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Pathway-selective insulin resistance where insulin fails to suppress hepatic glucose production but promotes liver fat storage may underlie glucose and lipid abnormalities after menopause. We tested the mechanisms by which estrogen treatment may alter the impact of a high-fat diet (HFD) when given at the time of ovariectomy (OVX) in mice. Female C57BL/6J mice underwent sham operation, OVX, or OVX with estradiol (E2) treatment and were fed an HFD. Hyperinsulinemic-euglycemic clamps were used to assess insulin sensitivity, tracer incorporation into hepatic lipids, and liver triglyceride export. OVX mice had increased adiposity that was prevented with E2 at the time of OVX. E2 treatment increased insulin sensitivity with OVX and HFD. In sham and OVX mice, HFD feeding induced fatty liver, and insulin reduced hepatic apoB100 and liver triglyceride export. E2 treatment reduced liver lipid deposition and prevented the decrease in liver triglyceride export during hyperinsulinemia. In mice lacking the liver estrogen receptor α, E2 after OVX limited adiposity but failed to improve insulin sensitivity, to limit liver lipid deposition, and to prevent insulin suppression of liver triglyceride export. In conclusion, estrogen treatment may reverse aspects of pathway-selective insulin resistance by promoting insulin action on glucose metabolism but limiting hepatic lipid deposition.

Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERα (ERα-AF2 ⁰) provided selective loss of function of nuclear ERα actions. In ERα-AF2 ⁰, the acceleration of endothelial repair in response to estrogen–dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF2 ⁰, whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles.

Estradiol is a major regulator of postnatal mammary gland development and thought to exert its effects through estrogen receptor α (ERα) expressed in the mammary gland stroma and epithelium. Previous studies, however, were confounded by the use of an ERα mutant strain that retains some of the protein with transactivation activity. Here, we use an $ER\\alpha^{-/-}$ mouse strain in which no ERα transcript can be detected to analyze mammary gland development in the complete absence of ERα signaling. The $ER\\alpha^{-/-}$ females show no development beyond a rudimentary ductal system. By grafting $ER\\alpha^{-/-}$ epithelium or stroma in combination with ERα WT stroma or epithelium, we show that the primary target for estradiol is the mammary epithelium, whereas a direct response of the mammary stroma is not required for mammary gland development to proceed normally. Mammary glands reconstituted with $ER\\alpha^{-/-}$ mammary epithelium exposed to pregnancy hormones show increased transcription of milk protein genes, indicating that ERα signaling is not an absolute requirement for a transcriptional response to pregnancy hormones. When $ER\\alpha^{-/-}$ mammary epithelial cells are in close vicinity to ERα WT cells, they proliferate and contribute to all aspects of mammary gland development, indicating that estradiol, like progesterone, orchestrates proliferation and morphogenesis by a paracrine mechanism, affecting nearby cells in the mammary epithelium.

Estrogen signaling is mediated by estrogen receptors α (ERα) and β (ERβ). Although a consensus has now been reached concerning many physiological functions of ERα, those of ERβ are still controversial: When housed and examined in two distant laboratories, mice originating from the same initial ERβ mutant exhibited widely different phenotypes, which were themselves different from the phenotype of another ERβ mutant previously generated in our laboratory. Because, in addition to a knockout insertion in exon 3, all these mouse mutants displayed alternative splicing transcripts, we have now constructed a ERβ mouse mutant (ERβSTL⁻/L⁻) in which exon 3 was cleanly deleted by Cre/LoxP-mediated excision and was devoid of any transcript downstream of exon 3. Both females and males were sterile. The histology of the ovary was mildly affected, and no histological defects were detected in other organs, neither in females nor in males. Our present results, which are in contrast with previously published data, suggest that, with the notable exception of male and female reproduction, ERβ is not required in the mouse for the development and homeostasis of the major body systems.

Oestrogen receptor α (ERα) is a transcription factor with ligand-independent and ligand-dependent activation functions (AF)-1 and -2. Oestrogens control postnatal mammary gland development acting on a subset of mammary epithelial cells (MECs), termed sensor cells, which are ERα-positive by immunohistochemistry (IHC) and secrete paracrine factors, which stimulate ERα-negative responder cells. Here we show that deletion of AF-1 or AF-2 blocks pubertal ductal growth and subsequent development because both are required for expression of essential paracrine mediators. Thirty percent of the luminal cells are ERα-negative by IHC but express Esr1 transcripts. This low level ERα expression through AF-2 is essential for cell expansion during puberty and growth-inhibitory during pregnancy. Cell-intrinsic ERα is not required for cell proliferation nor for secretory differentiation but controls transcript levels of cell motility and cell adhesion genes and a stem cell and epithelial mesenchymal transition (EMT) signature identifying ERα as a key regulator of mammary epithelial cell plasticity. Oestrogen receptors α (ERα) are expressed in a subset of mammary epithelial cells. Here, the authors identify cells with low-ERα protein levels and show that distinct cell populations have distinct requirements for the AF1 and AF2 domains of the ERα, and ERα acts in a biphasic manner dependent on developmental stage.

17β-Estradiol (E2) regulates estrogen receptor-α (ERα) target gene transcription through the two independent activation functions (AFs), AF1 and AF2, located in the N-terminal and ligand binding domain of ERα, respectively. We previously reported that ERα is required for the E2 atheroprotective action as well as for its accelerative action on endothelial healing, but its AF1 function is dispensable. Here, we investigated the role of ERαAF2 in these two major beneficial actions of E2 by electively targeting ERαAF2 (named ERαAF2°). Our results prove four points. (i) Compared with WT ERα, the ability of ERαAF2° to stimulate the C3 complement or the estrogen response element-thymidine kinase promoter in two cell lines was dramatically decreased, confirming the importance of AF2 in the E2-induced transcriptional activity of ERα. (ii) The uterotrophic action of E2 was totally absent in ERαAF2° mice, showing the crucial role of ERαAF2 in E2-induced uterus hyperplasia. (iii) ERαAF2 was dispensable for the accelerative action of E2 on endothelial healing, underlining the functionality of ERαAF2° in vivo. (iv) Finally, the atheroprotective effect of E2 was abrogated in ERαAF2° LDL-r−/− mice. Thus, whereas ERαAF1 and ERαAF2 are both required for the uterotrophic action of E2, we show that only ERαAF2 is necessary for its atheroprotective effect.

Beyond the key role in reproductive and cognitive functions, estrogens have been shown to protect against neurodegeneration associated with acute and chronic injuries of the adult brain. Current hypotheses reconcile this activity with a direct effect of 17β-estradiol (E2) on neurons. Here we demonstrate that brain macrophages are also involved in E2action on the brain. Systemic administration of hormone prevents, in a time- and dose-dependent manner, the activation of microglia and the recruitment of peripheral monocytes induced by intraventricular injection of lipopolysaccharide. This effect occurs by limiting the expression of neuroinflammatory mediators, such as the matrix metalloproteinase 9 and lysosomal enzymes and complement C3 receptor, as well as by preventing morphological changes occurring in microglia during the inflammatory response. By injecting lipopolysaccharide in estrogen receptor (ER)-null mouse brains, we demonstrate that hormone action is mediated by activation of ER α but not of ER β. The specific role of ER α is further confirmed by comparing the effects of ERs on the matrix metalloproteinase 9 promoter activity in transient transfection assays. Finally, we report that genetic ablation of ER α is associated with a spontaneous reactive phenotype of microglia in specific brain regions of adult ER α-null mice. Altogether, these results reveal a previously undescribed function for E2in brain and provide a mechanism for its beneficial activity on neuroinflammatory pathologies. They also underline the key role of ER α in brain macrophage reactivity and hint toward the usefulness of ER α-specific drugs in hormone replacement therapy of inflammatory diseases.

Full-length 66-kDa estrogen receptor α (ERα) stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal domain and AF-2 in the ligand binding domain. Another physiologically expressed 46-kDa ERα isoform lacks the N-terminal A/B domains and is consequently devoid of AF-1. Previous studies in cultured endothelial cells showed that the N-terminal A/B domain might not be required for estradiol (E2)-elicited NO production. To evaluate the involvement of ERα AF-1 in the vasculoprotective actions of E2, we generated a targeted deletion of the ERα A/B domain in the mouse. In these ERαAF-1° mice, both basal endothelial NO production and reendothelialization process were increased by E2 administration to a similar extent than in control mice. Furthermore, exogenous E2 similarly decreased fatty streak deposits at the aortic root from both ovariectomized 18-week-old $ER\\alpha - 1^{ + / + } \\,LDLr^{ - / - }$ (low-density lipoprotein receptor) and ERαAF-1° $LDLr^{ - / - }$ mice fed with a hypercholesterolemic diet. In addition, quantification of lesion size on en face preparations of the aortic tree of 8-month-old ovariectomized or intact female mice revealed that ERα AF-1 is dispensable for the atheroprotective action of endogenous estrogens. We conclude that ERα AF-1 is not required for three major vasculoprotective actions of E2, whereas it is necessary for the effects of E2 on its reproductive targets. Thus, selective ER modulators stimulating ERα with minimal activation of ERα AF-1 could retain beneficial vascular actions, while minimizing the sexual effects.

We previously generated and characterized a genuine estrogen receptor (ER) β-null mouse line (named ERβ STᴸ⁻/ᴸ⁻) and showed that ERβ STᴸ⁻/ᴸ⁻ mice were sterile, due to an ovulation impairment in females and to an unknown reason in males, as their reproductive organs and spermatozoid motility appeared normal. We report here an assessment of the sexual behavior of ERβ STᴸ⁻/ᴸ⁻ null mice. We found that ERβ STᴸ⁻/ᴸ⁻ males display mildly impaired sexual behavior and that ERβ STᴸ⁻/ᴸ⁻ females are significantly less receptive and less attractive than wild-type (WT) females. Decreased attractivity is also exhibited by ERβAF2 ⁰ but not by ERβAF1 ⁰ mutant females (females devoid of either AF2 or AF1 activation function of ERβ). Interestingly, by using an odor preference test, we have determined that the low attractiveness of ERβ STᴸ⁻/ᴸ⁻ and ERβAF2 ⁰ females is related to a deficiency of a volatile chemosignal.

Estrogens are powerful modulators of neuronal physiology and in humans may affect a broad range of functions, including reproductive, emotional, and cognitive behaviors. We studied the contribution of estrogen receptors (ERs) in modulation of emotional processes and analyzed the effects of deleting ERα or ERβ in mice. Behavior consistent with increased anxiety was observed principally in ERβ mutant females and was associated with a reduced threshold for the induction of synaptic plasticity in the basolateral amygdala. Local increase of 5-hydroxytryptamine 1a receptor expression in medial amygdala may contribute to these changes. Our data show that, particularly in females, there is an important role for ERβ-mediated estrogen signaling in the processing of emotional behavior.