Adipose tissue is responsive to estrogen and expresses both estrogen receptor alpha and beta. To test the hypothesis that the estrogenic soy isoflavone genistein can have effects on adipose tissue, juvenile or adult C57/BL6 mice were ovariectomized and given daily injections of vehicle, 17beta-estradiol (5 microg/kg.d) or genistein (8-200 mg/kg.d) sc for 21-28 d. To test effects of dietary genistein, 25- to 27-d-old mice were fed diets containing 0-1500 parts per million (ppm) genistein for 12 d. Mice were killed and fat pads weighed. Parametrial fat pads were used for morphometric and Northern analysis. Genistein injections decreased adipose weight and adipocyte circumference at higher doses; effects in adult and juvenile mice were similar. Genistein decreased lipoprotein lipase mRNA, which may be a critical aspect of its adipose effects. Juveniles fed 500-1500 ppm dietary genistein had dose-responsive decreases in fat pad weights of 37-57%, compared with controls; 300 ppm genistein did not cause decreases. Genistein doses of 300, 500, 1000, and 1500 ppm produced serum genistein concentrations of 1.02 +/- 0.14 microM, 1.79 +/- 0.32 microM, 2.55 +/- 0.18 microM, and 3.81 +/- 0.39 microM, respectively. These results indicate dietary genistein at 500-1500 ppm produces antilipogenic effects in mice at serum levels that humans are realistically exposed to.

Adipose tissue deposition is highly responsive to estrogen; ovariectomy increases adipose deposition, and estrogen replacement reverses this. Estrogen receptor alpha (ERalpha) plays a major role in adipose tissue. ERalpha knockout (alphaERKO) mice show an increase in adipose tissue of over a 100 % compared to wild-type mice. However, alphaERKO mice undergo a 10-fold increase in 17beta-estradiol (E2), and persistent or even increased signaling through ERbeta could be a factor in obesity of alphaERKO mice. To test the hypothesis that ERbeta plays a role in adipose tissue, adult female alphaERKO mice were ovariectomized or sham-ovariectomized and fed a phytoestrogen-free diet. Ovariectomized mice were treated with vehicle or E2, and bodyweights and food consumption were measured. Mice were killed after 28 days and inguinal and parametrial fat pads collected. Sham-ovariectomized alphaERKO mice had increased body weight, ovariectomized alphaERKO mice showed a 6 % decrease, and E2 replacement restored body weight to sham levels. Fat pads of ovariectomized alphaERKO mice showed 45 % and 16 % decreases in weight and adipocyte circumference, respectively, compared to sham-ovariectomized or E2-replaced ovariectomized alphaERKO mice. Ovariectomized alphaERKO mice showed a trend towards decreased feed consumption that did not reach significance. Blood glucose levels were lower both before and after glucose injection in ovariectomized compared to sham alphaERKO mice, and E2 treatment reversed this. Insulin levels following glucose challenge were lower in ovariectomized compared to sham-ovariectomized alphaERKO mice, indicating that ovariectomy ameliorated the glucose intolerance and insulin resistance in alphaERKO mice. Immunohistochemical analysis revealed strong staining for ERbeta in adipose tissue. These observations indicate that removing E2/ERbeta signaling in alphaERKO mice by ovariectomy decreases body and fat-pad weights and adipocyte size, while improving insulin and glucose metabolism. ERbeta mediated effects on adipose tissue are opposite those of ERalpha, although E2 effects on adipose tissue are predominately through ERalpha.

The soy phytoestrogen, genistein, induces thymic atrophy when administered to ovariectomized mice by injection or in the diet. Injected genistein also causes decreased humoral immunity, but the effects of genistein on cellmediated immunity have not been addressed. Here we examined effects of injected and dietary genistein on cell-mediated immune responses. Female C57BL/6 mice (25-to 27-days-old) were ovariectomized, then placed on phytoestrogen-free feed 5 days later. Seven days after ovariectomy, they were given daily subcutaneous injections of either dimethylsulfoxide (DMSO) or genistein (8, 20, 80 mg/kg) for 28 days; some mice were given 80 mg/ kg genistein plus the anti-estrogen ICI 182,780 (5 mg/ kg/week). Cell-mediated immune response was tested by analyzing the delayed-type hypersensitivity (DTH) response to a hapten, 4-hydroxy-3-nitrophenyl acetyl succinimide (NP-O-SU), at the end of treatment. Reversibility of the effects of genistein was tested by measuring the DTH response in mice that were given genistein (20 or 80 mg/kg) for 28 days, then allowed to recover for 28 days. To determine if dietary genistein could affect cell-mediated immunity, mice ovariectomized as above were fed genistein at 0, 1000 or 1500 parts per million (ppm) for 28 days. There was a 46-67% decrease in the DTH response in the footpads of mice injected with 8-80 mg/kg genistein compared with controls (P<0·05 vs control for all treatment groups); these effects were reversible. On histopathological examination of the feet, there was decreased cell infiltration in genistein-treated animals compared with controls, and the numbers of CD4 + and CD8 + T cells in popliteal lymph nodes were reduced. The effects of genistein are mediated through both estrogen receptor (ER) and non-ER pathways, as the anti-estrogen ICI 182,780 only partially blocked the effects of genistein on the DTH response. Dietary genistein (1000 or 1500 ppm) decreased cellmediated immunity while producing serum genistein concentrations in the physiological range for humans under certain nutritional conditions. Further work is needed to determine if dietary genistein and phytoestrogen exposure can produce effects on cell-mediated immunity in humans or other animals under various nutritional conditions.

Equol, a metabolite of the phytoestrogen daidzein, is present at significant levels in some humans who consume soy and in rodents fed soy-based diets. Equol is estrogenic in vitro, but there have been limited studies of its activity in vivo. We evaluated equol effects on reproductive and non-reproductive endpoints in mice. Ovariectomized age-matched (30-day-old) female C57BL/6 mice were fed phytoestrogen-free diets and given a racemic mixture of equol by daily injections (0, 4, 8, 12, or 20 mg [kg body weight](-1) day(-1)) or in the diet (0, 500, or 1,000 ppm) for 12 days. Mice were killed, and serum concentrations of total and aglycone equol were measured. Total serum equol concentrations ranged from 1.4 to 7.5 microM with increasing doses of injected equol, but uterine weight increased significantly only at 12 and 20 mg (kg body weight)(-1) day(-1). Dietary equol at 500 or 1,000 ppm produced total serum equol concentrations of 5.9 and 8.1 microM, respectively, comparable with those in rodents consuming certain high-soy chows; the proportion of equol present as the free aglycone was much lower with dietary administration than injections, which may be a factor in the greater biological effects induced by injections. Dietary equol did not significantly increase uterine weight. Increasing dietary and injected equol doses caused a dose-dependent increase in vaginal epithelial thickness. Uterine epithelial proliferation was increased by equol injections at 8-20 mg (kg body weight)(-1) day(-1) and 1,000 ppm dietary equol. Neither dietary nor injected equol decreased thymic or adipose weights. In conclusion, equol is a weak estrogen with modest effects on endpoints regulated by estrogen receptor alpha when present at serum levels seen in rodents fed soy-based diets, but quantities present in humans may not be sufficient to induce estrogenic effects, although additive effects of equol with other phytoestrogens may occur.

Gender influences mediated by 17␤-estradiol (E2) have been associated with susceptibility to and severity of autoimmune diseases such as diabetes, arthritis, and multiple sclerosis. In this regard, we have shown that estrogen receptor-␣ (Esr1) is crucial for the protective effect of 17␤-estradiol (E2) in murine experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis. The expression of estrogen receptors among various immune cells (eg, T and B lymphocytes, antigen-presenting cells) suggests that the therapeutic effect of E2 is likely mediated directly through specific receptor binding. However, the target immune cell populations responsive to E2 treatment have not been identified. In the current study, we induced EAE in T-cell-deficient, severe combined immunodeficient mice or in immunocompetent mice with encephalitogenic T cells from wildtype Esr1؉/؉ or Esr1 knockout (Esr1؊/؊) donors and compared the protective E2 responses. The results showed that E2-responsive, Esr1؉/؉ diseaseinducing encephalitogenic T cells were neither necessary nor sufficient for E2-mediated protection from EAE. Instead, the therapeutic response appeared to be mediated through direct effects on nonlymphocytic, E2-responsive cells and down-regulation of the inflammatory response in the central nervous system. These results provide the first demonstration that the protective effect of E2 on EAE is not mediated directly through E2-responsive T cells and raise the alternative possibility that nonlymphocytic cells such as macrophages, dendritic cells, or other nonlymphocytic cells are primarily responsive to E2 treatment in