27-Hydroxycholesterol Promotes Adiposity and Mimics Adipogenic Diet-Induced Inflammatory Signaling

Abstract

27-Hydroxycholesterol (27HC) is an abundant cholesterol metabolite and has detrimental effects on the cardiovascular system, whereas its impact on adiposity is not well known. In this study, we found that elevations in 27HC cause increased body weight gain in mice fed a high-fat/high-cholesterol diet in an estrogen receptor α–dependent manner. Regardless of diet type, body fat mass was increased by 27HC without changes in food intake or fat absorption. 27HC did not alter energy expenditure in mice fed a normal chow diet and increased visceral white adipose mass by inducing hyperplasia but not hypertrophy. Although 27HC did not augment adipocyte terminal differentiation, it increased the adipose cell population that differentiates to mature adipocytes. RNA sequencing analysis revealed that 27HC treatment of mice fed a normal chow diet induces inflammatory gene sets similar to those seen after high-fat/high-cholesterol diet feeding, whereas there was no overlap in inflammatory gene expression among any other 27HC administration/diet change combination. Histological analysis showed that 27HC treatment increased the number of total and M1-type macrophages in white adipose tissues. Thus, 27HC promotes adiposity by directly affecting white adipose tissues and by increasing adipose inflammatory responses. Lowering serum 27HC levels may lead to an approach targeting cholesterol to prevent diet-induced obesity.

Obesity is one of the main risk factors influencing cardiovascular disease worldwide in both men and women and at all ages, with a sexual dimorphism in body composition (1, 2). Women prior to menopause and men preferentially accumulate subcutaneous white adipose tissues (SWATs) and visceral white adipose tissues (VWATs), respectively; menopause in females favors the shift toward VWAT accumulation (3, 4). Estrogens suppress adipocyte differentiation and protect against adiposity and body weight gain (5). In men, estrogens are also synthesized locally by aromatase-mediated conversion of testosterone. Therefore, estrogens may play important roles in the regulation of fat deposition and in the development of the fat tissues in males (6, 7). The estrogen receptors (ERα and ERβ) are members of the nuclear receptor superfamily and are present in adipocytes. Patients with estrogen insensitivity/resistance syndrome and with a loss of function of ERα or aromatase are obese (8–10), and both male and female ERα-deficient mice have increased adipose tissue mass and adipocyte populations, even when food intake does not change (6, 7, 11), indicating that ERα is the important isoform in the regulation of adipose tissue by estrogen.

Oxysterols are metabolites of cholesterol that are produced in peripheral tissues as a means to eliminate cholesterol (12–14). The most prevalent oxysterol in the circulation is 27-hydroxycholesterol (27HC); serum concentrations of 27HC correlate well with those of cholesterol and also with age (12, 15). The enzymes that generate and catabolize 27HC, sterol 27-hydroxylase (CYP27A1) and oxysterol 7α-hydroxylase (CYP7B1), respectively, are primarily expressed in the liver, but also in peripheral tissues, such as adipose, to a lesser extent (16, 17). Previously, we found that 27HC directly binds to ERα and ERβ, and it inhibits the beneficial effects afforded by estrogens to the cardiovascular system and bone mineral density (18–20). Upon binding, 27HC induces a unique conformational change to ER and shows antagonistic or agonistic estrogenic effects in a tissue-dependent manner (18, 21). Thus, 27HC is the first identified, endogenously produced selective ER modulator (SERM). Furthermore, in addition to its estrogen-dependent actions, 27HC induces inflammatory responses in vascular endothelial cells in an estrogen-independent but ER-dependent manner (19).

In the current study, we investigated how 27HC impacts body weight and body fat regulation and discovered that elevated 27HC levels in mice increase body weight gain in response to a high-fat/high-cholesterol (HFHC) diet and increase adiposity independently of diet. Based on these findings, we propose a mechanism through which 27HC acts as a SERM to influence adiposity.

Materials and Methods

Animal models

All animal experiments were approved by the Institutional Animal Care and Use Committees at the University of Houston and UT Southwestern. Six-month-old Cyp7b1^−/−, ERα-null (Esr1^−/−), LXRα/β-null (Nr1h3^−/−Nr1h2^−/−), or wild-type mice, all on a C57BL/6 background, were fed ad libitum with an HFHC diet (Teklad 88137) or standard normal chow (NC; Teklad 2016) (19). Two weeks prior to the experiments, all female mice were ovariectomized and implanted with a subcutaneous slow release pellet of vehicle or 17β-estradiol (E₂; 6 μg/d, Innovative Research of America) (19). Body weight was recorded weekly. Total body fat and total lean mass were analyzed at the beginning and termination of the experiments by nuclear magnetic resonance (minispec mq spectrometer, Brucker). The administration of 27HC or 7-keto cholesterol (20 mg/kg body weight) was performed by subcutaneous injection (19) every 2 days. Transcript abundance was evaluated in mouse liver or white adipose tissue (WAT) by quantitative RT-PCR (qRT-PCR) (19, 22).

Cholesterol, triglyceride, 27HC, and estradiol analyses

Plasma concentrations of total cholesterol, triglyceride, 27HC, and E₂ and liver concentrations of cholesterol and triglycerides were evaluated as previously described (18, 22). Adipocyte triglyceride levels were measured by colorimetric enzymatic assays (Roche Diagnostics) by using repeated freeze-thaw followed by 1% Triton X-100 lipid extraction of cultured cells (23). Triglyceride levels were normalized to total protein content with a Bio-Rad protein assay kit.

Metabolic assessment

Food intake was measured weekly from individually housed mice. For lipid absorption measurements, stools were collected from individually housed mice during 72 hours. Lipid content of diets and stools was determined as previously described (22). Calorimetry measurements were performed by using CLAMS system metabolic cages (Columbus Instruments) as described previously (22). Following the administration of 27HC or vehicle on an HFHC or an NC diet for 8 weeks, the mice were individually housed in calorimeter chambers containing water and food (HFHC vs NC) and allowed to acclimate for 1 day during a 12-hour light/12-hour dark cycle. Data collection followed for the next 3 days. To avoid potential effects due to handling, 27HC/vehicle administration was not performed during the data collection period.

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 WAT (GWAT) and inguinal WAT (IgWAT) after hematoxylin and eosin staining (Thermo Fisher Scientific). ImageJ software was used to determine the diameter of each cell from five independent images, in which >150 cells were included. To evaluate the cell number in WAT, DNA was extracted from WAT (24) and its abundance was measured with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Immunohistochemistry was performed on 6-μm tissue sections. The sections were rehydrated, treated with citrate buffer (pH 6.0) at 95°C for 10 minutes, and blocked with 10% goat serum (Life Technologies) and an avidin/biotin blocking kit (Vector Laboratories). Then, the sections were incubated with anti-CD68 (Abcam, ab125212) (25), anti-NOS2 (Abcam, ab15323) (26), or anti-CD206 (Abcam, ab64693) (27). After washing and blocking with 3% hydrogen peroxidase (Thermo Fisher Scientific), the samples were incubated with biotinylated secondary antibody (Vector Laboratories) (28), visualized with an ABC kit (Vector Laboratories) and DAB Quanto chromogen (Thermo Fisher Scientific), and counterstained with hematoxylin.

Stromal vascular cell isolation and adipocyte differentiation

Stromal vascular (SV) cells were isolated from adipose tissues of 3-month-old male mice. The adipose pads were isolated and incubated with 0.1% type 2 collagenase and 2% BSA in DMEM at 37°C for 1 hour. Mature adipocytes were removed by centrifugation and aspiration. Then SV cells were washed with 10% fetal bovine serum in DMEM, placed through a 100-μm mesh filter, and plated onto a six-well cell culture plate (2 × 10⁶ cells per well). To differentiate to mature adipocytes, cells were treated with 10% fetal bovine serum–DMEM containing 10 μg/mL insulin for 2 days. The medium was replaced every 48 hours for an additional 6 days. The remaining cells were dissolved and triglyceride content was measured for mature adipocyte content. In separate experiments, cells were collected to assay mRNA expression of mature adipocyte markers by qRT-PCR.

qRT-PCR

mRNA abundance was evaluated by qRT-PCR using previously described approaches (19, 22).

RNA sequencing

Total RNAs were extracted from SV cells with a QIAcube automated RNA purification robot (Qiagen), and the RNA sequencing analysis was performed at the Whole-Genome Sequencing Core at the University of Houston. Sample quality was measured by an Agilent Bioanalyzer system, and precise RNA quantification was determined by using RiboGreen reagents and a fluorescence plate reader. Samples with the RNA integrity number ≥4 (scale of 1 to 10, with 10 representing the highest integrity and quality) were forwarded for library construction and sequencing. Libraries were prepared by using a NuGen Ovation universal RNA kit followed by rRNA depletion. Libraries were sequenced by an Illumina NextSeq 500 genome analyzer, and the sequenced data were paired-end and second stranded. The quality of obtained data was validated by analyzing the sequenced libraries using FastQC (version 0.11.6) (29), and all libraries passed the checkpoints of (i) basic statistics, (ii) per base sequence quality, (iii) per tile sequence quality, (iv) per sequence quality scores, (v) per base N content, (vi) sequence duplication levels, and (vii) adapter content (data not shown). Additionally, the uniform distribution of the reads across all libraries was confirmed by generating the corresponding box plots. Then, the data were mapped to the Mus musculus Ensembl genome, GRCm38.p5, using HiSat2 and default parameters (30). SAM files were sorted and converted to BAM files using SamTools (version 1.3.1), and raw counts per gene from the mapped reads were obtained from StringTie results using the “prepDE” python code (31). Differential expression analysis was performed using DESeq2 package (version 1.20) (32). Genes with significant difference by a P value ≤0.05 and log₂(fold change) ≥1 were identified by pairwise comparison between sample groups. Pathway analysis and functional classification were performed by using the DAVID functional annotation clustering tool with medium classification stringency and Metascape gene annotation and analysis tool (http://metascape.org) with a P value ≤0.01 (33, 34). Pathways with a Benjamini-corrected P value <0.05 were considered significant.

Statistical analysis

All data are expressed as mean ± SEM. A two-tailed Student t test or ANOVA was used to assess differences between two groups or among more than two groups, respectively, with Newman–Keuls post hoc testing following ANOVA. P values <0.05 were considered significant.

Results

27HC increases body weight in response to an adipogenic diet

One of the major protective effects of estrogen/ER in cardiovascular and metabolic diseases is the prevention of body weight gain. To determine the impact of 27HC on the regulation of body weight gain by estrogens, ovariectomized Cyp7b1^−/− and Cyp7b1^+/+ mice received either E₂ or vehicle and were fed either an HFHC diet (21% milk fat, 0.2% cholesterol) or corresponding NC diet (4.0% milk fat, <0.04% cholesterol) for 8 weeks. Mice null for CYP7B1 have elevated 27HC levels similar to HFHC diet–fed wild-type mice, in both the circulation and in tissues, but entirely normal plasma cholesterol and triglyceride levels (Table 1) (19). Wild-type mice treated with vehicle showed modest body weight gain on an NC diet and predictably marked body weight gain on an HFHC diet (Fig. 1A). Treatment with E₂, which caused elevated but still physiologically relevant circulating E₂ levels (Table 1), blunted body weight gain for wild-type mice on an HFHC diet, indicative of the potent capacity for estrogen to regulate body weight gain. In contrast, the body weight gain that occurred on an HFHC diet in Cyp7b1^−/− mice was not attenuated by E₂. There was no difference in the body weight gain between Cyp7b1^−/− and Cyp7b1^+/+ mice with an NC diet (Fig. 1A). Similar to the results from female mice without E₂, there was no difference in body weight between Cyp7b1^−/− and Cyp7b1^+/+ male mice fed with an NC diet; however, Cyp7b1^−/− male mice gained more body weight when fed an HFHC diet compared with Cyp7b1^+/+ male mice (Fig. 1B). To evaluate the direct effect of 27HC on body weight gain, C57BL/6 mice were administered 27HC or vehicle and fed an NC or HFHC diet. As we confirmed previously (18, 19), 27HC administration increased circulating 27HC levels within the physiological range but did not change circulating cholesterol levels (Table 2). As observed with Cyp7b1^−/− and Cyp7b1^+/+ male mice on an HFHC diet (Fig. 1B), mice on an HFHC diet gained more body weight when they were treated with 27HC starting from 6 weeks (Fig. 1C). There was no difference in body weight gain between the two groups fed an NC diet. To determine whether the effect of 27HC on body weight gain in response to the HFHC diet is ERα-dependent, male ERα-null or LXRα/β-null mice were also tested. As previously reported (11), ERα-null mice had elevated body weight compared with wild-type mice at the start of the experiment. 27HC treatment did not stimulate additional body weight gain in ERα-null mice (Fig. 1D); however, ERα-null mice still retained the capacity to gain body weight (data not shown). Despite previous reports that 27HC acts as a potential agonist for the oxysterol receptor LXR in a tissue-specific manner (18, 35), treatment of LXRα/β-null mice with 27HC increased body weight gain more than in vehicle-treated mice (Fig. 1D). These results indicate that 27HC increased body weight gain in response to HFHC diet feeding both in male and female mice, and the effect of 27HC is ERα-dependent but not LXR-dependent.

Table 1.

Plasma Cholesterol, 27HC, Triglyceride, and E₂ (for Females) Levels of Cyp7b1^+/+ and Cyp7b1^−/− Mice Used in the Experiments (n = 6 to 11)

	Diet	Cholesterol, mg/dL	27HC, ng/mL	TGs, mg/dL	E2, ng/mL
Male
Cyp7b1+/+	NC	220.2 ± 37.5	75.5 ± 9.6	175.2 ± 18.0
	HFHC	443.5 ± 34.4a	188.0 ± 8.3a	206.6 ± 9.0a
Cyp7b1−/−	NC	241.5 ± 18.1	412.6 ± 25.1b	169.5 ± 13.5
	HFHC	453.2 ± 29.1a	617.6 ± 38.0a,b	220.4 ± 11.9a
Female
Cyp7b1+/+; Veh.	NC	141.2 ± 21.3	82.6 ± 15.4	133.1 ± 16.9	11.4 ± 3.1
	HFHC	225.7 ± 23.3a	203.2 ± 37.0a	184.1 ± 6.5a	8.6 ± 0.8
Cyp7b1+/+; E2	NC	124.5 ± 9.2	56.1 ± 5.3c	153.0 ± 5.7c	45.4 ± 9.2c
	HFHC	191.9 ± 19.8a	87.9 ± 11.1a,c	215.5 ± 11.2a,c	79.0 ± 25.5c
Cyp7b1−/−; Veh.	NC	159.0 ± 11.7	201.4 ± 26.8b	145.5 ± 4.0	11.7 ± 1.9
	HFHC	239.4 ± 21.9a	295.5 ± 32.3a,b	187.9 ± 7.0a	10.8 ± 3.1
Cyp7b1−/−; E2	NC	124.0 ± 6.5	202.4 ± 20.4b	144.9 ± 16.3	94.0 ± 33.7c
	HFHC	238.9 ± 14.8a	266.9 ± 18.1a,b	226.6 ± 20.0a,c	100.9 ± 14.6c

Abbreviations: TG, triglyceride; Veh., vehicle.

^a

P < 0.05 vs NC.

^b

P < 0.05 vs wild type.

^c

P < 0.05 vs vehicle.

Figure 1.

27HC increases body weight in response to an HFHC diet. (A) Female Cyp7b1^+/+ and Cyp7b1^−/− mice (n = 6 to 8 per group) were ovariectomized, implanted with placebo or E₂ pellets, and fed an NC or HFHC diet for 8 wk. Then body weights of the mice were measured for 8 wk. Data are provided in scatterplots. Mean values are indicated by the long horizontal line, and SEM by the short horizontal line. (B) Body weights of male Cyp7b1^+/+ and Cyp7b1^−/− mice (n = 8 to 11 per group) fed an NC or HFHC diet for 8 wk. (C) Male C57BL/6 mice were treated with 27HC for 8 wk (n = 6 to 7). (D) Body weights of male ERα-null (Esr1^−/−) and LXRα/β-null (Nr1h3^−/−Nr1h2^−/−) mice treated with vehicle or 27HC. The mice were fed with an HFHC diet for 8 wk (n = 6 to 7 per group). ^★P < 0.05.

Table 2.

Plasma and Hepatic Parameters of C57BL/6 Male Mice With Vehicle or 27HC Administration (n = 6 to 7)

	NC		HFHC
	Vehicle	27HC	Vehicle	27HC
Plasma
Cholesterol, mg/dL	116.5 ± 23.0	124.1 ± 18.1	191.3 ± 8.4a	187.1 ± 8.2a
27HC, ng/mL	118.8 ± 7.2	290.5 ± 20.2b	211.1 ± 17.8a	602.9 ± 47.3a,b
TGs, mg/dL	70.8 ± 5.9	71.1 ± 5.6	138.2 ± 9.7a	136.0 ± 11.9a
Fasting glucose, mg/mL	103.1 ± 2.8	103.8 ± 5.0	112.4 ± 6.5a	115.1 ± 6.4a
Fasting insulin, ng/mL	0.335 ± 0.077	0.322 ± 0.048	0.568 ± 0.062a	0.574 ± 0.082a
Liver
Cholesterol, mg/dL	6.21 ± 0.29	6.56 ± 0.60	24.0 ± 1.89a	22.2 ± 2.65a
27HC, ng/mL	3.88 ± 0.67	12.0 ± 0.16b	8.37 ± 2.07a	13.0 ± 0.28b
TGs, mg/dL	71.4 ± 6.7	61.2 ± 8.1	234.8 ± 21.0a	220.7 ± 25.3a

Abbreviation: TG, triglyceride.

^a

P < 0.05 vs NC.

^b

P < 0.05 vs vehicle control.

27HC increases body fat mass

Because estrogen/ER decreases satiety (11) and 27HC modulates ER function, the effect of 27HC on body weight gain in response to an HFHC diet may be due to the effects caused by feeding behavior or metabolic alteration (36). Therefore, the effects of 27HC on calorie intake and fat absorption were evaluated by measuring food consumption and lipid excretion in feces, respectively. In the NC-fed groups, there was no difference in food intake or fat absorption between 27HC-treated and control mice (Fig. 2A and 2B). The HFHC diet increased fat absorption compared with NC, but, again, there was no difference in food intake or fat absorption between the 27HC and vehicle groups, indicating that although lipid contents in feces do not necessarily represent caloric contents, 27HC administration does not affect food intake or calorie absorption under our experimental conditions. Next, we measured total body fat mass by use of mouse nuclear magnetic resonance spectroscopy. Although there was no difference in body weight between 27HC- and vehicle-treated mice fed an NC diet, 27HC-treated mice had more body fat mass than did control mice (Fig. 2C). On an HFHC diet, control mice gained more fat mass than did the NC-fed mice, and 27HC-treated mice gained further body fat mass in parallel with the body weight gain. These findings indicate that 27HC increases body fat mass regardless of diet type.

Figure 2.

27HC increases fat mass. (A) Food intake and (B) fat absorption were measured on individually housed mice. Each dot represents data from an individual mouse. (C) Body fat and lean mass were measured at the end of the experiment. (D) Oxygen consumption (VO₂) , (E) physical activity, and (F) respiratory exchange ratio (VCO₂/VO₂) were measured for male mice treated with 27HC for 8 wk in metabolic cages. In (D), the areas under the curve (AUC) are also shown. n = 5 to 6. ^★P < 0.05 vs vehicle; ^†P < 0.05 vs NC.

To further investigate the effects of 27HC on metabolism, 27HC- and vehicle-treated mice fed with an NC or an HFHC diet were housed in metabolic cages and assayed for oxygen consumption, physical activity, and respiratory exchange rates. There was no difference in O₂ consumption between 27HC- and vehicle-treated groups in the NC-fed mice (Fig. 2D), despite the increased body fat mass (Fig. 2C). In the HFHC diet groups, 27HC-treated mice had less O₂ consumption in the dark (active) cycle compared with vehicle-treated mice. Given that physical activity shows a similar trend as O₂ consumption (Fig. 2E) and also that the mice had more body weight than did vehicle-treated mice at the time when the metabolic measurements started (Fig. 1C and data not shown), it is plausible that 27HC-treated mice on an HFHC diet are physically less active owing to heavier body weight and therefore consume less O₂. Indeed, there was no difference in the respiratory exchange ratio between 27HC- and vehicle-treated mice in any diet groups (Fig. 2F). Although it is still possible that 27HC can alter metabolism in the HFHC diet groups, these results suggest that 27HC directly affects peripheral tissues to increase body fat.

27HC increases VWAT

Liver and adipose tissues are major contributors to body fat mass. To examine the effect of 27HC on liver fat content, biochemical, histological, and gene expression analyses were performed. Liver 27HC levels were elevated by 27HC administration, although there was no difference in liver cholesterol or triglyceride levels between 27HC- and vehicle-treated mice (Table 2). These results are consistent with the histological observation that 27HC treatment did not increase the size of lipid droplets in liver (Fig. 3A and 3B). Additionally, lipogenic gene expression was not affected by 27HC, although mRNA expression of Scd-1 and Srebp-1c was elevated in the HFHC diet–fed mice (Fig. 3C). Thus, the effect of 27HC on body fat mass increase is not due to effects on the liver.

Figure 3.

27HC does not increase hepatic lipogenesis. (A and B) Representative images of liver samples stained with (A) hematoxylin and eosin or (B) Oil Red O. Original magnification, ×400. (C) Fold changes of the mRNA abundance of hepatic lipogenesis genes (n = 5 to 6 per group). ^†P < 0.05 vs NC feeding.

Next, the effect of 27HC on adipose tissue mass was examined for brown adipose tissue (BAT), IgWAT, and GWAT. As shown in Fig. 4A, 8 weeks of 27HC administration did not affect BAT or IgWAT mass. In contrast, 27HC increased GWAT mass in both the NC and HFHC groups. In 1-year-old male Cyp7b1^−/− and Cyp7b1^+/+ mice fed an NC diet, Cyp7b1^−/− mice had increased IgWAT and GWAT mass compared with Cyp7b1^+/+ mice; in contrast, there was no difference in the BAT mass (Fig. 4B). We also confirmed that the effect of 27HC on GWAT mass occurs in an ERα-dependent, but LXR-independent, manner, because 27HC changed GWAT mass in LXRα/β-null mice, but not in ERα-null mice (Fig. 4C and 4D). Additionally, the mRNA expression of mature adipocyte markers aP2, lipoprotein lipase, and PPARγ2 was increased in GWAT for 27HC-treated mice fed either an NC or a HFHC diet (Fig. 4E). These findings indicate that 27HC treatment preferentially increases GWAT mass.

Figure 4.

27HC increases visceral fat. (A–D) Ratio of adipose tissue weight to body weight in (A) wild-type (WT), (B) Cyp7b1^−/−, (C) ERα-null, or (D) LXRα/β-null mice. In (B), the mice fed with normal chow at 12 mo of age were used (n = 7 to 8). (E) Gene expression analyses by qRT-PCR using IgWAT and GWAT collected from vehicle- or 27HC-treated mice fed NC or HFHC diets (n = 6 to 7). ^★P < 0.05 vs vehicle control, except for (B) (^★P < 0.05 vs WT).

27HC induces adipose hyperplasia and adipose tissue expression of inflammatory genes, similar to that induced by an HFHC diet challenge

Increased WAT mass occurs via hypertrophy and/or hyperplasia in the tissue. Having determined that GWAT mass was increased by 27HC, we next measured the cell size of GWAT in histological sections from mice treated with 27HC for 8 weeks. As expected, HFHC diet–fed mice had larger adipose cell size in GWAT compared with NC-fed mice (Fig. 5A). Administration of 27HC did not alter cell size in GWAT, either in the NC diet or HFHC diet groups. Next, DNA content in GWAT and IgWAT from the HFHC diet–fed mice was measured to evaluate the relative numbers of adipocytes. DNA content in both GWAT and IgWAT was increased by 27HC with an abundant increase in GWAT (Fig. 5B), suggesting that the increased adiposity in GWAT was a consequence of hyperplasia rather than hypertrophy of adipose cells. To examine whether 27HC treatment alters the adipose tissue cell population, SV compartments, which contain the cells that differentiate into mature adipocytes (37), were taken from IgWAT of Cyp7b1^−/− and Cyp7b1^+/+ mice and cultured to differentiate them into mature adipocytes ex vivo. Isolated SV cells were cultured under the same culture conditions for all groups with excess volume of culture media. Endogenous CYP7B1 abundance in the cells did not affect levels of 27HC in the culture media under the experimental conditions, as we observed for other cell types (18). Therefore, the difference in cellular triglyceride content and mature adipocyte-marker expression reflects the difference in the adipocyte tissue cell population in SV compartments. The SV cells from Cyp7b1^−/− mice showed more mature adipocyte-marker expression than did those from wild-type mice (Fig. 5C), suggesting that 27HC increases the number of cells that can be differentiated to mature adipocytes in the SV compartments. To assess the effect of 27HC on adipocyte cell differentiation, we isolated SV compartments from GWAT or IgWAT of wild-type mice and cultured them ex vivo in the presence of 27HC, E₂, or vehicle control. Consistent with the gene expression results (Fig. 5C), insulin induced adipocyte differentiation as determined by measuring cellular lipid content (Fig. 5D). In contrast, treatment with 27HC or E₂ did not increase cellular lipid content in IgWAT and GWAT, indicating that 27HC does not affect preadipocyte-to-mature adipocyte differentiation, which is consistent with a previous report (16). These findings indicate that 27HC induces hyperplasia in GWAT by increasing the number of cells that give rise to mature adipocytes.

Figure 5.

27HC induces hyperplasia and inflammatory gene expression in WAT. (A) Adipocyte cell size in GWAT from vehicle- and 27HC-treated mice (n = 7). ^†P < 0.05 vs NC. (B) Adipose DNA content per individual tissue (n = 6). *P < 0.05 vs vehicle control. (C) mRNA abundance in adipocytes differentiated ex vivo from SV cells obtained from IgWAT of Cyp7b1^−/− or Cyp7b1^+/+ [wild-type (WT)] mice (n = 6 to 8); ^★P < 0.05 vs no insulin; ^†P < 0.05 vs WT. (D) SV cells from GWAT or IgWAT of male C57BL/6 mice were differentiated ex vivo with compounds for 6 d. Then intracellular triglyceride (TG) was measured (n = 4). ^★P < 0.05 vs vehicle without insulin. (E) Heat map of inflammatory gene expression created using RNA sequencing data (n = 3). (F) Venn diagram of genes induced by each treatment.

To elucidate the mechanism by which 27HC increases adiposity, RNA sequencing analysis was performed to profile gene expression in SV compartments from mice treated with 27HC and fed either an NC or an HFHC diet. We validated the quality of the data and also confirmed the uniform distribution of the reads across all libraries (data not shown). Mice in the HFHC diet group showed increased expression of pathways related to inflammation and immune responses compared with the NC diet group (Fig. 5E), similar to the changes observed in mature adipose tissue (38). The administration of 27HC to NC-fed mice also enriched genes related with inflammatory responses. Additionally, the HFHC vs NC diet group and the 27HC vs vehicle in an NC diet group shared gene clusters related to inflammatory and immune responses. In contrast, there was no overlap in inflammatory genes between any other comparison groups, suggesting that 27HC induces the same set of inflammatory genes as does the HFHC diet challenge.

27HC increases total and M1 macrophages in WAT

Inflammation in adipose tissues is critically involved in adipose tissue development (5, 38), and 27HC increases vascular inflammation and circulating levels of the inflammatory cytokines TNFα and IL-1β through its action on ERs (19). To elucidate the effect of 27HC on adipose inflammation, macrophage content in GWAT was analyzed by immunohistochemistry using markers for total (CD68), M1 (NOS2), and M2 (CD206) macrophages (39). The treatment groups were NC diet–fed mice treated with vehicle or 27HC and HFHC diet–fed mice treated with vehicle. Treatment with 27HC increased the number of CD68-positive (Fig. 6A) and NOS2-positive (Fig. 6B) macrophages; in contrast, it decreased the number of CD206-positive macrophages (Fig. 6C). As predicted (40, 41), the HFHC diet also caused the similar changes as did the 27HC treatment on an NC diet in total, M1, and M2 macrophages. Because M1 macrophages produce proinflammatory cytokines, to examine whether the increase in total and M1 macrophages resulted in the expression of inflammatory cytokines, gene expression of Il-1β and Tnfα, which were also observed in the SV compartments from mice treated with 27HC (Fig. 5E), was analyzed in WAT from mice treated with 27HC. Treatment of mice with 27HC increased expression of Il-1β and Tnfα in adipose tissue regardless of diet (Fig. 6D). Furthermore, treatment of mice with 7-keto cholesterol, which does not affect ER activity (18), did not increase the expression of these genes in GWAT (Fig. 6E), indicating that the effect was 27HC specific. These results indicate that 27HC increases the number of total and M1 macrophages in WAT and also induces inflammatory cytokine expression in the same tissue.

Figure 6.

27HC increases total and M1 macrophages in WAT. (A–C) Representative images of GWAT samples stained with (A) anti-CD68, (B) anti-NOS2, or (C) anti-CD206. Arrows point to positive staining. Scale bars, 100 μm. (D) Gene expression analyses by qRT-PCR using IgWAT and GWAT collected from vehicle- or 27HC-treated mice fed NC or HFHC diets (n = 6 to 7). ^★P < 0.05 vs vehicle control. (E) Fold changes of the mRNA abundance of Il-1β and Tnfα genes in GWAT from vehicle-, 27HC-, or 7-keto cholesterol (7KC)–treated mice fed an NC diet (n = 4 to 5 per group).

Discussion

Although there is no clear evidence that menopause directly causes an increase in body weight, it does promote changes in body fat distribution (42). Estrogens protect against the accumulation of visceral fat, and the loss of estrogens with menopause is associated with an increase in central fat. In this study, 27HC administration for 8 weeks promoted body fat accumulation via ERα actions without altering food intake, fat absorption, or fasting plasma glucose levels (Fig. 2; Table 2). Because it takes at least 3 months of E₂ treatment to alter fasting glucose levels and to develop glucose intolerance and insulin resistance (43), it is still possible that 27HC induced small, undetectable metabolic changes that if maintained over a long period could result in glucose intolerance and insulin resistance (36). This notion is also supported by the finding that 27HC administration for 8 weeks only increased VWAT (Fig. 4A), whereas long-term 27HC elevation by the genetic deletion of Cyp7b1 increased both GWAT and IgWAT (Fig. 4B). If so, then the change in obesity would come earlier than changes in glucose intolerance and insulin resistance, which thereby might be secondary effects of the obesity caused by elevated 27HC levels.

The effect of 27HC on body weight gain and WAT mass was dependent on ERα, but not LXR (Fig. 1D and Fig. 4C and 4D). LXR deficiency protects from body weight gain during HFHC diet feeding (22). LXR agonists induce hepatic steatosis, which was not seen in the mice with 27HC administration in the current study (Fig. 3), further supporting the idea that the effect of 27HC on body weight gain and obesity is LXR-independent. Adipocyte-specific ERα deletion eliminates the beneficial effects of estrogen and increases inflammation in adipose tissue in male mice (5). Although 27HC blocked the effect of E₂ on body weight gain in female mice (Fig. 1A), 27HC may have specific effects on WAT that are mediated by ERα, similar to what is seen in the cardiovascular system where 27HC has opposing effects to E₂ on inflammatory responses in vascular endothelial cells and macrophages (19). High-fat diet feeding induces hyperplasia of VWAT, but not SWAT, whereas estrogen induces hyperplasia of both in response to a high-fat diet (44). In female mice, ovariectomy decreases hyperplasia in SWAT, whereas it does not affect high-fat diet–induced hyperplasia in VWAT. In contrast, we found that 27HC induced hyperplasia in GWAT and IgWAT, suggesting that ER is also involved in the hyperplasia of VWAT in an estrogen-independent manner. Interestingly, a synthetic SERM tamoxifen induces acute loss of fat mass followed by de novo adipogenesis in VWAT (45). GWAT mass was increased by 27HC regardless of diet type, suggesting that 27HC increases the capacity of fat storage in WAT, which may be a reason, at least a part, that 27HC treatment did not increase fat content in the liver regardless of the increase in body weight. There are anatomical differences between mice and humans, and it should be taken into consideration whether the GWAT in mice is comparable in terms of location and function to the VWAT in human, although GWAT is most frequently used in the literature (4). Further research is warranted to define the effects of 27HC on the adipose tissue in human.

Interestingly, administration of 27HC to NC diet–fed mice induced similar inflammatory gene sets as those observed after HFHC diet feeding (Fig. 5F). 27HC also increased total and M1 macrophages in GWAT (Fig. 6). Increased inflammatory responses in GWAT is critically involved in pathological WAT expansion (38); therefore, these results suggest that 27HC mimics the inflammatory signaling that occurs as a consequence of adipogenic diet challenge. Further investigations into how 27HC increases proinflammatory M1 macrophages in adipose tissue and the precise mechanism by which 27HC induces inflammatory responses in WAT are warranted.

In this study, we show a direct link between 27HC and adipose tissue function. Further studies to define the precise mechanism by which 27HC induces adiposity may lead to novel approaches to regulate diet-induced obesity by controlling adipose tissue distribution.

Acknowledgments

We thank Dr. David Mangelsdorf for LXRα/β-null mice, Dr. Jeffery McDonald for 27HC measurements, Drs. Sujash Chatterjee and Yinghong Pan for the RNA sequencing, Kevin Dao and Dr. Wanfu Wu for the histology, and Drs. Kai Sun, Steven Kliewer, Jan-Ake Gustafsson, David Stewart, Philip Shaul, and their laboratory members for helpful comments and suggestions.

Financial Support: This work was supported by a Japan Society for Menopause and Women’s Health (JSMWH) Bayer grant (to S.H.), National Institute of Environmental Health Sciences (NIEHS) Division of Intramural Research Grant 1ZIAES070065 (to K.S.K), National Institutes of Health Grant HL127037 (to M.U.), and American Diabetes Association Grant 7-11-JF-46 (to M.U.).

Author Contributions: A.A., T.I., and M.U. designed, executed, and interpreted the data; W.-R.L. performed cell culture studies; J.U. and L.B. performed histology and biochemical analyses; A.A. and S.H. performed RNA sequencing; K.S.K. supplied the Esr1^−/− mice and contributed to the design of the mouse studies and manuscript preparation; M.U. conceived and supervised the project; and A.A. and M.U. wrote the paper.

Additional Information

Disclosure Summary: The authors have nothing to disclose.

Data Availability:

The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

Abbreviations:

Abbreviations:
 
• 27HC

  27-hydroxycholesterol

 
• BAT

  brown adipose tissue

 
• E₂

  17β-estradiol

 
• ER

  estrogen receptor

 
• GWAT

  gonadal white adipose tissue

 
• HFHC

  high-fat, high-cholesterol

 
• IgWAT

  inguinal white adipose tissue

 
• NC

  normal chow

 
• qRT-PCR

  quantitative RT-PCR

 
• SERM

  selective estrogen receptor modulator

 
• SV

  stromal vascular

 
• SWAT

  subcutaneous white adipose tissue

 
• VWAT

  visceral white adipose tissue

 
• WAT

  white adipose tissue

Author notes

A.A. and T.I. contributed equally to this study.