Genistein and exercise do not improve cardiovascular risk factors in ...

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May 8, 2013 - ABSTRACT. Objective To investigate the effect of either genistein, or exercise, or both, on parameters that are indicators of cardiovascular ...
CLIMACTERIC 2014;17:136–147

Genistein and exercise do not improve cardiovascular risk factors in the ovariectomized rat L. Al-Nakkash*, T. Janjulia*, K. Peterson*, D. Lucy*, D. Wilson*, A. Peterson*, W. Prozialeck† and T. L. Broderick* *Department of Physiology, Midwestern University, Arizona College of Osteopathic Medicine, Glendale, AZ; †Department of Pharmacology, Midwestern University, Chicago College of Osteopathic Medicine, Downers Grove, IL, USA Key words: GENISTEIN, EXERCISE, OVARIECTOMIZED, BLOOD PRESSURE

ABSTRACT Objective To investigate the effect of either genistein, or exercise, or both, on parameters that are indicators of cardiovascular health. Methods We investigated the effect of genistein treatment (300 mg genisten/kg body weight/day), or exercise training, or combined genistein and exercise training, for a period of 6 weeks on physical characteristics, cardiovascular plasma markers, blood pressure, aortic morphology, cardiac structure and oxidative stress in the ovariectomized (OVX) Sprague–Dawley rat. Comparisons were made with intact rats. Results Ovariectomy (compared to intact) resulted in significant decreases in uterine weight (6-fold, p ⬍ 0.0001), insulin levels (4-fold, p ⫽ 0.0214), insulin/glucose ratio (3-fold, p ⫽ 0.0029), and tumor necrosis factor-α plasma levels (2-fold, p ⬍ 0.0001). Similarly, aortic blood pressure was significantly increased (by 8%, p ⬍ 0.0033) in OVX rats, without changes in aortic luminal or wall dimensions. Heart surface area was significantly increased (by 16%, p ⫽ 0.0160) in OVX rats and this was without changes in non-protein thiol levels (a marker of oxidative stress). Physical characteristics were not altered by treatment with genistein, or genistein with exercise, with the exception of increased uterine weight in OVX rats treated under these same conditions. There were no effects of genistein or exercise on indices of blood pressure and aortic morphology in the OVX rat. However, right ventricular nuclei count was reduced in sedentary genistein-treated rats compared to non-treated control OVX rats. Conclusion Our results indicate that administration of genistein at this dose, treadmill running, or the combination of both, are not associated with any improvement in cardiovascular function and structure, and risk factors in an ovariectomy model of postmenopause.

INTRODUCTION The increased incidence of cardiovascular disease in postmenopausal women is related to a decrease in the synthesis of endogenous estrogen1. The protective effects of estrogen replacement therapy (ERT) on cardiovascular function are known2,3. Unfortunately, use of estrogen is restricted, given its carcinogenic effects1,4, thus, the requirement for an estrogen alternative is crucial. Genistein, a naturally occurring phytoestrogen, binds to estrogen receptors, exerts estrogenic effects, and has been proposed as a natural alternative to ERT5,6. Genistein has vasodilatory effects on the cardiovascular system: enhancing

coronary vasoreactivity7, relaxing mesenteric and coronary arteries in vitro8, and relaxing human forearm vasculature in vivo via an nitric oxide-dependent pathway9, and, yet, is without adverse effects on the female reproductive system10,11. We have previously shown that the effects of genistein on cardiovascular function are concentration- and route of administration-dependent. Daily subcutaneous injection with genistein (250 mg genistein/kg body weight, 2 days), is antiischemic and induces a dose-dependent increase in cardiac output in the ovariectomized (OVX) rat heart12. A 2-week treatment produces estrogen-like effects in the OVX rat: decreasing body weight, heart rate and aortic pulse pressure13. On the other hand, we have shown that in

Correspondence: Dr L. Al-Nakkash, Department of Physiology, Midwestern University, 19555 N. 59th Ave, Glendale, AZ 85308, USA

ORIGINAL ARTICLE © 2014 International Menopause Society DOI: 10.3109/13697137.2013.804503

Received 01-03-2013 Revised 16-04-2013 Accepted 08-05-2013

Genistein and exercise in ovariectomized rats non-ovariectomized rodents, a genistein-containing diet for 1 month (600 mg genistein/kg diet) induces beneficial effects on glucose metabolism, cardiovascular risk factors and aortic reactivity14. In postmenopausal women, moderate-intensity exercise training favorably improves metabolic and lipid profiles, maintains bone mass15, and reduces blood pressure and adiposity16. A common metabolic basis for the benefits of both exercise and genistein on cardiovascular function has been proposed. Both activate an oxytocin-mediated system, increasing cGMP and nitric oxide release from aortic vascular endothelial cells17; both prevent postmenopause- and age-related declines in health in rodents18,19, and both maintain bone mass and endothelial function18. However, the cooperative effects of genistein and exercise on common markers of cardiovascular health, cardiovascular structure and function in the OVX rat remain poorly understood. We examined the potential cooperative effects of genistein administration and exercise training on cardiac and vascular structure and dimensions, markers of cardiovascular health and cardiac oxidative stress, and aortic blood pressure. We chose a dose of genistein that closely matched our previous work, albeit for a longer duration (6 weeks). Genistein was administered via subcutaneous pellets, which release a constant daily amount of genistein (300 mg genistein/kg body weight/day). The exercise training regimen was designed to closely mimic the intensity recommended by the American College of Sports Medicine for exercise in the postmenopausal population20.

METHODS Animal model, treatment and exercise training protocol All animals used in this study were cared for in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (National Institute of Health, Publ. No. 85-23, 1986). Ovariectomized (OVX) and intact (INT) Sprague–Dawley female rats weighing 250–300 g (9–16 weeks of age) were purchased from Harlan (Indianapolis, IN, USA). Rats were randomly divided into exercise-trained (EX) and sedentary (SED) groups (Table 1). Ketamine (100 mg/kg)/xylazine (5 mg/kg) anesthetized rats in the SED and EX groups were surgically implanted subcutaneously at the nape of the neck, with either genistein (GEN) constant day-release (300 mg genistein/kg body weight) or vehicle control pellets (VEH). Pellets were purchased from Innovative Research of America (Sarasota, FL, USA). All animals were fed a genistein-free, specially formulated, casein-based diet (Dyets Inc, Bethlehem, PA, USA). Animals were exercised on an Eco-6M treadmill (Columbus Instruments, OH, USA) for 6 weeks, 5 days/week. The running protocol was as follows: week 1 ⫽ 15 m/min for 10 min, week 2 ⫽ 15 m/min for 15 min, week 3 ⫽ 15 m/min for 20 min, week 4 ⫽ 20 m/min for 25 min, and weeks 5–6 ⫽ 25 m/min

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Al-Nakkash et al. Table 1 Group assignments. Female Sprague–Dawley rats were randomized into a total of eight groups (n ⫽ 16–19/subgroup) according to presence/absence of ovaries, 6 weeks of exercise (EX) or sedentary(SED) lifestyle, and 6 weeks of 300 mg genistein/kg body weight/day genistein constant regime pellet supplementation (GEN) or empty control vehicle (VEH) Ovary status

Physical activity

Treatment

Intact (INT)

Sedentary (SED)

Vehicle (VEH) Genistein (GEN) Vehicle (VEH) Genistein (GEN) Vehicle (VEH) Genistein (GEN) Vehicle (VEH) Genistein (GEN)

Exercise (EX) Ovariectomized (OVX)

Sedentary (SED) Exercise (EX)

for 30 min. This protocol was as described previously, and follows the guidelines from the American College of Sports Medicine for moderate exercise in postmenopausal women21. After the 6-week study, rats were euthanized via inhalation with carbon dioxide followed by pneumothorax. Liver, heart, fat pad, uterine, tibia/femur were excised and weighed. Plasma was collected and stored at -80°C until used. In a separate series of animals, whole hearts were removed and frozen whole for morphometric analysis.

Measurement of blood pressure Measures of blood pressure (systolic, diastolic and mean arterial pressure, along with heart rate) were made at the start and end of the study, in restrained unanesthetized rats using a non-invasive pneumonic tail cuff device (NIBP-8, Columbus Instruments, Columbus, OH, USA).

Morphometric measures of thoracic aortae and cardiac tissue Freshly isolated pieces of aorta were embedded and flash frozen in Optimal Cutting Temperature compound (O.C.T., Tissue-Tek, Torrance, CA, USA). Frozen sliced sections of aorta (8–10 μm) were stained with a standard hematoxylin and eosin (H & E) protocol prior to performing the morphometric analyses to evaluate wall thickness. Briefly, sections were treated with hematoxylin, then rinsed with water rinse, Scott’s Solution, 95% and 200% ethanol, eosin, followed by histoclear (National Diagnostics, Atlanta, GA, USA). Images were taken at 20 ⫻ magnification with an Olympus Vanox-AHBS3 light microscope (Center Valley, PA, USA). Measures from the H & E-stained aortic ring sections were taken using Image J (NIH) of the inner luminal area and outer serosal area. From those measurements, the inner luminal diameter and outer serosal diameter were calculated and wall thickness determined as the difference

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Genistein and exercise in ovariectomized rats between the two. Averages of measurements were taken from six to eight separate sections of aorta per animal, and then averaged. For heart morphometric analyses, whole hearts were embedded in O.C.T. and subjected to the same protocol above. Whole sections of cardiac tissue were taken at 10–12-μM thickness, and measures made according to methods previously published22. Measures were taken of: whole heart surface area, intraventricular septum thickness, left ventricular surface area, right ventricular surface area, right ventricular nuclei count, left ventricular nuclei count, left ventricular cell area, right ventricular cell area. Cell area assessments were made with 100 ⫻ objective (an average was taken of five measures per heart), whereas nuclei counts were made using 40 ⫻ objective (duplicates were averaged per heart).

Determination of non-protein thiols Pulverized samples of frozen heart tissue were homogenized in 10 volumes of cold phosphate buffer (pH 6.8). For the determination of non-protein thiols, a portion of the homogenate was deproteinized by adding trichloroacetic acid to a final concentration of 5%, incubating at 0°C for 1 h, and centrifuging at 3000 ⫻ g for 10 min at 4°C. Aliquots (200 μl) of the deproteinized samples were drawn off, neutralized to pH 7.0 by the addition of 100 μl of 2 mol/l Tris base and then reacted with 2.5 ml of 5.5′dithio-bis (2-nitrobenzoic acid, 1.18 mg/ml) according to the method of Ellman23. Reduced glutathione (GSH) was used as a standard.

Measurement of plasma metabolites Cardiovascular markers were measured from plasma in postprandial rats using commercially available assay kits for glucose and non-esterified fatty acid (NEFA) (Wako Diagnostics, Richmond, VA, USA), insulin (Millipore rat/mouse ELISA, Temecula, CA, USA), cholesterol and triglycerides (Cayman Chemical, Ann Arbor, MI, USA), tumor necrosis factor (TNF)-α and interleukin (IL)-6 (Thermo Fisher Scientific, Rockford, IL, USA) and total C-reactive protein (BD Biosciences, San Jose, CA, USA).

Statistical analysis Statistical analysis was performed using a one-way analysis of variance (ANOVA) with Neuman–Keuls post test, or paired t-tests depending on the parameter. Two-tailed unpaired t-tests were performed on comparisons between individual groups in the intact with the OVX counterpart. Analyses were performed using Graphpad (Graphpad software, Inc.). Data are presented as mean ⫹ ⫺ standard error of the mean. Significance is shown as p ⬍ 0.05.

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RESULTS Effects on physical characteristics The effects of genistein treatment and exercise training, or both combined, on physical characteristics in OVX and intact rats are shown in Table 2. Confirming ovariectomy and loss of endogenous estrogen production, all ovariectomized rats exhibited reduced uterine weight when compared to intact rats. In the OVX group, however, genistein treatment significantly increased uterine weight in both sedentary (p ⬍ 0.0001) and exercise-trained groups (p ⫽ 0.0026) compared with their respective non-treated groups, but this increase remained lower compared with the uterine weight of intact groups. Body weight was significantly increased in the OVX VEH-SED group compared to intact counterparts (p ⫽ 0.0025). Similar results were obtained for heart weight/ body weight ratio (HW/BW, p ⫽ 0.0003). For all other parameters measured, there were no effects of either genistein or exercise, or the combination of genistein treatment and exercise training in OVX or intact rats.

Effects on plasma markers of cardiovascular health Measurement of plasma glucose levels showed no changes after the 6-week period of genistein treatment or exercise training, as illustrated in Figure 1. Plasma insulin was significantly decreased with genistein treatment (p ⫽ 0.0258) and exercise training (p ⫽ 0.0439) in intact rats, but unaffected by both genistein and exercise in combination. All groups of OVX rats had significantly lower insulin levels when compared to intact control rats (p ⫽ 0.0214) and there was no effect of genistein, exercise training, or of both combined. The insulin/glucose ratio reflected the changes observed with insulin. Figure 2 illustrates the effects of genistein and exercise training on common cardiovascular risk markers. There were no measurable changes in serum levels of cholesterol, NEFA, triglycerides or IL-6, with either genistein or exercise, or both, in intact and OVX rats. However, changes in levels of inflammatory markers were observed. Plasma levels of TNF-α were significantly increased in the intact SED rat compared to those following exercise training (p ⫽ 0.0192). In the OVX rat, plasma levels of this marker were significantly lower compared with their respective intact groups (i.e. OVX VEH-SED versus intact VEH-SED, p ⬍ 0.001). Plasma levels of C-reactive protein were also increased significantly by exercise in intact rats compared to their sedentary controls (p ⫽ 0.0027). Interestingly, genistein treatment reversed this exercise-mediated increase in C-reactive protein in the intact rat. Ovariectomy, genistein treatment and exercise did not induce any changes in the plasma levels of C-reactive protein in the OVX rat. Plasma levels of IL-6 were not altered under any of the experimental conditions.

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Climacteric 273 ⫹ ⫺ 16 306 ⫹ ⫺ 15 0.57 ⫹ ⫺ 0.06 0.96 ⫹ ⫺ 0.04 3.2 ⫹ ⫺ 0.01 9.22 ⫹ ⫺ 0.63 0.029 ⫹ ⫺ 0.001 0.80 ⫹ ⫺ 0.03 3.66 ⫹ ⫺ 0.06 0.60 ⫹ ⫺ 0.02 4.08 ⫹ ⫺ 0.06 0.235 ⫹ ⫺ 0.007 15.3 ⫹ ⫺ 2.7

258 ⫹ ⫺ 11 286 ⫹ ⫺ 12 0.65 ⫹ ⫺ 0.05 0.90 ⫹ ⫺ 0.03 3.2 ⫹ ⫺ 0.01 7.94 ⫹ ⫺ 0.44 0.028 ⫹ ⫺ 0.001 0.79 ⫹ ⫺ 0.02 3.60 ⫹ ⫺ 0.05 0.61 ⫹ ⫺ 0.02 3.98 ⫹ ⫺ 0.04 0.278 ⫹ ⫺ 0.006 14.7 ⫹ ⫺ 1.4

Start body weight, BW Final body weight Uterine weight Heart weight, HW HW/BW (⫻ 10⫺3) Liver weight, LW LW/BW Femur weight Femur length (cm) Tibia weight Tibia length (cm) HW/tibia length Fat pad weight

257 ⫹ ⫺ 15 300 ⫹ ⫺ 19 0.56 ⫹ ⫺ 0.04 0.89 ⫹ ⫺ 0.03 3.1 ⫹ ⫺ 0.01 8.88 ⫹ ⫺ 0.65 0.029 ⫹ ⫺ 0.001 0.74 ⫹ ⫺ 0.03 3.58 ⫹ ⫺ 0.05 0.56 ⫹ ⫺ 0.02 3.95 ⫹ ⫺ 0.05 0.226 ⫹ ⫺ 0.005 19.9 ⫹ ⫺ 3.4

SED

GEN

239 ⫹ ⫺ 12 278 ⫹ ⫺ 12 0.64 ⫹ ⫺ 0.04 0.90 ⫹ ⫺ 0.03 3.3 ⫹ ⫺ 0.01 8.49 ⫹ ⫺ 0.55 0.030 ⫹ ⫺ 0.001 0.82 ⫹ ⫺ 0.03 3.54 ⫹ ⫺ 0.05 0.58 ⫹ ⫺ 0.03 3.97 ⫹ ⫺ 0.05 0.225 ⫹ ⫺ 0.005 12.4 ⫹ ⫺ 2.0

EX 305 ⫹ ⫺ 15 † 342 ⫹ ⫺ 13 † 0.11 ⫹ ⫺ 0.01 0.93 ⫹ ⫺ 0.02 † 2.8 ⫹ ⫺ 0.01 8.91 ⫹ 0.42 ⫺ 0.026 ⫹ ⫺ 0.001 0.75 ⫹ ⫺ 0.02 3.65 ⫹ ⫺ 0.06 0.56 ⫹ ⫺ 0.02 4.02 ⫹ ⫺ 0.06 0.232 ⫹ ⫺ 0.006 18.3 ⫹ ⫺ 1.9

SED

VEH

VEH, vehicle-treated; GEN, genistein-treated; EX, exercised trained; SED, sedentary *, Significantly different from VEH-SED within that group, p ⬍ 0.05; †, significantly different from VEH-SED intacts, p ⬍ 0.05

EX

SED

Physical characteristic

VEH

Intact

299 ⫹ ⫺ 13 337 ⫹ ⫺ 10 0.13 ⫹ ⫺ 0.01 1.01 ⫹ ⫺ 0.02 3.0 ⫹ ⫺ 0.01 8.69 ⫹ ⫺ 0.32 0.026 ⫹ 0.001 ⫺ 0.84 ⫹ ⫺ 0.03 3.70 ⫹ 0.05 ⫺ 0.63 ⫹ ⫺ 0.02 4.18 ⫹ 0.05 ⫺ 0.242 ⫹ ⫺ 0.005 16.1 ⫹ 2.2 ⫺

EX

290 ⫹ ⫺ 13 349 ⫹ ⫺ 13 * 0.18 ⫹ ⫺ 0.01 0.99 ⫹ ⫺ 0.03 2.9 ⫹ ⫺ 0.01 8.91 ⫹ ⫺ 0.37 0.026 ⫹ ⫺ 0.001 0.75 ⫹ ⫺ 0.04 3.63 ⫹ ⫺ 0.03 0.57 ⫹ ⫺ 0.02 4.02 ⫹ ⫺ 0.04 0.246 ⫹ ⫺ 0.006 18.2 ⫹ ⫺ 2.0

SED

Ovariectomized GEN

273 ⫹ ⫺ 12 319 ⫹ ⫺ 10 * 0.15 ⫹ ⫺ 0.01 0.95 ⫹ ⫺ 0.02 3.0 ⫹ ⫺ 0.01 8.54 ⫹ ⫺ 0.44 0.027 ⫹ ⫺ 0.001 0.82 ⫹ ⫺ 0.03 3.65 ⫹ ⫺ 0.06 0.60 ⫹ ⫺ 0.03 4.06 ⫹ ⫺ 0.07 0.233 ⫹ ⫺ 0.004 13.8 ⫹ ⫺ 1.6

EX

Table 2 Effect of genistein supplementation and exercise on physical characteristics in female intact and ovariectomized rats. Data are given as mean ⫹ ⫺ standard error of the mean. All weights are expressed in grams. n ⫽ 16–19/group

Genistein and exercise in ovariectomized rats Al-Nakkash et al.

139

Genistein and exercise in ovariectomized rats

Al-Nakkash et al. Intact

(a)

OVX

Glucose (ng/dl)

0.15

0.1

0.05

0

(b)

1.75

Insulin (ng/ml)

1.5 1.25 1

*

0.75

* *

0.5

*

*

*

0.25 0

(c) Insulin/Glucose

20 15 10

*

*

GenSed

VehEx

*

5

*

* *

0 VehSed

GenEx

VehSed

GenSed

VehEx

GenEx

Figure 1 Effect of genistein treatment, exercise or both, on plasma glucose and insulin levels in intact and ovariectomized (OVX) rats. (a) Glucose (ng/dl), n ⫽ 9 –10; (b) insulin (ng/l), n ⫽ 7–9; (c) insulin/glucose ratio, n ⫽ 7–9. The vehicle-sedentary group (Veh-Sed) is represented by open bars, the genistein-treated group (Gen-Sed) by solid bars, the exercise-trained group (Veh-Ex) by dotted bars, and the genistein-treated with * exercise-training group (Gen-Ex) by hashed bars. Values are reported as mean ⫹ ⫺ SEM. , Significantly different from intact Veh-Sed, p ⬍ 0.05

Effects on blood pressure The effects of genistein treatment and exercise on blood pressure are shown in Table 3. There was no effect of either genistein or exercise, or both on systolic pressure, diastolic pressure, mean arterial pressure, heart rate and calculated cardiac work in intact rats. Similarly, there was no effect of genistein or exercise, or both these parameters on OVX rats. However, ovariectomy alone resulted in significant increases in systolic pressure (p ⫽ 0.0049), diastolic pressure (p ⫽ 0.0090), and mean arterial pressure (p ⫽ 0.0033) compared to their intact counterparts.

Effects on aortic morphology Table 4 shows the effects of genistein and exercise on basic aortic morphology, expressed as serosal diameter, aortic

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luminal diameter, or aortic wall thickness. Consistent with our recent work in the intact mouse14, there was no effect of genistein treatment on aortic morphology. Neither genistein nor exercise training nor a combination of both had an effect on aortic serosal diameter, aortic luminal diameter, or aortic wall thickness in OVX or intact rats.

Effects on cardiac dimensions Basic cardiac structural parameters were measured in intact and OVX rats following the genistein treatment, exercise training, or both. As shown in Table 5, left ventricle luminal area was increased two-fold (p ⫽ 0.0436) in intact rats following exercise training, compared to their sedentary control counterparts. Moreover, in these hearts following exercise training, a concomitant significant increase in whole heart surface area was observed compared to their sedentary control counterparts

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Genistein and exercise in ovariectomized rats

Al-Nakkash et al. Intact

Cholesterol (mg/dl)

(a)

NEFA (mg/dl)

(b)

25 20 15 10 5 0 12 10 8 6 4 2 0 60 50 40 30 20 10 0

(d)

10

TNFα (mg/dl)

Triglycerides (mg/dl)

(c)

OVX

#

8 6

#

4



#¶ #¶

2 0

1500000

(f)

200 180 160 140 120 100 80 60 40 20 0

IL-6 (pg/ml)

Total CRP (ng/ml)

(e) ¶ 1000000

500000

vehsed

gensed

vehex

genex

vehsed

gensed

vehex

genex

Figure 2 Effect of genistein treatment, exercise or both, on plasma cholesterol, non-esterified fatty acid (NEFA), triglyceride, tumor necrosis factor alpha (TNFα), total C-reactive protein (CRP) and interleukin-6 (IL-6) levels in intact and ovariectomized (OVX) rats. (a) Cholesterol (mg/dl), n ⫽ 8 –10; (b) NEFA (mg/l), n ⫽ 7–10; (c) triglyceride (mg/dl); n ⫽ 9 –10; (d) TNFα (mg/dl);, n ⫽ 10; (e) total CRP (ng/ml); n ⫽ 8 –10; (f) IL-6 (pg/ml); n ⫽ 10. The vehicle-sedentary group (Veh-Sed) is represented by open bars, the genistein-treated group (Gen-Sed) by solid bars, the exercise-trained group (Veh-Ex) by dotted bars, and the genistein-treated with exercise-training group (Gen-Ex) by hashed bars. Values are #, Significantly different from intact Veh-Sed, p ⬍ 0.05; ¶, significantly different from intact equivalent, p ⬍ 0.05 reported as mean ⫹ ⫺ SEM.

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Table 3 The effects of genistein and exercise on blood pressure, heart rate, pulse pressure and cardiac work of intact and ovariectomized rats. Data are given as mean ⫹ ⫺ standard error of the mean. All weights are in grams. n ⫽ 14–18/group Intact

Ovariectomized

VEH Parameter Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Mean arterial pressure (mmHg) Pulse pressure (mmHg) Cardiac work (mmHg*beats/min) Heart rate (beats/min)

GEN

VEH

GEN

SED

EX

SED

EX

SED

EX

SED

EX

110 ⫹ ⫺2 79 ⫹ ⫺2 89 ⫹ ⫺2 31 ⫹ ⫺2 14 ⫹ ⫺1 443 ⫹ ⫺8

115 ⫹ ⫺ 2.0 83 ⫹ ⫺2 93 ⫹ ⫺2 33 ⫹ ⫺1 14 ⫹ ⫺1 440 ⫹ ⫺7

110 ⫹ ⫺3 81 ⫹ ⫺2 91 ⫹ ⫺2 30 ⫹ ⫺1 14 ⫹ ⫺1 454 ⫹ ⫺ 12

116 ⫹ ⫺2 85 ⫹ ⫺2 95 ⫹ ⫺2 31 ⫹ ⫺1 13 ⫹ ⫺1 438 ⫹ ⫺ 10

* 119 ⫹ ⫺3 * 87 ⫹ ⫺2 * 97 ⫹ ⫺2 33 ⫹ 2 ⫺ 1 15 ⫹ ⫺ 448 ⫹ ⫺5

121 ⫹ ⫺4 87 ⫹ ⫺3 98 ⫹ ⫺3 33 ⫹ ⫺2 15 ⫹ ⫺1 442 ⫹ ⫺5

118 ⫹ ⫺2 84 ⫹ ⫺2 95 ⫹ ⫺2 33 ⫹ ⫺1 15 ⫹ ⫺1 455 ⫹ ⫺8

118 ⫹ ⫺2 85 ⫹ ⫺2 96 ⫹ ⫺2 33 ⫹ ⫺1 15 ⫹ ⫺1 443 ⫹ ⫺7

VEH, vehicle-treated; GEN, genistein-treated; EX, exercised trained; SED, sedentary *, Denotes significantly different from VEH–SED intacts, p ⬍ 0.05

(p ⫽ 0.0198). No changes in cardiac morphology were seen in intact genistein-treated rats after exercise training. Hearts from sedentary OVX rats had a significantly greater whole heart surface area, compared to their intact counterparts (p ⫽ 0.0160). All other cardiac dimensions including right ventricular luminal area, left and right ventricular cell areas, and intraventricular septal thickness were not altered with genistein treatment, exercise training, or both interventions, as well as following ovariectomy. However, the nuclei count of the right ventricle was significantly decreased in OVX sedentary rats treated with genistein, compared to OVX sedentary control rats (p ⫽ 0.0250).

Effects on cardiac oxidative status Table 6 shows that there was no differences between intact and OVX hearts in non-protein thiol content. There were also no differences in cardiac non-protein thiol content following genistein treatment, exercise training, or with genistein in combination with exercise training.

similarities to estradiol and thus a potential use as a cardioprotective estrogen-mimic. In fact, genistein (100 μg/kg) has been shown to exert pharmacological post-conditioning with a similar potency to 17β-estradiol via an estrogen receptor, PI3K/Akt-mediated pathway25. In addition, regular exercise training of moderate intensity is well known to favorably improve several metabolic and cardiovascular outcomes in postmenopausal women15. With evidence indicating cooperative effects of genistein administration and exercise training on common markers of cardiovascular health, in this study we aimed to determine the effects of genistein treatment (300 mg/kg/body weight, 300G) in the presence of exercise training on physical parameters, blood pressure, aortic and cardiac structure, markers of cardiac stress, and serum markers of cardiovascular health in the ovariectomized rat model of menopause. We predicted that genistein treatment in combination with exercise training for a period of 6 weeks would have beneficial effects on several plasma and physiological markers of cardiovascular health in the OVX rat.

Effects on physical characteristics DISCUSSION Diets rich in soy have been associated with a reduction in the risk of cardiovascular disease24. Genistein has structural

Our data indicate that genistein had no effect on heart weight in OVX rats, whereas exercise training significantly increased heart weight in OVX rats (p ⫽ 0.0078). Our data are in accord with the findings of Gutkowska and colleagues26, demonstrating

Table 4 Effect of genistein supplementation and exercise on aortic dimensions in female intact and ovariectomized rats. Data are given as mean ⫹ ⫺ SEM for 8–12 rats/group Intact

Ovariectomized

VEH

Serosal diameter (μm) Luminal diameter (μm) Wall thickness (μm)

GEN

VEH

GEN

SED

EX

SED

EX

SED

EX

SED

EX

828 ⫹ ⫺ 17 748 ⫹ ⫺ 16 80 ⫹ ⫺3

839 ⫹ ⫺ 13 755 ⫹ ⫺ 12 83 ⫹ ⫺3

810 ⫹ ⫺ 18 727 ⫹ ⫺ 17 83 ⫹ ⫺2

846 ⫹ ⫺ 24 766 ⫹ ⫺ 21 80 ⫹ ⫺4

855 ⫹ ⫺ 21 20 774 ⫹ ⫺ 82 ⫹ 3 ⫺

874 ⫹ ⫺ 16 791 ⫹ ⫺ 16 83 ⫹ ⫺2

874 ⫹ ⫺ 17 791 ⫹ ⫺ 15 83 ⫹ ⫺3

845 ⫹ ⫺ 23 759 ⫹ ⫺ 24 83 ⫹ ⫺3

VEH, vehicle-treated; GEN, genistein-treated; EX, exercised trained; SED, sedentary

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487 ⫹ ⫺ 125 3 127 ⫹ ⫺ 111 ⫹ 9 ⫺ 138 ⫹ ⫺ 14 160 ⫹ ⫺ 20 98.8 ⫹ ⫺ 4.2 21 385 ⫹ ⫺ 943 1555 ⫹ ⫺ 72 0.9 4.9 ⫹ ⫺ 716 ⫹ ⫺ 229 110 ⫹ ⫺ 10 126 ⫹ ⫺ 19 154 ⫹ ⫺ 15 * 137 ⫹ ⫺ 13 97.4 ⫹ ⫺ 3.3 20 658 ⫹ ⫺ 824 1409 ⫹ ⫺ 44 5.1 ⫹ ⫺ 0.7 608 ⫹ ⫺ 138 137 ⫹ ⫺ 13 113 ⫹ ⫺8 215 ⫹ ⫺ 41 174 ⫹ ⫺8 89.1 ⫹ ⫺ 3.1 † 20 602 ⫹ ⫺ 653 1477 ⫹ ⫺ 59 4.2 ⫹ ⫺ 0.9 597 ⫹ ⫺ 161 127 ⫹ ⫺9 123 ⫹ ⫺ 18 173 ⫹ ⫺ 56 159 ⫹ ⫺8 96.7 ⫹ ⫺ 19.8 17 006 ⫹ ⫺ 2265 1369 ⫹ ⫺ 70 3.8 ⫹ ⫺ 0.6 521 ⫹ ⫺ 109 131 ⫹ ⫺ 14 103 ⫹ ⫺8 253 ⫹ ⫺ 80 165 ⫹ ⫺8 100.0 ⫹ ⫺ 2.5 19 524 ⫹ ⫺ 819 1340 ⫹ ⫺7 4.4 ⫹ ⫺ 0.7 * 808 ⫹ ⫺ 123 148 ⫹ 17 ⫺ 110 ⫹ ⫺ 10 260 ⫹ ⫺ 75 170 ⫹ ⫺5 94.7 ⫹ ⫺ 5.7 * 20 433 ⫹ ⫺ 638 1446 ⫹ ⫺ 68 4.0 ⫹ ⫺ 0.6 385 ⫹ ⫺ 156 126 ⫹ ⫺9 98 ⫹ ⫺ 14 191 ⫹ ⫺ 64 159 ⫹ ⫺6 94.3 ⫹ ⫺ 10.5 17 789 ⫹ ⫺ 785 1327 ⫹ ⫺ 42 5.5 ⫹ ⫺ 1.5 LV luminal area (mm2) LV nuclei count LV cell area (μm2) RV luminal area (mm2) RV nuclei count RV cell area (μm2) Whole heart surface area (mm2) IV septum area (mm2) % area fibrosis

VEH

Al-Nakkash et al.

VEH, vehicle-treated; GEN, genistein-treated; EX, exercised trained; SED, sedentary; LV, left ventricle; RV, right ventricle; IV, interventricular *, Significantly different from VEH-SED within that group, p ⬍ 0.05; †, significantly different from VEH-SED intact, p ⬍ 0.05

EX SED EX SED

GEN

EX

SED

VEH

614 ⫹ ⫺ 127 123 ⫹ ⫺9 118 ⫹ ⫺ 11 277 ⫹ ⫺ 96 146 ⫹ ⫺ 13 107.7 ⫹ ⫺ 10.9 20 586 ⫹ ⫺ 596 1490 ⫹ ⫺ 74 3.9 ⫹ ⫺ 0.9

SED

GEN Ovariectomized Intact

Table 5

Effect of genistein supplementation and exercise on cardiac structure in female intact and ovariectomized rats. Data are given as mean ⫹ ⫺ SEM for five rats per group

EX

Genistein and exercise in ovariectomized rats

an oxytocin receptor- and ANP-dependent increase in left ventricular mass in exercise-trained OVX rats. We postulate that the exercise-induced increase in heart weight in our study might be due to an increased left ventricular mass, associated with the increased work of the heart. Genistein significantly increased uterine weight in OVX rats (p ⫽ 0.0026), suggesting that genistein mimics estrogen in the OVX uterine tissue. This is consistent with the study of Nevala and colleagues27, whose evidence suggests that both dose and duration of genistein administration are important in determinations of change in uterine weight: a high dose of 25 mg genistein/kg body weight for 2 days increased uterine weight, whereas a lower dose of 2.5 mg genistein/kg body weight for 2 days had no effect on uterine weight. However, when the study duration was extended from 2 days to 2 weeks, both were effective in increasing uterine weight27. Whilst our data contradict other published work demonstrating a lack of effect of 0.4 mg or 1 μg genistein/day on uterine weight in OVX mice17,18, we hypothesize that those negative effects are likely due the low doses of genistein used. After menopause, estrogen deficiency leads to visceral obesity, which in turn leads to insulin sensitivity28. Similar to postmenopausal women, OVX rats have been shown to have measureable increases in adipose tissue29. In the current study, body weight and fat pad weight were measured in order to determine effects of genistein (and exercise) on these physiological parameters. We show that genistein treatment had no effect on body weight and fat pad weight in either OVX or intact rats, and this is likely due to its relatively low dose. In contrast, OVX mice fed genistein (1500 mg/kg) for 3 weeks had significantly decreased body and fat pad weights30, highlighting its previously reported lipolytic effect30. Thus, when comparing effects of genistein on body weight and fat pad weight, it is important to note the dose of genistein utilized, the route of administration (subcutaneous injection versus oral), duration of exposure to genistein and, lastly, the experimental model used (rats versus mice). Osteoporosis is a common health problem in older women; one-third of all women will likely experience a hip fracture by age 90 years31. Bones from postmenopausal women have a lower bone density compared to those of younger rats, indicating greater porosity with age32. Exercise training has been thought to increase bone formation by increasing bone mass and strength33. In fact, 10 weeks of exercise training in male rats increased femur weight compared to that of untrained rats34 and this is indicative of a beneficial effect of exercise training on bone metabolism. In accordance with that, a higher bone mass density has been confirmed after 4 weeks of exercise training compared to controls18. Furthermore, administration of a low dose of genistein (0.4 mg/day) has been shown to increase bone mineral density as compared to controls18, and combined intervention of genistein and exercise has been shown to prevent bone loss in OVX mice18. In contrast, our results show that tibia and femur lengths (and weights) remained unchanged after genistein treatment or exercise. This lack of effect of genistein on simple bone parameters could be due to differences in dose of genistein, different

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Genistein and exercise in ovariectomized rats

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Table 6 Effect of genistein supplementation and exercise on non-protein thiols (nmoles/g tissue) in female intact and ovariectomized rats. Data are given as mean ⫹ ⫺ SEM for 5–9 hearts per group Intact

Ovariectomized

VEH

Non-protein thiol

GEN

VEH

GEN

SED

EX

SED

EX

SED

EX

SED

EX

1515 ⫹ ⫺ 91

1492 ⫹ ⫺ 79

1485 ⫹ ⫺ 33

1572 ⫹ ⫺ 74

1432 ⫹ ⫺ 64

1399 ⫹ ⫺ 57

1487 ⫹ ⫺ 47

1390 ⫹ ⫺ 53

VEH, vehicle-treated; GEN, genistein-treated; EX, exercised trained; SED, sedentary

routes of administration (implantation vs. subcutaneous injection), or different species of animals (rats vs. mice).

Effects on aortic morphology and blood pressure Our results indicate that administration of 300G for 6 weeks had no effect on blood pressure measurements. This is consistent with previously published evidence showing a lack of effect of genistein on blood pressure measures; 0.2 mg genistein/kg body weight/day for 4 weeks had no effect on mean arterial pressure and heart rate in OVX rats35 and following the consumption of a 1-month genistein-containing diet (600 mg/kg food) in both male and female intact mice14. Conversely, dietary supplementation of genistein (2.0 g/kg) for 6 weeks has been shown to lower both systolic and diastolic blood pressures (without changing heart rate) in spontaneously hypertensive rats, effects proposed to be due to genisteinmediated increases in eNOS protein expression and decreased aortic wall thickness36. Interestingly, OVX animals have been shown to have a significantly increased mean arterial pressure as compared to sham-operated animals37. Indeed, our data also indicated significant increases in systolic blood pressure (p ⫽ 0.0049), diastolic blood pressure (p ⫽ 0.0090) and mean arterial pressure (p ⫽ 0.0033) in the OVX-SED group compared to their intact counterparts. Differences in our findings with some previously published studies could be explained by the following differences: genistein dose (300 mg/kg vs. 0.4 mg/kg), duration of the study (6 weeks vs. 3 weeks), and route of administration (genistein implantation vs. subcutaneous genistein injection). Differences in blood pressure measurements may also be explained by a differential effect of genistein in models of hypertension. We measured luminal and serosal aortic diameters in order to determine whether potential morphological changes may have associations with blood pressure parameters. Wall thickness was assessed because a thicker aortic wall is a risk factor for increased blood pressure38. Wall thickness remained unchanged after treating OVX and intact animals with either genistein, exercise, or both, which was consistent with our blood pressure data. Interestingly, the significant increases in systolic blood pressure, diastolic blood pressure and mean arterial pressure in the OVX controls compared to their intact counterparts were not associated with concomitant change in aortic wall thickness, indicating that other parameters/mechanism(s) are likely to be involved.

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In contrast, other studies have reported beneficial effects of exercise (60 min/day, 1.2 km/h, 8 weeks) in OVX rats, reducing arterial pressure and heart rate, increasing baroreflex sensitivity and reducing oxidative stress39. Our data, indicating a lack of effect of exercise training on systolic blood pressure, diastolic blood pressure and mean arterial pressure in both OVX and intact rats, could be explained in part by probable increased stress levels associated with forced treadmill running, as well as restraining the animals during blood pressure measurements. It is feasible that increased circulating cortisol levels, probably released due to the stress incurred during forced treadmill running, could have obstructed the potential beneficial effects of either genistein or exercise.

Effects on selected plasma metabolites Genistein has been shown to increase insulin sensitivity in postmenopausal women when administered at 54 mg/day for 24 weeks40 or when administered at 1 mg/kg body weight for 44 days in male Wistar rats41. Those findings are consistent with our previously published data demonstrating that dietary genistein (600 mg/kg food) for 1 month significantly increased (by 63%) serum insulin levels in intact male mice14. Several mechanisms have been postulated to explain genistein’s stimulatory action on insulin secretion, including inhibition of tyrosine kinase activity42 and activation of the cAMP/PKA ERK1/2 and PLC/PKC pathways43. Adipose tissue synthesizes and releases multiple cytokines that mediate several biological processes. Indeed, it has been reported that TNF-α is secreted from adipocytes and that a rise in TNF-α secretion increases hormone-sensitive lipase activity in adipocytes44. Further, recent work has demonstrated a strong association between exercise training and TNF-α secretion, with a significant increase in both TNF-α protein content and its mRNA expression in adipose tissue occurring after training45. This is expected since one hallmark of chronic endurance exercise training is enhanced lipolysis46. Our results showing that TNF-α secretion was elevated in the intact exercise-trained female rat compared to the respective sedentary rat (p ⫽ 0.0192) are consistent with the effects of endurance training45, although a recent report demonstrated no change in TNF-α secretion in male Wistar rats following daily swim training for a period of 8 weeks47. However, a significant increase in TNF-α secretion

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Genistein and exercise in ovariectomized rats was not detected in the intact genistein-treated group after exercise training. In this group, an increase in the secretion of this cytokine was expected considering that both genistein and estrogen stimulate lipolysis30. An interesting observation, on the other hand, is the dramatic reduction in TNF-α secretion in all ovariectomized rats, regardless of training and genistein treatment (i.e. intact VEH-SED versus OVX VEH-SED, p ⬍ 0.0001) and despite no change in fat pad content. This finding is in sharp contrast with the protective role of estrogen, in which a lack of ovarian function in the rat increases the inflammatory profile, including C-reactive protein, increasing the risk of cardiovascular and neuroinflammatory disorders48. While we cannot offer an explanation for this seemingly disparate effect of ovariectomy on TNF-α compared to previously published studies, we consider the lack of the effect of ovariectomy on fat pad weight to be a pivotal factor.

Effects on cardiac morphology and thiols Our data indicate that a 6-week moderate exercise regimen resulted in significant increase in left ventricular luminal area (p ⫽ 0.0436), with a concomitant increase in whole heart surface area (p ⫽ 0.0198) in intact vehicle control rats, but not in intact genistein-treated rats or any of the OVX rats. Interestingly, whole heart surface area was significantly greater in the OVX VEH-SED group compared to their intact counterparts (p ⫽ 0.0160). Our data suggest then that OVX VEH-SED rat hearts have a different baseline compared to their counterpart intact rats, and this dissimilarity could potentially play a role in the lack of effect of exercise on the OVX group. Previous evidence suggests that ovariectomy results in adverse effects on cardiac remodeling (cardiomyocyte hypertrophy, myocardial reparative interstitial fibrosis and vascularization impairment with loss of cardiomyocytes) in spontaneously hypertensive rats and that exercise training diminished these effects to some extent49. It is likely that the type of exercise training regimen is itself a determinant of the potential benefits of exercise to reverse or remodel OVX cardiac tissue, i.e. Marques and colleagues used a 13-week 60-min/day for 5 days/week exercise schedule49, and Gutkowska and colleagues utilized an 8-week, 15–60-min/day 5 days/week schedule26. Interestingly, Paquette and colleagues have shown that estrogen receptor gene expression in intact and OVX rat cardiac tissue in response to exercise training is divergent50. Thus, the effects of either exercise or ovariectomy on cardiac remodeling are

Al-Nakkash et al. interesting and relatively inconsistent and likely reflect variances in both the duration and vigor of the exercise training method and differences in the basal condition of intact and OVX models. Several previous studies suggest that genistein improves the antioxidant status in tissues, reflected by increases or prevention of loss of intracellular GSH levels, or by reduced GSSG to GSH ratios, or at restoring levels of TBARS. Our data failed to show an increase in cardiac TBARS with genistein. However, this does not suggest that the oxidative stress capacity of genistein-treated hearts is limited, because we recently reported that a 2-day treatment period increases tolerance of hearts to severe ischemia, in the absence of changes in myocardial levels of non-protein thiols12. More recently, we demonstrated estrogen-like effects of genistein (administered for 2 weeks via daily subcutaneous injections), again in OVX rats, that were similarly mediated in the absence of changes in cardiac oxidative stress, i.e. no effect on TBARS or non-protein thiols13. Cellular defenses against oxidative stress also include superoxide dismutase, catalase, cystathionine-γ-lyase and glutathione peroxidase, all of which are increased with either estrogen or genistein51–53. Thus, alternative mechanisms/pathways may mediate genistein’s actions, and may likely be dependent upon the dose utilized, duration and route of administration. This study demonstrated that OVX rats have significantly decreased uterine weight, plasma insulin levels, insulin/glucose ratio and plasma TNF-α levels, compared with intact counterparts. In addition, OVX rats have significantly increased mean arterial pressure that is independent of any structural changes in the thoracic aorta. We conclude that genistein (300 mg genistein/kg body weight/day), in addition to moderate exercise treadmill training, for a 6-week period, does not improve cardiovascular parameters and risk factors in an OVX rat model of menopause. Conflict of interest The authors report no confl ict of interest. The authors alone are responsible for the content and writing of this paper. Source of funding This work was supported by Soy Health Research Program (awarded to L. Al-Nakkash), and Midwestern University intramural funding awarded to L. Al-Nakkash and T. L. Broderick. T. Janjulia and D. Wilson were supported through the Department of Biomedical Sciences. Kurt Peterson was a recipient of the Midwestern University DO Summer Fellowship.

References 1. Dubey RK, Gillespie D, Imthurn B, Rosselli M, Jackson EK, Keller PJ. Phytoestrogens inhibit growth and MAP kinase activity in human aortic smooth muscle cells. Hypertension 1999; 33:177–82

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2. Scuteri A, Ferrucci L. Blood pressure, arterial function, structure, and aging: the role of hormonal replacement therapy in postmenopausal women. J Clin Hypertens 2003;5: 219–25

145

Genistein and exercise in ovariectomized rats 3. Li HF, Li W, Zheng TZ, Qu SY, Zhang CL. A study of the mechanisms involved in relaxation induced by 17-beta-estradiol in the isolated rabbit aorta. Arch Gynecol Obstet 2002;266:101–4 4. Hulley S, Grady D, Bush T, et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/progestin Replacement Study Research Group. JAMA 1998;280:605–13 5. Suetsugi M, Su L, Karlsberg K, Yuan YC, Chen S. Flavone and isoflavone phytoestrogens are agonists of estrogen-related receptors. Mol Cancer Res 2003;1:981–91 6. Cassidy A, Griffin B. Phyto-estrogens: a possible role in the prevention of CHD? Proc Nutri Soc 1999;58:193–9 7. Honore EK, Williams JK, Anthony MS, Clarkson TB. Soy isoflavones enhance coronary vascular reactivity in atherosclerotic female macaques. Fertil Steril 1997;67:148–54 8. Figtree GA, Griffiths H, Lu YQ, Webb CM, MacLeod K, Collins P. Plant-derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism. J Am Coll Cardiol 2000;35:1977–85 9. Walker HA, Dean TS, Sanders TA, Jackson G, Ritter JM, Chowienczyk P. The phytoestrogen genistein produces acute nitric oxide-dependent dilation of human forearm vasculature with similar potency to 17beta-estradiol. Circulation 2001; 103:258–62 10. Anthony MS, Clarkson TB, Hughes CL, Morgan TM, Burke GL. Soybean isoflavones improve cardiovasular risk factors without affecting the reproductive system or peripubertal rhesus monkeys. J Nutr 1996;126:43–50 11. Sliva D. Suppression of cancer invasiveness by dietary compounds. Mini Rev Med Chem 2008;8:677–88 12. Al-Nakkash L, Markus B, Bowden K, Batia L, Prozialeck WC, Broderick TL. Effects of acute and 2-day genistein treatment on cardiac function and ischemic tolerance in ovariectomized rats. Gender Med 2009;6:488–97 13. Al-Nakkash L, Markus B, Batia L, Prozialeck WC, Broderick TB. Genistein induces estrogen-like effects in ovariectomized rats but fails to increase cardiac GLUT4 and oxidative stress. J Med Food 2010;13:1–7 14. Al-Nakkash L, Martin JB, Petty D, et al. Dietary genistein induces sex-dependent effects on murine body weight, serum profiles, and vascular function of thoracic aortae. Gender Med 2012;9: 295–308 15. Wegge JK, Roberts CK, Ngo TH, Barnard RJ. Effect of diet and exercise intervention on inflammatory and adhesion molecules in postmenopausal women on hormone replacement therapy and at risk for coronary heart disease. Metabolism 2004;53:377–81 16. Staffileno BA, Braun LT, Rosenson RS. The accumulative effects of physical activity in hypertensive post-menopausal women. J Cardiovasc Risk 2001;8:283–90 17. Wang D, Gutkowska J, Marcinkiewicz M, Rachelska G, Jankowski M. Genistein supplementation stimulates the oxytocin system in the aorta of ovariectomized rats. Cardiovasc Res 2003;57:186–94 18. Wu J, Wang XX, Takasaki M, Ohta A, Higuchi M, Ishimi Y. Cooperative effects of exercise training and genistein adminstration on bone mass in ovariectomized mice. J Bone Miner Res 2001;16:1829–36 19. Ekskulkla S, Suksom D, Siriviriyakul P, Patumraj S. Increased NO bioavialability in aging male rats by genistein and exercise training: using 4,5-diaminofluorescein diacetate. Reprod Biol Endocrinol 2009;7:93 20. Kohrt WM, Bloomfield SA, Little KD, Nelson ME, Yingling VR. American College of Sports Medicine Position Stand: physical activity and bone health. Med Sci Sports Exerc 2004;36: 1985–96

146

Al-Nakkash et al. 21. Ramos JE, Al-Nakkash L, Peterson A, et al. The soy isoflavone genistein inhibits the reduction in Achilles tendon collagen content induced by ovariectomy in rats. Scand J Med Sci Sports 2012;22:e108–14 22. Gutkowska J, Broderick TB, Bogdan D, Wang D, Lavoie J-M, Jankowski M. Downregulation of oxytocin and natriuretic peptides in diabetes: possible implications in cardiomyopathy. J Physiol 2009;587:4725–36 23. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70–7 24. Bhathena SJ, Velasquez MT. Beneficial role of dietary phytoestrogens in obesity and diabetes. Am J Clin Nutr 2002;76: 1191–201 25. Tissier R, Waintraub X, Couvreur N, et al. Pharmacological postconditioning with the phytoestrogen genistein. J Mol Cell Cardiol 2007;42:79–87 26. Gutkowska J, Paquette A, Wang D, Lavoie JM, Jankowski M. Effect of exercise training on cardiac oxytocin and natriuretic peptide systems in ovariectomized rats. Am J Physiol Regul Integr Comp Physiol 2007;293:R267–75 27. Nevala R, Lassila M, Finckenberg P, Paukku K, Korpela R, Vapaatalo H. Genistein treatment reduces arterial contractions by inhibiting tyrosine kinases in ovariectomized hypertensive rats. Eur J Pharmacol 2002;452:87–96 28. Small CJ, Bloom SR. The therapeutic potential of gut hormone peptide YY3-36 in the treatment of obesity. Expert Opin Investig Drugs 2005;14:647–53 29. Orgaard A, Jensen L. The effects of soy isoflavones on obesity. Exp Biol Med 2008;233:1066–80 30. Kim H-K, Nelson-Dooley C, Della-Fera MA, et al. Genistein decreases food intake, body weight, and fat pad weight and causes adipose tissue apoptosis in ovariectomized female mice. J Nutr 2006;136:409–14 31. Riggs BL, Melton LR. Osteoporosis and age-related fracture syndromes. Ciba Foundation Symp 1988;134:129–42 32. Beyer R, Huang JC, Wilshire G. The effect of endurance exercise on bone dimensions, collagen, and calcium in the aged male rat. Exp Gerontol 1985;20:315–23 33. Steinberg ME, Trueta J. Effects of activity on bone growth and development in the rat. Clin Orthop Relat Res 1981;156:52–60 34. Raab DM, Smith EL, Crenshaw TD, Thomas DP. Bone mechanical properties after exercise training in young and old rats. J Appl Physiol 1990;68:130–4 35. Squadrito F, Altavilla D, Squadrito G, et al. Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats. Cardiovasc Res 2000;45:454–62 36. Si H, Liu D. Genistein, a soy phytoestrogen, upregulates the expression of human endothelial nitric oxide synthase and lowers blood pressure in spontaneously hypertensive rats. J Nutr 2008;138:297–304 37. Li H-F, Wang L-D, Qu S-Y. Phytoestrogen genistein decreases contractile response to aortic artery in vitro and arterial blood pressure in vivo. Acta Pharmacol Sin 2004;25:313–18 38. Kitayama J, Kitazono T, Ooboshi H, et al. Chronic administration of a tyrosine kinase inhibitor restores functional and morphological changes of the basilar artery during chronic hypertension. J Hypertens 2002;20:2205–11 39. Irigoyen MC, Paulini J, Flores LJ, et al. Exercise training improves baroreflex sensitivity associated with oxidative stress reduction in ovariectomized rats. Hypertension 2005;46:998–1003 40. Villa P, Costantini B, Suriano R, et al. The differential effect of the phytoestrogen genistein on cardiovascular risk factors in postmenopausal women: relationship with the metabolic status. J Clin Endocrinol Metab 2009;94:552–8

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Genistein and exercise in ovariectomized rats 41. Salih SM, Nallasamy P, Muniyandi P, Periyasami V, Ventkatraman AC. Genistein improves liver function and attenuates non-alcoholic fatty liver disease in a rat model of insulin resistance. J Diabetes 2009;1:278–87 42. Sorenson RL, Brelje TC, Roth C. Effect of tyrosine kinase inhibitors on islets of Langerhans: evidence for tyrosine kinases in the regulation of insulin secretion. Endocrinology 1994; 134:1955–8 43. Fu Z, Zhang W, Zhen W, et al. Genistein induces pancreatic B-cell proliferation through activation of multiple signaling pathways and prevents insulin-deficient diabetes in mice. Endocrinology 2010;151:3026–37 44. Patton JS, Shepard HM, Wilking H, et al. Interferons and tumor necrosis factors have similar catabolic effects on 3T3L1 cells. Proc Natl Acad Sci USA 1986;83:8313–17 45. Ito Y, Nomura S, Ueda H, et al. Exercise training increases membrane bound form of tumor necrosis factor-alpha receptors with decreases in the secretion of soluble forms of receptors in rat adipocytes. Life Sci 2002;71:601–9 46. Bukowiecki L, Lupien J, Follea N, Paradis A, Richard D, Leblanc J. Mechanism of enhanced lipolysis in adipose tissue of exercise-trained rats. Am J Physiol 1980;239:E422–9 47. Bonyadi M, Badalzadeh R, Mohammadi M, Poozesh S, Salehi I. The effect of regular training on plasma cytokines

Climacteric

Al-Nakkash et al.

48.

49.

50.

51.

52.

53.

response in healthy and diabeteic rats. Saudi Med J 2009; 30:1390–4 Benedusi V, Meda C, Della Torre S, Monteleone G, Vegeto E, Maggi A. A lack of ovarian function increases neuroinflammation in aged mice. Endocrinology 2012;153:2777–88 Marques CMM, Nascimento FAM, Mandarim-de-Lacerdo CA, Aguila MB. Exercise attenuates cardiovascular adverse remodelling in adult ovariectomized spontaneously hypertensive rats. Menopause 2006;13:87–95 Paquette A, Wang D, Gauthier M-S, et al. Specific adaptations of estrogen receptor α and β transcripts in liver and heart after endurance training in rats. Mol Cell Biochem 2007;306: 179–87 Suzuki K, Koike H, Matsui H, et al. Genistein, a soy isoflavone, induces glutathione peroxidase in the human prostate cancer cell lines LNCAP and PC-3. Int J Cancer 2002;99:846–52 Mahn K, Borras C, Knock GA, et al. Dietary soy isoflavoneinduced increases in antioxidant and eNOS gene expression lead to improved edothelial function and reduced blood pressure in vivo. FASEB J 2005;19:1755–7 Zhu X, Tang Z, Cong B, et al. Estrogens increase cystathionineγ-lyase expression and decrease inflammation and oxidative stress in the myocardium of ovariectomized rats. Menopause 2013 April 8. [Epub ahead of print]

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