Reduction of dendritic spines and elevation of GABAergic signaling in the brains of mice treated with an estrogen receptor ß ligand.

An estrogen receptor (ER) ß ligand (LY3201) with a preference for ERß over ERα was administered in s.c. pellets releasing 0.04 mg/d. The brains of these mice were examined 3 d after treatment had begun. Although estradiol-17ß is known to increase spine density and glutaminergic signaling, as measured by Golgi staining, a clear reduction in spines was evident on the dendritic branches in LY3201-treated mice but no morphological alteration and no difference in the number of dendritic spines on dendritic stems were observed. In the LY3201-treatment group, there was higher expression of glutamic acid decarboxylase (GAD) in layer V of cortex and in the CA1 of hippocampus, more GAD(+) terminals surrounding the pyramidal neurons and less glutamate receptor (NMDAR) on the neurons in layer V. There were no alterations in expression of Iba1 or in Olig2 or CNPase. However, GFAP(+) astrocytes were increased in the LY3201-treatment group. There were also more projections characteristic of activated astrocytes and increased expression of glutamine synthetase (GS). No expression of ERß was detectable in the nuclei of astrocytes. Clearly, LY3201 caused a shift in the balance between excitatory and inhibitory neurotransmission in favor of inhibition. This shift was due in part to increased synthesis of GABA and increased removal of glutamate from the synaptic cleft by astrocytes. The data reveal that treatment with a selective ERß agonist results in changes opposite to those reported in estradiol-17ß-treated mice and suggests that ERα and ERß play opposing roles in the brain.

Both liver-X receptor (LXR) isoforms control energy expenditure by regulating brown adipose tissue activity.

Brown adipocytes are multilocular lipid storage cells that play a crucial role in nonshivering thermogenesis. Uncoupling protein 1 (UCP1) is a unique feature of brown fat cells that allows heat generation on sympathetic nervous system stimulation. As conventional transcriptional factors that are activated in various signaling pathways, liver-X receptors (LXRs) play important roles in many physiological processes. The role of LXRs in the regulation of energy homeostasis remains unclear, however. Female WT, LXRαß(-/-), LXRα(-/-), and LXRß(-/-) mice were fed with either a normal diet (ND) or a high-carbohydrate diet (HCD) supplemented with or without GW3965-LXR agonist. LXRαß(-/-) mice exhibited higher energy expenditure (EE) as well as higher UCP1 expression in brown adipose tissue (BAT) compared with WT mice on the HCD. In addition, long-term treatment of WT mice with GW3965 showed lower EE at thermoneutrality (30 °C) and lower Ucp1 expression level in BAT. Furthermore, H&E staining of the BAT of LXRαß(-/-) mice exhibited decreased lipid droplet size compared with WT mice on the HCD associated with a more intense UCP1-positive reaction. Quantification of triglyceride (TG) content in BAT showed lower TG accumulation in LXRß(-/-) mice compared with WT mice. Surprisingly, GW3965 treatment increased TG content (twofold) in the BAT of WT and LXRα(-/-) mice but not in LXRß(-/-) mice. Furthermore, glucose transporter (GLUT4) in the BAT of LXRα(-/-) and LXRß(-/-) mice was sixfold and fourfold increased, respectively, compared with WT mice on the ND. These findings suggest that LXRα as well as LXRß could play a crucial role in the regulation of energy homeostasis in female mice and may be a potential target for the treatment of obesity and energy regulation.

Liver X receptors regulate de novo lipogenesis in a tissue-specific manner in C57BL/6 female mice.

The liver X receptors (LXRs) play a key role in cholesterol and bile acid metabolism but are also important regulators of glucose metabolism. Recently, LXRs have been proposed as a glucose sensor affecting LXR-dependent gene expression. We challenged wild-type (WT) and LXRαß(-/-) mice with a normal diet (ND) or a high-carbohydrate diet (HCD). Magnetic resonance imaging showed different fat distribution between WT and LXRαß(-/-) mice. Surprisingly, gonadal (GL) adipocyte volume decreased on HCD compared with ND in WT mice, whereas it slightly increased in LXRαß(-/-) mice. Interestingly, insulin-stimulated lipogenesis of isolated GL fat cells was reduced on HCD compared with ND in LXRαß(-/-) mice, whereas no changes were observed in WT mice. Net de novo lipogenesis (DNL) calculated from Vo(2) and Vco(2) was significantly higher in LXRαß(-/-) than in WT mice on HCD. Histology of HCD-fed livers showed hepatic steatosis in WT mice but not in LXRαß(-/-) mice. Glucose tolerance was not different between groups, but insulin sensitivity was decreased by the HCD in WT but not in LXRαß(-/-) mice. Finally, gene expression analysis of adipose tissue showed induced expression of genes involved in DNL in LXRαß(-/-) mice compared with WT animals as opposed to the liver, where expression of DNL genes was repressed in LXRαß(-/-) mice. We thus conclude that absence of LXRs stimulates DNL in adipose tissue, but suppresses DNL in the liver, demonstrating opposite roles of LXR in DNL regulation in these two tissues. These results show tissue-specific regulation of LXR activity, a crucial finding for drug development.

Participation of ERalpha and ERbeta in glucose homeostasis in skeletal muscle and white adipose tissue.

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 insulin-regulated glucose transporter (GLUT)4 is reduced by estrogen receptor (ER)beta agonists. In the present study, to investigate the relative contributions of ERalpha and ERbeta in glucose homeostasis, we examined the effects of tamoxifen (Tam) on GLUT4 expression in SM and WAT in wild-type (WT) and ER-/- mice. ERbeta-/- 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. ERalpha-/- 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 ERalpha-/- mice, Tam increased GLUT4 and improved insulin sensitivity. i.e., it behaved as an ERbeta antagonist in SM but had no effect on WAT. In ERbeta-/- mice, Tam did not affect GLUT4 in SM but acted as an ERalpha antagonist in WAT, decreasing GLUT4. Thus, in the interplay between ERalpha and ERbeta, ERbeta-mediated repression of GLUT4 predominates in SM but ERalpha-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. ERalpha predominates in WAT and ERbeta in SM.

Estrogen receptors: new players in diabetes mellitus.

Diabetes mellitus type 2 is a systemic disease characterized by imbalance of energy metabolism, which is mainly caused by inadequate insulin action. Recent data have revealed a surprising role for estradiol in regulating energy metabolism and opened new insights into the role of the two estrogen receptors, ERalpha and ERbeta, in this context. New findings on gene modulation by ERalpha and ERbeta of insulin-sensitive tissues indicate that estradiol participates in glucose homeostasis by modulating the expression of genes that are involved in insulin sensitivity and glucose uptake. Drugs that can selectively modulate the activity of either ERalpha or ERbeta in their interactions with target genes represent a promising frontier in diabetes mellitus coadjuvant therapy.

An ERß agonist induces browning of subcutaneous abdominal fat pad in obese female mice.

Estrogen, via estrogen receptor alpha (ERα), exerts several beneficial effects on metabolism and energy homeostasis by controlling size, enzymatic activity and hormonal content of adipose tissue. The actions of estrogen on sympathetic ganglia, which are key players in the browning process, are less well known. In the present study we show that ERß influences browning of subcutaneous adipose tissue (SAT) via its actions both on sympathetic ganglia and on the SAT itself. A 3-day-treatment with a selective ERß agonist, LY3201, induced browning of SAT in 1-year-old obese WT and ERα-/- female mice. Browning was associated with increased expression of ERß in the nuclei of neurons in the sympathetic ganglia, increase in tyrosine hydroxylase in both nerve terminals in the SAT and sympathetic ganglia neurons and an increase of ß3-adrenoceptor in the SAT. LY3201 had no effect on browning in young female or male mice. In the case of young females browning was already maximal while in males there was very little expression of ERß in the SAT and very little expression of the ß3-adrenoceptor. The increase in both sympathetic tone and responsiveness of adipocytes to catecholamines reveals a novel role for ERß in controlling browning of adipose tissue.

Insight into the mechanisms of action of estrogen receptor ß in the breast, prostate, colon, and CNS.

Estrogen and its receptors (ERs) influence many biological processes in physiology and pathology in men and women. ERs are involved in the etiology and/or progression of cancers of the prostate, breast, uterus, ovary, colon, lung, stomach, and malignancies of the immune system. In estrogen-sensitive malignancies, ERß usually is a tumor suppressor and ERα is an oncogene. ERß regulates genes in several key pathways including tumor suppression (p53, PTEN); metabolism (PI3K); survival (Akt); proliferation pathways (p45(Skp2), cMyc, and cyclin E); cell-cycle arresting factors (p21(WAF1), cyclin-dependent kinase inhibitor 1 (CDKN1A)), p27(Kip1), and cyclin-dependent kinases (CDKs); protection from reactive oxygen species, glutathione peroxidase. Because they are activated by small molecules, ERs are excellent targets for pharmaceuticals. ERα antagonists have been used for many years in the treatment of breast cancer and more recently pharmaceutical companies have produced agonists which are very selective for ERα or ERß. ERß agonists are being considered for preventing progression of cancer, treatment of anxiety and depression, as anti-inflammatory agents and as agents, which prevent or reduce the severity of neurodegenerative diseases.

Estrogen receptors and the metabolic network.

The metabolic syndrome has reached pandemic level worldwide, and evidence is that estradiol plays a key role in its development. The discovery of the second estrogen receptor, ERß, in tissues previously not considered targets of estradiol was a breakthrough in endocrinology. In the present review, we discuss how the presence of ERß and the previously described ERα in tissues involved in glucose and lipid homeostasis (brain, skeletal muscle, adipose tissue, pancreas, liver, and heart) may have important implications to risk factors associated with the metabolic syndrome. Imbalance of ERα/ERß ratio in this "metabolic network" may lead to the metabolic syndrome.

ERbeta: recent understanding of estrogen signaling.

The discovery of a second estrogen receptor, ERbeta, and the finding that 5alpha-androstane-3beta,17beta-diol (3betaAdiol) strongly binds to ERbeta, have opened up a new aspect of estrogen signaling. Some of the major shifts in our understanding come from finding ERbeta in tissues which do not express ERalpha but are estrogen-responsive; these were called sites of 'indirect estrogen action'. Two key sites that fall into this category are the brain and the prostate. Studies of ERbeta in the past 10 years have led us to hypothesize that estrogen signaling depends on the balance between ERalpha and ERbeta, and that inadequate predominance of one or the other isoform could lead to disease.

Muscle GLUT4 regulation by estrogen receptors ERbeta and ERalpha.

Estrogen is known to influence glucose homeostasis with dominant effects in the liver, but the role of estrogen receptors in muscle glucose metabolism is unknown. In the present study, we investigated the expression of the two estrogen receptors, ERalpha and ERbeta, and their influence on regulation of the glucose transporter, GLUT4, and its associated structural protein, caveolin-1, in mouse gastrocnemius muscle. Immunohistochemical analysis revealed that ERalpha and ERbeta are coexpressed in the nuclei of most muscle cells, and that their levels were not affected by absence of estradiol [in aromatase-knockout (ArKO) mice]. GLUT4 expression on the muscle cell membrane was not affected by loss of ERbeta but was extremely reduced in ERalpha(-/-) mice and elevated in ArKO mice. RT-PCR confirmed a parallel reduction in GLUT4 mRNA levels in ERalpha(-/-) mice. Upon treatment of ArKO mice with the ERbeta agonist 2,3-bis(4-hydroxyphenyl)propionitrile, GLUT4 expression was reduced. By immunofluorescence and Western blotting, caveolin-1 expression was higher in ArKO mice and lower in ERbeta(-/-) and ERalpha(-/-) mice than in WT littermates. GLUT4 and caveolin-1 were colocalized in WT and ArKO mice but not in ERbeta(-/-) and ERalpha(-/-) mice. These results reveal that ERalpha is a positive regulator of GLUT4 expression, whereas ERbeta has a suppressive role. Both ERbeta and ERalpha are necessary for optimal caveolin-1 expression. Taken together, these results indicate that colocalization of caveolin-1 and GLUT4 is not an absolute requirement for muscle glucose metabolism but that reduction in GLUT4 could be contributing to the insulin resistance observed in ERalpha(-/-) mice.

Lung dysfunction causes systemic hypoxia in estrogen receptor beta knockout (ERbeta-/-) mice.

Estrogen receptor beta (ERbeta) is highly expressed in both type I and II pneumocytes as well as bronchiolar epithelial cells. ERalpha is not detectable in the adult lung. Lungs of adult female ERbeta knockout (ERbeta-/-) mice have already been reported to have fewer alveoli and reduced elastic recoil. In this article, we report that, by 5 months of age, there are large areas of unexpanded alveoli in lungs of both male and female ERbeta-/- mice. There is increased staining for collagen and, by EM, abnormal clusters of collagen fibers are seen in the alveolar septa of ERbeta-/- mice. Immunohistochemical analysis and Western blotting with lung membrane fractions of ERbeta-/- mice revealed down-regulation of caveolin-1, increased expression of membrane type-1 metalloproteinase, matrix metalloproteinase 2 (active form), and tissue inhibitors of metalloproteinases 2. Hypoxia, measured by immunohistochemical analysis for hypoxia-inducible factor 1alpha and chemical adducts (with Hypoxyprobe), was evident in the heart, ventral prostate, periovarian sac, kidney, liver, and brain of ERbeta-/- mice under resting conditions. Furthermore, both male and female adult ERbeta-/- mice were reluctant to run on a treadmill and tissue hypoxia became very pronounced after exercise. We conclude that ERbeta is necessary for the maintenance of the extracellular matrix composition in the lung and loss of ERbeta leads to abnormal lung structure and systemic hypoxia. Systemic hypoxia may be responsible for the reported left and right heart ventricular hypertrophy and systemic hypertension in ERbeta-/- mice.

Role of estrogen receptor beta in uterine stroma and epithelium: Insights from estrogen receptor beta-/- mice.

In this study, we compared the uterine tissue of estrogen receptor (ER)beta(-/-) mice and their WT littermates for differences in morphology, proliferation [the percentage of labeled cells 2 h after BrdUrd injection and EGF receptor (EGFR) expression], and differentiation (expression of progesterone receptor, E-cadherin, and cytokeratins). In ovariectomized mice, progesterone receptor expression in the uterine epithelium was similar in WT and ERbeta(-/-) mice, but E-cadherin and cytokeratin 18 expression was lower in ERbeta(-/-) mice. The percentage of cells in S phase was 1.5% in WT mice and 8% in ERbeta(-/-) mice. Sixteen hours after injection of 17beta-estradiol (E(2)), the number of BrdUrd-labeled cells increased 20-fold in WT mice and 80-fold in ERbeta(-/-) mice. Although ERalpha was abundant in intact mice, after ovariectomy, ERalpha could not be detected in the luminal epithelium of either WT or ERbeta(-/-) mice. In both untreated and E(2)-treated mice, ERalpha and ERbeta were colocalized in the nuclei of many stromal and glandular epithelial cells. However, upon E(2) + progesterone treatment, ERalpha and ERbeta were not coexpressed in any cells. In WT mice, EGFR was located on the membranes and in the cytoplasm of luminal epithelium, but not in the stroma. In ERbeta(-/-) mice, there was a marked expression of EGFR in the nuclei of epithelial and stromal cells. Upon E(2) treatment, EGFR on cell membranes was down-regulated in WT but not in ERbeta(-/-) mice. These findings reveal an important role for ERbeta in response to E(2) and in the organization, growth, and differentiation of the uterine epithelium.