Layla Al-Nakkash

Estrogen-gut microbiome axis: Physiological and clinical implications.

Low levels of gonadal circulating estrogen observed in post-menopausal women can adversely impact a diverse range of physiological factors, with clinical implications for brain cognition, gut health, the female reproductive tract and other aspects of womens health. One of the principal regulators of circulating estrogens is the gut microbiome. This review aims to shed light on the role of the gut microbiota in estrogen-modulated disease. The gut microbiota regulates estrogens through secretion of β-glucuronidase, an enzyme that deconjugates estrogens into their active forms. When this process is impaired through dysbiosis of gut microbiota, characterized by lower microbial diversity, the decrease in deconjugation results in a reduction of circulating estrogens. The alteration in circulating estrogens may contribute to the development of conditions discussed herein: obesity, metabolic syndrome, cancer, endometrial hyperplasia, endometriosis, polycystic ovary syndrome, fertility, cardiovascular disease (CVD) and cognitive function. The bi-directional relationship between the metabolic profile (including estrogen levels) and gut microbiota in estrogen-driven disease will also be discussed. Promising therapeutic interventions manipulating the gut microbiome and the metabolic profile of estrogen-driven disease, such as bariatric surgery and metformin, will be detailed. Modulation of the microbiome composition subsequently impacts the metabolic profile, and vice versa, and has been shown to alleviate many of the estrogen-modulated disease states. Last, we highlight promising research interventions in the field, such as dietary therapeutics, and discuss areas that provide exciting unexplored topics of study.

Effects of acute and 2-day genistein treatment on cardiac function and ischemic tolerance in ovariectomized rats.

BACKGROUND Genistein, a naturally occurring isoflavonic phytoestrogen associated with reduced incidence of heart disease, may be a possible alternative treatment for postmenopausal women with heart disease. OBJECTIVE This study examined the effects of genistein on in vitro heart function and ischemic tolerance in ovariectomized (OVX) Sprague-Dawley rats. METHODS To examine the acute effects of genistein on cardiac function, isolated working hearts were perfused under aerobic conditions with increasing concentrations of genistein (10-150 microM). A separate group of OVX rats was used to assess ischemic tolerance: treated rats received genistein (250 mg/kg, dissolved in 200 microL dimethyl sulfoxide [DMSO]) injected once daily for 2 days, and control rats received DMSO only. After treatment, hearts were perfused for 30 minutes under aerobic conditions and then subjected to 20 minutes of global no-flow ischemia by clamping the preload and afterload lines, followed by 30 minutes of reperfusion. RESULTS Genistein was associated with improvements in mechanical function in OVX rat hearts (n = 5) with maximum increases in contractility (259 mm Hg/sec above baseline) and cardiac output (7 mL/min above baseline) observed with 30 microM of genistein (both, P < 0.05). Relative to baseline, genistein-treated hearts (n = 5) also had greater ischemic tolerance than did control hearts (n = 6) and significant improvements in mean (SEM) recovery of contractility (to 75.0% [9.7%] of preischemic function; P < 0.05) and cardiac output (to 48.8% [12.3%] of preischemic function; P < 0.05) after reperfusion. These effects occurred without significant changes in myocardial levels of nonprotein thiols or thiobarbituric acid reactive substances, although a reduction in mean glucose transporter protein 4 content (13.2% [2.7%]; P < 0.05) was observed in genistein-treated hearts. No significant changes in blood pressure were observed with genistein. CONCLUSIONS Despite the lack of significant changes in physical characteristics, 2-day treatment with genistein was associated with significant cardioprotective effects in OVX rats, suggesting a potential therapeutic role in postmenopausal women.

Stimulation of murine intestinal secretion by daily genistein injections: gender-dependent differences.

Background/Aims: The effect of daily injections with genistein (naturally occurring phytoestrogen) on intestinal chloride (Cl-) secretion was measured with Ussing chamber short circuit current (Isc, µA/cm2), in C57BL/6J male and female mice, using 600 mg/kg genistein/day (600G), 300 mg/kg genistein/day (300G), 150 mg/kg genistein/day (150G) or genistein-free vehicle control (0G) for 1- or 2-weeks. Methods and Results: Injecting with 600G elicited significant increases in basal Isc in females after 1-week (ñ70 µA/cm2, n=15, p < 0.05) and in males after 2-weeks (ñ80 µA/cm2, n=5, p < 0.05) compared to their 0G counterparts. Chloride-free ringer significantly reduced basal Isc by 65% in 600G males and 72% in 600G females, suggesting that Cl- was the major anion comprising the genistein-stimulated secretion. The forskolin-stimulated (10 µM) Isc was significantly inhibited by the CFTR chloride channel inhibitors, glibenclamide (500 µM) and CFTRinh-172 (100 µM) in 600G males and females, suggesting some contribution by genistein-dependent CFTR-mediated Cl- secretion. We found no associated changes in intestinal morphology, nor change in total CFTR protein with 600G. There was a 5% increase in apical/subapical ratio in 600G males compared to controls (no change in females). Conclusion: These data suggest that male and female mice both exhibit increased Cl- secretion with 600G, however, the mechanisms mediating this are gender-dependent.

Genistein Stimulates Jejunal Chloride Secretion via Sex-dependent, Estrogen Receptor or Adenylate Cyclase Mechanisms

Background/Aims: Daily subcutaneous injections with the phytoestrogen genistein, 600 mg/ kg genistein/day (600G) significantly increased intestinal chloride (Cl-) secretion (Isc, µA/cm2) in C57BL/6J female and male murine jejunum after 1-2-weeks treatment. Methods and Results: In 600G females, basolateral application of the adenylate cyclase inhibitor MDL-12330A (10 µM) significantly reduced basal and total Isc in the presence of forskolin (27 and 40% respectively, P &lt; 0.05), with no effect in 600G males, suggesting that 600G-mediated increases in Isc in females are due to an adenylate cyclase-dependent mechanism. Concomitant injections with the non-selective estrogen receptor (ER) antagonist ICI-182780 (25 mg/kg/day) resulted in a significant inhibition of basal Isc in males (38%, P &lt; 0.05), but was without effect in females (further reinforcing an ER-independent mechanism of action). The ERα-selective antagonist (MPP, 25 mg/kg/day) similarly significantly inhibited the basal Isc (37%, P &lt; 0.05) in males, whereas the ERβ-selective antagonist (PHTPP, 25 mg/kg/day) was without effect, suggesting that 600G-mediated increases in I sc in male mice are due to an ERα-dependent mechanism. Jejunum ERα/actin expression was significantly increased by 600G in males. Compared to intact mice, orchiectomy has differing effects on 600G-mediated basal Isc; castration (CAST) abolished the 600G-mediated increases in I sc, and ovariectomy (OVX) had no effect on the 600G-stimulated increases in I sc. Daily estradiol injections (10-20 mg/kg body weight estradiol (10E2 or 20E2) had no effect in intact females, whereas 10E2 significantly increased basal Isc in OVX females. Conclusion: These data suggest that daily estradiol and genistein injections have differential sex-dependent mechanisms of action on murine intestinal Cl- secretion.

Dietary genistein induces sex-dependent effects on murine body weight, serum profiles, and vascular function of thoracic aortae.

BACKGROUND The influence on, or interaction of, sex and dietary genistein on serum markers of cardiovascular health and cardiovascular function remain unclear. OBJECTIVES Our purpose was to examine the effects of a genistein-containing diet (600 mg/kg food) (600G) and a genistein-free diet (0G), on cardiovascular risk parameters of male and female mice. METHODS C57BL/6J mice were fed the diets for 1 month, after which time blood pressure, serum markers, and in vitro vascular reactivity was measured. RESULTS Males fed the 600G diet gained significantly less weight than males fed the 0G diet (by 1.71 g); diet had no effect on female weight gain. Males fed the 600G diet also exhibited significantly elevated serum insulin (2.9 [0.5] vs 1.8 [0.4] ng/dL), and decreased serum glucose (0.15 [0.01] vs 0.24 [0.02] ng/dL) levels, resulting in a significant increase in the ratio of insulin to glucose; insulin and glucose levels were not changed by dietary genistein in females. Arterial pressure measurements from 0G-fed males were lower than other groups. However, basal vascular reactivity of isolated aortic rings was significantly increased by the 600G diet in both males (from 0.55 [0.03] to 0.94 [0.18] g) and females (from 0.45 [0.04] to 0.78 [0.09] g). Aortic wall thickness was not affected by diet. Norepinephrine-mediated contractility was also greater in aortic rings of male and female mice fed the 600G diet, and differences from the 0G diet persisted in the presence of L-NG-nitroarginine methyl ester but were completely accounted for by increased basal reactivity. CONCLUSION Our data indicate that 1 month of a 600G or 0G diet significantly alters vascular function independent of sex. In contrast, sex-dependent differences exist in well-established serum markers of cardiovascular health and disease.

Genistein Induces Estrogen-Like Effects in Ovariectomized Rats but Fails to Increase Cardiac GLUT4 and Oxidative Stress

This study aimed to determine whether a 2-week genistein treatment induced estrogen-like effects in ovariectomized (OVX) Sprague-Dawley rats, after 2 weeks of subcutaneous genistein injections (250 mg/kg of body weight/day). Uterine weight, uterine-to-body weight ratio, femur weight, and femur-to-body weight ratio were all significantly increased with genistein in OVX rats. Body weight was significantly decreased with genistein in OVX rats. Genistein had no effect on the weights of heart, heart-to-body ratio, and fat pad but significantly decreased heart rate and pulse pressure. Genistein had no effect on cardiac GLUT4 protein, oxidative stress, plasma glucose, nonesterified fatty acids, or low-density lipoprotein levels; however, plasma insulin levels were significantly increased. Our results show that a 2-week genistein treatment produced favorable estrogen-like effects on some physical and physiological characteristics in OVX rats. However, based on our experimental conditions, the effects of genistein were not associated with changes in cardiac GLUT4 or oxidative stress.

Activation of CFTR by UCCF-029 and genistein

The mechanism of action of a novel CFTR activator UC(CF)-029 on NIH3T3 cells stably expressing DeltaF508-CFTR was investigated and its effects compared to those of genistein, a known CFTR activator. This study shows that UC(CF)-029 and genistein have differing efficacies. The efficacy of UC(CF)-029 in the presence of forskolin (10microM) is approximately 50% that of genistein; however, the EC(50)s for both drugs are comparable; 3.5microM for UC(CF)-029 and 4.4muM for genistein. Using NIH3T3 cells stably transfected with K1250A-CFTR we find that CFTR channel open time is unaffected by UC(CF)-029 or genistein, supporting the hypothesis that these compounds stabilize the open state by inhibiting ATP hydrolysis at NBD2. Our data suggest that the ability of UC(CF)-029 to augment DeltaF508-CFTR channel activity necessitates further interest.

Genistein treatment increases bone mass in obese, hyperglycemic mice.

Background Obesity and type 2 diabetes mellitus are associated with elevated risk of limb bone fracture. Incidences of these conditions are on the rise worldwide. Genistein, a phytoestrogen, has been shown by several studies to demonstrate bone-protective properties and may improve bone health in obese type 2 diabetics. Methods In this study, we test the effects of genistein treatment on limb bone and growth plate cartilage histomorphometry in obese, hyperglycemic ob/ob mice. Six-week-old ob/ob mice were divided into control and genistein-treated groups. Genistein-treated mice were fed a diet containing 600 mg genistein/kg for a period of 4 weeks. Cross-sectional geometric and histomorphometric analyses were conducted on tibias. Results Genistein-treated mice remained obese and hyperglycemic. However, histomorphometric comparisons show that genistein-treated mice have greater tibial midshaft diameters and ratios of cortical bone to total tissue area than the controls. Genistein-treated mice also exhibit decreased growth plate thickness of the proximal tibia. Conclusion Our results indicate that genistein treatment affects bone of the tibial midshaft in the ob/ob mouse, independent of improvements in the hyperglycemic state and body weight.

Genistein and exercise do not improve cardiovascular risk factors in the ovariectomized rat

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.

Dietary Genistein Rescues Reduced Basal Chloride Secretion in Diabetic Jejunum via Sex-Dependent Mechanisms.

Background/Aims: The goal of this study was to determine the effect of dietary genistein (naturally occurring phytoestrogen) on jejunal secretory function in a clinically relevant model of diabetes and obesity, the leptin-defIcient ob/ob mouse. Methods: We measured transepithelial short circuit current (Isc), across freshly isolated segments of jejunum from 12-week old male and female ob/ob and lean C57Bl/6J mice fed a genistein diet (600 mg genistein/kg diet) for 4-weeks. Separate segments of jejunum were frozen for western blot determination of key proteins involved in secretory transport. Results: Basal Isc was signifIcantly decreased (by 33%, P<0.05) in ob/ob females versus leans, and genistein-diet reversed this. Similarly, in males, basal Isc was decreased (by 47%, P<0.05) in ob/ob mice versus leans, and genistein-diet reversed this. Inhibition with either clotrimazole (100 µM, bilateral) or ouabain (100 µM, basolateral) was signifIcantly reduced in ob/ob mice compared to leans (P<0.05), and genistein-diet reversed clotrimazole-sensitive inhibition in ob/ob females, and reversed the ouabain-sensitive inhibition in males (indicating sex-dependent mechanisms). Our data suggested that PDE3 levels were dysregulated in ob/ob females and genistein reversed this. Expression of total CFTR (normalized to actin) was signifIcantly decreased ∼80% (P<0.05) in all ob/ob mice compared to leans, and genistein-diet was without effect. Expression of total NKCC1 (normalized to actin) was signifIcantly decreased ∼80% (P<0.05) in ob/ob male mice versus leans, and genistein-diet reversed this. Conclusions: Our data suggests that the reduced basal jejunal Isc in ob/ob female mice is a consequence of reduced CFTR expression, decreased activities of the basolateral KCa channel and Na+/K+-ATPase, and in male mice reduced basal jejunal Isc is a consequence of reduced CFTR and NKCC1 expression, along with decreased activities of the basolateral KCa channel and Na+/K+-ATPase. Genistein-diet has beneficial effects on basal Isc mediated by sex-dependent mechanisms in diabetic mice: in females via increased KCa-sensitive Isc and in males via increased Na+/K+-ATPase activity and increased NKCC1 expression. Improved understanding of intestinal dysfunctions in the ob/ob jejunum, may allow for the development of novel drug targets to treat obesity and diabetes, and may also be of benefit in CF-related diabetes.