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In female mammals, the cessation of ovarian functions is associated with significant metabolic alterations, weight gain, and increased susceptibility to a number of pathologies associated with ageing. The molecular mechanisms triggering these systemic events are unknown because most tissues are responsive to lowered circulating sex steroids. As it has been demonstrated that isoform alpha of the estrogen receptor (ERα) may be activated by both estrogens and amino acids, we test the metabolic effects of a diet enriched in specific amino acids in ovariectomized (OVX) mice. This diet is able to block the OVX-induced weight gain and fat deposition in the liver. The use of liver-specific ERα KO mice demonstrates that the hepatic ERα, through the control of liver lipid metabolism, has a key role in the systemic response to OVX. The study suggests that the liver ERα might be a valuable target for dietary treatments for the post-menopause. Menopause is associated with metabolic changes and an increased risk of a number of chronic diseases. Here, the authors show in a mouse model of menopause that an amino acid-enriched diet rescues liver lipid metabolism and prevents weight gain in a manner dependent on hepatic ERα. [ABSTRACT FROM AUTHOR]

Background: Microglia are resident myeloid cells of the central nervous system (CNS) that are maintained by self-renewal and actively participate in tissue homeostasis and immune defense. Under the influence of endogenous or pathological signals, microglia undertake biochemical transformations that are schematically classified as the pro-inflammatory M1 phenotype and the alternatively activated M2 state. Dysregulated proliferation of M1-activated microglia has detrimental effects, while an increased number of microglia with the alternative, pro-resolving phenotype might be beneficial in brain pathologies; however, the proliferative response of microglia to M2 signals is not yet known. We thus evaluated the ability of interleukin-4 (IL-4), a typical M2 and proliferative signal for peripheral macrophages, to induce microglia proliferation and compared it with other proliferative and M2 polarizing stimuli for macrophages, namely colony-stimulating factor-1 (CSF-1) and the estrogen hormone, 17β-estradiol (E2).Methods: Recombinant IL-4 was delivered to the brain of adult mice by intracerebroventricular (i.c.v.) injection; whole brain areas or ex vivo-sorted microglia were analyzed by real-time PCR for assessing the mRNA levels of genes related with cell proliferation (Ki67, CDK-1, and CcnB2) and M2 polarization (Arg1, Fizz1, Ym-1) or by FACS analyses of in vivo BrdU incorporation in microglia. Primary cultures of microglia and astrocytes were also tested for proliferative effects.Results: Our results show that IL-4 only slightly modified the expression of cell cycle-related genes in some brain areas but not in microglia, where it strongly enhanced M2 gene expression; on the contrary, brain delivery of CSF-1 triggered proliferation as well as M2 polarization of microglia both in vivo and in vitro. Similar to IL-4, the systemic E2 administration failed to induce microglia proliferation while it increased M2 gene expression.Conclusions: Our data show that, in contrast to the wider responsiveness of peripheral macrophages, microglia proliferation is stimulated by selected M2 polarizing stimuli suggesting a role for the local microenvironment and developmental origin of tissue macrophages in regulating self-renewal following alternative activating stimuli. [ABSTRACT FROM AUTHOR]

Abstract: In recent years several studies demonstrated the presence of estrogen receptors in mammalian tissues and significantly improved our understanding of their ability to control biological processes in reproductive as well as non-reproductive organs. Considering the manifold mechanisms and organs that are involved in estrogen action and the implication of estrogens in human female physiology, innovative approaches are required to shed light on the widespread activities of estrogen receptors in woman physiology. This is particularly relevant for the definition of novel, more efficacious hormonal replacement therapies or for the evaluation of the risk associated with the exposure to endocrine disruptors. The introduction of genetic engineering and the development and application of in vivo imaging techniques offer new tools for pre-clinical studies. The generation of the ERE-Luc mouse, a reporter animal developed for in vivo studies of the estrogen receptor activity, allows assessing the activity state of the ER signaling pathway in all target tissues and organs at once, under physiological stimuli or as a result of a pharmacological treatment. This review summarizes the main steps in the generation and appraisal of the estrogen receptor reporter mouse ERE-Luc, designed for in vivo molecular imaging studies, and describes examples demonstrating the suitability of the ERE-Luc model for drug development and for the investigation of the effects of endogenous, environmental, and dietary estrogens in vivo. This article is part of a Special Issue entitled ‘Phytoestrogens’. [Copyright &y& Elsevier]

The article presents a study whcih investigates cell proliferation in reproductive and nonreproductive tissues in mice using a novel transgenic mouse. The study made use of the bioluminescence-based in vivo imaging to monitor the proliferative effects of treatments. The result of the study shows that there was a large increase in luciferase activity underlying the hormone's proliferative effect.

In the liver of female mice, the transcriptional activity of estrogen receptor (ER) α oscillates in phase with the 4-d-long estrous cycle. Here systemic, genome-wide analysis demonstrates that ER tetradian oscillation is necessary to generate pulses of expression in genes for fatty acid and cholesterol synthesis. This ER-dependent metabolic programming changes with pregnancy and after cessation of ovarian function due to age or surgical menopause, suggesting that ER signaling is optimized to coordinate liver functions with the energetic requirements of each reproductive stage. Alterations of amplitude and frequency of the tetradian cycle, as observed after surgical menopause, age, or specific ablation of the hepatic lgf-1 gene, are associated with liver fat deposition. Appropriate hormone replacement therapy reinstating the oscillatory activity of liver ER prevents the effect of surgical menopause on fat deposition in liver. [ABSTRACT FROM AUTHOR]

Estrogen receptors have long been known to be expressed in several brain areas in addition to those directly involved in the control of reproductive functions. Investigations in humans and in animal models suggest a strong influence of estrogens on limbic and motor functions, yet the complexity and heterogeneity of neural tissue have limited our approaches to the full understanding of estrogen activity in the central nervous system. The aim of this Study was to examine the transcriptional activity of estrogen receptors in the brain of male and female mice. Exploiting the ERE-Luc reporter mouse, we set up a novel, bioluminescence-based technique to study brain estrogen receptor transcriptional activity. Here we show, for the first time, that estrogen receptors are similarly active in male and female brains and that the estrous us cycle affects estrogen receptor activity in regions of the central nervous system not known to be associated with reproductive functions. Because of its reproducibility and sensitivity, this novel bioluminescence application stands as a candidate as an innovative methodology for the study and development of drugs targeting brain estrogen receptors. [ABSTRACT FROM AUTHOR]

During the past decade remarkable progress in molecular genetics and the possibility of manipulating cells so that the expression of genes can directly ‘report’ on drug activity has produced major changes in drug development strategies. The recent description and pharmacological validation of reporter mice for in vivo analysis of hormone receptor activity opens new horizons for drug discovery. These novel animal models, in association with in vivo imaging technologies, provide a global view of the target tissues of drug action following acute and repeated drug treatment, thus enabling the prediction of potential side-effects in the early phase of preclinical studies. It is anticipated that further improvements of transgene architecture will lead to models that combine pharmacokinetic, pharmacodynamic and toxicological studies in a single step, which should provide a tremendous saving in time and, paradoxically, the number of animals to be sacrificed in the development of novel pharmacologically active molecules. [Copyright &y& Elsevier]

Highlights: [•] We used the ERE–luciferase mouse to assess real-time estrogen receptor activation. [•] We demonstrate temporally regulated peri-wound activation of estrogen signalling. [•] This signal is localised to keratinocytes and infiltrating dermal cells. [•] Using pharmacological agonists we reveal specific differences in active signalling. [Copyright &y& Elsevier]

Recent evidence highlights the peroxisome proliferator-activated receptors (PPARs) as critical neuroprotective factors in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). To gain new mechanistic insights into the role of these receptors in the context of ALS, here we investigated how PPAR transcriptional activity varies in hSOD1G93A ALS transgenic mice.Wedemonstrate that PPAR-αdriven transcription selectively increases in the spinal cord of symptomatic hSOD1G93A mice. This phenomenon correlates with the upregulation of target genes, such as lipoprotein lipase and glutathione S-transferase -α2, which are implicated in scavenging lipid peroxidation by-products. Such events are associated with enhanced PPARγ immunoreactivity withinmotorneuronal nuclei. This observation, and the fact that PPARγ displays increased responsiveness in cultured hSOD1G93A motor neurons, points to a role for this receptor in neutralizing deleterious lipoperoxidation derivatives within the motor cells. Consistently, in both motor neuron-like cultures and animal models, we report that PPARγ is activated by lipid peroxidation end products, such as 4-hydroxynonenal, whose levels are elevated in the cerebrospinal fluid and spinal cord from ALSpatients.Wepropose that the accumulation of critical concentrations of lipid peroxidation adducts during ALS progression leads to the activation of PPARγ in motor neurons. This in turn triggers self-protective mechanisms that involve the up-regulation of lipid detoxification enzymes, such as lipoprotein lipase and glutathione S-transferaseα-2. Our findings indicate that anticipating natural protective reactions by pharmacologically modulating PPARγ transcriptional activity may attenuate attenuate neurodegeneration by limiting the damage induced by lipid peroxidation derivatives. [ABSTRACT FROM AUTHOR]

Abstract: Detailed knowledge of drug metabolism is relevant information provided by preclinical drug development research. Oxidative enzymes such as those belonging to P450 family of cytochromes (CYP) play a prominent role in drug metabolism. Here, we propose an innovative method based on bioluminescence in vivo imaging which has the potential to simplify the in vivo measurement of CYP activity also providing a dynamic measure of the effects of a drug on a specific P450 enzyme complex in a living mouse. The method is based on a pro-luciferin which can be converted into the active luciferase substrate by a specific P450 activity. The pro-luciferin is administered to a luciferase reporter mouse which produces luminescent signals in relation to the cytochrome activity present in each tissue. The photon emission generated can be easily localized and quantified by optical imaging. To demonstrate the validity of the system, we pharmacologically induced hepatic Cyp3a in the reporter mouse and proved that pro-luciferin administration generates a Cyp3a selective signal in the chest area that can be efficiently detected by optical imaging. The kind of tool generated has the potential to be exploited for the study of additional CYPs. [Copyright &y& Elsevier]

Abstract: Cadmium (Cd), an environmental and dietary contaminant, has been described to mimic the effects of 17β-estradiol (E2) in selected model systems when studied as an inorganic salt. However, inorganic Cd salts do not represent the main form of Cd exposure in general human populations. The aims of this study were to compare the estrogen-like effects and the bioavailability of dietary Cd to inorganic CdCl2. Adult ovariectomized ERE-luc reporter mice were administered two bread based diets containing different concentrations of Cd (17.57 and 49.22μg/kg, corresponding to oral intakes of 1.8 and 5.1μg/kg body weight (bw) per day, respectively), inorganic CdCl2 (1μg/kg bw per day by gavage) or E2 (5μg/kg bw per day pellet) for 21 days. The effects on estrogen signaling were investigated by studying the uterine weights, luciferase activation, and expression of endogenous estrogen target genes. The uterine weight was significantly increased by both CdCl2 and E2 but not by the Cd containing diets. All treatments modulated the expression of luciferase and the endogenous estrogen target genes; however, there was no consistent overlap between the responses triggered by the bread diets and the responses stimulated by CdCl2 or E2. Oral exposure to Cd was calculated and the concentrations in liver and kidneys quantified to estimate the amount of absorbed Cd retained in tissues. The results suggest significantly lower absorption and/or tissue retention of dietary Cd compared to CdCl2 following oral exposure. Altogether, our results support previous reports on in vivo estrogenicity of CdCl2 but do not suggest the same activity for diet bound Cd. This study calls for caution when extrapolating results from pure compound studies (e.g. estrogenicity of CdCl2) to dietary exposure scenarios (e.g. estrogenicity of diet bound Cd). Further basic research is needed on the mechanisms of interaction between Cd and the estrogen signaling, biologically active species of Cd, and biomarkers of estrogen-like effects of Cd in vivo before human health risk assessment on the hormone disruptive effects of Cd can be carried out. [Copyright &y& Elsevier]

Steroid hormone receptors function classically in the nucleus as transcription factors. However, recent data indicate that there are also non-nuclear subpopulations of steroid hormone receptors, including estrogen receptors (ERs), that mediate membrane-initiated signaling of unclear basis and significance. Here we have shown that an estrogen-dendrimer conjugate (EDC) that is excluded from the nucleus stimulates endothelial cell proliferation and migration via ERalpha, direct ERalpha-Galphai interaction, and endothelial NOS (eNOS) activation. Analysis of mice carrying an estrogen response element luciferase reporter, ER-regulated genes in the mouse uterus, and eNOS enzyme activation further indicated that EDC specifically targets non-nuclear processes in vivo. In mice, estradiol and EDC equally stimulated carotid artery reendothelialization in an ERalpha- and G protein-dependent manner, and both agents attenuated the development of neointimal hyperplasia following endothelial injury. In contrast, endometrial carcinoma cell growth in vitro and uterine enlargement and MCF-7 cell breast cancer xenograft growth in vivo were stimulated by estradiol but not EDC. Thus, EDC is a non-nuclear selective ER modulator (SERM) in vivo, and in mice, non-nuclear ER signaling promotes cardiovascular protection. These processes potentially could be harnessed to provide vascular benefit without increasing the risk of uterine or breast cancer. [ABSTRACT FROM AUTHOR]

Abstract: Background: Because of the complexity of estrogen receptor (ER) physiological activity, the interaction of pure isoflavones or soy-based diets on ER needs to be clearly demonstrated. Objectives: To investigate the effects of the administration of isoflavones as a pure compound or as a component of diet on the ER transcriptional activity in adult mice. Methods: Effects of acute (6 h) and chronic (21 days) oral administration of soy milk, pure genistein and a mix of genistein and daidzein was studied in living ERE-Luc mice. In this animal model, the synthesis of luciferase is under the state of ER transcriptional activity. Luciferase activity was measured in living mice by daily bioluminescence imaging sessions and in tissue extracts by enzymatic assay. Results: Acute, oral administration of genistein or soymilk caused a significant increase of ER activity in liver. In a 20 day long treatment, soymilk was more potent than genistein in liver and appeared to extend its influence on ER transcriptional activity in other tissues, such as the digestive tract. A mixture of pure genistein and daidzein at the same concentration as in soymilk failed to induce significant changes during acute and chronic studies suggesting an important, uncharacterized role of the soymilk matrix. Consistent with this observation, synergistic effects of the matrix plus isoflavones were observed in MCF-7 cells stably transfected with the ERE-luc construct. Conclusions: This study underlines the limitations of the analysis of single food components in the evaluation of their effects on estrogen receptor activity and advocates the necessity to use complex organisms for the full comprehension of the effects of compounds altering the endocrine balance. [Copyright &y& Elsevier]