Rhein protects against obesity and related metabolic disorders through liver X receptor-mediated uncoupling protein 1 upregulation in brown adipose tissue

Abstract

Liver X receptors (LXRs) play important roles in regulating cholesterol homeostasis, and lipid and energy metabolism. Therefore, LXR ligands could be used for the management of metabolic disorders. We evaluated rhein, a natural compound from Rheum palmatum L., as an antagonist for LXRs and investigated its anti-obesity mechanism in high-fat diet-fed mice. Surface plasmon resonance assays were performed to examine the direct binding of rhein to LXRs. LXR target gene expression was assessed in 3T3-L1 adipocytes and HepG2 hepatic cells in vitro. C57BL/6J mice fed a high-fat diet were orally administered with rhein for 4 weeks, and then the expression levels of LXR-related genes were analyzed. Rhein bound directly to LXRs. The expression levels of LXR target genes were suppressed by rhein in 3T3-L1 and HepG2 cells. In white adipose tissue, muscle and liver, rhein reprogrammed the expression of LXR target genes related to adipogenesis and cholesterol metabolism. Rhein activated uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) in wild-type mice, but did not affect UCP1 expression in LXR knockout mice. In HIB-1B brown adipocytes, rhein activated the UCP1 gene by antagonizing the repressive effect of LXR on UCP1 expression. This study suggests that rhein may protect against obesity and related metabolic disorders through LXR antagonism and regulation of UCP1 expression in BAT.

Keywords: LXR; UCP1; antagonist; diet-induced obesity; rhein..

Figure 1

Rhein binds directly to liver X receptors (LXRs). (A) to (F) Specific binding affinities to the ligand-binding domains (LBDs) of LXRs were analyzed by surface plasmon resonance (SPR) assays. The photographs were obtained after injection of a series of concentrations of GW3965, puerarin or rhein in phosphate-buffered saline (PBS) containing 0.2% dimethyl sulfoxide (DMSO) over the immobilized LXRα-LBD surface (A to C) or the LXRβ-LBD surface (D to F) on a CM5 sensor chip. The K_D values of rhein for LXRα and LXRβ were 46.7 μM and 31.97 μM, respectively. (G) and (H) Specific binding affinities against thyroid hormone receptors (TRs) were analyzed by SPR assays. The photographs were obtained after injection of a series of concentrations of rhein and T3 in PBS containing 0.2% DMSO over the immobilized TRα surface (G) or TRβ surface (H) on a CM5 sensor chip. For the SPR assay, ligand binding was measured at a flow rate of 30 μL/min for 2 min, and dissociation was initiated upon replacement of the analyte with running buffer. The response units (RU) were corrected for non-specific binding to a blank flow channel (relative response). RH, rhein.

Figure 2

Rhein decreases liver X receptor (LXR) target gene expression in vitro. Rhein decreased the expression of LXR target genes in 3T3-L1 adipocytes (A) and HepG2 cells (B). White bars, 3T3-L1 adipocytes or HepG2 cells treated with dimethyl sulfoxide (DMSO); black bars, 3T3-L1 adipocytes or HepG2 cells were treated with 1 μM GW3965 (GW); striped bars, 3T3-L1 adipocytes or HepG2 cells were treated with 1 μM GW3965 and 25 μM rhein (RH). mRNA expression levels were estimated by quantitative real-time PCR. The results are shown as mRNA levels relative to that treated with DMSO. Values are means ± standard deviation of three independent experiments.

Figure 3

Rhein decreases the expression levels of liver X receptor (LXR) target genes in white adipose tissue (WAT), muscle and liver in diet-induced obese mice. After administration of rhein for 4 weeks, total RNA was isolated from WAT, muscle and liver, and was subjected to quantitative real-time RT-PCR. Rhein decreased the expression of LXR target genes in WAT (A), muscle (B) and liver (C). The mRNA expression levels are shown as values in rhein-treated groups (striped bars, HF + RH) relative to the control (water) groups (black bars, HF). Values are means ± standard error of the mean for five mice per group. *P < 0.05, **P < 0.01.

Figure 4

Rhein activates uncoupling protein 1 (UCP1) gene expression in brown adipose tissue (BAT) through LXR antagonism. (A) Rhein increased the expression of UCP1 in BAT of high-fat diet-induced obese (DIO) wild-type mice. The mRNA expression levels are shown as values in rhein-treated groups (striped bars, HF + RH) relative to the control (water) groups (black bars, HF). Values are means ± standard error of the mean (SEM) for five mice per group. **P < 0.01. (B) Rhein did not affect UCP1 expression in BAT of high-fat DIO LXR knockout (KO) mice. mRNA expression levels are shown as values in rhein-treated groups (striped bars, HF + RH) relative to the control (water) groups (black bars, HF). Values are means ± SEM for five mice per group. (C) mRNA expression levels of UCP1 in differentiated HIB-1B cells. HIB-1B cells were treated with forskolin (FSK, 10 μM), GW3965 (1 μM) or rhein (25 μM) for 6 h and mRNA expression was determined by quantitative real-time PCR. Values are means ± standard deviation of three independent experiments. (D) Differentiated HIB-1B cells were treated with the indicated compounds for 6 h and the nuclear extract (NE) was collected for electrophoretic mobility shift assays. Lane 1: probe only; lane 2: probe plus NE [dimethyl sulfoxide (DMSO)]; lane 3: probe plus NE (DMSO) plus 100 × competing probe; lane 4: probe plus NE (1 μM GW3965); lane 5: probe plus NE (1 μM GW3965 plus 25 µM rhein); lane 6: probe plus NE (1 μM GW3965 plus 10 µM FSK); lane 7: probe plus NE (1 µM GW3965 plus 25 µM rhein plus 10 µM FSK). (E) Transmission electron microscopy images of BAT (magnification, 2550×) from high-fat DIO mice treated with water (HF) or rhein (HF + RH). Arrows indicate the mitochondria (M) and lipid droplets (L). (F) Number of mitochondria per unit of nuclear DNA in BAT of high-fat DIO mice treated with rhein (striped bars) or water (black bars). Values are means ± SEM for five mice per group. *P < 0.05.