A Novel Aryl Hydrocarbon Receptor Antagonist HBU651 Ameliorates Peripheral and Hypothalamic Inflammation in High-Fat Diet-Induced Obese Mice

Abstract

Obesity is a chronic peripheral inflammation condition that is strongly correlated with neurodegenerative diseases and associated with exposure to environmental chemicals. The aryl hydrocarbon receptor (AhR) is a ligand-activated nuclear receptor activated by environmental chemical, such as dioxins, and also is a regulator of inflammation through interacting with nuclear factor (NF)-κB. In this study, we evaluated the anti-obesity and anti-inflammatory activity of HBU651, a novel AhR antagonist. In BV2 microglia cells, HBU651 successfully inhibited lipopolysaccharide (LPS)-mediated nuclear localization of NF-κB and production of NF-κB-dependent proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. It also restored LPS-induced mitochondrial dysfunction. While mice being fed a high-fat diet (HFD) induced peripheral and central inflammation and obesity, HBU651 alleviated HFD-induced obesity, insulin resistance, glucose intolerance, dyslipidemia, and liver enzyme activity, without hepatic and renal damage. HBU651 ameliorated the production of inflammatory cytokines and chemokines, proinflammatory Ly6c^high monocytes, and macrophage infiltration in the blood, liver, and adipose tissue. HBU651 also decreased microglial activation in the arcuate nucleus in the hypothalamus. These findings suggest that HBU651 may be a potential candidate for the treatment of obesity-related metabolic diseases.

Keywords: antagonist; aryl hydrocarbon receptor (AhR); high-fat diet; hypothalamus; inflammation.

Figure 1

Effects of AhR antagonists on inflammatory cytokines and mitochondrial activities. (A) Dose-dependent AhR antagonist activity assay using AhR-dependent luciferase (AhRE-luciferase) assay. The cells were pretreated with AhR antagonists for 1 h, stimulated with 50 pM TCDD for 24 h, and assayed for luciferase activity. (B–D) Murine BV2 microglial cells were pre-treated with 10 μM HBU651 (HBU) or 1 μM CH223191 (CH) for 24 h, stimulated with 100 ng/mL LPS for 4 h, and harvested for analysis. Realtime qRT-PCR for TNF-α (B), IL-1β (C), and IL-6 (D) mRNA levels. Data are expressed as the mean ± SEM (n = 5). ^## p < 0.01, ^### p < 0.001 vs. Control (CTL), and * p < 0.05, ** p < 0.01 vs. LPS-treated DMSO. (E) Effect of HBU651 on LPS-induced nuclear localization of NF-κB. Murine BV2 microglial cells were pre-treated with 10 μM HBU651 or 1 μM CH223191 for 24 h, prior to 100 ng/mL LPS stimulation for 4 h. BV2 cells were immunostained with NF-κB p65/RELA antibody (green) and DAPI (blue) for nuclei (Merge). Cell images were enlarged to visualize the nuclear localization of NF-κB (Enlarged). Representative confocal micrograph images of BV2 cells are shown (scale bar = 20 μm).

Figure 2

Effects of AhR antagonists on mitochondrial function. Murine BV2 microglial cells on a 96 well plate were pre-treated with 10 μM HBU651 (HBU) or 1 μM CH223191 (CH) for 24 h, stimulated with 100 ng/mL LPS for 4 h, and harvested for mitochondrial activity analysis. (A) intracellular ATP content, (B) TMRE-mediated mitochondrial membrane potential, and (C) DCF-DA-based total ROS generation. (D) Mito-Sox-based mitochondrial superoxide generation. Data are expressed as the mean ± SEM (n = 5). ^# p < 0.05, ^### p < 0.001 vs. Control (CTL), and ** p < 0.01 vs. LPS-treated DMSO.

Figure 3

Effects of HBU651 on metabolic phenotypes in HFD-induced obese mice. (A) Experimental scheme in vivo, (B) Body weight (BW, g) at the end of the experiment, (C) Changes of BW, (D) Oral calories intake, (E) Epididymal white adipose tissue (EWAT) weight, (F) Liver weight, (G) Fasting blood glucose (FBG), (H) Fasting insulin, (I) Homeostatic model assessment of insulin resistance (HOMA-IR), (J) Time-course changes of blood glucose in the oral glucose tolerance test (OGTT), (K) Area under the curve (AUC) of OGTT. Data are expressed as means ± SEM (n = 5). ^### p < 0.001 vs. NC, and * p < 0.05, ** p < 0.01 vs. HFD-control. NC, normal chow; Control, HFD-control; HBU10, HFD plus HBU651 10 mg/kg; HBU30, HFD plus HBU651 30 mg/kg.

Figure 4

Effects of HBU651 on the lipid and biochemical profiles in the blood of HFD-induced obese mice. (A) Total-cholesterol (Total-C), (B) LDL-cholesterol (LDL-C), (C) HDL-cholesterol (HDL-C), (D) Free fatty acid (FFA), (E) Phospholipids (PL), (F) Triglyceride (TG), (G,H) Liver function marker enzyme activities of AST (G) and ALT (H), (I) Creatinine, (J,K) ELISA. Serum protein levels of TNF-α (J) and MCP-1 (K). Data are expressed as means ± SEM (n = 5). ^# p < 0.05, ^### p < 0.001 vs. NC, and * p < 0.05, ** p < 0.01 vs. HFD-control. NC, normal chow; Control, HFD-control; HBU10, HFD plus HBU651 10 mg/kg; HBU30, HFD plus HBU651 30 mg/kg.

Figure 5

Effects of HBU651 on blood inflammatory monocytes and adipose tissue macrophages (ATM) in HFD-induced obese mice. (A) Flow cytometry of blood Ly6c^low monocytes (Mo), and (B) blood Ly6c^high monocytes, (C) Percentage of Ly6c^low monocytes, and (D) Ly6c^high monocytes, (E) Flow cytometry of CD45⁺, CD11b⁺, and F4/80⁺ ATM, and (F) CD11c⁺ inflammatory ATM, (G) Percentage of total ATMs, and (H) CD11c⁺ inflammatory ATM. Data are expressed as the mean ± SEM (n = 5). ^## p < 0.01, ^### p < 0.001 vs. NC, and * p < 0.05, ** p < 0.01 vs. HFD-control. NC, normal chow; Control, HFD-control; HBU10, HFD plus HBU651 10 mg/kg; HBU30, HFD plus HBU651 30 mg/kg.

Figure 6

Effects of HBU651 on histological analysis and inflammatory gene expression of the liver in HFD-induced obese mice. (A) Liver H&E staining and F4/80 immunostaining, Arrows indicate F4/80 positive cells. (B) Hepatic lipid droplet (LD) area. (C–G) Realtime qRT-PCR of hepatic mRNA for F4/80 (C), TNF-α (D), IL-1β (E), IL-6 (F), and MCP-1 (G). Data are expressed as the mean ± SEM (n = 5). ^### p < 0.001 vs. NC, and * p < 0.05, *** p < 0.001 vs. HFD-control. NC, normal chow; Control, HFD-control; HBU10, HFD plus HBU651 10 mg/kg; HBU30, HFD plus HBU651 30 mg/kg.

Figure 7

Effects of HBU651 on the histological analysis and inflammatory gene expression of the epididymal fat in HFD-induced obese mice. (A) EWAT H&E staining and F4/80 immunostaining, arrows indicate F4/80 positive cells. (B) Adipocyte size of the EWAT, (C–G) Realtime qRT-PCR of EWAT mRNA for F4/80 (C), TNF-α (D), IL-1β (E), IL-6 (F), and MCP-1 (G). Data are expressed as means ± SEM (n = 5). ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. NC, and * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HFD-control. NC, normal chow; Control, HFD-control; HBU10, HFD plus HBU651 10 mg/kg; HBU30, HFD plus HBU651 30 mg/kg.

Figure 8

HBU651 attenuates HFD-induced hypothalamic inflammation. (A,B) Representative images and higher magnification inset of the hypothalamus of HFD-fed mice. Hypothalamic sections were immunostained with GFAP (A) or Iba-1 (B) antibodies. The arcuate nucleus (ARC) area (arrow, inset box) and the third ventricle (3V) are indicated. Box area in ARC has been enlarged to view the cellular morphology. (C,D) Quantification of astrocytes (GFAP-positive cells) (C) and activated microglia (Iba-1 positive cells) (D) in the ARC. Data are expressed as means ± SEM (n = 5). ^### p < 0.001 vs. NC, and ** p < 0.01. *** p < 0.001 vs. HFD-control. NC, normal chow; Control, HFD-control; HBU10, HFD plus HBU651 10 mg/kg; HBU30, HFD plus HBU651 30 mg/kg.