Estrogen receptors (ER) are important regulators of metabolic diseases such as obesity and insulin resistance (IR). While ERα seems to have a protective role in such diseases, the function of ERβ is not clear. To characterize the metabolic function of ERβ, we investigated its molecular interaction with a master regulator of insulin signaling/glucose metabolism, the PPARγ, in vitro and in high-fat diet (HFD)-fed ERβ -/- mice (βERKO) mice. Our in vitro experiments showed that ERβ inhibits ligand-mediated PPARγ-transcriptional activity. That resulted in a blockade of PPARγ-induced adipocytic gene expression and in decreased adipogenesis. Overexpression of nuclear coactivators such as SRC1 and TIF2 prevented the ERβ-mediated inhibition of PPARγ activity. Consistent with the in vitro data, we observed increased PPARγ activity in gonadal fat from HFD-fed βERKO mice. In consonance with enhanced PPARγ activation, HFD-fed βERKO mice showed increased body weight gain and fat mass in the presence of improved insulin sensitivity. To directly demonstrate the role of PPARγ in HFD-fed βERKO mice, PPARγ signaling was disrupted by PPARγ antisense oligonucleotide (ASO). Blockade of adipose PPARγ by ASO reversed the phenotype of βERKO mice with an impairment of insulin sensitization and glucose tolerance. Finally, binding of SRC1 and TIF2 to the PPARγ-regulated adiponectin promoter was enhanced in gonadal fat from βERKO mice indicating that the absence of ERβ in adipose tissue results in exaggerated coactivator binding to a PPARγ target promoter. Collectively, our data provide the first evidence that ERβ-deficiency protects against diet-induced IR and glucose intolerance which involves an augmented PPARγ signaling in adipose tissue. Moreover, our data suggest that the coactivators SRC1 and TIF2 are involved in this interaction. Impairment of insulin and glucose metabolism by ERβ may have significant implications for our understanding of hormone receptor-dependent pathophysiology of metabolic diseases, and may be essential for the development of new ERβ-selective agonists.

We have previously reported epithelial cellular hyperplasia in ventral prostates (VP) of mice lacking estrogen receptor β (ERβ). To investigate the causes of this phenomenon, we measured cellular proliferation and apoptosis in VP of ERβ -/- and WT mice. With BrdUrd labeling, the number of proliferating cells was 3.6-fold higher in ERβ -/- mice. There was also a decrease in apoptosis as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay and an increase in expression of the anti-apoptotic bcl-2. The state of differentiation of the epithelial cells of the VP was studied by immunohistochemical staining, Western blotting, and fluorescence-activated cell sorting (FACS). In ERβ -/- mouse VP, the number of p63-positive cells (basal phenotype) was 2.6-fold higher, and expression level of cytokeratin (CK) 8, a luminal cell marker, was lower. FACS analysis with p63 showed that in WT mice the ratio of basal to intermediate/luminal cell populations expressing p63 was 1:2.5, whereas in ERβ -/- mice it was 1:9. The expression of basal/intermediate marker CK 19 in three FACS areas, g1, g2, and g3, gated according to cellular size and granularity, was 1:0.6:2 in WT and 1:4:6.7 in ERβ -/- mice, showing a shift of CK 19-positive cells toward a cell population of intermediate size and granularity. We conclude that, in ERβ -/- mouse VP, there is increased epithelial proliferation, decreased apoptosis, and accumulation of incompletely differentiated cells in an intermediate pool. The continued proliferation of intermediate cells leads to the prostatic epithelial hyperplasia observed in the absence of ERβ signaling.

Estrogens, which are stimulators of growth of both the normal breast and malignant breast, mediate their effects through two estrogen receptors (ER), namely ERA and ERB. ERA mediates the proliferative effect of estrogen in breast cancer cells, whereas ERB seems to be antiproliferative. We engineered ERA-positive T47D breast cancer cells to express ERB in a Tet-Off-regulated manner. These cells were then injected orthotopically into severe combined immunodeficient mice, and the growth of the resulting tumors was compared with tumors resulting from injecting the parental T47D cells that do not express ERB. The presence of ERB resulted in a reduction in tumor growth. Comparison of the ERB-expressing and non-ERB-expressing tumors revealed that the expression of ERB caused a reduction in the number of intratumoral blood vessels and a decrease in expression of the proangiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived growth factor B (PDGFB). In cell culture, with the Tet-Off-regulated ERB-expressing cells, expression of ERB decreased expression of VEGF and PDGFB mRNA under normoxic as well as hypoxic conditions and reduced secreted VEGF and PDGFB proteins in cell culture medium. Transient transfection assays with 1,026 bp VEGF and 1,006 bp PDGFB promoter constructs revealed a repressive effect of ERB at the promoter level of these genes. Taken together, these data show that introduction of ERB into malignant cells inhibits their growth and prevents tumor expansion by inhibiting angiogenesis. (Cancer Res 2006; 66(23): 11207-13)

Glucose uptake and homeostasis are regulated mainly by skeletal muscle (SM), white adipose tissue (WAT), pancreas, and the liver. Participation of estradiol in this regulation is still under intense investigation. We have demonstrated that, in SM of male mice, expression of the insulinregulated glucose transporter (GLUT)4 is reduced by estrogen receptor (ER)␤ agonists. In the present study, to investigate the relative contributions of ER␣ and ER␤ in glucose homeostasis, we examined the effects of tamoxifen (Tam) on GLUT4 expression in SM and WAT in wild-type (WT) and ERϪ/Ϫ mice. ER␤Ϫ/Ϫ mice were characterized by fasting hypoglycemia, increased levels of SM GLUT4, pancreatic islet hypertrophy, and a belated rise in plasma insulin in response to a glucose challenge. ER␣Ϫ/Ϫ mice, on the contrary, were hyperglycemic and glucose intolerant, and expression of SM GLUT4 was markedly lower than in WT mice. Tam had no effect on glucose tolerance or insulin sensitivity in WT mice. In ER␣Ϫ/Ϫ mice, Tam increased GLUT4 and improved insulin sensitivity. i.e., it behaved as an ER␤ antagonist in SM but had no effect on WAT. In ER␤Ϫ/Ϫ mice, Tam did not affect GLUT4 in SM but acted as an ER␣ antagonist in WAT, decreasing GLUT4. Thus, in the interplay between ER␣ and ER␤, ER␤-mediated repression of GLUT4 predominates in SM but ER␣-mediated induction of GLUT4 predominates in WAT. This tissue-specific difference in dominance of one ER over the other is reflected in the ratio of the expression of the two receptors. ER␣ predominates in WAT and ER␤ in SM.estrogen receptor-␣; estrogen receptor-␤; glucose transporter 4; tamoxifen FOR SEVERAL DECADES the multiple and sometimes contradictory effects of estrogens on human physiology and disease have puzzled endocrinologists. Even today, the modulatory effects of estrogen on glucose homeostasis are not completely understood. The effects of estrogen on gene regulation are mediated by two sometimes opposing forces, estrogen receptor (ER)␣ and ER␤ (20, 28), in both females and males (19,25). Recent experimental evidence has revealed that estradiol (E 2 ) is an important modulator in tissues previously not considered to be classical estrogen targets.In mice, insulin resistance develops when there is no estrogen (in aromatase-knockout mice) and when ER␣ is inactivated (ER␣Ϫ/Ϫ mice) (5,17,19). In women, a clear relationship between E 2 and glycemia has been observed during the menstrual cycle (32), in gestation (7), in gestational diabetes mellitus, and in polycystic ovarian syndrome (11). In normal males, because of the absence of estrogen cyclicity, the effect of estrogen on glycemia is less obvious, but men with hypoestrogenism or with mutations in the aromatase (13) or ER␣ (31) genes do develop insulin resistance.Insulin is the most important hormone for the maintenance of euglycemia. It regulates carbohydrate metabolism in the liver and glucose uptake in insulin-sensitive tissues, i.e., skeletal muscle (SM) and white adipose tissue (WAT) (12). On binding to its receptors on the cell membrane o...

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