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1 國科會專題研究計畫成果報告撰寫格式 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 ( 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 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

2 附件一 成果報告行政院國家科學委員會補助專題研究計畫 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 : 個別型計畫 整合型計畫計畫編號 :NSC B 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 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 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 二年後可公開查詢 執行單位 : 中華民國 100 年 7 月 31 日

3 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 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. 6 It was a different class of binding sites than the α 2 -adrenoceptors and specifically recognized imidazoline and guanidine groups. 7

4 Imidazoline receptor has been subclassed into two sites. The first are I 1 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 I 2 binding sites that show a high affinity for imidazoline and guanidine derivatives and a medium affinity for imidazolidine derivatives. 8-9 Moreover, I 3 binding sites have been proposed. 10 Imidazoline receptor localizes in both central and peripheral nervous systems, and other tissues such as kidney, prostate, stomach, heart, liver, placenta, and colon. 8 Functionally, peripheral imidazoline receptors mediate the movement of smooth muscle, stimulate insulin release and regulate the renal excretion of sodium, potassium and water. 11 Some α 2 -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 I 1 receptor agonist-moxonidine. 14 It can decrease sympathetic nervous activity and improve insulin resistance in the spontaneously hypertensive obese rat model 15, as well as in the obese hypertensive patients. 16 However, the pharmacological role of imidazoline I 2 receptor agonist on T2DM or metabolic syndrome remains unclear. Besides, little is known about the I 1 or I 2 receptor expression in female genital organs until now. II. Glucosamine and Insulin resistance

5 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 Female gender is associated with the age-related increase in the risk of knee OA 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 era 1 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, 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

6 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 IR 22 because of the decrease of glycogen synthesis and on glucose uptake in adipocytes and skeletal muscle as well as on insulin production of pancreatic β-cells. 28, 33 Some clinical studies showed the deleterious effects of GlcN on glucose metabolism, whiles some found no effects of GlcN on glucose metabolism 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 Experiment animals

7 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 43, Institutes of Health as well as the guidelines of the Animal Welfare Act. 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

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

9 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 ±13.23 g in OVX +GlcN group versus ±8.04 g in SHAM group and ±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

10 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).

11 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

12 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

13 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. References 1. Reaven GM. Why Syndrome X? From Harold Himsworth to the insulin resistance syndrome. Cell Metab 2005;1: Bentley-Lewis, R., Koruda, K., Seely, E.W.: The metabolic syndrome in women. Nat Clin Pract Endocrinol Metab. 3: , 2007.

14 3. Ferrara, C.M. et al.: Differences in adipose tissue metabolism between postmenopausal and perimenopausal women. J Clin Endocrinol Metab 87: , Carr, M.C.: The emergence of the metabolic syndrome with menopause. J Clin Endocrinol Metab 88: , Reid, J.L., Panfilov, V., MacPhee, G., Elliott, H.L.: Clinical pharmacology of drugs acting on imidazoline and adrenergic receptors. Studies with clonidine, moxonidine, rilmenidine, and atenolol. Ann. N. Y. Acad. Sci. 763: , Bousquet, P., Feldman, J., Schwartz, J.: Central cardiovascular effects of alpha adrenergic drugs: differences between catecholamines and imidazolines. J Pharmacol Exp Ther 1984, 230: Dardonville, C., Rozas, I., Callado, L.F., Meana, J.J.: I(2)-imidazoline binding site affinity of a structurally different type of ligands. Bioorg Med Chem 10: , Michel, M.C., Ernsberger, P.: Keeping an eye on the I site: imidazoline preferring receptors, Trends Pharmacol. Sci. 13: , Eglen, R. M., Hudson, A. L., Kendall, D. A., Nutt, D. J., Morgan, N. G.; Wilson, V. G., Dillon, M. P.: Seeing through a glass darkly : casting light on imidazoline I sites. Trends Pharmacol Sci 19: , Raasch, W., Schafer, U., Chun, J., Dominiak, P.: Biological significance of agmatine, an endogenous ligand at imidazoline binding sites, Br J Pharmacol. 133: , Schulz, A. Hasselblatt, A.: Dual action of clonidine on insulin release:suppression, but stimulation when alpha 2-adrenoceptors are blocked. Naunyn-Schmiedeberg s Arch.

15 Pharmacol. 340: , Dunne, M.J. et al.: Potassium channels, imidazolines, and insulin-secreting cells, Ann. N. Y. Acad. Sci. 763: , Derosa, G., Cicero, A.F., D'Angelo, A., Fogari, E., Salvadeo, S., Gravina, A., Ferrari, I., Fassi, R., Fogari, R.: Metabolic and antihypertensive effects of moxonidine and moxonidine plus irbesartan in patients with type 2 diabetes mellitus and mild hypertension: a sequential, randomized, double-blind clinical trial. Clin Ther 29: , Koletsky, R.J., Velliquette, R.A., Ernsberger, P.: The role of I (1)-imidazoline receptors and alpha (2)-adrenergic receptors in the modulation of glucose and lipid metabolism in the SHROB model of metabolic syndrome X. Ann N Y Acad Sci 1009: , Dostrovsky NR, Towheed TE, Hudson RW, Anastassiades TP. The effect of glucosamine on glucose metabolism in humans: a systematic review of the literature. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 2011;19: Kennedy J. Herb and supplement use in the US adult population. Clin Ther 2005;27: Stumpf JL, Lin SW. Effect of glucosamine on glucose control. Ann Pharmacother 2006;40: Arden N, Nevitt MC. Osteoarthritis: epidemiology. Best Pract Res Clin Rheumatol 2006;20: Kellgren JH, Moore R. Generalized osteoarthritis and Heberden's nodes. Br Med J 1952;1:

16 20. Oliveria SA, Felson DT, Reed JI, Cirillo PA, Walker AM. Incidence of symptomatic hand, hip, and knee osteoarthritis among patients in a health maintenance organization. Arthritis Rheum 1995;38: NIH Consensus Statement on total knee replacement. NIH Consens State Sci Statements 2003;20: Buse MG. Hexosamines, insulin resistance, and the complications of diabetes: current status. Am J Physiol Endocrinol Metab 2006;290:E1-E Otsuki M, Kasayama S, Morita S, et al. Menopause, but not age, is an independent risk factor for fasting plasma glucose levels in nondiabetic women. Menopause 2007;14: Bonora E, Targher G, Alberiche M, et al. Homeostasis model assessment closely mirrors the glucose clamp technique in the assessment of insulin sensitivity: studies in subjects with various degrees of glucose tolerance and insulin sensitivity. Diabetes Care 2000;23: Dorum A, Tonstad S, Liavaag AH, Michelsen TM, Hildrum B, Dahl AA. Bilateral oophorectomy before 50 years of age is significantly associated with the metabolic syndrome and Framingham risk score: a controlled, population-based study (HUNT-2). Gynecol Oncol 2008;109: Bryzgalova G, Lundholm L, Portwood N, et al. Mechanisms of antidiabetogenic and body weight-lowering effects of estrogen in high-fat diet-fed mice. Am J Physiol Endocrinol Metab 2008;295:E Riant E, Waget A, Cogo H, Arnal JF, Burcelin R, Gourdy P. Estrogens protect against high-fat diet-induced insulin resistance and glucose intolerance in mice. Endocrinology

17 2009;150: Anello M, Spampinato D, Piro S, Purrello F, Rabuazzo AM. Glucosamine-induced alterations of mitochondrial function in pancreatic beta-cells: possible role of protein glycosylation. Am J Physiol Endocrinol Metab 2004;287:E Brady MJ, Kartha PM, Aysola AA, Saltiel AR. The role of glucose metabolites in the activation and translocation of glycogen synthase by insulin in 3T3-L1 adipocytes. J Biol Chem 1999;274: Ciaraldi TP, Carter L, Nikoulina S, Mudaliar S, McClain DA, Henry RR. Glucosamine regulation of glucose metabolism in cultured human skeletal muscle cells: divergent effects on glucose transport/phosphorylation and glycogen synthase in non-diabetic and type 2 diabetic subjects. Endocrinology 1999;140: Robinson KA, Sens DA, Buse MG. Pre-exposure to glucosamine induces insulin resistance of glucose transport and glycogen synthesis in isolated rat skeletal muscles. Study of mechanisms in muscle and in rat-1 fibroblasts overexpressing the human insulin receptor. Diabetes 1993;42: Vosseller K, Wells L, Lane MD, Hart GW. Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes. Proc Natl Acad Sci U S A 2002;99: D'Alessandris C, Andreozzi F, Federici M, et al. Increased O-glycosylation of insulin signaling proteins results in their impaired activation and enhanced susceptibility to apoptosis in pancreatic beta-cells. FASEB J 2004;18:

18 34. Biggee BA, Blinn CM, Nuite M, Silbert JE, McAlindon TE. Effects of oral glucosamine sulphate on serum glucose and insulin during an oral glucose tolerance test of subjects with osteoarthritis. Ann Rheum Dis 2007;66: Monauni T, Zenti MG, Cretti A, et al. Effects of glucosamine infusion on insulin secretion and insulin action in humans. Diabetes 2000;49: Pham T, Cornea A, Blick KE, Jenkins A, Scofield RH. Oral glucosamine in doses used to treat osteoarthritis worsens insulin resistance. Am J Med Sci 2007;333: Albert SG, Oiknine RF, Parseghian S, Mooradian AD, Haas MJ, McPherson T. The effect of glucosamine on Serum HDL cholesterol and apolipoprotein AI levels in people with diabetes. Diabetes Care 2007;30: Muniyappa R, Karne RJ, Hall G, et al. Oral glucosamine for 6 weeks at standard doses does not cause or worsen insulin resistance or endothelial dysfunction in lean or obese subjects. Diabetes 2006;55: Scroggie DA, Albright A, Harris MD. The effect of glucosamine-chondroitin supplementation on glycosylated hemoglobin levels in patients with type 2 diabetes mellitus: a placebo-controlled, double-blinded, randomized clinical trial. Arch Intern Med 2003;163: Tannis AJ, Barban J, Conquer JA. Effect of glucosamine supplementation on fasting and non-fasting plasma glucose and serum insulin concentrations in healthy individuals. Osteoarthritis Cartilage 2004;12: Yu JG, Boies SM, Olefsky JM. The effect of oral glucosamine sulfate on insulin sensitivity in

19 human subjects. Diabetes Care 2003;26: Chien CS, Cheng SC, Wu HT, Tsao CW, Cheng JT. Insulin resistance induced by glucosamine in fructose-fed rats. Horm Metab Res 2009;41: Ho M, Chen Y, Liao H, et al. Simvastatin increases osteoblasts and osteogenic proteins in ovariectomized rats. Eur J Clin Invest 2009;39: Saengsirisuwan V, Pongseeda S, Prasannarong M, Vichaiwong K, Toskulkao C. Modulation of insulin resistance in ovariectomized rats by endurance exercise training and estrogen replacement. Metabolism 2009;58: Cheng JT, Liu IM, Chi TC, Tzeng TF. Release of beta-endorphin by prostaglandin E2 to lower plasma glucose in streptozotocin-induced diabetic rats. Horm Metab Res 2001;33: Cheng JT, Liu IM, Chi TC, Tzeng TF, Lu FH, Chang CJ. Plasma glucose-lowering effect of tramadol in streptozotocin-induced diabetic rats. Diabetes 2001;50: Cheng JT, Liu IM, Tzeng TF, Tsai CC, Lai TY. Plasma glucose-lowering effect of beta-endorphin in streptozotocin-induced diabetic rats. Horm Metab Res 2002;34: Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28: Liu IM, Chen WC, Cheng JT. Mediation of beta-endorphin by isoferulic acid to lower plasma glucose in streptozotocin-induced diabetic rats. J Pharmacol Exp Ther 2003;307: Wu HT, Chang CK, Tsao CW, et al. Insulin resistance without obesity induced by cotton

20 pellet granuloma in mice. Lab Invest 2009;89: Withers DJ, Gutierrez JS, Towery H, et al. Disruption of IRS-2 causes type 2 diabetes in mice. Nature 1998;391: Milburn JL, Jr., Hirose H, Lee YH, et al. Pancreatic beta-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids. J Biol Chem 1995;270: Joost HG. Pathogenesis, risk assessment and prevention of type 2 diabetes mellitus. Obes Facts 2008;1: 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.

21 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,

22 ***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.

23 EES苟EE胃里E軍區倡 ZagA Figure 1 # # 句 SHAM SHAM+GlcN ovx OVX+GlcN B 句 50 句 C 50 i SHAM SHAM+OlcN ovx OVX+OlcN 咀 UE,44置 的 SHAM SHAM+GlcN 'VX VX+GlcN

24 + 蜘甜甜 Figure 2 A B 35., ** ** ** ** 3D. i 司 E, 3OOOO ** -E Ea 于 SHAM -o- SHAM 咱, ovx ' >E 扭曲 2 甘曲 旱 i 10 曲 -a-fe---aa SHAM SHAM+OlcN ovx OVX+OlcN a 3D 60 ' 120 (min) C D NCS OE 岫曲 -:::-St!AM *** -0- SHAM+OlcN...ovx -.-OVX+OlcN ** E' 司的 E 曲曲 EZ 30 閣 r. 圓 mee--m 圓圓 ** 2.00 " 百 EZL 25OOOO *** 可.00 ω2 可 ' A,li E州雷 d.omo o 120 (min) SHAM SHAM+GlcN vx VX+GlcN

25 htde z. FM的===-MoAO苟EEE=-oFigure 3 A 可 Ho o *** 30 ** 60 ~SHAM -s-sham+glcn - 倉 -OVX -.-OVX+GlcN ** ** 90 可 20 Time (Min) B ==的EE圖 " ω可 2000 *** 可 SHAM SHAM+GlcN OVX OVX+GlcN

26 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 Western blot

27

28 View Letter Date: Sep 26, 2011 To: From: Subject: "Fong-Ming Chang" "Menopause" MENO Decision Sep 26, 2011 RE: MENO-D R1, 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 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 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. Your username is: ****** Your password is: ****** With Kind Regards, Isaac Schiff, MD Editor-in-Chief, Menopause Menopause - The Journal of The North American Menopause Society

29 附件二 可供推廣之研發成果資料表 可申請專利 可技術移轉日期 : 年月日 國科會補助計畫 計畫名稱 : 計畫主持人 : 計畫編號 : 學門領域 : 技術 / 創作名稱 發明人 / 創作人 中文 : (100~500 字 ) 技術說明 英文 : 可利用之產業及可開發之產品 技術特點 推廣及運用的價值 1. 每項研發成果請填寫一式二份, 一份隨成果報告送繳本會, 一份送貴單位研發成果推廣單位 ( 如技術移轉中心 )

30 2. 本項研發成果若尚未申請專利, 請勿揭露可申請專利之主要內容 3. 本表若不敷使用, 請自行影印使用