女性代謝症候群之分子醫學研究---動物模式的建立與賀爾蒙治療藥物對於Imidazolline Receptor之機轉探討

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一、說明國科會基於學術公開之立場，鼓勵一般專題研究計畫主持人發表其研究成果，但主持人對於研究成果之內容應負完全責任。計畫內容及研究成果如涉及專利或其他智慧財產權、違異現行醫藥衛生規範、影響公序良俗或政治社會安定等顧慮者，應事先通知國科會不宜將所繳交之成果報告蒐錄於學門成果報告彙編或公開查詢，以免造成無謂之困擾。另外，各學門在製作成果報告彙編時，將直接使用主持人提供的成果報告，因此主持人在繳交報告之前，應對內容詳細校對，以確定其正確性。本格式說明僅為統一成果報告之格式，以供撰寫之參考，並非限制研究成果之呈現方式。精簡報告之篇幅（不含封面之頁數）以4 至 10 頁為原則，完整報告之篇幅則不限制頁數。成果報告繳交之期限及種類（精簡報告、完整報告或期中報告等），應依本會補助專題研究計畫作業要點及專題研究計畫經費核定清單之規定辦理。二、內容格式：依序為封面、中英文摘要、目錄（精簡報告得省略）、報告內容、參考文獻、計畫成果自評、可供推廣之研發成果資料表、附錄。 (一)報告封面：請至本會網站（http：//www.nsc.gov.tw）下載製作（格式如附件一）。 (二)中、英文摘要及關鍵詞(keywords)。 (三)報告內容：請包括前言、研究目的、文獻探討、研究方法、結果與討論（含結論與建議）…等。若該計畫已有論文發表者，可以A4 紙影印，作為成果報告內容或附錄，並請註明發表刊物名稱、卷期及出版日期。若有與執行本計畫相關之著作、專利、技術報告、或學生畢業論文等，請在參考文獻內註明之，俾可供進一步查考。 (四)頁碼編寫：請對摘要及目錄部分用羅馬字 I 、II、 III……標在每頁下方中央；報告內容至附錄部分請以阿拉伯數字1.2.3.……順序標在每頁下方中央。 (五)附表及附圖可列在文中或參考文獻之後，各表、圖請說明內容。 (六)計畫成果自評部份，請就研究內容與原計畫相符程度、達成預期目標情況、研究成果之學術或應用價值、是否適合在學術期刊發表或申請專利、主要發現或其他有關價值等，作一綜合評估。 (七)可供推廣之研發成果資料表：凡研究性質屬

應用研究

及

技術發展

之計畫，請依本會提供之表格（如附件二），每項研發成果填寫一份。三、計畫中獲補助國外或大陸地區差旅費、出席國際學術會議差旅費或國際合作研究計畫差旅費者，須依規定撰寫心得報告，以附件方式併同成果報告繳交，並請於成果報告封面註記。四、打字編印注意事項 1. 用紙使用A4 紙，即長 29.7 公分，寬 21 公分。 2. 格式中文打字規格為每行繕打（行間不另留間距），英文打字規格為Single Space。 3. 字體報告之正文以中英文撰寫均可。在字體之使用方面，英文使用 Times New Roman Font，中文使用標楷體，字體大小請以 12 號為主。

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附件一

行政院國家科學委員會補助專題研究計畫

5 成果報告

期中進度報告

（計畫名稱）

女性代謝症候群之分子醫學研究：

動物模式的建立與賀爾蒙治療藥物對於Imidazolline Receptor 之機轉探討

計畫類別：5 個別型計畫 □ 整合型計畫

計畫編號：NSC 97－2314－B－006－022－MY3

執行期間： 97 年 08 月 01 日至 100 年 07 月 31 日

計畫主持人：張峰銘教授

共同主持人：

計畫參與人員：鄭瑞棠教授、康琳、陳明輝

成果報告類型(依經費核定清單規定繳交)：□精簡報告 5完整報告

本成果報告包括以下應繳交之附件：

□赴國外出差或研習心得報告一份

□赴大陸地區出差或研習心得報告一份

5出席國際學術會議心得報告及發表之論文各一份

□國際合作研究計畫國外研究報告書一份

處理方式：除產學合作研究計畫、提升產業技術及人才培育研究計畫、

列管計畫及下列情形者外，得立即公開查詢

□涉及專利或其他智慧財產權，□一年5二年後可公開查詢

執行單位：

中華民國 100 年 7 月 31 日

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Introduction

The metabolic syndrome, a highly prevalent health problem in the modern era, is a

combination of risk factors, including abdominal obesity, dyslipidemia, glucose intolerance, and hypertension. The clustering of these factors is often attributed to Gerald Reaven, who

popularized the term ‘Syndrome X’ in 1988.1 The aggregation of these features into a single

entity provides clinicians with a tool by which they can identify a significant segment of the population at increased risk for developing type 2 diabetes mellitus (T2DM) as well as increased the morbidity and mortality of cardiovascular disease (CVD).2 In addition, there are gender

differences in metabolic diseases. For example, CVD is a more significant cause of morbidity and mortality in women than in men, and women have unique risk factors for metabolic syndrome, such as pregnancy-related weight gain, hormonal contraceptive use, polycystic ovary syndrome, gestational diabetes, preeclampsia, and the menopause.2

The menopause promotes a change in body fat distribution to increase central adiposity and subsequently enhance the likelihood of satisfying the metabolic syndrome criteria.3 Insulin

resistance increases with age and with increased abdominal obesity, but the mechanism by which the menopause modifies these increases is still unclear.4 The menopause has been reported to be

associated with an increased incidence of hypertension, lower HDL, and higher LDL levels. 5

I. Imidazoline receptor:

Imidazoline receptor represents a new class of receptors which are thought to mediate the central antihypertensive action of clonidine and analogues. 6It was a different class of binding sites than the α2-adrenoceptors and specifically recognized imidazoline and guanidine groups. 7

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Imidazoline receptor has been subclassed into two sites. The first are I1 binding sites that have a

high affinity for imidazolidine derivatives such as clonidine or moxonidine, medium affinity for imidazoline derivatives such as idazoxan or phentolamine and low affinity for guanidine

derivatives such as amiloride or guanabenz.8 The second are I2 binding sites that show a high

affinity for imidazoline and guanidine derivatives and a medium affinity for imidazolidine derivatives.8-9 Moreover, I3 binding sites have been proposed.10

Imidazoline receptor localizes in both central and peripheralnervous systems, and other tissues such as kidney, prostate, stomach, heart, liver, placenta, and colon.8Functionally,

peripheral imidazoline receptors mediate the movement of smooth muscle, stimulate insulin release and regulate the renal excretion of sodium, potassium and water.11Some α2-adrenergic

antagonists with a moeity of imidazoline enhanced insulin secretion in vivo and in vitro.12 The mechanisms of this action are mentioned to work by blocking ATP-regulated potassium channels in pancreatic beta cells, resulting in membrane depolarisation.13 For this reason, imidazoline

compounds have been used as adjuncts in the treatment of T2DM. For example, some studies have shown the metabolic and antihypertensive effects of selective imidazoline I1 receptor

agonist-moxonidine.14 It can decrease sympathetic nervous activity and improve insulin resistance in the spontaneously hypertensive obese rat model15, as well as in the obese

hypertensive patients.16 However, the pharmacological role of imidazoline I2 receptor agonist on T2DM or metabolic syndrome remains unclear. Besides, little is known about the I1 or I2 receptor

expression in female genital organs until now.

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Glucosamine (GlcN) is a popular nutritional supplement or medication used to treat osteoarthritis (OA). In the United States, GlcN is used by over five million people annually, making it the fourth most commonly used herbal/dietary supplement.15-17 Female gender is

associated with the age-related increase in the risk of knee OA.18-21 Post-menopausal women have

more chance not only to suffer from OA, but also to receive total knee arthroplasty due to advanced OA.21 Therefore, most of those who take GlcN for treatment of OA are

post-menopausal women.

The metabolic syndrome, a combination of risk factors including abdominal obesity, dyslipidemia, glucose intolerance, and hypertension, is a highly prevalent health problem in the modern era1 due to the higher risk for developing type 2 diabetes mellitus (T2DM) as well as

increased the morbidity and mortality of cardiovascular disease.2 Now insulin resistance (IR) is a

well-known hallmark of T2DM and metabolic syndrome.22 Menopause promotes a change in

body fat distribution to increase central adiposity, subsequently enhances the likelihood of satisfying the metabolic syndrome criteria.4, 23-25 It had been demonstrated that menopause, but

not age, is an independent risk factor for the elevation of fasting plasma glucose levels in nondiabetic women.23 Some studies also reported that the prevalence of IR increased in the

menopausal women.24 In addition, Dorum et al revealed that bilateral oophorectomy before 50

years of age was significantly associated with T2DM and metabolic syndrome.25 In animal

studies, ovariectomy was associated with increased risk of diabetes, whereas estrogen administration protected against diabetes and increased the insulin response to glucose.26,27

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Therefore, ovarian hormone has protecting effect for IR and decreases the risk of T2DM and metablic syndrome; however, the mechanisms are still unclear.

A number of studies have demonstrated that GlcN causes IR22 because of the decrease of

glycogen synthesis 28-32 and on glucose uptake in adipocytes and skeletal muscle 29-32 as well as

on insulin production of pancreatic β-cells.28, 33 Some clinical studies showed the deleterious

effects of GlcN on glucose metabolism,34-36 whiles some found no effects of GlcN on glucose

metabolism.37-41 Chien et al. also demonstrated that GlcN augmented IR in male fructose-fed

T2DM rats, but not in normal male wistar rats.42 GlcN may adversely affect glucose metabolism

and augment IR; however, the related mechanisms are unclear till now. More studies are needed, particularly focusing on the groups at higher risk for impairments in glucose homeostasis. Since women after menopause or receiving bilateral oophorectomy have higher risk for IR and the higher chance to take GlcN for OA, it is of importance to study on the influence of GlcN in women without the protection of ovarian hormone. In this study, we used ovariectomized (OVX) rats, a rat model of ovarian hormone deficiency,43,44 to investigate whether ovarian hormone has

the protecting effect for GlcN-induced IR in female rats and the underlying mechanisms of this protecting effect.

Methods

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Female Sprague-Dawley rats at the age of 12-week-old, were purchased from the Animal Center of National Cheng Kung University Medical College. Rats were housed in a

temperature-controlled room (25±1˚C) and keep on a 12:12 light-dark cycle (light on at 0600 h). Food and water were available ad libitum throughout the experiment. Rats were randomly assigned to either sham operation (SHAM) or bilateral ovariectomy (OVX). Surgical procedures were performed under sodium pentobarbital (Sigma-Aldrich, St. Louis, MO, USA) anaesthesia (30 mg/kg intraperitoneally [i.p.] injected) through bilateral skin incision at the lower back. Experiments were performed 12 weeks after the surgical procedure. All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health as well as the guidelines of the Animal Welfare Act.43, 45-47

Intraperitoneal glucose tolerance test (IPGTT)

In the experiment of IPGTT, we divided the female rats into 4 groups: (1) Sham-operated group (SHAM), (2) SHAM with 750 mg/kg/day GlcN (Sigma-Aldrich, St. Louis, MO, USA) i.p. injected for 14 days (SHAM+GlcN) , (3) OVX group (OVX), (4) OVX with 750 mg/kg/day GlcN i.p. injected for 14 days (OVX+GlcN) (n=9-10 in each group). IPGTT were preformed after fasting for 6 hours. Blood samples for measurement of plasma glucose and insulin were taken from the femoral vein of rats before glucose load (1 mg/kg, i.p.) at time 0, and at 30, 60, 90 and 120 min thereafter.45-47

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Determination of plasma glucose and insulin level

The plasma glucose levels were determined by a commercialized glucose kit reagent (Biosystems S.S., Barcelona, Spain), using an analyzer (Quik-Lab, Ames, Miles Inc., Elkhart, Indiana, USA). Insulin ELISA kit (Mercodia AB, Uppsala, Sweden) was used to detect the levels of plasma insulin, according to the manufacture’s protocols.

Determination of insulin resistance in animals

The homeostatic model assessment (HOMA) is a method used to quantify insulin resistance and beta-cell function.48 After the concentrations of plasma glucose and insulin were measured,

we used the clinical homeostasis model assessment-insulin resistance (HOMA-IR) and glucose-insulin index to evaluate IR and compare the differences in each group. We obtained HOMA-IR index from the two parameters in a formula: HOMA-IR = Fasting glucose (mmol/L) x fasting insulin (pmol/ml)/22.5.24

The glucose-insulin index was calculated as the product of the glucose and insulin areas under the curve (AUC).38

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Data were expressed as the mean±SEM for the number (n) of animals in each group as indicated in the figures. Statistical differences among groups were determined by using repeated-measure analysis of variance (ANOVA). The Dunnett’s range post hoc comparisons were used to determine the source of significant differences where appropriate. P<0.05 was considered statistically significant.

Results

Effects on body weight

Rats receiving ovariectomy led to significantly body weight gain compared with

sham-operated rats at the end of experiment (379.05±7.23 g in OVX group and 414.25±13.23 g in OVX +GlcN group versus 265.41±8.04 g in SHAM group and 297.05±12.43 g in

SHAM+GlcN group, P<0.05). The increased body weight in ovariectomized rats was compatible with result in previous study which meant the success of animal model.43

Baseline glucose, insulin, and HOMA-IR

The fasting plasma glucose level increased in the OVX+GlcN group only. Ovariectomy and GlcN alone did not increase fasting plasma glucose level. The fasting glucose level was higher in

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the OVX+GlcN group (132±3.5 mg/dl) compared with the SHAM group (120±1.6 mg/dl)

(P<0.05) and the SHAM+GlcN group (120±1.3 mg/dl) (P<0.05). Though the glucose level in the OVX group (126±4.0 mg/dl) mild increased, it did not reach statistically significant compared with all the other groups (Fig. 1A). The fasting plasma insulin level was significantly higher in OVX+GlcN group (1543±399 pmol/l) than all the other groups (P<0.05) (Fig. 1B). The fasting HOMA-IR (76.43±20.85) was significantly higher in OVX+GlcN group than all the other groups (P<0.05) (Fig. 1C).

The changes in plasma glucose and insulin during IPGTT

In IPGTT test (Fig. 2A), at 30, 60, 90 and 120 min after glucose load, the elevation of plasma glucose was significantly higher in OVX+GlcN group (all P<0.01), implying that the glucose utility was more decreased in OVX+GlcN group.50 The plasma glucose levels of other

three groups during IPGTT had no significant differences. Also, in OVX+GlcN group, the AUC of plasma glucose concentrations during IPGTT was markedly higher than that of other three groups (Fig. 2B). In addition, at 30, 60, 90 and 120 min after glucose load, plasma insulin levels were significantly higher in OVX+GlcN group comparing with other three groups (P<0.001 at 30 min, P<0.01 at 60, 90 and 120 min) (Fig. 2C). There was no significant difference in the plasma insulin levels of other three groups during IPGTT. The AUC for plasma insulin concentrations during IPGTT was obviously higher in OVX+GlcN group (Fig. 2D) (P<0.001 comparing with SHAM, SHAM+GlcN, and OVX groups).

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HOMA-IR and glucose-insulin index in IPGTT

HOMA-IR was only significantly elevated in OVX+GlcN group (Fig. 3A) (P<0.001 at 30 min, P<0.01 at 0, 60, 90 and 120 min). The HOMA-IR of other three groups in IPGTT had no significant difference. Neither OVX nor GlcN treatment only induced IR. The glucose-insulin index was calculated as the product of the glucose and insulin AUC. The glucose-insulin index only significantly increased in the OVX+GlcN group (Fig. 3B) (P<0.001). Our results indicated GlcN-induced IR was only found in OVX rats.

Discussion

In the present study, we found the fasting plasma glucose levels only elevated in OVX+GlcN group, and the fasting plasma insulin level was more significantly higher in OVX+GlcN group. In addition, HOMA-IR was significantly elevated in OVX+GlcN group, implying IR was only induced by GlcN in the female rats without the protection of ovarian hormone. The result indicated that the ovariectomized rats can nearly compensate the elevated plasma glucose to just mild elevation by increase in insulin secretion, however, IR developed confirmed by HOMA-IR. In IPGTT test, plasma glucose, insulin, HOMA-IR and glucose-insulin index elevated significantly after intraperitoneal glucose load. The compensation of increase insulin secretion could not overcome the intraperitoneal glucose load, leading to the increase

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plasma glucose level and finally the increase HOMA-IR and glucose-insulin index. These results indicated that in OVX+GlcN group, the rats could maintain mildly elevated fasting blood glucose level by the compensation of hyperinsulinemia, but the compensation mechanism could not sustain normal blood glucose level after glucose challenge. Our results revealed the protecting effect of ovarian hormone against GlcN-induced IR.

IR is a characteristic feature of T2DM and metabolic syndrome.22 The insulin resistant state

persists when islet hyperplasia and hyperinsulinemia compensate to maintain normoglycemia, but T2DM develops upon failure of the β-cells to secrete sufficient insulin to satisfy the rising insulin demand.51,52 In our study, we found that there was only with mild elevated blood glucose but a

significant difference in the fasting insulin level in the OVX+GlcN group because of the compensation mechanism. However, the compensation mechanism could not sustain normal blood glucose level after glucose challenge in the OVX+GlcN group only. Our study was the first one demonstrated that under the ovarian hormone deficient state, administration with GlcN induced hyperinsulinemia and finally led to IR. Persistence of this insulin resistant state may cause the development of T2DM. Obesity is also a risk factor of IR.53 In our study, rats receiving

ovariectomy led to significantly body weight gain compared with sham-operated rats at the end of experiment. Neither SHAM versus SHAM+GlcN groups nor OVX versus OVX+GlcN groups showed significant differences in body weight, implying that GlcN treatment did not cause body weight gain either in the SHAM or OVX rats. The daily diet and water intake were nearly the same between groups (data not shown). Our result indicated that IR was resulted from GlcN

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under the state of ovarian hormone deficiency, but not body weight increase after ovariectomy.

Previous clinical studies about the effects of GlcN in glucose metabolism focused on both men and women, but not on post-menopausal women or women received bilateral oophorectomy. Due to the higher prevalence of OA in the post-menopausal women and the higher chance to take GlcN, it is very important to clarify the insulin resistant effects of GlcN. According to our results, we suggest it should be more cautious to use GlcN in women after menopause or bilateral

oophorectomy. Those who receive GlcN after menopause or bilateral oophorectomy should watch for their blood glucose level, especially the blood glucose level after meal but not the fasting glucose level.

Conclusion

In conclusion, our results show that in OVX rats, GlcN administration increase the risk of IR. The underlying mechanisms require further studies.

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Acknowledgements

We are grateful to Miss M.Y. Wang, Y.P. Lin, Y.S. Lin and Mr. M.H. Chen for their assistance in this study.

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Figure legends

Figure 1. The level of fasting plasma glucose and insulin. (A) The fasting plasma glucose level increased in the OVX+GlcN group only. Ovariectomy and GlcN alone did not increase fasting plasma glucose level. The fasting glucose level was mild higher in the OVX+GlcN group. (B) The fasting plasma insulin level was significantly higher in OVX+GlcN group than all the other groups. #P <0.05. (C) The fasting HOMA-IR was significantly higher in OVX+GlcN group than all the other groups. *P <0.05 versus SHAM, SHAM+GlcN, and OVX groups.

Figure 2. The level of plasma glucose and insulin in IPGTT. (A) The level of glucose in IPGTT only significantly elevated in OVX+GlcN group at 30, 60, 90 and 120 min after glucose load. (B) The AUC for plasma glucose concentrations during IPGTT showed markedly elevation in

OVX+GlcN group. (C) The level of insulin elevated only in the OVX+GlcN group during fasting (time 0), as well as at 30, 60, 90 and 120 min after glucose load. (D) The AUC for plasma insulin concentrations during IPGTT showed markedly elevation in OVX+GlcN group. **P <0.01, ***P<0.001 versus SHAM, SHAM+GlcN, and OVX groups.

Figure 3. HOMA-IR and glucose-insulin index in IPGTT. (A) HOMA-IR was only significantly elevated in OVX+GlcN group. The HOMA-IR of other three groups in IPGTT had no significant difference. (B) The glucose-insulin index only significantly increased in the OVX+GlcN group. Our result indicated only the OVX+GlcN group developed insulin resistance. **P <0.01,

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***P<0.001 versus SHAM, SHAM+GlcN, and OVX groups.

Figure 4. In RT-PCR analysis, the expression of I1 receptors was shown in both uterus and ovaries of female rats. However, in western blot analysis, the expression of I1 receptors in ovary was less than that in uterus. Further investigation would be performed to determine the different findings in RT-PCR and Western blot.

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#

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Figure 4. RT-PCR

Marker： 50bps ladder 1：Uterus ＋ I1 primer 2：Uterus ＋ β-actin primer 3：Ovary ＋ I1 primer 4：Ovary ＋ β-actin primer

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View Letter

Date: Sep 26, 2011

To: "Fong-Ming Chang" [email protected]

From: "Menopause" [email protected]

Subject: MENO Decision

Sep 26, 2011

RE: MENO-D-11-00235R1 , entitled "Glucosamine-induced insulin resistance in ovariectomized rats is relevant to decreasing the expression of glucose transport protein subtype 4 in skeletal muscle and increasing the size of pancreatic islets"

Dear Dr. Chang:

I am pleased to inform you that your manuscript has now been accepted for publication in Menopause - The Journal of The North American Menopause Society. All manuscript materials will be forwarded immediately to the production staff for placement in Volume 19 Issue 5 which is the May 2012 issue. Your manuscript will also appear on the Menopause web site in the Publish Ahead of Print section approximately 12-15 weeks from the date of acceptance after proofs have been finalized and approved.

The journal has added to the Table of Contents a summary of each article. Because you know the material best, we ask that you respond to this email with 1-2 summary sentences about your article to be included on the table of contents page.

Thank you for submitting your interesting and important work to the journal.

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With Kind Regards,

Isaac Schiff, MD

Editor-in-Chief, Menopause

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