f zu{ g } 15 í } 24 2011 06 11 Çf Journal of Clinical Rehabilitative Tissue Engineering Research June 11, 2011 Vol.15, No.24 í ü ÎÎ Œ Ù q * i ¾ i Íl Establishment of rat models of polysystic ovarian syndrome characterized by skeletal muscle insulin resistance Wang Kai-jun, Wang Luan, Ma Rui-xin, Zhao Wen-juan Wang Kai-jun, Studying for master s degree, Department of wkjabc2@163.com Correspondence to: Ma Rui-xin, Doctor, Associate chief physician, Associate professor, ruixinma@yahoo. com Correspondence to: Zhao Wen-juan, Master, Chief physician, Professor, zhaowenjuan@ medmail.com.cn Supported by: Public Domain Science and Technology Plan Program of Qingdao City, No. 09-1-1-1-nsh* Received: 2011-03-19 Accepted: 2011-05-10 Abstract BACKGROUND: Studies have demonstrated that insulin resistance plays an important role in the occurrence and development of polycystic ovarian syndrome (PCOS). Establishing rat models of PCOS characterized by skeletal muscle insulin resistance is the basis for studying PCOS. OBJECTIVE: To establish the ideal rat models of PCOS characterized by skeletal muscle insulin resistance. METHODS: Female Sprague-Dawley rats, aged 89 weeks, were randomized to model group and control group with 20 animals in each group. Insulin and human chorionic gonadotrophin were administered to the model group rats which were fed with high fat diet and 50 g/l glucose solution. The rats in the control group were given normal diet and injected normal saline daily. After 6 weeks of treatment, the body weight was measured and then all of rats were sacrificed. Ovaries were taken out for histological observation. Serum concentrations of estradiol, testosterone, progestogen, follicle-stimulating hormone and luteinizing hormone were detected. Fasting blood glucose and insulin levels were measured for evaluating the insulin resistance. The expression of glucose transporter 4 in the skeletal muscle was detected by immunohistochemical staining. RESULTS AND CONCLUSION: The ovarian size in the model group was larger than that in the control group. The ovaries of the model group showed multiple follicular cysts. The fasting blood glucose, fasting insulin, testosterone and luteinizing hormone levels in the model group were respectively higher than those in the control group. While, the expression of glucose transporter 4 in PCOS rats was significantly lower than that of the control group. In model group, the intracytoplasmic granules of glucose transporter 4 were mostly located in the cytoplasm, however, in the control group, they were near the muscle cellullar membrane. Therefore, the administration of insulin and human chorionic gonadotrophin combined with high fat diet and 50 g/l glucose solution is an ideal method to establish the rat models of PCOS with significant muscle insulin resistance. Wang KJ, Wang L, Ma RX, Zhao WJ. Establishment of rat models of polysystic ovarian syndrome characterized by skeletal muscle insulin resistance.zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(24): 4400-4404. [http://www.crter.cn http://en.zglckf.com] u{ÿ Œ í ü qöm ö ²zf n k q í ü ÎÎ Œ Ùg u{ ooq u rq ±gk q í ü ÎÎ Œ Ù q ¹k Ú SD Ä Ùà Ég d oœ Šüt q xîœ Ê 50 g/l» d q mkr ÊÉ» ³ 6 ý Ùí ƒz d ž t ؃m { ž Œ Îp d ÎÎ f ±² q 4 Ÿ² ƒp d dº ±² q 4 Â È Å²ÎÎ Œ ² ± ù Œ Šüt q ÊxÎŒÊ 50 g/l í ü ÎÎ Œ Ù ±gk q ºÀ í ü Ùg Œ ÎÎ ±² q 4 Ù doi:10.3969/j.issn.1673-8225.2011.24.008 i ¾ i Íl. í ü ÎÎ Œ Ù q [J].f zu{ g 2011 15(24):4400-4404. [http://www.crter.org http://cn.zglckf.com] 0 í ü (polycystic ovary syndrome, PCOS) Ú q¼é öx mooi xä ² ígg g g Ú föoi g6%~10% [1] Ž1980 Burghen}Ë ÇŒ (insulin resistance, IR)õ PCOSq öo²zx u{ x 60%~80%qPCOS d90%q PCOS Œ [2] u{ÿ Œ PCOSqöm ö ²zf n [3] ömœ q Í [4-5] ÎÎ Œ nqg Å i ƒ¼œ Î q 85%nÎÎ [6] ÎÎ g ± Ä ÎÎ Œ qpcos Ù u{pcosqöo Í g ƒpîî Œ q ±²ƒ4(glucose transporter 4, 4400
i ¾ }. í ü ÎÎ Œ Ù q GLUT4)v oœ Î q q u { x GLUT4qÉ Ÿ² ùœ Œ Érº [7-8] Í nœ Šüt (human chorionic gonadotropin, HCG)q ÊxÎŒÊ ö50 g/l PCOS Ù ³ ² zœ g ö ÎÎ Œ GLUT4qŸ² À z qùå 1 à dùg Í Âö p200910/201003 Å ç àçãùgf ÍÙg Ÿ SPF ¹k ÚSDÄ Ù 40ù ƒ«200~220 g «Ž Ò ç ù ú SCXKÒ20080002 ÙgÊ»j (20±2) 12 h d ŽnÊ ² É Ê ³Ê ü É g w ä üg60% q«22% Œ 10% â ¼qºw É8% ÎŒÊ ü É g w äüg40% Œ 40% q«13% ºw É7% g Ï y Ïöy Œ (~ N) HCG ÎŒÊ ž Äp t í Î öøƒm Ïr Œ ELISA Ïr GLUT4 ƒ ³ž y ˆ x ¹ú j hjä hjí ï äsˆ mgç y ¹ú ˆ R&D ¹ú äsë mg  ¹ú ˆ ¹ú ÍÉ ö Í Ù²»3 dý à Ég d 20ù õdporesky [9] nø ² oîœê 50 g/l }1~10 µq q tœ Ï n0.5 IU ² ² ( Ø0.5 IU)Ž5.0 IU } 11~22 µq q tœ 6.0 IU ü ohcg 3.0 IU( p 0.2 mlmkr ) ¼Œq ü1 d o ³Ê ³Ên }1~10 µq 0.2 mlmkr }11~22 ü²ø 1 0.2 mlmkr }22 ý ž n d ž q mkr ž ˆn50 g/l g ³Ên e Ù»Ž}6 zƒ«1 p È30 dý e Ù ²Ÿ ³ Œ f l { ž ž p}6 22:00 yé zƒ«ý ž { ž ýj Ø sýåœd ž 3 000 r/miny ýn ž ˆ 20 À~ n n o ž Äp t í Î öøƒm n ELISA { Œ nz Œ (homestasis model assessment for insulin resistance, HOMA-IR) zœ z [10] HOMA-IR= { ž Œ (miu/l) { ž (mmol/l)/22.5 í q sýåœd žýè ŒŽ Ù ²³ öœí ôãÿå qœ º ƒ ˆ ƒzé 10%n uœâ Éf ² Ÿ À º í ö À o ä ÎÎ fglut4qÿ² É Ù ƒzé 10%n 48 hý j Œ pn ² uœâ Áíý²ŸŒœ ä ñ ²Ÿ GLUT4 o ä [11] À ÙÎÎ fglut4qÿ² É n É GLUT4Ÿ²qÅz ²Ÿ É Åz ~ À ( 40) { à ² 20f ± É GLUT4q  g ~ g qåz Åzq ígglut4qåz 20f q ígglut4qåz ²Â g ²Ÿ É º g q g Ùqƒ«ä ³ Œ f ä í ƒ xöok ž ä { ž ö{ Œ HOMA-IR ÎÎ GLUT4qŸ² www.crter.org { œ Þ ÛÚ 1981 ± { x À Ôd wkjabc2@163. com þ š šì { œ Þ ruixinma@ yahoo.com ò Ø Ì { œ Þ zhaowenjuan@ medmail.com.cn f É ú:r318 h u:b ú:1673-8225 (2011)24-04400-05 2011-03-19 º 2011-05-10 (20110319002/WLM es) ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 4401
www.crter.org i ¾ }. í ü ÎÎ Œ Ù q É É nspss 13.0±z²Ÿ É Í xx _ ±sÿx e  ±nt Í P < 0.05g i 2 2.1 ð ͺ 40ù Ù ³ }21 ùœ ƒž r1ù º 39ù Ù² É 2.2 { ð ³ Ð d Ù ƒ«i(p > 0.05) ³ ý Ùƒ«± d Ø(P < 0.01) Ÿ1 Ÿ 1 ÍÐýe Ùƒ«q ä Table 1 Changes of body weight in two groups (x _ ±s, g) Group n Before experiment a P < 0.01, vs. control group After experiment Changes Control 20 209.50±8.96 273.25±7.98 63.75±8.73 Model 19 208.76±9.03 305.21±10.40 a 96.45±9.80 a 2.3 { Ä Ù Ù qüfâ Ã¼Ä ä ³ qœ ŽÂ³ q Œk j ä ² ³ Œ f l À ù ö Ð Œ ² ö Œ ² ¾ d ŒÂ ² ±o ö  xq Œgg ö ý x 3y Œ ù Íf 17ù( 90%)Ÿjg ä q Œ x Ù ö í ù 2ù( 10%)v Ù d Ù 2.4 ük ÆhΫƒ öj Ù í ƒz d qp í q Ÿ ñ º à f qí d Ùí Ó ŸÅ À 1 2 Figure 2 Histological image of ovarian in the two groups after different treatments (Hematoxylin-eosin staining, 400) 2 Ùí (, 400) 1 2 x qí È Œ à Ž1~3 Ê m ñ ؃ d ù ؃ üö  qí í ¼È Œ g7~10 Ê Ù ù íd í Œ ¾ ² 2.5 Ùž t ؃m q öøƒm í Î q Îp d (P < 0.01) Äp í Î d  i(p > 0.05) Ÿ2 Ÿ 2 ³ ý Ùž Table 2 Comparison of serum hormone level between two groups Group n E 2 (ng/l) T (μg/l) P (μg/l) Control 20 20.91±5.67 25.17±7.90 34.39±3.17 Model 19 25.22±7.62 51.21±10.10 a 35.17±4.63 Group n LH (IU/L) FSH (IU/L) LH/FSH Control 20 3.04±0.66 1.15±0.50 2.64±0.46 Model 19 11.94±2.62 a 1.69±0.52 7.07±0.84 a a P < 0.01, vs. control group; E 2 : estradiol; T: testosterone; P: progestogen; LH: luteinizing hormone; FSH: follicle stimulating hormone 2.6 à cà HOMA-IR Ù { ž { Œ öhoma-ir Îp d (P < 0.05 P < 0.01) Ÿ3 Ÿ 3 ³ ýe Ù{ ž { Œ öz Œ q ± Table 3 Comparison of insulin resistance of FBG, FINS, and HOMA-IR between two groups Group n FBG (mmol/l) FINS (miu/l) HOMA-IR Control 20 4.85±0.55 12.29±3.34 2.65±0.53 Model 19 5.45±0.96 b 25.00±4.67 a 6.61±0.96 a 4402 Figure 1 Histological image of ovarian in the two groups after different treatments (Hematoxylin-eosin staining, 40) 1 Ùí (, 40) a P < 0.01, b P < 0.05, vs. control group; FBG: fasting blood glucose; FINS: fasting insulin; HOMA-IR: homestasis model assessment for insulin resistance 2.7 { ¾GLUT4 { o ä x ÙÎÎ Œf Ø qglut4â r
i ¾ }. í ü ÎÎ Œ Ù q g dg É pœ«f Œ d ÙÎÎ Œfù Ø qglut4â rg É xå²îî Œ ² gg 3 É x ÙÎÎ GLUT4Ÿ² qåz µ ƒp d (P < 0.01) xºglut4ÿ²q ƒp d Ÿ4 3 Figure 3 Expression of glucose transporter 4 in skeletal muscle in two groups (Immunohistochemical staining, 100) 3 ÙÎÎ ±²ƒ 4 qÿ² ( o ä 100) Ÿ 4 ÙÎÎ ±²ƒ 4 Ÿ² q É Table 4 Comparison of image analysis of glucose transporter 4 expression Group n Area density (%) Absorbance value Control 20 51.36±4.40 1.72±0.08 Model 19 41.55±3.80 a 1.53±0.07 a a P < 0.01, vs. control group PCOS Ú q¼é x m o o öoig6%~10% g g g ÎÄ žo À í IR PCOSföm Íqu{ Ùp PCOSqöo Í ÎÎ Œ nqg Å i ƒ¼œ Î q 85%n ÎÎ ÎÎ g ± Ä Î Î Œ qpcos Ù u{pcosq öo Í g IR ƒ¼mk qœ ² Œ Ìn q Œ  g r qž ƒx ØŒ qé Î Œ žo [12] ÎŒ žoù³²x ² ŽÎ Ä žo Ží Á Œ ùr  ³²Œ mà í Ø ŒrmÄ Î Œ ü qœ Ÿ Ä Í n Žº éî ÎÄ žoù Í Œü ük q ˆ yt é Ä n ÎqŒ ù ²Øƒm www.crter.org ùr í í ¼ 17-αˆä n ؃ m qéü nžä ü [13] HCG ؃m q g Œ Íí È Œq dé ÂÍ È Œq º í qî n Œ qéü xø ŠüˆnŒ HCG(Poresky ) opcosqöo²z Äx à Œ q zœ g Ã Í ÙÎŒÊÉ4~6 ù z ùåqœ Ù [14-15] Í Poresky ²Ÿo Ù oîœê» d n ý Ž}6 v Íz qœ g z o Œ Œ q g ³ ²zfŒ Ï ² Ž Í Ùömƒž Ž r Ù o 50 g/l x ³Ê Íf u 1ù Ù rœ ( ÃŒ Ï Ø ƒž Ž) ý u{ x Ùí ƒz À ù í È Œ Ã Ê m ؃à ² ³ Œ f x í ž t ؃m Î x g }üpcosq äg ÎŒ ¾ rð zirq  [16] º «n «Äx ³ np Íu{ u{ÿ HOMA-IR ÎŒ ¾ qrº [17] Í n zœ x ÙqHOMA-IR Îp d ýqpcos³ ü rmo± qžœ g² u{œ PCOSöm ö ²zq Í o u GLUT4 yÿ509f q q r É «45 000~55 000 g p Œ qîî Œ [18] Œ Î g ƒp Œ¼q«¼ Œ ƒ üý ö Ê Š Ž ÿglut4q ý Œ z Ù Œ ü GLUT4±ƒŽ Œ d Ø ü öm ± ²Ž Œ¼ý ñ [8] rð gpcos Œ g öm Œ nq ºÅ íîî Œ Œ ÎÎ ¹ x qg ÎÎ Œ¼qGLUT4 ±²Ž Œ¼qºÀ [19] u{ ömœ fqglut4ùçjx Å Å GLUT4q¼ É gì Œ Î ý Œ¼ ý Œ q±ƒ à ÿglut4qg Œ ü ü à GLUT4 ± Í ŽGLUT4 mrna qÿ qã Zierath} [20] u{öj ÎÎ À ÅpÎ Œ f ŽGLUT4 ƒ ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 4403
www.crter.org ±ƒãv d mk qœ Î ö Íf Ù Œ üîœêé Ž rmîœ žo o ä ÙÎÎ ŒfGLUT4qŸ² É x ÙÎ Î ŒfqGLUT4Ÿ² ƒp d dœ É ± ÙqÎÎ Œ GLUT4±ƒ ² Ž ±²Ãv ž éî Ž Ífk i HOMA-IRn{ ž ö{ Œ ~ Ͳzf ž ³ q Ùg ž žùœ ² Œ q ö q x ~HOMA-IR ÎŒ ¾ Í qrº º Œ ¹x º zœ ù À ùx qö Œ qz Í º PCOSr qí ƒ ok xöž ä { ž { Œ HOMA-IR Îp d ü Œ o ä x ÙÎÎ ŒGLUT4Ÿ²± d d É pœ«¼ x ÙÎÎ Œ Í Ë o±gk q í ü ÎÎ Œ Ù ý Ùg q u u{pcos ömîî Œ qé Í 4 õ h [1] Reckelhoff JF. Polycystic ovary syndrome: androgens and hypertension. Hypertension. 2007;49(6):1220-1221. [2] Li JY, Chen MD. Zhonghua Neifenmi Daixie Zazhi. 2010;26(9): 803-806.,Âü³. í ü q ž~ ž~ooè Â Ä ² í ü (AE-PCOS) ~qº [J]. fé¼é x,2010,26(9):803-806. [3] Slaeh AM, khalil HS. Review of nonsurgical and surgical treatment and the role of insulin-sensitizing agents in the management of infertile women with polycystic ovary syndrome. Acta Obstet Gynecol Scand. 2004;83(7):614-621. [4] Mukherjee S, Maitra A. Molecular & genetic factors contributing to insulin resistance in polycystic ovary syndrome. Indian J Med Res. 2010;131:743-760. [5] Rajkhowa M, Brett S, Cuthbertson DJ, et al. Insulin resistance in polycystic ovary syndrome is associated with defective regulation of ERK1/2 by insulin in skeletal muscle in vivo. Biochem J. 2009; 418(3):665-671. [6] Li HB, Ge YK, Zheng XX, et al. Salidroside stimulated glucose uptake in skeletal muscle cells by activating AMP-activated protein kinase. Eur J Pharmacol. 2008;588(2-3):165-169. [7] Huang S, Czech MP. The GLUT4 glucose transporter. Cell Metab. 2007;5(4):237-252. [8] Wston RT, Pessin JE. Intracellular Organization of Insulin Signaling and GLUT4 Translocation. Recent Progress in Hormone Research. 2001;56:175-193. [9] Poretsky L, Clemons J, Bogovich K. Hyperinsulinemia and human chorionic gonadotropin synergistically promote the growth of ovarian follicular cysts in rats. Metabolism. 1992;41(8):903-910. [10] Li GW. Laonian duo Qiguan Jibing Zazhi. 2004;3(1):11-12. ~.Œ öºg n[j]. fé oo,2004,3(1):11-12. i ¾ }. í ü ÎÎ Œ Ù q [11] Wang D, Li JL, Jiang LD. Zhonghua Zhongyiyao Zazhi. 2007;22 (12): 884-888. id,,. dï 2 oœ ÙTNF-α öglu T4 fÿ²q [J].féfç,2007,22(12): 884-888. [12] Mlinar B, Marc J, Janez A, et al. Molecular mechanisms of insulin resistance and associated diseases. Clinica Chimica Acta. 2007; 375(1-2):20-35. [13] Nisenblat V, Norman RJ. Androgens and polycystic ovary syndrome. Curr Opin Endocrinol Diabetes Obes. 2009;16(3): 224-231. [14] Liu LP, Wu J, Wang JQ, et al. Anhui Yiyao. 2009;13(10): 1171-1173. Êh,,i Å,}.ÎŒ» ÙŒ [J]. ç, 2009,13 (10):1171-1173. [15] Liu J, Tang H, Niu L, et al. Upregulation of Tanis mrna expression in the liver is associated with insulin resistance in rats. Tohoku J Exp Med. 2009;219(4):307-310. [16] Kim JK. Hyperinsulinemic-euglycemic clamp to assess insulin sensitivity in vivo. Methods Mol Biol. 2009;560:221-238. [17] Jia WP, Chen L, Xiang KS, et al. Zhonghua Neifenmi Daixie Zazhi. 2001;17(5):268-271. «~,Â,È,}. ÎŒ - ¾ q [J]. fé¼é x,2001,17(5):268-271. [18] Nia JB, Roland G, David EJ. Regulated transport of the glucose transporter GLUT4.Nature Reviews/ Molecular Cell Biology. 2002;4(3):267. [19] Sun Y, Bilan PJ, Liu Z, et al. Rab8A and Rab13 are activated by insulin and regulate GLUT4 translocation in muscle cells. Proc Natl Acad Sci U S A. 2010;107(46):19909-19914. [20] Zierath J R, Krook A, Wallberg-Henriksson H. Insulin action in skeletal muscle from patients with NIDDM. Mol Cell Biochem. 1998;182:153-160. Ž Èq «Ù õ ÞÁ mâ ( 09-1-1-17-nsh) «h þ c Ø Â nd ÛÚ ÛÚ À òc ìn dþ c Ø þ d ÌrÀ{ ß À jãà ½ Ò ͽÅÍ Ò½í d k  tì «Þ Á w Çx d Ë Æ Pubmedc c ccnki á Æ Å Èá 201012 Çw qœ ük c c Ýc ¾c ~ 4r Çw q polycystic ovary syndrome, insulin resistance, muscle, glucose transporter 4r Ç Ôd u ƒ «Œ ük È d u œðòj Œ ük t î y (Poresky ) ¾s Ÿ { x i Ýd {  òƒ Œ ük ük ì œ ò ¾ Ý d 4404