively (p < 0.01). Thus, aged flies did not compensate for dilutions of their food. We then analyzed yeast intake by self selecting flies. Figure 2B shows that flies compensated for a 2 fold dilution of their food when they were 1 day old. They did not compensate for food dilutions when they were 10, 20 or 30 day old. These results indicated that regulation of yeast intake changed between 1 and 10 days of age.

Figure 3 analyses intake data according to the nutritional geometrical model developed by Simpson andRaubenheimer (1997, 1999). This representation relates sucrose and yeast intakes under different conditions. Figure 3 first shows that flies under self selection conditions had a marked preference for sucrose. The sucrose: yeast ratio of ingested food was 1:3 to 1:5 and this value was not affected by ageing. Figure 3 also shows that self selecting flies and flies fed on a pure sucrose diet ingested similar amounts of sucrose, suggesting that flies had a better control of their sucrose yeast intake than of their yeast intake. Thus, the additional intake of yeast was not accompanied by a decreased intake of sucrose.

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Flies fed on a mixed diet have no choice. They are forced to ingest yeast and sucrose in proportions that are different from those of self selecting flies. Figure 3 shows that flies fed on mixed diets ingested less sucrose than self selecting flies (p < 0.01) and two times more yeast (p < 0.01). Very similar results were reported by [START_REF] Lee | Ageing and reproduction in Drosophila: new insights from nutritional geometry[END_REF] for female flies.

Taken together these results indicated that flies did not regulate their daily calorie intake and responded to specific nutrients. They had a marked preference for sucrose and they regulated their intake of sucrose more tightly than their intake of yeast. This study further shows that ageing had little influence on feeding preferences, nutrient intake targets, and the capacity of the flies to respond to mixed diets.

The limit to compensatory feeding

Is there a limit to compensatory feeding when flies are exposed to highly diluted sources of food? To answer this question, we exposed 1 day old flies to serial dilutions of a 10S feeding solution and measured their daily intakes of liquid. Figure 4A shows that flies compensated for 2 to 3 fold dilutions of a 10S solution. They did not compensate for a 5 fold dilution. Thus, compensatory feeding was limited at large dilutions of a sucrose solution. As a consequence, the daily intake of sucrose is a saturable function of the dietary sucrose (Figure 4B). The curve is usually interpreted as feeding compensation; i.e. flies increase their feeding in response to food dilution. It can also be viewed as a saturable phenomenon. For instance, flies fed on concentrated sucrose solutions could be rapidly satiated and could stop feeding.

Feeding flies on dilute solutions did not promote longevity.

The existence of compensatory feeding mechanisms calls for a reassessment of the protocols of dietary restriction. It is well known that diluting food (provided as a semi solid medium) increases longevity probably because flies are dietary restricted. The previous results (Figures 2 and3) indicated that flies fed on a liquid 5S5Y diet ingested just the same amount of nutrients as flies fed on a liquid 10S10Y diet, meaning that they were not dietary restricted. As a consequence their longevities should not be modified by a change in diet from 10S10Y to 5S5Y. Figure 5 shows that, as expected, longevities were identical under the two conditions. [START_REF] Lee | Ageing and reproduction in Drosophila: new insights from nutritional geometry[END_REF] recently reported that feeding flies on liquid solutions under self selection conditions promoted longevity. We therefore asked whether flies responded to self selection conditions when nutrients were presented as semi solid food. Figure 6 shows the results of an experiment in which flies were given the choice to feed on separate patches of 10% yeast and 10% sucrose or forced to feed on patches of mixed 10% sucrose/10% yeast.

Self selection conditions promote longevity

It shows that flies under self selection conditions had an increased median longevity. The maximum longevity was not affected however. We also observed that the first deaths occurred soon after the start of the experiments, which suggests that flies experienced more stressful conditions. It should be noted that in these experiments, flies did not sit on food and had to climb to the top of the tubes in order to reach their food. As a consequence, weak flies have probably less chance to survive than under standard conditions.

Discussion.

This study analyses the feeding behaviour and feeding preferences by male Drosophila and their relationships to longevity In a key paper, [START_REF] Lee | Ageing and reproduction in Drosophila: new insights from nutritional geometry[END_REF] showed that female flies regulate their intake of proteins and carbohydrates separately. Under self selection conditions, they select a low yeast diet that maximises fitness. This study shows that male flies have a similar behaviour.

Male flies have a 3 to 5 fold preference for sucrose when sucrose and yeast are presented separately. When there is no choice, male flies control the sucrose intake more tightly than the yeast intake.

One objective of this study was to determine whether flies changed their feeding behaviour with age. Analysis of the data using the nutrient geometrical framework [START_REF] Simpson | Geometric analysis of macronutrient selection in the rat[END_REF][START_REF] Simpson | Assuaging nutritional complexity: a geometrical approach[END_REF][START_REF] Lee | Ageing and reproduction in Drosophila: new insights from nutritional geometry[END_REF] indicates that flies do not modify their preference for sucrose and their responses to mixed diets. Flies respond to a dilution of a sucrose diet by compensatory feeding and this response is little affected by ageing.

A change in the regulation of yeast intake was observed as flies reach 10 days of age. Young, 1 day old, flies respond to a dilution of a yeast solution by compensatory feeding. Older (10, 20 or 30 day old) flies do not (Figure 2). In other words, yeast intake by young flies saturates. As a consequence, compensatory feeding is observed when a yeast solution is diluted. Yeast intake by aged flies does not saturate. The more yeast is provided to aged flies, the more yeast is ingested. Compensatory feeding was observed however when aged flies were fed with mixed diets (Figure 2D). Thus, the presence of sucrose corrected for the changes in yeast intake.

Limits to compensation

This study also confirms previous reports [START_REF] Carvalho | Compensatory ingestion upon dietary restriction in Drosophila melanogaster[END_REF][START_REF] Ja | Prandiology of Drosophila and the CAFÉ assay[END_REF][START_REF] Lee | Ageing and reproduction in Drosophila: new insights from nutritional geometry[END_REF] showing that flies respond to a dilution of their diets by compensatory feeding.

We further show that compensation was nearly complete over a large range of sucrose concentrations, from 3% to 10%. Flies exposed to lower concentrations of sucrose did not compensate for the dilution of food. The reason for this lack of compensation is not known.

We observed however that flies fed on a diluted 2% sucrose solution ingest about 1.2 µl of water solution per day. This volume is 3 times larger than the water content of a fly (0.4 mg assuming an average wet weight of 0.6 mg and a mean water content of 69%, [START_REF] Gibbs | Evolution of water balance in the genus Drosophila[END_REF]. How flies handle such a water load is not known. Water balance in Drosophila has been studied in relation to desiccation resistance (Giggs andMatzkin, 2001, Albers and[START_REF] Albers | Osmotic regulation in adult Drosophila melanogaster during dehydration and rehydration[END_REF]. The responses to large water loads have not yet been studied. In heamatophagous insects, feeding triggers a rapid diuresis which is used to eliminate the excess of water [START_REF] Maddrell | The regulation of haemolymph potassium activity during initiation and maintenance of diuresis in fed Rhodnius proxilus[END_REF]. It is of interest to note that flies fed on dilute solutions lay down numerous liquid droplets on the wall of the culture tubes, which indicates diuresis. Thus, water intake may limit the intake of nutrients when flies are fed on highly diluted solutions.

Influence of food presentation.

Dietary restriction, which increases longevity in Drosophila, is usually achieved by diluting food [START_REF] Partridge | Dietary restriction in Drosophila[END_REF]. This and previous studies [START_REF] Ja | Prandiology of Drosophila and the CAFÉ assay[END_REF][START_REF] Ja | Prandiology of Drosophila and the CAFÉ assay[END_REF][START_REF] Lee | Ageing and reproduction in Drosophila: new insights from nutritional geometry[END_REF][START_REF] Wong | Quantification of food intake in Drosophila[END_REF] clearly indicate that food presentation modifies the effect of dietary restriction.

(i) semi solid vs. liquid diet. The feeding behaviour of flies fed on agar based food was analyzed in great details by [START_REF] Wong | Quantification of food intake in Drosophila[END_REF]. Briefly, flies fed on semi solid food do not change their feeding behaviour after switching to diluted food [START_REF] Skorupa | Dietary composition specifies consumption, obesity, and lifespan in Drosophila melanogaster[END_REF][START_REF] Wong | Quantification of food intake in Drosophila[END_REF]. They can be dietary restricted. Their longevity is usually increased as compared to flies fed on richer diets. The feeding behaviour of flies fed on liquid food is different. Flies respond to a dilution of their liquid food by compensatory feeding. They cannot be dietary restricted. Their longevity is not modified by a two fold dilution of their food.

It should be noted that different sources of yeast have been used in the two types of experiments. Soluble yeast extracts are used in capillary feeding experiments such as the ones presented here. Heat inactivated lyophilized yeast is used to prepare agar based food mixtures. [START_REF] Wong | Quantification of food intake in Drosophila[END_REF] recently reported that flies on agar based media containing yeast extracts decrease their feeding frequency as the concentration of yeast extract is increased. They do not when they are fed on agar based media containing lyophilized yeast.

Thus, the source of yeast probably contributed to the different behaviours of flies exposed to liquid or agar based diets. Results presented here do show that flies which compensate for different quality of their foods and which cannot be dietary restricted, do not get any benefit from a dilution of their food. longevity of female flies fed on liquid food [START_REF] Lee | Ageing and reproduction in Drosophila: new insights from nutritional geometry[END_REF]. This study reports similar results for male flies fed on agar based food. Self selecting flies which have the choice to feed on separate sources of sucrose and yeast are longer lived than flies which are forced to ingest sucrose and yeast in a 1/1 ratio.

Conclusions.

This study shows that the feeding behaviour of Drosophila is far more complex than usually considered. Flies sense sucrose and yeast nutrients independently and adapt to changes in the quantity and presentation of their foods. This study, together with recent studies [START_REF] Lee | Ageing and reproduction in Drosophila: new insights from nutritional geometry[END_REF][START_REF] Skorupa | Dietary composition specifies consumption, obesity, and lifespan in Drosophila melanogaster[END_REF][START_REF] Wong | Quantification of food intake in Drosophila[END_REF] stresses that a careful analysis of feeding behaviour is necessary before drawing any conclusion the relationship between diet and longevity.

Legends to the Figures Figure 1. Correcting for evaporative losses

Flies (3 to 9 per tube) were fed with a capillary filled with a 5S solution and total liquid intakes were measured (Panel A). Daily intakes were calculated from the slopes of the representations and plotted against the number of flies used (panel B). A control experiment using fly free tubes was also performed. The daily evaporation rate was estimated to 2 µl/day. Means ± SEM are indicated (n = 6 -13).

Figure 2. Feeding preferences are independent of age.

Flies were fed with different diets as indicated and the daily intakes of nutrients were determined. Flies used at 1, 10, 20 and 30 days as indicated. Filled bars refer to the richest dietary conditions (10S10Y, 10S-10Y or 10S). Open bars refer to the poorest diets (5S5Y, 5S-5Y or 5S). All graphs have been drawn to the same scale to highlight differences.

A. Sucrose intake by self selecting flies. A two way ANOVA indicated a statistically significant effect of the sucrose concentration (p = 0.002); Age had no effect. Mean intakes were computed using pooled data were 51.9 ± 3.1 µg (n = 63) and 43.5 ± 1.9 µg (n = 72) for flies fed on 10S-10Y and 5S-5Y diets respectively. Thus, flies responded to the diluted solution of sucrose by increasing their intake of sucrose solution. Yet, compensation was not complete and flies on a 5S-5Y diet got less sucrose than flies on the 10S-10Y diet.

B. Yeast intake by self selecting flies. A two way ANOVA indicated statistically significant effects of yeast concentration and age (both p< 0.0001). Yeast intake by 1 day old flies fed on 10S-10Y or 5S-5Y diets were not different. Young flies largely compensated for a two fold dilution of their yeast diet. Yeast intake by older flies (10, 20 or 30 days) was significantly larger (p<0.001) when flies were fed on a 10Y diet. Thus aged flies lose their capacity to compensate for a two fold dilution of their yeast diet.

C. Sucrose intake by flies on pure sucrose diets. A two way ANOVA indicated that age and the concentration of sucrose had no effect. Mean intakes were computed for pooled data. They were 39.5 ± 2.0 µg (n = 78) and 43.9 ± 1.9 µg (n = 70) for flies fed on 10S and 5S diets respectively. Thus, flies fully compensated for a two fold dilution of a pure sucrose diet. D. Sucrose intake by flies on mixed diets. A two way ANOVA indicated that food concentration and age had no effect. Mean intakes were computed for pooled data. They were 27.9 ± 2.7 µg (n = 63) and 34.7 ± 1.8 µg (n = 52) for flies fed on 10S10Y and 5S5Y diets respectively. Thus, flies compensated for a two fold dilution of a mixed 10S10Y diet.

Note that in these experiments, yeast and sucrose were mixed in the same solution. As a consequence flies ingested identical quantities of yeast and sucrose. Flies of different ages were fed on diets consisting of different proportions of sucrose and yeast (10Y, 5S5Y, 10S10Y, 5S-5Y, 10S-10Y, 5S, 10S and 10Y as indicated) and the daily intakes of sucrose and yeast were determined. Data corresponding to the different ages (0, 10, 20, 30 days) are clustered into intake spaces. Numbers on their sides indicate the corresponding ages. The thin lines represent the nutritional rails for different yeast to sucrose ratios. Self selecting flies had a 3 to 5 fold preference for sucrose. This preference and the intake targets were not modified as fly age from 1 day to 30 days. Flies were fed on a mixed 10S10Y diet under "choice" (10S-10Y) or "no choice" (10S10Y) conditions and survivorship curves were determined using conditions defined as Protocol B in the Materials and Method section. Median and maximum longevities were 41 days and 81 days under "no choice" conditions. They were 55 days and 83 days under "choice" conditions. Sample sizes were 192 and 119 under the "no choice" and "choice" conditions respectively. P < 0.01 using the log rank test.

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Figure 3 .

 3 Figure 3. Analysis of feeding data using the nutritional geometrical framework.

Figure 4 .

 4 Figure 4. Limits to compensatory feeding A. Flies were fed on serial dilutions of 10S solution and daily intakes of solution were determined. Means of 9-12 experiments are indicated. Error bars were smaller than the size of the symbols. The dotted line represents the volumes of solution that had to be ingested in order to maintain a daily intake of 36 µg, i.e. to fully compensate for food dilution. B. Sucrose intake is a non linear, saturable function of dietary sucrose. Daily intakes of sucrose were calculated from the data presented in panel A. Means ± SEM are shown. The sucrose intake curve can be viewed as a saturation phenomenon (upper arrow, flies avoid highly concentrated solutions) or as feeding compensation (lower arrow, flies increase their feeding in response to food dilution).

Figure 5 .

 5 Figure 5. The beneficial effect of dietary restriction is lost when flies are fed on liquid diets. Survivorship curves of flies fed on liquid 10S10Y (N = 122, median: 26 days) and 5S5Y (N = 129, median: 31 days) diets. P = 0.80 using the Log rank test.

Figure 6 .

 6 Figure 6. Self selecting flies are longer living.

Acknowledgements

This study was funded by the INSERM, ANR and the University of Nice Sophia Antipolis. We are grateful to anonymous reviewers for their comment of on a previous version of this paper.


Anke Thielemann

Prof. Dr Wolfgang Wuttke

Marcus Wuttke

Dana Seidlova-Wuttke

Comparison of urodynamic effects of phytoestrogens equol, puerarin and genistein with these of estradiol 17β in ovariectomized rats

Keywords: Urinary incontinence, bladder, urethra, pressure, urethral closure, equol, genistein, puerarin

Comparison of urodynamic effects of phytoestrogens equol, puerarin and genistein with these of estradiol 17β

in ovariectomized rats

Introduction

Urinary incontinence is a typical but often hidden complaint of postmenopausal women [START_REF] Irwin | Population-based survey of urinary incontinence, overactive bladder, and other lower urinary tract symptoms in five countries: results of the EPIC study[END_REF]. While urge incontinence is based on nervous disregulation (so-called overactive or unstable bladder), stress incontinence is based on an insufficient closure mechanism of the urethral sphincter during increased intraabdominal pressure [START_REF] Abrams | The standardisation of terminology in lower urinary tract function: report from the standardisation sub-committee of the International Continence Society[END_REF]. According to [START_REF] Swanson | Urinary incontinence: common problem among women over 45[END_REF] menopause is mostly associated with mixed incontinence in around 50%, stress incontinence in 34% and urge incontinence in 15% of cases. Since the Women's Health Initiative (WHI) trial was stopped prematurely, hormone replacement therapy (HRT) for treatment of climacteric complaints is widely criticized [START_REF] Ness | Use of hormone replacement therapy by postmenopausal women after publication of the Women's Health Initiative Trial[END_REF]. Treatment of women suffering from urinary incontinence with estrogens is also controversially discussed [START_REF] Hendrix | Effects of estrogen with and without progestin on urinary incontinence[END_REF][START_REF] Kim | Is estrogen for urinary incontinence good or bad?[END_REF][START_REF] Tseng | Randomized comparison of tolterodine with vaginal estrogen cream versus tolterodine alone for the treatment of postmenopausal women with overactive bladder syndrome[END_REF]. The growing insecurity of women whether or not to practice HRT has increased the request for plant substances as hormone alternatives [START_REF] Nelson | Nonhormonal therapies for menopausal hot flashes: systematic review and meta-analysis[END_REF]. However, effects of plant substances such as the soy derived isoflavones which have structural similarity with 17βestradiol (E2) are not understood in detail. Genistein (Gen) is the main soy isoflavone and has the highest estrogenic potency of all soy constituents as tested in cell biological experiments [START_REF] Chen | Effects of genistein, resveratrol, and quercetin on steroidogenesis and proliferation of MA-10 mouse Leydig tumor cells[END_REF]. Effects of E2 in the urethra and urinary bladder are exerted via both estrogen receptor (ER) subtypes as the presence of both the ER and ERß has been demonstrated in the urinary tract [START_REF] Tikkanen | Dietary soy-derived isoflavone phytoestrogens. Could they have a role in coronary heart disease prevention?[END_REF]. Equol (Eq) is a biologically active metabolite of daidzein (Daid), which is another main isoflavone of soy extracts. Eq is exclusively produced by intestinal bacteria and only about one third of people possess the proper intestinal flora [START_REF] Setchell | The clinical importance of the metabolite equol-a clue to the effectiveness of soy and its isoflavones[END_REF]. Puer is an isoflavone derived from the root of Pueraria lobata, widely known as Kudzu in traditional Chinese medicine. [START_REF] Boue | Evaluation of the estrogenic effects of legume extracts containing phytoestrogens[END_REF] demonstrated the highest estrogenic activity from all Kudzu constituents for Puer. Since Puer is the glycosylated form of Daid it must be deglycosylated by intestinal bacteria, therefore, a depot effect of Puer may be assumed [START_REF] Park | Intestinal bacteria activate estrogenic effect of main constituents puerarin and daidzin of Pueraria thunbergiana[END_REF]. Data regarding the therapy of urinary incontinence in menopause with phytoestrogens is rare. [START_REF] Manonai | The effect of a soy-rich diet on urogenital atrophy: a randomized, cross-over trial[END_REF]) demonstrated a significant increase of complaints regarding urge incontinence and vaginal atrophy after a 12 week soy-free diet of 36 climacteric Thai women. A soy-rich diet however, in contrast could not relieve the pertained complaints. [START_REF] Tomaszewski | Effect of 17 beta-estradiol and phytoestrogen daidzein on the proliferation of pubocervical fascia and skin fibroblasts derived from women suffering from stress urinary incontinence[END_REF] showed that fibroblasts out of the pubocervical fascia of climacteric women have a higher proliferation activity under E2 and Daid compared to skin fibroblasts It is known that a dedicate equilibration between detrusor muscle pressure of the urinary bladder and the sphincter pressure of the urethra are of importance for continence [START_REF] Delancey | Anatomy and physiology of urinary continence[END_REF][START_REF] Delancey | Pathophysiology of adult urinary incontinence[END_REF][START_REF] Keane | Urinary incontinence: anatomy, physiology and pathophysiology[END_REF]. A good animal model to collect urodynamic data was shown previously to be the ovariectomized (ovx) rat in which irregular contractions of the detrusor of the bladder occurred such that the intravesicular pressure often exceeded the urethral pressure which resulted in urine leakage [START_REF] Seidlova-Wuttke | Urodynamic effects of estradiol (E2) in ovariectomized (ovx) rats[END_REF]. Hence, this overactivity of the bladder of ovx rats is reminiscent of the instable bladder in women suffering from urge incontinence [START_REF] Keane | Urinary incontinence: anatomy, physiology and pathophysiology[END_REF]. In our earlier study E2 treatment of ovx animals both increased intravesicular and intraurethral pressures but in the urethra to a higher degree than in the bladder. Therefore, unwilling voiding during the filling periods of the urinary bladder occurred much less frequently and at lower volumes in the estrogen treated animals [START_REF] Seidlova-Wuttke | Urodynamic effects of estradiol (E2) in ovariectomized (ovx) rats[END_REF]. In the present study we investigated the effects of Gen, Eq and Puer on urethral and urinary bladder pressures during and after 2 artificial filling periods and compared them with those in ovx but otherwise untreated and with those in E2 treated animals.

Material and Methods

The study was performed as part of a larger trial designed to investigate a number of endpoints in chronically substituted ovariectomized rats. Permission to perform experiments was obtained from the Regional Administrative Authority in Braunschweig (AZ 33.42502-032/06). Sixty 3 months old female Sprague Dawley rats with an average weight of 227 g were Puerchased from Winkelmann (Borchen, Germany). Rats were kept under standard conditions in the animal experimental facilities of the University of Goettingen (6 rats per Makrolon ® IV cage, 20° C, 50% relative humidity, light on from 6 am to 6 pm, water and food ad libitum). After a two week acclimatization period on a soy-and phytoestrogen-free diet, all experimental rats were ovariectomized under isoflurane anaesthesia. From this point in time, rats in groups of 12 animals were kept under special diet supplied by Ssniff (Ssniff Spezialdiaeten GmbH, Soest, Germany). In control group Co-SF, feeding with soy-and phytoestrogen-free pellets continued. All other groups received pelleted food supplemented with the phytoestrogens to be tested. On the basis of the twice weekly measured food intake per cage the approximate daily intake of each individual rat and consequently the amount of test substances could be calculated (Table I).

Urodynamic procedures

After 12 weeks of food supplementation, rats were anaesthetised with isoflurane and the urodynamic experiment was performed. In a supine position, the bladder was catheterized transurethrally using a biluminal catheter (CS-14402: Pediatric Two-Lumen Central Venous Catheterization Set with Blue FlexTip ® Catheter: 4 Fr. x 5-1/8'' (13 cm), Arrow). As soon as the catheter reached the bladder during catheterization, residual urine volume was released. Both openings of the biluminal catheter were connected to electromechanical pressure monitoring devices and in that way to a computer for recording measured values. The proximal catheter opening, positioned in the bladder, was connected to an infusion pump with saline solution. Correct position of proximal catheter opening was checked by automatic urine release and decreasing pressure values down to zero after catheterization. The distal catheter opening was positioned in the urethra in juxtaposition to the internal sphincter. Correct positioning was ensured by reaching highest possible pressure values during catheter insertion. Having ensured correct catheter positioning, the urodynamic experiment was started by infusion of 0.5 ml saline solution over 30s. Infusion was then stopped for 60s. A second infusion period of 0.5 ml saline solution over 30s succeeded. The experiment ended with a 120s observation period. Pressure values were monitored over the entire time span of 240s. Animals were sacrificed after another life span of 2 weeks, uteri were removed and wet weight was determined. Blood was collected for later determination of E2 concentrations by a commercially available assay (Elecsys Roche, Mannheim, Germany).

Analysis of urodynamic data

Analysis of Urodynamic data was performed with GraphPad Prism TM Version 4.00 for Windows (GraphPad Software, San Diego, California, USA). Figure 2 to 6 show that the experiment consisted of different periods. The 2 infusion periods lasted 30 seconds and in between these 2 infusion periods 2 times 30 seconds relaxation was allowed. Following the 2 nd infusion period, recording of intravesicular (vP) and intraurethral pressure (uP) commenced for four 30 second periods. During these periods, the mean values of 2 second time segments per group were calculated and shown in Fig. 2 to 6. In addition, the differences between urethral and intravesicular pressures (ΔuP -vP) were calculated to demonstrate putative beneficial effects of the different treatments. Hence, a large difference between these two pressures indicated highest continence whereas low differences between these two pressures or even negative differences were indicative of an incontinent situation. Pressure values were analysed in 2-sec intervals and the means over 30-sec periods were calculated followed by a one-way ANOVA using Dunnett's post test for significance. A pvalue of less than 0.05 was considered significant.

Results

Average body weight (BW) of animals at the beginning of the experiment was 227,29 ± 2,79 g SD. Mean daily food intake per animal was significantly decreased under Eq, Puer and E2 supplementation (Table I) in comparison to animals fed with unsubstituted chow and this resulted in significantly decreased body weights (Table II) . This effect was not seen in the Gen treated animals. Supplementation with E2 resulted in serum E2 concentrations of 40.2 4.6 pg/ml vs undetectable levels in the control animals. The E2 treated animals had highest uterine weights compared to the control group (Fig. 1) and increased uterine weights were also recorded under Gen and Puer but not under Eq supplementation. Fig. 2 details mean bladder and urethra pressures during and after the 2 filling periods in the control animals (Fig. 2a) and in the E2 treated rats (Fig. 2b). In comparison to the control group urethral pressure curves under E2 supplementation were consistently higher than the intravesicular pressures except during the 2 nd filling period when the mean urinary bladder pressure exceeded the urethral pressure. In the control animals urethral and vesicular pressures were significantly lower than in E2 treated animals during the first filling period and the values overlapped at many time points as indicated by the frequent overlapping of the SD. During and after the 2 nd filling period the means of uP and vP were almost identical in control animals whereas bladder pressure in the E2 treated rats dropped dramatically to values well below those recorded in the urethra at all times after the 2 nd filing period. In Fig. 3, bladder and urethral pressure changes during the experiment's course are shown. In comparison to the control group Co-SF (see Fig. 2a), urethral pressures in the phytoestrogen treated animals fluctuate less during the infusion times and were similar to those observed in the E2 treated animals. Therefore, as in the E2 treated animals pressures in the urethra were usually higher than in the bladder. Fig. 4 depicts mean urethral pressures per 30s interval. At each time point they were significantly higher under supplementation with E2, Puer, Gen and Eq in all study periods compared to the control group. In Fig. 5 mean bladder pressures per 30s intervals are shown. Under E2 and phytoestrogen supplementation, bladder pressures were significantly higher compared to control group in the first study phases, whereas values dropped significantly below control group's values in the relaxation phases at the experiment's end. Fig. 6 details mean differences of pressures in urethra minus those in the bladder in 30s intervals. In cases of similar or even negative values the bladder pressures were higher than in the urethral which facilitated unwilling voiding. In all study phases values for these differences were lowest in the control animals and significantly higher in the E2 and isoflavones treated animals.

Discussion

Purpose of our experiments was to determine the influence of the phytoestrogens Eq, Puer and Gen on the urodynamic profile of ovariectomized rats. Thus, consequences for therapy of urinary incontinence after menopause may be determined. Special interest was focussed on previously not studied urethral pressures (uP) and the ovariectomized rats were used as they constitute an established model for menopause research [START_REF] Wu | Ovarian aging and menopause: current theories, hypotheses, and research models[END_REF]. Uterine weights as measurement for estrogenic effects in ovariectomized rats are proposed in the OECD guidelines regarding as the uterotrophy assay [START_REF] Owens | The OECD program to validate the rat uterotrophic bioassay: an overview[END_REF] In our experiments, uterine wet weights were not only significantly increased in the Estradiol group, but also in animals treated with Puer, Gen and Eq. However, increase in those groups was lower than under E2 and increase under Eq was not statistically significant. [START_REF] Zheng | Estrogen-like effects of puerarin and total isoflavones from Pueraria lobata[END_REF] supplemented different doses of Puer orally in mice and ovx rats for 5 to 9 days to determine estrogen-like effects of Puer. Estrogenic effects were quantified by vaginal cytology, uterine and ovarian weights and a dose-dependent stimulation of uterine weight and vaginal cornification was demonstrated. Vaginal weight of mice previously treated with E2 decreased under Puer administration; on this basis [START_REF] Zheng | Estrogen-like effects of puerarin and total isoflavones from Pueraria lobata[END_REF] concluded that Puer has estrogen-agonistic effects in estrogen depleted and estrogen-antagonistic effects in E2 substituted animals. The estrogen-agonistic characteristics of Puer in this study are comparable to the results of our trial. Ovx is known to result in obesity [START_REF] Wade | Gonadal effects on food intake and adiposity: a metabolic hypothesis[END_REF] and this can be prevented by estrogens. This is confirmed in the present study and also the phytoestrogens had an obesity preventive effect which was strongest for Puer>Eq>Gen whereby the effects of Gen and Eq were not statistically significant. The observation that Gen has a relatively strong uterotrophic effect but a minor obesity preventive effect is of interest and points to some tissue specificity of this isoflavone. Puer with a molecular weight (MW) of 416 is the glycosylated form of Daid (MW 254) and is deglycosylated by the intestinal flora [START_REF] Rong | Identification of isoflavones in the roots of Pueraria lobata[END_REF]. Hence, the active principle is Daid and the mean daily uptake of 44 mg Puer would amount to 27 mg Daid after deglycosylation. These amounts had similar uterotrophic effects as Gen but were much more effective to prevent obesity. If this difference occurs also in postmenopausal women a Puer/Daid containing preparation would be preferable to Gen containing products. Catheterizing of the urinary bladder with the biluminal catheter of which one lumen was located within the bladder the other within the sphincter of the urethra, allowed recording of intravesicular and intraurethral pressure during two filling periods. This was elaborated in previous experiments [START_REF] Seidlova-Wuttke | Urodynamic effects of estradiol (E2) in ovariectomized (ovx) rats[END_REF]. As demonstrated there and confirmed in the present experiments E2 had significant tonus stabilizing effects on both urethral and vesicular pressures, which were both low in the ovariectomized animals. The present study shows that from the 3 phytoestrogens tested, Puer had the strongest estrogenic activity in urinary bladder/urethral system. By investigating isoflavones contained in Pueraria lobata (Kudzu) with high-performance liquid chromatography coupled to photodiode array and to mass spectroscopy, [START_REF] Rong | Identification of isoflavones in the roots of Pueraria lobata[END_REF] showed that Puer is the glycosylated form of Daid and they concluded that estrogenic activity occurs only after deglycosylation by gut bacteria which takes time. It may therefore be assumed that Puer has a depot effect. Such a depot effect of Puer would be a possible explanation for the stronger estrogenic effects in the urinary tract in comparison to Gen or Eq. As such depot effect cannot be assumed for Eq and Gen, the difference in pressure levels may be explained in this way. Voiding is the result of bladder pressure exceeding urethral pressure and this may occur unwillingly which results in incontinence. Hence, the differences between urethral minus bladder pressure are indicators of continence, if negative or close to zero, i.e. overlapping, unwilling voiding may be the result. As illustrated paradigmatically in Fig. 2 vP and uP in ovx control animals are considerably lower than in E2 treated animals which confirms our previous observation. The increased bladder and urethral pressure are most likely due to increased muscular activity as a higher muscle mass of the detrusor and sphincter and higher blood supply was reported in ovx-E2 in comparison to untreated animals [START_REF] Aikawa | The effect of ovariectomy and estradiol on rabbit bladder smooth muscle contraction and morphology[END_REF]Lin et al., 2006a;Lin et al., 2006b;Lin et al., 2006c). It appears that the phytoestrogens have similar, though weaker effects in the detrusor than in the sphincter apparatus. Studies of estrogen effects in the urethra and the urinary bladder of animal origin are scarce. Morphological changes in response to ovariectomy and E2 replacement have been reported. For functional studies most authors used either isolated urinary bladder tissue strips or intact bladders of ovariectomized (ovx) rats and rabbits, both kept under in vitro conditions, and these few experiments indicated that E2 affects the function of the voiding mechanisms. It was shown that detrusor contractility in rodents and its innervation are sensitive to estrogen deficiency. Estrogen withdrawal causes an over reactivity of urinary bladder tissue, which can be "smoothened" by estrogens, and it appears that ß2or 3-adrenergic receptor subtypes may be involved in this process. This effect may involve sympathetic and parasympathetic preganglionic neurons innervating the bladder, which express the estrogen receptor of the -and ß-subtype. Owing to the presence of ERß in the urothelium and ER in the fibroblasts and detrusor muscle cells, a direct action of estrogens on these cell types is also likely and, indeed, histological changes in ovx estrogen-replaced animals have been demonstrated when compared to ovx controls. In isolated rabbit bladder preparations weak estrogens such as genistein caused also contractile response induced by muscarinergic/purinergic receptor activation [START_REF] Ratz | Differential effects of sex hormones and phytoestrogens on peak and steady state contractions in isolated rabbit detrusor[END_REF] and in a recent publication it was shown that detrusor overactivity in ovx rats was also reduced by an application of a mixture of commercially available isoflavones given orally at approximately the same amounts as in the present study [START_REF] Okada | Dietary soy isoflavone replacement improves detrusor overactivity of ovariectomized rats with altered connexin-43 expression in the urinary bladder[END_REF]. The temporal aspects of bladder and urethral pressures during and after the 2 filling periods under the different treatments deserve also some discussion. The vP was similarly high throughout the initial 30 sec filling phase and the following 60 sec in the treated groups. Even though the absolute values for uP and vP were significantly higher in the E2 treated animals during the 2 nd 30 sec filling period which filled the bladder maximally with 1 ml, the ΔuP-vP was lowest in all groups, hence in many animals the vP was higher than the uP which indicated incontinence. Interestingly the ΔuP-vP in all following phases (in which the bladder remained filled) remained low or even negative indicative for incontinence. This was neither seen in the E2 nor in the phytoestrogens treated animals in which the vPs decreased progressively while the uPs stayed stable. Hence, the ΔuP-vP stayed high indicating continence. It was also interesting to note that the magnitude of ΔuP-vP after the 2 nd filling period did not differ significantly. Using estrogen receptor binding assays, it was repeatedly shown that isoflavones have a slightly higher affinity to ERß then to ER [START_REF] Kuiper | Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta[END_REF][START_REF] Mueller | Phytoestrogens and their human metabolites show distinct agonistic and antagonistic properties on estrogen receptor alpha (ERalpha) and ERbeta in human cells[END_REF] though their transactivational efficacy was similarly strong via both ER subtypes [START_REF] Mueller | Phytoestrogens and their human metabolites show distinct agonistic and antagonistic properties on estrogen receptor alpha (ERalpha) and ERbeta in human cells[END_REF]. Dominant occurrence of ERβ in urethral epithelium of adult rats was shown by [START_REF] Makela | Differential expression of estrogen receptors alpha and beta in adult rat accessory sex glands and lower urinary tract[END_REF] using immunohistochemistry and Northern Blot [START_REF] Salmi | Co-localization of androgen receptor with estrogen receptor beta in the lower urinary tract of the male rat[END_REF] found that ERβ is predominant in urethral muscle of rats. Therefore, increased urethral pressures under E2 and isoflavone supplementation might be an effect of their affinity to ERβ. As a result of high affinity, muscle tone of the urethral sphincter might rise and lead to increased uPs. [START_REF] Strauss | Genistein exerts estrogen-like effects in male mouse reproductive tract[END_REF] investigated estrogen-like effects of Gen on the reproductive tract of male mice. Newborn and adult mice received Gen injections over 9 days. Control group received s.c. E2. Histology and quantitative RNA amount in urethral epithelium showed an increase in c-fos, which is interpreted as typical estrogenic effect. E2like effects of Gen on urethral epithelium were thus shown. These results of [START_REF] Strauss | Genistein exerts estrogen-like effects in male mouse reproductive tract[END_REF] fit to our findings in which Gen significantly increased uPs in an estrogen-like way.

In the urinary tract our study showed strong E2-like effects under all isoflavones with some minor quantitative differences. Differences in the urethral pressure minus bladder pressure indication for the functionality of the urethral closure mechanism. In literature, the incompetence of the urethral closure mechanism is seen as a major factor for incontinence [START_REF] Keane | Urinary incontinence: anatomy, physiology and pathophysiology[END_REF]. For all tested compounds, E2 and the phytoestrogens, a significant increase in the difference of pressures (ΔuP-vP) could be shown. Since a higher uP level can improve urethral closure, an improved urethral closure mechanism under E2 and the phytoestrogens can be postulated. In control animals, vesicular pressure often exceeded urethral pressure such that a negative difference between the two pressures resulted which indicated an incontinence situation. This was obvious during both filling periods as well as in each of the periods after the second filling period. Our results do not indicate the location of action of E2 and of the phytoestrogens. Presence of estrogen receptors was not only shown for genitourinary tract but also for parts of the central and peripheral nervous system involved in micturition [START_REF] Vanderhorst | Estrogen receptor-alpha immunoreactivity in parasympathetic preganglionic neurons innervating the bladder in the adult ovariectomized cat[END_REF] and [START_REF] Woolley | Effects of estrogen in the CNS[END_REF]. Therefore, pressure changes due to investigated substance in our study may reflect the total activity of the estrogens in all of the estrogen sensitive structures involved in regulation of micturition. Taken together our data indicate urethral affects of E2 and of the phytoestrogens on continence behaviour of ovx rats. Future clinical studies need to be performed to test whether such effects are also mediated in incontinent postmenopausal women. In summary, in the present experiments we demonstrated that oral treatment of ovariectomized rats over a period of 3 months with E2 resulted in massively increased uterine weights and reduced obesity. Similarly, though less potently, Gen and Puer had significant stimulatory effects on the uterine weights.

Figure Legends

Fig. 1 Effects of 14-week feeding with soy-free food (Co-SF), or food containing Eq, Gen, Puer or E2 on uterine wet weight of ovx rats. Means and SD are shown. * p<0.05 vs. Co-SF Fig. 2 Mean bladder (vP) and urethral pressures (uP) in ovx animals kept under a) soy-free (Co-SF) or b) E2 enriched diet. The vertical lines show the segments (intervals) during which means of all values were calculated. Means SD are shown.

Fig. 3

Mean vesicular pressure (vP) and urethra pressure (uP) in ovx animals kept under Eq, Gen or Puer enriched diet for 3 mo. Time flow after retrograde infusion of 0.5 ml saline solution at 0s and 90 s over 30 s (details see legend Fig. 1).For the sale of clarity only means are shown.

Fig. 4

Mean urethra pressures (uP) in experiment at phases of 30s; Means and SD are shown; in some cases SD were so small that they cannot be seen. * p<0.05 compared to control group Co-SF Fig. 5 Mean vesicular pressures (vP) in experiment at phases of 30s; Means and SD are shown; in some cases SD were so small that they cannot be seen. * p<0.05 compared to control group Co-SF Fig. 6 Mean differences of pressures in urethra minus bladder (ΔuP-vP) in experiment at phases of 30s; a large difference indicates larger urethral than bladder pressure which suggests a higher continence. Consequently, a low or negative value indicates a higher predisposition for incontinence. In some cases SD were so small that they cannot be seen. Means and SD are shown; * p<0.05 compared to control group Co-SF Table 1 Mean daily food and substance intake, mean body weight (BW) at the end of the experiment.

  Fig.1

Fig. 2b

  Fig.2b

  Fig.2b

Table 1

 1

			Mean substance	Mean BW at the end
			intake	of the experiment
		Mean food intake	per animal per	(starting BW: 227,3
	Group	per animal per day	day	 0,9)
	Co-SF	16.7  0,6 g		325,4  5,7 g
	Equol	15,0  0,5 g *	16,0  0,18 mg	307,3  5,0 g
	Genistein	16.5  0,5 g	16.5  0,50 mg	322,3  7,7 g
	Puerarin	14.7  0,4 g *	44.2  1,20 mg	271,8  4,0 g*
	E2	14,0  0,5 g *	0.06  0,002 mg	278,3  5,3 g *
	Mean daily food and substance intake, mean body weight (BW) at the end
	of the experiment. * p<0.05 vs. Co-SF

Acknowledgement: This work was in part funded by the European network of Excellence CASCADE (Grant # Food- CT-2004-506319)