Sex-based differences in obesity-related hepatic malignancies suggest the protective roles of estrogen. Using a preclinical model, we dissected estrogen receptor (ER) isoform-driven molecular responses in high-fat diet (HFD)-induced liver diseases of male and female mice treated with or without an estrogen agonist by integrating liver multi-omics data. We found that selective ER activation recovers HFD-induced molecular and physiological liver phenotypes. HFD and systemic ER activation altered core liver pathways, beyond lipid metabolism, that are consistent between mice and primates. By including patient cohort data, we uncovered that ER-regulated enhancers govern central regulatory and metabolic genes with clinical significance in metabolic dysfunction-associated steatotic liver disease (MASLD) patients, including the transcription factor TEAD1. TEAD1 expression increased in MASLD patients, and its downregulation by short interfering RNA reduced intracellular lipid content. Subsequent TEAD small molecule inhibition improved steatosis in primary human hepatocyte spheroids by suppressing lipogenic pathways. Thus, TEAD1 emerged as a new therapeutic candidate whose inhibition ameliorates hepatic steatosis.

ABSTRACT Introduction The occurrence of obesity-related hepatic malignancies differs between sexes, suggesting the involvement of sex hormones. Female sex hormones maintain cell homeostasis through estrogen receptor (ER) signaling and protect from developing nonalcoholic fatty liver disease (NAFLD) in mice and humans. Rationale To understand recovery from high-fat diet (HFD)-induced liver disease in males upon estrogen treatment, we comprehensively characterized molecular changes in the liver upon selective activation of estrogen receptors (ERs) to identify novel therapeutic targets downstream of estrogen signaling. Methods To dissect hepatic ER isoform-driven responses, we integrated liver transcriptomes from female and male HFD mice treated with or without four different estrogen agonists, along with multiomics data, including bulk, single-cell and spatial transcriptomics, chromatin profiling, machine learning models and advanced microscopy. Patient cohorts and primary human hepatocyte spheroids datasets were included. Results Only males developed liver steatosis. We found that selective activation of either ERα or ERβ reduced HFD-induced hepatic steatosis in male mice. Systemic ER activation restored HFD-induced aberrant gene expression of cellular processes across liver cell types, including hepatocytes. Profiling of marked histones revealed that ER activation modulated promoter and enhancer sites and identified 68 estrogen-sensitive enhancer-gene pairs. Most of these genes were similarly deregulated in human nonalcoholic fatty liver disease (NAFLD) patients, including the transcription factor TEAD1 . TEAD1 expression increased in NAFLD patients, and inhibiting TEAD ameliorated steatosis in spheroids by suppressing lipogenic pathways. Conclusions Systemic activation of ERα or ERβ modulates molecular pathways in the liver to counteract NAFLD. Our study identified TEAD1 as a key ER-sensitive gene and suggested that its inhibition poses a therapeutic strategy to combat NAFLD without the undesired side effects elicited by estrogen signaling. Clinical research relevance We identified drug targets downstream of estrogen signaling, including TEAD1, and demonstrate that TEAD inhibition improves steatosis by suppressing lipogenic pathways. Basic research relevance The targeted activation of nuclear ERs recovers high-fat diet-induced molecular and physiological liver phenotypes by remodeling core pathways beyond lipid metabolism. ER-responsive enhancers regulate central metabolic genes of clinical significance in NAFLD patients, highlighting the potential impact of this research on understanding liver cell plasticity. HIGHLIGHTS steatosis in livers of high-fat diet (HFD) male mice was effectively reduced by selective activation of estrogen receptors (ERα and ERβ) with four different agonists. ER agonist treatments successfully reversed HFD-induced changes in gene regulation and expression, revealing new treatment targets involving previously unconnected molecular pathways. estrogen-sensitive enhancers regulated important genes, including TEAD1, emerging as pivotal NAFLD regulators significantly impacting metabolic processes. high TEAD1 gene expression in NAFLD patients correlated with disease severity, underscoring its clinical significance in disease progression. inhibiting TEAD with small molecules alleviated steatosis by suppressing lipogenic pathways, resembling some of the same beneficial effects as estrogen treatment.

Objective The prevalence of overweight and obesity among children has drastically increased during the last decades and maternal obesity has been demonstrated as one of the ultimate factors. Nutrition-stimulated transgenerational regulation of key metabolic genes is fundamental to the developmental origins of the metabolic syndrome. Fetal nutrition may differently influence female and male offspring. Methods Mice dam were fed either a control diet or a high-fat diet (HFD) for 6-week prior mating and continued their respective diet during gestation and lactation. At weaning, female and male offspring were fed the HFD until sacrifice. White (WAT) and brown (BAT) adipose tissues were investigated in vivo by nuclear magnetic resonance at two different timepoints in life (midterm and endterm) and tissues were collected at endterm for lipidomic analysis and RNA sequencing. We explored the sex-dependent metabolic adaptation and gene programming changes by maternal HFD in visceral AT (VAT), subcutaneous AT (SAT) and BAT of offspring. Results We show that the triglyceride profile varies between adipose depots, sexes and maternal diet. In female offspring, maternal HFD remodels the triglycerides profile in SAT and BAT, and increases thermogenesis and cell differentiation in BAT, which may prevent metabolic complication later in life. Male offspring exhibit whitening of BAT and hyperplasia in VAT when born from high-fat mothers, with impaired metabolic profile. Maternal HFD differentially programs gene expression in WAT and BAT of female and male offspring. Conclusion Maternal HFD modulates metabolic profile in offspring in a sex-dependent manner. A sex- and maternal diet-dependent gene programming exists in VAT, SAT, and BAT which may be key player in the sexual dimorphism in the metabolic adaptation later in life.

Male and female offspring of obese mothers are known to differ extensively in their metabolic adaptation and later development of complications. We investigate the sex-dependent responses in obese offspring mice with maternal obesity, focusing on changes in liver glucose and lipid metabolism. Here we show that maternal obesity prior to and during gestation leads to hepatic steatosis and inflammation in male offspring, while female offspring are protected. Females from obese mothers display important changes in hepatic transcriptional activity and triglycerides profile which may prevent the damaging effects of maternal obesity compared to males. These differences are sustained later in life, resulting in a better metabolic balance in female offspring. In conclusion, sex and maternal obesity drive differently transcriptional and posttranscriptional regulation of major metabolic processes in offspring liver, explaining the sexual dimorphism in obesity-associated metabolic risk.

With the increasing prevalence of obesity in women of reproductive age, there is an urgent need to understand the metabolic impact on the fetus. Sex-related susceptibility to liver diseases has been demonstrated but the underlying mechanism remains unclear. Here we report that maternal obesity impacts lipid metabolism differently in female and male offspring. Males, but not females, gained more weight and had impaired insulin sensitivity when born from obese mothers compared to control. Although lipid mass was similar in the livers of female and male offspring, sex-specific modifications in the composition of fatty acids, triglycerides and phospholipids was observed. These overall changes could be linked to sex-specific regulation of genes controlling metabolic pathways. Our findings revised the current assumption that sex-dependent susceptibility to metabolic disorders is caused by sex-specific postnatal regulation and instead we provide molecular evidence supporting in utero metabolic adaptations in the offspring of obese mothers.

Fat distribution, on top of general obesity, contributes to the severity of histologic features in patients with nonalcoholic fatty liver diseases (NAFLD); and visceral obesity has been correlated to fatty liver diseases. Therefore, investigation of fat distribution in vivo could be a good predictor of fatty liver risks in obesity. Fatty acids composition is a key player in hepatic dysfunctions and cardiovascular risk in obesity. Because fatty acids can damage biological membranes, fatty acid accumulation in the liver may be partially responsible for the functional and morphological changes that are observed in NAFLD. Fatty acids stored into triglycerides are lipid species that act as signaling molecules and therefore are key regulators of posttranslational regulation of biological functions such as lipid homeostasis and lipotoxicity. Here, we describe magnetic resonance methods to investigate in vivo whole-body fat distribution and hepatic liver fatty acid composition in order to directly assess the liver metabolic status and may allow to anticipate liver diseases.

Background and Aims: With the increasing prevalence of obesity in women in reproductive age, there is an urgent need to further understand the metabolic impact on the fetus. We aim to characterize the sexual dimorphism in offspring in response to maternal obesity with a special focus on hepatic lipid species and the pathophysiological outcomes. We aim to better understand the mechanisms whereby maternal obesity affects the adiposity and lipid composition differently in female (F) and male (M) offspring.

There is a strong association between obesity and colorectal cancer (CRC), especially in men, whereas estrogen protects against both the metabolic syndrome and CRC. Colon is the first organ to respond to high-fat diet (HFD), and estrogen receptor beta (ERβ) can attenuate CRC development. How estrogen impacts the colon under HFD and related sex differences has, however, not been investigated. To dissect this, mice were fed control diet or HFD for 13 weeks and administered receptor-selective estrogenic ligands for the last three weeks. We recorded impact on metabolism, colon crypt proliferation, macrophage infiltration, and the colon transcriptome. We found clear sex differences in the colon transcriptome and in the impact by HFD and estrogens, including on clock genes. ERα-selective activation reduced body weight and generated systemic effects, whereas ERβ-selective activation had local effects in the colon, attenuating HFD-induced macrophage infiltration and epithelial cell proliferation. We here demonstrate how HFD and estrogens modulate the colon microenvironment in a sex- and ER-specific manner.

In this communication, we aim to summarize the role of estrogen receptor beta (ERβ) in lipid metabolism in the main metabolic organs with a special focus on sex differences. The action of ERβ is tissue-specific and acts in a sex-dependent manner, emphasizing the necessity of developing sex- and tissue-selective targeting drugs in the future.

Background and Aims: Estrogens are important regulators of metabolic diseases including obesity and insulin resistance. The physiological and genomic signaling of estrogens is mainly mediated through the estrogen receptors (ER)α and ERβ. Although the role of ERβ in metabolism has recently gained attention, it is still unclear. However, no data report the different response to pharmaceutical activation of ERβ between sexes.

Epidemiological studies highlight a strong association between obesity and colorectal cancer (CRC), especially in men. Estrogen, on the other hand, is associated with protection against both the metabolic syndrome and CRC. The colon is the first organ to respond to a high-fat diet (HFD), and estrogen receptor beta (ERβ) in the intestine appears to prevent CRC. How estrogen impacts the colon under HFD condition has, however, not been investigated. Estrogen can act through three different receptors (ERα, ERβ, GPER1) which all may impact metabolism. In an effort to dissect this, we fed mice a control diet or a high-fat diet (HFD) for 13 weeks and administered receptor-selective estrogenic ligands for the last three weeks. We recorded corresponding physiological impact on fat distribution, fasting glucose, colon crypt proliferation and immune cell infiltration, and the colon transcriptome response. We identify clear sex-differences at the transcriptome level, both at base line and after HFD and ligand treatments. An unexpected observation was the significant sex-differences and impact by HFD and estrogens on circadian clock gene expression, such as Npas2 and Arntl (Bmal1) , in the colon. Both sexes also exhibited an increased infiltration of F4/80+ macrophages as a result of HFD. In males, but not females, this was accompanied by changes in colonic epithelial cell proliferation. ERα-selective PPT treatment had significant systemic effects, reducing body weight in both sexes, whereas ERβ-selective DPN treatment did not impact body weight, but reduced infiltration of F4/80+ macrophages in colon of both sexes and attenuated HFD-induced proliferation of male colon crypts. Both ERα and ERβ activation contributed to circadian clock gene regulations. We detail for the first time how HFD and estrogens modulate the colon transcriptome and physiology in a sex and ER-specific manner.

The response to overfeeding is sex dependent, and metabolic syndrome is more likely associated to obesity in men or postmenopausal women than in young fertile women. We hypothesized that obesity-induced metabolic syndrome is sex dependent due to a sex-specific regulation of the fatty acid (FA) synthesis pathways in liver and white adipose depots. We aimed to identify distinctive molecular signatures between sexes using a lipidomics approach to characterize lipid species in liver, perigonadal adipose tissue, and inguinal adipose tissue and correlate them to the physiopathological responses observed. Males had less total fat but lower subcutaneous on visceral fat ratio together with higher liver weight and higher liver and serum triglyceride (TG) levels. Males were insulin resistant compared to females. Fatty acid (FA) and TG profiles differed between sexes in both fat pads, with longer chain FAs and TGs in males compared to that in females. Remarkably, hepatic phospholipid composition was sex dependent with more abundant lipotoxic FAs in males than in females. This may contribute to the sexual dimorphism in response to obesity towards more metaflammation in males. Our work presents an exhaustive novel description of a sex-specific lipid signature in the pathophysiology of metabolic disorders associated with obesity in ob / ob mice. These data could settle the basis for future pharmacological treatment in obesity.

Estrogen exerts its action through the binding to two major receptors, estrogen receptor (ER)α and β. Recently, the beneficial role of selective ERβ activation in the regulation of metabolic homeostasis in obesity has been demonstrated, but its importance is still controversial. However, no data are available regarding possible gender differences in response to pharmaceutical activation of ERβ. Male mice were fed a control diet (CD) or a high fat diet (HFD) before being treated with the ERβ selective ligand, 4-(2-(3-5-dimethylisoxazol-4-yl)-1H-indol-3yl)phenol (DIP) in the same conditions as in our recently published paper in female mice. Magnetic resonance imaging and spectroscopy were performed repeatedly in vivo after 6 weeks of diet and after 2 weeks of DIP. Adipose tissue distribution and hepatic triglycerides composition were quantified. HFD-treated males showed a feminization of their fat distribution towards more subcutaneous fat depots and increase total fat content and visceral adipose tissue showed clear browning sites after DIP. Hepatic lipid composition was modified by DIP, with less saturated and more unsaturated lipids and an improved insulin sensitivity. Finally, brown adipose tissue size expended after DIP, due to an increase of the size of the lipid droplets. Our data demonstrate that selective activation of ERβ exerts a tissue-specific and sex-dependent response to metabolic adaptation to overfeeding. Most importantly, together with our previously published results in females, the current findings support the concept that sex should be considered in the future development of obesity-moderating drugs.

OBJECTIVE:Estrogens play a key role in the distribution of adipose tissue and have their action by binding to both estrogen receptors (ER), α and β. Although ERβ has a role in the energy metabolism, limited data of the physiological mechanism and metabolic response involved in the pharmacological activation of ERβ is available. METHODS:For clinical relevance, non-ovariectomized female mice were subjected to high fat diet together with pharmacological (DIP - 4-(2-(3,5-dimethylisoxazol-4-yl)-1H-indol-3-yl)phenol) interventions to ERβ selective activation. The physiological mechanism was assessed in vivo by magnetic resonance imaging and spectroscopy, and oral glucose and intraperitoneal insulin tolerance test before and after DIP treatment. Liver and adipose tissue metabolic response was measured in HFD + vehicle and HFD + DIP by stable isotope, RNA sequencing and protein content. RESULTS:HFD-fed females treated with DIP had a tissue-specific response towards ERβ selective activation. The metabolic profile showed an improved fasting glucose level, insulin sensitivity and reduced liver steatosis. CONCLUSIONS:Our data demonstrate that selective activation of ERβ exerts a tissue-specific activity which promotes a beneficial effect on whole body metabolic response to obesity.

Abstract Recent epidemiological studies highlight the strong association between high body mass index (BMI) and enhanced risk of colon cancer. This association appears stronger in men than in women, indicating a possible protective role of estrogens in this context. The biological action of the estrogens is mediated by specific transcription factors, the estrogen receptors (ERs). ERβ is the main ER in the intestine; and it is expressed in both epithelial cells and immune cells. Anti-inflammatory and anti-tumorigenic effects of ERβ in colitis-associated neoplasia have been demonstrated. Several rodent models for obesity show an impaired intestinal epithelial barrier function and an increase of pro-inflammatory markers in the colon. Moreover, in murine colitis models, high fat diet (HFD) exacerbates colonic inflammation. However, the specific molecular mechanisms linking obesity to increased colon cancer development, where estrogen signaling may be involved, is not clear. We hypothesize that ERβ-selective agonists can oppose the HFD-mediated inflammatory pro-tumorigenic colonic signalling in a gender-specific manner during obesity, and thereby protect against colorectal cancer. Our study is focused on the initiation of colon inflammation during obesity and aims to investigate the impact of ERβ activation in this context. To address this question, male and female mice were fed a HFD for 26 weeks and treated with a ERβ-selective agonist for 3 weeks prior sacrifice. Stool pellets were regularly collected. Animals were sacrificed; colon samples and caecal contents were collected for further analysis. We show that the expression pattern of inflammatory genes in colon is modified by the ERβ-selective agonist in HFD fed mice. Moreover the ERβ-selective agonist appears to induce gender-specific changes of the microbiota of HFD-fed mice. Our results show that an ERβ-selective agonist can modulate the colon microenvironment during obesity in mice and will provide a better comprehension of the molecular mechanisms leading to obesity-associated colonic inflammation. Citation Format: Amena Archer, Linnea Pettersson, Marcela González-Granillo, Christina Savva, Marion Korach-André, Cecilia Williams. Role of estrogen signaling in colon microenvironment during obesity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4401. doi:10.1158/1538-7445.AM2017-4401

Both functional ovaries and estrogen replacement therapy (ERT) reduce the risk of type 2 diabetes (T2D). Understanding the mechanisms underlying the antidiabetic effects of 17β-estradiol (E2) may permit the development of a molecular targeting strategy for the treatment of metabolic disease. This study examines how the promotion of insulin sensitivity and weight loss by E2 treatment in high-fat-diet (HFD)-fed mice involve several anti-adipogenic processes in the visceral adipose tissue. Magnetic resonance imaging (MRI) revealed specific reductions in visceral adipose tissue volume in HFD+E2 mice, compared with HFD mice. This loss of adiposity was associated with diminished visceral adipocyte size and reductions in expression of lipogenic genes, adipokines and of the nuclear receptor nr2c2/tr4. Meanwhile, expression levels of adipose triglyceride lipase/pnpla2 and leptin receptor were increased. As mRNA levels of stat3, a transcription factor involved in brown adipose tissue differentiation, were also increased in visceral adipose, the expression of other brown adipose-specific markers was assessed. Both expression and immunohistochemical staining of ucp-1 were increased, and mRNA levels of dio-2, and of adrβ3, a regulator of ucp-1 expression during the thermogenic response, were increased. Furthermore, expression of cpt-1b, a brown adipose-specific gene involved in fatty acid utilization, was also increased. Methylation studies demonstrated that the methylation status of both dio-2 and adrβ3 was significantly reduced. These results show that improved glycemic control and weight loss due to E2 involve anti-adipogenic mechanisms which include suppressed lipogenesis and augmented fatty acid utilization, and in addition, the activation of brown adipose tissue-specific gene expression in association with E2-dependent epigenetic modifications in these genes.

The liver X receptors (LXR) are crucial regulators of metabolism. After ligand binding, they regulate gene transcription and thereby mediate changes in metabolic pathways. Modulation of LXR and their downstream targets has appeared to be a promising treatment for metabolic diseases especially atherosclerosis and cholesterol metabolism. However, the complexity of LXR action in various metabolic tissues and the liver side effect of LXR activation have slowed down the interest for LXR drugs. In this review, we summarized the role of LXR in the main metabolically active tissues with a special focus on obesity and associated diseases in mammals. We will also discuss the dual interplay between the two LXR isoforms suggesting that they may collaborate to establish a fine and efficient system for the maintenance of metabolism homeostasis.

The liver X receptors (LXR)α and LXRβ are transcription factors belonging to the nuclear receptor family, which play a central role in metabolic homeostasis, being master regulators of key target genes in the glucose and lipid pathways. Wild-type (WT), LXRα(-/-), and LXRβ(-/-) mice were fed a chow diet with (treated) or without (control) the synthetic dual LXR agonist GW3965 for 5 wk. GW3965 raised intrahepatic triglyceride (TG) level but, surprisingly, reduced serum TG level through the activation of serum lipase activity. The serum TG reduction was associated with a repression of both catecholamine-stimulated lipolysis and relative glucose incorporation into lipid in isolated adipocytes through activation of LXRβ. We also demonstrated that LXRα is required for basal (nonstimulated) adipocyte metabolism, whereas LXRβ acts as a repressor of lipolysis. On the contrary, in skeletal muscle (SM), the lipogenic and cholesterol transporter LXR target genes were markedly induced in WT and LXRα(-/-) mice and to a lesser extent in LXRβ(-/-) mice following treatment with GW3965. Moreover, TG content was reduced in SM of LXRβ(-/-) mice, associated with increased expression of the main TG-lipase genes Hsl and Atgl. Energy expenditure was increased, and a switch from glucose to lipid oxidation was observed. In conclusion, we provide evidence that LXR might be an essential regulator of the lipid balance between tissues to ensure appropriate control of the flux of fuel. Importantly, we show that, after chronic treatment with GW3965, SM becomes the target tissue for LXR activation, as opposed to liver, in acute treatment.