The role of receptor-interacting protein 140 in the accumulation of fat in ovariectomised rats

Editorial Manager(tm) for Obesity Surgery Manuscript Draft

Manuscript Number: OBSU-D-10-00133R1

Title: The role of receptor-interacting protein 140 in the accumulation of fat in ovariectomised rats Article Type: Research - Basic Science

Keywords: Receptor-interacting protein 140, Ovariectomy, White adipose tissue Corresponding Author: Dr. Pao-Yun Cheng, Ph.D.

Corresponding Author's Institution: China Medical University First Author: Won-Hsiung Liu

Order of Authors: Won-Hsiung Liu; Yen-Mei Lee; Kwok-Keung Lam; Yuh-Fung Chen; Jhi-Joung Wang;

Mao-Hsiung Yen; Pao-Yun Cheng, Ph.D.

Abstract: Background Receptor-interacting protein 140 (RIP140) is a corepressor for nuclear

receptors with an important role in the inhibition of energy expenditure. Postmenopausal women have increased white adipose tissue (WAT), and excessive accumulation of adipose tissue (obesity) implies a health risk. The aim of the present work was to investigate the time-course of RIP140 expression in WAT during the development of ovariectomy (OVX)-induced obesity in rats.

Methods OVX was performed in female Sprague-Dawley (SD) rats 8 weeks old. Body weight and food intake were determined once a week. WAT of sham-operated, OVX, and OVX plus 17β-estradiol therapy (OVX/E2) female SD rats was weighed and used to analyse RIP140 and uncoupling protein 1 (UCP-1) expression by Western blot.

Results Food intake and body weight were significantly increased during the 2-8 weeks after OVX.

Even though body weight increased until sacrifice, food intake progressively decreased from 9 to 16 weeks after ovariectomy in rats. Meanwhile, increased WAT mass and decreased RIP140 expression in WAT were observed in OVX rats. In contrast, the expression of UCP-1, a key target gene of RIP140, in WAT of OVX rats was significantly higher than in sham-operated rats. All of these alterations caused by OVX were mostly reversed by the replacement of 17β-estradiol.

Conclusions The down-regulation of RIP140 in WAT may play a compensatory role in OVX-induced obesity in rat.

Response to Reviewers: We appreciate the comments of the Reviewer and believe that our manuscript has been improved by attention to them. The followings are our responses to the specific issues raised:

Reviewer #1

1.This is a very interesting paper that suggests that weight gain in ovariectomised rats can be reversed by estrogen and that compensatory mechanisms can be measured at a molecular level. Specifically RIP140 an inhibitor of UCP-1 falls in the setting of weight gain allowing increased thermogenesis. As such this is an interesting paper and is worthy of submission it has no therapeutic implications. I am curious as to why the authors used Western Blot vs RT PCR. Second there needs to be more discussion as to how the WAT was collected. I have difficulty understanding how this can be adequately quantities without MR. Please comment

Response: Thanks for your constructive comments.

1.The western blot is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. Thus, it was used to investigate the expression of RIP140 and UCP-1 proteins in white adipose tissue in this study and others (Debevec et al., 2007).

2.Total body MRI which offers the most accurate currently available method of assessing body composition components, however, the amount of adipose tissue can be shown by the weight of adipose tissue in many studies (Remesar et al., 2002; Meli et al., 2004). In this study the white adipose tissue was collected as follow: The rats were anaesthetised with Zoletil (15 mg/kg, i.p.) and blood was collected by intra-abdominal aortic artery. Rats were then skinned, and the main adipose tissue masses were carefully dissected, weighed, frozen and stored for further analyses (del Mar Grasa et al., 2001).

The subcutaneous white adipose tissue was obtained from the inguinal areas of each rat. Please see the Methods section.

Reference:

1.Debevec D, Christian M, Morganstein D, Seth A, Herzog B, Parker M, White R. Receptor interacting protein 140 regulates expression of uncoupling protein 1 in adipocytes through specific peroxisome proliferator activated receptor isoforms and estrogen-related receptor alpha. Mol Endocrinol.

2007;21:1581-1592.

2.del Mar Grasa M, Cabot C, Adán C, de Matteis R, Esteve M, Cinti S, Fernández JA, López, Remesar X, Alemany A. Corticosteroid-binding globulin synthesis and distribution in rat white adipose tissue. Mol Cell Biochem. 2001;228:25-31.

3.Remesar X, Fernández-López JA, Blay MT, Savall P, Salas A, Díaz-Silva M, Esteve M, Grasa MM, Alemany M. Effect of oral oleoyl-estrone on adipose tissue composition in male rats. International Journal of Obesity. 2002; 26: 1092-1102.

4.Meli R, Pacilio M, Raso GM, Esposito E, Coppola A, Nasti A, Di Carlo C, Nappi C, Di Carlo R. Estrogen and raloxifene modulate leptin and its receptor in hypothalamus and adipose tissue from

ovariectomized rats. Endocrinology. 2004 ;145:3115-3121.

Reviewer #2

A well written paper with sound methodology. I am however not sure why such a very basic science paper is submitted to an obesity surgery journal.

Minor comments

Please omit any extrapolations in your discussions to human application. You have no basis for this.

This remains a rat study, elegant as it my be. You must also take great caution to try and relate this to human female menopause-in humans this is a very gradual process that encompasses the

perimenopause in times of time frame. What you created in your rats is "iatrogenic" or instant surgical menopause.

Response: Thanks for your constructive comments. We have deleted the extrapolations in our discussion to human. In addition, we also agree with the reviewer's opinion. In clinical practice, menopause is diagnosed retrospectively when the absence of regular menses lasts for at least 12 months (Lund, 2008). It is due to the aging of the ovary resulting from the exhaustion of ovarian follicles (Wise et al., 1996) causing infertility and a progressive loss of its hormonal activity (Greendale and Sowers, 1997). Loss of ovarian estrogen production is the key pathophysiological event

responsible for the consequences of menopause. The incidence of obesity is higher in post-menopausal women than in age-matched pre-menopausal women; this increase is thought to be associated with the decline of estrogen levels after menopause. In fact, estrogen replacement therapy is an effective

treatment to reduce body weight and fat accumulation in post-menopausal women (Svendsen et al.

1995). In rats, estrogen treatment reduces energy intake (Donohoe and Stevens 1983), and it has been demonstrated that ovariectomy (OVX) induces an increase in body weight, which can be reversed with estrogen replacement treatment (Wade et al. 1985). Thus, estrogen insufficiency in human can be modeled using ovariectomised rats. This model is characterized by mild obesity and is useful to study how hypoestrogenism alters adiposity in many studies (Ainslie et al., 2001；Meli et al., 2004).

References:

1.Ainslie DA, Morris MJ, Wittert G, Turnbull H, Proietto J, Thorburn AW. Estrogen deficiency causes central leptin insensitivity and increased hypothalamic neuropeptide Y. Int J Obes Relat Metab Disord.

2001; 25(11):1680-1688.

2.Donohoe TP, Stevens R. Effects of ovariectomy, estrogen treatment and CI-628 on food intake and body weight in female rats treated neonatally with gonadal hormones. Physiol. Behav. 1983; 31:325- 329.

3.Gotoh K, Masaki T, Chiba S, Higuchi K, Kakuma T, Shimizu H, Mori M, Sakata T, Yoshimatsu H.

Hypothalamic neuronal histamine signaling in the estrogen deficiency-induced obesity. J Neurochem.

2009;110:1796-1805

4.Greendale GA, Sowers M. The menopause transition. Endocrinol. Metab. Clin. North Am. 1997;

26:261-277.

5.Lund KJ. Menopause and the menopausal transition. Med. Clin. North Am. 2008; 92:1253-1271.

6.Meli R, Pacilio M, Raso GM, Esposito E, Coppola A, Nasti A, Di Carlo C, Nappi C, Di Carlo R. Estrogen and raloxifene modulate leptin and its receptor in hypothalamus and adipose tissue from

ovariectomized rats. Endocrinology. 2004; 145:3115-3121.

7.Svendsen O. L., Hassager C. and Christiansen C. Age- and menopause- associated variations in body composition and fat distribution in healthy women as measured by dual-energy X-ray absorptiometry.

Metabolism 1995; 44: 369-373.

8.Wade GN, Gray JM, Bartness TJ. Gonadal influences on adiposity. Int. J. Obes. 1985; 9:83-92.

9.Wise PM, Krajnak KM, Kashon ML. Menopause: the aging of multiple pacemakers. Science 1996; 273:

67-70.

The role of receptor-interacting protein 140 in the accumulation of fat in

ovariectomised rats

Won-Hsiung Liu

, Yen-Mei Lee

, Kwok-Keung Lam

, Yuh-Fung Chen

, Jhi-Joung

Wang

, Mao-Hsiung Yen

, Pao-Yun Cheng

a

Department of Pediatrics, Chi-Mei Foundation Hospital, Tainan, Taiwan;

b

Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan;

c

Department of Pharmacology, Taipei Medical College, Taipei;

Department of

Anesthesiology, Catholic Mercy Hospital, Hsin-Chu, Taiwan;

Graduate Institute of

Chinese Pharmaceutical Sciences, China Medical University, Taichung;

Department

of Medical Research, Chi-Mei Medical Center, Tainan, Taiwan

＊ Address correspondence to: Pao-Yun Cheng, PhD, Graduate Institute of Chinese Pharmaceutical Sciences, China Medical University, No.91 Hsueh-Shih Road,

Taichung, Taiwan 40402, R.O.C; E-mail address:[email protected]

＊ Short title: The receptor-interacting protein 140 in ovariectomised rats

*Clean Manuscript

Abstract

Background Receptor-interacting protein 140 (RIP140) is a corepressor for nuclear

receptors with an important role in the inhibition of energy expenditure.

Postmenopausal women have increased white adipose tissue (WAT), and excessive

accumulation of adipose tissue (obesity) implies a health risk. The aim of the present

work was to investigate the time-course of RIP140 expression in WAT during the

development of ovariectomy (OVX)-induced obesity in rats.

Methods OVX was performed in female Sprague-Dawley (SD) rats 8 weeks old.

Body weight and food intake were determined once a week. WAT of sham-operated,

OVX, and OVX plus 17β-estradiol therapy (OVX/E2) female SD rats was weighed

and used to analyse RIP140 and uncoupling protein 1 (UCP-1) expression by Western

blot.

Results Food intake and body weight were significantly increased during the 2-8

weeks after OVX. Even though body weight increased until sacrifice, food intake

progressively decreased from 9 to 16 weeks after ovariectomy in rats. Meanwhile,

increased WAT mass and decreased RIP140 expression in WAT were observed in

OVX rats. In contrast, the expression of UCP-1, a key target gene of RIP140, in WAT

of OVX rats was significantly higher than in sham-operated rats. All of these

alterations caused by OVX were mostly reversed by the replacement of 17β-estradiol.

Conclusions The down-regulation of RIP140 in WAT may play a compensatory role in

OVX-induced obesity in rat.

Introduction

Energy homeostasis is a highly regulated process that requires precise control of

food intake and energy expenditure [1]. The major and most efficient storage of

energy occurs in the form of triglycerides in white adipose tissue (WAT), and it is now

clear that the adipocyte itself is an endocrine cell; therefore, altered adipocyte

function can alter systemic energy balance [2, 3]. Evidence from both human and

animal experiments suggests that oestrogen plays an important role in the regulation

of WAT. Postmenopausal women have increased WAT, and oestrogen therapy

decreases WAT levels compared with untreated postmenopausal women [4, 5].

Animal experiments have also shown that ovariectomy (OVX) of rodents increases

WAT and that oestrogen replacement decreases WAT [6, 7]. Excessive accumulation

of adipose tissue (obesity) implies a health risk. It leads to various chronic morbidities,

including insulin resistance, type II diabetes, and cardiovascular disease [8-10]. These

observations highlight the importance of understanding the molecular and

physiological mechanisms that underlie menopause-associated obesity and metabolic

dysregulation. Currently, however, these mechanisms remain unclear.

Receptor-interacting protein 140 (RIP140) is a nuclear protein of 1158 amino

acids [11]. The RIP140 gene is expressed in many tissues; it is localised in specific

cell types and subject to developmental control. The highest levels of expression are

detected in metabolic tissues, including adipose tissue, liver and muscle, and in the

ovary [12]. The expression of RIP140 is regulated by a number of hormones,

including oestrogen [13] and vitamin D [14]. It is also regulated during adipogenesis,

as exemplified in 3T3-L1 cells, where there is a progressive increase in expression

during differentiation in response to oestrogen-related receptor signalling [12, 15].

The physiological importance of RIP140 was evaluated using mice devoid of the

RIP140 gene (RIPKO). RIP140-null mice accumulate markedly less fat in their

adipose tissue, are resistant to high-fat diet-induced obesity and exhibit improved

glucose tolerance and insulin sensitivity [12, 16, 17]. These changes are associated

with increased expression of metabolic genes in WAT and muscle, resulting in an

increased metabolic rate and energy expenditure [12, 18]. Along these lines, in the

absence of RIP140, the expression of uncoupling protein 1 (UCP-1) is elevated in

adipocytes derived from WAT, with increased energy dissipation facilitating the

process of thermogenesis as well as maintaining the redox balance and reducing the

generation of reactive oxygen species [16, 19, 20]. Therefore, the aim of this study

was to analyse the influence of menopause-associated obesity on RIP140 protein

expression in the WAT of OVX rats.

Material and methods

Animal preparation

Female Sprague-Dawley (SD) rats (8 weeks old) were obtained from the

National Laboratory Animal Breeding and Research Center of the National Science

Council, Taiwan. Handling of the animals was in accordance with the Guide for the

Care and Use of Laboratory Animals published by the US National Institutes of

Health (NIH Publication No. 85-23, revised 1996). This study was approved by the

National Defense Medical Center Institutional Animal Care and Use Committee,

Taiwan. Rats were anaesthetised with Zoletil (15 mg/kg, i.p.) and underwent bilateral

ovariectomy at 8 weeks of age. Small incisions were made bilaterally on the sides of

the back to expose the ovaries retroperitoneally. The ovaries were clamped and

removed, and the uterine tubes were ligated. The muscle and skin were then sutured.

The sham procedure consisted of anaesthesia, visualisation of the ovaries via incisions

into the abdominal cavity, and closure of the wounds.

Experimental groups

One week after the operation, the rats were randomly divided into three groups:

(1) sham group, rats that had undergone sham operation (n=18); (2) OVX group,

OVX rats injected intramuscularly (IM) with the vehicle for oestrogen for 15 weeks,

beginning 1 week after ovariectomy (n=18); (3) OVX/E2 group, OVX rats injected

with 17β-estradiol (E2; 50 μg/kg/day, IM, once daily) for 15 weeks, beginning 1 week after ovariectomy (n=18). To investigate the time-courses of RIP140 and UCP-1

expression in WAT, rats were anaesthetised with Zoletil (15 mg/kg, i.p.) and killed at

4, 12 or 16 weeks after ovariectomy (n=6 for each time point tested). Blood samples

were collected by intra-abdominal aortic puncture. Rats were then skinned, and

subcutaneous WAT was carefully excised and weighted. The subcutaneous white

adipose tissue was obtained from the inguinal areas of each rat [21]. Then the WAT

was snap-frozen in liquid nitrogen and stored at -80°C for later analysis.

Measurements of food intake and body weight

Each rat was housed in a single cage. Food intake was measured every week

when replacing food. Food intake rate was determined by measuring the loss of food

from the food container during the feeding period. Throughout the experiment period,

body weight was monitored once a week.

Western blot analysis

The adipose tissue (0.3 g) obtained from each animal was ground in a mortar

containing liquid nitrogen. The powdered tissue was then suspended in 1 mL of lysis

buffer [20 mM Tris-HCl (pH 7.5), 10 mM NaF, 150 mM NaCl, 1% Nonidet P-40, 1

mM phenylmethylsulphonylfluoride (PMSF), 1 mM Na

VO

, leupeptin, and 10 μg/ml

trypsin inhibitor] and agitated at 4°C for 1 h. After 1 h, cell lysates were obtained by

centrifugation at 100,000 × g for 15 min at 4°C. Protein concentration was determined

using the BCA protein assay kit (Pierce, Rockford, IL, USA) according to the

manufacture's instructions. For Western blot analysis, 100 μg protein of the tissue

lysate was dissolved in Laemmli sample buffer, boiled for 5 min, and subjected to

SDS-PAGE (10 or 8% polyacrylamide). The proteins were transferred from gel to

nitrocellulose membrane at 250 mA for 2 h at room temperature. The membrane was

then blocked with 1x TBST and 5% nonfat dried milk for 40 min at room temperature

and probed with rabbit polyclonal RIP140 antibody (1:2000; Affinity Bioreagents,

Golden, CO) or rabbit polyclonal UCP-1 antibody (1:1000; Chemicon International)

in 1x TBST, 5% nonfat dried milk, and 0.1% Tween 20 at 4°C overnight. The

secondary antibody (anti-rabbit or anti-goat IgG-horseradish peroxidase conjugate;

1:2000 dilution; Cell Signaling) was incubated for 1 h at room temperature.

Subsequently, the blot was extensively washed with TBST, developed using an

enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions, and exposed to X-ray film (Kodak, Rochester, NY, USA).

To verify that blots were loaded with equal amounts of protein lysates, they were also

incubated in the presence of the antibody against β-actin (Sigma-Aldrich Corp.). The

density of the respective bands was quantified by densitometric scanning of the blots

using Image-Pro software (Media Cybermetrics, Inc.).

Statistical analysis

All measurements are expressed as group mean ± SEM. Statistical evaluation

was performed with one-factor analysis of variance followed by the Tukey–Kramer

test. A P value of less than 0.05 was deemed statistically significant.

Results

Effect of ovariectomy (OVX) and 17β-estradiol (E2) substitution on body weight

The mean body weights of the rats in the three groups are given in Fig. 1. At the

time of surgery, all rats had similar body weight (240.7 ± 1.5 g). The control group

gained ~60 g during the 16-week study period, reaching a mean weight of 296 ± 4 g

after 16 weeks. The OVX rats gained significantly more, reaching 422.2 ± 15.9 g after

16 weeks, whereas the OVX/E2 rats gained much less (final weight 281.67 ± 6.5 g)

than the OVX group (P < 0.05).

Effect of ovariectomy and 17β-estradiol substitution on food intake

Food intake in all rats was measured once a week. As shown in Fig. 2, the food

intake of rats within each group did not differ at the time of operation. The control

rats ate 120 ± 3 g/rat/week 16 weeks after OVX. The OVX rats consumed

significantly more (148 ± 2 g/rat/week) 2 weeks after OVX and reached a maximum

(163.9 ± 3.9 g/rat/week) at 8 weeks after surgery, and then progressively decreased to

the same level as the control group. During the entire experiment, the OVX/E2 rats

consumed a similar level as the control rats.

Effects of ovariectomy and 17β-estradiol substitution on WAT mass

WAT mass was examined at the end of the study by dissection of the adipose

tissue. As shown in Fig. 3, the amount of WAT was significantly increased in the

OVX rats compared to the OVX/E2 rats (P < 0.05). The 17β-estradiol–substituted

OVX/E2 rats had significantly less WAT than the OVX rats (P < 0.05).

RIP140 protein expression in WAT

To evaluate whether accumulation of WAT after OVX was caused by the effect

of RIP140, we measured the expression of RIP140 protein in WAT. As shown in Fig.

4, the expression of RIP140 protein in WAT compared with control rats was

significantly lower in OVX rats (P < 0.05), whereas OVX/E2 rats expressed more

RIP140 than the OVX rats (P < 0.05).

UCP-1 protein expression in WAT

To clarify whether the biological effect of RIP140 remained throughout the

experimental period, we evaluated the expression of UCP-1, a key target gene of

RIP140, in WAT. As shown in Fig. 5, the expression of UCP-1 in the WAT of OVX

rats was significantly higher than in controls (P < 0.05), whereas OVX/E2 rats

exhibited similar UCP-1 expression to control rats (P < 0.05).

Discussion

This is the first in vivo study demonstrating the time-course of changes of

RIP140 protein expression in WAT of OVX-induced obese rats. RIP140 plays an

important role in the inhibition of energy expenditure. In the OVX group, a marked

reduction in oestrogen level led to increased body weight and WAT mass and

decreased RIP140 protein expression in WAT, accompanied by the up-regulation of

UCP-1 protein expression. Replacement of 17β-estradiol significantly reversed these

alterations. These results imply that the abnormal lipid accumulation of subcutaneous

WAT cannot be accounted for by the up-regulation of RIP140 protein in WAT in

OVX-induced obesity. On the contrary, the reduced RIP140 protein expression under

OVX suggests a compensatory mechanism to accelerate energy expenditure and

reduce fat accumulation in obese states.

Many pieces of evidence suggest that body fat content is controlled, at least

partially, by the metabolism of adipose tissue itself. First, most of the candidate genes

for obesity have important roles in adipocytes [22]. Second, many mouse models that

are prone or resistant to obesity are created by transgenic modification of adipose

tissue [23]. In these transgenic models, metabolic changes in white but not in brown

adipose tissue are mostly responsible for the altered accretion of body fat,

highlighting the importance of lipid metabolism in adipocytes of the white fat. In this

study, ovariectomised rats had increased adiposity compared with rats with intact

ovaries (Fig. 3). Meanwhile, 17β-estradiol treatment in ovariectomised rats

significantly reduced white adipose mass. These data confirm the report by D’Eon et

al. [24] and suggest that oestrogen plays important roles in adipose tissue biology and

in the prevention of obesity, possibly via decreased expression of lipogenic genes in

adipose tissue.

The evidence emerging from both in vivo studies and cellular systems has

identified a role for RIP140 as an important regulatory factor in many metabolic

processes. RIP140-knockout mice are leaner than their control littermates, even when

challenged by high-fat feeding [12]. Interestingly, this phenotype is not linked to a

defect in adipogenesis. Meanwhile, the WAT of these animals expresses high levels of

UCP-1 [12], a protein normally confined to the brown adipose tissue (BAT), where it

contributes to adaptive thermogenesis. In addition to repressing the uncoupling of

respiration, RIP140 inhibits other aspects of energy expenditure in the adipose tissue

by repressing genes implicated in fatty acid oxidation, mitochondrial biogenesis and

oxidative phosphorylation [17]. In primary adipocytes from RIP140-null mice,

increased metabolic rate and release of chemical energy in the form of heat have been

observed [16]. However, no studies have analysed the expression of RIP140 in

obesity induced by ovarectomy in rats. We observed a down-regulation of RIP140

protein levels in OVX-induced obese rats compared to sham rats (Fig. 4).

Supplementation with 17β-estradiol reversed this alteration. These findings suggest

that the down-regulation of RIP140 may be a compensatory effect to counteract

obesity. This speculation is supported by a report maintaining that both reduced and

increased body weight are associated with compensatory changes in energy

expenditure [25].

UCP-1 is a proton transporter that uncouples oxidative metabolism from ATP

synthesis and dissipates energy through heat generation [26]. UCP-1 was once

thought to be expressed only in BAT; recently, it was reported that UCP-1 mRNA and

protein are also detectable in the WAT of mouse and human [27, 28]. A number of

studies have demonstrated that UCP-1 expression is regulated by both nuclear

receptors and alterations in the levels of intracellular cAMP, with coregulators such as

PGC-1α acting as key factors required for the integration of different signalling

pathways [29, 30]. Recent studies have shown that UCP-1 is a key target gene of

RIP140 in adipocytes [12, 16]. In the present study, we demonstrated that the

expression of UCP-1 was significantly increased in WAT from OVX rats, compared

with that from sham rats (Fig. 5). These changes were reversed by supplementation

with 17β-estradiol. These findings further corroborate the notion that the

down-regulation of RIP140 may be a compensatory mechanism to counteract fat

accumulation via subsequent up-regulation of UCP-1 expression, increasing the

energy expenditure in WAT.

In summary, oestrogen regulates the expression and activity of many genes

involved in the control of energy homeostasis. A number of factors may be involved

in the OVX-induced changes in RIP140 expression. In our study, body weight and

WAT mass were profoundly increased in OVX rats. RIP140 inhibits energy

expenditure in adipose tissue by repressing metabolic genes. In OVX rats, the

expression of RIP140 was decreased in WAT, accompanied by increased UCP-1.

Therefore, the down-regulation of RIP140 protein in WAT seems to rule out increased

RIP140 as a causative role in OVX-induced obesity. It is possible that the gene

expression changes we observed reflect a compensatory mechanism to accelerate

energy expenditure and reduce fat accumulation in OVX-induced obesity. Further

studies are needed to clarify the possible molecular mechanism of the decrease of

RIP140 expression in WAT of OVX rats.

Acknowledgements

This work was supported by a research grants from the National Science Council

(NSC 97-2320-B-039-032), the China Medical University (CMU 96-262) and the Chi

Mei Medical Center (CMNDMC9803), Taiwan.

Disclosure Statement

The authors have nothing to disclose.

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Legends

Figure 1 Body weight of the rats in the three different groups was monitored weekly.

Sham, sham operated; OVX, ovariectomised without oestrogen treatment; OVX/E2,

ovariectomised with 17β-estradiol (E2) supplementation. Data are expressed as mean

± SEM (n = 6 in each). *P ＜ 0.05 for OVX vs. sham group.

P ＜ 0.05 for OVX/E2 vs. OVX group.

Figure 2 Food intake of the rats in the three different groups was monitored weekly.

Sham, sham operated; OVX, ovariectomised without oestrogen treatment; OVX/E2,

ovariectomised with 17β-estradiol (E2) supplementation. Data are expressed as mean

± SEM (n = 6 in each). *P ＜ 0.05 for OVX vs. sham group.

P ＜ 0.05 for OVX/E2 vs. OVX group.

Figure 3 Weight of white adipose tissue in 16-week-old rats in the sham, OVX, and

OVX/E2 groups. The amount of white adipose tissue is expressed as percent of body

weight. Data are expressed as mean ± SEM (n = 6 in each). *P ＜ 0.05 for OVX vs.

sham group.

P ＜ 0.05 for OVX/E2 vs. OVX group.

Figure 4 Time-course of changes of RIP140 protein expression in white adipose tissue

of the sham, OVX, and OVX/E2 groups. Depicted are a typical display of RIP140

protein expression (upper panel) and the statistical analysis of the changes of RIP140

levels (lower panel). Data are expressed as mean ± SEM (n = 6 in each). *P ＜ 0.05

for OVX vs. sham group.

P ＜ 0.05 for OVX/E

vs. OVX group.

Figure 5 Time-course of changes of UCP-1 protein expression in white adipose tissue

of the sham, OVX, and OVX/E2 groups. Depicted are a typical display of UCP-1

protein expression (upper panel) and the statistical analysis of the changes of UCP-1

levels (lower panel). Data are expressed as mean ± SEM (n = 6 in each). *P ＜ 0.05

for OVX vs. sham group.

P ＜ 0.05 for OVX/E

vs. OVX group.

Figure 1

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Figure 5

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