Soy isoflavone-caused shunting of the corticosteroidogenesis pathways in andropausal subjects: Top-down impulse for the optimal supplementation design.

In a series of our previous works, we revealed the beneficial effects of applied soy isoflavones (genistein or daidzein) on the wide context of corticosteroidogenesis in vivo, in a rat model of the andropause. Soy isoflavones decreased the circulating levels of pituitary adrenocorticotropic hormone, inhibited aldosterone secretion, as well as corticosterone production and secretion, but stimulated dehydroepiandrosterone secretion, all in andropausal rats. In vitro studies indicate that the mechanism underlying these hormonal changes relies on inhibition of the pituitary tyrosine kinase and adrenocortical 3ß-hydroxysteroid dehydrogenase enzymes by soy isoflavones. Although the clinical studies are in their infancy, the opinion is that genistein and daidzein have therapeutic potential for the safe treatment of ageing-caused androgen deprivation and glucocorticoid excess with related metabolic/hemodynamic issues in males. Our accumulated experience and knowledge in the field of biomedical effects of plant polyphenols have provided a platform for potential recommending the agenda to organize and accelerate experimental research aimed at producing the optimal supplementation. We hypothesize that an in vivo approach should first be exploited in the sequence of investigative steps, followed by in vitro studies and synchronously conducted molecular docking analyses. In vivo research, besides establishing the margin of exposure safety or adjustment of the correct polyphenol dose, enables identification and quantification of the metabolites of applied polyphenols in the blood. Subsequent in vitro exploitation of the metabolites and related docking analyses provide clarification of the molecular mechanisms of action of applied polyphenols. Chemical modification of the polyphenol structure or coupling it with nanoparticles might be the next step in optimizing the design of supplementation. Selected, intact or chemically-modified polyphenol molecules should be included in preclinical studies on a more closely-related species, while clinical studies would finally assess the safety and effectiveness of a polyphenol-based remedial strategy. The final supplement represents a product of an appropriate technological process, conducted in accordance with the recommendations derived from the preceding research.

Citrus Flavanones Upregulate Thyrotroph Sirt1 and Differently Affect Thyroid Nrf2 Expressions in Old-Aged Wistar Rats.

A growing population of elderly people consume citrus flavanones, naringenin, and hesperetin in the form of fruits or juices. Flavanones are bioactives with potent antioxidant properties and have potential in slowing down the aging process. Because flavanones exert controversial effects on pituitary-thyroid functioning, our study on the old-aged rat model aimed to elucidate the mechanism by which naringenin and hesperetin affect this axis. Naringenin and hesperetin increased the Sirt1 mRNA level by 91 and 71% (p < 0.05), which was followed by increased Sirt1 expression by 20 and 15% (p < 0.05), respectively. Only naringenin decreased thyroid-stimulating hormone expression by 20% (p < 0.05). Thyroid peroxidase protein expression was upregulated after naringenin or hesperetin by 62 and 43% (p < 0.05), respectively. Naringenin lowered mRNA levels of Tpo, Sod1, Sod2, Cat, and Nrf2 by 50, 32, 45, 35, and 42% (p < 0.05), respectively, and increased Gpx by 54% (p < 0.05), while hesperetin decreased Sod1 and Sod2 mRNA levels by 46 and 55% (p < 0.05), respectively. Naringenin increased the protein expressions of Nrf2 and SOD2 by 58 and 50% (p < 0.05), respectively, and decreased SOD1 expression by 48% (p < 0.05), while hesperetin protein decreased expressions of SOD1 and Nrf2 by 63 and 32% (p < 0.05), respectively. Altogether, our findings suggest that citrus flavanones contribute to restoring the impaired thyroid functioning in the old-aged rats.

The adrenal cortex after estradiol or daidzein application in a rat model of the andropause: Structural and hormonal study.

INTRODUCTION AND AIM: Daidzein application may represent an effective and less harmful alternative to indicated, classical estrogenization of ageing men. The aim of this study was to perform structural and hormonal analysis of the adrenal cortex, after estradiol or daidzein supplementation in a rat model of the andropause. MATERIAL AND METHODS: Middle-aged Wistar rats were divided into sham operated (SO; n = 8), orchidectomized (Orx; n = 8), estradiol treated orchidectomized (Orx + E; n = 8) and daidzein treated orchidectomized (Orx + D; n = 8) groups. Estradiol (0.625 mg/kg b.m./day) or daidzein (30 mg/kg b.m./day) were administered subcutaneously for three weeks, while the SO and Orx groups received the vehicle alone. Set objectives were achieved using stereology, histochemistry/immunohistochemistry, immunoassays and ultrastructural analysis. RESULTS: Both estradiol and daidzein treatment significantly increased volumes of the zona glomerulosa cell and nuclei, but decreased circulating aldosterone levels. Estradiol markedly increased volumes of the zona fasciculata cell and nuclei in parallel with significant decrease of the adrenal tissue level of corticosterone, while daidzein significantly decreased both the adrenal and circulating levels of corticosterone. Serum DHEA level and volumes of the zona reticularis cell and nuclei significantly increased upon estradiol treatment, whereas daidzein even stronger increased the circulating level of DHEA. Shunting of the corticosteroidogenesis pathways towards adrenal androgens production, after the treatments, corresponded to the ultrastructural findings and zonal capillary network rearrangements. CONCLUSIONS: Given the coherence of its effects and relative safety, daidzein could be the remedy of choice for the treatment of ageing-caused androgen deprivation and the hypothalamo-pituitary-adrenal axis hyperfunction/related metabolic issues in males.

An Extremely Low Frequency Magnetic Field and Global Cerebral Ischemia Affect Pituitary ACTH and TSH Cells in Gerbils.

The neuroendocrine system can be modulated by a magnetic field and cerebral ischemia as external and internal stressors, respectively. This study deals with the separate or combined effects of an extremely low frequency (ELF) magnetic field (50 Hz, average magnetic field of 0.5 mT) for 7 days and global cerebral ischemia for 10 min on the morpho-functional features of pituitary adrenocorticotrophic (ACTH) and thyrotrophic (TSH) cells in 3-month-old gerbils. To determine the immediate and delayed effects of the applied stressors, measurements were made on the 7th and 14th days after the onset of the experiment. The ELF magnetic field and 10-min global cerebral ischemia, separately and particularly in combination, decreased (P < 0.05) the volume density of ACTH cells, while only in combination were intracellular ACTH content and plasma ACTH concentration increased (P < 0.05) on day 7. The ELF magnetic field elevated serum TSH concentration on day 7 and intracellular TSHß content on day 14 (P < 0.05). Also, 10-min global cerebral ischemia alone increased serum TSH concentration (P < 0.05), while in combination with the ELF magnetic field it elevated (P < 0.05) intracellular TSHß content on day 14. In conclusion, an ELF magnetic field and/or 10-min global cerebral ischemia can induce immediate and delayed stimulation of ACTH and TSH synthesis and secretion. Bioelectromagnetics. 2020;41:91-103. © 2019 Bioelectromagnetics Society.

Daidzein upregulates anti-aging protein Klotho and NaPi 2a cotransporter in a rat model of the andropause.

In a rat model of the andropause we aimed to examine the influence of daidzein, soy isoflavone, on the structure and function of parathyroid glands (PTG) and the expression levels of some of the crucial regulators of Ca2+ and Pi homeostasis in the kidney, and to compare these effects with the effects of estradiol, serving as a positive control. Middle-aged (16-month-old) male Wistar rats were divided into the following groups: sham-operated (SO), orchidectomized (Orx), orchidectomized and estradiol-treated (Orx+E; 0.625mg/kg b.w./day, s.c.) as well as orchidectomized and daidzein-treated (Orx+D; 30mg/kg b.w./day, s.c.) group. Every treated group had a corresponding control group. PTH serum concentration was decreased in Orx+E and Orx+D groups by 10% and 21% (p<0.05) respectively, in comparison with the Orx. PTG volume was decreased in Orx+E group by 16% (p<0.05), when compared to the Orx. In Orx+E group expression of NaPi 2a was lower (p<0.05), while NaPi 2a abundance in Orx+D animals was increased (p<0.05), when compared to Orx. Expression of PTH1R was increased (p<0.05) in Orx+E group, while in Orx+D animals the same parameter was decreased (p<0.05), in comparison with Orx. Klotho expression was elevated (p<0.05) in Orx+D rats, in regard to Orx. Orx+D induced reduction in Ca2+/creatinine and Pi/creatinine ratio in urine by 32% and 16% (p<0.05) respectively, in comparison with Orx. In conclusion, presented results indicate the more coherent beneficial effects of daidzein compared to estradiol, on disturbed Ca2+ and Pi homeostasis, and presumably on bone health, in the aging male rats.

The phytoestrogen genistein prevents trabecular bone loss and affects thyroid follicular cells in a male rat model of osteoporosis.

As a major phytoestrogen of soy, genistein effectively prevents bone loss in both humans and rat models of osteoporosis. However, although the bone-sparing effects of genistein are achieved directly through estrogen receptors, its mode of action on bone by modulation of other endocrine functions is not entirely clear. Thus, thyroid hormones and calcitonin (CT) have an essential influence on bone metabolism. Besides its action on bones, in this study we examined the effect of genistein on the activity of two different endocrine cell populations, thyroid follicular and C-cells. Fifteen-month-old Wistar rats were either bilaterally orchidectomized (Orx) or sham-operated (SO). Two weeks after surgery, half of the Orx rats were treated chronically with 30 mg kg-1 b.w. genistein (Orx + G) subcutaneously (s.c.) every day for 3 weeks, while the remaining Orx rats and the SO rats were given the same volume of sterile olive oil to serve as controls. For histomorphometrical analysis of the trabecular bone microarchitecture an ImageJ public domain image processing programme was used. Thyroid sections were analysed histologically and stereologically after visualization of follicular and C-cells by immunohistochemical staining for thyroglobulin and CT. Thyroid follicular epithelium, interstitium, colloid and CT-immunopositive C-cells were examined morphometrically. Serum concentrations of osteocalcin (OC), triiodothyronine (T3 ), thyroxine (T4 ) and CT were determined as well as urinary calcium (Ca2+ ) concentrations. Genistein treatment significantly increased cancellous bone area (B.Ar), trabecular thickness (TbTh) and trabecular number (TbN) (P < 0.05), but trabecular separation (Tb.Sp) was decreased (P < 0.05) compared with control Orx rats. In the thyroid, genistein treatment significantly elevated the relative volume density (Vv) of the follicular cells (P < 0.05) compared with Orx, whereas Vv of the colloid was lower (P < 0.05) than in the Orx. Evaluation of the biochemical parameters showed significant reductions in serum OC, T3 , T4 and urinary Ca2+ concentrations (P < 0.05), compared with Orx rats. These data indicate that genistein treatment improves the trabecular microarchitecture of proximal tibia, induces histomorphometrical changes in thyroid glands, and decreases circulating thyroid hormone levels in orchidectomized rat model of male osteoporosis.

Soy Phytoestrogens Do Not Fully Reverse Changes in Rat Pituitary Castration Cells: Unbiased Stereological Study.

The aim of the study was to examine the potential of the principal soy isoflavones, genistein and daidzein, or isoflavone rich soy extract to recover pituitary castration cells in orchidectomized adult male rats in comparison with the effects of estradiol. Two weeks post orchidectomy (Orx), animals received estradiol-dipropionate, genistein, daidzein or soy extract subcutaneously for 3 weeks. Control sham-operated (So) and Orx rats received just the vehicle. Changes in the volumes of pars distalis, of individual follicle-stimulating hormone (FSH) and luteinizing hormone (LH) containing cells, their volume, numerical density and number were determined by unbiased design-based stereology. The intracellular content of ßFSH and ßLH was estimated by relative intensity of fluorescence (RIF). Orchidectomy increased all examined stereological parameters and RIF. Compared to Orx, estradiol increased the volume of pars distalis, but reversed RIF and all morphometric parameters of gonadotropes to the level of So rats, except their number. Treatments with purified isoflavones and soy extract decreased RIF to the control So level, expressing an estradiol-like effect. However, the histological appearance and morphometrical features of gonadotropes did not follow this pattern. Genistein increased the volume of pars distalis, decreased the volume density of LH-labeled cells and raised the number of gonadotropes. Daidzein decreased the cell volume of gonadotropic cells but increased their number and numerical density. Soy extract induced an increase in number and numerical density of FSH-containing cells. Therefore, it can be concluded that soy phytoestrogens do not fully reverse the Orx-induced changes in pituitary castration cells. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Effects of age and soybean isoflavones on hepatic cholesterol metabolism and thyroid hormone availability in acyclic female rats.

Soy-food and its isoflavones, genistein (G) and daidzein (D), were reported to exert mild cholesterol-lowering effect, but the underlying mechanism is still unclear. In this research, first we studied age-related alterations in hepatic cholesterol metabolism of acyclic middle-aged (MA) female rats. Then we tested if purified isoflavones may prevent or reverse these changes, and whether putative changes in hepatic thyroid hormone availability may be associated with this effect. Serum and hepatic total cholesterol (TChol), bile acid and cholesterol precursors, as well as serum TSH and T4 concentrations, hepatic deiodinase (Dio) 1 enzyme activity and MCT8 protein expression were determined by comparing data obtained for MA with young adult (YA) intact (IC) females. Effects of subcutaneously administered G or D (35mg/kg) to MA rats were evaluated versus vehicle-treated MA females. MA IC females were characterized by: higher (p<0.05) serum TChol, lower (p<0.05) hepatic TChol and its biosynthetic precursors, lower (p<0.05) hepatic 7α-hydroxycholesterol but elevated (p<0.05) 27- and 24-hydroxycholesterol in comparison to YA IC. Both isoflavone treatments decreased (p<0.05) hepatic 27-hydroxycholesterol, G being more effective than D, without affecting any other parameter of Chol metabolism. Only G elevated hepatic Dio1 activity (p<0.05). In conclusion, age-related hypercholesteremia was associated with lower hepatic Chol synthesis and shift from main neutral (lower 7α-hydroxycholesterol) to alternative acidic pathway (higher 27-hydroxycholesterol) of Chol degradation to bile acid. Both isoflavones lowered hepatic 27-hydroxycholesterol, which may be considered beneficial. Only G treatment increased hepatic Dio1 activity, thus indicating local increase in thyroid hormones, obviously insufficient to induce prominent cholesterol-lowering effect.

Testosterone supplementation, glucocorticoid milieu and bone homeostasis in the ageing male.

Male ageing is entwined with a continuous fall in free testosterone levels, which contributes to the pathogenesis of bone loss. Glucocorticoid excess, either dependent on the ageing process or iatrogenically induced, was found to additionally impair the bone structure and metabolism. Cautious testosterone supplementation in this respect may positively affect the glucocorticoid milieu and bone homeostasis, while testosterone-induced changes in the glucocorticoid output could serve as a determinant of bone-related therapeutic outcome. Namely, bone mineral content/density, the parameters of trabecular bone structure as well as bone strength are enhanced, serum calcitonin levels tend to increase, while serum osteocalcin, serum parathyroid hormone and urinary calcium decrease, all upon testosterone administration to the ageing male. In parallel, testosterone application decreases glucocorticoid secretion in the animal models of male ageing, while clinical data in this field are still inconsistent. Importantly, a physiological link exists between testosterone-induced changes in glucocorticoid levels and the tendency of bone status improvement in the ageing male. We believe that the assessment of circulating adrenocorticotropic hormone concentrations together with glucocorticoid levels, reflecting the hypothalamic-pituitary-adrenal axis feedback loop operativeness during testosterone supplementation, represents a well-balanced bone-related therapeutic update.

Citrus flavanones naringenin and hesperetin improve antioxidant status and membrane lipid compositions in the liver of old-aged Wistar rats.

This study aimed to investigate effects of citrus flavanones naringenin (NAR) and hesperetin (HES) on liver antioxidant status and membrane phospholipid composition in 24-month-old rats. NAR and HES (15mg/kg) were administrated orally to male Wistar rats, once per day, for 4weeks. Control group received either vehicle (sunflower oil) or remained intact. The results showed decreased (p<0.05) activity of antioxidant enzymes (AOE), specifically catalase (CAT), superoxide dismutase (SOD) 1 and glutathione reductase (GR) in the liver of intact control old-aged rats in comparison to young intact controls. Flavanone administration to old-aged males increased (p<0.05) examined AOE activities in comparison to vehicle-administered animals. Namely, NAR was more potent in comparison to HES regarding the increase (p<0.05) in activities of examined antioxidant enzymes (SOD 1 and 2, glutathione peroxidase-GPx and GR) and the liver glutathione (GSH), while HES elevated (p<0.05) only activity of CAT and GR. Both flavanones significantly decreased (p<0.05) TBARS and improved (p<0.05) membrane phospholipid composition in favor of n-3 PUFA and n-6/n-3 PUFA ratio. Both flavanones did not affect liver histology and reduced (p<0.05) alanine aminotransferase and aspartate aminotransferase levels in serum. The results of this study indicate beneficial potential of citrus flavanones in the old-aged rat liver.

Tamoxifen stimulates calcitonin-producing thyroid C-cells and prevents trabecular bone loss in a rat model of androgen deficiency.

Thyroid C-cells produce calcitonin (CT), a hypocalcemic hormone, that acts as an inhibitor of bone resorption. In this study, we investigated the effects of tamoxifen (TAM) as a selective estrogen receptor modulator on thyroid C-cells, trabecular bone and biochemical markers of bone metabolism in an animal model of androgen deficiency, represented by middle-aged orchidectomized (Orx) rats. Fifteen-month-old male Wistar rats were divided into: Orx and sham-operated (SO) groups. Rats from one Orx group were injected subcutaneously with TAM citrate (Orx + TAM; 0.3 mg kg(-1) b.w.), while the rats from SO and a second Orx group received vehicle alone, once a day for 3 weeks. The peroxidase-antiperoxidase method was applied for localization of CT in C-cells. Thyroid C-cells were morphometrically and ultrastructurally analyzed. An ImageJ image-processing program was used to measure bone histomorphometric parameters. Blood serum samples were analyzed for CT, osteocalcin (OC), calcium (Ca2+ ) and phosphorus (P). Urinary Ca2+ concentrations were measured. TAM treatment significantly increased thyroid C-cell volume (Vc ) and serum CT when compared with vehicle-treated Orx rats. Analysis of trabecular microarchitecture of the tibia showed that administration of TAM significantly increased cancellous bone area, trabecular thickness and trabecular number, whereas trabecular separation was significantly decreased compared with vehicle-treated Orx rats. Serum OC and urinary Ca2+ concentrations were significantly lower in comparison with the control Orx group. These results indicate that in our rat model of androgen deficiency, TAM stimulated calcitonin-producing thyroid C-cells and increased trabecular bone mass.

Diosgenin does not express estrogenic activity: a uterotrophic assay.

This study assessed the effects of diosgenin on estrogenic activity using a uterotrophic assay. Immature female rats received diosgenin orally at doses of 200, 100, or 20 mg/kg body mass; and 17α ethynylestradiol at doses of 1 or 0.3 µg/kg, daily, for 3 consecutive days from day 19 to day 21. Controls were distributed among 2 groups: an intact control group and a vehicle control group. Animals were sacrificed 24 h after the last application of diosgenin, estradiol, or vehicle (22nd day of life). Uterine wet weight, stereological and histomorphometrical changes, immunohistochemical expression of estrogen receptor alpha (ERα), progesterone receptor (PR), and the expression of lactoferrin (LF) were examined. Diosgenin did not affect the uterine wet weight, epithelium height, volume densities of endometrium, endometrial epithelia, number of endometrial glands, or histological appearance of vaginal epithelia. ERα, PR, and LF immunostaining intensity were not altered in the animals that received diosgenin. High-potency reference ER agonist 17α-ethynylestradiol induced a significant increase in all of the measured parameters, and as expected, decreased ERα immunostaining intensity. Based on these data, it can be concluded that diosgenin, at doses of 20-200 mg/kg, did not act as an estrogen agonist in the immature rat uterotrophic assay.

Soy extract-dependent changes in morphofunctional parameters of the pituitary corticotropes in adult rats.

Numerous studies have established a link between estrogen levels and activity of the hypothalamic pituitary adrenal (HPA) system. Considering the "weak estrogenic" effect of soy isoflavones, this study was designed to evaluate the influence of soy extract treatment on some morphofunctional parameters of rat pituitary corticotropes in vivo. Adult male orchidectomized Wistar rats received estradiol-dipropionate or soy extract in oil/ethanol solvent subcutaneously for 3 weeks. Both controls, sham-operated (So) and orchidectomized (Orx) rats, were injected with solvent, in the same regime. Changes in pituitary volume, total volume, total number and volume of individual corticotropes were evaluated stereologically, while ACTH levels were determined biochemically. In comparison with So rats, estradiol treatment provoked increases (p<0.05) of: ACTH level (166%), pituitary weight (167%) and volume (102%), total volume (20%) and total number of corticotropes (18%). In comparison to Orx, following estradiol treatment elevation (p<0.05) of: ACTH level (69%), pituitary weight (131%) and volume (82%), total (30%) and individual volume (29%) of corticotropes was observed. Soy extract treatment led to enhancement (p<0.05) of: ACTH level (28%), total (25%) and individual volume (20%) of corticotropes. It can be concluded that soy extract acts in a similar way to estradiol, but the increased activity of corticotropes was weaker.

Suppressive effects of genistein and daidzein on pituitary-thyroid axis in orchidectomized middle-aged rats.

High intake of soybean phytoestrogens, isoflavones genistein (G) and daidzein (D), has been associated with health benefits. However, isoflavones were reported to affect adversely thyroid function in the presence of other goitrogenic factors. As the thyroid gland becomes functionally impaired with age, we examined whether supplementary doses of G or D would affect morphology and function of pituitary-thyroid axis in middle-aged male rats. Sixteen-month-old orchidectomized Wistar rats were treated with 10 mg/kg of either G or D, while the control sham-operated and orchidectomized group received just the vehicle for three weeks. The animals were fed soy-free diet with increased iodine content, and killed 24 h after the last treatment. Their pituitaries and thyroids were excised and prepared for further immunohistochemical and morphometric investigation. The concentrations of thyroid-stimulating hormone (TSH), total T(4) and T(3), in the serum were determined. In both isoflavone-treated groups, pituitary TSH-immunopositive cells had increased cellular volume and relative volume density (P < 0.05), as well as increased serum TSH levels (P < 0.05) in comparison to the controls; their thyroid tissue was characterized by increased volume of thyroglobulin-immunopositive epithelium (P < 0.05), epithelial height and index of activation rate (P < 0.05), while the volume of luminal colloid, and total serum T(4) and T(3) levels decreased (P < 0.05) in comparison to the controls. In conclusion, this study provides the first direct evidence that both G and D can induce microfollicular changes in the thyroid tissue and reduce the level of thyroid hormones in Orx middle-aged male rats, a model of andropause. This reduction consequently led to a feedback stimulation of pituitary TSH cells. The detected stimulatory effect was higher in the daidzein-treated rats.

Subcutaneously administrated genistein and daidzein decrease serum cholesterol and increase triglyceride levels in male middle-aged rats.

Nutritional supplements containing soybean phytoestrogens, the isoflavones genistein (G) and daidzein (D), are increasingly used as alternative therapy for osteoporosis, cancer, and cardiovascular and other diseases with a frequency that increases with advancing age. In this study we examined the effects of subcutaneous administration of either G or D on serum lipid levels in orchidectomized (Orx) and intact (IA) middle-aged male rats, which are experimental models of andropause. Sixteen-month-old Wistar rats were treated with 10 mg/kg and 30 mg/kg of either G or D. The control groups received testosterone, estradiol, or vehicle for 3 weeks, after which the total serum cholesterol (TC), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), and total triglycerides (TT) were measured. Compared with the matching vehicle-treated controls, the higher doses of G and D and testosterone treatment significantly (P < 0.05) lowered the TC and lipoprotein cholesterol levels. The greatest effect was observed regarding LDL-C in both Orx and IA males after G and D treatments, in which LDL-C decreased by more than 30%. The lower isoflavone doses induced a significant cholesterol-lowering effect (P < 0.05) only in the Orx group. Like the estradiol treatment, the higher doses of G and D increased the TT levels in both rat models by more than 50% (P < 0.05). The lower doses of isoflavones increased TT only in the Orx group. In male middle-aged rats, injections of higher doses of G and D decreased the serum cholesterol levels, as did testosterone injection, and brought about an increase in serum triglycerides similar to that observed after estradiol treatment.