Nongenomic effects of estrogen receptor α (ERα) signaling have been described for decades. Several distinct animal models have been generated previously to analyze the nongenomic ERα signaling (eg, membrane-only ER, and ERαC451A). However, the mechanisms and physiological processes resulting solely from nongenomic signaling are still poorly understood. Herein, we describe a novel mouse model for analyzing nongenomic ERα actions named H2NES knock-in (KI). H2NES ERα possesses a nuclear export signal (NES) in the hinge region of ERα protein resulting in exclusive cytoplasmic localization that involves only the nongenomic action but not nuclear genomic actions. We generated H2NESKI mice by homologous recombination method and have characterized the phenotypes. H2NESKI homozygote mice possess almost identical phenotypes with ERα null mice except for the vascular activity on reendothelialization. We conclude that ERα-mediated nongenomic estrogenic signaling alone is insufficient to control most estrogen-mediated endocrine physiological responses; however, there could be some physiological responses that are nongenomic action dominant. H2NESKI mice have been deposited in the repository at Jax (stock no. 032176). These mice should be useful for analyzing nongenomic estrogenic responses and could expand analysis along with other ERα mutant mice lacking membrane-bound ERα. We expect the H2NESKI mouse model to aid our understanding of ERα-mediated nongenomic physiological responses and serve as an in vivo model for evaluating the nongenomic action of various estrogenic agents.

Abstract 27-Hydroxycholesterol (27HC) is the most abundant oxysterol in circulation and metabolized by a P450 enzyme CYP7B1. Its levels closely correspond to those of cholesterol in the body. In addition, previously it was found that 27HC is an endogenous selective estrogen receptor modulator (SERM), which links cholesterol metabolism to estrogen receptor actions (1). Brown adipose tissue (BAT) is the primary source of energy expenditure and energy homeostasis, as well as body temperature maintenance. While previously it was believed that BAT activity is limited to neonates and young children, it is now recognized that BAT is also active in adult humans and its function is impaired by metabolic diseases such as obesity. BAT is also a secretory organ and produces brown adipokines, although the exact function of BAT and adipokines from this tissue in obesity has not been completely understood. Recently, it was reported that 27HC plays an important role in obesity and augments body weight gain in response to a high fat, high cholesterol (HFHC) diet by increasing pre-adipocyte population in the white adipose tissue. 27HC mimics the effects by HFHC diet-feeding on white adipose tissue, such as promoting the inflammation and macrophage infiltration (2). In this study, we explored the effect of 27HC on BAT morphology and function. First, we compared the morphology of BAT from wild-type mice and Cyp7b1-/- mice that have elevated levels of 27HC using H&E staining. Interestingly, brown adipocytes from Cyp7b1-/- mice were larger in cell size than those from wild-type mice, and the cells were mostly unilocular compared to the multilocular cells from wild-type mice, indicating the transition toward a “whitening” phenotype. Next, We treated mice fed a normal chow or a HFHC diet with 27HC or vehicle control for 8 weeks to examine the direct effect by 27HC on BAT. Similar to the phenotype in Cyp7b1-/-mice, 27HC increased the “whitening” of BAT regardless of the diet. We also determined the gene expression of brown adipocyte markers such as UCP1, PGC1a, and DIO2, and found that 27HC significantly decreased the expression of the BAT markers regardless of the diet, confirming the “whitening” observed in the morphology. Moreover, the energy expenditure in mice treated with 27HC was decreased compared to the vehicle control on a HFHC diet, suggesting that 27HC also alters BAT function. These results show that 27HC causes the whitening of BAT, and shed light on the important role of 27HC in brown adipose tissue function. Future experiments will be warranted toward further understanding of the role of 27HC in BAT function. Reference:(1) Umetani, Michihisa, et al. Nature medicine 13.10 (2007): 1185. (2) Asghari, Arvand, et al. Endocrinology 160.10 (2019): 2485-2494.