ACh (nitric oxide, NO) (1) 17β -E 2

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1 Acta Physiologica Sinica, October 25, 2005, 57 (5): β- * β- (17beta-estradiol, 17β-E 2 ) (insulin resistance, IR) Sprague-Dawley 8 IR 17β-E 2 (30 µg/kg) IR HE (L-phenylephrine, PE) ACh(sodium nitroprusside, SNP) (nitric oxide synthase, NOS) N- -L- (N-nitrl-L-arginine methyl ester, L- NAME) + +17β-E 2 ACh (nitric oxide, NO) (1) 17β -E 2 IR (2) (3) + NO PE ACh 17β-E 2 L-NAME 17β-E 2 (4) SNP PE 17β-E 2 IR NOS NO IR Q463 17β-estradiol protects against injury of aortic relaxation and contraction in ovariectomized rats with insulin resistance induced by fructose ZHOU Shou-Hong, LING Hong-Yan, TIAN Shao-Wen, LIU Xian-Qing, WANG Bing-Xiang, HU Bi * Department of Physiology, School of Medicine, Nanhua University, Hengyang , China Abstract: The purpose of the present study was to investigate the effect of 17β-estradiol (17β-E 2 ) on the structure and relaxation and contraction activity of thoracic aortas in ovariectomized rats with insulin resistance induced by fructose. Ovariectomized mature female Sprague-Dawley rats were fed with high fructose diet for 8 weeks to induce insulin resistance. Physiological dose of 17β-E 2 (30 µg/kg) was injected subcutaneously every day for 8 weeks. Systolic blood pressure (SBP) was measured by use of tail-cuff. Serum nitric oxide (NO), estradiol (E 2 ), fasting blood sugar (FBS) and fasting serum insulin (FSI) were measured respectively in each group. The insulin sensitive index (ISI) was calculated. The thoracic aortas were fixed in formalin, sliced and HE dyed. The structure of thoracic aortas, lumen breadth, media thickness, media thickness/lumen breadth ratio and media cross-section area were measured. The contraction response of thoracic aorta rings induced by L-phenylephrine (PE) and the relaxation response of thoracic aorta rings induced by ACh and sodium nitroprusside (SNP) were measured. To explore the mechanism, nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME) was used. The results obtained are as follows: (1) 17β-E 2 protected against the effect of high fructose diet, which caused an increase in SBP, hyperinsulinemia and a decrease in ISI in ovariectomized rats. (2) The structure of thoracic aortas had no significant difference among the groups. (3) Compared with the ovariectomized group (OVX) or fructose fed group (F), serum nitric Received Accepted This work was supported by the Natural Science Foundation of Hunan Province (No.01JJY2147). * Corresponding author. Tel: ; Hu50Bi@hotmail.com

2 628 Acta Physiologica Sinica, October 25, 2005, 57(5): oxide was significantly reduced, the contraction response of thoracic aorta rings to PE was enhanced and the relaxation response to ACh was depressed significantly in ovariectomized+fructose fed group (OVX+F). The effect of high fructose was reversed by 17β-E 2. After pretreatment with L-NAME, the effect of 17β-E 2, which enhanced the relaxation response of thoracic aorta rings to ACh in ovariectomized+fructose+17β-e 2 group (OVX+F+E 2 ), was partly blocked. (4) The relaxation response of thoracic aorta rings to SNP had no significant difference among the groups. (5) The contraction response of thoracic aorta rings without endothelium to PE had no significant difference among the groups. These findings suggest that 17β-E 2 may provide protection against the effect of high fructose diet, which causes hypertension, dysfunction of endothelial cells and insulin resistance. The mechanism of this effect of 17β-E 2 could be partly associated with the increase of NO by NOS pathway, or associated with the decrease in the level of systolic blood pressure and serum insulin, and the improvement of insulin resistance. Key words: estradiol; fructose; ovariectomized; insulin resistance; aorta; endothelium (insulin resistance, IR) [1] [2,3] Reaven IR (syndrome of insulin resistance, SIR) [4] IR IR [5] Horton [6] Sprague-Dawley (SD) SIR SD SIR Hulman [7] SIR Galipeau [8] SIR? IR? 17β-E 2 IR IR (HX- ) ECG-6511 KOHDEN HITACH717 γ PIPS-2020 BL420 (estradiol, E 2 ) (nitric oxide, NO) 17β- (17betaestradiol, 17β-E 2 )(L-phenylephrine, PE) ACh(sodium nitroprusside, SNP) N- -L- (N-nitro-L-arginine methyl ester, L- NAME) KCl Sigma NaCl NaHCO 3 D- 1.2 SD (220±20) g 7 (normal control group 20 NC) (sham operation control group 20 SC) (ovariectomized group 20 OVX) (fructose fed group 20 F) + (ovariectomized+fructose fed group 20, OVX+F) + + (ovariectomized + fructose fed+17β-estradiol group 20, OVX+F+E 2 ) + + (ovariectomized+fructose fed+vehicle control group 20, OVX+F+V) 12 h (22±3) 6~ % (30 mg/kg)

3 : 17β- 629 SC 8 OVX 8 OVX+F 8 OVX+F+E β-E 2 1 µg/µl 30 µg/kg ( Horvath [9] [10] ) 8 OVX+F+V 8 F 8 NC 8 Reaven [11,12] 60% 11% 29% 1.4 E 2 NO 8 ( ) (systolic blood pressure, SBP) h 1% (30 mg/kg) 16 E 2 NO 550 nm 0.5 cm NO HITACH717 (fasting blood sugar, FBS); (fasting serum insulin, FSI) [13] (insulin sensitive index, ISI = ln(fbs SI)) ISI 4.88 IR 1.5 [14] ( 1 cm) 10% 7 µm HE / 1.6 [15] (8 / ) 95% O 2 5% CO 2 K-H 3 mm K-H(37 ) 95% O 2 5% CO 2 BL g 1 h 15 min 100 mmol/l KCl 1 µmol/l PE 10 µmol/l ACh 10% (1) ACh PE ( mol/l) K-H ACh mol/l ACh OVX+F+ E 2 L-NAME ( mmol/l) 30 min ACh (2)SNP PE ( mol/l) K-H SNP mol/l SNP(3) PE K- H PE mol/l PE (4) PE (3) K-H 3~5 PE ( mol/l) KCl (100 mol/l) (IC 50 ) Log (PD 2 ) 1.7 mean± SD (ANOVAoneway) q P< β-E 2 SBP E 2 FBS FSI ISI NO 1 SC NC F

4 630 Acta Physiologica Sinica, October 25, 2005, 57(5): SBP E 2 FBS FSI ISI NO OVX SC E 2 (P<0.01) SBP FBS FSI ISI NO SC OVX+F F E 2 NO ISI ( P<0.01) SBP FBS FSI ( P<0.01 P<0.05 P<0.01); OVX +F OVX NO ISI ( P<0.01) SBP FBS FSI ( P<0.01 P<0.05 P<0.01); OVX+F+E 2 OVX+F E 2 NO ISI ( P<0.01) SBP FBS FSI ( P<0.01 P<0.05 P<0.01); OVX+F+V OVX+F β-E β-E 2 ACh 2 NS SC F OVX ACh (PD ± ± ± ±0.18); OVX+F OVX F ACh - PD 2 (3.20±0.14)( P <0.01); OVX+F+E 2 OVX+F ACh- PD 2 (6.21±0.25) (P<0.01); OVX+F+E 2 L-NAME ( mmol/l) ACh - PD β-E 2 SBP FBS FSI ISI NO Table 1. Effects of 17β-E 2 and high fructose diet on SBP, FBS, FSI, ISI and NO of rats Group SBP (mmhg) E 2 (pmol/l) FBS (mmol/l) FSI (mmol/l) ISI ( ln(fbs FSI)) NO (µmol/l) (n=12) (n=12) (n=12) (n=12) (n=12) (n=12) NC ± ± ± ± ± ±5.64 SC ± ± ± ± ± ±10.80 OVX ± ± ± ± ± ±7.90 F ± ± ± ± ± ±4.20 OVX+F ± ± ± ± ± ±1.80 OVX+F+E ± ± ± ± ± ±7.20 OVX+F+V ± ± ± ± ± ±3.80 NC, normal control group; SC, sham operation control group; OVX, ovariectomized group; F, fructose fed group; OVX+F, ovariectomized +fructose fed group; OVX+F+E 2, ovariectomized+fructose fed+17β-estradiol group; OVX+F+V, ovariectomized+fructose fed+vehicle control group. Values are mean±sd. P<0.01 compared with NC; P<0.05 P<0.01 compared with F group; + P<0.05, ++ P<0.01 compared with OVX group; P<0.05 P<0.01 compared with OVX+F group β-E 2 Table 2. Effects of 17β-E 2 and high fructose diet on the structure of thoracic aortas of rats Group Lumen (µm) Media (µm) Media/Lumen (%) Media cross-section (10 3, µm 2 ) (n=12) (n=12) (n=12) (n=12) NC ± ± ± ±80.50 SC ± ± ± ± OVX ± ± ± ±68.42 F ± ± ± ±85.75 OVX+F ± ± ± ±96.72 OVX+F+E ± ± ± ±74.58 OVX+F+V ± ± ± ±62.36 NC, normal control group; SC, sham operation control group; OVX, ovariectomized group; F, fructose fed group; OVX+F, ovariectomized+fructose fed group; OVX+F+E 2, ovariectomized+fructose fed+17β-estradiol group; OVX+F+V, ovariectomized+fructose fed+vehicle control group. Values are mean±sd.

5 : 17β β-E 2 Fig.1. Effects of 17β-E 2 and high fructose diet on the structure of thoracic aorta of rats (HE stain). NC, normal control group; SC, sham operation control group; OVX, ovariectomized group; F, fructose fed group; OVX+F, ovariectomized+fructose fed group; OVX+F+E 2, ovariectomized+fructose fed+17β-estradiol group; OVX+F+V, ovariectomized+fructose fed+vehicle control group. Scabe bar, 100 µm β-E 2 ACh L-NAME OVX+F+E 2 ACh Fig.2. Effects of 17β-E 2 and high fructose diet on relaxation response of thoracic aorta rings to ACh in each group and effect of L-NAME on relaxation response of thoracic aorta rings to ACh in OVX+F+E 2 group. NC, normal control group; SC, sham operation control group; OVX, ovariectomized group; F, fructose fed group; OVX+F, ovariectomized+fructose fed group; OVX+F+E 2, ovariectomized+fructose fed+17β-estradiol group; OVX+F+V, ovariectomized+fructose fed+vehicle control group. Results are expressed as mean±sd. n=8. P<0.01 compared with F group; P<0.05, P<0.01 compared with OVX+F group; P<0.05 compared with OVX+F+E 2 group. (5.06±0.17)(P<0.05); OVX+F+V OVX+F ACh (PD ± ±0.14) β-E 2 SNP 3 SNP (NS SC F OVX OVX+F OVX+F+E 2 OVX+F+V PD ±0.31

6 632 Acta Physiologica Sinica, October 25, 2005, 57(5): β-E 2 SNP Fig.3. Effects of 17β-E 2 and high fructose diet on relaxation response of thoracic aorta rings to SNP in each group. NC, normal control group; SC, sham operation control group; OVX, ovariectomized group; F, fructose fed group; OVX+F, ovariectomized+fructose fed group; OVX+F+E 2, ovariectomized+fructose fed+17β-estradiol group; OVX+F+V, ovariectomized+fructose fed+vehicle control group. Results are expressed as mean±sd. n= β-E 2 PE Fig. 4. Effects of 17β-E 2 and high fructose diet on the contraction response of thoracic aorta rings to PE in each group. NC, normal control group; SC, sham operation control group; OVX, ovariectomized group; F, fructose fed group; OVX+F, ovariectomized+ fructose fed group; OVX+F+E 2, ovariectomized+fructose fed+17β-estradiol group; OVX+F+V, ovariectomized+fructose fed+vehicle control group. Results are expressed as mean±sd. n=8. P<0.05 compared with F group; P<0.05 compared with OVX+F group β-E 2 PE Fig.5. Effects of 17β-E 2 and high fructose diet on the contraction response of thoracic aorta rings (without endothelium) to PE in each group. NC, normal control group; SC, sham operation control group; OVX, ovariectomized group; F, fructose fed group; OVX+F, ovariectomized+fructose fed group; OVX+F+E 2, ovariectomized+fructose fed+17β-estradiol group; OVX+F+V, ovariectomized+fructose fed +vehicle control group. Results are expressed as mean±sd. n= ± ± ± ± ± ±0.22) β-E 2 PE 4 NS SC F OVX PE (PD 2

7 : 17β ± ± ± ±0.23); OVX+F OVX F PE - PD 2 (7.15±0.37)( P < 0.05); OVX+F+E 2 OVX+F PE - PD 2 (6.43±0.22)(P< 0.05); OVX+F+V OVX+F PE (PD ± ±0.37) β-E 2 PE 5 PE (NS SC F OVX OVX+F OVX+F+E 2 OVX+F+V PD ± ± ± ± ± ± ±0.22) 3 IR IR SIR [16,17] IR [7,8] IR IR IR IR IR 17β-E 2 IR 17β-E 2 IR IR IR IR [16] 17β-E 2 ( 8 ) ACh ACh 17β-E 2 (nitric oxide synthase, NOS) L-NAME NO NO NO NO 17β-E 2 L- NAME + +E 2 ACh + 17β-E 2 L-NAME 17β-E 2 NO [10,18-20] NOS mrna NO Tsang [21] 17β-E 2 L mrna Jiang [22] 17β-E 2 17β-E 2 NOS NO 17β-E 2 L NO SNP 17β-E 2 PE α 1 Ca 2+ Ca 2+ Ca 2+

8 634 Acta Physiologica Sinica, October 25, 2005, 57(5): [23] PE 17β-E 2 PE PE PE PE 17β-E 2 NOS NO 17β-E 2 IR NOS NO IR ( ET-1,TXA 2 ) IR Garcia- Arencibia [24] 17β-E 2 U-937 mrna IR 17β-E 2 IR [25] 1 Sowers JR, Frohlich ED. Insulin and insulin resistance: impact on blood pressure and cardiovascular disease. Med Clin North Am 2004; 88(1): Robinson LE, Graham TE. Metabolic syndrome, a cardiovascular disease risk factor: role of adipocytokines and impact of diet and physical activity. Can J Appl Physiol 2004; 29(6): Smith DO, LeRoith D. Insulin resistance syndrome, prediabetes, and the prevention of type 2 diabetes mellitus. Clin Cornerstone 2004; 6(2): Reaven GM. Syndrome X. Blood Press Suppl 1992; 4: Ludwig DS. Diet and development of the insulin resistance syndrome. Asia Pac J Clin Nutr 2003; 12 suppl: S4. 6 Horton TJ, Gayles EC, Prach PA, Koppenhafer TA, Pagliassotti MJ. Female rats do not develop sucrose-induced insulin resistance. Am J Physiol 1997; 272(5 Pt 2): R1571- R Hulman S, Falkner B. The effect of excess dietary sucrose on growth, blood pressure, and metabolism in developing Sprague-Dawley rats. Pediatr Res 1994; 36(1 Pt 1): Galipeau D, Verma S, McNeill JH. Female rats are protected against fructose-induced changes in metabolism and blood pressure. Am J Physiol Heart Circ Physiol 2002; 283(6): H2478-H Horvath KM, Hartig W, Van der Veen R, Keijser JN, Mulder J, Ziegert M, Van der Zee EA, Harkany T, Luiten PG. 17betaestradiol enhances cortical cholinergic innervation and preserves synaptic density following excitotoxic lesions to the rat nucleus basalis magnocellularis. Neuroscience 2002; 110(3): Wang TH ( ), Fu XD, Yang D, Tan Z, Pan JY. Membrance estrogen receptor mediates the rapid nongenomi cactivation of endothelial nitric oxide synthase by estrogen. Acta Physiol Sin () 2003; 55(2): (Chinese, English abstract). 11 Hwang IS, Ho H, Hoffman BB, Reaven GM. Fructose-induced insulin resistance and hypertension in rats. Hypertension 1987; 10(5): Reaven GM, Ho H, Hoffmann BB. Somatostatin inhibition of fructose- induced hypertension. Hypertension 1989; 14(2): Li GW( ), Pan XR. A novel index of detecting insulin sensitivity of population. Chin J Intern Med ( ) 1993; 37 (1): (Chinese, English abstract). 14 Wen JS ( ), Hu b, Long G, Duan ZL, Ling HY, Zhou SH. Effect of rosiglitazone on blood glucose, blood lipid and vascular remodeling in rats fed with high-fructose diet. Chin Pharmacol Bull () 2004; 20(5): (Chinese, English abstract). 15 Kuang W ( ), Chen YY, Shen YL, Xia Q. Effect of iron on vasoconstriction in the isolated rat aorta. Acta Physiol Sin () 2003; 55(3): (Chinese, English abstract). 16 Dall Aglio E, Tosini P, Zavaroni I, Olivetti G, Reaven GM. Comparison of the metabolic changes in rats with hyperten-

9 : 17β- 635 sion secondary to fructose feeding or renal artery stenosis. Am J Hypertens 1995; 8(5 Pt 1): Takiuchi S, Fujii H, Kamide K, Horio T, Nakatani S, Hiuge A, Rakugi H, Ogihara T, Kawano Y. Plasma asymmetric dimethylarginine and coronary and peripheral endothelial dysfunction in hypertensive patients. Am J Hypertens 2004; 17(9): Ogando D, Farina M, Ribeiro ML, Perez Martinez S, Cella M, Rettori V, Franchi A. Steroid hormones augment nitric oxide synthase activity and expression in rat uterus. Reprod Fertil Dev 2003; 15(5): Wang S ( ), Fan ZZ, He RR. 17β-estradiol inhibits carotid sinus baroreflex in male rats. Acta Physiol Sin () 2000; 52(6): (Chinese, English abstract). 20 Wang TH ( ), Yang D, Liu PQ, Gong SZ, LU W, Pan JY. 17β-estradiol induced nitric oxide release in vascular endothelial cells. Acta Physiol Sin () 2000; 52(6): (Chinese, English abstract). 21 Tsang SY, Yao X, Wong CM, Chan FL, Chen ZY, Huang Y. Differential regulation of K + and Ca 2+ channel gene expression by chronic treatment with estrogen and tamoxifen in rat aorta. Eur J Pharmacol 2004; 483(2-3): Jiang C, Poole-Wilson PA, Sarrel PM, Mochizuki S, Collins P, MacLeod KT. Effect of 17beta-oestradiol on contraction, Ca 2+ current and intracellular free Ca 2+ in guinea pig isolated cardiac myocytes. Br J Pharmacol 1992; 106(3): Cao CM ( ), Ye S, Yu H, Xu QS, Ye ZG, Shen YL, Lu Y, Xia Q. Interleukin-2 induced endothelium-dependent relaxation of rat thoracic aorta. Acta physiol Sin () 2003; 55(1): (Chinese, English abstract). 24 Garcia-Arencibia M, Molero S, Davila N, Carranza MC, Calle C. 17beta -Estradiol transcriptionally represses human insulin receptor gene expression causing cellular insulin resistance. Leuk Res 2005; 29(1): Louet JF, LeMay C, Mauvais-Jarvis F. Antidiabetic actions of estrogen: insight from human and genetic mouse models. Curr Atheroscler Rep 2004; 6(3):