ACh (nitric oxide, NO) (1) 17β -E 2

Acta Physiologica Sinica, October 25, 2005, 57 (5): 627-635 http://www.actaps.com.cn 627 17β- * 421001 17β- (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 421001, 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 2005-01-24 Accepted 2005-08-05 This work was supported by the Natural Science Foundation of Hunan Province (No.01JJY2147). * Corresponding author. Tel: +86-734-8282753; E-mail: Hu50Bi@hotmail.com

628 Acta Physiologica Sinica, October 25, 2005, 57(5): 627-635 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 1 1.1 (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 140 3 (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) 6~8 1.3 1 1% (30 mg/kg)

: 17β- 629 SC 8 OVX 8 OVX+F 8 OVX+F+E 2 8 17β-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) 12 12 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 BL420 1.0 g 1 h 15 min 100 mmol/l KCl 1 µmol/l PE 10 µmol/l ACh 10% (1) ACh PE (1 10 6 mol/l) K-H ACh 1 10 9 1 10 8 1 10 7 1 10 6 1 10 5 1 10 4 mol/l ACh OVX+F+ E 2 L-NAME (1 10 5 mmol/l) 30 min ACh (2)SNP PE (1 10 6 mol/l) K-H SNP 1 10 10 1 10 9 1 10 8 1 10 7 1 10 6 1 10 5 mol/l SNP(3) PE K- H PE 1 10 9 1 10 8 1 10 7 1 10 6 1 10 5 1 10 4 mol/l PE (4) PE (3) K-H 3~5 PE (1 10 6 mol/l) KCl (100 mol/l) (IC 50 ) Log (PD 2 ) 1.7 mean± SD (ANOVAoneway) q P<0.05 2 2.1 17β-E 2 SBP E 2 FBS FSI ISI NO 1 SC NC F

630 Acta Physiologica Sinica, October 25, 2005, 57(5): 627-635 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 2.2 17β-E 2 2 1 2.3 17β-E 2 ACh 2 NS SC F OVX ACh (PD 2 6.19±0.27 6.18±0.21 6.21±0.30 6.13±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 (1 10 5 mmol/l) ACh - PD 2 1. 17β-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 118.16±5.42 71.25±16.14 4.72±0.31 15.97±4.21 4.32±0.21 39.54±5.64 SC 121.38±4.27 66.21±12.42 4.86±0.48 16.23±6.34 4.37±0.34 39.96±10.80 OVX 120.35±3.67 14.23±5.36 4.93±0.39 15.34±4.12 4.33±0.26 22.70±7.90 F 120.97±5.18 65.41±14.25 5.04±0.27 18.65±6.34 4.54±0.44 36.50±4.20 OVX+F 164.72±6.54 ++ 15.78±6.85 ++ 7.30±1.25 + 67.01±11.42 ++ 6.19±0.46 ++ ++ 7.40±1.80 OVX+F+E 2 121.31±4.12 69.71±17.35 5.12±0.64 17.48±8.47 4.49±0.35 36.51±7.20 OVX+F+V 166.48±5.04 17.46±6.93 7.32±1.06 68.17±14.69 6.21±0.40 15.10±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. 2. 17β-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 1651.5±91.4 97.6±9.3 5.90±0.45 571.85±80.50 SC 1687.3±114.9 104.7±11.5 6.20±0.66 595.31±105.57 OVX 1635.2±86.4 95.5±9.3 5.84±0.41 555.96±68.42 F 1692.8±97.1 103.5±9.1 6.11±0.50 605.35±85.75 OVX+F 1681.6±103.4 99.2±10.5 5.90±0.61 587.64±96.72 OVX+F+E 2 1630.1±86.5 95.5±10.8 5.86±0.43 558.17±74.58 OVX+F+V 1626.8±84.7 96.9±8.9 5.96±0.40 551.49±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.

: 17β- 631 1. 17β-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. 2. 17β-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 2 3.12±0.16 3.20±0.14) 2.4 17β-E 2 SNP 3 SNP (NS SC F OVX OVX+F OVX+F+E 2 OVX+F+V PD 2 7.20±0.31

632 Acta Physiologica Sinica, October 25, 2005, 57(5): 627-635 3. 17β-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=8. 4. 17β-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. 5. 17β-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=8. 7.20±0.24 7.13±0.19 7.06±0.27 7.03±0.30 7.28±0.28 7.06±0.22) 2.5 17β-E 2 PE 4 NS SC F OVX PE (PD 2

: 17β- 633 6.46±0.27 6.48±0.14 6.46±0.16 6.59±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 2 7.14± 0.28 7.15±0.37) 2.6 17β-E 2 PE 5 PE (NS SC F OVX OVX+F OVX+F+E 2 OVX+F+V PD 2 6.99±0.25 7.00±0.21 7.22±0.33 6.89±0.17 7.13±0.31 7.20±0.23 7.06±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+

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