Molecular and Cellular Endocrinology 387 (2014) 19–34

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H NMR metabolic profiling analysis offers evaluation of Nilestriol treatment in ovariectomised rats Yan-Ru Liu a,b, Rong-Qing Huang b,⇑, Bing-Kun Xiao b, Jian-Yun Yang b, Jun-Xing Dong a,b,⇑ a b

School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, No. 103, Wenhua Rd, Shenhe District, Shenyang 110016, PR China Beijing Institute of Radiation Medicine, No. 27, Taiping Road, Haidian District, Beijing 100850, PR China

a r t i c l e

i n f o

Article history: Received 7 August 2013 Received in revised form 11 February 2014 Accepted 14 February 2014 Available online 22 February 2014 Keywords: Nilestriol Metabonomic Ovariectomy 1 H NMR Metabolic profiling

a b s t r a c t Nilestriol (NIL) has been applied to treat menopausal dysfunctions, yet its mechanism has remained unknown. To understand the relationship between the changes in homeostatic metabolites and ovarian oestrogen deficiency syndromes after NIL treatment, proton Nuclear Magnetic Resonance (1H NMR)based metabonomic technologies were used to analyse a rat model of oestrogen deficiency. An orthogonal partial least-squares regression (OPLS) differentiation model was used on 12-week metabolic analyses of ovariectomised (OVX) rats treated or mock treated with NIL. Furthermore, data analysis using Chenomx software quantified results to identify the most significantly altered metabolite concentrations, allowing for metabolic explanations of the effects of NIL therapies. In this study, PLS results revealed that there are considerably distinct differences between treatment groups. Additionally, a total of 45 metabolites shown to have a high variation between groups were selected for target quantification. Using a one-way LSD ANOVA analysis, 32 metabolite concentrations were significantly altered in the OVX group. A total of 21 metabolites were altered significantly in the NIL-treatment group but later returned to normal. According to the OPLS VIP calculation, the metabolites most affected by NIL treatment were mostly involved in insulin resistance. In addition, abnormal concentration changes in lactate in the NIL-treatment group and 3-indoxylsulfate in the OVX group were observed. To our knowledge, this study is the first to address the molecular mechanism of NIL from a metabonomic perspective, and, more specifically, to establish a catalogue of endo-molecular changes effected by NIL in the regulation of oestrogen deficiency disorder. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Abbreviations: ACC, acetyl-CoA; AGAT, arginine-glycine amidinotransferase; ANOVA, analysis of variance; CPMG, Carr–Purcell–Meiboom–Gill; CKD, chronic kidney disease; DAAO, D-amino acid oxidase; DDC (or AAAD), amino acid decarboxylase; GABA, c-aminobutyric acid; GAD, glutamic decarboxylase; GCS, glycine cleavage enzyme; GS, glutamine synthetase; HDC, histidine decarboxylase; IR, insulin resistance; LDH, lactate dehydrogenase; LSD, least significant different; MAT, methionine adenosyltransferase; MeSe, methionine synthase; NIL, Nilestriol; NMR, Nuclear Magnetic Resonance; OPLS, orthogonal partial least-squares regression; OVX, ovariectomy; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; SAH, S-adenosylhomocysteine; SAHase, S-adenosylhomocysteinase; SAM, S-adenosylmethionine; SPSS, statistical package for social science program; TCA, Tricarboxylic acid cycle; TMA, trimethylamine; TMAO, trimethylamine oxide; TPH, tryptophan hydroxylase; TST, tail skin temperature; VIP, Variable Importance Plotz; WET, water suppression enhanced through T1 effects. ⇑ Corresponding authors at: Beijing Institute of Radiation Medicine, No. 27 Taiping Road, Haidian District, Beijing 100850, P.R. China. Tel./fax: +86 10 66931341. E-mail addresses: [email protected] (R.-Q. Huang), [email protected] (J.-X. Dong). http://dx.doi.org/10.1016/j.mce.2014.02.007 0303-7207/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

Menopause is an oestrogen dysfunction endocrine disorder (Taylor and Manson, 2011) and is characterised by psychological and physiological symptoms. Over the past decades, several studies have been performed in an attempt to reveal the mechanisms of menopause. Hormone replacement therapy with oestrogen and progesterone is currently the most successful approach. However, in response to reports outlining the negative outcomes of menopause hormone therapy from the Women’s Health Initiative, the number of women receiving conventional oestrogen treatments has declined dramatically. Accordingly, a series of oestrogen types have been designed to mitigate the side effects of oestrogen (Barros et al., 2008). The drug known as Nilestriol (NIL), 17a-ethynyl-3-cyclopentyloxyestra1,3,5(10)-triene-16a, or CCE3, represents a class of chemical analogues of oestrogen characterised by long-lasting effects and delayed release. The ethynyl substitute at the 17a-carbon

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enhanced oestrogenic activities while the cyclopentyl ether group attached to the C3 position increased hydrophobicity, which benefited intestinal absorption (Liu et al., 2000). NIL has a lower endometrial hyperplasia effect than classical oestrogens due to the short duration it binds to the oestrogen receptor (Xu et al., 1998). NIL appears particularly useful in the treatment of atherosclerosis and seems to improve menopause symptoms. Experimental data have suggested that NIL could treat osteoporosis or hypertension better than estriol (Yang et al., 2003); Deng et al. demonstrated that NIL was also beneficial in the treatment of coronary heart disease of mid-aged women through improvement of the metabolism of serum lipids and inhibition of lipid peroxidation (Deng et al., 1999). Metabonomic technology is focused on the dynamic biological and biochemical changes of small molecule metabolites processed in complex bio-systems, and it provides a global path to understanding, in an untargeted or targeted way, when combined with chemometrics (Banos et al., 2008). Nuclear Magnetic Resonance (NMR) spectroscopy has been used as a powerful tool to investigate disease states of organisms. It is a non-destructive pre-treatment process and can simultaneously detect the spectra of multiple metabolites (Weljie et al., 2006). After untargeted studies, Chenomx-targeted profiling was used to fit the disease-associated spectral areas and quantify them. Instead of binning spectral areas, target profiling depends on the spectra libraries of pure compounds determined under comparable experimental conditions and suited to different pH conditions (Villa et al., 2007). Some studies have been conducted to elucidate the relationship between age-related syndromes and endo-metabolite imbalance via the metabonomic approach (Bernini et al., 2011; Holmes et al., 2008; Zhu et al., 2010). Although these studies have found some connections between serum or urine metabolites and menopause under physiological or pathologic states, e.g., osteoporosis (Long et al., 2009; Xue et al., 2011), obesity (Ma et al., 2011), and breast cancer (Odunsi et al., 2005), the ability of metabonomics to clearly assess integral menopause risks remains ambiguous. This study was intended to test the assumption that menopausal insulin resistance is responsive to interruptions in the metabolite pathway. Ovariectomy was performed on adult female SD rats to mimic the lifespan of middle-aged women who suffer from ovarian hormone deficiency accompanied by various syndromes (Kalu, 1991; Nawata et al., 1995; Reckelhoff and Fortepiani, 2004). A metabolite-profiling fingerprint was then prepared to clarify the connections between those syndromes. Moreover, some biomarkers can be selected to reveal which was the most important or the most responsible metabolic pathway. This study also used body weight, tail skin temperature and endometrial pathological examination to try to understand how pathological states are reflected in abnormal metabolite profiles. Additionally, this study aimed to identify metabolite changes to explain how NIL confers its beneficial effects to the endocrine system using a novel chemometric diagnostic model which may help determine normal, oestrogen-loss or treatment groups.

2. Materials and methods 2.1. Animals Twenty 6–8 weeks old pathogen-free female Sprague–Dawley rats were obtained from the National Beijing Animal Centre, were housed individually in metabolic cages under a 12 h light/dark cycle at 23 ± 2 °C and 55 ± 10% relative humidity and were acclimatised for 5 days before treatment. Food and drinking water were provided ad libitum. Body weights were recorded daily. Experimental protocols followed the guidelines of the Guide for the Care and

Use of Laboratory Animals and were approved by the Laboratory Animal Care and Use Committee of the Beijing Institute of Radiation Medicine. 2.2. Experimental protocol 2.2.1. Model establishment 15 of the 20 rats were either bilaterally ovariectomised (OVX) or sham ovariectomised under aseptic conditions. Subjects were anesthetised with 2% phenobarbital sodium and then underwent surgery as described by Larson and Carroll (2007). Immediately after OVX surgery, penicillin-G was administered intramuscularly to subjects for 3 days to prevent infection. After recovery for 7 days, experimental sessions were commenced following the protocol outlined in Fig. 1a. All the subjects were weighed every week during the experiment. 2.2.2. Drug administration The 20 rats in this study were divided into four treatment groups, with five rats per group: control (no surgery, mock treatment with saline), sham (sham operation, mock treatment with saline), OVX group (underwent bilateral ovariectomy, mock treatment with saline), and OVX + NIL (underwent bilateral ovariectomy and treatment with NIL). All treatments were carried out in the morning to minimise their effect on the circadian rhythm of the rats. Starting 7 days after the operation, OVX + NIL rats were dosed with NIL at 1.5 mg/kg/week for the entire 12-week experiment (Shen et al., 2010; Song and Zhang, 2011; Xu et al., 1998). NIL solution was prepared using a sterile 0.9% saline solution so that each rat received the same volume of NIL solutions or vehicle for reinstatement. 2.2.3. Temperature recording Temperature recording tests were performed before the surgery, in the 1st, 6th and the last week of treatment. Following a previously reported procedure (Munehiko Ikeda et al., 2008), rats were kept in a porous-column shaped container (diameter  length = 5.5 cm  22 cm) and acclimated approximately 15 min prior to temperature recording test so that the tail skin temperature (TST) was stable. The TST was measured at 5 min intervals by a thermo tracer (NS 9300, NEC San-ei Instruments, Ltd., Japan) for 6 h under the stable temperature of the laboratory environment (23 ± 2 °C). The tail skin temperature data were collected approximately 2 cm from the base of the tail. Image Processor Pro II software (version 4.0.5, NEC San-ei Instruments, Ltd., Japan) was used to prepare a statistical analysis and determine average TST of each 20-rat experimental run. 2.3. Sample collection Urine samples were collected during the pre-surgery week, week 1, 2, 4, 6, 8, 10 and 12. Urine samples were collected for 24 h intervals in 20 ml epoxy resin tubes containing 1 ml of 0.1% sodium azide (Alfa Aesar, Lancashire, England) over gel ice packs (TECHNI ICE™, Australia). Urine samples were processed by centrifugation at 12,000 rpm for 10 min. The supernatants were collected and stored at 20 °C until NMR analysis. After 12 weeks of treatment, all rats were sacrificed and blood was drawn from each inferior vena cava. Blood samples were allowed to clot and serum was obtained by centrifugation at 12,000 rpm for 10 min. All serum was sterile and kept at 80 °C prior to NMR assays. At the end of experiments, the rats were anaesthetised by 2% sodium phenobarbital solution with no restraint and one third of the uterus was dissected. After the adherent fat tissue was removed, the uterine sections were immersed in 10% neutral

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(a)

(b)

(c)

Fig. 1. Experimental summary and index measurements. (a) Experimental timelines. Each bar represents one week. (b) Body weight alterations in female rats over 13 weeks following OVX. Values are mean value ± s.e.m (n = 5) for each group. (c) Effects of NIL (1.5 mg/kg) treatment on tail skin temperature (TST) of OVX rats over 13 weeks. Control, sham and OVX rats were administered same volume of saline. Data are presented as the mean ± s.e.m. *Above a single bar indicates a significant difference from the control group (p < 0.05); **indicates that p < 0.01. #above a single bar indicates a significant difference from the NIL group (p < 0.05); ##indicates that p < 0.01.

buffered formaldehyde solution (Sigma, St. Louis, Missouri, USA), and the tissues were dehydrated and embedded using a routine procedure. The tissue sections were stained with hematoxylin–eosin (H&E) for histological examination under a light microscope. 2.4. 1H NMR urinary and serum analysis Urine samples were mixed with an equal volume of sodium phosphate buffer (pH 7.4, 0.4 M) (Sigma, St. Louis, Missouri, USA) and centrifuged. The urine supernatants were combined with an internal standard D2O solution of (2, 2-dimethyl-2-silapentane-5sulfonic acid, (DSS, Chenomx Inc., Edmonton, Alberta, Canada) to make a final DSS concentration of 0.5 mM, which served as an internal reference standard (d0.00). The supernatants of the serum sample were added to the DSS solution so that DSS was diluted to a concentration of 1.0 mM. As above, DSS served as the internal standard reference (d0.00) (Dieterle et al., 2011; Shearer et al., 2008; Slupsky et al., 2010). NMR spectra were obtained on a JEOL ECA 400 NMR spectrometer (JEOL, Tokyo, Japan). 1D NOESY spectra were obtained for biofluids analysis. To suppress the large residual water resonance or wide protein resonance, the WET and CPMG pulse sequences were used. 1H NMR spectra were acquired using the following

parameters: spectra width 4800 Hz, 64 K complex data points, 4 s of acquisition time, 12.0 ppm spectral width, 64 transients, mixtime 0.3 s, relaxation delay time 1.2 s, temperature 293 K, and 96 (urine) or 120 (serum) scans. The data were zero-filled by a factor of 2 and the free induction decays (FIDs) were multiplied by an exponential weighting function equivalent to a line boarding of 0.5 Hz prior to Fourier transformation (FT). The 1H NMR spectra were manually phased using Delta software. The baseline was then corrected and referenced to DSS (d0.00). The chemical shift range of d0.0–10.0 for NMR spectra was reduced corresponding to 0.02 ppm for urine or 0.01 ppm for serum per interval by MestReNova software (version 6.0.2, Mestrelab Research S.L., Santiago de Compostela, Spain). All spectral regions were normalised to the DSS methyl peak area of the spectra to account for any significant concentration differences after the bin processing and so that the particular resonance in the spectrum could be measured absolutely. The region d4.60–5.20 was suppressed to prevent variation in water signals. 1H NMR signal matches were referenced by metabolite databases such as Human Metabolome Database (HMDB), serum metabolome database (SMDB) or PubChem. The data were saved as an ASCII text file that was then converted to .xls format (Microsoft Excel 2003, Microsoft, Redmond, WA, USA) for multivariate statistical analysis (Liu et al., 2010).

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2.5. Statistics analysis of weight and tail temperature indicators

3.2. Effects of NIL on rat tail skin temperature

Weight and temperature data were analysed using one-way analysis of variance (ANOVA), followed by post hoc least significant difference (LSD) tests via SPSS software (version 19.0, IBM Corporation, USA). A p value of 1.0), which indicated that these metabolites play an important role in the metabolic changes caused by OVX (Table 2). Serum glucose (VIP = 3.12), urine citrate (VIP = 3.13) and urine creatinine (VIP = 1.73) were ranked as the most important variables, all of which are regulated by insulin concentration. Certain metabolites, such as urine taurine (VIP = 2.85), serum lactate (VIP = 1.88), betaine (VIP = 1.22) and glutamine (VIP = 1.18) were associated with the glycolysis, one-carbon unit or homocysteine pathways.

The 12-week urinary concentration data were reported as relative concentrations represented by the ratios of the data to the sum of all detectable metabolites. Following the post hoc LSD analysis, the results indicated that the concentration of 23 of the 30 urine metabolites of the OVX group changed significantly when compared with the control or sham group. Compounds that showed a substantial increase in concentration included 3-indoxylsulfate, allantoin, betaine, carnitine, creatinine, glutamine, glycine, hippurate, lysine, methylhistidine and b-alanine. However, 2-oxoglutarate, acetate, citrate, fumarate, methionine, N,N-dimethylglycine, succinate, taurine and TMAO exhibited a decrease in relative concentration (Fig. 5c and d). Following NIL treatment, the concentration of 8 of these 23 metabolites returned to normal: 3-indoxylsulfate, betaine, creatine, fumarate, glycine, methionine, TMAO and b-alanine. Among the remaining metabolites, 12 metabolites had clear trends against the OVX group. Meanwhile, carnitine and hippurate did not change significantly when compared with the OVX group. It should be noted that the lactate concentration in either the serum or urine samples of the NIL group, showed a trend of increasing concentration with serum and urine approximately 3-fold and 10-fold higher than in control or OVX groups, respectively.

3.5. Target metabolites profiling analysis

4. Discussion

Metabolites that had proton resonances were identified by comparison to standard 1H NMR spectra in the Chenomx software public metabolite databases. Fig. 4 shows the serum and urine metabolite identification map of the 12-week serum and urine control samples. To further substantiate the metabolite changes and the impact of NIL in the arrest of OVX syndromes, a quantitative analysis of these markers was employed, resulting in a mean of 45 compounds being verified and classified into five related metabolic pathways. When the 12-week serum concentration data were subjected to the LSD post hoc test, 20 of the 30 metabolites in the OVX group showed apparent differences with the control or sham groups (Fig. 5a and b). The concentration of 3-hydroxybutyrate, alanine, formate, glutamine, taurine and threonine was significantly reduced compared with acetate, betaine, carnitine, choline, creatine, creatinine, glycine, glucose, glutamate, histidine, lysine, ornithine and proline. After NIL dosage, a total of 17 metabolites returned to levels seen in the control groups: 3-hydroxybutyrate, acetate, alanine, betaine, choline, creatine, creatinine, formate, glucose, glutamine, glycine, histidine, lysine, ornithine, proline, taurine and threonine. In addition, three metabolites (3-hydroxybutyrate, creatinine and taurine) showed no difference in the control (or sham) vs. OVX groups. As for other compounds, carnitine and glutamate concentrations exhibited a trend of improvement, but were still different from the control or sham group. In other cases, carnitine concentration was clearly different from that of the OVX group.

Women have battled with menopause disorder for decades. Abnormal insulin level is the result of menopause-related disorders that manifest from age-related ovarian dysfunction. Research on metabolic abnormalities revealed that there are several menopausal syndromes that have been attributed to insulin resistance (IR), such as obesity, atherosclerosis, cardiovascular diseases and some cancers (Kim et al., 2011). Prediction or diagnosis of menopausal syndromes has depended on the measurement of biochemical and histopathological indices. However, it is now widely recognised that menopause is a complex disease in which the many relationships between these syndromes remain unknown. Although some studies have employed various metabolomic analytical techniques to investigate menopause related obesity and osteoporosis, there is still a pressing need to identify biomarkers that respond according to different menopause syndromes such as hot flashes, depression, insomnia, atherosclerosis and gout (Long et al., 2009; Ma et al., 2011, 2013a,b; Odunsi et al., 2005; Xue et al., 2011; Zhu et al., 2010). This NMR-metabolic profiling study revealed significant changes to 32 of 45 metabolites measured in serum and urine samples in OVX rats in comparison with control or sham rats. With NIL therapy, the concentration of 12 of these metabolites returned to levels seen in control groups. These metabolic mechanisms were elucidated by metabonomic technologies. Therefore, our research has attempted to further explain these connections.

Table 2 VIP values of the serum and urine samples from OPLS models. No.

1 2 3 4 5 6 7 8

Compound name

Alanine Betaine Citrate Creatinine Glucose Glutamine Lactate Taurine

OVX vs. control (or sham)

Nil vs. control (or sham)

Serum

All groups Urine

Serum

Urine

Serum

Urine

(1)H NMR metabolic profiling analysis offers evaluation of Nilestriol treatment in ovariectomised rats.

Nilestriol (NIL) has been applied to treat menopausal dysfunctions, yet its mechanism has remained unknown. To understand the relationship between the...
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