Impact of 27-Hydrocholesterol on Brown Adipose Tissue at the Single Cell Level

Linh Bui, Arvand Asghari, Michihisa UmetaniCenter for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Texas, USA

**The authors hereby, declare that they have no potential conflict of interests.

Introduction27-Hydroxycholesterol (27HC), a cholesterol metabolite, is producedfrom cholesterol by CYP27A1 enzyme, and is metabolized by CYP7B1enzyme. 27HC is the most abundant oxysterol in circulation and its levelclosely matches the levels of cholesterol in the body. 27HC is the firstidentified endogenous selective estrogen receptor modulator (SERM)and is known to exert its effects on various tissues (e.g. cardiovasculartissue, breast tissue, and brain) through affecting estrogen receptorsignaling. Previously we found that 27HC induces the weight gain in thewhite adipose tissue (WAT) in presence of high fat high cholesterol diet.Moreover, we found that 27HC can increase the fat percentage of thebody and the inflammation in the adipose tissue regardless of the diet.

Moreover, our previous studies showed that 27HC might also affect the brown adipose tissue (BAT) in the mice. Brown adipose tissue is the primary source of energy expenditure and energy homeostasis, as well as body temperature maintenance. In our previous studies we noticed that upon treatment of mice with 27HC in presence of high fat high cholesterol diet, the mice activity and energy expenditure decreases which suggests that it might be through the effects of 27HC on BAT. We checked the morphology of the BAT in presence of 27HC and found that 27HC treatment leads to an increase in the size of the brown adipose tissue and a change toward white-like state, which is called the whitening of the BAT. Following this finding, we decided to look deeper at the effects of 27HC on the brown adipose tissue, by performing qPCR experiments on this tissue upon 27HC treatment and using single cell RNA-sequencing (scRNA-seq) to determine the different cell populations in the BAT and how 27HC might affect the composition of these various cell groups in the brown adipose tissue. BAT consists of multiple cell types including brown adipocytes, immune cells, and vascular cells, and the exact function of BAT in the level of each cell type is largely unknown.

MethodsTo examine the effect of 27HC on cellular diversity and gene expression in the BAT, we will use the single cell RNA-sequencing approach to first identify the different cell populations in BAT by analyzing each cell marker gene expression and then to compare the changes in cell diversity and gene expressions upon elevation of 27HC levels. This will be done using 27HC-catabolizing enzyme Cyp7b1-/-mice. Briefly, 8-12 weeks old wild type and Cyp7b1 mice will be sacrificed, brown adipose tissues will be collected, and single cells will be isolated using the proper digestion method. Following the isolation of the single cells, cells will be sent out for library preparations and single cell sequencing. The data from scRNA-seq will be analyzed using available bioinformatic tools and packages in R, such as SURET. We will also use histology and qPCR to collect more data and confirm the results from our scRNA-seq analysis. Histology: Histology specimens were paraffin embedded for adipose tissues or frozen for all other tissues. Adipocyte cell size was measured in histological sections of gonadal white adipose (GWAT) and brown adipose (BAT) tissues after Hematoxylin and Eosin staining (ThermoFisher Scientific). ImageJ software was used to determine the diameter of each cell from five independent pictures, in which more than 150 cells were included. To evaluate the cell number in GWAT, DNA was extracted from GWAT and its abundance was measured with a Nanodrop 2000 spectrophotometer (ThermoFischer). Quantitative RT-PCR: mRNA abundance of BAT markers as well as some inflammation markers was evaluated by qRT-PCR.

Results and Discussion

Figure 1. Different gene expressions in BAT

Conclusion and Future Directions

Study Design

SACRIFICE

scRNA-seq

BAT

Wild Type Mice

Cyp7b1-/-Mice

Single Cell Isolation

Figure 1. 27-HC is the first identified endogenous SERM and most prevalent oxysterol in circularion. Some of the functions of 27-HC in various tissues and the metabolizing and catabolizing enzymes of 27-HC are shown here

Figure 2. 27-HC and Body Weight Gain

Following our finding that increased levels of 27HC cause the “whitening” of brown adipose tissue, we also checked the expression of the BAT markers using qPCR to confirm our findings from the histology studies. The results showed a decrease in the expression of all of the BAT gene markers, namely UCP1, DIO2, and PGC1, which confirms that 27HC is inducing the whitening of the BAT and moves us one step closer to confirming our hypothesis that the observed decrease in energy expenditure of the mice upon 27HC treatment, is because of the effects of 27HC on the BAT.

We also did a bioinformatic analysis on the publicly available data from brown adipocytes scRNA-seq where we showed that both estrogen receptor and CYP27A1 gene are expressed in brown adipocytes, suggesting that 27HC is locally produced in BAT and that can affect the local estrogen receptors in the tissue.

Figure 2. The “whitening” of the BAT in 27HC treatment.

Normal 27HCCurrently, there is no available scRNA-seq dataset from BAT except for that using brown adipocyte only. Therefore, our proposed study will provide a fundamental resource for BAT biology and enables us to identify the cells responsible for the 27HC action in BAT and explore the changes in BAT following 27HC up-regulation, all for the first time.

Up to this point, we confirmed that 27HC is affecting the brown adipose tissue morphology and function by manipulating the expression of BAT markers. The next step in our study is to use scRNA-seq to find the effects of the 27HC on the BAT cell population and the gene expression of each of these cell populations. Up to now, we are refining the process of single cell isolation because it is the hardest part in the project and needs modifications to different digestion protocols in order to produce the desired results. As soon as we can finalize our digestion and single cell isolation protocols, we will move forward with the scRNA-seq experiment.

Acknowledgements: Funding support from Summer Undergraduate Research Fellowship (SURF) to LB and NIH HL127037 to MUReferences:• Arvand Asghari, Tomonori Ishikawa, Shiro Hiramitsu, Wan-Ru Lee, Junko Umetani, Linh

Bui, Kenneth S Korach, Michihisa Umetani, 27-Hydroxycholesterol Promotes Adiposity andMimics Adipogenic Diet-Induced Inflammatory Signaling, Endocrinology, Volume 160,Issue 10, October 2019, Pages 2485–2494, https://doi.org/10.1210/en.2019-00349